Table of contents


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Editorial

Empowering citizen scientists p795

doi:10.1038/nmeth.3577

Scientists should consider engaging more with the DIYbio community.


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This Month

The Author File: Daniel J. Müller p797

Vivien Marx

doi:10.1038/nmeth.3557

A new way to quantify ligand-binding interactions of individual membrane proteins.


Points of Significance: Bayesian networks pp799 - 800

Jorge López Puga, Martin Krzywinski & Naomi Altman

doi:10.1038/nmeth.3550


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Correspondence

Validating subcellular thermal changes revealed by fluorescent thermosensors pp801 - 802

Shigeki Kiyonaka, Reiko Sakaguchi, Itaru Hamachi, Takashi Morii, Takenao Yoshizaki & Yasuo Mori

doi:10.1038/nmeth.3548

See also: Correspondence by Suzuki et al. | Correspondence by Baffou et al.


The 105 gap issue between calculation and measurement in single-cell thermometry pp802 - 803

Madoka Suzuki, Vadim Zeeb, Satoshi Arai, Kotaro Oyama & Shin'ichi Ishiwata

doi:10.1038/nmeth.3551

See also: Correspondence by Baffou et al. | Correspondence by Kiyonaka et al.



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Research Highlights

Building lasers inside cells p805

Optical microresonators form stand-alone intracellular lasers.

Algae are the best engineers of optogenetic inhibitors pp806 - 807

Anion channelrhodopsins, recently discovered in cryptophyte algae, can be used as highly efficient optogenetic tools to inhibit neurons.

Where the G-quadruplexes are pp806 - 807

A new sequencing method identifies G-quadruplexes genome wide.

Navigating the negative-mode proteome p808

A negative-mode mass spectrometry approach characterizes intractable parts of the proteome.

Stop the presses p811

A self-cleaving degradation tag allows researchers to quickly and reversibly shut down protein production.

Micro-C maps of genome structure p812

A method using micrococcal nuclease for chromatin fragmentation gives a high-resolution view of 3D genome structure.

RNA catch and release p813

The binding of single RNA molecules to individual proteins can be observed in the subcellular compartments of living cells.

Methods in Brief

Tools in Brief

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Technology Feature

Mapping proteins with spatial proteomics pp815 - 819

Vivien Marx

doi:10.1038/nmeth.3555

A number of techniques address the location of proteins within cells.


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News and Views

Removing roadblocks to deep sequencing of modified RNAs pp821 - 822

Jeremy E Wilusz

doi:10.1038/nmeth.3516

By removing modified nucleotides that block reverse transcriptase, two methods have now made tRNAs amenable to RNA-seq.

See also: Brief Communication by Zheng et al. | Article by Cozen et al.


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Brief Communications

Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach pp823 - 826

Raj Chari, Prashant Mali, Mark Moosburner & George M Church

doi:10.1038/nmeth.3473

Assessing the activity of >1,000 single guide RNAs (sgRNAs) on >1,000 genetic loci with two Cas9 orthologs provides insight into sgRNA design and epigenetic parameters for optimal activity.


Ultrafast, temporally stochastic STED nanoscopy of millisecond dynamics pp827 - 830

Jale Schneider, Jasmin Zahn, Marta Maglione, Stephan J Sigrist, Jonas Marquard, Jakub Chojnacki, Hans-Georg Kräusslich, Steffen J Sahl, Johann Engelhardt & Stefan W Hell

doi:10.1038/nmeth.3481

Temporally stochastic STED nanoscopy with electro-optical deflector–based scanning allows ultrafast super-resolution imaging. The method was used to monitor vesicles and viruses moving at ~2 μm/s with no motion blur.


EEG and functional ultrasound imaging in mobile rats pp831 - 834

Lim-Anna Sieu, Antoine Bergel, Elodie Tiran, Thomas Deffieux, Mathieu Pernot, Jean-Luc Gennisson, Mickaël Tanter & Ivan Cohen

doi:10.1038/nmeth.3506

Functional ultrasound imaging and electroencephalography are combined to assess brain activity in mobile rats. The methodology is applied to the analysis of theta rhythms in a maze task and of epileptic seizures.


Efficient and quantitative high-throughput tRNA sequencing pp835 - 837

Guanqun Zheng, Yidan Qin, Wesley C Clark, Qing Dai, Chengqi Yi, Chuan He, Alan M Lambowitz & Tao Pan

doi:10.1038/nmeth.3478

The combination of an engineered demthylase and a highly processive reverse transcriptase during tRNA library preparation for high-throughput sequencing allows comprehensive profiling of the small RNAs.

See also: News and Views by Wilusz


Inferring transient particle transport dynamics in live cells pp838 - 840

Nilah Monnier, Zachary Barry, Hye Yoon Park, Kuan-Chung Su, Zachary Katz, Brian P English, Arkajit Dey, Keyao Pan, Iain M Cheeseman, Robert H Singer & Mark Bathe

doi:10.1038/nmeth.3483

Single-particle dynamics are analyzed with hidden Markov modeling in combination with Bayesian model selection. This method can annotate both diffusive and directed motion states with single-step resolution.


Phenolyzer: phenotype-based prioritization of candidate genes for human diseases pp841 - 843

Hui Yang, Peter N Robinson & Kai Wang

doi:10.1038/nmeth.3484

The Phenolyzer software provides prioritized candidate gene lists based on disease and phenotype terms that are entered by users as free text.


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Articles

Imaging G protein–coupled receptors while quantifying their ligand-binding free-energy landscape pp845 - 851

David Alsteens, Moritz Pfreundschuh, Cheng Zhang, Patrizia M Spoerri, Shaun R Coughlin, Brian K Kobilka & Daniel J Müller

doi:10.1038/nmeth.3479

A force-distance curve–based atomic force microscopy method is developed and applied to both image and quantify the ligand-binding free-energy landscape of single protease-activated receptor-1 (PAR1) molecules under physiologically relevant conditions.


Photoactivatable genetically encoded calcium indicators for targeted neuronal imaging pp852 - 858

Shai Berlin, Elizabeth C Carroll, Zachary L Newman, Hitomi O Okada, Carson M Quinn, Benjamin Kallman, Nathan C Rockwell, Shelley S Martin, J Clark Lagarias & Ehud Y Isacoff

doi:10.1038/nmeth.3480

The photoactivatable calcium sensors reported in this paper allow simultaneous highlighting of cellular morphology and recording of calcium activity, which is demonstrated in neuronal cultures, in Drosophila and in zebrafish.


ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space pp859 - 865

Ashwin Chari, David Haselbach, Jan-Martin Kirves, Juergen Ohmer, Elham Paknia, Niels Fischer, Oleg Ganichkin, Vanessa Möller, Jeremiah J Frye, Georg Petzold, Marc Jarvis, Michael Tietzel, Clemens Grimm, Jan-Michael Peters, Brenda A Schulman, Kai Tittmann, Jürgen Markl, Utz Fischer & Holger Stark

doi:10.1038/nmeth.3493

ProteoPlex optimizes buffer conditions for the isolation and purification of macromolecular complexes. The concurrent complex stabilization is beneficial for structure determination using X-ray crystallography or cryo-electron microscopy.


Mutational interference mapping experiment (MIME) for studying RNA structure and function pp866 - 872

Redmond P Smyth, Laurence Despons, Gong Huili, Serena Bernacchi, Marcel Hijnen, Johnson Mak, Fabrice Jossinet, Li Weixi, Jean-Christophe Paillart, Max von Kleist & Roland Marquet

doi:10.1038/nmeth.3490

Functionally important residues in a long RNA can be identified by mutational interference mapping experiment (MIME), a method which uses random mutagenesis of RNA followed by selection for function and high-throughput sequencing.


Real-time imaging of brain activity in freely moving rats using functional ultrasound pp873 - 878

Alan Urban, Clara Dussaux, Guillaume Martel, Clément Brunner, Emilie Mace & Gabriel Montaldo

doi:10.1038/nmeth.3482

A miniaturized ultrasound probe enables functional brain imaging in freely behaving rats. The large field of view and deep penetration makes this technique complementary to optical imaging approaches.


ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments pp879 - 884

Aaron E Cozen, Erin Quartley, Andrew D Holmes, Eva Hrabeta-Robinson, Eric M Phizicky & Todd M Lowe

doi:10.1038/nmeth.3508

ARM-seq enables enhanced sequencing of modified tRNAs and tRNA fragments. Treatment of RNA with the demethylase AlkB prior to reverse transcription removes three ‘hard-stop’ modifications, allowing for discovery of modified tRNA fragments and their precursors by RNA sequencing.

See also: News and Views by Wilusz


Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells pp885 - 892

Daniel Paull, Ana Sevilla, Hongyan Zhou, Aana Kim Hahn, Hesed Kim, Christopher Napolitano, Alexander Tsankov, Linshan Shang, Katie Krumholz, Premlatha Jagadeesan, Chris M Woodard, Bruce Sun, Thierry Vilboux, Matthew Zimmer, Eliana Forero, Dorota N Moroziewicz, Hector Martinez, May Christine V Malicdan, Keren A Weiss, Lauren B Vensand, Carmen R Dusenberry, Hannah Polus, Karla Therese L Sy, David J Kahler, William A Gahl, Susan L Solomon, Stephen Chang, Alexander Meissner, Kevin Eggan & Scott A Noggle

doi:10.1038/nmeth.3507

A modular, robotic system for reprogramming to induced pluripotent stem cells in high-throughput and automated fashion.


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Corrigendum

Corrigendum: Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns p893

Stefanie Hampel, Phuong Chung, Claire E McKellar, Donald Hall, Loren L Looger & Julie H Simpson

doi:10.1038/nmeth0915-893a


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Errata

Erratum: Rational design of a high-affinity, fast, red calcium indicator R-CaMP2 p893

Masatoshi Inoue, Atsuya Takeuchi, Shin-ichiro Horigane, Masamichi Ohkura, Keiko Gengyo-Ando, Hajime Fujii, Satoshi Kamijo, Sayaka Takemoto-Kimura, Masanobu Kano, Junichi Nakai, Kazuo Kitamura & Haruhiko Bito

doi:10.1038/nmeth0915-893b


Erratum: Whole-body immunoPET reveals active SIV dynamics in viremic and antiretroviral therapy–treated macaques p893

Philip J Santangelo, Kenneth A Rogers, Chiara Zurla, Emmeline L Blanchard, Sanjeev Gumber, Karen Strait, Fawn Connor-Stroud, David M Schuster, Praveen K Amancha, Jung Joo Hong, Siddappa N Byrareddy, James A Hoxie, Brani Vidakovic, Aftab A Ansari, Eric Hunter & Francois Villinger

doi:10.1038/nmeth0915-893c


Erratum: Multitarget super-resolution microscopy with high-density labeling by exchangeable probes p893

Tai Kiuchi, Makio Higuchi, Akihiro Takamura, Masahiro Maruoka & Naoki Watanabe

doi:10.1038/nmeth0915-893d


Erratum: Power and sample size p893

Martin Krzywinski & Naomi Altman

doi:10.1038/nmeth0915-893e


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Application Note

InnoStamp 40 and InnoScan 1100AL: a complete automated platform for microstructured cell arrays

Adriana Lagraulet, Julie Foncy, Benjamin Berteloite, Aurore Esteve, Marie-Charline Blatche, Laurent Malaquin & Christophe Vieu


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