Comparison of isolation methods using commercially available kits for obtaining extracellular vesicles from cow milk

Extracellular vesicles (EV) are important for delivering biologically active substances to facilitate cell-to-cell communication. Milk-derived EV are widely known because of their potential for immune enhancement. However, procedures for isolating milk-derived EV have not been fully established. To obtain pure milk-derived EV and accurately reveal their function, such procedures must be established. The aim of the present study was to compare methods using commercially available kits for isolating milk-derived EV. Initially, we investigated procedures to remove casein, which is the major obstacle in determining milk-derived EV purity. We separated whey using centrifugation only, acetic acid precipitation, and EDTA precipitation. Then, we isolated milk-derived EV by ultracentrifugation, membrane affinity column, size exclusion chromatography (SEC), polymer-based isolation, or phosphatidylserine-affinity isolation. Using EV count per milligram of protein, which is a good indicator of purity, we determined that acetic acid precipitation was the best method for removing casein. Using nanoparticle tracking analysis, protein quantity analysis, and RNA quantity analysis, we comprehensively compared each isolation method for its purity and yield. We found that SEC-based qEV column (Izon Science) could collect purer milk-derived EV at higher quantities. Thus, a combination of acetic acid precipitation and qEV can effectively isolate high amounts of pure extracellular vesicles from bovine milk.     

Dynamic Power Integrity Control of ATE for Eliminating Overkills and Underkills in Device Testing

This paper proposes a power integrity control technique for dynamically controlling power supply voltage fluctuations for a device under test (DUT), and demonstrates its effectiveness for eliminating the overkills/underkills due to the difference of power supply impedance between an automatic test equipment (ATE) and a practical operating environment of the DUT. The proposed method injects compensation currents into the power supply nodes on the ATE system in a feed-forward manner such that the ATE power supply waveform matches with the one on the customer’s operating environment of the DUT. A method for calculating the compensation current is also described. Experimental results show that the proposed method can emulate the power supply voltage waveform under a customer’s operating condition and eliminate 95 % of overkills/underkills in the maximum operating frequency testing with 105 real silicon devices. Limitations and applications of the proposed method are also discussed.     

Route guidance by a car navigation system based on augmented reality

Car navigation systems play a prominent role in road traffic safety and traffic regulation. However, it is necessary to improve the route guidance of car navigation systems so that they accurately and quickly recognize small differences in location. Thus, to increase the ease of understanding and safety of car navigation systems, navigation systems based on augmented reality have been proposed for providing guidance at road intersections. We are currently developing a car navigation system based on augmented reality, called AR‐Navi. We investigated designs for the display of road intersection guidance that can be easily understood even when limited information is available and proposed a “best shot” display method that does not use moving images. In addition, we implemented a prototype system that includes these methods and conducted driving experiments on public roads to evaluate the ease of understanding and safety of AR‐Navi. Using the evaluation results, we confirmed that the ease of understanding and safety is similar in the case of AR‐Navi and CG‐Navi. We also clarified the characteristics of AR‐Navi. © 2012 Wiley Periodicals, Inc. Electr Eng Jpn, 180(2): 43–54, 2012; Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/eej.22278     

Augmented reality-based block piling game with superimposed collapse prediction

Understanding what cannot be seen is difficult. Physical behavior can be explained on the basis of physical theories even if the behavior cannot be observed. Explanation of what is physically happening in the real world would become easy, however, if annotations were superimposed on the real objects. Herein, the authors demonstrate how an understanding of a physical event can be facilitated by overlapping a real-world situation with a simulation that predicts a future state. This idea is demonstrated in a game application in which a player stacks blocks into a pile until it collapses. In general, it is easy to estimate whether a block on the edge of a table will fall or not. However, it is more difficult to predict whether a stack of many blocks will collapse, and in what manner the stack will collapse. Even though previous research has demonstrated that the problem of how two-dimensionally stacked blocks collapse can be reduced to solving a sequence of convex quadratic programs, algorithms for convex quadratic programs require massive computational resources. Hence, the authors developed a fast and new algorithm based on a linear program. The proposed algorithm realizes real-time simulation based on physics that superimposes predicted collapse. The block that is predicted to fall is superimposed on the real block with a lit background projection. The system was evaluated in an experiment, and superimposed augmented reality annotation was observed to be efficient. The system was also demonstrated in game contests and received positive feedback and comments.     

Nanoporous structure of the cell walls of polycarbonate foams

Using CO₂ to prepare microcellular polycarbonate foams resulted in a pore diameter of 45 nm and a pore density of 10⁸ cm^?2 on the walls of microscale cells, which created nano/micro foams with an open cell structure. In this study, the craze nucleation theory and the bubble nucleation theory of foaming were combined to explain the mechanism of the foaming-induced nanopores (microvoids) on the cell walls. In the foaming process, the strain energy was developed in the cell walls by bubble nucleation and growth. With large strain energy, a nanoporous structure of the cell walls was formed by initiation of crazing. Because the foaming temperature affected the strain energy in the cell wall, the temperature became a key factor of forming microcellular structure as well as the nanopores on the cell walls. Our experimental results showed that the diameter and density of the nanopores were determined by the competitive movements between chain stretching and relaxation. Furthermore, certain solvents, such as acetone, were found to increase the nanopore density of the walls by exploiting the plasticization effect of the solvent on the reduction of surface tension and viscosity.     

Preparation of (110)-oriented SrTiO3 film on quartz glass by laser chemical vapor deposition

SrTiO₃ (STO) film was prepared on quartz glass by laser chemical vapor deposition at a deposition temperature (T_dep) ranged from 760 to 1104 K. Effect of the T_dep on the orientation, crystallinity, texture, and microstructure of the STO film was investigated. As the T_dep was increased, the preferred orientation of the STO film tended to be (110)-orientated with corresponding texture coefficient (TC) on the (110) reflection enhanced from 2.3 to 6; meanwhile, the full width at half maximum of the ω-scan on the (110) reflection decreased from 0.85° to 0.59°. The (110)-oriented grains were in wedge shape about 60 × 150 nm in size, which tended to be flat at an elevated T_dep of 1104 K.     

Isolation and Characterization of Two Types of Xyloglucanases from a Phytopathogenic Fungus,Verticillium dahliae

Xyloglucan is a major hemicellulosic component in plant cell walls. Phytopathogenic fungi secrete cell wall-degrading enzymes on their infection to hosts, while the nature of the cell wall-lytic enzymes of such fungi are yet to be fully understood. Verticillium dahliae is a soil-borne fungus that causes vascular wilt diseases in a variety of commercially important crops worldwide. We purified two types of xyloglucanases, XEG12A and XEG74B, from the culture of naturally isolated Verticillium dahliae strain 2148. XEG12A showed a molecular size of 23 kDa with its maximal activity at pH 7.5. XEG12A specifically hydrolyzed xyloglucan with no activity on other β-glucans. XEG74B had a molecular size of 110 kDa with its optimum pH at 6.0. XEG74B primarily hydrolyzed xyloglucan, with a slight activity on β-1,3-1,4-glucan. Analysis of hydrolytic products of xyloglucanooligasaccharide (XXXGXXXG) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the both enzymes cleaved β-1,4-glucosidic linkage at the position of unbranched chain, while XEG74B showed a little fluctuation with the cleavage site. Both enzymes did not hydrolyzed xyloglucanoheptasaccharide (XXXG) at all. N-Terminal and internal amino acid sequencing of the enzymes revealed that XEG12A and XEG74B belonged to Glycoside Hydrolase (GH) Families 12 and 74, respectively. Based on these results we concluded that V. dahliae XEG12A and XEG74B were xyloglucan-specific endo-β-1,4-glucanases (EC 3.2.1.151).