Measuring EGFR Separations on Cells with ∼10 nm Resolution via Fluorophore Localization Imaging with Photobleaching

Detecting receptor dimerisation and other forms of clustering on the cell surface depends on methods capable of determining protein-protein separations with high resolution in the ∼10-50 nm range. However, this distance range poses a significant challenge because it is too large for fluorescence resonance energy transfer and contains distances too small for all other techniques capable of high-resolution in cells. Here we have adapted the technique of fluorophore localisation imaging with photobleaching to measure inter-receptor separations in the cellular environment. Using the epidermal growth factor receptor, a key cancer target molecule, we demonstrate ∼10 nm resolution while continuously covering the range of ∼10-80 nm. By labelling the receptor on cells expressing low receptor numbers with a fluorescent antagonist we have found inter-receptor separations all the way up from 8 nm to 59 nm. Our data are consistent with epidermal growth factor receptors being able to form homo-polymers of at least 10 receptors in the absence of activating ligands. © 2013 Needham et al.

Increasing the open-circuit voltage of photoprotein-based photoelectrochemical cells by manipulation of the vacuum potential of the electrolytes.

The innately highly efficient light-powered separation of charge that underpins natural photosynthesis can be exploited for applications in photoelectrochemistry by coupling nanoscale protein photoreaction centers to man-made electrodes. Planar photoelectrochemical cells employing purple bacterial reaction centers have been constructed that produce a direct current under continuous illumination and an alternating current in response to discontinuous illumination. The present work explored the basis of the open-circuit voltage (V(OC)) produced by such cells with reaction center/antenna (RC-LH1) proteins as the photovoltaic component. It was established that an up to ~30-fold increase in V(OC) could be achieved by simple manipulation of the electrolyte connecting the protein to the counter electrode, with an approximately linear relationship being observed between the vacuum potential of the electrolyte and the resulting V(OC). We conclude that the V(OC) of such a cell is dependent on the potential difference between the electrolyte and the photo-oxidized bacteriochlorophylls in the reaction center. The steady-state short-circuit current (J(SC)) obtained under continuous illumination also varied with different electrolytes by a factor of ~6-fold. The findings demonstrate a simple way to boost the voltage output of such protein-based cells into the hundreds of millivolts range typical of dye-sensitized and polymer-blend solar cells, while maintaining or improving the J(SC). Possible strategies for further increasing the V(OC) of such protein-based photoelectrochemical cells through protein engineering are discussed.

Numerical investigation of the active role of the actin cytoskeleton in the compression resistance of cells.

Numerous in-vitro studies have established that cells react to their physical environment and to applied mechanical loading. However, the mechanisms underlying such phenomena are poorly understood. Previous modelling of cell compression considered the cell as a passive homogenous material, requiring an artificial increase in the stiffness of spread cells to replicate experimentally measured forces. In this study, we implement a fully 3D active constitutive formulation that predicts the distribution, remodelling, and contractile behaviour of the cytoskeleton. Simulations reveal that polarised and axisymmetric spread cells contain stress fibres which form dominant bundles that are stretched during compression. These dominant fibres exert tension; causing an increase in computed compression forces compared to round cells. In contrast, fewer stress fibres are computed for round cells and a lower resistance to compression is predicted. The effect of different levels of cellular contractility associated with different cell phenotypes is also investigated. Highly contractile cells form more dominant circumferential stress fibres and hence provide greater resistance to compression. Computed predictions correlate strongly with published experimentally observed trends of compression resistance as a function of cellular contractility and offer an insight into the link between cell geometry, stress fibre distribution and contractility, and cell deformability. Importantly, it is possible to capture the behaviour of both round and spread cells using a given, unchanged set of material parameters for each cell type. Finally, it is demonstrated that stress distributions in the cell cytoplasm and nucleus computed using the active formulation differ significantly from those computed using passive material models.

The influence of 1D, meso- and crystal structures on charge transport and recombination in solid-state dye-sensitized solar cells

We have prepared single crystalline SnO2 and ZnO nanowires and polycrystalline TiO2 nanotubes (1D networks) as well as nanoparticle-based films (3D networks) from the same materials to be used as photoanodes for solid-state dye-sensitized solar cells. In general, superior photovoltaic performance can be achieved from devices based on 3-dimensional networks, mostly due to their higher short circuit currents. To further characterize the fabricated devices, the electronic properties of the different networks were measured via the transient photocurrent and photovoltage decay techniques. Nanowire-based devices exhibit extremely high, light independent electron transport rates while recombination dynamics remain unchanged. This indicates, contrary to expectations, a decoupling of transport and recombination dynamics. For typical nanoparticle-based photoanodes, the devices are usually considered electron-limited due to the poor electron transport through nanocrystalline titania networks. In the case of the nanowire-based devices, the system becomes limited by the organic hole transporter used. In the case of polycrystalline TiO2 nanotube-based devices, we observe lower transport rates and higher recombination dynamics than their nanoparticle-based counterparts, suggesting that in order to improve the electron transport properties of solid-state dye-sensitized solar cells, single crystalline structures should be used. These findings should aid future design of photoanodes based on nanowires or porous semiconductors with extended crystallinity to be used in dye-sensitized solar cells. © 2013 The Royal Society of Chemistry.

Optimizing the energy offset between dye and hole-transporting material in solid-state dye-sensitized solar cells

The power-conversion efficiency of solid-state dye-sensitized solar cells can be optimized by reducing the energy offset between the highest occupied molecular orbital (HOMO) levels of dye and hole-transporting material (HTM) to minimize the loss-in-potential. Here, we report a study of three novel HTMs with HOMO levels slightly above and below the one of the commonly used HTM 2,2′,7,7′- tetrakis(N,N-di-p-methoxyphenylamino)-9,9′- spirobifluorene (spiro-OMeTAD) to systematically explore this possibility. Using transient absorption spectroscopy and employing the ruthenium based dye Z907 as sensitizer, it is shown that, despite one new HTM showing a 100% hole-transfer yield, all devices based on the new HTMs performed worse than those incorporating spiro-OMeTAD. We further demonstrate that the design of the HTM has an additional impact on the electronic density of states present at the TiO2 electrode surface and hence influences not only hole- but also electron-transfer from the sensitizer. These results provide insight into the complex influence of the HTM on charge transfer and provide guidance for the molecular design of new materials. © 2013 American Chemical Society.

Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment.

In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN), the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103⁺CX₃CR1⁻CD11c⁺ dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.