Potent Activation of Human but Not Mouse TRPA1 by JT010

Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1), which is involved in inflammatory pain sensation, is activated by endogenous factors, such as intracellular Zn²⁺ and hydrogen peroxide, and by irritant chemical compounds. The synthetic compound JT010 potently and selectively activates human TRPA1 (hTRPA1) among the TRPs. Therefore, JT010 is a useful tool for analyzing TRPA1 functions in biological systems. Here, we show that JT010 is a potent activator of hTRPA1, but not mouse TRPA1 (mTRPA1) in human embryonic kidney (HEK) cells expressing hTRPA1 and mTRPA1. Application of 0.3–100 nM of JT010 to HEK cells with hTRPA1 induced large Ca²⁺ responses. However, in HEK cells with mTRPA1, the response was small. In contrast, both TRPA1s were effectively activated by allyl isothiocyanate (AITC) at 10–100 μM. Similar selective activation of hTRPA1 by JT010 was observed in electrophysiological experiments. Additionally, JT010 activated TRPA1 in human fibroblast-like synoviocytes with inflammation, but not TRPA1 in mouse dorsal root ganglion cells. As cysteine at 621 (C621) of hTRPA1, a critical cysteine for interaction with JT010, is conserved in mTRPA1, we applied JT010 to HEK cells with mutations in mTRPA1, where the different residue of mTRPA1 with tyrosine at 60 (Y60), with histidine at 1023 (H1023), and with asparagine at 1027 (N1027) were substituted with cysteine in hTRPA1. However, these mutants showed low sensitivity to JT010. In contrast, the mutation of hTRPA1 at position 669 from phenylalanine to methionine (F669M), comprising methionine at 670 in mTRPA1 (M670), significantly reduced the response to JT010. Moreover, the double mutant at S669 and M670 of mTRPA1 to S669E and M670F, respectively, induced slight but substantial sensitivity to 30 and 100 nM JT010. Taken together, our findings demonstrate that JT010 potently and selectively activates hTRPA1 but not mTRPA1.     

Visualizing Intramolecular Dynamics of Membrane Proteins

Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.     

Super-vector coding features extracted from both depth buffer and view-normal-angle images for part-based 3D shape retrieval

We have witnessed 3D shape models abundant in many application fields including 3D CAD/CAM, augmented/mixed reality (AR/MR), and entertainment. Creating 3D shape models from scratch is still very expensive. Efficient and accurate methods for shape retrieval is essential for 3D shape models to be reused. To retrieve similar 3D shape models, one must provide an arbitrary 3D shape as a query. Most of the research on 3D shape retrieval has been conducted with a “whole” shape as a query (aka whole-to-whole shape retrieval), while a “part” shape (aka part-to-whole shape retrieval) is more practically requested as a query especially by mechanical engineering with 3D CAD/CAM applications. A “part” shape is naturally constructed by a 3D range scanner as an input device. In this paper, we focus on the efficient method for part-to-whole shape retrieval where the “part” shape is assumed to be given by a 3D range scanner. Specifically, we propose a Super-Vector coding feature with SURF local features extracted from the View-Normal-Angle image, or the image synthesized by taking account of the angle between the view vector and the surface normal vector, together with the depth-buffered image, for part-to-whole shape retrieval. In addition, we propose a weighted whole-to-whole re-ranking method taking advantage of global information based on the result of part-to-whole shape retrieval. Through experiments we demonstrate that our proposed method outperforms the previous methods with or without re-ranking.     

Psychological Stress Exacerbates Inflammation of the Ileum via the Corticotropin-Releasing Hormone-Mast Cell Axis in a Mouse Model of Eosinophilic Enteritis

The effects of psychological stress on eosinophilic gastrointestinal disorders have not been elucidated. This study investigated the effects of psychological stress in a mouse model of eosinophilic enteritis (EoN). BALB/c mice were treated with ovalbumin (OVA) to create an EoN model and subjected to either water avoidance stress (WAS) or sham stress (SS). Microscopic inflammation, eosinophil and mast cell counts, mRNA expression, and protein levels of type 2 helper T cell (Th2) cytokines in the ileum were compared between groups. We evaluated ex vivo intestinal permeability using an Ussing chamber. A corticotropin-releasing hormone type 1 receptor (CRH-R1) antagonist was administered before WAS, and its effects were analyzed. WAS significantly increased diarrhea occurrence and, eosinophil and mast cell counts, and decreased the villus/crypt ratio compared to those in the SS group. The mRNA expression of CRH, interleukin IL-4, IL-5, IL-13, eotaxin-1, and mast cell tryptase β2 significantly increased, and the protein levels of IL-5, IL-13, and OVA-specific immunoglobulin E (IgE) also significantly increased in the WAS group. Moreover, WAS significantly increased the intestinal permeability. The CRH-R1 antagonist significantly inhibited all changes induced by WAS. Psychological stress exacerbated ileal inflammation via the CRH-mast cell axis in an EoN mouse model.     

Bile Acid–Drug Interaction via Organic Anion-Transporting Polypeptide 4C1 Is a Potential Mechanism of Altered Pharmacokinetics of Renally Excreted Drugs

Patients with liver diseases not only experience the adverse effects of liver-metabolized drugs, but also the unexpected adverse effects of renally excreted drugs. Bile acids alter the expression of renal drug transporters, however, the direct effects of bile acids on drug transport remain unknown. Renal drug transporter organic anion-transporting polypeptide 4C1 (OATP4C1) was reported to be inhibited by chenodeoxycholic acid. Therefore, we predicted that the inhibition of OATP4C1-mediated transport by bile acids might be a potential mechanism for the altered pharmacokinetics of renally excreted drugs. We screened 45 types of bile acids and calculated the IC₅₀, K_i values, and bile acid–drug interaction (BDI) indices of bile acids whose inhibitory effect on OATP4C1 was >50%. From the screening results, lithocholic acid (LCA), glycine-conjugated lithocholic acid (GLCA), and taurine-conjugated lithocholic acid (TLCA) were newly identified as inhibitors of OATP4C1. Since the BDI index of LCA was 0.278, LCA is likely to inhibit OATP4C1-mediated transport in clinical settings. Our findings suggest that dose adjustment of renally excreted drugs may be required in patients with renal failure as well as in patients with hepatic failure. We believe that our findings provide essential information for drug development and safe drug treatment in clinics.     

Bone Tissue Engineering in Rat Calvarial Defects Using Induced Bone-like Tissue by rhBMPs from Immature Muscular Tissues In Vitro

This study aimed to induce bone-like tissue from immature muscular tissue (IMT) in vitro using commercially available recombinant human bone morphogenetic protein (rhBMP)-2, rhBMP-4, and rhBMP-7, and then implanting this tissue into a calvarial defect in rats to assess healing. IMTs were extracted from 20-day-old Sprague-Dawley (SD) fetal rats, placed on expanded polytetrafluoroethylene (ePTFE) with 10 ng/μL each of rhBMP-2, BMP-4, and BMP-7, and cultured for two weeks. The specimens were implanted into calvarial defects in 3-week-old SD rats for up to three weeks. Relatively strong radiopacity was observed on micro-CT two weeks after culture, and bone-like tissue, comprising osteoblastic cells and osteoids, was partially observed by H&E staining. Calcium, phosphorus, and oxygen were detected in the extracellular matrix using an electron probe micro analyzer, and X-ray diffraction patterns and Fourier transform infrared spectroscopy spectra of the specimen were found to have typical apatite crystal peaks and spectra, respectively. Furthermore, partial strong radiopacity and ossification were confirmed one week after implantation, and a dominant novel bone was observed after two weeks in the defect site. Thus, rhBMP-2, BMP-4, and BMP-7 differentiated IMT into bone-like tissue in vitro, and this induced bone-like tissue has ossification potential and promotes the healing of calvarial defects. Our results suggest that IMT is an effective tissue source for bone tissue engineering.     

Methodology to optimize radiation protection in radioactive waste disposal after closure of a disposal facility based on probabilistic approach

We propose a methodology to optimize radiation protection in radioactive waste disposal after the closure of a disposal facility based on a probabilistic approach. In this methodology, a set of alternative options of the disposal system design with the associated uncertainties estimated through a probabilistic approach are developed. Then, the methodology evaluates the advantages and disadvantages of each option for the optimization of radiation protection, in which it is possible to determine the compliance with a dose constraint of 0.3 mSv/y and to collect information for making a decision on the optimization. In particular, the obtained information on the exposure, which is the mode and width of the dose distribution, can be converted into information on engineering measures and site conditions for the options. This process allows us to discuss which option should be selected as the optimal option by considering the balance between the exposure and the engineering, economic, and social feasibility of the option. This methodology is helpful for providing clear reasons why an optimal disposal system design is selected by combining quantitative information on the exposure and the feasibility of each option.     

Features of a control blade degradation observed in situ during severe accident conditions in boiling water reactors

In the present paper, new results using in situ video are presented regarding boiling water reactor (BWR) control blade degradation up to 1750 K at the beginning of a nuclear severe accident. Energy-dispersive X-ray spectrometry (EDS) mapping indicated stratification of the absorber blade melt with formation of a chromium and boride-enriched layer. High-content-B- and C-containing material with increased melting temperature acted like a shielding and was found to prevent further relocation of control blade claddings. The interacted layers around the B₄C-granules prevented direct steam attack of residual B₄C. The results provide new insights for understanding of the absorber blade degradation mechanism under reducing conditions specific to Fukushima Dai-Ichi Unit 2 resulting from prolonged steam starvation.