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  • Publisher Correction: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study
    Nat. Methods (IF 26.919) Pub Date : 2018-10-16
    Björn Hellenkamp, Sonja Schmid, Olga Doroshenko, Oleg Opanasyuk, Ralf Kühnemuth, Soheila Rezaei Adariani, Benjamin Ambrose, Mikayel Aznauryan, Anders Barth, Victoria Birkedal, Mark E. Bowen, Hongtao Chen, Thorben Cordes, Tobias Eilert, Carel Fijen, Christian Gebhardt, Markus Götz, Giorgos Gouridis, Enrico Gratton, Taekjip Ha, Pengyu Hao, Christian A. Hanke, Andreas Hartmann, Jelle Hendrix, Lasse L. Hildebrandt, Verena Hirschfeld, Johannes Hohlbein, Boyang Hua, Christian G. Hübner, Eleni Kallis, Achillefs N. Kapanidis, Jae-Yeol Kim, Georg Krainer, Don C. Lamb, Nam Ki Lee, Edward A. Lemke, Brié Levesque, Marcia Levitus, James J. McCann, Nikolaus Naredi-Rainer, Daniel Nettels, Thuy Ngo, Ruoyi Qiu, Nicole C. Robb, Carlheinz Röcker, Hugo Sanabria, Michael Schlierf, Tim Schröder, Benjamin Schuler, Henning Seidel, Lisa Streit, Johann Thurn, Philip Tinnefeld, Swati Tyagi, Niels Vandenberk, Andrés Manuel Vera, Keith R. Weninger, Bettina Wünsch, Inna S. Yanez-Orozco, Jens Michaelis, Cl..

    Publisher Correction: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark studyPublisher Correction: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study, Published online: 16 October 2018; doi:10.1038/s41592-018-0193-xPublisher Correction: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

    更新日期:2018-10-16
  • Transparent Danionella translucida as a genetically tractable vertebrate brain model
    Nat. Methods (IF 26.919) Pub Date : 2018-10-15
    Lisanne Schulze, Jörg Henninger, Mykola Kadobianskyi, Thomas Chaigne, Ana Isabel Faustino, Nahid Hakiy, Shahad Albadri, Markus Schuelke, Leonard Maler, Filippo Del Bene, Benjamin Judkewitz

    Understanding how distributed neuronal circuits integrate sensory information and generate behavior is a central goal of neuroscience. However, it has been difficult to study neuronal networks at single-cell resolution across the entire adult brain in vertebrates because of their size and opacity. We address this challenge here by introducing the fish Danionella translucida to neuroscience as a potential model organism. This teleost remains small and transparent even in adulthood, when neural circuits and behavior have matured. Despite having the smallest known adult vertebrate brain, D. translucida displays a rich set of complex behaviors, including courtship, shoaling, schooling, and acoustic communication. In order to carry out optical measurements and perturbations of neural activity with genetically encoded tools, we established CRISPR–Cas9 genome editing and Tol2 transgenesis techniques. These features make D. translucida a promising model organism for the study of adult vertebrate brain function at single-cell resolution.

    更新日期:2018-10-16
  • Guide Swap enables genome-scale pooled CRISPR–Cas9 screening in human primary cells
    Nat. Methods (IF 26.919) Pub Date : 2018-10-08
    Pamela Y. Ting, Albert E. Parker, J. Scott Lee, Chris Trussell, Orzala Sharif, Fabio Luna, Glenn Federe, S. Whitney Barnes, John R. Walker, Julie Vance, Mu-Yun Gao, Heath E. Klock, Scott Clarkson, Carsten Russ, Loren J. Miraglia, Michael P. Cooke, Anthony E. Boitano, Peter McNamara, John Lamb, Christian Schmedt, Jennifer L. Snead

    CRISPR–Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells—an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR–Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.

    更新日期:2018-10-09
  • Publisher Correction: Image Data Resource: a bioimage data integration and publication platform
    Nat. Methods (IF 26.919) Pub Date : 2018-10-04
    Eleanor Williams, Josh Moore, Simon W Li, Gabriella Rustici, Aleksandra Tarkowska, Anatole Chessel, Simone Leo, Bálint Antal, Richard K Ferguson, Ugis Sarkans, Alvis Brazma, Rafael E Carazo Salas, Jason R Swedlow

    Publisher Correction: Image Data Resource: a bioimage data integration and publication platformPublisher Correction: Image Data Resource: a bioimage data integration and publication platform, Published online: 04 October 2018; doi:10.1038/s41592-018-0169-xPublisher Correction: Image Data Resource: a bioimage data integration and publication platform

    更新日期:2018-10-04
  • Engineering wild bacteria
    Nat. Methods (IF 26.919) Pub Date : 2018-10-01
    Nicole Rusk

    Engineering wild bacteriaEngineering wild bacteria, Published online: 01 October 2018; doi:10.1038/s41592-018-0160-6Engineering wild bacteria

    更新日期:2018-10-02
  • Lighting up proteins
    Nat. Methods (IF 26.919) Pub Date : 2018-10-01
    Rita Strack

    Lighting up proteinsLighting up proteins, Published online: 01 October 2018; doi:10.1038/s41592-018-0156-2Lighting up proteins

    更新日期:2018-10-02
  • A tissue-to-organelle view of cellular proteins
    Nat. Methods (IF 26.919) Pub Date : 2018-10-01
    Vesna Todorovic

    A tissue-to-organelle view of cellular proteinsA tissue-to-organelle view of cellular proteins, Published online: 01 October 2018; doi:10.1038/s41592-018-0163-3By coupling multiplex iterative indirect immunofluorescence imaging with computer vision methods, researchers can detect at least 40 different proteins with subcellular resolution.

    更新日期:2018-10-02
  • Single-particle analysis for fluorescence nanoscopy
    Nat. Methods (IF 26.919) Pub Date : 2018-10-01
    Mark Bates

    Single-particle analysis for fluorescence nanoscopySingle-particle analysis for fluorescence nanoscopy, Published online: 01 October 2018; doi:10.1038/s41592-018-0151-7Building on methods from electron microscopy, researchers are applying nanoscale fluorescence imaging to address questions in structural biology.

    更新日期:2018-10-02
  • Phototoxicity revisited
    Nat. Methods (IF 26.919) Pub Date : 2018-10-01

    Phototoxicity revisitedPhototoxicity revisited, Published online: 01 October 2018; doi:10.1038/s41592-018-0170-4As microscopy methods for studying biology in living samples advance and demand for them grows, assessment of light damage caused by imaging becomes increasingly important.

    更新日期:2018-10-02
  • Bridget Carragher
    Nat. Methods (IF 26.919) Pub Date : 2018-10-01
    Vivien Marx

    Bridget CarragherBridget Carragher, Published online: 01 October 2018; doi:10.1038/s41592-018-0147-3Speeding up spot-to-plunge in cryo-EM and how to keep a lab talking.

    更新日期:2018-10-02
  • Coloring electron microscopy connectomes
    Nat. Methods (IF 26.919) Pub Date : 2018-10-01
    Ada Yee

    Coloring electron microscopy connectomesColoring electron microscopy connectomes, Published online: 01 October 2018; doi:10.1038/s41592-018-0166-0FluoEM aligns axon reconstructions from fluorescence and 3D electron microscopy on the same tissue to allow multicolor labeling of distant inputs.

    更新日期:2018-10-02
  • Better base editors
    Nat. Methods (IF 26.919) Pub Date : 2018-10-01
    Nicole Rusk

    Better base editorsBetter base editors, Published online: 01 October 2018; doi:10.1038/s41592-018-0154-4Better base editors

    更新日期:2018-10-02
  • Deep generative models of genetic variation capture the effects of mutations
    Nat. Methods (IF 26.919) Pub Date : 2018-09-24
    Adam J. Riesselman, John B. Ingraham, Debora S. Marks

    The functions of proteins and RNAs are defined by the collective interactions of many residues, and yet most statistical models of biological sequences consider sites nearly independently. Recent approaches have demonstrated benefits of including interactions to capture pairwise covariation, but leave higher-order dependencies out of reach. Here we show how it is possible to capture higher-order, context-dependent constraints in biological sequences via latent variable models with nonlinear dependencies. We found that DeepSequence (https://github.com/debbiemarkslab/DeepSequence), a probabilistic model for sequence families, predicted the effects of mutations across a variety of deep mutational scanning experiments substantially better than existing methods based on the same evolutionary data. The model, learned in an unsupervised manner solely on the basis of sequence information, is grounded with biologically motivated priors, reveals the latent organization of sequence families, and can be used to explore new parts of sequence space.

    更新日期:2018-09-25
  • Reducing effects of particle adsorption to the air–water interface in cryo-EM
    Nat. Methods (IF 26.919) Pub Date : 2018-09-24
    Alex J. Noble, Hui Wei, Venkata P. Dandey, Zhening Zhang, Yong Zi Tan, Clinton S. Potter, Bridget Carragher

    Most protein particles prepared in vitreous ice for single-particle cryo-electron microscopy (cryo-EM) are adsorbed to air–water or substrate–water interfaces, which can cause the particles to adopt preferred orientations. By using a rapid plunge-freezing robot and nanowire grids, we were able to reduce some of the deleterious effects of the air–water interface by decreasing the dwell time of particles in thin liquid films. We demonstrated this by using single-particle cryo-EM and cryo-electron tomography (cryo-ET) to examine hemagglutinin, insulin receptor complex, and apoferritin.

    更新日期:2018-09-25
  • Inferring single-trial neural population dynamics using sequential auto-encoders
    Nat. Methods (IF 26.919) Pub Date : 2018-09-17
    Chethan Pandarinath, Daniel J. O’Shea, Jasmine Collins, Rafal Jozefowicz, Sergey D. Stavisky, Jonathan C. Kao, Eric M. Trautmann, Matthew T. Kaufman, Stephen I. Ryu, Leigh R. Hochberg, Jaimie M. Henderson, Krishna V. Shenoy, L. F. Abbott, David Sussillo

    Neuroscience is experiencing a revolution in which simultaneous recording of thousands of neurons is revealing population dynamics that are not apparent from single-neuron responses. This structure is typically extracted from data averaged across many trials, but deeper understanding requires studying phenomena detected in single trials, which is challenging due to incomplete sampling of the neural population, trial-to-trial variability, and fluctuations in action potential timing. We introduce latent factor analysis via dynamical systems, a deep learning method to infer latent dynamics from single-trial neural spiking data. When applied to a variety of macaque and human motor cortical datasets, latent factor analysis via dynamical systems accurately predicts observed behavioral variables, extracts precise firing rate estimates of neural dynamics on single trials, infers perturbations to those dynamics that correlate with behavioral choices, and combines data from non-overlapping recording sessions spanning months to improve inference of underlying dynamics.

    更新日期:2018-09-18
  • Label-free prediction of three-dimensional fluorescence images from transmitted-light microscopy
    Nat. Methods (IF 26.919) Pub Date : 2018-09-17
    Chawin Ounkomol, Sharmishtaa Seshamani, Mary M. Maleckar, Forrest Collman, Gregory R. Johnson

    Understanding cells as integrated systems is central to modern biology. Although fluorescence microscopy can resolve subcellular structure in living cells, it is expensive, is slow, and can damage cells. We present a label-free method for predicting three-dimensional fluorescence directly from transmitted-light images and demonstrate that it can be used to generate multi-structure, integrated images. The method can also predict immunofluorescence (IF) from electron micrograph (EM) inputs, extending the potential applications.

    更新日期:2018-09-18
  • Template-free 2D particle fusion in localization microscopy
    Nat. Methods (IF 26.919) Pub Date : 2018-09-17
    Hamidreza Heydarian, Florian Schueder, Maximilian T. Strauss, Ben van Werkhoven, Mohamadreza Fazel, Keith A. Lidke, Ralf Jungmann, Sjoerd Stallinga, Bernd Rieger

    Methods that fuse multiple localization microscopy images of a single structure can improve signal-to-noise ratio and resolution, but they generally suffer from template bias or sensitivity to registration errors. We present a template-free particle-fusion approach based on an all-to-all registration that provides robustness against individual misregistrations and underlabeling. We achieved 3.3-nm Fourier ring correlation (FRC) image resolution by fusing 383 DNA origami nanostructures with 80% labeling density, and 5.0-nm resolution for structures with 30% labeling density.

    更新日期:2018-09-18
  • Three-photon imaging of mouse brain structure and function through the intact skull
    Nat. Methods (IF 26.919) Pub Date : 2018-09-10
    Tianyu Wang, Dimitre G. Ouzounov, Chunyan Wu, Nicholas G. Horton, Bin Zhang, Cheng-Hsun Wu, Yanping Zhang, Mark J. Schnitzer, Chris Xu

    Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-μm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.

    更新日期:2018-09-11
  • COMRADES determines in vivo RNA structures and interactions
    Nat. Methods (IF 26.919) Pub Date : 2018-09-10
    Omer Ziv, Marta M. Gabryelska, Aaron T. L. Lun, Luca F. R. Gebert, Jessica Sheu-Gruttadauria, Luke W. Meredith, Zhong-Yu Liu, Chun Kit Kwok, Cheng-Feng Qin, Ian J. MacRae, Ian Goodfellow, John C. Marioni, Grzegorz Kudla, Eric A. Miska

    The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.

    更新日期:2018-09-11
  • Terminal exon characterization with TECtool reveals an abundance of cell-specific isoforms
    Nat. Methods (IF 26.919) Pub Date : 2018-09-10
    Andreas J. Gruber, Foivos Gypas, Andrea Riba, Ralf Schmidt, Mihaela Zavolan

    Sequencing of RNA 3′ ends has uncovered numerous sites that do not correspond to the termination sites of known transcripts. Through their 3′ untranslated regions, protein-coding RNAs interact with RNA-binding proteins and microRNAs, which regulate many properties, including RNA stability and subcellular localization. We developed the terminal exon characterization (TEC) tool (http://tectool.unibas.ch), which can be used with RNA-sequencing data from any species for which a genome annotation that includes sites of RNA cleavage and polyadenylation is available. We discovered hundreds of previously unknown isoforms and cell-type-specific terminal exons in human cells. Ribosome profiling data revealed that many of these isoforms were translated. By applying TECtool to single-cell sequencing data, we found that the newly identified isoforms were expressed in subpopulations of cells. Thus, TECtool enables the identification of previously unknown isoforms in well-studied cell systems and in rare cell types.

    更新日期:2018-09-11
  • The whole fly brain in detail
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31
    Nina Vogt

    The whole fly brain in detailThe whole fly brain in detail, Published online: 31 August 2018; doi:10.1038/s41592-018-0125-9The whole fly brain in detail

    更新日期:2018-09-04
  • Expansion of the transgenic toolkit in mouse
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31
    Nina Vogt

    Expansion of the transgenic toolkit in mouseExpansion of the transgenic toolkit in mouse, Published online: 31 August 2018; doi:10.1038/s41592-018-0124-xExpansion of the transgenic toolkit in mouse

    更新日期:2018-09-04
  • Three tissues to gastrulation
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31
    Tal Nawy

    Three tissues to gastrulationThree tissues to gastrulation, Published online: 31 August 2018; doi:10.1038/s41592-018-0128-6Three tissues to gastrulation

    更新日期:2018-09-04
  • Easing the burden of code review
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31

    Easing the burden of code reviewEasing the burden of code review, Published online: 31 August 2018; doi:10.1038/s41592-018-0137-5Nature Methods is one of several Nature journals undertaking a trial with Code Ocean, a cloud-based reproducibility platform, to make it easier to peer review computational code.

    更新日期:2018-09-01
  • Detecting acetylcholine
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31
    Nina Vogt

    Detecting acetylcholineDetecting acetylcholine, Published online: 31 August 2018; doi:10.1038/s41592-018-0131-yA genetically encoded sensor based on G-protein-coupled receptors can detect the neurotransmitter acetylcholine in vitro and in vivo.

    更新日期:2018-09-01
  • Detecting repeated cancer evolution from multi-region tumor sequencing data
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31
    Giulio Caravagna, Ylenia Giarratano, Daniele Ramazzotti, Ian Tomlinson, Trevor A. Graham, Guido Sanguinetti, Andrea Sottoriva

    Recurrent successions of genomic changes, both within and between patients, reflect repeated evolutionary processes that are valuable for the anticipation of cancer progression. Multi-region sequencing allows the temporal order of some genomic changes in a tumor to be inferred, but the robust identification of repeated evolution across patients remains a challenge. We developed a machine-learning method based on transfer learning that allowed us to overcome the stochastic effects of cancer evolution and noise in data and identified hidden evolutionary patterns in cancer cohorts. When applied to multi-region sequencing datasets from lung, breast, renal, and colorectal cancer (768 samples from 178 patients), our method detected repeated evolutionary trajectories in subgroups of patients, which were reproduced in single-sample cohorts (n = 2,935). Our method provides a means of classifying patients on the basis of how their tumor evolved, with implications for the anticipation of disease progression.

    更新日期:2018-09-01
  • Comprehensive mapping of ubiquitination
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31
    Allison Doerr

    Comprehensive mapping of ubiquitinationComprehensive mapping of ubiquitination, Published online: 31 August 2018; doi:10.1038/s41592-018-0122-zComprehensive mapping of ubiquitination

    更新日期:2018-09-01
  • Single-cell genomics to guide human stem cell and tissue engineering
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31
    J. Gray Camp, Damian Wollny, Barbara Treutlein

    To understand human development and disease, as well as to regenerate damaged tissues, scientists are working to engineer certain cell types in vitro and to create 3D microenvironments in which cells behave physiologically. Single-cell genomics (SCG) technologies are being applied to primary human organs and to engineered cells and tissues to generate atlases of cell diversity in these systems at unparalleled resolution. Moving beyond atlases, SCG methods are powerful tools for gaining insight into the engineering and disease process. Here we discuss how scientists can use single-cell sequencing to optimize human cell and tissue engineering by measuring precision, detecting inefficiencies, and assessing accuracy. We also provide a perspective on how emerging SCG methods can be used to reverse-engineer human cells and tissues and unravel disease mechanisms.

    更新日期:2018-09-01
  • Improving probes for super-resolution
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31
    Regan P. Moore, Wesley R. Legant

    Improving probes for super-resolutionImproving probes for super-resolution, Published online: 31 August 2018; doi:10.1038/s41592-018-0120-1Chemically modified DNA aptamers enable quantitative super-resolution imaging.

    更新日期:2018-09-01
  • CDeep3M—Plug-and-Play cloud-based deep learning for image segmentation
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31
    Matthias G. Haberl, Christopher Churas, Lucas Tindall, Daniela Boassa, Sébastien Phan, Eric A. Bushong, Matthew Madany, Raffi Akay, Thomas J. Deerinck, Steven T. Peltier, Mark H. Ellisman

    As biomedical imaging datasets expand, deep neural networks are considered vital for image processing, yet community access is still limited by setting up complex computational environments and availability of high-performance computing resources. We address these bottlenecks with CDeep3M, a ready-to-use image segmentation solution employing a cloud-based deep convolutional neural network. We benchmark CDeep3M on large and complex two-dimensional and three-dimensional imaging datasets from light, X-ray, and electron microscopy.

    更新日期:2018-09-01
  • A path to predict RNA tertiary structures
    Nat. Methods (IF 26.919) Pub Date : 2018-08-31
    Lei Tang

    A path to predict RNA tertiary structuresA path to predict RNA tertiary structures, Published online: 31 August 2018; doi:10.1038/s41592-018-0133-9A high-throughput platform allows biophysical measurements to probe RNA tertiary folding.

    更新日期:2018-09-01
  • Publisher Correction: Calling cell biologists to try cryo-ET
    Nat. Methods (IF 26.919) Pub Date : 2018-08-29
    Vivien Marx

    Publisher Correction: Calling cell biologists to try cryo-ETPublisher Correction: Calling cell biologists to try cryo-ET, Published online: 29 August 2018; doi:10.1038/s41592-018-0135-7Publisher Correction: Calling cell biologists to try cryo-ET

    更新日期:2018-08-30
  • XCMS-MRM and METLIN-MRM: a cloud library and public resource for targeted analysis of small molecules
    Nat. Methods (IF 26.919) Pub Date : 2018-08-27
    Xavier Domingo-Almenara, J. Rafael Montenegro-Burke, Julijana Ivanisevic, Aurelien Thomas, Jonathan Sidibé, Tony Teav, Carlos Guijas, Aries E. Aisporna, Duane Rinehart, Linh Hoang, Anders Nordström, María Gómez-Romero, Luke Whiley, Matthew R. Lewis, Jeremy K. Nicholson, H. Paul Benton, Gary Siuzdak

    We report XCMS-MRM and METLIN-MRM (http://xcmsonline-mrm.scripps.edu/ and http://metlin.scripps.edu/), a cloud-based data-analysis platform and a public multiple-reaction monitoring (MRM) transition repository for small-molecule quantitative tandem mass spectrometry. This platform provides MRM transitions for more than 15,500 molecules and facilitates data sharing across different instruments and laboratories.

    更新日期:2018-08-27
  • Trac-looping measures genome structure and chromatin accessibility
    Nat. Methods (IF 26.919) Pub Date : 2018-08-27
    Binbin Lai, Qingsong Tang, Wenfei Jin, Gangqing Hu, Darawalee Wangsa, Kairong Cui, Benjamin Z. Stanton, Gang Ren, Yi Ding, Ming Zhao, Shuai Liu, Jiuzhou Song, Thomas Ried, Keji Zhao

    Long-range chromatin interactions play critical roles in genome organization and regulation of transcription. We now report transposase-mediated analysis of chromatin looping (Trac-looping) for simultaneous detection of multiscale genome-wide chromatin interactions among regulatory elements and chromatin accessibility. With this technique, a bivalent oligonucleotide linker is inserted between two interacting regions such that the chromatin interactions are captured without prior chromatin fragmentation and proximity-based ligation. Application of Trac-looping to human CD4+ T cells revealed substantial reorganization of enhancer–promoter interactions associated with changes in gene expression after T cell receptor stimulation.

    更新日期:2018-08-27
  • Author Correction: Genetically engineered cerebral organoids model brain tumor formation
    Nat. Methods (IF 26.919) Pub Date : 2018-08-22
    Shan Bian, Marko Repic, Zhenming Guo, Anoop Kavirayani, Thomas Burkard, Joshua A. Bagley, Christian Krauditsch, Jürgen A. Knoblich

    Author Correction: Genetically engineered cerebral organoids model brain tumor formationAuthor Correction: Genetically engineered cerebral organoids model brain tumor formation, Published online: 22 August 2018; doi:10.1038/s41592-018-0118-8Author Correction: Genetically engineered cerebral organoids model brain tumor formation

    更新日期:2018-08-22
  • Rapid and efficient induction of functional astrocytes from human pluripotent stem cells
    Nat. Methods (IF 26.919) Pub Date : 2018-08-20
    Isaac Canals, Aurélie Ginisty, Ella Quist, Raissa Timmerman, Jonas Fritze, Giedre Miskinyte, Emanuela Monni, Marita G. Hansen, Isabel Hidalgo, David Bryder, Johan Bengzon, Henrik Ahlenius

    The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease.

    更新日期:2018-08-20
  • Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells
    Nat. Methods (IF 26.919) Pub Date : 2018-08-20
    Bruno Di Stefano, Mai Ueda, Shan Sabri, Justin Brumbaugh, Aaron J. Huebner, Anna Sahakyan, Kendell Clement, Katie J. Clowers, Alison R. Erickson, Keiko Shioda, Steven P. Gygi, Hongcang Gu, Toshi Shioda, Alexander Meissner, Yasuhiro Takashima, Kathrin Plath, Konrad Hochedlinger

    Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.

    更新日期:2018-08-20
  • Modified aptamers enable quantitative sub-10-nm cellular DNA-PAINT imaging
    Nat. Methods (IF 26.919) Pub Date : 2018-08-20
    Sebastian Strauss, Philipp C. Nickels, Maximilian T. Strauss, Vilma Jimenez Sabinina, Jan Ellenberg, Jeffrey D. Carter, Shashi Gupta, Nebojsa Janjic, Ralf Jungmann

    Although current implementations of super-resolution microscopy are technically approaching true molecular-scale resolution, this has not translated to imaging of biological specimens, because of the large size of conventional affinity reagents. Here we introduce slow off-rate modified aptamers (SOMAmers) as small and specific labeling reagents for use with DNA points accumulation in nanoscale topography (DNA-PAINT). To demonstrate the achievable resolution, specificity, and multiplexing capability of SOMAmers, we labeled and imaged both transmembrane and intracellular targets in fixed and live cells.

    更新日期:2018-08-20
  • Publisher Correction: Single-DNA electron spin resonance spectroscopy in aqueous solutions
    Nat. Methods (IF 26.919) Pub Date : 2018-08-14
    Fazhan Shi, Fei Kong, Pengju Zhao, Xiaojun Zhang, Ming Chen, Sanyou Chen, Qi Zhang, Mengqi Wang, Xiangyu Ye, Zhecheng Wang, Zhuoyang Qin, Xing Rong, Jihu Su, Pengfei Wang, Peter Z. Qin, Jiangfeng Du

    Publisher Correction: Single-DNA electron spin resonance spectroscopy in aqueous solutions Publisher Correction: Single-DNA electron spin resonance spectroscopy in aqueous solutions, Published online: 14 August 2018; doi:10.1038/s41592-018-0119-7 Publisher Correction: Single-DNA electron spin resonance spectroscopy in aqueous solutions

    更新日期:2018-08-14
  • A proximity-tagging system to identify membrane protein–protein interactions
    Nat. Methods (IF 26.919) Pub Date : 2018-08-13
    Qiang Liu, Jun Zheng, Weiping Sun, Yinbo Huo, Liye Zhang, Piliang Hao, Haopeng Wang, Min Zhuang

    The communication between cells and between cellular organelles is often controlled by the interaction of membrane proteins. Although many methods for the detection of protein–protein interactions (PPIs) exist, membrane PPIs remain difficult to detect. Here we developed a proximity-based tagging system, PUP-IT (pupylation-based interaction tagging), to identify membrane protein interactions. In this approach, a small protein tag, Pup, is applied to proteins that interact with a PafA-fused bait, enabling transient and weak interactions to be enriched and detected by mass spectrometry. Pup does not diffuse from the enzyme, which allows high-specificity labeling. We applied this approach to CD28, a critical costimulatory receptor for T lymphocyte activation, and identified known CD28 binding partners and multiple potential interacting proteins. In addition, we demonstrated that this method can identify the interaction between a cell surface receptor and its ligand.

    更新日期:2018-08-13
  • The GAGOme: a cell-based library of displayed glycosaminoglycans
    Nat. Methods (IF 26.919) Pub Date : 2018-08-13
    Yen-Hsi Chen, Yoshiki Narimatsu, Thomas M. Clausen, Catarina Gomes, Richard Karlsson, Catharina Steentoft, Charlotte B. Spliid, Tobias Gustavsson, Ali Salanti, Andrea Persson, Anders Malmström, Daniel Willén, Ulf Ellervik, Eric P. Bennett, Yang Mao, Henrik Clausen, Zhang Yang

    Glycosaminoglycans (GAGs) are essential polysaccharides in normal physiology and disease. However, understanding of the contribution of specific GAG structures to specific biological functions is limited, largely because of the great structural heterogeneity among GAGs themselves, as well as technical limitations in the structural characterization and chemical synthesis of GAGs. Here we describe a cell-based method to produce and display distinct GAGs with a broad repertoire of modifications, a library we refer to as the GAGOme. By using precise gene editing, we engineered a large panel of Chinese hamster ovary cells with knockout or knock-in of the genes encoding most of the enzymes involved in GAG biosynthesis, to generate a library of isogenic cell lines that differentially display distinct GAG features. We show that this library can be used for cell-based binding assays, recombinant expression of proteoglycans with distinct GAG structures, and production of distinct GAG chains on metabolic primers that may be used for the assembly of GAG glycan microarrays.

    更新日期:2018-08-13
  • Generation and post-injury integration of human spinal cord neural stem cells
    Nat. Methods (IF 26.919) Pub Date : 2018-08-06
    Hiromi Kumamaru, Ken Kadoya, Andrew F. Adler, Yoshio Takashima, Lori Graham, Giovanni Coppola, Mark H. Tuszynski

    Spinal cord neural stem cells (NSCs) have great potential to reconstitute damaged spinal neural circuitry, but they have yet to be generated in vitro. We now report the derivation of spinal cord NSCs from human pluripotent stem cells (hPSCs). Our observations show that these spinal cord NSCs differentiate into a diverse population of spinal cord neurons occupying multiple positions along the dorso-ventral axis, and can be maintained for prolonged time periods. Grafts into injured spinal cords were rich with excitatory neurons, extended large numbers of axons over long distances, innervated their target structures, and enabled robust corticospinal regeneration. The grafts synaptically integrated into multiple host intraspinal and supraspinal systems, including the corticospinal projection, and improved functional outcomes after injury. hPSC-derived spinal cord NSCs could enable a broad range of biomedical applications for in vitro disease modeling and constitute an improved clinically translatable cell source for ‘replacement’ strategies in several spinal cord disorders.

    更新日期:2018-08-06
  • Single-DNA electron spin resonance spectroscopy in aqueous solutions
    Nat. Methods (IF 26.919) Pub Date : 2018-08-06
    Fazhan Shi, Fei Kong, Pengju Zhao, Xiaojun Zhang, Ming Chen, Sanyou Chen, Qi Zhang, Mengqi Wang, Xiangyu Ye, Zhecheng Wang, Zhuoyang Qin, Xing Rong, Jihu Su, Pengfei Wang, Peter Z. Qin, Jiangfeng Du

    Magnetic resonance spectroscopy of single biomolecules under near-physiological conditions could substantially advance understanding of their biological function, but this approach remains very challenging. Here we used nitrogen-vacancy centers in diamonds to detect electron spin resonance spectra of individual, tethered DNA duplexes labeled with a nitroxide spin label in aqueous buffer solutions at ambient temperatures. This work paves the way for magnetic resonance studies on single biomolecules and their intermolecular interactions in native-like environments.

    更新日期:2018-08-06
  • Ilaria Testa
    Nat. Methods (IF 26.919) Pub Date : 2018-07-31
    Vivien Marx

    Ilaria TestaIlaria Testa, Published online: 31 July 2018; doi:10.1038/s41592-018-0078-zProbes, optics for super-resolution microscopy and life in a creative country.

    更新日期:2018-07-31
  • Probes for molecular crowding
    Nat. Methods (IF 26.919) Pub Date : 2018-07-31
    Rita Strack

    Probes for molecular crowdingProbes for molecular crowding, Published online: 31 July 2018; doi:10.1038/s41592-018-0098-8Self-assembled nanoparticles give insight into the regulation of macromolecular crowding.

    更新日期:2018-07-31
  • Quanti.us: a tool for rapid, flexible, crowd-based annotation of images
    Nat. Methods (IF 26.919) Pub Date : 2018-07-31
    Alex J. Hughes, Joseph D. Mornin, Sujoy K. Biswas, Lauren E. Beck, David P. Bauer, Arjun Raj, Simone Bianco, Zev J. Gartner

    We describe Quanti.us, a crowd-based image-annotation platform that provides an accurate alternative to computational algorithms for difficult image-analysis problems. We used Quanti.us for a variety of medium-throughput image-analysis tasks and achieved 10–50× savings in analysis time compared with that required for the same task by a single expert annotator. We show equivalent deep learning performance for Quanti.us-derived and expert-derived annotations, which should allow scalable integration with tailored machine learning algorithms.

    更新日期:2018-07-31
  • Finally: development of humanized lymph nodes
    Nat. Methods (IF 26.919) Pub Date : 2018-07-31
    Alexandre P. A. Theocharides, Markus G. Manz

    Finally: development of humanized lymph nodesFinally: development of humanized lymph nodes, Published online: 31 July 2018; doi:10.1038/s41592-018-0080-5Overexpression of mouse thymic-stromal-cell-derived lymphopoietin in immune-compromised mice that harbor a reconstituted human immune system rescues lymph node formation and enhances adaptive immune responses.

    更新日期:2018-07-31
  • Water content, not stiffness, dominates Brillouin spectroscopy measurements in hydrated materials
    Nat. Methods (IF 26.919) Pub Date : 2018-07-31
    Pei-Jung Wu, Irina V. Kabakova, Jeffrey W. Ruberti, Joseph M. Sherwood, Iain E. Dunlop, Carl Paterson, Peter Török, Darryl R. Overby

    Water content, not stiffness, dominates Brillouin spectroscopy measurements in hydrated materialsWater content, not stiffness, dominates Brillouin spectroscopy measurements in hydrated materials, Published online: 31 July 2018; doi:10.1038/s41592-018-0076-1Water content, not stiffness, dominates Brillouin spectroscopy measurements in hydrated materials

    更新日期:2018-07-31
  • Optimal experimental design
    Nat. Methods (IF 26.919) Pub Date : 2018-07-31
    Byran Smucker, Martin Krzywinski, Naomi Altman

    Optimal experimental designOptimal experimental design, Published online: 31 July 2018; doi:10.1038/s41592-018-0083-2Customize the experiment for the setting instead of adjusting the setting to fit a classical design.

    更新日期:2018-07-31
  • Tumor genetic analysis from single-cell RNA-seq data
    Nat. Methods (IF 26.919) Pub Date : 2018-07-31
    Tal Nawy

    Tumor genetic analysis from single-cell RNA-seq dataTumor genetic analysis from single-cell RNA-seq data, Published online: 31 July 2018; doi:10.1038/s41592-018-0089-9Tumor genetic analysis from single-cell RNA-seq data

    更新日期:2018-07-31
  • Self-assembled ‘silicages’
    Nat. Methods (IF 26.919) Pub Date : 2018-07-31
    Lei Tang

    Self-assembled ‘silicages’Self-assembled ‘silicages’, Published online: 31 July 2018; doi:10.1038/s41592-018-0088-xSelf-assembled ‘silicages’

    更新日期:2018-07-31
  • Artifact-free high-density localization microscopy analysis
    Nat. Methods (IF 26.919) Pub Date : 2018-07-30
    Richard J. Marsh, Karin Pfisterer, Pauline Bennett, Liisa M. Hirvonen, Mathias Gautel, Gareth E. Jones, Susan Cox

    High-density analysis methods for localization microscopy increase acquisition speed but produce artifacts. We demonstrate that these artifacts can be eliminated by the combination of Haar wavelet kernel (HAWK) analysis with standard single-frame fitting. We tested the performance of this method on synthetic, fixed-cell, and live-cell data, and found that HAWK preprocessing yielded reconstructions that reflected the structure of the sample, thus enabling high-speed, artifact-free super-resolution imaging of live cells.

    更新日期:2018-07-31
  • Addendum: Biotinylation by antibody recognition—a method for proximity labeling
    Nat. Methods (IF 26.919) Pub Date : 2018-07-27
    Daniel Z Bar, Kathleen Atkatsh, Urraca Tavarez, Michael R Erdos, Yosef Gruenbaum, Francis S Collins

    In the version of this article initially published, the introduction focused on methods for characterizing the nuclear envelope and did not include a comprehensive overview of proximity-based methods, some of which have similarly utilized antibody-conjugated peroxidase for proximity labeling by biotin deposition. The authors apologize for the omission of the following references: Kotani, N. et al., Proc. Natl Acad. Sci. USA 105, 7405–7409 (2008); Hashimoto, N. et al., Proteomics 12, 3154–3163 (2012); Li, X. W. et al., J. Biol. Chem. 289, 14434–14447 (2014); and Rees, J. S. et al., Curr. Protoc. Protein Sci. 80, 19.27.1–19.27.18 (2015).

    更新日期:2018-07-27
  • Induction of myelinating oligodendrocytes in human cortical spheroids
    Nat. Methods (IF 26.919) Pub Date : 2018-07-25
    Mayur Madhavan, Zachary S. Nevin, H. Elizabeth Shick, Eric Garrison, Cheryl Clarkson-Paredes, Molly Karl, Benjamin L. L. Clayton, Daniel C. Factor, Kevin C. Allan, Lilianne Barbar, Tanya Jain, Panagiotis Douvaras, Valentina Fossati, Robert H. Miller, Paul J. Tesar

    Cerebral organoids provide an accessible system for investigations of cellular composition, interactions, and organization but have lacked oligodendrocytes, the myelinating glia of the central nervous system. Here we reproducibly generated oligodendrocytes and myelin in ‘oligocortical spheroids’ derived from human pluripotent stem cells. Molecular features consistent with those of maturing oligodendrocytes and early myelin appeared by week 20 in culture, with further maturation and myelin compaction evident by week 30. Promyelinating drugs enhanced the rate and extent of oligodendrocyte generation and myelination, and spheroids generated from human subjects with a genetic myelin disorder recapitulated human disease phenotypes. Oligocortical spheroids provide a versatile platform for studies of myelination of the developing central nervous system and offer new opportunities for disease modeling and therapeutic development.

    更新日期:2018-07-25
  • Genetically engineered cerebral organoids model brain tumor formation
    Nat. Methods (IF 26.919) Pub Date : 2018-07-23
    Shan Bian, Marko Repic, Zhenming Guo, Anoop Kavirayani, Thomas Burkard, Joshua A. Bagley, Christian Krauditsch, Jürgen A. Knoblich

    Brain tumors are among the most lethal and devastating cancers. Their study is limited by genetic heterogeneity and the incompleteness of available laboratory models. Three-dimensional organoid culture models offer innovative possibilities for the modeling of human disease. Here we establish a 3D in vitro model called a neoplastic cerebral organoid (neoCOR), in which we recapitulate brain tumorigenesis by introducing oncogenic mutations in cerebral organoids via transposon- and CRISPR–Cas9-mediated mutagenesis. By screening clinically relevant mutations identified in cancer genome projects, we defined mutation combinations that result in glioblastoma-like and central nervous system primitive neuroectodermal tumor (CNS-PNET)-like neoplasms. We demonstrate that neoCORs are suitable for use in investigations of aspects of tumor biology such as invasiveness, and for evaluation of drug effects in the context of specific DNA aberrations. NeoCORs will provide a valuable complement to the current basic and preclinical models used to study brain tumor biology.

    更新日期:2018-07-24
  • Strelka2: fast and accurate calling of germline and somatic variants
    Nat. Methods (IF 26.919) Pub Date : 2018-07-16
    Sangtae Kim, Konrad Scheffler, Aaron L. Halpern, Mitchell A. Bekritsky, Eunho Noh, Morten Källberg, Xiaoyu Chen, Yeonbin Kim, Doruk Beyter, Peter Krusche, Christopher T. Saunders

    We describe Strelka2 (https://github.com/Illumina/strelka), an open-source small-variant-calling method for research and clinical germline and somatic sequencing applications. Strelka2 introduces a novel mixture-model-based estimation of insertion/deletion error parameters from each sample, an efficient tiered haplotype-modeling strategy, and a normal sample contamination model to improve liquid tumor analysis. For both germline and somatic calling, Strelka2 substantially outperformed the current leading tools in terms of both variant-calling accuracy and computing cost.

    更新日期:2018-07-18
  • An enhanced CRISPR repressor for targeted mammalian gene regulation
    Nat. Methods (IF 26.919) Pub Date : 2018-07-16
    Nan Cher Yeo, Alejandro Chavez, Alissa Lance-Byrne, Yingleong Chan, David Menn, Denitsa Milanova, Chih-Chung Kuo, Xiaoge Guo, Sumana Sharma, Angela Tung, Ryan J. Cecchi, Marcelle Tuttle, Swechchha Pradhan, Elaine T. Lim, Noah Davidsohn, Mo R. Ebrahimkhani, James J. Collins, Nathan E. Lewis, Samira Kiani, George M. Church

    The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB–MeCP2, to nuclease-dead Cas9. We demonstrate the system’s superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.

    更新日期:2018-07-18
  • Active PSF shaping and adaptive optics enable volumetric localization microscopy through brain sections
    Nat. Methods (IF 26.919) Pub Date : 2018-07-16
    Michael J. Mlodzianoski, Paul J. Cheng-Hathaway, Shane M. Bemiller, Tyler J. McCray, Sheng Liu, David A. Miller, Bruce T. Lamb, Gary E. Landreth, Fang Huang

    Application of single-molecule switching nanoscopy (SMSN) beyond the coverslip surface poses substantial challenges due to sample-induced aberrations that distort and blur single-molecule emission patterns. We combined active shaping of point spread functions and efficient adaptive optics to enable robust 3D-SMSN imaging within tissues. This development allowed us to image through 30-μm-thick brain sections to visualize and reconstruct the morphology and the nanoscale details of amyloid-β filaments in a mouse model of Alzheimer’s disease.

    更新日期:2018-07-18
  • High-precision automated reconstruction of neurons with flood-filling networks
    Nat. Methods (IF 26.919) Pub Date : 2018-07-16
    Michał Januszewski, Jörgen Kornfeld, Peter H. Li, Art Pope, Tim Blakely, Larry Lindsey, Jeremy Maitin-Shepard, Mike Tyka, Winfried Denk, Viren Jain

    Reconstruction of neural circuits from volume electron microscopy data requires the tracing of cells in their entirety, including all their neurites. Automated approaches have been developed for tracing, but their error rates are too high to generate reliable circuit diagrams without extensive human proofreading. We present flood-filling networks, a method for automated segmentation that, similar to most previous efforts, uses convolutional neural networks, but contains in addition a recurrent pathway that allows the iterative optimization and extension of individual neuronal processes. We used flood-filling networks to trace neurons in a dataset obtained by serial block-face electron microscopy of a zebra finch brain. Using our method, we achieved a mean error-free neurite path length of 1.1 mm, and we observed only four mergers in a test set with a path length of 97 mm. The performance of flood-filling networks was an order of magnitude better than that of previous approaches applied to this dataset, although with substantially increased computational costs.

    更新日期:2018-07-18
  • A synthetic-diploid benchmark for accurate variant-calling evaluation
    Nat. Methods (IF 26.919) Pub Date : 2018-07-16
    Heng Li, Jonathan M. Bloom, Yossi Farjoun, Mark Fleharty, Laura Gauthier, Benjamin Neale, Daniel MacArthur

    Existing benchmark datasets for use in evaluating variant-calling accuracy are constructed from a consensus of known short-variant callers, and they are thus biased toward easy regions that are accessible by these algorithms. We derived a new benchmark dataset from the de novo PacBio assemblies of two fully homozygous human cell lines, which provides a relatively more accurate and less biased estimate of small-variant-calling error rates in a realistic context.

    更新日期:2018-07-18
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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