Hot off the presses! Jul 01 Nat Methods

The Jul 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:

• Summer reading: science in fiction
  - Nat Methods 6(7):471 (2009)
  Though somewhat rare, there are a few good fiction books to be found with refreshingly realistic biologists as central characters in laboratory settings.
• MoDIL: detecting small indels from clone-end sequencing with mixtures of distributions
  - Nat Methods 6(7):473-474 (2009)
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• Limitations and possibilities of small RNA digital gene expression profiling
  - Nat Methods 6(7):474-476 (2009)
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• RNAiCut: automated detection of significant genes from functional genomic screens
  - Nat Methods 6(7):476-477 (2009)
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• Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates
  - Nat Methods 6(7):477-478 (2009)
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• A question of culture
  - Nat Methods 6(7):481 (2009)
  Two groups report culture conditions for long-term in vitro growth of intestinal tissue from the mouse.
• Fluorescent proteins: into the infrared
  - Nat Methods 6(7):482-483 (2009)
  An engineered infrared fluorescent protein is the first member of a new class of genetically encodable probes, with special advantages over visible-wavelength fluorescent proteins for in vivo imaging.
• Going with the skewed flow
  - Nat Methods 6(7):482-483 (2009)
  Computational and experimental biologists teamed up to develop a new software tool to analyze the rich data generated by new and powerful flow cytometers.
• News in brief
  - Nat Methods 6(7):483 (2009)
  
• Grafting as a potent molecular tool
  - Nat Methods 6(7):484 (2009)
  Grafting two transgenic plants triggers lateral gene transfer at the graft site but does not elicit long-distance transport of DNA into the scion or root of the graft.
• Fluorescent false neurotransmitters
  - Nat Methods 6(7):486 (2009)
  A fluorescent probe designed to incorporate a fluorophore into the structure of a neurotransmitter finds activity-dependent heterogeneity in dopamine release at individual synapses.
• Will planets reveal the light of their life?
  - Nat Methods 6(7):487 (2009)
  Optical signatures from organic chemicals may help scientists detect traces of life on other planets.
• Finding multiple needles in one immune haystack
  - Nat Methods 6(7):489-490 (2009)
  An approach using multiple fluorochrome combinations allows the simultaneous detection of many T-cell populations within a single blood sample.
• Downhill protein folding under pressure
  - Nat Methods 6(7):490-491 (2009)
  Sub-microsecond, downhill-reaction protein folding can be investigated by a method to generate large and fast pressure drops. The approach is complementary to nanosecond temperature-jump methods and could provide new insights into the biophysics of protein folding.
• Agouti C57BL/6N embryonic stem cells for mouse genetic resources
  - Nat Methods 6(7):493-495 (2009)
  We report the characterization of a highly germline competent C57BL/6N mouse embryonic stem cell line, JM8. To simplify breeding schemes, the dominant agouti coat color gene was restored in JM8 cells by targeted repair of the C57BL/6 nonagouti mutation. These cells provide a robust foundation for large-scale mouse knockout programs that aim to provide a public resource of targeted mutations in the C57BL/6 genetic background.
• Simultaneous detection of many T-cell specificities using combinatorial tetramer staining
  - Nat Methods 6(7):497-499 (2009)
  The direct detection of antigen-specific T cells using tetramers of soluble peptide–major histocompatibilty complex (pMHC) molecules is widely used in both basic and clinical immunology. However, the number of specificities that can be assessed simultaneously has been a major limitation. Here we describe and validate a method using combinations of fluorescent pMHC tetramers to simultaneously detect and enrich for many (15) T-cell specificities in a single human blood sample.
• Protein interaction platforms: visualization of interacting proteins in yeast
  - Nat Methods 6(7):500-502 (2009)
  Here we describe the protein interaction platform assay, a method for identifying interacting proteins in Saccharomyces cerevisiae. This assay relies on the reovirus scaffolding protein NS, which forms large focal inclusions in living cells. When a query protein is fused to NS and potential interaction partners are fused to a fluorescent reporter, interactors can be identified by screening for yeast that display fluorescent foci.
• Quantitative analysis of gene expression in a single cell by qPCR
  - Nat Methods 6(7):503-506 (2009)
  We developed a quantitative PCR method featuring a reusable single-cell cDNA library immobilized on beads for measuring the expression of multiple genes in a single cell. We used this method to analyze multiple cDNA targets (from several copies to several hundred thousand copies) with an experimental error of 15.9% or less. This method is sufficiently accurate to investigate the heterogeneity of single cells.
• Filter-based hybridization capture of subgenomes enables resequencing and copy-number detection
  - Nat Methods 6(7):507-510 (2009)
  To exploit contemporary sequencing technologies for targeted genetic analyses, we developed a hybridization enrichment strategy for DNA capture that uses PCR products as subgenomic traps. We applied this strategy to 115 kilobases of the human genome encompassing 47 genes implicated in cardiovascular disease. Massively parallel sequencing of captured subgenomic libraries interrogated 99.8% of targeted nucleotides 20 times (40,000-fold enrichment), enabling sensitive and specific detection of sequence variation and copy-number variation.
• In vivo fluorescence imaging with high-resolution microlenses
  - Nat Methods 6(7):511-512 (2009)
  Micro-optics are increasingly used for minimally invasive in vivo imaging, in miniaturized microscopes and in lab-on-a-chip devices. Owing to optical aberrations and lower numerical apertures, a main class of microlens, gradient refractive index lenses, has not achieved resolution comparable to conventional microscopy. Here we describe high-resolution microlenses, and illustrate two-photon imaging of dendritic spines on hippocampal neurons and dual-color nonlinear optical imaging of neuromuscular junctions in live mice.
• 'Injecting' yeast
  - Nat Methods 6(7):513-514 (2009)
  Yeast is a powerful genetic model system, but its rigid cell wall has prohibited microinjection. Using microfabricated channels to constrain the fission yeast Schizosaccharomyces pombe, we sheared local regions of individual cells with a piezoelectric unit. The cells remained viable, we detected actin patches in the cell after introduction of fluorescent phalloidin into the medium, and the cytokinetic ring was disrupted after injection of the myosin II inhibitor blebbistatin.
• Reaching the protein folding speed limit with large, sub-microsecond pressure jumps
  - Nat Methods 6(7):515-519 (2009)
  Biomolecules are highly pressure-sensitive, but their dynamics upon return to ambient pressure are often too fast to observe with existing approaches. We describe a sample-efficient method capable of large and very fast pressure drops (<1 nanomole, >2,500 atmospheres and <0.7 microseconds). We validated the method by fluorescence-detected refolding of a genetically engineered lambda repressor mutant from its pressure-denatured state. We resolved barrierless structure formation upon return to ambient pressure; we observed a 2.1 0.7 microsecond refolding time, which is very close to the 'speed limit' for proteins and much faster than the corresponding temperature-jump refolding of the same protein. The ability to experimentally perform a large and very fast pressure drop opens up a new region of the biomolecular energy landscape for atomic-level simulation.
• Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers
  - Nat Methods 6(7):520-526 (2009)
  The use of fluorescently labeled major histocompatibility complex multimers has become an essential technique for analyzing disease- and therapy-induced T-cell immunity. Whereas classical major histocompatibility complex multimer analyses are well-suited for the detection of immune responses to a few epitopes, limitations on human-subject sample size preclude a comprehensive analysis of T-cell immunity. To address this issue, we developed a combinatorial encoding strategy that allows the parallel detection of a multitude of different T-cell populations in a single sample. Detection of T cells from peripheral blood by combinatorial encoding is as efficient as detection with conventionally labeled multimers but results in a substantially increased sensitivity and, most notably, allows comprehensive screens to be performed. We obtained proof of principle for the feasibility of large-scale screening of human material by analysis of human leukocyte antigen A3–restricted T! -cell responses to known and potential melanoma-associated antigens in peripheral blood from individuals with melanoma.
• Doxycycline-dependent photoactivated gene expression in eukaryotic systems
  - Nat Methods 6(7):527-531 (2009)
  High spatial and temporal resolution of conditional gene expression is typically difficult to achieve in whole tissues or organisms. We synthesized two reversibly inhibited, photoactivatable ('caged') doxycycline derivatives with different membrane permeabilities for precise spatial and temporal light-controlled activation of transgenes based on the 'Tet-on' system. After incubation with caged doxycycline or caged cyanodoxycycline, we induced gene expression by local irradiation with UV light or by two-photon uncaging in diverse biological systems, including mouse organotypic brain cultures, developing mouse embryos and Xenopus laevis tadpoles. The amount of UV light needed for induction was harmless as we detected no signs of toxicity. This method allows high-resolution conditional transgene expression at different spatial scales, ranging from single cells to entire complex organisms.
• Mapping the structure and conformational movements of proteins with transition metal ion FRET
  - Nat Methods 6(7):532-537 (2009)
  Visualizing conformational dynamics in proteins has been difficult, and the atomic-scale motions responsible for the behavior of most allosteric proteins are unknown. Here we report that fluorescence resonance energy transfer (FRET) between a small fluorescent dye and a nickel ion bound to a dihistidine motif can be used to monitor small structural rearrangements in proteins. This method provides several key advantages over classical FRET, including the ability to measure the dynamics of close-range interactions, the use of small probes with short linkers, a low orientation dependence, and the ability to add and remove unique tunable acceptors. We used this 'transition metal ion FRET' approach along with X-ray crystallography to determine the structural changes of the gating ring of the mouse hyperpolarization-activated cyclic nucleotide–regulated ion channel HCN2. Our results suggest a general model for the conformational switch in the cyclic nucleotide–binding si! te of cyclic nucleotide–regulated ion channels.
• Genomics: catch me if you can
  - Nat Methods 6(7):539-544 (2009)
  Next-generation sequencing has made decoding entire genomes cheaper and faster. But what about those researchers who only want to sequence a small section of a genome or focus on a couple thousand specific exons? A wave of new technologies has recently emerged that should help these scientists target their sequencing efforts to sequences of interest.
• Corrigendum: A HUPO test sample study reveals common problems in mass spectrometry–based proteomics
  - Nat Methods 6(7):546 (2009)
  Introduction Alexander W Bell, Eric W Deutsch, Catherine E Au, Robert E Kearney, Ron Beavis, Salvatore Sechi, Tommy Nilsson, John J M Bergeron & HUPO Test Sample Working Group ADVERTISEMENT Nat. Methods 6, 423–430 (2009); published online 17 May 2009; corrected after print 29 June 2009. In the version of this article initially published, the author name Steven A. Carr was spelled incorrectly, and the name of an organization described in the text, the HUPO Proteomics Standards Initiative (PSI), was given incorrectly. These errors have been corrected in the PDF and HTML versions of this article.
• Erratum: Transposon-mediated genome manipulation in vertebrates
  - Nat Methods 6(7):546 (2009)
  Introduction Zoltán Ivics, Meng Amy Li, Lajos Mátés, Jef D Boeke, Andras Nagy, Allan Bradley & Zsuzsanna Izsvák ADVERTISEMENT Nat. Methods 6, 415–422 (2009); published online 28 May 2009; corrected after print 11 June 2009. In the version of this article initially published, a part of Figure 1b was incorrectly labeled. The error has been corrected in the HTML and PDF versions of the article.