The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen

Protocollagen, a non-hydroxylated form of collagen, was extracted with cold 0.1 N acetic acid from embryonic tendon cells incubated with α,α′-dipyridyl and the protein was purified by controlled proteolytic digestion. The resulting modified protocollagen was shown to consist of polypeptides the same size as α1 and α2 chains of collagen and had a thermal transition by optical rotation similar to collagen. The T_m however was 24°, a value which was 15° lower than the T_m of an hydroxylated form of collagen from the same source. The results suggest that hydroxylated proline increases the thermal stability of collagen.     

Design – an inappropriate concept in evolutionary theory*

The concept of accident in evolution refers to causes which are stochastic with respect to selective demands arising from the external environment and acting on the organism, while the concept of design refers to causes which meet the requirement of these selective demands. The condition ‘with respect to selective demands’ is generally forgotten so that evolutionary changes are described as being design modifications. Design is an invalid synonym for adaptation. Further it implies a designer and has been used by some authors since before Darwin to argue that design in organisms demonstrates the existence of a designer and hence a plan. Yet if evolution depends on two simultaneously acting causes, one of which is accidental, then the process of evolution and all attributes of organisms are accidental. The concept of design is inappropriate in biology and should be eliminated from all biological explanations.     

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases^1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field^6-21 (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting^22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)^24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies^4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.     

Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen ^1-7. The performance of SOFCs and the rates of many chemical and energy transformation processes in energy storage and conversion devices in general are limited primarily by charge and mass transfer along electrode surfaces and across interfaces. Unfortunately, the mechanistic understanding of these processes is still lacking, due largely to the difficulty of characterizing these processes under in situ conditions. This knowledge gap is a chief obstacle to SOFC commercialization. The development of tools for probing and mapping surface chemistries relevant to electrode reactions is vital to unraveling the mechanisms of surface processes and to achieving rational design of new electrode materials for more efficient energy storage and conversion². Among the relatively few in situ surface analysis methods, Raman spectroscopy can be performed even with high temperatures and harsh atmospheres, making it ideal for characterizing chemical processes relevant to SOFC anode performance and degradation^8-12. It can also be used alongside electrochemical measurements, potentially allowing direct correlation of electrochemistry to surface chemistry in an operating cell. Proper in situ Raman mapping measurements would be useful for pin-pointing important anode reaction mechanisms because of its sensitivity to the relevant species, including anode performance degradation through carbon deposition^{8, 10, 13, 14} ("coking") and sulfur poisoning^{11, 15} and the manner in which surface modifications stave off this degradation¹⁶. The current work demonstrates significant progress towards this capability. In addition, the family of scanning probe microscopy (SPM) techniques provides a special approach to interrogate the electrode surface with nanoscale resolution. Besides the surface topography that is routinely collected by AFM and STM, other properties such as local electronic states, ion diffusion coefficient and surface potential can also be investigated^17-22. In this work, electrochemical measurements, Raman spectroscopy, and SPM were used in conjunction with a novel test electrode platform that consists of a Ni mesh electrode embedded in an yttria-stabilized zirconia (YSZ) electrolyte. Cell performance testing and impedance spectroscopy under fuel containing H₂S was characterized, and Raman mapping was used to further elucidate the nature of sulfur poisoning. In situ Raman monitoring was used to investigate coking behavior. Finally, atomic force microscopy (AFM) and electrostatic force microscopy (EFM) were used to further visualize carbon deposition on the nanoscale. From this research, we desire to produce a more complete picture of the SOFC anode.     

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK₂ complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.     

Hydrophobic salt-modified Nafion for enzyme immobilization and stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells. In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes. In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion. Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells. This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.     

四种口腔修复材料对细胞的毒性分析

目的:研究纯钛、钛合金、钴铬合金和镍铬合金四种非贵金属口腔修复材料对L-929成纤维细胞的毒性作用。方法:应用口腔修复材料制备材料浸提液处理培养L-929细胞,采用AnnexinV-FITC试剂盒比较细胞凋亡水平改变,采用Western blot检测细胞凋亡相关基因的表达。结果:纯钛与钛合金诱导L-929细胞凋亡的水平与对照组没有统计学差异(P>0.05);钴铬合金和镍铬合金材料浸提液可引起L-929细胞凋亡增加(P<0.05)。结论:纯钛与钛合金材料对口腔黏膜细胞的毒性作用相较钴铬合金和镍铬合金材料低,更具安全性。     

Development of Whispering Gallery Mode Polymeric Micro-optical Electric Field Sensors

Optical modes of dielectric micro-cavities have received significant attention in recent years for their potential in a broad range of applications. The optical modes are frequently referred to as "whispering gallery modes" (WGM) or "morphology dependent resonances" (MDR) and exhibit high optical quality factors. Some proposed applications of micro-cavity optical resonators are in spectroscopy¹, micro-cavity laser technology², optical communications^3-6 as well as sensor technology. The WGM-based sensor applications include those in biology⁷, trace gas detection⁸, and impurity detection in liquids⁹. Mechanical sensors based on microsphere resonators have also been proposed, including those for force^10,11, pressure¹², acceleration¹³ and wall shear stress¹⁴. In the present, we demonstrate a WGM-based electric field sensor, which builds on our previous studies^15,16. A candidate application of this sensor is in the detection of neuronal action potential.The electric field sensor is based on polymeric multi-layered dielectric microspheres. The external electric field induces surface and body forces on the spheres (electrostriction effect) leading to elastic deformation. This change in the morphology of the spheres, leads to shifts in the WGM. The electric field-induced WGM shifts are interrogated by exciting the optical modes of the spheres by laser light. Light from a distributed feedback (DFB) laser (nominal wavelength of ~ 1.3 μm) is side-coupled into the microspheres using a tapered section of a single mode optical fiber. The base material of the spheres is polydimethylsiloxane (PDMS). Three microsphere geometries are used: (1) PDMS sphere with a 60:1 volumetric ratio of base-to-curing agent mixture, (2) multi layer sphere with 60:1 PDMS core, in order to increase the dielectric constant of the sphere, a middle layer of 60:1 PDMS that is mixed with varying amounts (2% to 10% by volume) of barium titanate and an outer layer of 60:1 PDMS and (3) solid silica sphere coated with a thin layer of uncured PDMS base. In each type of sensor, laser light from the tapered fiber is coupled into the outermost layer that provides high optical quality factor WGM (Q ~ 10⁶). The microspheres are poled for several hours at electric fields of ~ 1 MV/m to increase their sensitivity to electric field.     

Throughflow centrality is a global indicator of the functional importance of species in ecosystems

To better understand and manage complex systems like ecosystems it is critical to know the relative contribution of the system components to the system function. Ecologists and social scientists have described a diversity of ways that individuals can be important; This paper makes two key contributions to this research area. First, it shows that throughflow (T_j), the total energy or matter entering or exiting a system component, is a global indicator of the relative contribution of the component to the whole system activity. It is global because it includes the direct and indirect exchanges among community members. Further, throughflow is a special case of Hubbell status or centrality as defined in social science. This recognition effectively joins the concepts, enabling ecologists to use and build on the broader centrality research in network science. Second, I characterize the distribution of throughflow in 45 empirically-based trophic ecosystem models. Consistent with theoretical expectations, this analysis shows that a small fraction of the system components are responsible for the majority of the system activity. In 73% of the ecosystem models, 20% or less of the nodes generate 80% or more of the total system throughflow. Four or fewer nodes are required to account for 50% of the total system activity and are thus defined as community dominants. 121 of the 130 dominant nodes in the 45 ecosystem models could be classified as primary producers, dead organic matter, or bacteria. Thus, throughflow centrality indicates the rank power of the ecosystems components and shows the concentration of power in the primary production and decomposition cycle. Although these results are specific to ecosystems, these techniques build on flow analysis based on economic input–output analysis. Therefore these results should be useful for ecosystem ecology, industrial ecology, the study of urban metabolism, as well as other domains using input–output analysis.     

Controlling the Morphology and Efficiency of Hybrid ZnO:Polythiophene Solar Cells Via Side Chain Functionalization

The efficiency of polymer – metal oxide hybrid solar cells depends critically on the intimacy of mixing of the two semiconductors. The effect of side chain functionalization on the morphology and performance of conjugated polymer:ZnO solar cells is investigated. Using an ester‐functionalized side chain poly(3‐hexylthiophene‐2,5‐diyl) derivative (P3HT‐E), the nanoscale morphology of ZnO:polymer solar cells is significantly more intimately mixed compared to ZnO:poly(3‐hexylthiophene‐2,5‐diyl) (ZnO:P3HT), as evidenced experimentally from a 3D reconstruction of the phase separation using electron tomography. Photoinduced absorption reveals nearly quantitative charge generation for the ZnO:P3HT‐E blend but not for ZnO:P3HT, consistent with the results obtained from solving the 3D diffusion equation for excitons formed in the polymer within the two experimental ZnO morphologies. For thin ZnO:P3HT‐E active layers (～50 nm) this yields a significant improvement of the solar cell performance. For thicker cells, however, the reduced hole mobility and a reduced percolation of ZnO pathways hinders charge carrier collection, limiting the power conversion efficiency.