Research articles

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.

Although never demonstrated in humans, exchange of the antigen-binding regions of IgG4 antibodies with different specificities could complicate certain antibody therapies. Labrijn et al. show that Fab-arm exchange occurs in patients and propose that a single mutation can inhibit the process.

The safety of induced pluripotent stem cells seems to depend on how they were generated. Miura et al. examine the effects of the c-myc transgene, tissue of origin and selection method on the tumor-forming propensity of iPS-cell derivatives.

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.

Although multiple reaction monitoring (MRM) mass spectrometry holds considerable promise for quantifying candidate protein biomarkers in blood, transferability of MRM assays between laboratories has never been shown. Addona et al. assess the reproducibility, dynamic range and limits of detection and quantification of MRM across multiple sites.

Until now, determining the sequences recognized by an RNA-binding protein has been time and labor intensive. Ray et al. use a custom pool of >210,000 oligos that encode linear and stem-loop RNAs to rapidly determine the sequences bound by nine RNA-binding proteins.

Forrester et al. adapt the biotin switch technique for studying reversible protein modification by nitric oxide, by using resin-assisted capture of S-nitrosothiols. This enables more sensitive detection of larger S-nitrosylated proteins and can be coupled with quantitative mass spectrometry to study the kinetics of denitrosylation.

Traditional methods for modifying viruses to produce live attenuated vaccines are being updated with molecularly targeted approaches. Perez et al. attenuate influenza A viruses by introducing miRNA target sites into the coding region of the viral genome.

Transfection of siRNAs and miRNAs into cells has been observed to generate unexpected effects in the form of gene upregulation. By statistically analyzing published transfection experiments, Khan et al. explain these effects with a model that the transfected RNAs compete with miRNAs naturally expressed by the cell.

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.

The delivery to primary cells is a major challenge in the application of siRNAs in biological research. Using a fusion protein consisting of a double-stranded RNA binding domain and a protein transduction domain, Eguchi et al. are able to transfect siRNAs into a wide variety of cells with very high efficiency without cytotoxicity.

Strategies for allele-specific knockdown of the mutant genes in triplet-repeat disorders have relied on point or deletion mutations that differ among affected individuals. Hu et al. show that antisense oligomers can selectively recognize expanded CAG repeats in mRNAs, allowing silencing of mutant but not wild-type alleles.

Hosaka et al. show that selection of bacteria for antibiotic resistance can be used to discover new antibacterials. Some of the mutant strains they generated, which bear mutations in RNA polymerase and in a ribosomal protein, produce a previously unknown class of antibacterial called piperidamycin.

Single-nucleotide polymorphisms in microRNA target sites can disrupt the effects of the microRNA. Kim and Bartel use sequencing to investigate this phenomenon on a large scale and find that such polymorphisms generate gene-regulatory diversity in mice.

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.

Judson et al. show that microRNAs specific to mouse embryonic stem cells can substitute for the reprogramming factor cMyc in the generation of induced pluripotent stem cells. The development of reprogramming methods that do not rely on transgenes may facilitate clinical translation of this technology.