Confocal/TEM overlay microscopy: a simple method for correlating confocal and electron microscopy of cells expressing GFP/YFP fusion proteins.

Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-microm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.     

Electrical behavior of droplet interface bilayer networks: experimental analysis and modeling

Aqueous droplets submerged in an oil-lipid mixture become enclosed by a lipid monolayer. The droplets can be connected to form robust networks of droplet interface bilayers (DIBs) with functions such as a biobattery and a light sensor. Such DIB networks might be used as model systems for the study of membrane-based biological phenomena. In this study, we develop and experimentally validate an electrical modeling approach for DIB networks by applying it to describe the current flow through a simple network containing protein pores and blocking molecules. We demonstrate the use of SPICE (Simulation Program with Integrated Circuit Emphasis) for simulating the electrical behavior of DIB networks. The modular and scalable nature of DIB networks should enable a straightforward extension of the analysis presented in this paper to large, complex networks.     

Virtual cell and tissue dynamics of ectopic activation of the ventricles

Cardiac ventricular cells and tissues are normally excitable, and are activated by propagating waves of excitation that are initiated in the specialized pacemaking region of the heart. However, isolated or repetitive activity can be initiated at abnormal (ectopic) sites in the ventricles. To trigger an endogenous ectopic beat, there must be a compact focus of cells with changed membrane excitation parameters and kinetics, which initiate activity by after-depolarizations triggered by propagating activity, or that have bifurcated into autorhythmicity. This ectopic focus needs to be large enough, and adequately coupled, to drive the surrounding tissue. We investigate the initiation of ectopic excitation in computational models of human ventricular cells triggered by after-depolarizations and by up/down regulation of specific membrane conductance systems, and the propagation and evolution of ectopic activity in homogeneous or heterogeneous and isotropic, anisotropic, or orthotropic tissues.     

Global Semigroup of Conservative Solutions of the Nonlinear Variational Wave Equation

We prove the existence of a global semigroup for conservative solutions of the nonlinear variational wave equation u _tt − c(u)(c(u)u _x ) _x  = 0. We allow for initial data u| _{t = 0} and u _t | _t=0 that contain measures. We assume that 0 < k^-1 \leqq c(u) \leqq k{0 < \kappa^{-1} \leqq c(u) \leqq \kappa}. Solutions of this equation may experience concentration of the energy density (u_t²+c(u)²u_x²)dx{(u_t^2+c(u)^2u_x^2){\rm d}x} into sets of measure zero. The solution is constructed by introducing new variables related to the characteristics, whereby singularities in the energy density become manageable. Furthermore, we prove that the energy may focus only on a set of times of zero measure or at points where c′(u) vanishes. A new numerical method for constructing conservative solutions is provided and illustrated with examples.     

Hydrogen bond-mediated recognition of the chemical warfare agent soman (GD)

NMR titration studies in acetonitrile-d(3)/DMSO-d(6) mixtures demonstrate that diindolylurea-based receptors can form complexes with the organophosphorus nerve agent soman in organic solution.     

Palladium-catalyzed substitution and cross-coupling of benzylic fluorides

Benzylic fluorides are suitable substrates for Pd(0)-catalyzed Tsuji-Trost substitution using carbon, nitrogen, oxygen, and sulfur nucleophiles and for cross-coupling with phenylboronic acid. For the bifunctional substrate 4-chlorobenzyl fluoride, fine-tuning of the reaction conditions allows for the regioselective displacement of either the chlorine or fluorine substituent. The leaving group ability of fluoride vs other groups displaced in substitution is CF(3)CO(2) ≈ p-NO(2)C(6)H(4)CO(2) ≈ OCO(2)CH(3) > F > CH(3)CO(2), a ranking similar to allylic fluorides under Pd catalysis.     

Comprehensive numerical analysis of a three-pass bead-in-slot weld and its critical validation using neutron and synchrotron diffraction residual stress measurements

The current paper presents a finite element simulation of the residual stress field associated with a three pass slot weld in an AISI 316LN austenitic stainless steel plate. The simulation is split into uncoupled thermal and mechanical analyses which enable a computationally less expensive solution. A dedicated welding heat source modelling tool is employed to calibrate the ellipsoidal Gaussian volumetric heat source by making use of extensive thermocouple measurements and metallographic analyses made during and after welding. The mechanical analysis employs the Lemaitre–Chaboche mixed hardening model. This captures the cyclic mechanical response which a material undergoes during the thermo-mechanical cycles imposed by the welding process. A close examination of the material behaviour at various locations in the sample during the welding process, clearly demonstrates the importance of defining the correct hardening and high temperature softening behaviour. The simulation is validated by two independent diffraction techniques. The well-established neutron diffraction technique and a very novel spiral slit X-ray synchrotron technique were used to measure the residual stress–strain field associated with the three-pass weld. The comparison between the model and the experiment reveals close agreement with no adjustable parameters and clearly validates the used modelling procedure.     

Untersuchung von Luft

Ohne Zusammenfassung     

Determination of loads applied perpendicularly to the outer boundary of a ring using coefficients of influence

A procedure is explained to determined the amount of several pairs of diametrical loads applied to the outside boundary of a ring when stresses at selected points of the inside or outside boundaries are known. Coefficients of influence are used, following an approach similar to the one presented in a previous paper. Examples of application are given and the possible increase in precision is shown when the number of points of measurements is larger than the number of loads to be determined.