Planar Support Research Articles

Chapter 9 - Critical Step-by-Step Approaches Toward Correlative Fluorescence/Soft X-Ray Cryo-Microscopy of Adherent Mammalian Cells

Soft X-ray cryo-microscopy/tomography with its extraordinary capability to map vitreous cells with high absorption contrast in their full three-dimensional extent, and at a resolution exceeding super-resolution fluorescence microscopy, is a valuable tool for integrative structural cell biology. Focusing on cell biological applications, its ongoing methodological development gained momentum by combining it with fluorescence cryo-microscopy, thus correlating highly resolved structural and specific information in situ. In this chapter, we provide a basic description of the techniques, as well as an overview of equipment and methods available to carry out correlative soft X-ray cryo-tomography experiments on frozen-hydrated cells grown on a planar support. Our aim here is to suggest ways that biologically representative data can be recorded to the highest possible resolution, while also keeping in mind the limitations of the technique during data acquisition and analysis. We have written from our perspective as electron cryo-microscopists/structural cell biologists who have experience using correlative fluorescence/cryoXM/T at synchrotron beamlines presently available for external users in Europe (HZB TXM at U41-FSGM, BESSY II, Berlin/Germany; Carl Zeiss TXMs at MISTRAL, ALBA, Barcelona/Spain, and B24, DLS, Oxfordshire, UK).

Theoretical Analysis and Verification of the Single-Surface Double-Layer Cable Architectural System

In order to meet the needs of large span project for Vegetable expo, supporting building system consisting of prestressed single-surface Double-layer Cable was put forward. Based on the assumption analytical solution of the double-layer cable planar support structure system was given. Using ANSYS finite element software simulation analysis on the double-layer cable system was carried out, the discrete finite element solution was acquired, and by comparing the analytical solution was verified to be accurate. Finally, the construction methods and means of monitoring during construction were given, and these can provide references for design and construction of similar projects.

Methanation on mass-selected Ru nanoparticles on a planar SiO2 model support: The importance of under-coordinated sites

Mass-selected Ru nanoparticles were deposited onto planar SiO2 support and their capability for the methanation reaction investigated. The catalytic activity for the methanation reaction at 100mbar under hydrogen rich conditions (1:99 CO/H2 ratio) was measured as a function of particle size. We found that the Turnover Frequency increased with nanoparticle diameter in the range 4–10nm.As the TOF decreases over reaction repetition, we investigated the possible reasons and ruled out carbon deposition as a cause for activity loss. As no particle sintering was observed, we invoked surface restructuring as a possible cause.

Progress Towards Wendelstein 7-X

The Wendelstein 7-X (W7-X) experimental device is presently being built at the Max-Planck-Institute for Plasma Physics (IPP) in Greifswald, Germany.The modularity of the machine is achieved using five identical modules which form a torus of 16 meters outer diameter. Each module consists of a plasma vessel, ten non-planar main field coils, four planar coils and an outer vessel sector with ports. To achieve steady-state operation, the coils are superconducting. Two different plasma heating systems, ECRH and NBI, are planned for the first operation phase of W7-X.Module-based assembly is a new approach for large fusion machines. There are three main assembly steps of the core machine. First, half-modules are assembled from a plasma vessel section, five non-planar and two planar coils and related support structures. In the second step, two half-modules are connected to form a module. Then the five modules are connected, after which the outer vessel is closed.The paper reports on about the latest progress in assembly, highlighting the most challenging tasks, describes future work leading to the start of W7-X commissioning in 2014.

Poly(Lactide-co -Glycolide) Nanoparticles, Layer by Layer Engineered for the Sustainable Delivery of AntiTNF-α

A strategy of encapsulation of the antiTNF-α antibody on top of poly(lactide-co-glycolide) nanoparticles (PLGA NPs) is presented on the basis of the complexation of antiTNF-α with alginate (Alg) and subsequent assembly layer by layer with poly(L-lysine) (PLL). The assembly of the antiTNF-α/Alg complex with PLL and its stability in PBS and lysozymes are monitored on a planar support using a quartz crystal microbalance with dissipation. The assembly of the antiTNF-α/Alg complex on PLGA NPs is followed by zeta potential measurements. AntiTNF-α release from the PLGA NPs is measured in PBS at 37 and 60 °C and in the HepG2 cell line following NP uptake, using the Q-ADA kit detection kit. The release follows first-order kinetics with an initial burst. Intracellular release of antiTNF-α is confirmed by confocal Raman microscopy.

Distance Measurements on a Dual-Labeled TOAC AChR M2δ Peptide in Mechanically Aligned DMPC Bilayers via Dipolar Broadening CW-EPR Spectroscopy

A membrane alignment technique has been used to measure the distance between two TOAC nitroxide spin labels on the membrane-spanning M2δ, peptide of the nicotinic acetylcholine receptor (AChR), via CW-EPR spectroscopy. The TOAC-labeled M2δ peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 14.6 Å from a dual TOAC-labeled AChR M2δ peptide at positions 7 and 13 that closely matches with the 14.5 Å distance obtained from a model of the labeled AChR M2δ peptide. In addition, the angles determining the relative orientation of the two nitroxides have been determined, and the results compare favorably with molecular modeling. The global analysis of the data from the aligned samples gives much more precise estimates of the parameters defining the geometry of the two labels than can be obtained from a randomly dispersed sample.

Path-based supports for hypergraphs

A path-based support of a hypergraph H is a graph with the same vertex set as H in which each hyperedge induces a Hamiltonian subgraph. While it is NP-hard to decide whether a path-based support has a monotone drawing, to determine a path-based support with the minimum number of edges, or to decide whether there is a planar path-based support, we show that a path-based tree support can be computed in polynomial time if it exists.

On the equilibria of finely discretized curves and surfaces

Our goal is to identify the type and number of static equilibrium points of solids arising from fine, equidistant $n$-discretrizations of smooth, convex surfaces. We assume uniform gravity and a frictionless, horizontal, planar support. We show that as $n$ approaches infinity these numbers fluctuate around specific values which we call the imaginary equilibrium indices associated with the approximated smooth surface. We derive simple formulae for these numbers in terms of the principal curvatures and the radial distances of the equilibrium points of the solid from its center of gravity. Our results are illustrated on a discretized ellipsoid and match well the observations on natural pebble surfaces.

ChemInform Abstract: Simulating the Complexities of Heterogeneous Catalysis with Model Systems: Case Studies of SiO2 Supported Pt‐Group Metals

AbstractReview: 105 refs.

Simulating the Complexities of Heterogeneous Catalysis with Model Systems: Case studies of SiO2 Supported Pt-Group Metals

Model catalyst surfaces, consisting of vapor-deposited metal nanoparticles supported on a planar oxide support, can help to link reactivity studies on well-defined single crystal surfaces with those conducted on high-surface area supported catalysts. When coupled with near atmospheric pressure kinetic and spectroscopic techniques, these well-defined model catalyst surfaces represent a useful approach to combine the power of surface analytical techniques with reactivity studies under relevant reaction conditions. Here, we review recent results of our investigations characterizing the physical and catalytic properties of Pt/SiO2 and Rh/SiO2 model catalyst surfaces. As will be discussed, the model catalyst approach can help simulate the complexities of catalytic reactions on supported catalysts, helping to provide insights into the role of particle size, particle morphology, and surface adsorbates in dictating the observed structure-sensitivity (activity and selectivity) during reactions at near atmospheric pressures.

On Planar Supports for Hypergraphs

A graph G is a support for a hypergraph H = (V, S) if the vertices of G correspond to the vertices of H such that for each hyperedge Si ? S the subgraph of G induced by Si is connected. G is a planar support if it is a support and planar. Johnson and Pollak [11] proved that it is NPcomplete to decide if a given hypergraph has a planar support. In contrast, there are lienar time algorithms to test whether a given hypergraph has a planar support that is a path, cycle, or tree. In this paper we present an e?cient algorithm which tests in polynomial time if a given hypergraph has a planar support that is a tree where the maximal degree of each vertex is bounded. Our algorithm is constructive and computes a support if it exists. Furthermore, we prove that it is already NP-hard to decide if a hypergraph has a 2-outerplanar support.

Guided self-assembly of microgels: from particle arrays to anisotropic nanostructures

We investigate the guided large scale self-assembly of microgels consisting of N-vinylcaprolactam (VCL), N-isopropylacrylamide (NiPAAm) and acetoacetoxyethyl methacrylate (AAEM) using nanostructured substrates fabricated via controlled wrinkling. Depending on the type of microgel, the arrangement in the grooves of the substrates influences the particle shape and the overall pattern formation. Thus we obtain straight lines composed of single particles as well as zigzag-structures. In an additional processing step, we use the nanostructured substrates as stamps to transfer the formed microgel patterns onto a planar support. Subsequent UV-crosslinking of the microgel strings yields anisotropic particles.

Electrocatalysis at Microelectrodes: Geometrical Considerations

The influence of the shape of individual electrocatalytically active, axisymmetric particles supported on an inactive substrate, on the current associated with heterogeneous redox reactions involving solution-phase species has been examined theoretically. In agreement with previous work, the diffusion-limited currents, ilim, for a disk and supported truncated spheres of the same area including a full sphere are very similar (within ca. 10%). Large enhancements in the predicted values of ilim were found, however, for spheroids of the prolate type, as the aspect ratio was increased. Analyses of arrays of electrocatalytically active disks embedded in a planar inactive support in the presence of a diffusion boundary layer of well-defined thickness, for example, a rotating disk electrode, revealed that values of ilim very close to those expected for fully catalytic surfaces could be achieved for disk coverages on the order of 1%. Similar conclusions were made for corresponding arrays of small disks embedded in microdisks or spherical inactive supports in quiescent media. This effect might explain observations made for iron porphyrins adsorbed on graphite surfaces in aqueous electrolytes, for which the experimentally determined onset for oxygen reduction is found to be at potentials well ahead of the onset of the voltammetric wave associated with the conversion of the inactive (ferric) to the active (ferrous) form of the macrocycle.

Small Molecule Macroarray Construction via Palladium-Mediated CarbonCarbon Bond-Forming Reactions: Highly Efficient Synthesis and Screening of Stilbene Arrays

Arrays of stilbenes have been prepared in high purity by using Heck reactions on planar cellulose support, and demonstrate the compatibility of the small-molecule macroarray platform (see figure) with metal-mediated reactions for the first time. Biological screening of the stilbene library revealed several compounds with the ability to inhibit quorum sensing responses in bacteria, expanding the activity of this compound class.

Use of magnetic field for addressing, grafting onto support and actuating permanent magnetic filaments applied to enhanced biodetection

A simple method combining magnetic field and microparticles has been designed for assembling, locating and grafting permanent microfilaments onto a silanised glass surface. Filaments orthogonal to a glass support are implemented by assembling magnetic carboxylatex particles under magnetic field. They exhibit an aspect ratio up to 14. Carboxyl moieties on beads and silanised glass are activated using the carbodiimide method. A further reaction with long chain diamines allows the formation of permanent filaments covalently anchored onto the glass support. Grafted microfilaments can withstand intense washing with detergent solutions under ultrasound. 500 μm diameter filament spots are formed using ferromagnetic tips connected to an electromagnet. After their formation, filaments can be actuated by an external magnetic field: their orientation is tunable in real time within a 2π solid angle. Filaments can be biofunctionalised in order to perform biomolecular recognition on the support. We show that nucleic acid probes immobilised onto these filaments hybridize with labelled oligonucleotide targets. Due to an increase of the specific surface, the fluorescence signal of targets is 3-fold higher with filaments than with a planar glass support biofunctionalised with the same oligonucleotides. Our methodology thus provides an easy way to create filaments usable for both actuation and biodetection onto a planar support. We expect our strategy to be compatible with the formation of nanofilaments using nanoparticles.

Functional fibrous polypropylene solid support and its application in solid phase peptide synthesis and cell specific binding

Surfaces of polypropylene (PP) fabrics were modified through thermal initiated radical graft polymerization with acrylic acid and poly (ethylene glycol) di-acrylate (PEG-DIA). Various polymerization conditions were studied to determine the optimal parameters leading to the best surface functionality. Carboxylic acid groups on the functionalized PP support were further derivatized for solid phase peptide synthesis. Peptide ligands LHPQF and sppLDI were successfully synthesized through simple coupling reactions utilizing Fmoc chemistry. Specific binding of Streptavidin–Alkaline phosphatase conjugate and Jurkat (T lymphoblastic) cell were successfully achieved on the novel functional PP substrates. The good geometrical flexibility, mechanical stability, chemical reactivity and biocompatibility of this novel planar support make it very promising substrate for various biotechnological applications.

Synthesis of Surface-Anchored DNA−Polymer Bioconjugates Using Reversible Addition−Fragmentation Chain Transfer Polymerization

We report here an approach to grafting DNA-polymer bioconjugates on a planar solid support using reversible addition-fragmentation chain transfer (RAFT) polymerization. In particular, a trithiocarbonate compound as the RAFT chain transfer agent (CTA) is attached to the distal point of a surface-immobilized oligonucleotide. Initiation of RAFT polymerization leads to controlled growth of polymers atop DNA molecules on the surface. Growth kinetics of poly(monomethoxy-capped oligo(ethylene glycol) methacrylate) atop DNA molecules is investigated by monitoring the change of polymer film thickness as a function of reaction time. The reaction conditions, including the polymerization temperature, the initiator concentration, the CTA surface density, and the selection of monomers, are varied to examine their impacts on the grafting efficiency of DNA-polymer conjugates. Comparing to polymer growth atop small molecules, the experimental results suggest that DNA molecules significantly accelerate polymer growth, which is speculated as a result of the presence of highly charged DNA backbones and purine/pyrimidine moieties surrounding the reaction sites.

Patterned Arrays of Vertically Aligned Carbon Nanotube Microelectrodes on Carbon Films Prepared by Thermal Chemical Vapor Deposition

A straightforward procedure is described for preparation of arrays of microdisk electrodes comprising bundles of vertically aligned carbon nanotubes (VACNTs). The arrays are fabricated by thermal chemical vapor deposition synthesis directly on a planar carbon film support. Use of standard micro- and nanolithography procedures for patterning the bilayer catalyst spots enables arrays to be grown with controlled electrode diameters and spacings. The minimum accessible VACNT bundle diameter, and hence microelectrode diameter, is 2 microm. After insulating the arrays with SU-8 epoxy and exposing the VACNT ends by polishing or treating with O2 plasma, the microdisk electrodes exhibit attractive electrochemical properties.

Encapsulation of Uncharged Water-Insoluble Organic Substance in Polymeric Membrane Capsules via Layer-by-Layer Approach

We report a general and versatile method for the encapsulation of electrically uncharged organic substance in polymeric capsules by using a layer-by-layer (LbL) approach. Electrical charge was induced on the surface of pyrene (uncharged organic substance) with an amphiphilic surfactant (sodium dodecyl sulfate, SDS) by micellar solubilization. The SDS micellar solution of pyrene in water was then deposited on a flat substrate as well as colloidal particles with chitosan as an oppositely charged polyelectrolyte. Pyrene was used as a model drug because it displayed intrinsic fluorescence that allowed us to monitor LbL growth by fluorescence and under confocal laser scanning microscopy (CLSM). To examine the proof of concept, multilayers were coated on the planar support by the LbL method. UV-vis spectroscopy showed regular growth of each layer deposited. Thin film formation was evidenced by scanning electron microscopy. The LbL method was extended to particles where fluorescence spectroscopy revealed LbL growth and transmission electron microscopy (TEM) provided evidence of particle coating. The quantification of dye in each deposited layer further proved LbL growth. The removal of sacrificial core provided thin capsules. The capsules were characterized by TEM and CLSM. The capsules showed potential as a drug delivery system, which is suggested by the slow release of entrapped dye by concentration-dependent diffusion in isotonic saline solution. The kinetics of desorption of pyrene from this thin film was modeled by a pseudo-second-order model.

Probing the function of ionotropic and G protein-coupled receptors in surface-confined membranes

This article reports on recent electrical and optical techniques for investigating cellular signaling reactions in artificial and native membranes immobilized on solid supports. The first part describes the formation of planar artificial lipid bilayers on gold electrodes, which reveal giga-ohm electrical resistance and the insertion and characterization of ionotropic receptors therein. These membranes are suited to record a few or even single ion channels by impedance spectroscopy. Such tethered membranes on planar arrays of microelectrodes offer mechanically robust, long-lasting measuring devices to probe the influence of different chemistries on biologically important ionotropic receptors and therefore will have a future impact to probe the function of channel proteins in basic science and in biosensor applications. In a second part, we present complementary approaches to form inside-out native membrane sheets that are immobilized on micrometer-sized beads or across submicrometer-sized holes machined in a planar support. Because the native membrane sheets are plasma membranes detached from live cells, these approaches offer a unique possibility to investigate cellular signaling processes, such as those mediated by ionotropic or G protein-coupled receptors, with original composition of lipids and proteins.