Hot off the presses! Oct 01 Nat Methods

The Oct 01 issue of the Nat Methods is now up on Pubget (About Nat Methods): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• In celebration of methods
  - Nat Methods 6(10):687 (2009)
  As evidenced by the cake adorning the cover, Nature Methods is five years old. To celebrate this anniversary, we look at methodological development and its role in scientific inquiry.
• Online image analysis software for photoactivation localization microscopy
  - Nat Methods 6(10):689-690 (2009)
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• My5C: web tools for chromosome conformation capture studies
  - Nat Methods 6(10):690-691 (2009)
  I want to purchase this article Register now Price: US$18 In order to purchase this article you must be a registered user. I want to subscribe to Nature Methods Select this option to purchase a personal subscription to Nature Methods.
• Two photons as exciting as one
  - Nat Methods 6(10):693 (2009)
  Channelrhodopsin-2 can be efficiently activated by infrared two-photon excitation light and stimulates action potentials in cultured neurons.
• 'Blue' lighting cell signaling research
  - Nat Methods 6(10):694-695 (2009)
  By fusing a light-sensitive domain of an oat plant protein to Rac1, researchers created a genetically encoded protein fusion that can be reversibly activated with blue light and control cell movement—an attractive alternative to current caging tools.
• A leader anyone can follow
  - Nat Methods 6(10):694-695 (2009)
  The development of leader sequences that stimulate mRNA translation in a species-independent manner could offer new possibilities for eukaryotic protein production and proteomic research.
• News in brief
  - Nat Methods 6(10):695 (2009)
  
• Illuminating lipids
  - Nat Methods 6(10):696 (2009)
  Visualization of choline-containing phospholipids in cells and in vivo is made possible by the metabolic incorporation of a choline analog with an alkyne handle for click chemistry–based labeling.
• Engineering bacteria in yeast
  - Nat Methods 6(10):698 (2009)
  Bacterial genomes shuttled into yeast can be easily altered before transplantation back into bacteria.
• Technical matters: method, knowledge and infrastructure in twentieth-century life science
  - Nat Methods 6(10):701-705 (2009)
  Conceptual breakthroughs in science tend to garner accolades and attention. But, as the invention of tissue culture and the development of isotopic tracers show, innovative methods open up new fields and enable the solution of longstanding problems.
• Seeing things: from microcinematography to live cell imaging
  - Nat Methods 6(10):707-709 (2009)
  From histology to microcinematography, from cytochemistry to live cell imaging, the history of visualization technology in the life sciences may be understood as a series of cycles of action and reaction between static and dynamic modes of representing life.
• Is sequencing enlightenment ending the dark age of the transcriptome?
  - Nat Methods 6(10):711-713 (2009)
  Sequencing-based technologies for RNA discovery are playing a key role in deciphering the transcriptome and hold the potential to provide us with a census of RNAs and their functions.
• Engineered fluorescent proteins: innovations and applications
  - Nat Methods 6(10):713-717 (2009)
  Despite expansion of the fluorescent protein and optical highlighter palette into the orange to far-red range of the visible spectrum, achieving performance equivalent to that of EGFP has continued to elude protein engineers.
• Comparative analysis to guide quality improvements in proteomics
  - Nat Methods 6(10):717-719 (2009)
  The potential of mass spectrometry–based proteomics to advance biology and biomedicine is nearly unlimited but so is its potential for generating bad data. Apart from the pursuit of technological progress in protocols and instruments, stringent comparative analyses of different approaches are critical for fully developing the discipline.
• From information to knowledge: new technologies for defining gene function
  - Nat Methods 6(10):721-723 (2009)
  A wide range of methodology will be needed to bridge the gap between genome sequence and mechanistic understanding in biology. Recent advances in high-throughput genetic screening address this task.
• Five years of Methods
  - Nat Methods 6(10):724-725 (2009)
  Introduction A fun selection of papers published in Nature Methods since 2004. Fastest citation rate Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Nat. Methods 5, 621–628 (2008). Cloonan, N. et al. Nat. Methods 5, 613–619 (2008). Some methods take a field by storm, and RNA-seq is one of those. The year 2008 saw several high-profile papers describing transcriptome profiling with second-generation sequencing platforms. The analysis of mammalian cell transcripts by Barbara Wold's group was received with instant interest by our readers as was a related paper by another group. Most surprising Fortin, D.L. et al. Nat. Methods 5, 331–338 (2008). Imbuing cellular proteins with new characteristics generally requires the introduction of engineered genes or proteins. But Richard Kramer and colleagues showed that attaching a photoisomerizable small molecule to endogenous K+ channels in neurons allows specific light-mediated control of their activity. Fastest to publication Xu, R.H. et al. Nat. Methods 2, 185–190 (2005). This report from James Thomson's group on proliferation mechanisms in human embryonic stem cells not only made culturing them easier but also sped through the review process to make it online just about a month after submission. Seen by the most economists Choi, W. et al. Nat. Methods 4, 717–719 (2007). It seems impossible to predict where a news article about a paper published in Nature Methods will appear. This was aptly demonstrated when an article by Michael Feld and colleagues on a new microscopy method called tomographic phase microscopy was covered in the 16 August 2007 issue of The Economist. Most 'insulting' Nishimura, N. et al. Nat. Methods 3, 99–108 (2006). Authors and editors try to avoid the appearance of insults in papers. But the January 2006 issue of Nature Methods not only had a paper by David Kleinfeld and colleagues devoted to targeted insults but also featured it on the cover. The laser-based insults were designed to model strokes in mice, though. Leaving the least trace Yusa, K., Rad, R., Takeda, J. & Bradley, A. Nat. Methods 6, 363–369 (2009). Joining the race to make induced pluripotent stem cells without permanent genome modification, this paper reports on the use of the piggyBac transposon to transiently express the four reprogramming factors and then remove them from the genome without a trace. Top article Irizarry, R.A. et al. Nat. Methods 2, 345–350 (2005). Frustration with irreproducible DNA microarray results begged the question: is this an inherent problem of the technology? In 2005 a consortium said no, showing that data collected on three platforms in ten labs agreed well. Our citation numbers indicate that the community eagerly received this message. Most aptly named Rust, M.J., Bates, M. & Zhuang, X. Nat. Methods 3, 793–795 (2006). Nature Methods published a paper describing a super-resolution imaging method on 9 August 2006, one day before a competing paper was published in Science. The name of the method, 'STORM', seemed to embody the level of interest these two papers generated in the community. Simplest fishing tool Vigneault, F., Sismour, A.M. & Church, G.M. Nat. Methods 5, 777–779 (2008). To fish out microRNAs from a sample of interest, you need good tools, and the less complicated the better. This paper reported a method to prepare preadenylated barcoded oligonucleotides, which could be used for efficient capture and multiplex analysis of microRNA from biological samples. Smallest tools Ebert, M.S., Neilson, J.R. & Sharp, P.A. Nat. Methods 4, 721–726 (2007). Loss-of-function phenotypes for microRNAs were still rare in 2007, explaining the appeal of microRNA sponges. These 'sponge' transcripts, containing multiple binding sites for a microRNA, soak it up and derepress its target, thus creating a miRNA knockdown phenotype. Among the most deadly Herm-Göz, A. et al. Nat. Methods 4, 1003–1005 (2007). Armstrong, C.M. & Goldberg, D.E. Nat. Methods 4, 1007–1009 (2007). Quod licet Homo non licet Apicomplexa—or maybe it is. Two groups showed that an inducible protein destabilization strategy developed for mammalian cells could be adapted for Apicomplexa to study proteins in these parasites that cause malaria and encephalitis. Most expensive instrument Hura, G.L. et al. Nat. Methods 6, 606–612 (2009). Structural biology is relying more and more on that most expensive of instruments, the synchrotron. John Tainer and colleagues used synchrotron radiation to rapidly carry out small-angle X-ray scattering (SAXS)-based analyses of folds and oligomeric states for large numbers of proteins. Most chemical Hennig, A., Bakirci, H. & Nau, W.M. Nat. Methods 4, 629–632 (2007). Examples of how chemistry provides useful tools for biological applications can be found in many of our issues. One such paper describes a new principle for label-free enzymatic assays, taking advantage of macrocycles that reversibly form complexes with fluorescent dyes and enzymatic products. Fastest amplification Atarashi, R. et al. Nat. Methods 4, 645–650 (2007). Atarashi, R. et al. Nat. Methods 5, 211–212 (2008). The protein misfolding cyclic amplification (PMCA) assay is widely used to detect the protease-resistant prion protein responsible for 'mad cow' and other prion diseases. With several improvements to the protocol, Byron Caughey and colleagues made this assay faster and more sensitive. Among the sweetest Structurally speaking, sugars are complex molecules, but they are also predictable in their protein binding preferences. With an evanescent-field method to detect glycans bound to arrayed lectins, Jun Hirabayashi and colleagues showed that they could not only profile glycans but do so quantitatively. Kuno, A. et al. Nat. Methods 2, 851–856 (2005).
• Staging worms for next-generation analysis
  - Nat Methods 6(10):727-728 (2009)
  Automated stage-specific sorting of Caenorhabditis elegans embryos enables next-generation molecular and biochemical analysis of development.
• Nanoscale 3D cellular imaging by axial scanning transmission electron tomography
  - Nat Methods 6(10):729-731 (2009)
  Electron tomography provides three-dimensional structural information about supramolecular assemblies and organelles in a cellular context, but image degradation, caused by scattering of transmitted electrons, limits applicability in specimens thicker than 300 nm. We found that scanning transmission electron tomography of 1,000-nm-thick samples using axial detection provided resolution comparable to that of conventional electron tomography. We demonstrated the method by reconstructing a human erythrocyte infected with the malaria parasite Plasmodium falciparum.
• Somatic cell nuclear transfer in zebrafish
  Siripattarapravat K Pinmee B Venta PJ Chang CC Cibelli JB - Nat Methods 6(10):733-735 (2009)
  We developed a method for somatic cell nuclear transfer in zebrafish using laser-ablated metaphase II eggs as recipients, the micropyle for transfer of the nucleus and an egg activation protocol after nuclear reconstruction. We produced clones from cells of both embryonic and adult origins, although the latter did not give rise to live adult clones.
• Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis
  Vinkenborg JL Nicolson TJ Bellomo EA Koay MS Rutter GA Merkx M - Nat Methods 6(10):737-740 (2009)
  We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn2+ binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn2+ was buffered at 0.4 nM in pancreatic cells, and we found substantially higher Zn2+ concentrations in insulin-containing secretory vesicles.
• Proteomics strategy for quantitative protein interaction profiling in cell extracts
  - Nat Methods 6(10):741-744 (2009)
  We report a proteomics strategy to both identify and quantify cellular target protein interactions with externally introduced ligands. We determined dissociation constants for target proteins interacting with the ligand of interest by combining quantitative mass spectrometry with a defined set of affinity purification experiments. We demonstrate the general utility of this methodology in interaction studies involving small-molecule kinase inhibitors, a tyrosine-phosphorylated peptide and an antibody as affinity ligands.
• Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression
  - Nat Methods 6(10):745-751 (2009)
  Caenorhabditis elegans is one of the most prominent model systems for embryogenesis, but collecting many precisely staged embryos has been impractical. Thus, early C. elegans embryogenesis has not been amenable to most high-throughput genomics or biochemistry assays. To overcome this problem, we devised a method to collect staged C. elegans embryos by fluorescence-activated cell sorting (eFACS). In a proof-of-principle experiment, we found that a single eFACS run routinely yielded tens of thousands of almost perfectly staged 1-cell stage embryos. As the earliest embryonic events are driven by posttranscriptional regulation, we combined eFACS with second-generation sequencing to profile the embryonic expression of small, noncoding RNAs. We discovered complex and orchestrated changes in the expression between and within almost all classes of small RNAs, including microRNAs and 26G-RNAs, during embryogenesis.
• A Flp-nick system to study repair of a single protein-bound nick in vivo
  - Nat Methods 6(10):753-757 (2009)
  We present the Flp-nick system, which allows introduction of a protein-bound nick at a single genomic site in Saccharomyces cerevisiae and thus mimics a stabilized topoisomerase I–DNA cleavage complex. We took advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp recombinase recognition target site that has been integrated in the yeast genome. The genetic requirement for cells to cope with this insult is the same as for cells treated with camptothecin, which traps topoisomerase I–DNA cleavage complexes genome-wide. Hence, a single protein-bound nick is enough to kill cells if functional repair pathways are lacking. The Flp-nick system can be used to dissect repair, checkpoint and replication fork management pathways activated by a single genomic insult, and it allows the study of events at the damage site, which so far has been impossible to address.
• An approach for extensibly profiling the molecular states of cellular subpopulations
  - Nat Methods 6(10):759-765 (2009)
  Microscopy often reveals the existence of phenotypically distinct cellular subpopulations. However, additional characterization of observed subpopulations can be limited by the number of biomolecular markers that can be simultaneously monitored. Here we present a computational approach for extensibly profiling cellular subpopulations by freeing one or more imaging channels to monitor additional probes. In our approach, we trained classifiers to re-identify subpopulations accurately based on an enhanced collection of phenotypic features extracted from only a subset of the original markers. Then we constructed subpopulation profiles step-wise from replicate experiments, in which cells were labeled with different but overlapping marker sets. We applied our approach to identify molecular differences among subpopulations and to identify functional groupings of markers, in populations of differentiating mouse preadipocytes, polarizing human neutrophil-like cells and dividing! human cancer cells.
• Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms
  - Nat Methods 6(10):767-772 (2009)
  Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how microorganisms cause disease. Here we present a method (Tn-seq) for accurately determining quantitative genetic interactions on a genome-wide scale in microorganisms. Tn-seq is based on the assembly of a saturated Mariner transposon insertion library. After library selection, changes in frequency of each insertion mutant are determined by sequencing the flanking regions en masse. These changes are used to calculate each mutant's fitness. Using this approach, we determined fitness for each gene of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. A genome-wide screen for genetic interactions of five query genes identified both alleviating and aggravating interactions that could be divided into seven distinct categories. Owing to the wide activity of the Mariner transposon, Tn-seq has t! he potential to contribute to the exploration of complex pathways across many different species.
• Getting inside their minds
  - Nat Methods 6(10):773-781 (2009)
  Neuroscientists are taking advantage of powerful new tools for fluorescence imaging that enable detailed visualization of the structure and activity of neuronal circuits within the living brain.