Observation of the Decay B0→D±D*∓

journal articl

Case Marking and Word Order in the Finnish Language

2009-03departmental bulletin pape

<研究ノート>標準化にともなう企業の能力構築の研究―移動体通信産業における知識と引用のネットワークの分析―

本研究では、後発企業が技術スピルオーバーによって、どのように知識を獲得し蓄積するのかを検討する。より具体的には、既存の有力な標準化の推進企業から後発企業への技術的リーダーシップの移転のプロセスを検討することによって、標準化にともなう技術のスピルオーバーにおいて、後発企業がいかに知識の獲得・強化できるのかを明らかにする。本研究では、技術仕様に関して宣言される必須特許（SEP：standard essential patent）と独自特許（Non-SEP）の分析を行った。その結果、補完的企業である、ある後発の半導体サプライヤーは、既存の有力な標準化の推進者からの必須特許の引用を通じて、システム知識を構築・強化し、それによって先発企業を圧倒してきたことが明らかとなった。この発見は、標準化の推進企業の知識マネジメントについての議論を拡張するとともに、実践的な示唆を提供すると期待される。departmental bulletin pape

Crystallization, Reentrant Melting, and Resolubilization of Virus Nanoparticles

Crystallization is a fundamental and ubiquitous process that is well understood in the case of atoms or small molecules, but its outcome is still hard to predict in the case of nanoparticles or macromolecular complexes. Controlling the organization of virus nanoparticles into a variety of 3D supramolecular architectures is often done by multivalent ions and is of great interest for biomedical applications such as drug or gene delivery and biosensing, as well as for bionanomaterials and catalysis. In this paper, we show that slow dialysis, over several hours, of wild-type Simian Virus 40 (wt SV40) nanoparticle solution against salt solutions containing MgCl₂, with or without added NaCl, results in wt SV40 nanoparticles arranged in a body cubic center crystal structure with <i>Im</i>3<i>m</i> space group, as a thermodynamic product, in coexistence with soluble wt SV40 nanoparticles. The nanoparticle crystals formed above a critical MgCl₂ concentrations. Reentrant melting and resolubilization of the virus nanoparticles took place when the MgCl₂ concentrations passed a second threshold. Using synchrotron solution X-ray scattering we determined the structures and the mass fraction of the soluble and crystal phases as a function of MgCl₂ and NaCl concentrations. A thermodynamic model, which balances the chemical potentials of the Mg²⁺ ions in each of the possible states, explains our observations. The model reveals the mechanism of both the crystallization and the reentrant melting and resolubilization and shows that counterion entropy is the main driving force for both processes

Scaffold Properties Are a Key Determinant of the Size and Shape of Self-Assembled Virus-Derived Particles

Controlling the geometry of self-assembly will enable a greater diversity of nanoparticles than now available. Viral capsid proteins, one starting point for investigating self-assembly, have evolved to form regular particles. The polyomavirus SV40 assembles from pentameric subunits and can encapsidate anionic cargos. On short ssRNA (≤814 nt), SV40 pentamers form 22 nm diameter capsids. On RNA too long to fit a <i>T</i> = 1 particle, pentamers forms strings of 22 nm particles and heterogeneous particles of 29–40 nm diameter. However, on dsDNA SV40 forms 50 nm particles composed of 72 pentamers. A 7.2-Å resolution cryo-EM image reconstruction of 22 nm particles shows that they are built of 12 pentamers arranged with <i>T</i> = 1 icosahedral symmetry. At 3-fold vertices, pentamers each contribute to a three-helix triangle. This geometry of interaction is not seen in crystal structures of <i>T</i> = 7 viruses and provides a structural basis for the smaller capsids. We propose that the heterogeneous particles are actually mosaics formed by combining different geometries of interaction from <i>T</i> = 1 capsids and virions. Assembly can be trapped in novel conformations because SV40 interpentamer contacts are relatively strong. The implication is that by virtue of their large catalog of interactions, SV40 pentamers have the ability to self-assemble on and conform to a broad range of shapes

Analysis of the particles.

<p>A,B-Proteins were separated by elecrtrophoresis in Tricine buffer on 16% polyacrylamide gel. Western blotting was performed with polyclonal antibody against histone H3 (Upstate) (A) and against VP1 (B). M–size marker; 1–Nuclear extracts used in the packaging reaction; 2–In vitro assembled particles, fraction 9 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000765#pone-0000765-g004" target="_blank">Fig. 4</a>. 3–wild type SV40. C-Analysis of the packaged DNA. Particles purified by ultrafiltration were treated with Tris base (200 mM) in presence of 25 mM EGTA and 25 mM DTT for 1 hr at 37°C. DNA was extracted by phenol-chloroform treatment in presence of 1% SDS, and analyzed by Southern blotting with pGL3-control as a probe.</p

SV40 triggers reversible changes in phospho-Ezrin, dependent on PDK1 function.

<p>(A) Cortical actin alterations occur in A431 cells following incubation with SV40 for 10 min, as visualized with phalloidin staining in fixed cells. (B) Phosphorylated ERM proteins become inactive shortly after SV40 treatment. A431 cells were treated with SV40 for the indicated time points, fixed and immunostained with an antibody against phosphorylated T567 of ERM proteins. p-ERM disappears as soon as 5 min after incubation with SV40 and it resumes after 1 h. (C) A phosphomimetic mutant form of Ezrin, T567D, fails to fall off the membrane following SV40 treatment. A431 cells were transfected with EzrinT567D-GFP, treated with SV40 for 15 min, and subsequently fixed and tested for the GFP signal localization. (D) Expression of both a phosphomimetic and an inactive Ezrin mutant form negatively correlates with SV40 infection. Large populations of A431 cells were transfected with EzrinT567D, EzrinT567A or wild-type Ezrin GFP constructs and subjected to SV40. Cells that were both transfected (GFP signal) and infected (T-antigen signal) were scored and compared with the expected number emerging from a random occurrence of the two signals. Negative log2 ratio values represent an anti-correlation, which demonstrates inhibition of infection by the transfected construct (p-value 6.8×10⁻³ and 0.05 for the T567D and T567A scores, respectively). (E, F) Inhibition of PDK1 function leads to increased levels of p-ERM. The PDK1 inhibitor was applied onto A431 cells for 1.5 h before the addition of SV40 for another 15 min. Levels of p-ERM were assessed using either immunofluorescence in fixed cells (E) or immunoblotting (F). Quantification of two different blots was performed using the ImageJ software. (G) Inhibition of PDK1 function blocks increased levels of infection as caused by Ezrin knockdown. A431 cells were subjected to siRNA against Ezrin or control siRNA, and subsequently treated with the PDK1 inhibitor 1.5 h prior to SV40 infection, or left untreated. Percentage of infection was calculated from a large number of cells. p-values: 1×10⁻⁴ (PDK1 inhibitor), 5.4×10⁻³ (Ezrin siRNA), 5.8×10⁻⁴ (Ezrin siRNA, PDK1 inhibitor).</p

Parameters affecting the in vitro packaging reaction.

<p>Effect of temperature (A) and concentration of monovalent salts (B) present during the re-association reaction (step B) on the yield, measured as titer of luc transducing units.</p

A model for the <i>in vitro</i> assembly reaction.

<p>The addition of DTT to nuclear extracts (A) leads to disassembly of the VLPs. Following the addition of supercoiled DNA (B) pentamers bind along each DNA molecule. This increases the local concentration of VP1, facilitating concerted assembly. Reassembly may be facilitated by presence of chaperones in the nuclear extracts. Assembly is accompanied by DNA condensation, presumably via the action of topo II. In Step C the capsids are stabilized at pH 5.2.</p

SV40 at its entry triggers the upregulation of a number of integrins on the cell surface.

<p>(A) Cell surface N-glycoproteins that are significantly altered in cell surface abundance upon exposure to SV40 are visualized with a network view. The glycoproteins whose abundance was either increased (yellow) or decreased (blue) during exposure to SV40 (and which either did not change during exposure to VSV, or changed in the opposite direction compared to SV40) are depicted as nodes in the network. The different shades represent different degrees of relative abandunce (log2 values). The remaining nodes in the network are the hits from the RNAi screen (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055799#pone-0055799-g002" target="_blank">Figure 2A</a>), which either increased (green) or decreased (red) SV40 infection upon siRNA knockdown. For the common hits in the CSC and RNAi screens, the node border represents the RNAi phenotype (ITGA6, ITGB6 and CD47 were CSC-hits but gave no RNAi phenotype when tested). The grey connecting lines between nodes illustrate protein interactions, which were assessed using the STRING database with a combined score of at least 0.9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055799#pone.0055799-Szklarczyk1" target="_blank">[45]</a>, and were visualized using Cytoscape (<a href="http://www.cytoscape.org" target="_blank">www.cytoscape.org</a>) and the Cerebral plugin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055799#pone.0055799-Barsky1" target="_blank">[46]</a>.</p