Cell-free protein expression systems in microdroplets: stabilization of interdroplet bilayers

Cell-free protein expression with bacterial lysates has been demonstrated to produce soluble proteins in microdroplets. However, droplet assays with expressed membrane proteins require the presence of a lipid bilayer. A bilayer can be formed in between lipid-coated aqueous droplets by bringing these into contact by electrokinetic manipulation in a continuous oil phase, but it is not known whether such interdroplet bilayers are compatible with high concentrations of biomolecules. In this study we have characterized the lifetime and the structural integrity of interdroplet bilayers by measuring the bilayer current in the presence of three different commercial cell-free expression mixtures and their individual components. Samples of pure proteins and of a polymer were included for comparison. It is shown that complete expression mixtures reduce the bilayer lifetime to several minutes or less, and that this is mainly due to the lysate fraction itself. The fraction that contains the molecules for metabolic energy generation does not reduce the bilayer lifetime but does give rise to current steps that are indicative of lipid packing defects. Gel electrophoresis confirmed that proteins are only present at significant amounts in the lysate fractions and, when supplied separately, in the T7 enzyme mixture. Interestingly, it was also found that pure-protein and pure-polymer solutions perturb the interdroplet bilayer at higher concentrations; 10% (w/v) PEG 8000 and 3 mM lysozyme induce large bilayer currents without a reduction in bilayer lifetime, whereas 3 mM albumin causes rapid bilayer failure. It can therefore be concluded that the high protein content of the lysates and the presence of PEG polymer, a typical lysate supplement, compromise the structural integrity of interdroplet bilayers. However, we established that the addition of lipid vesicles to the cell-free expression mixture stabilizes the interdroplet bilayer, allowing the exposure of interdroplet bilayers to cell-free expression solutions. Given that cell-free expressed membrane proteins can insert in lipid bilayers, we envisage that microdroplet technology may be extended to the study of in situ expressed membrane receptors and ion channel

The death of massive stars - II. Observational constraints on the progenitors of type Ibc supernovae

The progenitors of many type II core-collapse supernovae have now been identified directly on pre-discovery imaging. Here we present an extensive search for the progenitors of type Ibc supernovae in all available pre-discovery imaging since 1998. There are 12 type Ibc supernovae with no detections of progenitors in either deep ground-based or Hubble Space Telescope archival imaging. The deepest absolute BVR magnitude limits are between -4 and -5. We compare these limits with the observed Wolf-Rayet population in the Large Magellanic Cloud and estimate a 16 per cent probability we have failed to detect such a progenitor by chance. Alternatively the progenitors evolve significantly before core-collapse or we have underestimated the extinction towards the progenitors. Reviewing the relative rates and ejecta mass estimates from lightcurve modelling of Ibc SNe, we find both incompatible with Wolf-Rayet stars with initial masses >25Msun being the only progenitors. We present binary evolution models that fit these observational constraints. Stars in binaries with initial masses <20Msun lose their hydrogen envelopes in binary interactions to become low mass helium stars. They retain a low mass hydrogen envelope until approximately 10,000 years before core-collapse; hence it is not surprising that galactic analogues have been difficult to identify.Comment: Accepted by MNRAS. 31 pages, 12 figures, 8 table

On the Majorana representation of the optical Dirac equation

We consider the representations of the optical Dirac equation, especially ones where the Hamiltonian is purely real-valued. This is equivalent, for Maxwell's equations, to the Majorana representation of the massless Dirac (Weyl) equation. We draw analogies between the Dirac, chiral and Majorana representations of the Dirac and optical Dirac equations, and derive two new optical Majorana representations. Just as the Dirac and chiral representations are related to optical spin and helicity states, these Majorana representations of the optical Dirac equation are associated with the linear polarisation of light. This provides a means to compare electron and electromagnetic wave equations in the context of classical field theory.Comment: 17 pages. For special issue of J Phys A in honour of Michael Berr

Adhesion signalling complexes

SummaryIntercellular communication in metazoa not only requires autocrine, paracrine and exocrine signalling systems, but it also relies on the structural and positional information encoded in extracellular matrices (ECMs). Most cells in tissues are structurally and functionally integrated with their surrounding ECM in a highly organised manner involving thousands of dynamic connections. On the intracellular face of these linkages, adhesion receptors — principally integrins and syndecans — link the cytoskeleton to the plasma membrane and compartmentalise cytoplasmic signalling events, whereas at the extracellular face the same receptors direct and organise the deposition of the ECM itself. Adhesion receptors transduce mechanical force bidirectionally across the plasma membrane by tethering variably deformable ECMs to the contractile cytoskeleton (Figure 1), and they translate the topography and composition of the ECM into chemical signals that determine behaviour. The membrane-proximal functions of adhesion receptors in turn trigger distal processes within cells, such as alterations in the direction of cell movement and the regulation of gene transcription, and long-range effects outside cells, such as the construction of ECM networks and consequent shaping of higher-order tissue structure. Given the diverse and fundamental roles attributed to adhesion, it is understandable that adhesion receptor engagement has been reported to alter the flux through virtually all major signalling pathways

Forest management and wildfire risk in inland northwest

This brief reports the results of a mail survey of forest landowners in northeastern Oregon conducted in the fall of 2012 by the Communities and Forests in Oregon (CAFOR) Project at the University of Colorado and the University of New Hampshire in cooperation with Oregon State University College of Forestry Extension. The mail survey--a follow-up to a telephone survey conducted for the counties of Baker, Union, and Wallowa in the fall of 2011 -was administered to understand who constituted forest landowners in these three coun¬ties and their perceptions about forest management on both public and private land, as well as risks to forests in the area and the actions they have taken to reduce those risks. The respondents indicated that they perceive wildfire as the greatest threat to their lands, and they consider cooperation with neighbors as very or extremely important for land management. Forest landowners believe public lands are managed poorly and see a greater risk of wildfire occurring on neighboring public land than on their own land. Their opinions on land management are not strongly related to background factors or ideology (for example, gender, age, political party, wealth) but may be heavily influenced by personal experience with wildfire

p190RhoGAP is the convergence point of adhesion signals from α5β1 integrin and syndecan-4

The fibronectin receptors α5β1 integrin and syndecan-4 cocluster in focal adhesions and coordinate cell migration by making individual contributions to the suppression of RhoA activity during matrix engagement. p190Rho–guanosine triphosphatase–activating protein (GAP) is known to inhibit RhoA during the early stages of cell spreading in an Src-dependent manner. This paper dissects the mechanisms of p190RhoGAP regulation and distinguishes the contributions of α5β1 integrin and syndecan-4. Matrix-induced tyrosine phosphorylation of p190RhoGAP is stimulated solely by engagement of α5β1 integrin and is independent of syndecan-4. Parallel engagement of syndecan-4 causes redistribution of the tyrosine-phosphorylated pool of p190RhoGAP between membrane and cytosolic fractions by a mechanism that requires direct activation of protein kinase C α by syndecan-4. Activation of both pathways is necessary for the efficient regulation of RhoA and, as a consequence, focal adhesion formation. Accordingly, we identify p190RhoGAP as the convergence point for adhesive signals mediated by α5β1 integrin and syndecan-4. This molecular mechanism explains the cooperation between extracellular matrix receptors during cell adhesion

Beagle 2: Seeking the signatures of life on Mars

ESA's Beagle 2 lander will land on Mars to search for signatures of present and past life. A Gas Analysis Package (GAP) with a mass spectrometer, XRF, Mossbauer, stereo cameras, microscope, environmental sensors, rock corer/grinder, and a Mole attachment are on the lander

Molecular genetic differentiation in earthworms inhabiting a heterogeneous Pb-polluted landscape

A Pb-mine site situated on acidic soil, but comprising of Ca-enriched islands around derelict buildings was used to study the spatial pattern of genetic diversity in Lumbricus rubellus. Two distinct genetic lineages ('A' and 'B'), differentiated at both the mitochondrial (mtDNA COII) and nuclear level (AFLPs) were revealed with a mean inter-lineage mtDNA sequence divergence of approximately 13%, indicative of a cryptic species complex. AFLP analysis indicates that lineage A individuals within one central 'ecological island' site are uniquely clustered, with little genetic overlap with lineage A individuals at the two peripheral sites. FTIR microspectroscopy of Pb-sequestering chloragocytes revealed different phosphate profiles in residents of adjacent acidic and calcareous islands. Bioinformatics found over-representation of Ca pathway genes in ESTPb libraries. Subsequent sequencing of a Ca-transport gene, SERCA, revealed mutations in the protein's cytosolic domain. We recommend the mandatory genotyping of all individuals prior to field-based ecotoxicological assays, particularly those using discriminating genomic technologies