Letters in 2010

DNA molecules that are read by a next-generation sequencer have been discarded after the sequencing reaction, until now. By retrieving oil-emulsified beads encapsulating DNA with a known sequence, Matzas et al. obtain high-quality input material for DNA synthesis.

Long DNA molecules, such as those encoding genes, can be assembled from short oligonucleotides created on a microarray. Kosuri et al. improve the fidelity and scalability of this process, enabling synthesis of 40 antibody fragments having repetitive regions and other challenging sequence features.

Nanoparticles are under study for pulmonary drug delivery and are continually in contact with the lungs through air pollution. Choi et al. study the effects of size, charge and chemical composition on the behavior of nanoparticles in the rat lung.

Adaptation of insect pests to tolerate Bacillus thuringiensis (Bt) insecticidal proteins threatens to reduce the efficacy of Bt crops. Evidence from an extensive four-year field trial indicates that the release of sterile pink bollworm moths suppresses the emergence of resistance to transgenic Bt cotton, while helping to eradicate the pest.

The simultaneous detection of multiple mRNA species in thick tissues or whole-mount embryos has remained technically challenging. Choi et al. present a method based on the triggered polymerization of RNA stem-loop structures that allows the distribution of up to five mRNAs in intact zebrafish embryos to be imaged at the same time.

The culture of dedifferentiated plant cells to produce commercially important chemicals has met with limited success. Lee et al. demonstrate the potential of innately undifferentiated cells from Taxus cuspidata as an industrial source of the anticancer drug paclitaxel.

The typical therapeutic antibody binds only two target antigen molecules during its lifetime. Igawa et al. describe a method for engineering antibody recycling in vivo, suggesting an approach to reduce the size and/or frequency of dosage with therapeutic antibodies.

Biomechanical forces may be an effective approach for controlling the behavior of stem cells in vitro. Holst et al. show that the elasticity of a tropoelastin matrix expands hematopoietic stem and progenitor cells.

Fordyce et al. measure the sequence specificity and affinity of DNA-binding proteins using an improved microfluidic device. They characterize binding of 28 yeast transcription factors, several of which had proved intractable to previous approaches.

Isolation of antigen-specific antibodies or antibody fragments, whether through B-cell immortalization or recombinant libraries, generally requires laborious screening. Reddy et al. circumvent this step using high-throughput sequencing of plasma cells and bioinformatic analysis of the variable-gene repertoire.

Recombinant glycoproteins produced in animal cell lines often bear the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc). Ghaderi et al. show that two monoclonal antibodies in clinical use differ with respect to addition of Neu5Gc and propose that drug developers should consider the consequences of the Neu5Gc modification.

Efforts to study ubiquitination have been hampered by the large size of ubiquitin relative to other post-translational modifications. Xu et al. use a monoclonal antibody specific for the adduct left after proteolysis of ubiquitinated proteins to analyze the differential regulation of ubiquitination at distinct sites within the same proteins.

Although general inhibitors of the ubiquitin-proteasome system have been reported, compounds targeting specific ubiquitylation enzymes should be beneficial in clinical applications and basic research. Orlicky et al. present an allosteric inhibitor specific to the yeast SCF^Cdc4 E3 ligase that prevents binding of the target protein to the WD40 domain of the complex.

The ability to control the activity of cellular signaling pathways is useful for dissecting their functions. Using a short protein insert, Karginov et al. engineer protein kinases that can be specifically activated by the small-molecule rapamycin.

Although general inhibitors of the ubiquitin-proteasome system have been reported, compounds targeting specific ubiquitylation enzymes should be beneficial in clinical applications and basic research. Aghajan et al. present an inhibitor of the yeast SCF^Met30 E3 ligase that prevents the binding of the Met30 F-box protein to the core ligase complex.

Complex combinations of simultaneous stimuli control cellular responses, and cross-talk between signaling pathways makes it even more difficult to predict these responses. Chatterjee et al. demonstrate in human platelets that neural networks trained with measurements of pairwise stimuli can predict combinations of many signaling inputs.

Live attenuated viruses make more effective vaccines than newer protein subunit or recombinant DNA vaccines, but the traditional passaging methods used to generate them often fail to produce a suitable mutant. Mueller et al. improve the production of live attenuated influenza virus by extensive manipulation of codon-pair bias across the genome, which minimizes the risk of reversion to a virulent form.

The development of fully defined culture conditions for human embryonic stem cells (hESCs) should enhance experimental reproducibility, reduce unwanted contaminants and facilitate scale-up production. Melkoumian et al. show that a substrate made of peptides derived from extracellular matrix proteins supports long-term culture of hESCs and differentiation to cardiomyocytes.

The development of fully defined culture conditions for human embryonic stem cells (hESCs) should enhance experimental reproducibility, reduce unwanted contaminants and facilitate scale-up production. Rodin et al. show that a substrate made of human recombinant laminin-511 supports long-term culture of hESCs and human induced pluripotent stem cells.

The spread of influenza virus strains resistant to the current generation of anti-viral drugs makes the identification of new druggable targets and lead compounds of prime importance. Kao et al. show that the influenza A nucleoprotein can be targeted by a small molecule that protects mice from lethal viral challenges.