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The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.
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Núcleo Celular , Genoma , Genoma/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismoRESUMEN
For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.
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Metadatos , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Aplicaciones Móviles , Lenguajes de Programación , Programas Informáticos , Animales , Línea Celular , Biología Computacional/métodos , Humanos , Procesamiento de Imagen Asistido por Computador , Ratones , Reconocimiento de Normas Patrones Automatizadas , Control de Calidad , Reproducibilidad de los Resultados , Interfaz Usuario-Computador , Flujo de TrabajoRESUMEN
Although the nuclear pore complex (NPC) is best known for its primary function as the key regulator of molecular traffic between the cytoplasm and the nucleus, a growing body of experimental evidence suggests that this structure participates in a considerably broader range of cellular activities on both sides of the nuclear envelope. Indeed, the NPC is emerging as an important regulator of gene expression through its influence on the internal architectural organization of the nucleus and its apparently extensive involvement in coordinating the seamless delivery of genetic information to the cytoplasmic protein synthesis machinery.
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Transporte Activo de Núcleo Celular/fisiología , Regulación de la Expresión Génica , Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular/genética , Animales , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Humanos , Modelos Biológicos , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiologíaRESUMEN
A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.
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Microscopía , Estándares de Referencia , Reproducibilidad de los ResultadosAsunto(s)
Núcleo Celular/ultraestructura , Cromatina/ultraestructura , Procesamiento de Imagen Asistido por Computador/normas , Informática Médica/estadística & datos numéricos , Metadatos/estadística & datos numéricos , Microscopía/métodos , Programas Informáticos , Humanos , Procesamiento de Imagen Asistido por Computador/métodosAsunto(s)
Metadatos , Microscopía/instrumentación , Microscopía/métodos , Microscopía/normas , Animales , Investigación Biomédica/organización & administración , Calibración , Recolección de Datos , Minería de Datos/normas , Humanos , Control de Calidad , Reproducibilidad de los Resultados , Sociedades Científicas , Programas Informáticos , Integración de Sistemas , Interfaz Usuario-ComputadorAsunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/instrumentación , Microscopía/métodos , Microscopía/normas , Investigación Biomédica/organización & administración , Calibración , Humanos , Metadatos , Control de Calidad , Reproducibilidad de los Resultados , Programas InformáticosAsunto(s)
Biología Computacional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Metadatos , Algoritmos , Animales , Congresos como Asunto , Microscopía por Crioelectrón/métodos , Minería de Datos/métodos , Bases de Datos Factuales , Diagnóstico por Imagen/métodos , Humanos , Microscopía , Programas InformáticosRESUMEN
RATIONALE: In peptide quantification by liquid chromatography/mass spectrometry (LC/MS), the optimization of multiple reaction monitoring (MRM) parameters is essential for sensitive detection. We have compared different approaches to build MRM assays, based either on flow injection analysis (FIA) of isotopically labelled peptides, or on the knowledge and the prediction of the best settings for MRM transitions and collision energies (CE). In this context, we introduce MRMOptimizer, an open-source software tool that processes spectra and assists the user in selecting transitions in the FIA workflow. METHODS: MS/MS spectral libraries with CE voltages from 10 to 70 V are automatically acquired in FIA mode for isotopically labelled peptides. Then MRMOptimizer determines the optimal MRM settings for each peptide. To assess the quantitative performance of our approach, 155 peptides, representing 84 proteins, were analysed by LC/MRM-MS and the peak areas were compared between: (A) the MRMOptimizer-based workflow, (B1) the SRMAtlas transitions set used 'as-is'; (B2) the same SRMAtlas set with CE parameters optimized by Skyline. RESULTS: 51% of the three most intense transitions per peptide were shown to be common to both A and B1/B2 methods, and displayed similar sensitivity and peak area distributions. The peak areas obtained with MRMOptimizer for transitions sharing either the precursor ion charge state or the fragment ions with the SRMAtlas set at unique transitions were increased 1.8- to 2.3-fold. The gain in sensitivity using MRMOptimizer for transitions with different precursor ion charge state and fragment ions (8% of the total), reaches a ~ 11-fold increase. CONCLUSIONS: Isotopically labelled peptides can be used to optimize MRM transitions more efficiently in FIA than by searching databases. The MRMOptimizer software is MS independent and enables the post-acquisition selection of MRM parameters. Coefficients of variation for optimal CE values are lower than those obtained with the SRMAtlas approach (B2) and one additional peptide was detected. Copyright © 2017 John Wiley & Sons, Ltd.
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Cromatografía Liquida/métodos , Fragmentos de Péptidos/análisis , Espectrometría de Masas en Tándem/métodos , Células Cultivadas , Bases de Datos Factuales , Células Dendríticas/química , Humanos , Iones/análisis , Iones/química , Modelos Lineales , Fragmentos de Péptidos/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , TripsinaRESUMEN
TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.
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Cápside/química , Cápside/inmunología , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Inmunidad Innata/inmunología , Retroviridae/inmunología , Factores de Restricción Antivirales , Proteínas Portadoras/genética , Línea Celular , Activación Enzimática , Células HEK293 , VIH-1/química , VIH-1/inmunología , Humanos , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/metabolismo , Unión Proteica , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Retroviridae/química , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Data-independent acquisition (DIA) offers several advantages over data-dependent acquisition (DDA) schemes for characterizing complex protein digests analyzed by LC-MS/MS. In contrast to the sequential detection, selection, and analysis of individual ions during DDA, DIA systematically parallelizes the fragmentation of all detectable ions within a wide m/z range regardless of intensity, thereby providing broader dynamic range of detected signals, improved reproducibility for identification, better sensitivity, and accuracy for quantification, and, potentially, enhanced proteome coverage. To fully exploit these advantages, composite or multiplexed fragment ion spectra generated by DIA require more elaborate processing algorithms compared to DDA. This review examines different DIA schemes and, in particular, discusses the concepts applied to and related to data processing. Available software implementations for identification and quantification are presented as comprehensively as possible and examples of software usage are cited. Processing workflows, including complete proprietary frameworks or combinations of modules from different open source data processing packages are described and compared in terms of software availability and usability, programming language, operating system support, input/output data formats, as well as the main principles employed in the algorithms used for identification and quantification. This comparative study concludes with further discussion of current limitations and expectable improvements in the short- and midterm future.
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Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Proteómica/métodos , Programas Informáticos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
As tryptic peptides and metabolites are not equally distributed along the mass range, the probability of cross fragment ion interference is higher in certain windows when fixed Q1 SWATH windows are applied. We evaluated the benefits of utilizing variable Q1 SWATH windows with regards to selectivity improvement. Variable windows based on equalizing the distribution of either the precursor ion population (PIP) or the total ion current (TIC) within each window were generated by an in-house software, swathTUNER. These two variable Q1 SWATH window strategies outperformed, with respect to quantification and identification, the basic approach using a fixed window width (FIX) for proteomic profiling of human monocyte-derived dendritic cells (MDDCs). Thus, 13.8 and 8.4% additional peptide precursors, which resulted in 13.1 and 10.0% more proteins, were confidently identified by SWATH using the strategy PIP and TIC, respectively, in the MDDC proteomic sample. On the basis of the spectral library purity score, some improvement warranted by variable Q1 windows was also observed, albeit to a lesser extent, in the metabolomic profiling of human urine. We show that the novel concept of "scheduled SWATH" proposed here, which incorporates (i) variable isolation windows and (ii) precursor retention time segmentation further improves both peptide and metabolite identifications.
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Células Dendríticas/química , Péptidos/orina , Proteoma/aislamiento & purificación , Proteómica/métodos , Programas Informáticos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Cromatografía de Fase Inversa , Humanos , Datos de Secuencia Molecular , Cultivo Primario de Células , Proteolisis , Proteómica/instrumentación , Espectrometría de Masas en Tándem/instrumentación , Tripsina/químicaRESUMEN
Data-independent acquisition LC-MS/MS techniques complement supervised methods for peptide quantification. However, due to the wide precursor isolation windows, these techniques are prone to interference at the fragment ion level, which, in turn, is detrimental for accurate quantification. The nonoutlier fragment ion (NOFI) ranking algorithm has been developed to assign low priority to fragment ions affected by interference. By using the optimal subset of high-priority fragment ions, these interfered fragment ions are effectively excluded from quantification. NOFI represents each fragment ion as a vector of four dimensions related to chromatographic and MS fragmentation attributes and applies multivariate outlier detection techniques. Benchmarking conducted on a well-defined quantitative data set (i.e., the SWATH Gold Standard) indicates that NOFI on average is able to accurately quantify 11-25% more peptides than the commonly used Top-N library intensity ranking method. The sum of the area of the Top3-5 NOFIs produces similar coefficients of variation as compared to that with the library intensity method but with more accurate quantification results. On a biologically relevant human dendritic cell digest data set, NOFI properly assigns low-priority ranks to 85% of annotated interferences, resulting in sensitivity values between 0.92 and 0.80, against 0.76 for the Spectronaut interference detection algorithm.
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Algoritmos , Cromatografía Liquida/métodos , Fragmentos de Péptidos/análisis , Proteoma/análisis , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Benchmarking , Células Dendríticas/química , Células HeLa , Humanos , Iones , Datos de Secuencia Molecular , Cultivo Primario de Células , Proteolisis , Proteoma/químicaRESUMEN
Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry (MS)-based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize MS coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilization and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA (radioimmunoprecipitation assay) buffer, was shown to be the method of choice based on total protein extraction and on the solubilization and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than that by 10% trichloroacetic acid (TCA)/acetone, allowing in excess of 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate into the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6 to 11% more distinct peptides and 14 to 19% more total proteins identified than using 0.5M triethylammonium bicarbonate alone, with the greatest increase (34%) for hydrophobic proteins.
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Métodos Analíticos de la Preparación de la Muestra/métodos , Células Dendríticas/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Acetona/química , Tampones (Química) , Precipitación Química , Células Dendríticas/citología , Humanos , Monocitos/citología , Octoxinol/química , Desnaturalización Proteica , Transporte de Proteínas , Ensayo de Radioinmunoprecipitación , Solubilidad , Ácido Tricloroacético/químicaRESUMEN
Open and practical exchange, dissemination, and reuse of specimens and data have become a fundamental requirement for life sciences research. The quality of the data obtained and thus the findings and knowledge derived is thus significantly influenced by the quality of the samples, the experimental methods, and the data analysis. Therefore, a comprehensive and precise documentation of the pre-analytical conditions, the analytical procedures, and the data processing are essential to be able to assess the validity of the research results. With the increasing importance of the exchange, reuse, and sharing of data and samples, procedures are required that enable cross-organizational documentation, traceability, and non-repudiation. At present, this information on the provenance of samples and data is mostly either sparse, incomplete, or incoherent. Since there is no uniform framework, this information is usually only provided within the organization and not interoperably. At the same time, the collection and sharing of biological and environmental specimens increasingly require definition and documentation of benefit sharing and compliance to regulatory requirements rather than consideration of pure scientific needs. In this publication, we present an ongoing standardization effort to provide trustworthy machine-actionable documentation of the data lineage and specimens. We would like to invite experts from the biotechnology and biomedical fields to further contribute to the standard.
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Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled "Enabling Global Image Data Sharing in the Life Sciences," which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data (arXiv:2401.13023 [q-bio.OT], https://doi.org/10.48550/arXiv.2401.13023). In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges, and democratize access to common practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.