Identification of heparin-binding proteins expressed on Trichosporon asahii cell surface.

Trichosporon asahii is a pathogenic yeast that cause trichosporonosis. T. asahii exhibits several colony morphologies, such as white (W)- or off-white (O)-type, which may affect virulence. In this study, we compared the expression pattern of heparin-binding proteins in various colony morphologies and identified heparin-binding protein in T. asahii. Surface plasmon resonance analysis revealed that cell surface molecules attached more strongly to heparin in W- than O-type cells. We purified and identified a heparin-binding protein strongly expressed in W-type cells using heparin-Sepharose beads, named it heparin-binding protein 1 (HepBP1), and expressed Flag-tagged HepBP1 in mammalian cells. The heparin-binding ability of Flag-tagged HepBP1 was confirmed by pulldown assay using heparin-Sepharose beads. Thus, HepBP1 is a heparin-binding protein on T. asahii cell surface. These results suggest that several T. asahii cell surface proteins interact with glycosaminoglycans; therefore, they could contribute to infection.

Quantitative and Qualitative Analyses of Mass Spectra of OEL Materials by Artificial Neural Network and Interface Evaluation: Results from a VAMAS Interlaboratory Study.

Quantitative analysis of binary mixtures of tris(2-phenylpyridinato)iridium(III) (Ir(ppy)3) and tris(8-hydroxyquinolinato)aluminum (Alq3) by using an artificial neural network (ANN) system to mass spectra was attempted based on the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study (TW2 A31) to evaluate matrix-effect correction and to investigate interface determination. Monolayers of binary mixtures having different Ir(ppy)3 ratios (0, 0.25, 0.50, 0.75, and 1.00), and the multilayers containing these mixtures and pure samples were measured using time-of-flight secondary ion mass spectrometry (ToF-SIMS) with different primary ion beams, OrbiSIMS (SIMS with both Orbitrap and ToF mass spectrometers), laser desorption ionization (LDI), desorption/ionization induced by neutral clusters (DINeC), and X-ray photoelectron spectroscopy (XPS). The mass spectra were analyzed using a simple ANN with one hidden layer. The Ir(ppy)3 ratios of the unknown samples and the interfaces of the multilayers were predicted using the simple ANN system, even though the mass spectra of binary mixtures exhibited matrix effects. The Ir(ppy)3 ratios at the interfaces indicated by the simple ANN were consistent with the XPS results and the ToF-SIMS depth profiles. The simple ANN system not only provided quantitative information on unknown samples, but also indicated important mass peaks related to each molecule in the samples without a priori information. The important mass peaks indicated by the simple ANN depended on the ionization process. The simple ANN results of the spectra sets obtained by a softer ionization method, such as LDI and DINeC, suggested large ions such as trimers. From the first step of the investigation to build an ANN model for evaluating mixture samples influenced by matrix effects, it was indicated that the simple ANN method is useful for obtaining candidate mass peaks for identification and for assuming mixture conditions that are helpful for further analysis.

Interaction of Host Proteins with Cell Surface Molecules of the Pathogenic Yeast Trichosporon asahii.

Trichosporon asahii is an invasive pathogenic yeast that infects immunocompromised hosts. Several virulence factors contribute to the fungal infection; however, the factors that contribute to the occurrence of T. asahii infections remain unclear. Since adhesins are typical virulence factors reported for pathogenic fungi, we looked for host proteins that interact with the T. asahii cell surface. T. asahii and Candida albicans were used for screening using a pull-down assay with fetal bovine serum. Serum albumin and elongation factor 2 were identified as the yeast-binding serum proteins. Additionally, we investigated the interactions of the cell surface-associated molecules (CSM) of T. asahii with vitronectin (VTN), fibronectin, fetuin-A, and alpha-1antitrypsin (AAT). The surface plasmon resonance (SPR) method was used to examine the interaction between CSM and human proteins. On the other hand, the pull-down assay was used to examine the interaction between human proteins and the T. asahii cell surface. Serum albumin, AAT, and VTN were found to interact with T. asahii in both SPR and pull-down assays. This study identified several proteins that interact with T. asahii, suggesting that these proteins play a role in infection mechanisms.

Unsaturated fatty acid salts remove biofilms on dentures.

Candidiasis-causing Candida sp. forms biofilms with various oral bacteria in the dentures of the elderly, making it harder to kill and remove the microorganism due to the extracellular polymeric substances. We found that biofilms on dentures can effectively be removed by immersion in an unsaturated fatty acid salt solution. Using optical coherence tomography to observe the progression of biofilm removal by the fatty acid salt solution, we were able to determine that the removal was accompanied by the production of gaps at the interface between the biofilm and denture resin. Furthermore, microstructural electron microscopy observations and time-of-flight secondary ion mass spectrometry elucidated the site of action, revealing that localization of the fatty acid salt at the biofilm/denture-resin interface is an important factor.

Evaluation of Time-of-Flight Secondary Ion Mass Spectrometry Spectra of Peptides by Random Forest with Amino Acid Labels: Results from a Versailles Project on Advanced Materials and Standards Interlaboratory Study.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the identification of peptide sample TOF-SIMS spectra by machine learning. More than 1000 time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra of six peptide model samples (one of them was a test sample) were collected using 27 TOF-SIMS instruments from 25 institutes of six countries, the U. S., the U. K., Germany, China, South Korea, and Japan. Because peptides have systematic and simple chemical structures, they were selected as model samples. The intensity of peaks in every TOF-SIMS spectrum was extracted using the same peak list and normalized to the total ion count. The spectra of the test peptide sample were predicted by Random Forest with 20 amino acid labels. The accuracy of the prediction for the test spectra was 0.88. Although the prediction of an unknown peptide was not perfect, it was shown that all of the amino acids in an unknown peptide can be determined by Random Forest prediction and the TOF-SIMS spectra. Moreover, the prediction of peptides, which are included in the training spectra, was almost perfect. Random Forest also suggests specific fragment ions from an amino acid residue Q, whose fragment ions detected by TOF-SIMS have not been reported, in the important features. This study indicated that the analysis using Random Forest, which enables translation of the mathematical relationships to chemical relationships, and the multi labels representing monomer chemical structures, is useful to predict the TOF-SIMS spectra of an unknown peptide.

Adherence and Cytotoxicity of Candida spp. to HaCaT and A549 cells.

Candida species are opportunistic fungal pathogens that cause superficial or invasive infections. Recently, the incidence of infection by non-Candida albicans species, especially Candida glabrata, has increased. In this study, we analyzed the adhesion and cytotoxicity of various Candida spp. that are part of the normal human microbiota. C. albicans adheres well to cell culture plates and to cultured cells. C. glabrata selectively adheres to epithelial cells rather than to cell culture plates. Candida parapsilosis insufficiently adheres to confluent monolayers of human lung epithelial A549 and keratinocyte HaCaT cells. We then analyzed the cytotoxicity of C. albicans and C. glabrata, which adhered well to epithelial cells. C. glabrata has been found to cause more damage to A549 cells than to HaCaT cells, suggesting that resident Candida spp. have distinct cytotoxic effects in different tissues. It is important to clarify the properties of Candida spp. as there is evidence that normal microbiota can cause infections. Our data suggest that it is necessary to use appropriate cell lines for characterizing the adherence and cytotoxicity of Candida spp.

Hemagglutination ability and hemolytic activity of Trichosporon asahii.

Trichosporon asahii is a human fungal pathogen that causes deep-seated infections in immunocompromised patients. While the pathogenic mechanisms of T. asahii remain unknown, our previous studies indicate that adherent colony morphologies were generated from parent strains, which may contribute to their pathogenicity. In the present study, we analyzed the hemolytic and hemagglutination activities of T. asahii. We report that T. asahii cells demonstrate hemagglutination and hemolytic activities, and that cell surface molecules play a role in the hemagglutination activity of adherent strains. These observations suggest that hemagglutination and hemolysis may be one of the pathogenic mechanisms of T. asahii.

[Identification of Heparin-binding Proteins on the Cell Surface of Cryptococcus neoformans].

Interactions between virulence factors of pathogens and host responses play an important role in the establishment of infection by microbes. We focused on interactions between Cryptococcus neoformans proteins and heparin, which is abundant on host epithelial cells. Surface proteins were extracted and analyzed. Fractions from anion-exchange column chromatography interacted with heparin in surface plasmon resonance analyses. Heparin-binding proteins were purified and then separated by gel electrophoresis; and were identified as transaldolase, glutathione-disulfide reductase, and glyoxal oxidase. These results imply that multifunctional molecules on C. neoformans cells, such as those involved in heparin binding, may play roles in adhesion that trigger responses in the host.

Topical Application of Josamycin Inhibits Development of Atopic Dermatitis-Like Skin Lesions in NC/Nga Mice.

BACKGROUND: Patients with atopic dermatitis (AD) have superficial skin colonization by Staphylococcus aureus and an increased number of T helper type 2 (Th2) cells in their peripheral blood. Our previous study showed that josamycin, a macrolide antibiotic, had excellent bactericidal activity against S. aureus strains isolated from AD patients and simultaneously inhibited Th1 and Th2 cell development mediated by Langerhans cells. The purpose of the present study was to evaluate the effect of topical application of josamycin on AD-like skin lesions in NC/Nga mice. METHODS: Josamycin (0.1%) was topically administered to NC/Nga mice with AD-like skin lesions induced by 2, 4, 6-trinitrochlorobenzene (TNCB). The therapeutic effects of josamycin were assessed by measurement of the skin severity scores, histological changes in the lesioned skin, serum levels of total IgE, and expression of interferon (IFN)-γ and interleukin (IL)-4 in lymph nodes and skin lesions. RESULTS: Topical treatment with josamycin significantly suppressed the increase in the skin severity score in NC/Nga mice. This suppressive effect was equal to that of betamethasone, and was associated with a decrease in the density of cellular infiltration into the dermis, the mast cell count in the dermis and the serum IgE level. Furthermore, topical application of josamycin reduced the expression of IFN-γ and IL-4 in auricular lymph node cells and the skin lesions. CONCLUSION: The present results show that topical application of josamycin inhibits the development of AD-like skin lesions in NC/Nga mice. This suggests that topical application of josamycin to AD lesions colonized by S. aureus would be beneficial for control of AD by acting on superficially located S. aureus and by inhibiting the development of Th1 and Th2 cells.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Growth Inhibition of an Opportunistic Yeast Pathogen Trichosporon asahii by Staphylococcus epidermidis.

In the co-culture of Staphylococcus epidermidis and Trichosporon asahii, a fungal pathogen, it was observed that live S. epidermidis inhibited the growth of T. asahii. Soluble active anti-T. asahii substances were speculated to be produced by S. epidermidis in culture medium. Using 1H- and 13C-NMR spectra and electron ionization-high resolution mass spectrometry (HR-negative-FAB-MS), we separated the active molecule and identified it as lactic acid. Commercially available L-lactic acid and D-lactic acid inhibited the growth of T. asahii. These results show that metabolites from bacterial populations are involved in the interactions of pathogenic fungi. The use of antibacterial agents to treat primary diseases could lead to the disruption of normal microbial communities and could cause opportunistic infections such as trichosporonosis.

Cell surface hydrophobicity and colony morphology of Trichosporon asahii clinical isolates.

Trichosporon asahii is a pathogenic basidiomycetous yeast. Individual strains of T. asahii have different colony morphologies. However, it is not clear whether cell surface phenotypes differ among the colony morphologies. Here we characterized the cell surface hydrophobicity and analysed the carbohydrate contents of the cell surface polysaccharides in T. asahii clinical isolates with various colony morphologies. Among the three distinctive colony morphologies obtained from one clinical isolate, the white-type morphology exhibited higher hydrophobicity. The hydrophobicity of heat-killed T. asahii cells was greatly reduced after periodate oxidation of the cell surface carbohydrates. Furthermore, the cell wall and extracellular polysaccharide components differed among the morphologies. Our results suggest that T. asahii cell surface hydrophobicity is affected by cell surface carbohydrate composition. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of Macrolide Antibiotics on Th1 Cell and Th2 Cell Development Mediated by Langerhans Cells.

- Background: It is well known that Langerhans cells (LCs) work as the primary orchestrators in the polarization of the immune milieu towards a T helper type 1 (Th1) or a Th2 immune response. In this study, we investigated the effects of macrolide antibiotics on Th1 cell and Th2 cell development mediated by LCs. METHODS: LC-like dendritic cells (LDCs) were generated from mouse bone marrow cells and used as substitutes for LCs. Mice were primed with ovalbumin (OVA) peptide-pulsed LDCs, which had been treated with each macrolide antibiotic, via the hind footpad. After 5 days, the cytokine response in the popliteal lymph nodes was investigated by enzyme-linked immunosorbent assay. The expression of cell surface molecules on LDCs was investigated using reverse transcriptase polymerase chain reaction. RESULTS: Injection of OVA peptide-pulsed LDCs, which had been treated with josamycin or spiramycin, inhibited Th2 cell development as represented by down-regulation of interleukin (IL)-4 production as well as Th1 cell development as represented by down-regulation of interferon (IFN)-g production. This inhibition of Th1 cell and Th2 cell development was associated with suppression of CD86 and T-cell immunoglobulin and mucin domain-containing protein (TIM)-4 expression, respectively, in LDCs. Furthermore, Staphylococcus aureus strains isolated from skin lesions of patients with atopic dermatitis (AD) were more susceptible to josamycin than to spiramycin. CONCLUSIONS:   These results suggest that topical application of josamycin to AD lesions colonized with S. aureus would be beneficial for control of AD by acting on both superficial S. aureus and epidermal LCs, and inhibiting the development of Th2 cells.

Elucidation of Softening Mechanism in Rinse-Cycle Fabric Softeners. Part 2: Uneven Adsorption-The Key Phenomenon to the Effect of Fabric Softeners.

We investigated the actual factor determining the softening effect of a fabric softener. The adsorption area of the softener on model cotton cloths and yarns was identified using bromophenol blue. There was almost no softener at the cross-points of the yarns in the cloth samples or in the inner part of the yarns. The softening performance was better when there was less softener at the cross-points of the yarns than when the yarns were evenly covered by the softener. Thus we conclude that the presence of softener at the cross-points of yarns is not a vital factor in the softening effect. In addition, more softener was found on the outer part of the yarn than the inner part, indicating gradation in the adsorption pattern of the softener. Thus, we propose that more softener is adsorbed on the exposed part of the yarn in a cloth, and the formation of a hydrogen-bonding network containing bound water is inhibited, thus softening the outer part of the yarn. However, the presence of a small amount of softener in the inner part of the yarn preserves the hydrogen-bonding network. Favorable elasticity, or bounce, of the yarns and cloth is realized when an appropriate amount of softener is used. Excess softener would reach the inner part of the yarn, reducing the diameter of the core part of the yarn, making the cloth appear wilted.

Betamethasone, but Not Tacrolimus, Suppresses the Development of Th2 Cells Mediated by Langerhans Cell-Like Dendritic Cells.

It is well known that Langerhans cells (LCs) work as the primary orchestrators in the polarization of the immune milieu towards a T helper type 1 (Th1) or T helper type 2 (Th2) response. In this study, we investigated the effects of tacrolimus and betamethasone, each used as topical applications in atopic dermatitis (AD), on Th2 cell development mediated by LCs. LC-like dendritic cells (LDCs) were generated from mouse bone marrow cells and used as substitutes for LCs. Mice were primed with ovalbumin (OVA) peptide-pulsed LDCs, which had been treated with tacrolimus or betamethasone, via the hind footpad. After 5 d, the cytokine response in the popliteal lymph nodes was investigated by enzyme-linked immunosorbent assay. The expression of cell surface molecules on LDCs was investigated via reverse transcriptase polymerase chain reaction. Administration of OVA peptide-pulsed LDCs, which had been treated with betamethasone, inhibited Th2 cell development, as represented by the down-regulation of interleukin-4 production, and also inhibited Th1 cell development, represented by the down-regulation of interferon-γ production. However, tacrolimus-treated LDCs did not induce such inhibition of the development of Th1 and Th2 cells. The inhibition of Th1 and Th2 cell development was associated with the suppression of CD40 and T-cell immunoglobulin, and mucin domain-containing protein (TIM)-4 expression, respectively, in LDCs. These results suggest that the topical application of betamethasone to skin lesions of patients with AD acts on epidermal LCs, and may inhibit the development of Th2 cells, thus being of benefit for the control of AD.

Comparative Analysis of Extracellular Polymeric Substances from Cryptococcus gattii VGIIa Strain Isolated for the First Time in Japan.

Cryptococcus gattii and C. neoformans are pathogenic yeasts that cause meningoencephalitis. C. gattii has four molecular types: VGI, VGII, VGIII, and VGIV. Furthermore, three genotypes have been reported for VGII, and a high pathogenicity of the VGIIa genotype has been proposed. The VGIIa strain has been isolated from a patient in Japan, but little is known about the characteristics of the polysaccharides in this strain. In this study we examined the induction of interleukin-8（IL-8）transcriptional activation and compared the nuclear magnetic resonance（NMR）spectra of extracellular polymeric substances（EPSs）, mainly polysaccharides, from the VGIIa, VGIIb, and VGIIc genotypes. The induction of IL-8 by C. gattii EPSs was weaker than that by C. neoformans EPSs. The anomeric proton signals in the NMR spectra of EPSs obtained from VGII isolates were similar, and the polysaccharides were mainly mannose, xylose, galactose, and glucuronic acid. These results suggest that the extracellular polysaccharides from the VGIIa strain isolated in Japan are almost the same as those from other VGII strains.

Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population.

Cryptococcus neoformans var. neoformans (serotype D) represents about 30% of the clinical isolates in Europe and is present less frequently in the other continents. It is the prevalent etiological agent in primary cutaneous cryptococcosis as well as in cryptococcal skin lesions of disseminated cryptococcosis. Very little is known about the genotypic diversity of this Cryptococcus subtype. The aim of this study was to investigate the genotypic diversity among a set of clinical and environmental C. neoformans var. neoformans isolates and to evaluate the relationship between genotypes, geographical origin and clinical manifestations. A total of 83 globally collected C. neoformans var. neoformans isolates from Italy, Germany, France, Belgium, Denmark, Greece, Turkey, Thailand, Japan, Colombia, and the USA, recovered from different sources (primary and secondary cutaneous cryptococcosis, disseminated cryptococcosis, the environment, and animals), were included in the study. All isolates were confirmed to belong to genotype VNIV by molecular typing and they were further investigated by MLST analysis. Maximum likelihood phylogenetic as well as network analysis strongly suggested the existence of a recombinant rather than a clonal population structure. Geographical origin and source of isolation were not correlated with a specific MLST genotype. The comparison with a set of outgroup C. neoformans var. grubii isolates provided clear evidence that the two varieties have different population structures.

Switching of colony morphology and adhesion activity of Trichosporon asahii clinical isolates.

Trichosporon asahii is a pathogenic yeast that causes trichosporonosis, a deep-seated infection, in immunocompromised hosts. Pathogenic factors involved in this infection have not been investigated in detail, but morphological phenotype switching is thought to be important for T. asahii pathogenesis. Therefore, we analyzed adhesion, which may be a key early step in T. asahii infection, after morphological phenotype switching. T. asahii clinical isolates show several colony morphologies. In this study, colonies showing white-farinose (W), off-white-smooth (O), off-white-rugose (OR), smooth (S), and yellowish-white (Y) morphologies were obtained from three isolates and compared in an adhesion assay performed in cell culture dishes. At least one type of colony morphology from each clinical isolate adhered strongly to the culture dish surface, although the colony type that displayed strong adherence varied among the strains. Thus, morphological phenotype switching altered the adhesion of T. asahii strains.

CCL17 production by mouse langerhans cells stimulated with Staphylococcus aureus cell wall components.

Patients with atopic dermatitis (AD) show increased numbers of Th2 cells in their acute skin lesions and superficial skin colonization by Staphylococcus aureus. The purpose of this study was to clarify the effect of S. aureus cell wall components on Th2 chemokine production by murine Langerhans cells (LCs). Murine LCs were stimulated with peptidoglycan (PEG) and/or muramyldipeptide (MDP) for 24 or 48 h, and Th1 and Th2 chemokine production was investigated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). PEG-stimulation of LCs induced production of the Th2 chemokine CCL17 and this was enhanced in the presence of interleukin (IL)-4. A low-molecular weight PEG fragment, MDP, did not induce CCL17 production by LCs. However, when LCs were stimulated with PEG in combination with MDP, PEG-induced CCL17 production was synergistically enhanced by MDP. Furthermore, PEG- and MDP-induced CCL17 production was enhanced to a greater extent in the presence of IL-4. These results suggest that S. aureus colonization in AD patients may enhance the Th2-prone immune response through upregulation of CCL17 production by LCs, which would occur as a result of simultaneous stimulation with PEG and MDP from S. aureus in a Th2 environment.

Comparison of fetal toxicity of various multi-wall carbon nanotubes in mice.

The fetal toxicities of multi-wall carbon nanotubes (MWCNTs) with various sizes were compared in CD1(ICR) mice. MWCNTs were suspended in 2% sodium carboxymethyl cellulose solution in phosphate-buffered saline. On day 9 of gestation, dams were administered a single intraperitoneal dose of MWCNTs (4 mg/kg body weight), while dams in the control group were administered vehicle (10 mL/kg body weight). The rectal temperatures of the dams were monitored 2 h after administaration to asses statuses of the dams. The dams and fetuses were examined on day 18 of gestation. The number of live fetus per dam decreased in some MWCNTs-administered groups. The mean percentages of live fetuses in total implantations in the MWCNTs-administered groups markedly varied from 0% to 95%, and the highest mean percentage of live fetuses in the MWCNTs-administered group was equivalent to that of the control group. The decrease in live fetuses depended on an increased number of early dead fetuses. In the groups with markedly lowered rectal temperature after administration, the fetal loss were evident. The blood levels of interleukin-6 and/or monocyte chemoattractant protein-1 in dam 2 h after administration of MWCNTs markedlyr increased, especially in the goups with significant decrease in live fetuses. These results indicated a relationship between inflammation in the dam, which probabely depended on the particular length of the MWCNTs, and the fetal toxicioty of MWCNTs in mice.

Detection and characterization of plasminogen receptors on clinical isolates of Trichosporon asahii.

Trichosporon asahii is the major causative agent of deep-seated trichosporonosis. The virulence factors of this yeast pathogen remain uncharacterized. To investigate the pathogenicity of T. asahii, we focused on the interactions between surface molecules of the yeast and host biomolecules. We examined the ability of surface molecules to bind human plasminogen using clinical isolates of T. asahii. Living T. asahii cells accelerated the conversion of plasminogen to plasmin in a dose-dependent manner in the presence of tissue plasminogen activator. Extracts from cells using lithium chloride contained plasminogen-binding molecules based on surface plasmon resonance (SPR) analyses. In all strains tested, several of the fractions obtained using DEAE column chromatography bound and accelerated the conversion of plasminogen to plasmin. Based on far-Western blotting analyses, a common protein was identified within the four strains, which was identified as a hypothetical protein from genome analyses of T. asahii. blast searches suggested the protein might be heparinase, and heparinase activity was detected in the T. asahii extract. Furthermore, affinity chromatography using plasminogen as a ligand detected one protein band by SDS-PAGE, which was identified as thioredoxin-dependent peroxide reductase.SPR analyses suggested the presence of molecules on T. asahii cells that could bind plasminogen with differing affinities.