Nature Methods Contents: September 2015 Volume 12 pp 795 - 893

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Advertisement Nikon's A1rMP+ multiphoton system is now available with a New Dual IR beam option. With dual IR beams, users can now simultaneously excite two different fluorophores such as GFP and mCherry. This capability enables ultra-fast two-color multiphoton imaging, ideal for dynamic specimens. To learn more, please visit www.nikoninstruments.com/a1rmp TABLE OF CONTENTS September 2015 Volume 12, Issue 9 In This Issue Editorial This Month Correspondence Research Highlights Technology Feature News and Views Brief Communications Articles Corrigendum Errata Application Note Advertisement   Struggling with low protein expression?  Optimizing your expression vector may help. Express Cloning from GenScript is a convenient way to try new vectors.  >150 Free Vectors for mammalian, bacterial, yeast, or insect/baculovirus expression systems  $49, 2 days bundled with gene synthesis See all 150 Free Vectors>> Subscribe   Facebook   RSS   Recommend to library   Twitter   Advertisement EndoFectin™ Max Transfection Reagent  For difficult-to-transfect cells Try EndoFectin™Max — a new lipid-based transfection reagent. Superior transfection efficiency for a broad range of cell lines compared with Lipofectamine® 2000. Does not require removal of serum or culture medium, or washing or changing of medium after transfection. Low cytotoxicity. >> Request free sample    In This Issue Top In This Issue    Editorial Top Empowering citizen scientists   p795 doi:10.1038/nmeth.3577 Scientists should consider engaging more with the DIYbio community. This Month Top The Author File: Daniel J. Muller   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 and Naomi Altman doi:10.1038/nmeth.3550 Correspondence Top Validating subcellular thermal changes revealed by fluorescent thermosensors   pp801 - 802 Shigeki Kiyonaka, Reiko Sakaguchi, Itaru Hamachi, Takashi Morii, Takenao Yoshizaki et al. 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 and Shin'ichi Ishiwata doi:10.1038/nmeth.3551 See also: Correspondence by Baffou et al. | Correspondence by Kiyonaka et al. Reply to: "Validating subcellular thermal changes revealed by fluorescent thermosensors" and "The 105 gap issue between calculation and measurement in single-cell thermometry"   p803 Guillaume Baffou, Hervé Rigneault, Didier Marguet and Ludovic Jullien doi:10.1038/nmeth.3552 See also: Correspondence by Suzuki et al. | Correspondence by Kiyonaka et al. Research Highlights Top Building lasers inside cells Optical microresonators form stand-alone intracellular lasers. Algae are the best engineers of optogenetic inhibitors Anion channelrhodopsins, recently discovered in cryptophyte algae, can be used as highly efficient optogenetic tools to inhibit neurons. Where the G-quadruplexes are A new sequencing method identifies G-quadruplexes genome wide. Navigating the negative-mode proteome A negative-mode mass spectrometry approach characterizes intractable parts of the proteome. Stop the presses A self-cleaving degradation tag allows researchers to quickly and reversibly shut down protein production. Micro-C maps of genome structure A method using micrococcal nuclease for chromatin fragmentation gives a high-resolution view of 3D genome structure. RNA catch and release The binding of single RNA molecules to individual proteins can be observed in the subcellular compartments of living cells. Methods in Brief Deep learning to predict sequence specificity | Universal parts | Cardiovascular progenitors from human stem cells | Wireless pharmacology and optogenetics Tools in Brief Finding more ribozymes | Fluorescent proteins for diverse environments | A light switch for microtubule dynamics | Introducing the BioPlex network Methods JOBS of the week Environmental Chemistry University of Windsor Translational Bioinformatics Postdoc positions University of Hawaii Cancer Center Postdoctoral Position in Genomics and Molecular Biology at UCLA UCLA Advanced Research Assistant - Bespoke Gene Targeting Wellcome Trust Sanger Institute Assistant / Associate Professor, Bioinformatics The University of Nevada School Of Medicine More Science jobs from Methods EVENT X-Ray Methods in Structural Biology 12th Oct - 27th Oct 2015 Cold Spring Harbor, USA More science events from Technology Feature Top 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. News and Views Top 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. Brief Communications Top Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach   pp823 - 826 Raj Chari, Prashant Mali, Mark Moosburner and 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 et al. 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 et al. 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 et al. 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 et al. 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 and 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. Advertisement Are you sure that your CO2 incubator provides active humidity control? Technologies to provide precise, user-defined control of temperature and CO2 are widely available, but methods to control relative humidity (while preventing condensation) are still evolving.  Click the Baker logo to learn more about the available active humidity control technologies in CO2 incubators.   Articles Top 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 et al. 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 et al. 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 et al. 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 et al. 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 et al. 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 et al. 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 et al. doi:10.1038/nmeth.3507 A modular, robotic system for reprogramming to induced pluripotent stem cells in high-throughput and automated fashion. Advertisement Droplet Digital™ PCR (ddPCR™) is leading the way  More than 200 peer-reviewed publications have benefited from Droplet Digital PCR's unmatched ability to detect rare nucleic acid species. From tracking cancer biomarkers to quantifying rare mutations, gene expression, and miniscule viral loads, Droplet Digital PCR has led to more discoveries than any other digital PCR platform. Visit our ddPCR page for application guides, publication lists, and webinars that explore the world of ddPCR.   Corrigendum Top 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 et al. doi:10.1038/nmeth0915-893a Errata Top 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 et al. 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 et al. 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 and Naoki Watanabe doi:10.1038/nmeth0915-893d Erratum: Power and sample size   p893 Martin Krzywinski and Naomi Altman doi:10.1038/nmeth0915-893e Application Note Top InnoStamp 40™ and InnoScan 1100AL™: a complete automated platform for microstructured cell arrays    Adriana Lagraulet, Julie Foncy, Benjamin Berteloite, Aurore Esteve, Marie-Charline Blatche et al. 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