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In many sequencing applications, it is sufficient to sequence selected portions of a genome rather than the complete genome. Tewhey et al. describe an approach for massively parallel genome targeting that relies on PCR in microdroplets generated by a microfluidic device.
Kubota et al. describe a sensitive mass spectrometric method for generating signatures of kinase activity characteristic of specific cell types. They also identify kinases responsible for phosphorylating substrates of interest.
Tumors generate microenvironments that actively suppress the body's anti-tumor immune response. Kortylewski et al. restore immunity by delivering an siRNA against Stat3, a regulator of immune suppression, to myeloid and B cells using a CpG oligonucleotide that targets toll-like receptor 9.
Systemic delivery remains a major obstacle in therapeutic applications of siRNAs. Using RNA aptamer–siRNA chimeras with enhanced silencing activity and specificity, Dassie et al. achieve regression of xenograft prostate tumors by intraperitoneal injection.
Initiation of translation is an obstacle in the development of eukaryotic systems for cell-free protein synthesis. Mureev et al. describe a translational leader sequence that efficiently drives protein production in cell lysates from several eukaryotes and prokaryotes and use this sequence to develop a cell-free system based on Leishmania tarentolae.
Engineered metabolic pathways are usually devoid of the regulatory mechanisms characteristic of natural metabolism. Using pathways not normally found in E. coli, Dueber et al. show that synthetic scaffolds built using protein-protein interaction domains can boost yields of mevalonate and glucaric acid.
Although new metabolic pathways are generally introduced into bacteria on plasmids, this approach is limited by declining productivity after several generations. Tyo et al. describe a method for chromosome engineering that enables sustained production of a biopolymer or a nutraceutical.
Lipson et al. profile the yeast transcriptome using single-molecule sequencing. This approach avoids the inherent biases of the digestion, ligation and amplification steps in alternative methods based on microarrays or other sequencing technologies.
Although combinations of drugs are often more potent than single agents, they are also believed to induce worse side effects. By screening >94,000 drug pairs in vitro, Lehár et al. show that synergistic combinations tend to be more selective than single drugs and are therefore unlikely to cause synergistic side effects.
Drug resistance remains a major hurdle to effective cancer chemotherapy. MacDiarmid et al. show that bacterially derived minicells packaged with siRNAs reverse tumor drug resistance and that subsequent treatment with minicells loaded with cytotoxic drugs causes tumor stabilization or regression.
Pichia pastoris has been a workhorse of protein production for decades. De Schutter et al. present its genomic sequence, which will allow development of improved strains.
Ellis et al. describe a strategy for rationally assembling gene networks with predictable behaviors. Using mathematical models, they predict the responses of complex synthetic gene networks built from quantitatively characterized promoter libraries, and harness these networks to regulate an industrially relevant yeast phenotype.
Wollscheid et al. describe a multiplexed, mass spectrometry–based approach to catalog glycoproteins on the surfaces of live cells without the need for antibodies. They use it to monitor changes in the cell-surface glycoproteome during T-cell activation and the differentiation of embryonic stem cells to neural progenitors.
Knowing a drug's mode of action is invaluable for informing drug discovery efforts. Ho et al. construct an optimized yeast genomic library that enables rapid mutant cloning by complementation to guide mode-of-action studies.
Many enzymes in eukaryotic and prokaryotic proteomes have no known substrate. Bachovchin et al. use the fluorescence polarization signal of broad-spectrum, activity-based probes to find inhibitors of such enzymes in high-throughput screens.
Ball et al. exploit next-generation sequencing to detect methylation across the human genome. A targeted approach uses padlock probes and bisulfite-treated DNA, whereas an untargeted method relies on the methylation-sensitive restriction enzyme HpaII.
Although technically feasible, whole-genome analysis of cytosine methylation using bisulfite sequencing remains prohibitively expensive for large eukaryotic genomes. Deng et al. use 30,000 nondegenerate padlock probes to capture ∼66,000 bisulfite-converted sites in human CpG islands and compare their methylation in fibroblasts, embryonic stem cells and induced pluripotent stem cells.
Keng et al. characterize the mouse hepatocellular carcinoma genome by using tissue-specific recombinase expression to restrict mobilization of the Sleeping Beauty transposon to the liver. High throughput sequencing of >100,000 insertions sites in mouse tumor nodules identifies potential therapeutic targets.
Goraczniak et al. introduce a gene-silencing method that uses 'U1 adaptors' to block polyadenylation of pre-mRNA in the nucleus. Silencing is stronger when U1 adaptors are combined with siRNAs than when either is used alone.
Gnirke et al. present a bead-based method for capturing sequences of interest in the human genome for massively parallel sequencing. Using long, biotinylated RNA probes to pull down PCR-amplified DNA fragments, they demonstrate sequencing of 2.5 Mbs of exons in 1,900 genes.