Hot off the presses! Jul 01 Nat Meth

The Jul 01 issue of the Nat Meth is now up on Pubget (About Nat Meth): 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:

• Bioclouds
  - Nat Meth 7(7):481 (2010)
  Understanding how cloud computing can serve the scientific community is a research question in its own right and one that researchers—with the help of funders—should address.
• The author file: Vivek Jayaraman
  - Nat Meth 7(7):483 (2010)
  Fly brains light the way for neurobehavioral circuits.
• Primer-initiated sequence synthesis to detect and assemble structural variants
  Massouras A Hens K Gubelmann C Uplekar S Decouttere F Rougemont J Cole ST Deplancke B - Nat Meth 7(7):485-486 (2010)
  To the Editor: Structural variants constitute the largest portion of nucleotide variation in genomes, yet their comprehensive characterization based on high-throughput sequencing technologies is still challenging. Here we present primer-initiated sequence synthesis for genomes (PrInSeS-G), a software tool that detects and assembles sequence variants (1 base pair (bp) to |[sim]|10 kilobases (kb)) from single- or paired-end short reads.
• Nano-imaging of membrane topography affects interpretations in cell biology
  Jalink K van Rheenen J - Nat Meth 7(7):486 (2010)
  To the Editor: In their correspondence in Nature Methods, Adler et al. claim to present the first high-resolution topography of plasma membranes in living cells. Using hopping-probe ion conductance microscopy (HPICM), they show that the plasma membrane contains many subresolution membrane folds and point out serious implications for single-particle tracking studies.
• Advent of synthetic life
  - Nat Meth 7(7):487 (2010)
  Researchers synthesized the first functional synthetic bacterial genome; repeating this feat with different bacteria will require further methodological development.
• A toolset for the proficient geneticist
  - Nat Meth 7(7):488-489 (2010)
  New strategies expand the genetic toolkit for transgene expression, lineage tracing and mosaic analysis of gene function in flies and mammalian cells.
• Where do you come from?
  - Nat Meth 7(7):488-489 (2010)
  Live-cell time-lapse imaging of somatic cells undergoing reprogramming raises interesting questions about the mechanism of the process.
• News in brief
  - Nat Meth 7(7):489 (2010)
  Genomics The splicing code, decoded For many years, researchers have attempted to define the combinatorial rules that control alternative splicing. Barash et al. now report a code, implemented in a web tool, that predicts tissue-specific alternative splicing with high accuracy.
• Robotics on the nanoscale
  - Nat Meth 7(7):490 (2010)
  Nanoscale robots can be programmed to walk a DNA origami track.
• Searching for mismatches in a vast genomic landscape
  - Nat Meth 7(7):492 (2010)
  Raw data of millions of sequences used to assemble the reference genomes of ten organisms are analyzed in search of mismatches indicative of editing events. Findings include candidate sites for in vivo DNA and RNA editing, and a common sequencing error.
• Faster, sharper electron microscopy
  - Nat Meth 7(7):494 (2010)
  A technique combining laser and electron pulses is used to achieve nanometer and femtosecond resolution in biological imaging.
• Next-generation sequencing: adjusting to data overload
  Baker M - Nat Meth 7(7):495-499 (2010)
  To keep pace with accelerating sequencing machines, genomics researchers clean house and move toward the cloud.
• Illuminating eukaryotic transcription start sites
  Stamatoyannopoulos JA - Nat Meth 7(7):501-503 (2010)
  The initiation of transcription by RNA polymerase II is a defining event in eukaryotic gene regulation and represents the convergence of multiple cis- and trans-acting regulatory mechanisms. Genes are conventionally held to have a single canonical promoter, which in some cases may be accompanied by alternative promoters located at some distance (typically >500 base pairs (bp)) from the canonical promoter and one another.
• Watching the fly brain in action
  Clark DA de Vries SE Clandinin TR - Nat Meth 7(7):505-506 (2010)
  How does the brain transform sensory input into behavioral output? A strong approach to answering this question is to monitor activity from neurons in awake, behaving animals responding to sensory stimuli. Most of these experiments have been done in primates and rodents, and have identified brain regions and neural pathways involved in perception, sensorimotor integration and decision-making, among others.
• The inside view on plant growth
  Spalding EP - Nat Meth 7(7):506-507 (2010)
  Plant growth is a fine example of how a system in flux, such as a river, nonetheless presents a constant structure. From one day to the next, a plant meristem looks more or less the same despite being the site of prodigious cell division.
• Proteome-wide analysis of protein carboxy termini: C terminomics
  Schilling O Barré O Huesgen PF Overall CM - Nat Meth 7(7):508-511 (2010)
  As proteome-wide C-terminal sequence analysis has been largely intractable, we developed a polymer-based enrichment approach to profile protein C-terminal peptides by mass spectrometry and identified hundreds of C-terminal peptides in the Escherichia coli proteome. We isotopically labeled GluC protease–digested and undigested samples and identified GluC substrates and their cleavage sites by quantification of neo–C-terminal peptides. Our method thus enables global annotation of protein C-terminal posttranslational modifications, including proteolytic truncations.
• Complementary positional proteomics for screening substrates of endo- and exoproteases
  Van Damme P Staes A Bronsoms S Helsens K Colaert N Timmerman E Aviles FX Vandekerckhove J Gevaert K - Nat Meth 7(7):512-515 (2010)
  We describe a positional proteomics approach to simultaneously analyze N- and C-terminal peptides and used it to screen for human protein substrates of granzyme B and carboxypeptidase A4 in human cell lysates. This approach allowed comprehensive proteome studies, and we report the identification of 965 database-annotated protein C termini, 334 neo–C termini resulting from granzyme B processing and 16 neo–C termini resulting from carboxypeptidase A4 processing.
• Simultaneous intracellular chloride and pH measurements using a GFP-based sensor
  Arosio D Ricci F Marchetti L Gualdani R Albertazzi L Beltram F - Nat Meth 7(7):516-518 (2010)
  Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. We detected high chloride concentration in large dense-core exocytosis granules by targeting ClopHensor to these intracellular compartments.
• Estimating prion concentration in fluids and tissues by quantitative PMCA
  Chen B Morales R Barria MA Soto C - Nat Meth 7(7):519-520 (2010)
  Prions, the proteinaceous infectious agent responsible for prion diseases, can be detected with high sensitivity by protein misfolding cyclic amplification (PMCA) technology. Here we describe a quantitative PMCA procedure to calculate the concentration of very low levels of prions in biological samples. Using this procedure, we determined the quantities of misfolded prion protein (PrPSc) in brain, spleen, blood and urine of scrapie-affected hamsters.
• A paired-end sequencing strategy to map the complex landscape of transcription initiation
  - Nat Meth 7(7):521-527 (2010)
  Recent studies using high-throughput sequencing protocols have uncovered the complexity of mammalian transcription by RNA polymerase II, helping to define several initiation patterns in which transcription start sites (TSSs) cluster in both narrow and broad genomic windows. Here we describe a paired-end sequencing strategy, which enables more robust mapping and characterization of capped transcripts. We used this strategy to explore the transcription initiation landscape in the Drosophila melanogaster embryo. Extending the previous findings in mammals, we found that fly promoters exhibited distinct initiation patterns, which were linked to specific promoter sequence motifs. Furthermore, we identified many 5′ capped transcripts originating from coding exons; our analyses support that they are unlikely the result of alternative TSSs, but rather the product of post-transcriptional modifications. We demonstrated paired-end TSS analysis to be a powerful method to uncover ! the transcriptional complexity of eukaryotic genomes.
• Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan
  Plessy C Bertin N Takahashi H Simone R Salimullah M Lassmann T Vitezic M Severin J Olivarius S Lazarevic D Hornig N Orlando V Bell I Gao H Dumais J Kapranov P Wang H Davis CA Gingeras TR Kawai J Daub CO Hayashizaki Y Gustincich S Carninci P - Nat Meth 7(7):528-534 (2010)
  Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and noncoding RNAs with capped 5′ ends that vary in size. Methods to identify the 5′ ends of transcripts will facilitate the discovery of new promoters and 5′ ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nano–cap analysis of gene expression (nanoCAGE), a method that captures the 5′ ends of transcripts from as little as 10 ng of total RNA, and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5′ ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome.
• Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior
  Seelig JD Chiappe ME Lott GK Dutta A Osborne JE Reiser MB Jayaraman V - Nat Meth 7(7):535-540 (2010)
  Drosophila melanogaster is a model organism rich in genetic tools to manipulate and identify neural circuits involved in specific behaviors. Here we present a technique for two-photon calcium imaging in the central brain of head-fixed Drosophila walking on an air-supported ball. The ball's motion is tracked at high resolution and can be treated as a proxy for the fly's own movements. We used the genetically encoded calcium sensor, GCaMP3.0, to record from important elements of the motion-processing pathway, the horizontal-system lobula plate tangential cells (LPTCs) in the fly optic lobe. We presented motion stimuli to the tethered fly and found that calcium transients in horizontal-system neurons correlated with robust optomotor behavior during walking. Our technique allows both behavior and physiology in identified neurons to be monitored in a genetic model organism with an extensive repertoire of walking behaviors.
• cnvHap: an integrative population and haplotype–based multiplatform model of SNPs and CNVs
  Coin LJ Asher JE Walters RG El-Sayed Moustafa JS de Smith AJ Sladek R Balding DJ Froguel P Blakemore AI - Nat Meth 7(7):541-546 (2010)
  Although genome-wide association studies have uncovered single-nucleotide polymorphisms (SNPs) associated with complex disease, these variants account for a small portion of heritability. Some contribution to this 'missing heritability' may come from copy-number variants (CNVs), in particular rare CNVs; but assessment of this contribution remains challenging because of the difficulty in accurately genotyping CNVs, particularly small variants. We report a population-based approach for the identification of CNVs that integrates data from multiple samples and platforms. Our algorithm, cnvHap, jointly learns a chromosome-wide haplotype model of CNVs and cluster-based models of allele intensity at each probe. Using data for 50 French individuals assayed on four separate platforms, we found that cnvHap correctly detected at least 14% more deleted and 50% more amplified genotypes than PennCNV or QuantiSNP, with an 82% and 115% improvement for aberrations containing <10 probes! . Combining data from multiple platforms additionally improved sensitivity.
• Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution
  Fernandez R Das P Mirabet V Moscardi E Traas J Verdeil JL Malandain G Godin C - Nat Meth 7(7):547-553 (2010)
  Quantitative information on growing organs is required to better understand morphogenesis in both plants and animals. However, detailed analyses of growth patterns at cellular resolution have remained elusive. We developed an approach, multiangle image acquisition, three-dimensional reconstruction and cell segmentation–automated lineage tracking (MARS-ALT), in which we imaged whole organs from multiple angles, computationally merged and segmented these images to provide accurate cell identification in three dimensions and automatically tracked cell lineages through multiple rounds of cell division during development. Using these methods, we quantitatively analyzed Arabidopsis thaliana flower development at cell resolution, which revealed differential growth patterns of key regions during early stages of floral morphogenesis. Lastly, using rice roots, we demonstrated that this approach is both generic and scalable.
• Enhanced neuronal RNAi in C. elegans using SID-1
  Calixto A Chelur D Topalidou I Chen X Chalfie M - Nat Meth 7(7):554-559 (2010)
  We expressed SID-1, a transmembrane protein from Caenorhabditis elegans that is required for systemic RNA interference (RNAi), in C. elegans neurons. This expression increased the response of neurons to double-stranded (ds)RNA delivered by feeding. Mutations in the lin-15b and lin-35 genes enhanced this effect. Worms expressing neuronal SID-1 showed RNAi phenotypes when fed with bacteria expressing dsRNA for known neuronal genes and for uncharacterized genes with no previously known neuronal phenotypes. Neuronal expression of sid-1 decreased nonneuronal RNAi, suggesting that neurons expressing transgenic sid-1(+) served as a sink for dsRNA. This effect, or a sid-1(–) background, can be used to uncover neuronal defects for lethal genes. Expression of sid-1(+) from cell-specific promoters in sid-1 mutants results in cell-specific feeding RNAi. We used these strains to identify a role for integrin signaling genes in mechanosensation.
• Probabilistic density maps to study global endomembrane organization
  Schauer K Duong T Bleakley K Bardin S Bornens M Goud B - Nat Meth 7(7):560-566 (2010)
  We developed a computational imaging approach that describes the three-dimensional spatial organization of endomembranes from micromanipulation-normalized mammalian cells with probabilistic density maps. Applied to several well-known marker proteins, this approach revealed the average steady-state organization of early endosomes, multivesicular bodies or lysosomes, endoplasmic reticulum exit sites, the Golgi apparatus and Golgi-derived transport carriers in crossbow-shaped cells. The steady-state organization of each tested endomembranous population was well-defined, unique and in some cases depended on the cellular adhesion geometry. Density maps of all endomembrane populations became stable when pooling several tens of cells only and were reproducible in independent experiments, allowing construction of a standardized cell model. We detected subtle changes in steady-state organization induced by disruption of the cellular cytoskeleton, with statistical significance o! bserved for just 20 cells. Thus, combining micropatterning with construction of endomembrane density maps allows the systematic study of intracellular trafficking determinants.
• Corrigendum: A photoconvertible reporter of the ubiquitin-proteasome system in vivo
  - Nat Meth 7(7):567 (2010)
  Introduction Nat. Methods 7, 473–478 (2010); published online 9 May 2010; corrected after print 17 June 2010. ADVERTISEMENT In the version of this paper originally published, a reference to previous work on the use of Dendra2 as a reporter for protein stability in cultured cells should have been included (ref. 35). The error has been corrected in the PDF and HTML versions of the article.