High-throughput analysis of ligand-induced internalization of β2-adrenoceptors using the coiled-coil tag-probe method

Receptor internalization is a useful indicator of the activity of ligands. The N-terminus of the β(2)-adrenergic receptor expressed on the cell surface was labeled with fluorophores using a novel coiled-coil labeling system. Endocytosis of the receptors was automatically detected using a fluorescence image analyzer by evaluating (1) translocation of the receptor from cell-surface to intracellular regions and (2) acidification in endosomes. Both parameters increased upon agonist stimulation in a dose-dependent manner. The extent of endocytosis was significantly dependent on the agonist used, indicating the presence of a biased signaling for endocytosis. The receptor antagonists can also be screened by competitive inhibition of agonist-induced endocytosis. The image analysis approach has proven to be useful for high-throughput characterization and screening of GPCR ligands.     

Quantification of SOFC anode microstructure based on dual beam FIB-SEM technique

The three-dimensional microstructure of an SOFC anode is quantified using a dual beam focused ion beam scanning electron microscopy (FIB-SEM) system equipped with an energy dispersive X-ray spectroscopy (EDX) unit. The microstructure of the Ni-YSZ anode is virtually reconstructed in a computational field using a series of acquired two-dimensional SEM images. The three-phase boundary (TPB) density and tortuosity factors are carefully evaluated by applying two different evaluation methods to each parameter. The TPB density is estimated by a volume expansion method and a centroid method, while the tortuosity factors are evaluated by a random walk calculation and a lattice Boltzmann method (LBM). Estimates of each parameter obtained by the two methods are in good agreement with each other, thereby validating the reliability of the analysis methods proposed in this study.     

Beta tricalcium phosphate ceramics with controlled crystal orientation fabricated by application of external magnetic field during the slip casting process

Beta tricalcium phosphate (β-TCP) is a resorbable bioceramic that has hitherto been utilized in the medical field. Since it crystallizes in the anisotropic hexagonal system, properties such as chemical and physical ones are expected to depend on its crystal axis direction and/or on its crystal plane (anisotropy). Control of crystal orientation is thus important when used in polycrystalline form. Meanwhile, application of a strong magnetic field has been found to be a promising technique to control crystal orientation of anisotropic shape or structured crystals. In this work, we attempted to fabricate β-TCP ceramics with controlled crystal orientation by applying an external magnetic field during the slip casting process and subsequently sintering them at 1050 °C, below the β–α transition temperature. Application of a vertical magnetic field increased intensities of planes perpendicular to c-plane on the top surface, while a horizontal one with simultaneous mechanical mold rotation decreased it. These results indicated that crystal orientation of β-TCP ceramics were successfully controlled by the external magnetic field and together that the magnetic susceptibility of β-TCP is χ_c⊥ > χ_c//.     

Combined effects of hydrostatic pressure, temperature, and the addition of allyl isothiocyanate on inactivation of Escherichia coli

Combined effects of hydrostatic pressure, temperature, and the addition of allyl isothiocyanate (AIT) on the inactivation of Escherichia coli, including type O157, were investigated. Inactivation to undetectable levels by hydrostatic pressure alone requires 400 to 600 MPa. E. coli growth was delayed with increasing of applied pressure and the AIT concentration added subsequently. The antibacterial effects of AIT vapor increased on JCM 1649 and IFO 3301 after pressurization. The bactericidal effects of pressurization with the addition of AIT at 4 degrees C or 40 degrees C were greater than at 20 degrees C, and all bacteria tested were effectively killed at 200 or 250 MPa with 10 to 80 microg/ml of AIT. We tried to apply the combined treatment to a food product "Asazuke" (low salt vegetables), and it was confirmed that E. coli inoculated into the product was inactivated the same as in the in vitro test. We also studied the inactivation mechanism behind pressurization with AIT from the relationship between pressure resistance and precultivation temperature, and it was suggested that destruction of membrane structure caused bacterial kill.     

A Unique Amino Transfer Mechanism for Constructing the β‐Amino Fatty Acid Starter Unit in the Biosynthesis of the Macrolactam Antibiotic Cremimycin

Cremimycin is a 19‐membered macrolactam glycoside antibiotic based on three distinctive substructures: 1) a β‐amino fatty acid starter moiety, 2) a bicyclic macrolactam ring, and 3) a cymarose unit. To elucidate the biosynthetic machineries responsible for these three structures, the cremimycin biosynthetic gene cluster was identified. The cmi gene cluster consists of 33 open reading frames encoding eight polyketide synthases, six deoxysugar biosynthetic enzymes, and a characteristic group of five β‐amino‐acid‐transfer enzymes. Involvement of the gene cluster in cremimycin production was confirmed by a gene knockout experiment. Further, a feeding experiment demonstrated that 3‐aminononanoate is a direct precursor of cremimycin. Two characteristic enzymes of the cremimycin‐type biosynthesis were functionally characterized in vitro. The results showed that a putative thioesterase homologue, CmiS1, catalyzes the Michael addition of glycine to the β‐position of a non‐2‐enoic acid thioester, followed by hydrolysis of the thioester to give N‐carboxymethyl‐3‐aminononanoate. Subsequently, the resultant amino acid was oxidized by a putative FAD‐dependent glycine oxidase homologue, CmiS2, to produce 3‐aminononanoate and glyoxylate. This represents a unique amino transfer mechanism for β‐amino acid biosynthesis.     

Multipoint cure monitoring of CFRP laminates using a flexible matrix sensor

Since a carbon fiber reinforced plastic (CFRP) structure is complicated in the adoption of integral molding, local molding faults such as under curing and dry spots are liable to occur. To solve this problem, the distribution of the degree of curing for the entire composite structure must be measured. In the present study, we propose a patch-type flexible matrix sensor based on permittivity measurements. Multiple electrodes and wirings are readily fabricated simultaneously using a photolithographic process. Moreover, the sensor has only m + n wirings for m × n sensors, and is thereby suitable for multipoint cure monitoring. We also constructed a method for estimating the degree of curing considering the effect of frequency dependence of the permittivity of resin and viscosity variation due to temperature change. Experiments of multipoint cure monitoring are carried out using a CFRP plate and an actual aircraft structure. As a result, we confirmed the effectiveness of this method by comparing with results using a conventional differential scanning calorimeter.     

Development and Evaluation of a Robust Sandwich Immunoassay System Detecting Serum WFA-Reactive IgA1 for Diagnosis of IgA Nephropathy

Aberrant glycosylation of IgA1 is involved in the development of IgA nephropathy (IgAN). There are many reports of IgAN markers focusing on the glycoform of IgA1. None have been clinically applied as a routine test. In this study, we established an automated sandwich immunoassay system for detecting aberrant glycosylated IgA1, using Wisteria floribunda agglutinin (WFA) and anti-IgA1 monoclonal antibody. The diagnostic performance as an IgAN marker was evaluated. The usefulness of WFA for immunoassays was investigated by lectin microarray. A reliable standard for quantitative immunoassay measurements was designed by modifying a purified IgA1 substrate. A validation study using multiple serum specimens was performed using the established WFA-antibody sandwich automated immunoassay. Lectin microarray results showed that WFA specifically recognized N-glycans of agglutinated IgA1 in IgAN patients. The constructed IgA1 standard exhibited a wide dynamic range and high reactivity. In the validation study, serum WFA-reactive IgA1 (WFA+-IgA1) differed significantly between healthy control subjects and IgAN patients. The findings indicate that WFA is a suitable lectin that specifically targets abnormal agglutinated IgA1 in serum. We also describe an automated immunoassay system for detecting WFA+-IgA1, focusing on N-glycans.     

Effect of catch cup geometry on 3D boundary layer flow over the wafer surface in a spin coating

Recently, development of high technology has been required for the formation of thin uniform film in manufacturing processes of semiconductor as the semiconductor instruments become more sophisticated. Spin coating is usually used for spreading photoresist on a wafer surface. However, since rotating speed of the disk is very high in spin coating, the dropped photoresist scarers outward and reattaches on the film surface. A catch cup is set up outside the wafer in spin coating, and scattered photoresist mist is removed from the wafer edge by the exhaust flow generated at the gap between the wafer edge and the catch cup. In the dry process of a spin coating, it is a serious concern that the film thickness increases near the wafer edge in the case of low rotating speed. The purpose of this study is to make clear the effect of the catch cup geometry on the 3D boundary layer flow over the wafer surface and the drying rate of liquid film.     

Numerical study on particle motions in swirling flows in a cyclone separator

The purpose of this study is to establish the high-accurate prediction method of particle separation in a cyclone separator. Numerical simulation of the swirling flows in a cyclone separator is performed by using a large eddy simulation (LES) based on a Smagorinsky model. The validity of the simulation and the complicated flow characteristics are discussed by comparison with experimental results. Moreover, particle motions are treated by a Lagrangian method and are calculated with a one-way method. A performance for particle separation is predicted from the results of the particle tracing. As results of our investigation, the influences of the inserted height of the outlet pipe on the performance for particle separation of cyclone separator are shown.