Using structural information to change the phosphotransfer specificity of a two-component chemotaxis signalling complex.

addresses: Oxford Centre for Integrative Systems Biology, Department of Biochemistry, University of Oxford, Oxford, United Kingdom.notes: PMCID: PMC2817712types: Journal Article; Research Support, Non-U.S. Gov'tCopyright: © 2010 Bell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Two-component signal transduction pathways comprising histidine protein kinases (HPKs) and their response regulators (RRs) are widely used to control bacterial responses to environmental challenges. Some bacteria have over 150 different two-component pathways, and the specificity of the phosphotransfer reactions within these systems is tightly controlled to prevent unwanted crosstalk. One of the best understood two-component signalling pathways is the chemotaxis pathway. Here, we present the 1.40 A crystal structure of the histidine-containing phosphotransfer domain of the chemotaxis HPK, CheA(3), in complex with its cognate RR, CheY(6). A methionine finger on CheY(6) that nestles in a hydrophobic pocket in CheA(3) was shown to be important for the interaction and was found to only occur in the cognate RRs of CheA(3), CheY(6), and CheB(2). Site-directed mutagenesis of this methionine in combination with two adjacent residues abolished binding, as shown by surface plasmon resonance studies, and phosphotransfer from CheA(3)-P to CheY(6). Introduction of this methionine and an adjacent alanine residue into a range of noncognate CheYs, dramatically changed their specificity, allowing protein interaction and rapid phosphotransfer from CheA(3)-P. The structure presented here has allowed us to identify specificity determinants for the CheA-CheY interaction and subsequently to successfully reengineer phosphotransfer signalling. In summary, our results provide valuable insight into how cells mediate specificity in one of the most abundant signalling pathways in biology, two-component signal transduction

Manipulation of the spin helix in FeGe thin films and FeGe/Fe multilayers

Magnetic materials without structural inversion symmetry can display the Dzyaloshinskii-Moriya interaction, which manifests itself as chiral magnetic ground states. These chiral states can interact in complex ways with applied fields and boundary conditions provided by finite sample sizes that are of the order of the lengthscale of the chiral states. Here we study epitaxial thin films of FeGe with a thickness close to the helix pitch of the helimagnetic ground state, which is about 70 nm, by conventional magnetometry and polarized neutron reflectometry. We show that the helix in an FeGe film reverses under the application of a field by deforming into a helicoidal form, with twists in the helicoid being forced out of the film surfaces on the way to saturation. An additional boundary condition was imposed by exchange coupling a ferromagnetic Fe layer to one of the interfaces of an FeGe layer. This forces the FeGe spins at the interface to point in the same direction as the Fe, preventing node expulsion and giving a handle by which the reversal of the helical magnet may be controlled

Mid - infrared interferometry of massive young stellar objects II Evidence for a circumstellar disk surrounding the Kleinmann - Wright object

The formation scenario for massive stars is still under discussion. To further constrain current theories, it is vital to spatially resolve the structures from which material accretes onto massive young stellar objects (MYSOs). Due to the small angular extent of MYSOs, one needs to overcome the limitations of conventional thermal infrared imaging, regarding spatial resolution, in order to get observational access to the inner structure of these objects.We employed mid - infrared interferometry, using the MIDI instrument on the ESO /VLTI, to investigate the Kleinmann - Wright Object, a massive young stellar object previously identified as a Herbig Be star precursor. Dispersed visibility curves in the N- band (8 - 13 {\mu}m) have been obtained at 5 interferometric baselines. We show that the mid - infrared emission region is resolved. A qualitative analysis of the data indicates a non - rotationally symmetric structure, e.g. the projection of an inclined disk. We employed extensive radiative transfer simulations based on spectral energy distribution fitting. Since SED - only fitting usually yields degenerate results, we first employed a statistical analysis of the parameters provided by the radiative transfer models. In addition, we compared the ten best - fitting self - consistent models to the interferometric observations. Our analysis of the Kleinmann - Wright Object suggests the existence of a circumstellar disk of 0.1M\odot at an intermediate inclination of 76\circ, while an additional dusty envelope is not necessary for fitting the data. Furthermore, we demonstrate that the combination of IR interferometry with radiative transfer simulations has the potential to resolve ambiguities arising from the analysis of spectral energy distributions alone.Comment: 12 pages, 22 figures accepted for publication in A&

RAPID AND RELIABLE HEALING OF CRITICAL SIZE BONE DEFECTS WITH GENETICALLY MODIFIED SHEEP MUSCLE

Large segmental defects in bone fail to heal and remain a clinical problem. Muscle is highly osteogenic, and preliminary data suggest that autologous muscle tissue expressing bone morphogenetic protein-2 (BMP-2) efficiently heals critical size defects in rats. Translation into possible human clinical trials requires, inter alia, demonstration of efficacy in a large animal, such as the sheep. Scale-up is fraught with numerous biological, anatomical, mechanical and structural variables, which cannot be addressed systematically because of cost and other practical issues. For this reason, we developed a translational model enabling us to isolate the biological question of whether sheep muscle, transduced with adenovirus expressing BMP-2, could heal critical size defects in vivo. Initial experiments in athymic rats noted strong healing in only about one-third of animals because of unexpected immune responses to sheep antigens. For this reason, subsequent experiments were performed with Fischer rats under transient immunosuppression. Such experiments confirmed remarkably rapid and reliable healing of the defects in all rats, with bridging by 2 weeks and remodelling as early as 3-4 weeks, despite BMP-2 production only in nanogram quantities and persisting for only 1-3 weeks. By 8 weeks the healed defects contained well-organised new bone with advanced neo-cortication and abundant marrow. Bone mineral content and mechanical strength were close to normal values. These data demonstrate the utility of this model when adapting this technology for bone healing in sheep, as a prelude to human clinical trials

Chromosomal instability and lack of cyclin E regulation in hCdc4 mutant human breast cancer cells

INTRODUCTION: Cyclin E, a G(1 )cyclin essential for G(1)–S phase transition, is known to have a profound effect on tumorigenesis. Elevated levels of cyclin E have been associated with breast cancer, and chromosomal instability observed in breast cancer is suggested to be associated with constitutive expression of cyclin E. It was previously demonstrated that SUM149PT human breast cancer cells show very high levels of cyclin E expression by western analysis and that they express a nonfunctional cyclin E ubiquitin ligase due to a mutation in the F-box protein hCdc4. METHODS: We examined cyclin E expression in both MCF10A and SUM149PT cells using western blot analysis and flow cytometry. Immunofluorescence was utilized for the localization of cyclin E in both normal and breast cancer cells. In addition, array comparative genomic hybridization analysis was performed to compare chromosome copy number alterations with levels of cyclin E expression among a panel of breast cancer cell lines. RESULTS: SUM149PT cells overexpress cyclin E on a cell per cell basis for the duration of the cell cycle. High cyclin E levels are maintained throughout the S phase, and SUM149PT cells exhibit an S phase delay or arrest probably due to cyclin E overexpression. In addition, comparative genomic hybridization indicated that SUM149PT cells exhibit many chromosome copy number alterations, which may reflect prior or ongoing genomic instability. However, no direct correlation was observed between cyclin E levels and genomic copy number alteration in a panel of human breast cancer cell lines. CONCLUSIONS: Cyclin E is overexpressed at high levels throughout the cell cycle in SUM149PT cells, which is in stark contrast to cyclin E degradation observed in the mid to late S phase of normal cells. SUM149PT cells are unable to regulate cyclin E and also exhibit many copy number alterations. However, there was a lack of direct correlation between cyclin E overexpression and chromosomal instability across a panel of other breast cancer cell lines examined

Helical magnetic structure and the anomalous and topological Hall effects in epitaxial B20 Fe₁−yCoyGe films

Epitaxial films of the B20-structure compound Fe1−yCoyGe were grown by molecular beam epitaxy on Si (111) substrates. The magnetization varied smoothly from the bulklike values of one Bohr magneton per Fe atom for FeGe to zero for nonmagnetic CoGe. The chiral lattice structure leads to a Dzyaloshinskii-Moriya interaction (DMI), and the films' helical magnetic ground state was confirmed using polarized neutron reflectometry measurements. The pitch of the spin helix, measured by this method, varies with Co content y and diverges at y∼0.45. This indicates a zero crossing of the DMI, which we reproduced in calculations using first-principles methods. We also measured the longitudinal and Hall resistivity of our films as a function of magnetic field, temperature, and Co content y. The Hall resistivity is expected to contain contributions from the ordinary, anomalous, and topological Hall effects. Both the anomalous and topological Hall resistivities show peaks around y∼0.5. Our first-principles calculations show a peak in the topological Hall constant at this value of y, related to the strong spin polarization predicted for intermediate values of y. Our calculations predict half-metallicity for y=0.6, consistent with the experimentally observed linear magnetoresistance at this composition, and potentially related to the other unusual transport properties for intermediate value of y. While it is possible to reconcile theory with experiment for the various Hall effects for FeGe, the large topological Hall resistivities for y∼0.5 are much larger than expected when the very small emergent fields associated with the divergence in the DMI are taken into account

Homoclinic standing waves in focussing DNLS equations --Variational approach via constrained optimization

We study focussing discrete nonlinear Schr\"{o}dinger equations and present a new variational existence proof for homoclinic standing waves (bright solitons). Our approach relies on the constrained maximization of an energy functional and provides the existence of two one-parameter families of waves with unimodal and even profile function for a wide class of nonlinearities. Finally, we illustrate our results by numerical simulations.Comment: new version with revised introduction and improved condition (A3); 16 pages, several figure

Performance of a 229 Thorium solid-state nuclear clock

The 7.8 eV nuclear isomer transition in 229 Thorium has been suggested as an etalon transition in a new type of optical frequency standard. Here we discuss the construction of a "solid-state nuclear clock" from Thorium nuclei implanted into single crystals transparent in the vacuum ultraviolet range. We investigate crystal-induced line shifts and broadening effects for the specific system of Calcium fluoride. At liquid Nitrogen temperatures, the clock performance will be limited by decoherence due to magnetic coupling of the Thorium nucleus to neighboring nuclear moments, ruling out the commonly used Rabi or Ramsey interrogation schemes. We propose a clock stabilization based on counting of flourescence photons and present optimized operation parameters. Taking advantage of the high number of quantum oscillators under continuous interrogation, a fractional instability level of 10^{-19} might be reached within the solid-state approach.Comment: 28 pages, 9 figure