Hot off the presses! Apr 01 Nature methods

The Apr 01 issue of the Nature methods is now up on Pubget (About Nature 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:

• Cause and effect
  - Nature methods 7(4):243 (2010)
  The experimental tractability of biological systems makes it possible to explore the idea that causal relationships can be estimated from observational data.
• The author file: Philippe Bastiaens
  - Nature methods 7(4):245 (2010)
  Researchers watch proteins interact in real time.
• Predicting causal effects in large-scale systems from observational data
  - Nature methods 7(4):247-248 (2010)
  To the editor: Understanding cause-effect relationships between variables is of primary interest in many fields of science. The standard method for determining such relationships uses randomized controlled perturbation experiments.
• A method and server for predicting damaging missense mutations
  - Nature methods 7(4):248-249 (2010)
  To the editor: Applications of rapidly advancing sequencing technology exacerbate the need to interpret individual sequence variants. Sequencing of phenotyped clinical subjects will soon become a method of choice in studies of the genetic causes of Mendelian and complex diseases.
• Single-nucleotide evolutionary constraint scores highlight disease-causing mutations
  - Nature methods 7(4):250-251 (2010)
  To the editor: Identifying disease-causing genetic variants in individual human genomes is a major challenge, even in protein-coding exons (the 'exome'). Analysis of nucleotide-level sequence conservation may help address this challenge, on the assumption that purifying selection 'constrains' evolutionary divergence at phenotypically important nucleotides.
• Collective brain maps
  - Nature methods 7(4):253 (2010)
  A new study, pooling brain-imaging data from 35 centers across the world, shows the power of data sharing and demonstrates a universal architecture of functional connections in the human brain.
• The DNA-binding landscape
  - Nature methods 7(4):254-255 (2010)
  Visualizing binding data in the form of specificity landscapes yields new insight into the behavior of DNA-binding molecules.
• Fission yeast defies the code
  - Nature methods 7(4):254-255 (2010)
  A genome-wide map of nucleosome positions in Schizosaccharomyces pombe points to a positioning mechanism different from that of other organisms.
• News in brief
  - Nature methods 7(4):255 (2010)
  Stem cells From fibroblasts to neurons No biologist would be surprised these days if one cell type was converted to another by going through a pluripotent stage, nor if this reprogramming was done directly between two closely related cell lineages. But Vierbuchen et al. now demonstrate a way to directly convert fibroblasts into distantly related excitatory neurons.
• Glycan labeling, rerouted
  - Nature methods 7(4):256 (2010)
  Researchers develop a method to label GlcNAc residues of N-glycans in yeast with high specificity and efficiency.
• Tracking infection
  - Nature methods 7(4):258 (2010)
  A new cell-based fluorescent reporter system allows direct visualization of individual cells infected with hepatitis C virus in real time.
• Proteomics for exotic organisms
  - Nature methods 7(4):260 (2010)
  A creative application of protein labeling can probe uncharacterized genomes.
• Laser tricks without labels
  - Nature methods 7(4):261-266 (2010)
  Nonlinear optical microscopy lets researchers see chemical composition in living cells and organisms.
• The 'when and whereabouts' of injected pathogen effectors
  - Nature methods 7(4):267-269 (2010)
  Being in the right place at the right time is a straightforward recipe for success. This also holds true for many bacteria that use syringe-like injection devices, such as the type-3 secretion system (T3SS), to deliver into host cells cocktails of effector proteins that interfere with various cellular functions.
• Not all noise is waste
  - Nature methods 7(4):269-272 (2010)
  The dominance of deterministic thought in cell biology has obscured a fundamental property of living systems—variability. A cell population, however uniform, is ripe with heterogeneities.
• Toward atomic accuracy in RNA design
  - Nature methods 7(4):272-273 (2010)
  The amazingly diverse roles of RNA molecules in biology are attracting increasing attention. Their varied biological functions are a result of the diversity of three-dimensional structures they adopt.
• Probing cellular events, one quantum dot at a time
  - Nature methods 7(4):275-285 (2010)
  Monitoring the behavior of single molecules in living cells is a powerful approach to investigate the details of cellular processes. Owing to their optical, chemical and biofunctional properties, semiconductor quantum dot (QD) probes promise to be tools of choice in this endeavor. Here we review recent advances that allow ever more controlled experiments at the single-nanoparticle level in live cells. Several examples, related to membrane dynamics, cell signaling or intracellular transport, illustrate how single QD tracking can be readily used to decipher complex biological processes and address key concepts that underlie cellular organization and dynamics.
• Cell type–specific gene expression differences in complex tissues
  Shen-Orr SS Tibshirani R Khatri P Bodian DL Staedtler F Perry NM Hastie T Sarwal MM Davis MM Butte AJ - Nature methods 7(4):287-289 (2010)
  We describe cell type–specific significance analysis of microarrays (csSAM) for analyzing differential gene expression for each cell type in a biological sample from microarray data and relative cell-type frequencies. First, we validated csSAM with predesigned mixtures and then applied it to whole-blood gene expression datasets from stable post-transplant kidney transplant recipients and those experiencing acute transplant rejection, which revealed hundreds of differentially expressed genes that were otherwise undetectable.
• Atomic accuracy in predicting and designing noncanonical RNA structure
  Das R Karanicolas J Baker D - Nature methods 7(4):291-294 (2010)
  We present fragment assembly of RNA with full-atom refinement (FARFAR), a Rosetta framework for predicting and designing noncanonical motifs that define RNA tertiary structure. In a test set of thirty-two 6–20-nucleotide motifs, FARFAR recapitulated 50% of the experimental structures at near-atomic accuracy. Sequence redesign calculations recovered native bases at 65% of residues engaged in noncanonical interactions, and we experimentally validated mutations predicted to stabilize a signal recognition particle domain.
• Fluorescence fluctuations of quantum-dot sensors capture intracellular protein interaction dynamics
  Zamir E Lommerse PH Kinkhabwala A Grecco HE Bastiaens PI - Nature methods 7(4):295-298 (2010)
  We extend the in vitro principle of co-immunoprecipitation to quantify dynamic protein interactions in living cells. Using a multiresolution implementation of fluorescence correlation spectroscopy to achieve maximal temporal resolution, we monitored the interactions of endogenous bait proteins, recruited by quantum dots, with fluorescently tagged prey. With this approach, we analyzed the rapid physiological regulation of protein kinase A.
• Direct determination of molecular haplotypes by chromosome microdissection
  Ma L Xiao Y Huang H Wang Q Rao W Feng Y Zhang K Song Q - Nature methods 7(4):299-301 (2010)
  Direct observation of haplotypes is still technically challenging. Here we report a method for the determination of haplotypes through chromosome microdissection. We determined human long-range chromosomal haplotypes with more than 98.85% accuracy at 24,245 genome-wide heterozygous single-nucleotide polymorphism (SNP) loci.
• In vivo wide-area cellular imaging by side-view endomicroscopy
  Kim P Chung E Yamashita H Hung KE Mizoguchi A Kucherlapati R Fukumura D Jain RK Yun SH - Nature methods 7(4):303-305 (2010)
  In vivo imaging of small animals offers several possibilities for studying normal and disease biology, but visualizing organs with single-cell resolution is challenging. We describe rotational side-view confocal endomicroscopy, which enables cellular imaging of gastrointestinal and respiratory tracts in mice and may be extensible to imaging organ parenchyma such as cerebral cortex. We monitored cell infiltration, vascular changes and tumor progression during inflammation and tumorigenesis in colon over several months.
• Toxoplasma secreting Cre recombinase for analysis of host-parasite interactions
  Koshy AA Fouts AE Lodoen MB Alkan O Blau HM Boothroyd JC - Nature methods 7(4):307-309 (2010)
  We describe a Toxoplasma gondii strain that will permit the use of site-specific recombination to study the host-parasite interactions of this organism. This Toxoplasma strain efficiently injects a Cre fusion protein into host cells. In a Cre-reporter cell line, a single parasite invasion induced Cre-mediated recombination in 95% of infected host cells. By infecting Cre-reporter mice with these parasites, we also monitored host-cell infection in vivo.
• Identifying single-cell molecular programs by stochastic profiling
  Janes KA Wang CC Holmberg KJ Cabral K Brugge JS - Nature methods 7(4):311-317 (2010)
  Cells in tissues can be morphologically indistinguishable yet show molecular expression patterns that are remarkably heterogeneous. Here we describe an approach to comprehensively identify co-regulated, heterogeneously expressed genes among cells that otherwise appear identical. The technique, called stochastic profiling, involves repeated, random selection of very small cell populations via laser-capture microdissection followed by a customized single-cell amplification procedure and transcriptional profiling. Fluctuations in the resulting gene-expression measurements are then analyzed statistically to identify transcripts that are heterogeneously coexpressed. We stochastically profiled matrix-attached human epithelial cells in a three-dimensional culture model of mammary-acinar morphogenesis. Of 4,557 transcripts, we identified 547 genes with strong cell-to-cell expression differences. Clustering of this heterogeneous subset revealed several molecular 'programs' impl! icated in protein biosynthesis, oxidative-stress responses and NF-κB signaling, which we independently confirmed by RNA fluorescence in situ hybridization. Thus, stochastic profiling can reveal single-cell heterogeneities without the need to measure expression in individual cells.
• Protein folding stability and dynamics imaged in a living cell
  Ebbinghaus S Dhar A McDonald JD Gruebele M - Nature methods 7(4):319-323 (2010)
  Biomolecular dynamics and stability are predominantly investigated in vitro and extrapolated to explain function in the living cell. We present fast relaxation imaging (FreI), which combines fluorescence microscopy and temperature jumps to probe biomolecular dynamics and stability inside a single living cell with high spatiotemporal resolution. We demonstrated the method by measuring the reversible fast folding kinetics as well as folding thermodynamics of a fluorescence resonance energy transfer (FRET) probe-labeled phosphoglycerate kinase construct in two human cell lines. Comparison with in vitro experiments at 23–49 °C showed that the cell environment influences protein stability and folding rate. FReI should also be applicable to the study of protein-protein interactions and heat-shock responses as well as to comparative studies of cell populations or whole organisms.
• Imaging type-III secretion reveals dynamics and spatial segregation of Salmonella effectors
  Van Engelenburg SB Palmer AE - Nature methods 7(4):325-330 (2010)
  The type-III secretion system (T3SS) enables gram-negative bacteria to inject effector proteins into eukaryotic host cells. Upon entry, T3SS effectors work cooperatively to reprogram host cells, enabling bacterial survival. Progress in understanding when and where effectors localize in host cells has been hindered by a dearth of tools to study these proteins in the native cellular environment. We report a method to label and track T3SS effectors during infection using a split-GFP system. We demonstrate this technique by labeling three effectors from Salmonella enterica (PipB2, SteA and SteC) and characterizing their localization in host cells. PipB2 displayed highly dynamic behavior on tubules emanating from the Salmonella-containing vacuole labeled with both endo- and exocytic markers. SteA was preferentially enriched on tubules localizing with Golgi markers. This segregation suggests that effector targeting and localization may have a functional role during infection.