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Nature Methods

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https://www.readbyqxmd.com/read/28319114/internally-tagged-ubiquitin-a-tool-to-identify-linear-polyubiquitin-modified-proteins-by-mass-spectrometry
#1
Katarzyna Kliza, Christoph Taumer, Irene Pinzuti, Mirita Franz-Wachtel, Simone Kunzelmann, Benjamin Stieglitz, Boris Macek, Koraljka Husnjak
Ubiquitination controls a plethora of cellular processes. Modifications by linear polyubiquitin have so far been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (OTULIN) and a very few linear polyubiquitinated substrates have been identified. Current methods for studying lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present an approach to discovering linear polyubiquitin-modified substrates by combining a lysine-less internally tagged ubiquitin (INT-Ub...
March 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28319113/combinatorial-crispr-cas9-screens-for-de-novo-mapping-of-genetic-interactions
#2
John Paul Shen, Dongxin Zhao, Roman Sasik, Jens Luebeck, Amanda Birmingham, Ana Bojorquez-Gomez, Katherine Licon, Kristin Klepper, Daniel Pekin, Alex N Beckett, Kyle Salinas Sanchez, Alex Thomas, Chih-Chung Kuo, Dan Du, Assen Roguev, Nathan E Lewis, Aaron N Chang, Jason F Kreisberg, Nevan Krogan, Lei Qi, Trey Ideker, Prashant Mali
We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies...
March 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28319112/accurate-identification-of-single-nucleotide-variants-in-whole-genome-amplified-single-cells
#3
Xiao Dong, Lei Zhang, Brandon Milholland, Moonsook Lee, Alexander Y Maslov, Tao Wang, Jan Vijg
Mutation analysis in single-cell genomes is prone to artifacts associated with cell lysis and whole-genome amplification. Here we addressed these issues by developing single-cell multiple displacement amplification (SCMDA) and a general-purpose single-cell-variant caller, SCcaller (https://github.com/biosinodx/SCcaller/). By comparing SCMDA-amplified single cells with unamplified clones from the same population, we validated the procedure as a firm foundation for standardized somatic-mutation analysis in single-cell genomics...
March 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28319111/volumetric-two-photon-imaging-of-neurons-using-stereoscopy-vtwins
#4
Alexander Song, Adam S Charles, Sue Ann Koay, Jeff L Gauthier, Stephan Y Thiberge, Jonathan W Pillow, David W Tank
Two-photon laser scanning microscopy of calcium dynamics using fluorescent indicators is a widely used imaging method for large-scale recording of neural activity in vivo. Here, we introduce volumetric two-photon imaging of neurons using stereoscopy (vTwINS), a volumetric calcium imaging method that uses an elongated, V-shaped point spread function to image a 3D brain volume. Single neurons project to spatially displaced 'image pairs' in the resulting 2D image, and the separation distance between projections is proportional to depth in the volume...
March 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28288123/optogenetic-control-with-a-photocleavable-protein-phocl
#5
Wei Zhang, Alexander W Lohman, Yevgeniya Zhuravlova, Xiaocen Lu, Matthew D Wiens, Hiofan Hoi, Sine Yaganoglu, Manuel A Mohr, Elena N Kitova, John S Klassen, Periklis Pantazis, Roger J Thompson, Robert E Campbell
To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.
March 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/28288122/genetically-encoded-biosensors-for-visualizing-live-cell-biochemical-activity-at-super-resolution
#6
Gary C H Mo, Brian Ross, Fabian Hertel, Premashis Manna, Xinxing Yang, Eric Greenwald, Chris Booth, Ashlee M Plummer, Brian Tenner, Zan Chen, Yuxiao Wang, Eileen J Kennedy, Philip A Cole, Karen G Fleming, Amy Palmer, Ralph Jimenez, Jie Xiao, Peter Dedecker, Jin Zhang
Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging...
March 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/28288121/a-genetic-system-to-study-plasmodium-falciparum-protein-function
#7
Jakob Birnbaum, Sven Flemming, Nick Reichard, Alexandra Blancke Soares, Paolo Mesén-Ramírez, Ernst Jonscher, Bärbel Bergmann, Tobias Spielmann
Current systems to study essential genes in the human malaria parasite Plasmodium falciparum are often inefficient and time intensive, and they depend on the genetic modification of the target locus, a process hindered by the low frequency of integration of episomal DNA into the genome. Here, we introduce a method, termed selection-linked integration (SLI), to rapidly select for genomic integration. SLI allowed us to functionally analyze targets at the gene and protein levels, thus permitting mislocalization of native proteins, a strategy known as knock sideways, floxing to induce diCre-based excision of genes and knocking in altered gene copies...
March 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/28263961/power-analysis-of-single-cell-rna-sequencing-experiments
#8
Valentine Svensson, Kedar Nath Natarajan, Lam-Ha Ly, Ricardo J Miragaia, Charlotte Labalette, Iain C Macaulay, Ana Cvejic, Sarah A Teichmann
Single-cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, thereby revealing new cell types and providing insights into developmental processes and transcriptional stochasticity. A key question is how the variety of available protocols compare in terms of their ability to detect and accurately quantify gene expression. Here, we assessed the protocol sensitivity and accuracy of many published data sets, on the basis of spike-in standards and uniform data processing...
March 6, 2017: Nature Methods
https://www.readbyqxmd.com/read/28263960/visualization-and-analysis-of-single-cell-rna-seq-data-by-kernel-based-similarity-learning
#9
Bo Wang, Junjie Zhu, Emma Pierson, Daniele Ramazzotti, Serafim Batzoglou
We present single-cell interpretation via multikernel learning (SIMLR), an analytic framework and software which learns a similarity measure from single-cell RNA-seq data in order to perform dimension reduction, clustering and visualization. On seven published data sets, we benchmark SIMLR against state-of-the-art methods. We show that SIMLR is scalable and greatly enhances clustering performance while improving the visualization and interpretability of single-cell sequencing data.
March 6, 2017: Nature Methods
https://www.readbyqxmd.com/read/28263959/salmon-provides-fast-and-bias-aware-quantification-of-transcript-expression
#10
Rob Patro, Geet Duggal, Michael I Love, Rafael A Irizarry, Carl Kingsford
We introduce Salmon, a lightweight method for quantifying transcript abundance from RNA-seq reads. Salmon combines a new dual-phase parallel inference algorithm and feature-rich bias models with an ultra-fast read mapping procedure. It is the first transcriptome-wide quantifier to correct for fragment GC-content bias, which, as we demonstrate here, substantially improves the accuracy of abundance estimates and the sensitivity of subsequent differential expression analysis.
March 6, 2017: Nature Methods
https://www.readbyqxmd.com/read/28250468/drop-on-demand-sample-delivery-for-studying-biocatalysts-in-action-at-x-ray-free-electron-lasers
#11
Franklin D Fuller, Sheraz Gul, Ruchira Chatterjee, E Sethe Burgie, Iris D Young, Hugo Lebrette, Vivek Srinivas, Aaron S Brewster, Tara Michels-Clark, Jonathan A Clinger, Babak Andi, Mohamed Ibrahim, Ernest Pastor, Casper de Lichtenberg, Rana Hussein, Christopher J Pollock, Miao Zhang, Claudiu A Stan, Thomas Kroll, Thomas Fransson, Clemens Weninger, Markus Kubin, Pierre Aller, Louise Lassalle, Philipp Bräuer, Mitchell D Miller, Muhamed Amin, Sergey Koroidov, Christian G Roessler, Marc Allaire, Raymond G Sierra, Peter T Docker, James M Glownia, Silke Nelson, Jason E Koglin, Diling Zhu, Matthieu Chollet, Sanghoon Song, Henrik Lemke, Mengning Liang, Dimosthenis Sokaras, Roberto Alonso-Mori, Athina Zouni, Johannes Messinger, Uwe Bergmann, Amie K Boal, J Martin Bollinger, Carsten Krebs, Martin Högbom, George N Phillips, Richard D Vierstra, Nicholas K Sauter, Allen M Orville, Jan Kern, Vittal K Yachandra, Junko Yano
X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method...
February 27, 2017: Nature Methods
https://www.readbyqxmd.com/read/28250467/automated-synaptic-connectivity-inference-for-volume-electron-microscopy
#12
Sven Dorkenwald, Philipp J Schubert, Marius F Killinger, Gregor Urban, Shawn Mikula, Fabian Svara, Joergen Kornfeld
Teravoxel volume electron microscopy data sets from neural tissue can now be acquired in weeks, but data analysis requires years of manual labor. We developed the SyConn framework, which uses deep convolutional neural networks and random forest classifiers to infer a richly annotated synaptic connectivity matrix from manual neurite skeleton reconstructions by automatically identifying mitochondria, synapses and their types, axons, dendrites, spines, myelin, somata and cell types. We tested our approach on serial block-face electron microscopy data sets from zebrafish, mouse and zebra finch, and computed the synaptic wiring of songbird basal ganglia...
February 27, 2017: Nature Methods
https://www.readbyqxmd.com/read/28250466/motioncor2-anisotropic-correction-of-beam-induced-motion-for-improved-cryo-electron-microscopy
#13
Shawn Q Zheng, Eugene Palovcak, Jean-Paul Armache, Kliment A Verba, Yifan Cheng, David A Agard
No abstract text is available yet for this article.
February 27, 2017: Nature Methods
https://www.readbyqxmd.com/read/28218900/in-vivo-three-photon-imaging-of-activity-of-gcamp6-labeled-neurons-deep-in-intact-mouse-brain
#14
Dimitre G Ouzounov, Tianyu Wang, Mengran Wang, Danielle D Feng, Nicholas G Horton, Jean C Cruz-Hernández, Yu-Ting Cheng, Jacob Reimer, Andreas S Tolias, Nozomi Nishimura, Chris Xu
High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 150 neurons in the hippocampal stratum pyramidale at ∼1-mm depth within an intact mouse brain. Our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues...
February 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28218899/prospective-identification-of-hematopoietic-lineage-choice-by-deep-learning
#15
Felix Buggenthin, Florian Buettner, Philipp S Hoppe, Max Endele, Manuel Kroiss, Michael Strasser, Michael Schwarzfischer, Dirk Loeffler, Konstantinos D Kokkaliaris, Oliver Hilsenbeck, Timm Schroeder, Fabian J Theis, Carsten Marr
Differentiation alters molecular properties of stem and progenitor cells, leading to changes in their shape and movement characteristics. We present a deep neural network that prospectively predicts lineage choice in differentiating primary hematopoietic progenitors using image patches from brightfield microscopy and cellular movement. Surprisingly, lineage choice can be detected up to three generations before conventional molecular markers are observable. Our approach allows identification of cells with differentially expressed lineage-specifying genes without molecular labeling...
February 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28218898/detecting-dna-cytosine-methylation-using-nanopore-sequencing
#16
Jared T Simpson, Rachael E Workman, P C Zuzarte, Matei David, L J Dursi, Winston Timp
In nanopore sequencing devices, electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine (5-mC). Here we quantified the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. By using synthetically methylated DNA, we were able to train a hidden Markov model to distinguish 5-mC from unmethylated cytosine. We applied our method to sequence the methylome of human DNA, without requiring special steps for library preparation.
February 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28218897/mapping-dna-methylation-with-high-throughput-nanopore-sequencing
#17
Arthur C Rand, Miten Jain, Jordan M Eizenga, Audrey Musselman-Brown, Hugh E Olsen, Mark Akeson, Benedict Paten
DNA chemical modifications regulate genomic function. We present a framework for mapping cytosine and adenosine methylation with the Oxford Nanopore Technologies MinION using this nanopore sequencer's ionic current signal. We map three cytosine variants and two adenine variants. The results show that our model is sensitive enough to detect changes in genomic DNA methylation levels as a function of growth phase in Escherichia coli.
February 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28192420/atomic-resolution-structures-from-fragmented-protein-crystals-with-the-cryoem-method-microed
#18
M Jason de la Cruz, Johan Hattne, Dan Shi, Paul Seidler, Jose Rodriguez, Francis E Reyes, Michael R Sawaya, Duilio Cascio, Simon C Weiss, Sun Kyung Kim, Cynthia S Hinck, Andrew P Hinck, Guillermo Calero, David Eisenberg, Tamir Gonen
Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography...
February 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/28192419/seq-well-portable-low-cost-rna-sequencing-of-single-cells-at-high-throughput
#19
Todd M Gierahn, Marc H Wadsworth, Travis K Hughes, Bryan D Bryson, Andrew Butler, Rahul Satija, Sarah Fortune, J Christopher Love, Alex K Shalek
Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.
February 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/28068316/scalable-whole-genome-single-cell-library-preparation-without-preamplification
#20
Hans Zahn, Adi Steif, Emma Laks, Peter Eirew, Michael VanInsberghe, Sohrab P Shah, Samuel Aparicio, Carl L Hansen
Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations...
January 9, 2017: Nature Methods
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