Contribution of Invariant Natural Killer T Cells to the Clearance of Pseudomonas aeruginosa from Skin Wounds

Chronic infections are considered one of the most severe problems in skin wounds, and bacteria are present in over 90% of chronic wounds. Pseudomonas aeruginosa is frequently isolated from chronic wounds and is thought to be a cause of delayed wound healing. Invariant natural killer T (iNKT) cells, unique lymphocytes with a potent regulatory ability in various inflammatory responses, accelerate the wound healing process. In the present study, we investigated the contribution of iNKT cells in the host defense against P. aeruginosa inoculation at the wound sites. We analyzed the re-epithelialization, bacterial load, accumulation of leukocytes, and production of cytokines and antimicrobial peptides. In iNKT cell–deficient (Jα18KO) mice, re-epithelialization was significantly decreased, and the number of live colonies was significantly increased, when compared with those in wild-type (WT) mice on day 7. IL-17A, and IL-22 production was significantly lower in Jα18KO mice than in WT mice on day 5. Furthermore, the administration of α-galactosylceramide (α-GalCer), a specific activator of iNKT cells, led to enhanced host protection, as shown by reduced bacterial load, and to increased production of IL-22, IL-23, and S100A9 compared that of with WT mice. These results suggest that iNKT cells promote P. aeruginosa clearance during skin wound healing.     

Insights into Metabolite Diagnostic Biomarkers for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a persistent and unexplained pathological state characterized by exertional and severely debilitating fatigue, with/without infectious or neuropsychiatric symptoms, and with a minimum duration of 6 consecutive months. Its pathogenesis is not fully understood. There are no firmly established diagnostic biomarkers or treatment, due to incomplete understanding of the etiology of ME/CFS and diagnostic uncertainty. Establishing a biomarker for the objective diagnosis is urgently needed to treat a lot of patients. Recently, research on ME/CFS using metabolome analysis methods has been increasing. Here, we overview recent findings concerning the metabolic features in patients with ME/CFS and the animal models which contribute to the development of diagnostic biomarkers for ME/CFS and its treatment. In addition, we discuss future perspectives of studies on ME/CFS.     

Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B₄ (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.     

Preparation of polymeric nanocapsules via nano-sized poly(methoxydiethyleneglycol methacrylate) colloidal templates

Polymeric nanocapsules are attractive devices with a number of potential applications. In the present contribution we describe a method for nanocapsule preparation which is based on the formation of nanosized templates (mesoglobules, prepared from thermo-responsive poly(methoxydiethyleneglycol methacrylate)s, PDEGMA). These mesoglobules were coated with a cross-linked shell formed by pseudo-seeded radical polymerization of either N-isopropylacrylamide or 2-hydroxyethylmethacrylate in the presence of a cross-linking agent. Dissolution and removal of templates were achieved by extensive dialysis against water at temperatures below the LCST of PDEGMA. The obtained nanocapsules were visualized by transmission electron microscopy and their dimensions were determined by dynamic light scattering. The differences in the morphology of the nanocapsules were attributed to the different structures of the cross-linked membranes.     

Different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions: Microhardness and polarized light miscroscopy analyses

The aim of this study was to compare different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions by microhardness and polarized light microscopy analyses. One hundred blocks of bovine enamel were randomly divided into four groups (n = 25) according to the bacterial model for caries induction: (A) Streptococcus mutans, (B) S. mutans and Lactobacillus acidophilus, (C) S. mutans and L. casei, and (D) S. mutans, L. acidophilus, and L. casei. Within each group, the blocks were randomly divided into five subgroups according to the duration of the period of caries induction (4–20 days). The enamel blocks were immersed in cariogenic solution containing the microorganisms, which was changed every 48 h. Groups C and D presented lower surface hardness values (SMH) and higher area of hardness loss (ΔS) after the cariogenic challenge than groups A and B (P < 0.05). As regards lesion depth, under polarized light microscopy, group A presented significantly lower values, and groups C and D the highest values. Group B showed a higher value than group A (P < 0.05). Groups A and B exhibited subsurface caries lesions after all treatment durations, while groups C and D presented erosion‐type lesions with surface softening. The model using S. mutans, whether or not it was associated with L. acidophilus, was less aggressive and may be used for the induction of non‐cavitated enamel caries‐like lesions. The optimal period for inducing caries‐like lesions was 8 days. Microsc. Res. Tech. 78:444–451, 2015. © 2015 Wiley Periodicals, Inc.     

Application of full-factorial design for optimization of the moisture determination in wheat flour using infrared scales

We studied the parameters that may affect the analysis of the moisture content in wheat flour using infrared scales. The parameters were studied by one-factor at a time methodology and then, by full-factorial design (2³). The best condition using infrared scales occurred at 120 °C, with a sample weight of 1.5 g and an exposure time to infrared radiation of 6 min. We observed the interaction effect between the three factors studied. The analysis time was 60 times lower than classical method. The optimized method for determination of the moisture content in wheat flour using infrared scales was validated to range between 9 and 17 %.     

A two-way coupled multiscale model for predicting damage-associated performance of asphaltic roadways

This paper presents a quasi-static multiscale computational model with its verification and rational applications to mechanical behavior predictions of asphaltic roadways that are subject to viscoelastic deformation and fracture damage. The multiscale model is based on continuum thermo-mechanics and is implemented using a finite element formulation. Two length scales (global and local) are two-way coupled in the model framework by linking a homogenized global scale to a heterogeneous local scale representative volume element. With the unique multiscaling and the use of the finite element technique, it is possible to take into account the effect of material heterogeneity, viscoelasticity, and anisotropic damage accumulation in the small scale on the overall performance of larger scale structures. Along with the theoretical model formulation, two example problems are shown: one to verify the model and its computational benefits through comparisons with analytical solutions and single-scale simulation results, and the other to demonstrate the applicability of the approach to model general roadway structures where material viscoelasticity and cohesive zone fracture are involved.     

Micro cutting of ferrous materials using diamond tool under ionized coolant with carbon particles

A new coolant for micro cutting of ferrous materials using a diamond tool was proposed. The effects of the new coolant (ion-shot coolant) which consists of ionized coolant and carbon particles on two steel materials, a high carbon chromium steel and a pre-hardened mold steel were investigated. Also mechanism of the ion-shot cutting was discussed by the observation on material structure at the primary shear zone and frictional coefficient between diamond tool and ferrous material. The ion-shot coolant showed great potential for the used of the diamond tool on ferrous material in terms of tool wear and surface quality.     

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface‐active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino‐terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene‐disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low‐temperature tolerance of the yeast. Copyright © 2010 John Wiley & Sons, Ltd.     

Model analysis on dispersion characteristics of fine particles in Newtonian molten polymer

In this paper, a model estimating the time-dependent distribution of cluster size consisting of fine particles in Newtonian molten polymer was proposed. The present model for agglomerative suspension was developed based on Usui’s thixotropy model, derived by taking the balance between shear breakup, shear coagulation and Brownian coagulation processes. The analysis was applied to the experimental results for silica/(ethylene methyl-meta acrylate copolymer) suspensions. The time variations of cluster size distributions, of viscosities and of the mean numbers of particles in a cluster were calculated and the results were compared with experimental results for the cases of shear rates from 0.2 to 10 s^?1 when the solid volume fraction was set at 0.15. From the results, it was found that the present model can estimated the steady state values of the slurry viscosity and the mean particle numbers in a cluster as well as Usui’s original model. However, the model could not sufficiently express the time variation of rheological characteristics due to the over-estimation of the contribution of shear rate when the cluster size is small.