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Nature Methods

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https://www.readbyqxmd.com/read/29131164/an-improved-ms2-system-for-accurate-reporting-of-the-mrna-life-cycle
#1
Evelina Tutucci, Maria Vera, Jeetayu Biswas, Jennifer Garcia, Roy Parker, Robert H Singer
The MS2-MCP system enables researchers to image multiple steps of the mRNA life cycle with high temporal and spatial resolution. However, for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, and this confounds the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, which allows mRNA degradation while preserving single-molecule detection determined by single-molecule FISH (smFISH) or live imaging...
November 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/29131163/profiling-the-transcriptome-with-rna-spots
#2
Chee-Huat Linus Eng, Sheel Shah, Julian Thomassie, Long Cai
Single-molecule FISH (smFISH) has been the gold standard for quantifying individual transcript abundances. Here, we scale up multiplexed smFISH to the transcriptome level and profile 10,212 different mRNAs from mouse fibroblast and embryonic stem cells. This method, called RNA sequential probing of targets (SPOTs), provides an accurate, flexible, and low-cost alternative to sequencing for profiling transcriptomes.
November 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/29131162/the-3d-orbisims-label-free-metabolic-imaging-with-subcellular-lateral-resolution-and-high-mass-resolving-power
#3
Melissa K Passarelli, Alexander Pirkl, Rudolf Moellers, Dmitry Grinfeld, Felix Kollmer, Rasmus Havelund, Carla F Newman, Peter S Marshall, Henrik Arlinghaus, Morgan R Alexander, Andy West, Stevan Horning, Ewald Niehuis, Alexander Makarov, Colin T Dollery, Ian S Gilmore
We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 μm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters-gamma-aminobutyric acid, dopamine and serotonin-with high spectroscopic confidence in the mouse hippocampus...
November 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/29106405/isolation-and-3d-expansion-of-multipotent-sox9-mouse-lung-progenitors
#4
Massimo Nichane, Asif Javed, V Sivakamasundari, Monisha Ganesan, Lay Teng Ang, Petra Kraus, Thomas Lufkin, Kyle M Loh, Bing Lim
Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9(+) multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture...
November 6, 2017: Nature Methods
https://www.readbyqxmd.com/read/29083403/an-objective-comparison-of-cell-tracking-algorithms
#5
Vladimír Ulman, Martin Maška, Klas E G Magnusson, Olaf Ronneberger, Carsten Haubold, Nathalie Harder, Pavel Matula, Petr Matula, David Svoboda, Miroslav Radojevic, Ihor Smal, Karl Rohr, Joakim Jaldén, Helen M Blau, Oleh Dzyubachyk, Boudewijn Lelieveldt, Pengdong Xiao, Yuexiang Li, Siu-Yeung Cho, Alexandre C Dufour, Jean-Christophe Olivo-Marin, Constantino C Reyes-Aldasoro, Jose A Solis-Lemus, Robert Bensch, Thomas Brox, Johannes Stegmaier, Ralf Mikut, Steffen Wolf, Fred A Hamprecht, Tiago Esteves, Pedro Quelhas, Ömer Demirel, Lars Malmström, Florian Jug, Pavel Tomancak, Erik Meijering, Arrate Muñoz-Barrutia, Michal Kozubek, Carlos Ortiz-de-Solorzano
We present a combined report on the results of three editions of the Cell Tracking Challenge, an ongoing initiative aimed at promoting the development and objective evaluation of cell segmentation and tracking algorithms. With 21 participating algorithms and a data repository consisting of 13 data sets from various microscopy modalities, the challenge displays today's state-of-the-art methodology in the field. We analyzed the challenge results using performance measures for segmentation and tracking that rank all participating methods...
October 30, 2017: Nature Methods
https://www.readbyqxmd.com/read/29083402/inducible-and-multiplex-gene-regulation-using-crispr-cpf1-based-transcription-factors
#6
Y Esther Tak, Benjamin P Kleinstiver, James K Nuñez, Jonathan Y Hsu, Joy E Horng, Jingyi Gong, Jonathan S Weissman, J Keith Joung
Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 nuclease fused to transcriptional activation domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes...
October 30, 2017: Nature Methods
https://www.readbyqxmd.com/read/29083401/high-throughput-image-based-screening-of-pooled-genetic-variant-libraries
#7
George Emanuel, Jeffrey R Moffitt, Xiaowei Zhuang
We report a high-throughput screening method that allows diverse genotypes and corresponding phenotypes to be imaged in individual cells. We achieve genotyping by introducing barcoded genetic variants into cells as pooled libraries and reading the barcodes out using massively multiplexed fluorescence in situ hybridization. To demonstrate the power of image-based pooled screening, we identified brighter and more photostable variants of the fluorescent protein YFAST among 60,000 variants.
October 30, 2017: Nature Methods
https://www.readbyqxmd.com/read/29083400/localization-based-super-resolution-imaging-meets-high-content-screening
#8
Anne Beghin, Adel Kechkar, Corey Butler, Florian Levet, Marine Cabillic, Olivier Rossier, Gregory Giannone, Rémi Galland, Daniel Choquet, Jean-Baptiste Sibarita
Single-molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single-molecule-based super-resolution methodology that operates in standard multiwell plates and uses analysis based on high-content screening and data-mining software...
October 30, 2017: Nature Methods
https://www.readbyqxmd.com/read/29058722/deciphering-lipid-structures-based-on-platform-independent-decision-rules
#9
Jürgen Hartler, Alexander Triebl, Andreas Ziegl, Martin Trötzmüller, Gerald N Rechberger, Oana A Zeleznik, Kathrin A Zierler, Federico Torta, Amaury Cazenave-Gassiot, Markus R Wenk, Alexander Fauland, Craig E Wheelock, Aaron M Armando, Oswald Quehenberger, Qifeng Zhang, Michael J O Wakelam, Guenter Haemmerle, Friedrich Spener, Harald C Köfeler, Gerhard G Thallinger
We achieve automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry using decision rule sets embedded in Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2). Using various low- and high-resolution mass spectrometry instruments with several collision energies, we proved the method's platform independence. We propose that the software's reliability, flexibility, and ability to identify novel lipid molecular species may now render current state-of-the-art lipid libraries obsolete...
October 23, 2017: Nature Methods
https://www.readbyqxmd.com/read/29039417/protein-interaction-perturbation-profiling-at-amino-acid-resolution
#10
Jonathan Woodsmith, Luise Apelt, Victoria Casado-Medrano, Ziya Özkan, Bernd Timmermann, Ulrich Stelzl
The identification of genomic variants in healthy and diseased individuals continues to rapidly outpace our ability to functionally annotate these variants. Techniques that both systematically assay the functional consequences of nucleotide-resolution variation and can scale to hundreds of genes are urgently required. We designed a sensitive yeast two-hybrid-based 'off switch' for positive selection of interaction-disruptive variants from complex genetic libraries. Combined with massively parallel programmed mutagenesis and a sequencing readout, this method enables systematic profiling of protein-interaction determinants at amino-acid resolution...
October 16, 2017: Nature Methods
https://www.readbyqxmd.com/read/29039416/antibodies-to-biotin-enable-large-scale-detection-of-biotinylation-sites-on-proteins
#11
Namrata D Udeshi, Kayvon Pedram, Tanya Svinkina, Shaunt Fereshetian, Samuel A Myers, Ozan Aygun, Karsten Krug, Karl Clauser, Dominic Ryan, Tslil Ast, Vamsi K Mootha, Alice Y Ting, Steven A Carr
Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites identified compared to streptavidin-based enrichment of proteins...
October 16, 2017: Nature Methods
https://www.readbyqxmd.com/read/29039415/crispr-umi-single-cell-lineage-tracing-of-pooled-crispr-cas9-screens
#12
Georg Michlits, Maria Hubmann, Szu-Hsien Wu, Gintautas Vainorius, Elena Budusan, Sergei Zhuk, Thomas R Burkard, Maria Novatchkova, Martin Aichinger, Yiqing Lu, John Reece-Hoyes, Roberto Nitsch, Daniel Schramek, Dominic Hoepfner, Ulrich Elling
Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells...
October 16, 2017: Nature Methods
https://www.readbyqxmd.com/read/28945705/thiol-linked-alkylation-of-rna-to-assess-expression-dynamics
#13
Veronika A Herzog, Brian Reichholf, Tobias Neumann, Philipp Rescheneder, Pooja Bhat, Thomas R Burkard, Wiebke Wlotzka, Arndt von Haeseler, Johannes Zuber, Stefan L Ameres
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (s(4)U) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA...
September 25, 2017: Nature Methods
https://www.readbyqxmd.com/read/28945703/autofocusing-maldi-mass-spectrometry-imaging-of-tissue-sections-and-3d-chemical-topography-of-nonflat-surfaces
#14
Mario Kompauer, Sven Heiles, Bernhard Spengler
We describe an atmospheric pressure matrix-assisted laser desorption-ionization mass spectrometry imaging system that uses long-distance laser triangulation on a micrometer scale to simultaneously obtain topographic and molecular information from 3D surfaces. We studied the topographic distribution of compounds on irregular 3D surfaces of plants and parasites, and we imaged nonplanar tissue sections with high lateral resolution, thereby eliminating height-related signal artifacts.
September 18, 2017: Nature Methods
https://www.readbyqxmd.com/read/29039418/rapid-nonlinear-image-scanning-microscopy
#15
Ingo Gregor, Martin Spiecker, Roman Petrovsky, Jörg Großhans, Robert Ros, Jörg Enderlein
Image scanning microscopy (ISM) doubles the resolution of a conventional confocal microscope for super-resolution imaging. Here, we describe an all-optical ISM design based on rescanning microscopy for two-photon-excited fluorescence and second-harmonic generation that allows straightforward implementation into existing microscopes. The design offers improved sensitivity and high frame rates relative to those of existing systems. We demonstrate its utility using fixed and living specimens as well as collagen hydrogels...
November 2017: Nature Methods
https://www.readbyqxmd.com/read/28991892/scenic-single-cell-regulatory-network-inference-and-clustering
#16
Sara Aibar, Carmen Bravo González-Blas, Thomas Moerman, Vân Anh Huynh-Thu, Hana Imrichova, Gert Hulselmans, Florian Rambow, Jean-Christophe Marine, Pierre Geurts, Jan Aerts, Joost van den Oord, Zeynep Kalender Atak, Jasper Wouters, Stein Aerts
We present SCENIC, a computational method for simultaneous gene regulatory network reconstruction and cell-state identification from single-cell RNA-seq data (http://scenic.aertslab.org). On a compendium of single-cell data from tumors and brain, we demonstrate that cis-regulatory analysis can be exploited to guide the identification of transcription factors and cell states. SCENIC provides critical biological insights into the mechanisms driving cellular heterogeneity.
November 2017: Nature Methods
https://www.readbyqxmd.com/read/28991891/achieving-better-than-3-%C3%A3-resolution-by-single-particle-cryo-em-at-200-kev
#17
Mark A Herzik, Mengyu Wu, Gabriel C Lander
Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.
November 2017: Nature Methods
https://www.readbyqxmd.com/read/28967890/long-term-expansion-of-alveolar-stem-cells-derived-from-human-ips-cells-in-organoids
#18
Yuki Yamamoto, Shimpei Gotoh, Yohei Korogi, Masahide Seki, Satoshi Konishi, Satoshi Ikeo, Naoyuki Sone, Tadao Nagasaki, Hisako Matsumoto, Shigeo Muro, Isao Ito, Toyohiro Hirai, Takashi Kohno, Yutaka Suzuki, Michiaki Mishima
The stable expansion of tissue-specific stem cells in vitro has contributed to research on several organs. Alveolar epithelial type II (AT2) cells function as tissue stem cells in the lung, but robust models for studying human AT2 cells are lacking. Here we report a method for the efficient generation and long-term expansion of alveolar organoids (AOs) harboring SFTPC(+) alveolar stem cells derived from human induced pluripotent stem cells (hiPSCs). hiPSC-derived SFTPC(+) cells self-renewed, with transcriptomes and morphology consistent with those of AT2 cells, and were able to differentiate into alveolar epithelial type I (AT1)-like cells...
November 2017: Nature Methods
https://www.readbyqxmd.com/read/28967889/neubtracker-imaging-neurobehavioral-dynamics-in-freely-behaving-fish
#19
Panagiotis Symvoulidis, Antonella Lauri, Anca Stefanoiu, Michele Cappetta, Steffen Schneider, Hongbo Jia, Anja Stelzl, Maximilian Koch, Carlos Cruz Perez, Ahne Myklatun, Sabine Renninger, Andriy Chmyrov, Tobias Lasser, Wolfgang Wurst, Vasilis Ntziachristos, Gil G Westmeyer
A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior...
November 2017: Nature Methods
https://www.readbyqxmd.com/read/28967888/critical-assessment-of-metagenome-interpretation-a-benchmark-of-metagenomics-software
#20
Alexander Sczyrba, Peter Hofmann, Peter Belmann, David Koslicki, Stefan Janssen, Johannes Dröge, Ivan Gregor, Stephan Majda, Jessika Fiedler, Eik Dahms, Andreas Bremges, Adrian Fritz, Ruben Garrido-Oter, Tue Sparholt Jørgensen, Nicole Shapiro, Philip D Blood, Alexey Gurevich, Yang Bai, Dmitrij Turaev, Matthew Z DeMaere, Rayan Chikhi, Niranjan Nagarajan, Christopher Quince, Fernando Meyer, Monika Balvočiūtė, Lars Hestbjerg Hansen, Søren J Sørensen, Burton K H Chia, Bertrand Denis, Jeff L Froula, Zhong Wang, Robert Egan, Dongwan Don Kang, Jeffrey J Cook, Charles Deltel, Michael Beckstette, Claire Lemaitre, Pierre Peterlongo, Guillaume Rizk, Dominique Lavenier, Yu-Wei Wu, Steven W Singer, Chirag Jain, Marc Strous, Heiner Klingenberg, Peter Meinicke, Michael D Barton, Thomas Lingner, Hsin-Hung Lin, Yu-Chieh Liao, Genivaldo Gueiros Z Silva, Daniel A Cuevas, Robert A Edwards, Surya Saha, Vitor C Piro, Bernhard Y Renard, Mihai Pop, Hans-Peter Klenk, Markus Göker, Nikos C Kyrpides, Tanja Woyke, Julia A Vorholt, Paul Schulze-Lefert, Edward M Rubin, Aaron E Darling, Thomas Rattei, Alice C McHardy
Methods for assembly, taxonomic profiling and binning are key to interpreting metagenome data, but a lack of consensus about benchmarking complicates performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on highly complex and realistic data sets, generated from ∼700 newly sequenced microorganisms and ∼600 novel viruses and plasmids and representing common experimental setups. Assembly and genome binning programs performed well for species represented by individual genomes but were substantially affected by the presence of related strains...
November 2017: Nature Methods
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