Conformational Analysis of Misfolded Protein Aggregation by FRET and Live-Cell Imaging Techniques

Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.     

Thermoresponsive core–shell nanoparticles with cross-linked π-conjugate core based on amphiphilic block copolymers by RAFT polymerization and palladium-catalyzed coupling reactions

Well-defined amphiphilic block copolymers composed of S-vinyl sulfides and N-isopropyl acrylamide (NIPAM) were synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization. Thermoresponsive core–shell nanoparticles with cross-linked π-conjugate cores were obtained by in situ cross-linking reactions between 4-bromophenyl moieties in the block copolymers and diboronic acids or a diamine compound in the presence of a palladium catalyst following micelle formation in ethanol/H₂O or ethanol. We initially investigated RAFT polymerization of two S-vinyl sulfide derivatives, namely phenyl vinyl sulfide (PVS) and 4-bromophenyl vinyl sulfide (BPVS), using a dithiocarbamate-type chain transfer agent (CTA). Then, RAFT polymerization of NIPAM using poly(S-vinyl sulfide) macro-CTAs was conducted to synthesize the amphiphilic block copolymers. Suzuki and Buchwald-Hartwig coupling reactions were found to be effective in the preparation of core–shell nanoparticles with thermoresponsive shells and cross-linked optoelectronic cores. The resulting nanoparticles showed characteristic thermoresponsive properties, as confirmed by turbidity and dynamic light scattering measurements. Stable and uniform core cross-linked nanoparticles were successfully prepared by the in situ palladium-catalyzed coupling reactions, and the optoelectronic and thermoresponsive properties of the nanoparticles could be tuned depending on the nature of the difunctional coupling agents, reaction conditions, and comonomer composition of the block copolymers.     

Electrodeposition of platinum and silver into chemically-modified microporous silicon electrodes

ABSTRACT: Electrodeposition of platinum and silver into hydrophobic and hydrophilic microporous silicon layers was investigated using chemically-modified microporous silicon electrodes. Hydrophobic microporous silicon enhanced the electrodeposition of platinum in the porous layer. Meanwhile hydrophilic one showed that platinum was hardly deposited within the porous layer and a filmy growth of platinum on the top of the porous layer was observed. On the other hand, the electrodeposition of silver showed similar deposition behavior between these two chemically-modified electrodes. It was also found that the electrodeposition of silver started at the pore opening and grew toward the pore bottom, while a uniform deposition from the pore bottom was observed in platinum electrodeposition. These electrodeposition behaviors are explained on the basis of the both effect, the difference in overpotential for metal deposition on silicon and on the deposited metal, and displacement deposition rate of metal.     

Effects of Insert Metal Type on Interfacial Microstructure During Dissimilar Joining of TiAl Alloy to SCM440 by Friction Welding

Although the welding zone of direct bonding between a TiAl alloy and SCM440 can be obtained by friction welding, martensitic transformation and the formation of intermetallic compounds (IMCs) and cracks result in a lower tensile strength of the joints relative to those of other welding techniques. Insert metals were used as a buffer layer to relieve stress while increasing the bond strength. In this study, the microstructure and mechanical properties on welded joints of a TiAl alloy and SCM440 with various insert metals, were investigated. The TiAl/Cu/SCM440 and TiAl/Ni/SCM440 joints were fabricated using a servo-motor-type friction welding machine. As a result, it was confirmed that the formation of a welding flash was dependent on the insert metal type, and the strength of the base metal. At the TiAl/Cu/SCM440 interface, the formation of IMCs CuTiAl and Cu₂TiAl was observed at TiAl/Cu, while no IMC formation was observed at Cu/SCM440. On the other hand, at the TiAl/Ni/SCM440 interface, several IMCs with more than 100 μm thickness were found, and roughly two compositions, viz., Ti₂NiAl₃ and TiNi₂Al, were observed at the TiAl/Ni interface. At the Ni/SCM440 interface, 10 μm-thick FeNi and others were found.     

Structure Optimization of the Toxic Conformation Model of Amyloid β42 by Intramolecular Disulfide Bond Formation

Amyloid β (Aβ) oligomers play a critical role in the pathology of Alzheimer's disease. Recently, we reported that a conformation-restricted Aβ42 with an intramolecular disulfide bond through cysteine residues at positions 17/28 formed stable oligomers with potent cytotoxicity. To further optimize this compound as a toxic conformer model, we synthesized three analogues with a combination of cysteine and homocysteine at positions 17/28. The analogues with Cys-Cys, Cys-homoCys, or homoCys-Cys, but not the homoCys-homoCys analogue, exhibited potent cytotoxicity against SH-SY5Y and THP-1 cells even at 10 nM. In contrast, the cytotoxicity of conformation-restricted analogues at positions 16/29 or 18/27 was significantly weaker than that of wild-type Aβ42. Furthermore, thioflavin-T assay, non-denaturing gel electrophoresis, and morphological studies suggested that the majority of these conformation-restricted analogues exists in an oligomeric state in cell culture medium, indicating that the toxic conformation of Aβ42, rather than the oligomeric state, is essential to induce cytotoxicity.     

Influence of trimethylsilylation and detrimethylsilylation on the molecular weight of chitin: Evaluation of viscometry and gel permeation chromatography for molecular weight determination

In view of the high potential of trimethylsilylated chitin as a reaction precursor, the influence of trimethylsilylation and detrimethylsilylation on the molecular weight characteristics has been studied. Chitin was trimethylsilylated, and the product was detrimethylsilylated to regenerate chitin. The molecular weights of the original and regenerated chitins were determined by viscometry and GPC. The viscosity measurements, with either an Ubbelohde or a rotational viscometer, gave highly reproducible values, which were quite similar to each other. The molecular weight of the regenerated chitin was found to be a little over a half that of the original chitin despite after the silylation-desilylation reactions. GPC also supported a similarly low extent of main chain scission. These results indicate that the silylation-desilylation reactions are reproducible without a significant damage to the chitin main chain, and both viscometry and GPC have proved reliable in elucidating the molecular weight characteristics. Trimethylsilylation of chitin can thus be conveniently used as a key intermediate for further controlled modification reactions to prepare well-defined derivatives.     

Identification of Physiological and Toxic Conformations in Aβ42 Aggregates

Aggregation of the 42‐residue amyloid β‐protein (Aβ42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Despite numerous structural studies on Aβ aggregates, the relationship between tertiary structure and toxicity remains unclear. Our proline scanning and solid‐state NMR studies suggested that aggregates both of wild‐type Aβ42 and of E22K‐Aβ42 (one of the mutants related to cerebral amyloid angiopathy) contain two conformers: a major one with a turn at positions 25 and 26, and a minor one with a turn at positions 22 and 23. To identify the toxic conformer, the derivative Aβ42‐lactam(22K–23E), in which the side chains at positions 22 and 23 were covalently linked, was synthesized as a minor conformer surrogate, along with Aβ42‐lactam(25K–26E) as a major conformer surrogate. The Aβ42‐lactam(22K–23E) showed stronger aggregation, neurotoxicity, radical generation, and oligomerization than wild‐type Aβ42, whereas in Aβ42‐lactam(25K–26E) were weak. The transition from the physiological conformation with a turn at positions 25 and 26 to the toxic conformation with a turn at positions 22 and 23 might be a key event in the pathogenesis of AD.     

气体温度对冷喷涂7075铝合金涂层性能的影响

目的研究气体温度对冷喷涂7075铝合金涂层性能的影响,以期为制备高性能涂层提供重要参考。方法利用冷喷涂技术,在较高的工作气体压力(5 MPa)下,通过改变工作气体温度(450、500、550、600℃),采用氮气在纯铝基体上制备了大厚度7075铝合金涂层。采用光学显微镜(OM)、扫描电子显微镜(SEM)对涂层组织结构及孔隙率进行了分析测定,用维氏显微硬度法测定涂层硬度,通过拉伸实验测定涂层结合强度,研究了冷喷涂工作气体温度对7075铝合金涂层组织及沉积特性的影响。结果工作气体温度在450～550℃范围时,对涂层组织及力学性能影响有限。当工作气体温度为450℃时,涂层孔隙率为0.14%,显微硬度为117.8HV0.1,结合强度超过55.3 MPa。但过高的工作气体温度并不利于冷喷涂7075铝合金涂层的制备,当喷涂气体温度达到600℃时,涂层孔隙率升高到4.2%。结论仅采用廉价的氮气即可制备高致密度和高结合强度的冷喷涂7075涂层,当主气温度在450～550℃区间时,涂层微观组织和性能随气体温度变化不大,而过高的工作气体温度(600℃)并不利于冷喷涂7075涂层相关性能的提升。     

Motion Control of Caster-Type Passive Mobile Robot with Servo Brakes

In this paper, we introduce a passive mobile robot called prototype Caster-Type Passive Robot Porter (C-PRP), which is developed on the basis of a concept of passive robotics. This mobile robot consists of two casters with servo brakes and one passive rigid wheel. Prototype C-PRP has passive dynamics with respect to the force applied by a human and controls its appropriate motion with the servo brakes. We derive the feasible braking force/moment applied to the robot on the basis of the characteristics of the servo brakes. This paper especially focuses on a fundamental motion control algorithm based on the feasible braking force/moment. We realize the path tracking function and the collision avoidance function as examples by applying the proposed algorithm to prototype C-PRP. These functions are implemented to prototype C-PRP actually, and experimental results confirm its validity.