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Nature Methods

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https://www.readbyqxmd.com/read/29334379/highly-parallel-direct-rna-sequencing-on-an-array-of-nanopores
#1
Daniel R Garalde, Elizabeth A Snell, Daniel Jachimowicz, Botond Sipos, Joseph H Lloyd, Mark Bruce, Nadia Pantic, Tigist Admassu, Phillip James, Anthony Warland, Michael Jordan, Jonah Ciccone, Sabrina Serra, Jemma Keenan, Samuel Martin, Luke McNeill, E Jayne Wallace, Lakmal Jayasinghe, Chris Wright, Javier Blasco, Stephen Young, Denise Brocklebank, Sissel Juul, James Clarke, Andrew J Heron, Daniel J Turner
Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA...
January 15, 2018: Nature Methods
https://www.readbyqxmd.com/read/29334378/supermultiplexed-optical-imaging-and-barcoding-with-engineered-polyynes
#2
Fanghao Hu, Chen Zeng, Rong Long, Yupeng Miao, Lu Wei, Qizhi Xu, Wei Min
Optical multiplexing has a large impact in photonics, the life sciences and biomedicine. However, current technology is limited by a 'multiplexing ceiling' from existing optical materials. Here we engineered a class of polyyne-based materials for optical supermultiplexing. We achieved 20 distinct Raman frequencies, as 'Carbon rainbow', through rational engineering of conjugation length, bond-selective isotope doping and end-capping substitution of polyynes. With further probe functionalization, we demonstrated ten-color organelle imaging in individual living cells with high specificity, sensitivity and photostability...
January 15, 2018: Nature Methods
https://www.readbyqxmd.com/read/29334377/detecting-hierarchical-genome-folding-with-network-modularity
#3
Heidi K Norton, Daniel J Emerson, Harvey Huang, Jesi Kim, Katelyn R Titus, Shi Gu, Danielle S Bassett, Jennifer E Phillips-Cremins
Mammalian genomes are folded in a hierarchy of compartments, topologically associating domains (TADs), subTADs and looping interactions. Here, we describe 3DNetMod, a graph theory-based method for sensitive and accurate detection of chromatin domains across length scales in Hi-C data. We identify nested, partially overlapping TADs and subTADs genome wide by optimizing network modularity and varying a single resolution parameter. 3DNetMod can be applied broadly to understand genome reconfiguration in development and disease...
January 15, 2018: Nature Methods
https://www.readbyqxmd.com/read/29309061/giggle-a-search-engine-for-large-scale-integrated-genome-analysis
#4
Ryan M Layer, Brent S Pedersen, Tonya DiSera, Gabor T Marth, Jason Gertz, Aaron R Quinlan
GIGGLE is a genomics search engine that identifies and ranks the significance of genomic loci shared between query features and thousands of genome interval files. GIGGLE (https://github.com/ryanlayer/giggle) scales to billions of intervals and is over three orders of magnitude faster than existing methods. Its speed extends the accessibility and utility of resources such as ENCODE, Roadmap Epigenomics, and GTEx by facilitating data integration and hypothesis generation.
January 8, 2018: Nature Methods
https://www.readbyqxmd.com/read/29256494/biotinylation-by-antibody-recognition-a-method-for-proximity-labeling
#5
Daniel Z Bar, Kathleen Atkatsh, Urraca Tavarez, Michael R Erdos, Yosef Gruenbaum, Francis S Collins
The high-throughput detection of organelle composition and proteomic mapping of protein environment directly from primary tissue as well as the identification of interactors of insoluble proteins that form higher-order structures have remained challenges in biological research. We report a proximity-based labeling approach that uses an antibody to a target antigen to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues, which allows proteins in close proximity to the target antigen to be captured and identified by mass spectrometry...
December 18, 2017: Nature Methods
https://www.readbyqxmd.com/read/29256496/resolving-systematic-errors-in-widely-used-enhancer-activity-assays-in-human-cells
#6
Felix Muerdter, Łukasz M Boryń, Ashley R Woodfin, Christoph Neumayr, Martina Rath, Muhammad A Zabidi, Michaela Pagani, Vanja Haberle, Tomáš Kazmar, Rui R Catarino, Katharina Schernhuber, Cosmas D Arnold, Alexander Stark
The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated...
December 11, 2017: Nature Methods
https://www.readbyqxmd.com/read/29256495/mapping-the-3d-orientation-of-piconewton-integrin-traction-forces
#7
Joshua M Brockman, Aaron T Blanchard, Victor Pui-Yan, Wallace D Derricotte, Yun Zhang, Meredith E Fay, Wilbur A Lam, Francesco A Evangelista, Alexa L Mattheyses, Khalid Salaita
Mechanical forces are integral to many biological processes; however, current techniques cannot map the magnitude and direction of piconewton molecular forces. Here, we describe molecular force microscopy, leveraging molecular tension probes and fluorescence polarization microscopy to measure the magnitude and 3D orientation of cellular forces. We mapped the orientation of integrin-based traction forces in mouse fibroblasts and human platelets, revealing alignment between the organization of force-bearing structures and their force orientations...
December 11, 2017: Nature Methods
https://www.readbyqxmd.com/read/29256493/brca-deficient-mouse-mammary-tumor-organoids-to-study-cancer-drug-resistance
#8
Alexandra A Duarte, Ewa Gogola, Norman Sachs, Marco Barazas, Stefano Annunziato, Julian R de Ruiter, Arno Velds, Sohvi Blatter, Julia M Houthuijzen, Marieke van de Ven, Hans Clevers, Piet Borst, Jos Jonkers, Sven Rottenberg
Poly(ADP-ribose) polymerase inhibition (PARPi) is a promising new therapeutic approach for the treatment of cancers that show homologous recombination deficiency (HRD). Despite the success of PARPi in targeting HRD in tumors that lack the tumor suppressor function of BRCA1 or BRCA2, drug resistance poses a major obstacle. We developed three-dimensional cancer organoids derived from genetically engineered mouse models (GEMMs) for BRCA1- and BRCA2-deficient cancers. Unlike conventional cell lines or mammospheres, organoid cultures can be efficiently derived and rapidly expanded in vitro...
December 11, 2017: Nature Methods
https://www.readbyqxmd.com/read/29200199/programmable-full-adder-computations-in-communicating-three-dimensional-cell-cultures
#9
David Ausländer, Simon Ausländer, Xavier Pierrat, Leon Hellmann, Leila Rachid, Martin Fussenegger
Synthetic biologists have advanced the design of trigger-inducible gene switches and their assembly into input-programmable circuits that enable engineered human cells to perform arithmetic calculations reminiscent of electronic circuits. By designing a versatile plug-and-play molecular-computation platform, we have engineered nine different cell populations with genetic programs, each of which encodes a defined computational instruction. When assembled into 3D cultures, these engineered cell consortia execute programmable multicellular full-adder logics in response to three trigger compounds...
December 4, 2017: Nature Methods
https://www.readbyqxmd.com/read/29200198/netsig-network-based-discovery-from-cancer-genomes
#10
Heiko Horn, Michael S Lawrence, Candace R Chouinard, Yashaswi Shrestha, Jessica Xin Hu, Elizabeth Worstell, Emily Shea, Nina Ilic, Eejung Kim, Atanas Kamburov, Alireza Kashani, William C Hahn, Joshua D Campbell, Jesse S Boehm, Gad Getz, Kasper Lage
Methods that integrate molecular network information and tumor genome data could complement gene-based statistical tests to identify likely new cancer genes; but such approaches are challenging to validate at scale, and their predictive value remains unclear. We developed a robust statistic (NetSig) that integrates protein interaction networks with data from 4,742 tumor exomes. NetSig can accurately classify known driver genes in 60% of tested tumor types and predicts 62 new driver candidates. Using a quantitative experimental framework to determine in vivo tumorigenic potential in mice, we found that NetSig candidates induce tumors at rates that are comparable to those of known oncogenes and are ten-fold higher than those of random genes...
December 4, 2017: Nature Methods
https://www.readbyqxmd.com/read/29176591/identifying-metabolites-by-integrating-metabolome-databases-with-mass-spectrometry-cheminformatics
#11
Zijuan Lai, Hiroshi Tsugawa, Gert Wohlgemuth, Sajjan Mehta, Matthew Mueller, Yuxuan Zheng, Atsushi Ogiwara, John Meissen, Megan Showalter, Kohei Takeuchi, Tobias Kind, Peter Beal, Masanori Arita, Oliver Fiehn
Novel metabolites distinct from canonical pathways can be identified through the integration of three cheminformatics tools: BinVestigate, which queries the BinBase gas chromatography-mass spectrometry (GC-MS) metabolome database to match unknowns with biological metadata across over 110,000 samples; MS-DIAL 2.0, a software tool for chromatographic deconvolution of high-resolution GC-MS or liquid chromatography-mass spectrometry (LC-MS); and MS-FINDER 2.0, a structure-elucidation program that uses a combination of 14 metabolome databases in addition to an enzyme promiscuity library...
November 27, 2017: Nature Methods
https://www.readbyqxmd.com/read/29155427/pdb-wide-identification-of-biological-assemblies-from-conserved-quaternary-structure-geometry
#12
Sucharita Dey, David W Ritchie, Emmanuel D Levy
Protein structures are key to understanding biomolecular mechanisms and diseases, yet their interpretation is hampered by limited knowledge of their biologically relevant quaternary structure (QS). A critical challenge in inferring QS information from crystallographic data is distinguishing biological interfaces from fortuitous crystal-packing contacts. Here, we tackled this problem by developing strategies for aligning and comparing QS states across both homologs and data repositories. QS conservation across homologs proved remarkably strong at predicting biological relevance and is implemented in two methods, QSalign and anti-QSalign, for annotating homo-oligomers and monomers, respectively...
November 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/29131164/an-improved-ms2-system-for-accurate-reporting-of-the-mrna-life-cycle
#13
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/29106405/isolation-and-3d-expansion-of-multipotent-sox9-mouse-lung-progenitors
#14
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/29083402/inducible-and-multiplex-gene-regulation-using-crispr-cpf1-based-transcription-factors
#15
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/29083400/localization-based-super-resolution-imaging-meets-high-content-screening
#16
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/29298293/a-profusion-of-confusion-in-ngs-methods-naming
#17
James Hadfield, Jacques Retief
No abstract text is available yet for this article.
January 3, 2018: Nature Methods
https://www.readbyqxmd.com/read/29298292/imaging-organoids-a-bright-future-ahead
#18
Anne C Rios, Hans Clevers
Organogenesis, tissue homeostasis and organ function involve complex spatial cellular organization and tissue dynamics. The underlying mechanisms of these processes, and how they are disrupted in disease, are challenging to address in vivo and ethically impossible to study in human. Organoids, three-dimensional (3D) stem cell cultures that self-organize into ex vivo 'mini-organs', now open a new window onto cellular processes within tissue. Light microscopy is a powerful approach to probe the cellular complexity that can be modeled with organoids...
January 3, 2018: Nature Methods
https://www.readbyqxmd.com/read/29298291/chemical-biology-fats-as-research-subjects
#19
Vivien Marx
No abstract text is available yet for this article.
January 3, 2018: Nature Methods
https://www.readbyqxmd.com/read/29298290/motifstack-for-the-analysis-of-transcription-factor-binding-site-evolution
#20
Jianhong Ou, Scot A Wolfe, Michael H Brodsky, Lihua Julie Zhu
No abstract text is available yet for this article.
January 3, 2018: Nature Methods
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