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Editorial

Training the kit generation p983

doi:10.1038/nmeth.1804

Young scientists must learn not just how to use a kit, but how it works.


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This Month

The author file: Ernst Bamberg p985

Monya Baker

doi:10.1038/nmeth.1789

Fusing light-activated proteins for precise optogenetic control


Points of view: The design process p987

Bang Wong

doi:10.1038/nmeth.1783


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Correspondence

The Human Epigenome Browser at Washington University pp989 - 990

Xin Zhou, Brett Maricque, Mingchao Xie, Daofeng Li, Vasavi Sundaram, Eric A Martin, Brian C Koebbe, Cydney Nielsen, Martin Hirst, Peggy Farnham, Robert M Kuhn, Jingchun Zhu, Ivan Smirnov, W James Kent, David Haussler, Pamela A F Madden, Joseph F Costello & Ting Wang

doi:10.1038/nmeth.1772


Benchmarking a luciferase complementation assay for detecting protein complexes pp990 - 992

Patricia Cassonnet, Caroline Rolloy, Gregory Neveu, Pierre-Olivier Vidalain, Thibault Chantier, Johann Pellet, Louis Jones, Mandy Muller, Caroline Demeret, Guillaume Gaud, Françoise Vuillier, Vincent Lotteau, Fréderic Tangy, Michel Favre & Yves Jacob

doi:10.1038/nmeth.1773


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Research Highlights

Human oocytes: still in the game p995

Retaining the recipient oocyte genome after human somatic cell nuclear transfer permits development to the blastocyst stage and derivation of triploid human embryonic stem cell lines.

Nanoelectroporation pp996 - 997

Precise amounts of DNA and quantum dots can be moved into cells through tiny channels.

Sequencing for sugars pp996 - 997

The hypothesis that many glycans may have a regular sequence gains support, with new evidence of a sequence for the simplest proteoglycan, bikunin.

High-tech Petri dishes p999

A self-imaging Petri dish monitors cell cultures as they grow in the incubator.

Noncoding RNA's genomic hangouts p1000

Long noncoding RNA interactions with chromatin can be mapped genome-wide using biotinylated tiling oligos.

Wholesome proteomics p1002

Researchers pave the way to high-throughput studies of intact protein isoforms.

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Technology Feature

Faster frames, clearer pictures pp1005 - 1009

Monya Baker

doi:10.1038/nmeth.1777

Better-performing imaging sensors have arrived, but putting them to use is not easy.


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News and Views

The Lego-logic of optogenetics pp1011 - 1013

Thomas G Oertner

doi:10.1038/nmeth.1771

Daisy-chaining light-sensitive ion channels, pumps and fluorescent proteins extends the possibilities for control of neuronal activity.

See also: Article by Kleinlogel et al.


Recognizing heart cells in a crowd pp1013 - 1016

Timothy J Kamp

doi:10.1038/nmeth.1780

A cardiac-specific reporter genetically engineered into human embryonic stem cells allows the optimization of differentiation protocols and the identification of cell-surface markers—a welcome new tool to help isolate and define cardiac cell lineages.

See also: Brief Communication by Elliott et al.


Exciting times: bountiful data to facilitate studies of cis-regulatory control pp1016 - 1017

Anil Ozdemir & Angelike Stathopoulos

doi:10.1038/nmeth.1795

Chromatin immunoprecipitation and yeast one-hybrid systems are complementary approaches to identify protein-DNA interactions. Improvements to these methods now make them more versatile and high-throughput, and should lead to the generation of rich datasets for the study of gene regulation.

See also: Article by Reece-Hoyes et al. | Article by Hens et al. | Brief Communication by Reece-Hoyes et al. | Brief Communication by Gaudinier et al. | Brief Communication by Mazzoni et al.


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Perspective

Directed molecular evolution to design advanced red fluorescent proteins pp1019 - 1026

Fedor V Subach, Kiryl D Piatkevich & Vladislav V Verkhusha

doi:10.1038/nmeth.1776

In this Perspective the authors discuss strategies for the development of improved fluorescent proteins, with a focus on probes at the red end of the spectrum. They synthesize the literature on chromophore photochemistry and protein structure to identify residues for targeted mutagenesis, and consider improvements in molecular evolution methodologies to enable improved screening for desired probes.


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Analysis

Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging pp1027 - 1036

Graham T Dempsey, Joshua C Vaughan, Kok Hao Chen, Mark Bates & Xiaowei Zhuang

doi:10.1038/nmeth.1768

A quantitative characterization of the switching properties of 26 organic dyes relates these properties to the quality of localization-based super-resolution images they generate. The data are a useful resource for selecting dyes and point to avenues for future analysis.


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Brief Communications

NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes pp1037 - 1040

David A Elliott, Stefan R Braam, Katerina Koutsis, Elizabeth S Ng, Robert Jenny, Ebba L Lagerqvist, Christine Biben, Tanya Hatzistavrou, Claire E Hirst, Qing C Yu, Rhys J P Skelton, Dorien Ward-van Oostwaard, Sue Mei Lim, Ouda Khammy, Xueling Li, Susan M Hawes, Richard P Davis, Adam L Goulburn, Robert Passier, Owen W J Prall, John M Haynes, Colin W Pouton, David M Kaye, Christine L Mummery, Andrew G Elefanty & Edouard G Stanley

doi:10.1038/nmeth.1740

A human embryonic stem cell line expressing a fluorescent reporter of cardiac differentiation is described. The authors use this tool to optimize differentiation methods and to identify cell-surface markers in the cardiac lineage.

See also: News and Views by Kamp


Rapid empirical discovery of optimal peptides for targeted proteomics pp1041 - 1043

Andrew B Stergachis, Brendan MacLean, Kristen Lee, John A Stamatoyannopoulos & Michael J MacCoss

doi:10.1038/nmeth.1770

An empirical approach for identifying optimal proteotypic peptides and fragmentation patterns from in vitro–synthesized proteins, for targeted proteomics applications, is described.


Super-resolution 3D microscopy of live whole cells using structured illumination pp1044 - 1046

Lin Shao, Peter Kner, E Hesper Rego & Mats G L Gustafsson

doi:10.1038/nmeth.1734

Live-cell volumetric super-resolution imaging with 120-nm lateral and 360-nm axial resolution using structured-illumination microscopy at speeds of up to 5 s per cell volume over >50 time points captures fine cellular dynamics using only low illumination intensities.


Live-cell 3D super-resolution imaging in thick biological samples pp1047 - 1049

Francesca Cella Zanacchi, Zeno Lavagnino, Michela Perrone Donnorso, Alessio Del Bue, Laura Furia, Mario Faretta & Alberto Diaspro

doi:10.1038/nmeth.1744

The combination of light-sheet microscopy and localization-based super-resolution imaging allows deep subdiffraction resolution imaging in thick scattering specimens as demonstrated by three-dimensional super-resolution imaging of proteins in live 150-μm-diameter cell spheroids.


Yeast one-hybrid assays for gene-centered human gene regulatory network mapping pp1050 - 1052

John S Reece-Hoyes, A Rasim Barutcu, Rachel Patton McCord, Jun Seop Jeong, Lizhi Jiang, Andrew MacWilliams, Xinping Yang, Kourosh Salehi-Ashtiani, David E Hill, Seth Blackshaw, Heng Zhu, Job Dekker & Albertha J M Walhout

doi:10.1038/nmeth.1764

A sequence-verified collection of human transcription factors is reported. The authors used it in the enhanced yeast-one hybrid (eY1H) assay to map human gene regulatory networks. Also in this issue, Reece-Hoyes et al. describe the eY1H pipeline.

See also: News and Views by Ozdemir & Stathopoulos


Enhanced Y1H assays for Arabidopsis pp1053 - 1055

Allison Gaudinier, Lifang Zhang, John S Reece-Hoyes, Mallorie Taylor-Teeples, Li Pu, Zhijie Liu, Ghislain Breton, Jose L Pruneda-Paz, Dahae Kim, Steve A Kay, Albertha J M Walhout, Doreen Ware & Siobhan M Brady

doi:10.1038/nmeth.1750

An almost-complete, sequence-verified collection of Arabidopsis thaliana root stele transcription factors is reported. The authors use it in the enhanced yeast-one hybrid (eY1H) assay to map gene regulatory interactions in the plant. Also in this issue, Reece-Hoyes et al. describe the eY1H pipeline.

See also: News and Views by Ozdemir & Stathopoulos


Embryonic stem cell–based mapping of developmental transcriptional programs pp1056 - 1058

Esteban O Mazzoni, Shaun Mahony, Michelina Iacovino, Carolyn A Morrison, George Mountoufaris, Michael Closser, Warren A Whyte, Richard A Young, Michael Kyba, David K Gifford & Hynek Wichterle

doi:10.1038/nmeth.1775

Presented is a study of gene regulation during development using a combination of chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) and directed differentiation of mouse embryonic stem cells inducibly expressing epitope-tagged transcription factors.

See also: News and Views by Ozdemir & Stathopoulos


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Articles

Enhanced yeast one-hybrid assays for high-throughput gene-centered regulatory network mapping pp1059 - 1064

John S Reece-Hoyes, Alos Diallo, Bryan Lajoie, Amanda Kent, Shaleen Shrestha, Sreenath Kadreppa, Colin Pesyna, Job Dekker, Chad L Myers & Albertha J M Walhout

doi:10.1038/nmeth.1748

The authors describe the enhanced yeast one-hybrid platform for large-scale screening of protein-DNA interactions and test its performance by mapping Caenorhabditis elegans gene regulatory networks. Also in this issue, Hens et al. describe an alternative platform for this purpose and apply it to screen for transcription factor–DNA interactions in Drosophila melanogaster.

See also: News and Views by Ozdemir & Stathopoulos


Automated protein-DNA interaction screening of Drosophila regulatory elements pp1065 - 1070

Korneel Hens, Jean-Daniel Feuz, Alina Isakova, Antonina Iagovitina, Andreas Massouras, Julien Bryois, Patrick Callaerts, Susan E Celniker & Bart Deplancke

doi:10.1038/nmeth.1763

Described is a high-throughput yeast one-hybrid platform for mapping protein-DNA interactions and a sequence-verified clone collection of Drosophila transcription factors. Also in this issue, Reece-Hoyes et al. report enhanced yeast one-hybrid assays, an alternative system for large-scale protein-DNA interaction screens.

See also: News and Views by Ozdemir & Stathopoulos


A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening pp1071 - 1077

Kyoji Horie, Chikara Kokubu, Junko Yoshida, Keiko Akagi, Ayako Isotani, Akiko Oshitani, Kosuke Yusa, Ryuji Ikeda, Yue Huang, Allan Bradley & Junji Takeda

doi:10.1038/nmeth.1739

This paper describes a method to enrich for homozygous mutant mouse embryonic stem cells without an inherently selectable phenotype. It is used to construct a bank of homozygous and heterozygous mutant cells and should prove useful for cellular phenotypic screens.


A scalable pipeline for highly effective genetic modification of a malaria parasite pp1078 - 1082

Claudia Pfander, Burcu Anar, Frank Schwach, Thomas D Otto, Mathieu Brochet, Katrin Volkmann, Michael A Quail, Arnab Pain, Barry Rosen, William Skarnes, Julian C Rayner & Oliver Billker

doi:10.1038/nmeth.1742

Reported is a recombineering pipeline for efficient and large-scale gene modification in the mouse malarial parasite Plasmodium berghei.


A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins pp1083 - 1088

Sonja Kleinlogel, Ulrich Terpitz, Barbara Legrum, Deniz Gökbuget, Edward S Boyden, Christian Bamann, Phillip G Wood & Ernst Bamberg

doi:10.1038/nmeth.1766

Molecular engineering allows stoichiometric and co-localized expression of two optogenetic actuators, spaced by a fluorescent protein and an additional transmembrane helix in a single protein fusion. The method provides modular optogenetic tools for bidirectional membrane potential control or synergistic effects on neuronal activity.

See also: News and Views by Oertner


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Erratum

Erratum: Sorting out sequencing data p1089

Monya Baker

doi:10.1038/nmeth1211-1089


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