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Nature Methods

Joon Ho Kang, Teemu P Miettinen, Lynna Chen, Selim Olcum, Georgios Katsikis, Patrick S Doyle, Scott R Manalis
The monitoring of mechanics in a single cell throughout the cell cycle has been hampered by the invasiveness of mechanical measurements. Here we quantify mechanical properties via acoustic scattering of waves from a cell inside a fluid-filled vibrating cantilever with a temporal resolution of < 1 min. Through simulations, experiments with hydrogels and the use of chemically perturbed cells, we show that our readout, the size-normalized acoustic scattering (SNACS), measures stiffness. To demonstrate the noninvasiveness of SNACS over successive cell cycles, we used measurements that resulted in deformations of < 15 nm...
February 11, 2019: Nature Methods
George C Linderman, Manas Rachh, Jeremy G Hoskins, Stefan Steinerberger, Yuval Kluger
t-distributed stochastic neighbor embedding (t-SNE) is widely used for visualizing single-cell RNA-sequencing (scRNA-seq) data, but it scales poorly to large datasets. We dramatically accelerate t-SNE, obviating the need for data downsampling, and hence allowing visualization of rare cell populations. Furthermore, we implement a heatmap-style visualization for scRNA-seq based on one-dimensional t-SNE for simultaneously visualizing the expression patterns of thousands of genes. Software is available at https://github...
February 11, 2019: Nature Methods
Kimberly A Homan, Navin Gupta, Katharina T Kroll, David B Kolesky, Mark Skylar-Scott, Tomoya Miyoshi, Donald Mau, M Todd Valerius, Thomas Ferrante, Joseph V Bonventre, Jennifer A Lewis, Ryuji Morizane
Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls...
February 11, 2019: Nature Methods
Dhruva Katrekar, Genghao Chen, Dario Meluzzi, Ashwin Ganesh, Atharv Worlikar, Yu-Ru Shih, Shyni Varghese, Prashant Mali
We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.
February 8, 2019: Nature Methods
Yong Qian, Kiryl D Piatkevich, Benedict Mc Larney, Ahmed S Abdelfattah, Sohum Mehta, Mitchell H Murdock, Sven Gottschalk, Rosana S Molina, Wei Zhang, Yingche Chen, Jiahui Wu, Mikhail Drobizhev, Thomas E Hughes, Jin Zhang, Eric R Schreiter, Shy Shoham, Daniel Razansky, Edward S Boyden, Robert E Campbell
We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca2+ ) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca2+ imaging in combination with other optogenetic indicators and actuators...
January 21, 2019: Nature Methods
Vivien Marx
No abstract text is available yet for this article.
January 21, 2019: Nature Methods
Christian Munk, Eshita Mutt, Vignir Isberg, Louise F Nikolajsen, Janne M Bibbe, Tilman Flock, Michael A Hanson, Raymond C Stevens, Xavier Deupi, David E Gloriam
G-protein-coupled receptors (GPCRs) transduce physiological and sensory stimuli into appropriate cellular responses and mediate the actions of one-third of drugs. GPCR structural studies have revealed the general bases of receptor activation, signaling, drug action and allosteric modulation, but so far cover only 13% of nonolfactory receptors. We broadly surveyed the receptor modifications/engineering and methods used to produce all available GPCR crystal and cryo-electron microscopy (cryo-EM) structures, and present an interactive resource integrated in GPCRdb ( http://www...
January 21, 2019: Nature Methods
Mingzhi Lin, Edo Kussell
We present a robust, computationally efficient method ( ) for inferring the parameters of homologous recombination in bacteria, which can be applied in diverse datasets, from whole-genome sequencing to metagenomic shotgun sequencing data. Using correlation profiles of synonymous substitutions, we determine recombination rates and diversity levels of the shared gene pool that has contributed to a given sample. We validated the recombination parameters using data from laboratory experiments...
January 21, 2019: Nature Methods
Vasilis Ntranos, Lynn Yi, Páll Melsted, Lior Pachter
Single-cell RNA-seq makes it possible to characterize the transcriptomes of cell types across different conditions and to identify their transcriptional signatures via differential analysis. Our method detects changes in transcript dynamics and in overall gene abundance in large numbers of cells to determine differential expression. When applied to transcript compatibility counts obtained via pseudoalignment, our approach provides a quantification-free analysis of 3' single-cell RNA-seq that can identify previously undetectable marker genes...
January 21, 2019: Nature Methods
Francisco Romero-Ferrero, Mattia G Bergomi, Robert C Hinz, Francisco J H Heras, Gonzalo G de Polavieja
Understanding of animal collectives is limited by the ability to track each individual. We describe an algorithm and software that extract all trajectories from video, with high identification accuracy for collectives of up to 100 individuals. uses two convolutional networks: one that detects when animals touch or cross and another for animal identification. The tool is trained with a protocol that adapts to video conditions and tracking difficulty.
January 14, 2019: Nature Methods
Vivien Marx
No abstract text is available yet for this article.
January 14, 2019: Nature Methods
Carlotta Ronda, Sway P Chen, Vitor Cabral, Stephanie J Yaung, Harris H Wang
Engineering of microbial communities in open environments remains challenging. Here we describe a platform used to identify and modify genetically tractable mammalian microbiota by engineering community-wide horizontal gene transfer events in situ. With this approach, we demonstrate that diverse taxa in the mouse gut microbiome can be modified directly with a desired genetic payload. In situ microbiome engineering in living animals allows novel capabilities to be introduced into established communities in their native milieu...
January 14, 2019: Nature Methods
Marco Castello, Giorgio Tortarolo, Mauro Buttafava, Takahiro Deguchi, Federica Villa, Sami Koho, Luca Pesce, Michele Oneto, Simone Pelicci, Luca Lanzanó, Paolo Bianchini, Colin J R Sheppard, Alberto Diaspro, Alberto Tosi, Giuseppe Vicidomini
Image scanning microscopy (ISM) can improve the effective spatial resolution of confocal microscopy to its theoretical limit. However, current implementations are not robust or versatile, and are incompatible with fluorescence lifetime imaging (FLIM). We describe an implementation of ISM based on a single-photon detector array that enables super-resolution FLIM and improves multicolor, live-cell and in-depth imaging, thereby paving the way for a massive transition from confocal microscopy to ISM.
January 14, 2019: Nature Methods
Jonathan S Marvin, Benjamin Scholl, Daniel E Wilson, Kaspar Podgorski, Abbas Kazemipour, Johannes Alexander Müller, Susanne Schoch, Francisco José Urra Quiroz, Nelson Rebola, Huan Bao, Justin P Little, Ariana N Tkachuk, Edward Cai, Adam W Hantman, Samuel S-H Wang, Victor J DePiero, Bart G Borghuis, Edwin R Chapman, Dirk Dietrich, David A DiGregorio, David Fitzpatrick, Loren L Looger
In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors' original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.
January 2, 2019: Nature Methods
Uri Weill, Ido Yofe, Ehud Sass, Bram Stynen, Dan Davidi, Janani Natarajan, Reut Ben-Menachem, Zohar Avihou, Omer Goldman, Nofar Harpaz, Silvia Chuartzman, Kiril Kniazev, Barbara Knoblach, Janina Laborenz, Felix Boos, Jacqueline Kowarzyk, Shifra Ben-Dor, Einat Zalckvar, Johannes M Herrmann, Richard A Rachubinski, Ophry Pines, Doron Rapaport, Stephen W Michnick, Emmanuel D Levy, Maya Schuldiner
The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.
January 2, 2019: Nature Methods
Yicong Wu, Hari Shroff
In the version of this Perspective originally published, Fig. 4g included an incorrect inset adapted from a different figure than the main image in the panel. This error has been corrected in the PDF and HTML versions of the paper.
December 29, 2018: Nature Methods
Alexander H Tuttle, Vivek M Philip, Elissa J Chesler, Jeffrey S Mogil
In the version of this Comment originally published, the authors omitted a funding source. Grant 5 P50 DA039841 (to E.J.C.) from the US National Institute on Drug Abuse has been added to the Acknowledgements in the HTML and PDF versions of the paper.
December 21, 2018: Nature Methods
Tal Nawy
The originally published version of this Research Highlight incorrectly stated that Guo-Cheng Yuan is at the University of California at Los Angeles; the correct affiliation is Dana-Farber Cancer Institute. The text has been corrected in the HTML and PDF versions of the paper.
December 21, 2018: Nature Methods
Mridusmita Saikia, Philip Burnham, Sara H Keshavjee, Michael F Z Wang, Michael Heyang, Pablo Moral-Lopez, Meleana M Hinchman, Charles G Danko, John S L Parker, Iwijn De Vlaminck
We describe droplet-assisted RNA targeting by single-cell sequencing (DART-seq), a versatile technology that enables multiplexed amplicon sequencing and transcriptome profiling in single cells. We applied DART-seq to simultaneously characterize the non-A-tailed transcripts of a segmented dsRNA virus and the transcriptome of the infected cell. In addition, we used DART-seq to simultaneously determine the natively paired, variable region heavy and light chain amplicons and the transcriptome of B lymphocytes.
December 17, 2018: Nature Methods
Davide Gambarotto, Fabian U Zwettler, Maeva Le Guennec, Marketa Schmidt-Cernohorska, Denis Fortun, Susanne Borgers, Jörn Heine, Jan-Gero Schloetel, Matthias Reuss, Michael Unser, Edward S Boyden, Markus Sauer, Virginie Hamel, Paul Guichard
Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy...
December 17, 2018: Nature Methods
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