In vitro selection of proteins that undergo covalent labeling with small molecules by thiol-disulfide exchange by using ribosome display

There is a great deal of interest in proteins that can bind covalently to target molecules, as they allow unambiguous experiments by tight binding to molecules of interest. Here, we report the generation of proteins that undergo covalent labeling with small molecules through in vitro selection by using ribosome display. Selection was performed from a mutant library of the WW domain with a biotinylated peptide as its binding target, in which the biotin and the peptide are connected by a disulfide bond. After five rounds of selection, we identified mutants carrying a particular cysteine mutation. The binding target reacted specifically with the selected mutant, even in the presence of other proteins, and resulted in the generation of biotin- or peptide-labeled WW domains by thiol-disulfide exchange. When the mutant was fused to a protein of interest, the fusion protein was also labeled with biotin. Thus, the characteristics of the selected mutant should be suitable as a tag sequence that can be covalently labeled with small synthetic molecules. These results indicate that the rapid and efficient generation of such proteins is possible by ribosome display.     

Neuroprotective Effects of Liraglutide for Stroke Model of Rats

The number of diabetes mellitus (DM) patients is increasing, and stroke is deeply associated with DM. Recently, neuroprotective effects of glucagon-like peptide-1 (GLP-1) are reported. In this study, we explored whether liraglutide, a GLP-1 analogue exerts therapeutic effects on a rat stroke model. Wistar rats received occlusion of the middle cerebral artery for 90 min. At one hour after reperfusion, liraglutide or saline was administered intraperitoneally. Modified Bederson’s test was performed at 1 and 24 h and, subsequently, rats were euthanized for histological investigation. Peripheral blood was obtained for measurement of blood glucose level and evaluation of oxidative stress. Brain tissues were collected to evaluate the level of vascular endothelial growth factor (VEGF). The behavioral scores of liraglutide-treated rats were significantly better than those of control rats. Infarct volumes of liraglutide-treated rats at were reduced, compared with those of control rats. The level of derivatives of reactive oxygen metabolite was lower in liraglutide-treated rats. VEGF level of liraglutide-treated rats in the cortex, but not in the striatum significantly increased, compared to that of control rats. In conclusion, this is the first study to demonstrate neuroprotective effects of liraglutide on cerebral ischemia through anti-oxidative effects and VEGF upregulation.     

Development of thick-foil and fine-pitch GEMs with a laser etching technique

We have produced thick-foil and fine-pitch gas electron multipliers (GEMs) using a laser etching technique. To improve production yield we have employed a new material, liquid crystal polymer, instead of polyimide as an insulator layer. The effective gain of the thick-foil GEM with a hole pitch of , a hole diameter of , and a thickness of reached a value of 10⁴ at an applied voltage of 720 V. The measured effective gain of the thick-foil and fine-pitch GEM ( pitch, diameter, and thick) was similar to that of the thick-foil GEM. The gain stability was measured for the thick-foil and fine-pitch GEM, showing no significant increase or decrease as a function of elapsed time from applying the high voltage. The gain stability over 3 h of operation was about 0.5%. Gain mapping across the GEM showed a good uniformity with a standard deviation of about 4%. The distribution of hole diameters across the GEM was homogeneous with a standard deviation of about 3%. There was no clear correlation between the gain and hole diameter maps.     

Association of Ligamentum Flavum Hypertrophy with Adolescent Idiopathic Scoliosis Progression—Comparative Microarray Gene Expression Analysis

The role of the ligamentum flavum (LF) in the pathogenesis of adolescent idiopathic scoliosis (AIS) is not well understood. Using magnetic resonance imaging (MRI), we investigated the degrees of LF hypertrophy in 18 patients without scoliosis and on the convex and concave sides of the apex of the curvature in 22 patients with AIS. Next, gene expression was compared among neutral vertebral LF and LF on the convex and concave sides of the apex of the curvature in patients with AIS. Histological and microarray analyses of the LF were compared among neutral vertebrae (control) and the LF on the apex of the curvatures. The mean area of LF in the without scoliosis, apical concave, and convex with scoliosis groups was 10.5, 13.5, and 20.3 mm², respectively. There were significant differences among the three groups (p < 0.05). Histological analysis showed that the ratio of fibers (Collagen/Elastic) was significantly increased on the convex side compared to the concave side (p < 0.05). Microarray analysis showed that ERC2 and MAFB showed significantly increased gene expression on the convex side compared with those of the concave side and the neutral vertebral LF cells. These genes were significantly associated with increased expression of collagen by LF cells (p < 0.05). LF hypertrophy was identified in scoliosis patients, and the convex side was significantly more hypertrophic than that of the concave side. ERC2 and MAFB genes were associated with LF hypertrophy in patients with AIS. These phenomena are likely to be associated with the progression of scoliosis.     

Flow dynamics in Taylor–Couette flow reactor with axial distribution of temperature

In this study, the flow dynamics of a Taylor–Couette flow with an axial distribution of temperature was experimentally investigated. The flow can be classified into three patterns based on the balance between the centrifugal force and the buoyancy. If the buoyancy is dominant, global heat convection is observed instead of Taylor vortices (Case I). When the buoyancy is comparable to the centrifugal force, the Taylor vortices and global heat convection appear alternately (Case II). If the centrifugal force is sufficiently high to suppress the buoyancy, stable Taylor vortices are observed (Case III). The characteristics of the mixing/diffusion are investigated by conducting a decolorization experiment on a passive tracer. In Case II, the tracer is rapidly decolorized in the presence of the global heat convection instead of the Taylor vortices. This result implies that the interaction between the centrifugal force and the buoyancy would induce an anomalous transport. © 2017 American Institute of Chemical Engineers AIChE J, 64: 1075–1082, 2018     

Effective cache replacement policy for packet processing cache

Owing to the explosive increase in Internet traffic, increasing the throughput and energy efficiency of core routers is an important issue for future internet. Accessing the ternary content addressable memory (TCAM) in a core router can significantly degrade its throughput and energy efficiency owing to its low operating frequency and power consuming operations. To improve the throughput and energy‐efficiency of core routers, packet processing caches (PPCs) can be potentially used. PPCs can reduce the number of TCAM accesses by storing the TCAM lookup results in a fast and low‐energy cache memory and reusing them to process subsequent packets. To enhance the PPC performance, it is important to reduce the number of PPC misses. However, the PPC miss rate of the state‐of‐the‐art PPC is still high owing to its small capacity. In this study, we propose an effective cache replacement policy, called elevator cache (ELC) to improve the PPC miss rate. Our simulation results showed that the 8‐way ELC, which can be implementable at low hardware cost, reduced the number of PPC misses by 17.1% and 5.67% in comparison to the 4‐way least recently used (LRU) and the 4‐way ELC, respectively. Consequently, it was found that the 8‐way ELC can achieve a 1.21 times higher throughput (560 Gbps) while consuming 83% energy in comparison to conventional PPCs.     

Development of lithium vapor injector for boundary control

A lithium (Li) vapor injector for boundary control has been developed. A diverter covered with lithium is expected to reduce particle recycling. Recycling reduction is considered to be one of the triggers for the L-H transition. In this paper, the method of lithium dispersion is investigated under the assumption that the experiment is carried out in the Large Helical Device in National Institute for fusion Science, Japan (LHD). A performance test is performed on a prototype of the vapor injector. The amount of injected lithium was approximately 1% of the value expected from the vapor pressure data, due to the generation of lithium oxide. It is also found that nozzle temperature is quite important to suppress the Li dispersion.     

Prototype of Phase-Change Channel Transistor for Both Nonvolatile Memory and Current Control

We prototyped phase-change (PC) channel transistors and demonstrated two functions of nonvolatile memory and channel current control. We have developed prototype transistors that use a PC channel instead of a silicon channel. The PC material of a Ge₂Sb₂Te₅ thin film with a thickness of 50 nm was used. We demonstrated a memory function whereby we achieved a reversible change between the crystalline and amorphous phases by applying a source-drain (SD) voltage for Joule heating. In the experiment, the applied voltages for PC between amorphous and crystalline phases were from 5 to 8 V. Control of the channel current was realized by applying a gate bias. The SD current was suppressed to less than 1/20 of that at a gate bias of -3 V by applying a gate bias of 0-3 V     

Cell Profiling Based on Sugar-Chain–Cell Binding Interaction and Its Application to Typing and Quality Verification of Cells

Developing methods to determine cell type and cell state has been a significant challenge in the field of cancer diagnosis as well as in typing and quality verification for cultured cells. Herein, we report a cell profiling method based on binding interactions between cell-surface sugar-chain-binding proteins and sugar-chain-immobilized fluorescent nanoparticles (SFNPs), together with a method for cell typing and cell quality verification. Binding profiles of cells against sugar chains were analyzed by performing flow cytometry analysis with SFNPs. Discrimination analysis based on binding profiles could classify cell type and evaluate the quality of cultured cells. By applying our method to differentiated cells originating from conventional cell lines and also to mouse embryotic stem cells, we could detect the cells before and after differentiation. Our method can be utilized not only for the biofunctional analysis of cells but also for diagnosis of cancer cells and quality verification of cultured cells.