Host Cell Prediction of Exosomes Using Morphological Features on Solid Surfaces Analyzed by Machine Learning.

Exosomes are extracellular nanovesicles released from any cells and found in any body fluid. Because exosomes exhibit information of their host cells (secreting cells), their analysis is expected to be a powerful tool for early diagnosis of cancers. To predict the host cells, we extracted multidimensional feature data about size, shape, and deformation of exosomes immobilized on solid surfaces by atomic force microscopy (AFM). The key idea is combination of support vector machine (SVM) learning for individual exosome particles and their interpretation by principal component analysis (PCA). We observed exosomes derived from three different cancer cells on SiO2/Si, 3-aminopropyltriethoxysilane-modified-SiO2/Si, and TiO2 substrates by AFM. Then, 14-dimensional feature vectors were extracted from AFM particle data, and classifiers were trained in 14-dimensional space. The prediction accuracy for host cells of test AFM particles was examined by the cross-validation test. As a result, we obtained prediction of exosome host cells with the best accuracy of 85.2% for two-class SVM learning and 82.6% for three-class one. By PCA of the particle classifiers, we concluded that the main factors for prediction accuracy and its strong dependence on substrates are incremental decrease in the PCA-defined aspect ratio of the particles with their volume.

Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes.

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4 septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO2/Si3N4 septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.

Substrate-Induced Structure and Molecular Dynamics in a Lipid Bilayer Membrane.

The solid-substrate-dependent structure and dynamics of molecules in a supported lipid bilayer (SLB) were directly investigated via atomic force microscopy (AFM) and single particle tracking (SPT) measurements. The appearance of either vertical or horizontal heterogeneities in the SLB was found to be strongly dependent on the underlying substrates. SLB has been widely used as a biointerface with incorporated proteins and other biological materials. Both silica and mica are popular substrates for SLB. Using single-molecule dynamics, the fluidity of the upper and lower membrane leaflets was found to depend on the substrate, undergoing coupling and decoupling on the SiO2/Si and mica substrates, respectively. The anisotropic diffusion caused by the locally destabilized structure of the SLB at atomic steps appeared on the Al2O3(0001) substrate because of the strong van der Waals interaction between the SLB and the substrate. Our finding that the well-defined surfaces of mica and sapphire result in asymmetry and anisotropy in the plasma membrane is useful for the design of new plasma-membrane-mimetic systems. The application of well-defined supporting substrates for SLBs should have similar effects as cell membrane scaffolds, which regulate the dynamic structure of the membrane.

Fabrication of Au-Nanoparticle-Embedded Lipid Bilayer Membranes Supported on Solid Substrates.

We fabricated gold nanoparticle (Au-NP)-embedded supported lipid bilayers (SLBs) by two methods. In the vesicle-vesicle fusion method, vesicles with hydrophobized Au-NPs are ruptured and fused on SiO2/Si substrates. In the vesicle-membrane fusion method, SLBs without Au-NPs were preformed on the substrate and then vesicles with Au-NPs were fused into the preformed membranes. In the former method, Au-NP incorporation into the SLBs was observed as an increase in the membrane thickness in atomic force microscopy (AFM) images and directly observed by transmission electron microscopy. In the latter method, fusion of vesicles into the preformed membranes was confirmed by the fluorescent color change in the preformed membranes, and Au-NP incorporation was also confirmed by an increase in the membrane thickness in the AFM images. Key techniques for the successful vesicle-membrane fusion are hydrophobization of Au-NPs, approach control of vesicles by mixing the charged lipids, and destabilization of the lipid bilayers by adding lipids with a small polar headgroup.

Phase separation in lipid bilayer membranes induced by intermixing at a boundary of two phases with different components.

We have demonstrated that dynamic phase separation is induced by coalescence of two self-spreading supported lipid bilayers (SLBs) with different components. Coalescence between a phosphocholine/sphingolipid SLB and a phosphocholine/cholesterol one forms raft-like liquid ordered (Lo) domains, which can be observed by fluorescence microscopy at the boundary of two phases. This phase separation process indicates that lipid molecules, such as sphingolipid and cholesterol, are intermixed. When saturated phospholipid is used instead of sphingolipid, small Lo domains are formed. Cholesterol is harder to incorporate with domains of saturated phospholipid than that of sphingolipid. This technique is very useful for observation of lipid-lipid interactions.

Amino- and carboxyl-terminal domains of Filamin-A interact with CRMP1 to mediate Sema3A signalling.

Reorganization of the actin cytoskeleton is an early cellular response to various extracellular signals. Sema3A, a repulsive axon guidance molecule, induces the reorganization of actin cytoskeleton in the growth cones. Collapsin response mediator protein 1 (CRMP1) mediates the intracellular Sema3A signalling through its Ser522 phosphorylation. Here we show that UNC-33, CRMP1 C. elegans homologue, interacts with FLN-1, an actin-binding Filamin-A orthologue. In nematodes, this interaction participates in the projection of DD/VD motor neurons. CRMP1 binds both the actin-binding domain and the last immunoglobulin-like repeat of Filamin-A. The alanine mutants of Filamin-A or CRMP1 in their interacting residues suppress the Sema3A repulsion in neurons. Conversely, a phosphor-mimicking mutant CRMP1(Ser522Asp) enhances the Sema3A response. Atomic-force microscopy analysis reveals that the V-shaped Filamin-A changes to a condensed form with CRMP1(Ser522Asp). CRMP1(Ser522Asp) weakens the F-actin gelation crosslinked by Filamin-A. Thus, phosphorylated CRMP1 may remove Filamin-A from the actin cytoskeleton to facilitate its remodelling.

Protein adsorption to graphene surfaces controlled by chemical modification of the substrate surfaces.

We have investigated effects of the support substrate surfaces on properties of the attached graphene flakes by observing protein adsorption to the graphene surfaces on SiO2/Si substrates that are modified with self-assembled monolayers to control their hydrophilicity. Using atomic force microscopy operated in aqueous environment, we found that high-density clusters of agglomerated avidin molecules form on the graphene flakes in the areas supported by a hydrophobic substrate surface, whereas very low density of large avidin clusters form at the edge of graphene flakes in the area supported by a hydrophilic surface. These results demonstrate that hydrophilicity of the support surface affects hydrophilicity of the graphene surface also in aqueous environment and that surface modification of the support substrate is a useful technique to control protein adsorption phenomena on graphene surfaces for realization of high sensitive graphene biosensors.

Selective adsorption of protein molecules on phase-separated sapphire surfaces.

Site-selective adsorption of protein molecules was found on sapphire surfaces that exhibit a phase separation into two domains: weakly charged hydrophobic domain and negatively charged hydrophilic one. Ferritin and bovine serum albumin molecules, which are negatively charged in a buffer solution, are adsorbed to the hydrophobic domains. Avidin molecules, which are positively charged, are adsorbed to the other domain. Fibrinogen molecules, which consist of both negative and positive modules, are adsorbed to the whole sapphire surface. Hemoglobin molecules, whose net charge is almost zero, are also adsorbed to the whole surfaces. These results indicate that electrostatic double layer interaction is the primary origin of the observed selectivity. Dependence of protein adsorption or desorption behaviors on the pH value can also be interpreted by the proposed model.

Evolution of supported planar lipid bilayers on step-controlled sapphire surfaces.

Self-organized step/terrace structures on a sapphire surface were used to investigate interface properties between a solid surface and a supported planar lipid bilayer (SPB). We prepared random-stepped, single-stepped and multistepped sapphire surfaces. Some multistepped surfaces covered with crossing steps exhibit phase-separation into hydrophilic and hydrophobic domains. We studied evolution of self-spreading lipid bilayers that are subject to the atomic structures and chemical states on the surfaces. The growth direction of SPBs in the self-spreading method is regulated by the atomic steps. While the SPBs were apparently uniform after a 1 h self-spreading, a density gradient of the lipid molecules was observed even after 24 h spreading. We found that various patterns of the SPBs that depend on the density of the lipid molecules are self-assembled on the phase-separated surfaces. Although the SPB is supported on the sapphire surface via an about 1 nm water layer, the self-spreading direction and the morphology of the SPBs are affected by the atomic steps, whose height is much smaller than that of the water layer.

Recombinant nucleocapsid protein-based IgG enzyme-linked immunosorbent assay for the serological diagnosis of SARS.

The recombinant nucleocapsid protein (rNP) of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) was expressed in a baculovirus system. The purified SARS-CoV rNP was used as an antigen for detection of SARS-CoV antibodies in IgG enzyme-linked immunosorbent assay (ELISA). The ELISA was evaluated in comparison with neutralizing antibody assay and the authentic SARS-CoV antigen-based IgG ELISA. Two-hundred and seventy-six serum samples were collected from health care workers in a hospital in which a nosocomial SARS outbreak took place and used for evaluation. The SARS-CoV rNP-based IgG ELISA has 92% of sensitivity and specificity compared with the neutralizing antibody assay and 94% sensitivity and specificity compared with the authentic SARS-CoV antigen-based IgG ELISA. The results suggest that the newly developed SARS-CoV rNP-based IgG ELISA is a valuable tool for the diagnosis and seroepidemiological study of SARS. The SARS-CoV rNP-based IgG ELISA has an advantage over the conventional IgG ELISA in that the antigen can be prepared by laboratory workers without the risk of infection.

An experimental and theoretical study on the substituent effect of the permanganate oxidation of styrenes.

The kinetic data obtained for the cycloadditions of the permanganate ion to a series of styrene derivatives in dichloromethane solution in the presence of a quaternary ammonium ion were examined with two theoretical approaches, on the assumption that the reactions proceed via a concerted [3 + 2] mechanism. The semi-quantitative frontier molecular orbital analysis of the kinetic data shows a linear free energy relationship with better correlation than the Hammett plot with a values when the point for p-NO2 group is omitted. Further examination of the results of the FMO analysis reveals that the deviation of the point for p-nitrostyrene is attributed to the transition structure being more reactant-like than that of the other derivatives. The plot of log k2 vs. -deltaG++ calculated by the density functional theory (Becke3LYP) follows a straight line with the desired correlation for all the substituents. A marked tendency was observed for the MO calculations to underestimate the deltaG++ value for electron-withdrawing substituents when the calculation was carried out excluding the quaternary ammonium ion. This inconsistency was much improved by the calculations incorporating the quaternary ammonium ion. The actual values of deltaG++ obtained from the Eyring analysis are in good agreement with those calculated at the B3LYP/6-311 +G(d,p)//B3LYP/ LanL2DZ level.

Atomic structures of the Ge/Si(113)-(2 x 2) surface.

Based on scanning tunneling microscopy observations of the epitaxial growth of Ge on Si(113) and first-principles total energy and band calculations, we demonstrate that the Ge/Si(113)-(2 x 2) surface is made up of alternating [1;10]-oriented rows of rebonded atoms and tilted pentamers of five atoms, where each pentamer is stabilized by an interstitial atom at the subsurface. From the existence of stacking defects in rows of tilted pentamers observed at room temperature, we have deduced that at epitaxial temperatures the pentamers frequently change their tilting orientations between two minimum energy states.

Dephosphorylation failure of tyrosine-phosphorylated STAT1 in IFN-stimulated Sendai virus C protein-expressing cells.

Sendai virus C protein (SeV C) has been reported to counteract the antiviral activities of interferons (IFNs) by inhibiting the expression of IFN-stimulated gene products. In SeV C-expressing cells, formation of an active ISGF3 complex and translocation of STAT1 into the nucleus were not observed. STAT1 was continuously phosphorylated at tyrosine 701 by IFN signaling; however, its serine phosphorylation was suppressed. In addition, tyrosine-phosphorylated STAT1 grew to form abnormally huge complexes. These findings suggest that the counteraction of IFN in SeV C-expressing cells is caused by disordered phosphorylation and dephosphorylation of STAT1.

Semiquantitative FMO Analysis of Substituent Effect on the Reaction of Permanganate Ion with Unsymmetrical Alkenes.

The substituent effects on the reactions of permanganate ion with unsymmetrical alkenes are analyzed on the assumption of a concerted (3 + 2) cycloaddition model by using an equation obtained by approximation based on the FMO theory in which development and localization of the frontier molecular orbitals at the reaction sites with progress of the reaction are considered. The Hammett plots are successfully reproduced with the newly obtained rate data for the reactions of trans-chalcone and its derivatives and the data for methyl cinnamates, cinnamate ions, and alkyl vinyl ethers taken from the literature using FMO energies and orbital coefficients calculated by the PM3 method. It was indicated that a factor introduced to the basic equation in order to estimate the extent of localization of the molecular orbitals at the transition state is closely related to the position of the transition state along the reaction path.