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A combination of in vitro protein synthesis and microfluidics is used to measure protein-protein interactions between 43 proteins in Streptococcus pneumoniae. The method does not require expression within cells and is amenable to large-scale experiments.
An infrared laser is used to activate gene expression from a heat shock promoter in single cells in Caenorhabditis elegans, and is shown to be more effective and less detrimental to cells than a visible laser used for this purpose.
A web-based protein-protein interaction (PPI) analysis platform called PINA integrates PPI data from six public databases and provides tools to aid in the construction and analysis of PPI networks, including local recuration and annotation of existing records and manual addition of new records.
This variant ascertainment algorithm, or VAAL, uses short sequence reads of haploid bacterial genomes to first locally assemble the reads and then compare these assemblies to the reference genome. This allows VAAL to detect all types of variants ranging from single-nucleotide polymorphisms to large insertions or deletions.
High-throughput yeast two-hybrid screening is used to generate the largest C. elegans interactome resource available thus far. Using an empirical quality control framework presented in Venkatesan et al., also online, the data set is evaluated for quality and is used to estimate the total size of the worm interactome.
Different experimental designs for protein interaction mapping are modeled to compare their efficiency in completing an interactome map. Testing of the strategy that minimized the final experimental cost in an ongoing Drosophila melanogaster interactome project found 450 high-confidence interactions using only 47 microtiter plates.
A framework based on numerous empirical data, including protein-protein interaction reference sets, provides parameters for assessing the quality and coverage of protein-protein interaction datasets and estimation of the size of the human interactome. Braun et al., also in this issue, use the reference sets to help derive confidence scores for individual protein-protein interactions.
Use of the protein-protein interaction reference sets reported in this issue in Venkatesan et al. to benchmark four complementary protein-protein interaction assays, followed by the training of a logistic regression model, allows the assignment of standardized confidence scores to individual protein-protein interactions.
Massively parallel sequencing is a precise way to analyze copy-number variations given the right computational tools. An algorithm now facilitates the detection and fine mapping of copy-number gains and losses from millions of short sequence reads.
To study microRNA function in vivo, the authors optimize lentiviral-driven expression of microRNA target sequences in mice and show dose-dependent inhibition of microRNA-mediated regulation of reporter constructs as well as of natural microRNA targets. With the inhibition of a miR-223, they can phenocopy the knockout of this microRNA.
An efficient pipeline for mapping antibody epitopes is presented. Combining bacterial surface display of peptide libraries, flow cytometric sorting, and pyrosequencing, the approach is amenable to a high-throughput format and should find future application in whole-proteome studies.
A collection of 33,275 human Gateway entry clones and complementary in vitro protein expression methodologies are described that allow proteome-scale production of human proteins. This 'human protein factory' was validated by expression of 13,364 human proteins and assessment of activity in a variety of assays.
Extension of multicolor three-dimensional stochastic optical reconstruction microscopy (STORM) allows super-resolution fluorescence imaging of whole cells and quantitative characterization of subcellular structures and their spatial relationships. This was demonstrated by imaging the entire mitochondrial and tubulin networks in cells.
A simple modification to the optical configuration used for fluorescence photoactivation localization microscopy (FPALM) allows the fluorescence anisotropies of each individual molecule in a nanoscale image to be measured. The method was used to obtain position and orientation information for fluorescently labeled actin or hemagglutinin molecules in fixed fibroblasts.
The combination of a glass window placed on top of a mouse mammary gland with photoswitchable fluorescent protein labeling of implanted tumor cells allows tumor-cell tracking over multiple imaging sessions in orthotopic tumors. Results show the existence of two distinct microenvironments with different tumor-cell invasion and intravasation characteristics.
Co-patterning of a membrane protein bait and a fluorescently labeled prey is used to examine protein-protein interactions in a semiautomated fashion in living cells. Photobleaching experiments and single-molecule imaging further allow dynamic studies of the interaction.
The algorithm Sylamer finds over- or underrepresented nucleotide motifs, such as microRNA seeds, in a gene list ranked according to expression levels and thus establishes whether a microRNA is directly affecting gene expression.
The spatial organization of the genome within the eukaryotic cell nucleus is not random. Automated imaging of thousands of live yeast is now used to build high-resolution probabilistic maps of the locations occupied by individual loci.
Many different red fluorescent proteins display cytotoxicity substantially higher than EGFP when used for whole-cell labeling of bacterial and mammalian cells with standard high-level expression systems. An improved tetrameric red fluorescent protein called DsRed-Express2 allows high-level labeling with minimal cytotoxicity comparable to that of EGFP.
