Hot off the presses! May 01 Nat Methods

The May 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:

• Methods section remake
  - Nat Methods 6(5):313 (2009)
  Nature Methods follows in the footsteps of Nature by ushering in an Online Methods section, fully integrated with the paper, for all original research articles.
• Massively parallel exon capture and library-free resequencing across 16 genomes
  - Nat Methods 6(5):315-316 (2009)
  
• Lifeact cannot visualize some forms of stress-induced twisted f-actin
  - Nat Methods 6(5):317 (2009)
  
• Dawn on the mice
  - Nat Methods 6(5):319 (2009)
  By directly delivering light deep into the brain, scientists can now study the basis of neurological therapy and animal behavior.
• Capturing the human methylome
  - Nat Methods 6(5):320-321 (2009)
  Pairing bisulfite conversion of the human genome with targeted enrichment and high-throughput sequencing allows a quantitative assessment of DNA methylation at base-pair resolution.
• Sequence is not everything
  - Nat Methods 6(5):320-321 (2009)
  A new algorithm for identifying evolutionary constraint incorporates information on local DNA topology, and leads to the finding that this topology is conserved across species.
• News in brief
  - Nat Methods 6(5):321 (2009)
  
• Supercharging through the cell membrane
  - Nat Methods 6(5):322 (2009)
  Researchers show that superpositively charged GFP enters mammalian cells with ease and can be used as a nucleic acid delivery vehicle.
• A touching discovery
  - Nat Methods 6(5):324 (2009)
  Fake fingertips provide insights into how fingerprints help humans to feel fine details of surface texture.
• Protein production: no cells required
  - Nat Methods 6(5):326 (2009)
  DNA hydrogels improve the efficiency of cell-free protein production.
• Without a trace? PiggyBac-ing toward pluripotency
  - Nat Methods 6(5):329-330 (2009)
  A transposon-based approach has been added to the growing arsenal of technologies to produce transgene-free and potentially safer induced pluripotent stem cells.
• Imaging intracellular RNA distribution and dynamics in living cells
  - Nat Methods 6(5):331-338 (2009)
  Powerful methods now allow the imaging of specific mRNAs in living cells. These methods enlist fluorescent proteins to illuminate mRNAs, use labeled oligonucleotide probes and exploit aptamers that render organic dyes fluorescent. The intracellular dynamics of mRNA synthesis, transport and localization can be analyzed at higher temporal resolution with these methods than has been possible with traditional fixed-cell or biochemical approaches. These methods have also been adopted to visualize and track single mRNA molecules in real time. This review explores the promises and limitations of these methods.
• Super-resolution video microscopy of live cells by structured illumination
  - Nat Methods 6(5):339-342 (2009)
  Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.
• Enzymatic assembly of DNA molecules up to several hundred kilobases
  - Nat Methods 6(5):343-345 (2009)
  We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
• Single molecule–sensitive probes for imaging RNA in live cells
  - Nat Methods 6(5):347-349 (2009)
  To visualize native or non-engineered RNA in live cells with single-molecule sensitivity, we developed multiply labeled tetravalent RNA imaging probes (MTRIPs). When delivered with streptolysin O into living human epithelial cancer cells and primary chicken fibroblasts, MTRIPs allowed the accurate imaging of native mRNAs and a non-engineered viral RNA, of RNA co-localization with known RNA-binding proteins, and of RNA dynamics and interactions with stress granules.
• An ultramarine fluorescent protein with increased photostability and pH insensitivity
  - Nat Methods 6(5):351-353 (2009)
  We report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biological events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca2+ concentration and caspase-3 activation in the same apoptotic cells.
• Photoconversion in orange and red fluorescent proteins
  - Nat Methods 6(5):355-358 (2009)
  We found that photoconversion is fairly common among orange and red fluorescent proteins, as in a screen of 12 proteins, 8 exhibited photoconversion. Specifically, three red fluorescent proteins could be switched to a green state, and two orange variants could be photoconverted to a far-red state. The orange proteins are ideal for dual-probe highlighter applications, and they exhibited the most red-shifted excitation of all fluorescent proteins described to date.
• Universal sample preparation method for proteome analysis
  - Nat Methods 6(5):359-362 (2009)
  We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.
• Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon
  - Nat Methods 6(5):363-369 (2009)
  Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4 (Pou5f1), Sox2, Klf4 and Myc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we describe an efficient piggyBac transposon–based approach to generate integration-free iPSCs. Transposons carrying 2A peptide–linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. We removed transposons from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be identified by negative selection. piggyBac excised without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency! , such as pluripotency gene expression, teratoma formation and contribution to chimeras. piggyBac transposon–based reprogramming may be used to generate therapeutically applicable iPSCs.
• Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency
  - Nat Methods 6(5):370-376 (2009)
  Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome–specific mouse and human iPS cel! l lines with known mutations in MECP2.
• mRNA-Seq whole-transcriptome analysis of a single cell
  - Nat Methods 6(5):377-382 (2009)
  Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8–19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1-/- and Ago2-/- (Eif2c2-/-) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, w! ith 619 genes in common.
• Combined atomic force microscopy and side-view optical imaging for mechanical studies of cells
  - Nat Methods 6(5):383-387 (2009)
  The mechanical rigidity of cells and adhesion forces between cells are important in various biological processes, including cell differentiation, proliferation and tissue organization. Atomic force microscopy has emerged as a powerful tool to quantify the mechanical properties of individual cells and adhesion forces between cells. Here we demonstrate an instrument that combines atomic force microscopy with a side-view fluorescent imaging path that enables direct imaging of cellular deformation and cytoskeletal rearrangements along the axis of loading. With this instrument, we directly observed cell shape under mechanical load, correlated changes in shape with force-induced ruptures and imaged formation of membrane tethers during cell-cell adhesion measurements. Additionally, we observed cytoskeletal reorganization and stress-fiber formation while measuring the contractile force of an individual cell. This instrument can be a useful tool for understanding the role of me! chanics in biological processes.
• Proteins and proteomics: life on the surface
  - Nat Methods 6(5):389-393 (2009)
  Surface plasmon resonance sensing has entered the next phase of development as researchers advance array-based applications using the technique. Could these new approaches change the way scientists explore protein interactions?