Hot off the presses! Jun 01 Nat Methods

The Jun 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:

• No fruit fly an island?
  - Nat Methods 6(6):395 (2009)
  Methods to study the behavior of Drosophila sp. in the context of a group may deepen our understanding of the neural mechanisms underlying social behavior.
• Predicting microRNA targets and functions: traps for the unwary
  - Nat Methods 6(6):397-398 (2009)
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• Snapshots of the cell surface
  - Nat Methods 6(6):401 (2009)
  Researchers develop an approach to selectively isolate and profile cell-surface proteins by targeting the glycopeptides, a strategy that could be used to generate an atlas of cell-surface protein 'barcodes'.
• Micro-reprogramming
  - Nat Methods 6(6):402-403 (2009)
  Researchers use microRNAs to more efficiently generate induced pluripotent stem cells in the mouse.
• A functional blueprint of E. coli
  - Nat Methods 6(6):402-403 (2009)
  Researchers integrate proteomics data with genomic-context analysis and develop a protein-function prediction tool to annotate functional orphans in Escherichia coli.
• News in brief
  - Nat Methods 6(6):403 (2009)
  
• Nature's pH meter
  - Nat Methods 6(6):404 (2009)
  A new pH nanosensor changes color in acidic cell compartments by forming an unusual four-stranded DNA structure.
• TACLing rare genetic variants
  - Nat Methods 6(6):406 (2009)
  By combining methods for selective genome capture, allele enrichment and array resequencing, researchers create a pipeline for high-throughput variant detection.
• Flight patterns
  - Nat Methods 6(6):408 (2009)
  Flying animals ranging from bugs to bats use a common mechanism to maintain control in turns a discovery that reveals hidden advantages of flapping-wing flight.
• A stress test for mass spectrometry–based proteomics
  - Nat Methods 6(6):411-412 (2009)
  A multilaboratory study attempts to dispel some of the notions of the irreproducibility of mass spectrometry–based proteomics by pinpointing where the methodological problems are and where challenges remain.
• The ethomics era?
  - Nat Methods 6(6):413-414 (2009)
  Applying modern machine-vision techniques to the study of animal behavior, two groups developed systems that quantify many aspects of the complex social behaviors of Drosophila melanogaster. These software tools will enable high-throughput screens that seek to uncover the cellular and molecular underpinnings of behavior.
• Transposon-mediated genome manipulation in vertebrates
  - Nat Methods 6(6):415-422 (2009)
  Transposable elements are DNA segments with the unique ability to move about in the genome. This inherent feature can be exploited to harness these elements as gene vectors for genome manipulation. Transposon-based genetic strategies have been established in vertebrate species over the last decade, and current progress in this field suggests that transposable elements will serve as indispensable tools. In particular, transposons can be applied as vectors for somatic and germline transgenesis, and as insertional mutagens in both loss-of-function and gain-of-function forward mutagenesis screens. In addition, transposons will gain importance in future cell-based clinical applications, including nonviral gene transfer into stem cells and the rapidly developing field of induced pluripotent stem cells. Here we provide an overview of transposon-based methods used in vertebrate model organisms with an emphasis on the mouse system and highlight the most important considerations! concerning genetic applications of the transposon systems.
• A HUPO test sample study reveals common problems in mass spectrometry–based proteomics
  - Nat Methods 6(6):423-430 (2009)
  We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography–mass spectrometry–based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases ar! e needed for mass spectrometry–based proteomics.
• Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster
  - Nat Methods 6(6):431-434 (2009)
  We constructed Drosophila melanogaster bacterial artificial chromosome libraries with 21-kilobase and 83-kilobase inserts in the P[acman] system. We mapped clones representing 12-fold coverage and encompassing more than 95% of annotated genes onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using PhiC31 integrase to rescue mutations. They can be modified through recombineering, for example, to incorporate protein tags and assess expression patterns.
• A toolkit for high-throughput, cross-species gene engineering in Drosophila
  - Nat Methods 6(6):435-437 (2009)
  We generated two complementary genomic fosmid libraries for Drosophila melanogaster and Drosophila pseudoobscura that permit seamless modification of large genomic clones by high-throughput recombineering and direct transgenesis. The fosmid transgenes recapitulated endogenous gene expression patterns. These libraries, in combination with recombineering technology, will be useful to rescue mutant phenotypes, allow imaging of gene products in living flies and enable systematic analysis and manipulation of gene activity across species.
• TU-tagging: cell type–specific RNA isolation from intact complex tissues
  - Nat Methods 6(6):439-441 (2009)
  We found that the combination of spatially restricted uracil phosphoribosyltransferase (UPRT) expression with 4-thiouracil delivery can be used to label and purify cell type–specific RNA from intact complex tissues in Drosophila melanogaster. This method is useful for isolating RNA from cell types that are difficult to isolate by dissection or dissociation methods and should work in many organisms, including mammals and other vertebrates.
• Rapid creation and quantitative monitoring of high coverage shRNA libraries
  - Nat Methods 6(6):443-445 (2009)
  Short hairpin RNA libraries are limited by low efficacy of many shRNAs and by off-target effects, which give rise to false negatives and false positives, respectively. Here we present a strategy for rapidly creating expanded shRNA pools (approx30 shRNAs per gene) that are analyzed by deep sequencing (EXPAND). This approach enables identification of multiple effective target-specific shRNAs from a complex pool, allowing a rigorous statistical evaluation of true hits.
• Automated unrestricted multigene recombineering for multiprotein complex production
  - Nat Methods 6(6):447-450 (2009)
  Structural and functional studies of many multiprotein complexes depend on recombinant-protein overexpression. Rapid revision of expression experiments and diversification of the complexes are often crucial for success of these projects; therefore, automation is increasingly indispensable. We introduce Acembl, a versatile and automatable system for protein-complex expression in Escherichia coli that uses recombineering to facilitate multigene assembly and diversification. We demonstrated protein-complex expression using Acembl, including production of the complete prokaryotic holotranslocon.
• High-throughput ethomics in large groups of Drosophila
  - Nat Methods 6(6):451-457 (2009)
  We present a camera-based method for automatically quantifying the individual and social behaviors of fruit flies, Drosophila melanogaster, interacting in a planar arena. Our system includes machine-vision algorithms that accurately track many individuals without swapping identities and classification algorithms that detect behaviors. The data may be represented as an ethogram that plots the time course of behaviors exhibited by each fly or as a vector that concisely captures the statistical properties of all behaviors displayed in a given period. We found that behavioral differences between individuals were consistent over time and were sufficient to accurately predict gender and genotype. In addition, we found that the relative positions of flies during social interactions vary according to gender, genotype and social environment. We expect that our software, which permits high-throughput screening, will complement existing molecular methods available in Drosophila, ! facilitating new investigations into the genetic and cellular basis of behavior.
• Tissue tectonics: morphogenetic strain rates, cell shape change and intercalation
  - Nat Methods 6(6):458-464 (2009)
  The dynamic reshaping of tissues during morphogenesis results from a combination of individual cell behaviors and collective cell rearrangements. However, a comprehensive framework to unambiguously measure and link cell behavior to tissue morphogenesis is lacking. Here we introduce such a kinematic framework, bridging cell and tissue behaviors at an intermediate, mesoscopic, level of cell clusters or domains. By measuring domain deformation in terms of the relative motion of cell positions and the evolution of their shapes, we characterized the basic invariant quantities that measure fundamental classes of cell behavior, namely tensorial rates of cell shape change and cell intercalation. In doing so we introduce an explicit definition of cell intercalation as a continuous process. We mapped strain rates spatiotemporally in three models of tissue morphogenesis, gaining insight into morphogenetic mechanisms. Our quantitative approach has broad relevance for the precise c! haracterization and comparison of morphogenetic phenotypes.
• In vivo molecular imaging: the inside job
  - Nat Methods 6(6):465-469 (2009)
  In a short period of time, in vivo molecular imaging systems have become indispensable research tools in many clinical and basic research laboratories. But developers are now pushing the technology further in the hopes of making a new generation of platforms with greater accuracy and sensitivity for a wider array of applications.