Fabrication of a BOC-Protected 2-Hydroxyethyl Methacrylate Brush and Deprotection of the BOC Group to Control the Surface Hydrophilicity.

Fabrication of functional surfaces with designed patterns of different hydrophilicity has potential applications in active control of water droplets and water harvesting. For practical applications, the fabrication process needs to be applied to a large area in a cost-effective manner. Herein, we report the fabrication of a polymer brush of 2-(tert-butoxycarbonyloxy)ethyl methacrylate having a BOC-protected hydroxy group. The deprotection of the BOC group converts poly(2-(tert-butoxycarbonyloxy)ethyl methacrylate) (PBHEMA) into poly(2-hydroxyethyl methacrylate) (PHEMA) and hence changes the hydrophilicity. The chemical transformation changes the refractive index and thickness of the brush. This simple chemistry enables easy formation of the boundary of different hydrophilicity. Last, we demonstrate that the shape of the water droplet can be manipulated on the designed surface having different hydrophilicity.

Identification of fungi isolated from astronaut nasal and pharyngeal smears and saliva.

As part of a series of studies regarding the microbiota in manned space environments, we isolated the fungal strains from nasal and pharyngeal smears and saliva of 21 astronauts preflight, in-flight, and postflight. On the ground, 120 strains from 43 genera of environmental fungi were isolated from the astronauts. The dominant fungal genera were Cladosporium, Penicillium, and Aspergillus. Only 18 strains from four genera were isolated from the astronauts inside the International Space Station. These fungi are currently thought to be harmless, but regular screening and cleaning are necessary to prevent fungus-related health disorders.

Seven years of progress in determining fungal diversity and characterization of fungi isolated from the Japanese Experiment Module KIBO, International Space Station.

The International Space Station (ISS) is a closed facility that orbits the earth carrying not only its crew but also microorganisms. We have participated in microbiota analysis projects for the Japanese Experiment Module KIBO (ISS; operations nomenclature: Microbe-I, II, III, and IV) and were in charge of fungal screening. The interior of KIBO was sampled using swabs and microbe detection sheets (MDSs) for fungal detection. The dominant genera obtained by culture were Aspergillus and Penicillium. DNA analyses of the fungal biota using a clone library showed that KIBO was dominated by Malassezia, a fungal inhabitant of human skin. Three fungal species, Aspergillus sydowii, Penicillium palitans, and Rhodotorula mucilaginosa, which grew under microgravity in KIBO were observed under a field emission-scanning electron microscope on the ground. No novel phenotypic characteristics were noted. The results of antifungal susceptibility testing of all isolates did not differ significantly from previous reports of corresponding fungi. In Microbe-I (August 2009), MDSs were culture negative, while in the next stages the CFU of MDSs were 10 for Microbe-II (February 2011), 24 for Microbe-III (October 2012), and 151 for Microbe-IV (February 2015). These results indicated that fungi inside KIBO are increasing and expanding over time, and therefore continuous surveillance is crucial.

Investigation of the Physiological, Biochemical and Antifungal Susceptibility Properties of Candida auris.

BACKGROUND: Candida auris is an emerging pathogen associated with outbreaks in clinical settings. Isolates of the pathogen have been geographically clustered into four clades with high intra-clade clonality. Pathogenicity varies among the clades, highlighting the importance of understanding these differences. OBJECTIVES: To examine the physiological and biochemical properties of each clade of C. auris to improve our understanding of the fungus. METHODS: Optimal growth temperatures of four strains from three clades, East Asia, South Asia and South Africa, were explored. Moreover, assimilation and antifungal susceptibility properties of 22 C. auris strains from the three clades were studied. RESULTS: The optimal growth temperatures of all strains were 35-37 °C. Assimilation testing demonstrated that the commercial API ID 32 C system can be used to reliably identify C. auris based on the biochemical properties of the yeast. Notably, C. auris can be uniquely differentiated from commonly clinical fungi by its ability to assimilate raffinose and inability to utilize D-xylose, suggesting a useful simple screening tool. The antifungal susceptibility results revealed that all strains are resistant against fluconazole (minimal inhibitory concentration (MIC) 4 to > 64 µg/mL) and miconazole (MIC 8 to > 16 µg/mL), with strains from the Japanese lineage showing relatively lower MIC values (1-4 µg/mL). Conversely, itraconazole, voriconazole, amphotericin B, micafungin and caspofungin were active against most of the tested strains. On the clade level, East Asian strains generally showed lower MICs against azoles comparing to the other clades, while they displayed MICs against flucytosine higher than those of strains from South Africa and South Asia clades. CONCLUSION: Our data suggest a simple identification approach of C. auris based on its physiological and biochemical properties and highlight aspects of C. auris population from various clades.

Fast calculation method for parabolic-mirror-reflection holographic 3D display using wavefront segmentation.

Convex-parabolic-mirror reflection enables a very wide viewing zone in a holographic three-dimensional (3D) display. In this work, segmentation is introduced to reduce the calculation time of holograms in a convex-parabolic-mirror-reflection holographic 3D display. Wavefront segmentation can practically limit the lateral spread of the wavefront to be considered, which enables the application of geometrical approximation and conventional diffraction theories such as Fresnel diffraction. Thus, diffraction calculation via the convex parabolic mirror can be derived analytically and calculated rapidly using fast Fourier transform (FFT). Our proposed FFT-based method can calculate the diffraction integral 7000 times faster than our previous method, which involved calculating directly the diffraction integral without FFT. In addition, numerical simulation and an optical experiment are presented to verify our proposal.

DNA topoisomerase 2 gene polymorphism in dermatophytes.

BACKGROUND: Dermatophytes are a group of keratinophilic fungi of medical importance. Despite a relatively long history of molecular taxonomic studies, there is still a need for information on genetic polymorphism in wider variety of genomic loci. OBJECTIVES: Our goal was to study partial DNA topoisomerase 2 gene (TOP2) polymorphism in dermatophytes. METHODS: We performed DNA sequencing of TOP2 in 26 dermatophyte species along with ribosomal internal transcribed spacer (ITS) sequencing. RESULTS: The number of polymorphic sites in TOP2 data set was similar to that one in ITS data set. Nannizzia species formed paraphyletic group in TOP2 tree. Trichophyton simii was paraphyletic in concatenated TOP2-ITS tree, one of its two clades contained solely Iranian isolates. CONCLUSIONS: Our results revealed several unresolved problems in the taxonomy of dermatophytes, including probable polyphyly of the genus Nannizzia and the species T simii.

Epidemiological survey of onychomycosis pathogens in Japan by real-time PCR.

In Japan, an epidemiological survey of onychomycosis pathogens was performed using culture methods; however, the positive culture rate was 40% or less. As part of an epidemiological survey of dermatomycoses in Japan, we overcame this low positive rate by employing a real-time polymerase chain reaction (PCR) assay that allowed rapid and accurate detection and identification. In 2011, nail specimens were collected from patients at nine institutes in various prefectures in Japan and diagnosed as onychomycosis. For the detection and identification of the main pathogens causing onychomycosis, we performed real-time PCR using specific TaqMan® MGB probes and primer sets. Of the 496 onychomycosis samples, real-time PCR detected 382 cases (77.0%) caused by Trichophyton rubrum; 74 cases (15.0%) caused by Trichophyton interdigitale; and eight cases (1.6%) caused by Candida albicans. The real-time PCR positive rate was 96.2%. The most frequent pathogen was T. rubrum throughout life, with the number of patients affected peaking in the range of 60 to 69 years of age and no significant differences in the composition of causative pathogens by sex. We were able to detect and identify pathogens from almost all specimens and succeeded in analyzing the pathogens involved in onychomycosis cases in Japan. These data confirmed that our real-time PCR method was effective for detecting and identifying the main fungal pathogens from onychomycosis specimens.

Real-time interactive holographic 3D display with a 360° horizontal viewing zone.

To realize a real-time interactive holographic three-dimensional (3D) display system, we synthesize a set of 24 full high-definition (HD) binary computer-generated holograms (CGHs) based on a 3D fast-Fourier-transform-based approach. These 24 CGHs are streamed into a digital micromirror device (DMD) as a single 24-bit image at 60 Hz: 1440 CGHs are synthesized in less than a second. Continual updates of the CGHs displayed on the DMD and synchronization with a rotating mirror enlarges the horizontal viewing zone to 360° using a time-division approach. We successfully demonstrate interactive manipulation, such as object rotation, rendering mode switching, and threshold value alteration, for a medical dataset of a human head obtained by X-ray computed tomography.

The Molecular Identification and Antifungal Susceptibilities of Aspergillus Species Causing Otomycosis in Tochigi, Japan.

Aspergillus species are the most common pathogenic fungi involved in otomycosis, an infection of the outer ear canal. In this study, we examined the incidence of Aspergillus infections and the antifungal susceptibilities of 30 Aspergillus species isolates from patients with otomycosis who visited Saiseikai Utsunomiya Hospital between August 2013 and July 2016. Based on the morphological test results, the strains were identified as Aspergillus niger sensu lato (20 strains), A. terreus sensu lato (7 strains), and A. fumigatus sensu lato (3 strains). In contrast, the molecular identifications based on analyzing the isolates' partial ß-tubulin gene sequences revealed them to be A. niger sensu stricto (12 strains), A. tubingensis (8 strains), A. terreus sensu stricto (7 strains), and A. fumigatus sensu stricto (3 strains). The antifungal susceptibility test results indicated that strains of A. tubingensis and A. niger sensu stricto displayed lower susceptibilities to ravuconazole, compared with the other isolates. The Aspergillus strains from this study showed low minimum inhibitory concentrations toward the azole-based drugs efinaconazole, lanoconazole, and luliconazole. Therefore, these topical therapeutic agents may be effective for the treatment of otomycosis.

In Vitro Antifungal Activity of Novel Triazole Efinaconazole and Five Comparators against Dermatophyte Isolates.

The objective of this study was to assess the in vitro activity of the novel triazole antifungal drug, efinaconazole, and five comparators (luliconazole, lanoconazole, terbinafine, itraconazole, and fluconazole) against a large collection of Trichophyton interdigitale and Trichophyton rubrum clinical isolates. The geometric mean MICs were the lowest for luliconazole (0.0005 µg/ml), followed by lanoconazole (0.002 µg/ml), efinaconazole (0.007 µg/ml), terbinafine (0.011 µg/ml), itraconazole (0.095 µg/ml), and fluconazole (12.77 µg/ml). It appears that efinaconazole, lanoconazole, and luliconazole are promising candidates for the treatment of dermatophytosis due to T. interdigitale and T. rubrum.

Characterization of fungi isolated from the equipment used in the International Space Station or Space Shuttle.

As a part of a series of studies regarding the microbial biota in manned space environments, fungi were isolated from six pieces of equipment recovered from the Japanese Experimental Module "KIBO" of the International Space Station and from a space shuttle. Thirty-seven strains of fungi were isolated, identified and investigated with regard to morphological phenotypes and antifungal susceptibilities. The variety of fungi isolated in this study was similar to that of several previous reports. The dominant species belonged to the genera Penicillium, Aspergillus and Cladosporium, which are potential causative agents of allergy and opportunistic infections. The morphological phenotypes and antifungal susceptibilities of the strains isolated from space environments were not significantly different from those of reference strains on Earth.

Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.

Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.

Nucleotide sequence analysis of beta tubulin gene in a wide range of dermatophytes.

We investigated the resolving power of the beta tubulin protein-coding gene (BT2) for systematic study of dermatophyte fungi. Initially, 144 standard and clinical strains belonging to 26 species in the genera Trichophyton, Microsporum, and Epidermophyton were identified by internal transcribe spacer (ITS) sequencing. Subsequently, BT2 was partially amplified in all strains, and sequence analysis performed after construction of a BT2 database that showed length ranged from approximately 723 (T. ajelloi) to 808 nucleotides (M. persicolor) in different species. Intraspecific sequence variation was found in some species, but T. tonsurans, T. equinum, T. concentricum, T. verrucosum, T. rubrum, T. violaceum, T. eriotrephon, E. floccosum, M. canis, M. ferrugineum, and M. audouinii were invariant. The sequences were found to be relatively conserved among different strains of the same species. The species with the closest resemblance were Arthroderma benhamiae and T. concentricum and T. tonsurans and T. equinum with 100% and 99.8% identity, respectively; the most distant species were M. persicolor and M. amazonicum. The dendrogram obtained from BT2 topology was almost compatible with the species concept based on ITS sequencing, and similar clades and species were distinguished in the BT2 tree. Here, beta tubulin was characterized in a wide range of dermatophytes in order to assess intra- and interspecies variation and resolution and was found to be a taxonomically valuable gene.

The first case of human protothecosis caused by Prototheca zopfii in Japan.

This report describes a fatal case Prototheca zopfii genotype 2 infection in an immunosuppressed patient. The patient was a 62-year-old housewife who presented general malaise in April 2011. Hairy cell leukemia was highly suspected. Chemotherapy was started because the patient developed severe pancytopenia in October 2011. Itraconazole capsules (100 mg/day) and trimethoprim (320 mg/day) plus sulfamethoxazole (1600 mg/day) combinations were orally administered for prophylaxis of fungal infections. Of BacT/ALERT 3D FA aerobic culture bottles and FN anaerobic culture bottles, only FA aerobic blood culture bottles produced positive reactions when the patient developed fever in January 2012. Gram-staining of blood culture bottles revealed Gram-negative elliptical sporangia. Culturing on Sabouraud dextrose agar produced smooth and creamy white, yeast-like colonies. Partial DNA sequences of the nuclear 18S rDNA and 28S rDNA D1/D2 domains of the isolated strain were identical to those of P. zopfii genotype 2. The MICs and minimal lethal concentrations of antifungals revealed that it was susceptible to amphotericin B and itraconazole. The patient died, at which time plasma (1 â†’ 3)-ß-D-glucan was positive (131 pg/mL).

Molecular epidemiological investigation of Cryptococcus spp. carried by captive koalas (Phascolarctos cinereus) in Japan.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a systemic mycosis that infects a wide range of species. Recent molecular biological investigations have allowed for the genotyping of these species, providing more detailed information on their pathogenicity and infection routes. Koalas (Phascolarctos cinereus) are frequently colonized by Cryptococcus spp., but molecular epidemiological studies have yet to be conducted in Japan. Here, we conducted multi-locus sequence typing (MLST) analysis on Cryptococcus spp. colonization isolates obtained from all koalas kept in seven parks across Japan. Out of 46 koalas examined, 10 (22%) were positive for C. gattii and 3 (6.5%) were positive for C. neoformans. All C. gattii isolates belonged to molecular type VGI and were either sequence type (ST) 51 or a novel ST, and all C. neoformans isolates belonged to molecular type VNI and ST23. Despite the frequent movement of koalas between parks, the STs were relatively park-specific, suggesting that the floor of the rearing barns is a source of infection and may act as a reservoir. MLST analysis confirmed that C. gattii was transported, established, and spread by koalas in areas where C. gattii was not originally present. MLST analysis is considered useful in assessing the pathogenicity and tracing the transmission routes of Cryptococcus spp. carried by koalas.IMPORTANCEThis is the first study to conduct a multi-locus sequence typing analysis on Cryptococcus spp. carried by captive koalas in Japan. Cryptococcosis remains a globally high-fatality fungal infection in humans, and captive koalas are known to carry a high percentage of Cryptococcus spp. Through this research, the molecular types and transmission routes of Cryptococcus spp. carried by koalas have been elucidated, revealing the potential role of enclosure flooring as a reservoir. It has been confirmed that Cryptococcus gattii, which is not endemic in Japan, has become established through koalas and is spreading to new individuals in Japan. This study is believed to provide valuable insights into koala conservation and contribute to the One Health approach for Cryptococcosis, a zoonotic infection.

Induction of mucin and MUC5AC expression by the protease activity of Aspergillus fumigatus in airway epithelial cells.

Allergic bronchopulmonary mycosis, characterized by excessive mucus secretion, airflow limitation, bronchiectasis, and peripheral blood eosinophilia, is predominantly caused by a fungal pathogen, Aspergillus fumigatus. Using DNA microarray analysis of NCI-H292 cells, a human bronchial epithelial cell line, stimulated with fungal extracts from A. fumigatus, Alternaria alternata, or Penicillium notatum, we identified a mucin-related MUC5AC as one of the genes, the expression of which was selectively induced by A. fumigatus. Quantitative RT-PCR, ELISA, and histochemical analyses confirmed an induction of mucin and MUC5AC expression by A. fumigatus extracts or the culture supernatant of live microorganisms in NCI-H292 cells and primary cultures of airway epithelial cells. The expression of MUC5AC induced by A. fumigatus extracts diminished in the presence of neutralizing Abs or of inhibitors of the epidermal growth factor receptor or its ligand, TGF-α. We also found that A. fumigatus extracts activated the TNF-α-converting enzyme (TACE), critical for the cleavage of membrane-bound pro-TGF-α, and its inhibition with low-molecular weight inhibitors or small interfering RNA suppressed the expression of MUC5AC. The protease activity of A. fumigatus extracts was greater than that of other fungal extracts, and treatment with a serine protease inhibitor, but not with a cysteine protease inhibitor, eliminated its ability to activate TACE or induce the expression of MUC5AC mRNA in NCI-H292. In conclusion, the prominent serine protease activity of A. fumigatus, which caused the overproduction of mucus by the bronchial epithelium via the activation of the TACE/TGF-α/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.

Cryptococcus lacticolor sp. nov. and Rhodotorula oligophaga sp. nov., novel yeasts isolated from the nasal smear microbiota of Queensland koalas kept in Japanese zoological parks.

A total of 515 yeast strains were isolated from the nasal smears of Queensland koalas and their breeding environments in Japanese zoological parks between 2005 and 2012. The most frequent species in the basidiomycetous yeast biota isolated from koala nasal passages was Cryptococcus neoformans, followed by Rhodotorula minuta. R. minuta was the most frequent species in the breeding environments, while C. neoformans was rare. Seven strains representing two novel yeast species were identified. Analyses of the 26S rDNA (LSU) D1/D2 domain and nuclear ribosomal DNA internal transcribed spacer region sequences indicated that these strains represent new species with close phylogenetic relationships to Cryptococcus and Rhodotorula. A sexual state was not found for either of these two novel yeasts. Key phenotypic characters confirmed that these strains could be placed in Cryptococcus and Rhodotorula. The names Cryptococcus lacticolor sp. nov. (type strain TIMM 10013(T) = JCM 15449(T) = CBS 10915(T) = DSM 21093(T), DDBJ/EMBL/Genbank Accession No.; AB375774 (ITS) and AB375775 (26S rDNA D1/D2 region), MycoBank ID; MB 802688, Fungal Barcoding Database ID; 3174), and Rhodotorula oligophaga sp. nov. (type strain TIMM 10017(T) = JCM 18398(T) = CBS 12623(T) = DSM 25814(T), DDBJ/EMBL/Genbank Accession No.; AB702967 (ITS) and AB702967 (26S rDNA D1/D2 region), MycoBank ID; MB 802689, Fungal Barcoding Database ID; 3175) are proposed for these new species.

Incidence of pulmonary aspergillosis and correlation of conventional diagnostic methods with nested PCR and real-time PCR assay using BAL fluid in intensive care unit patients.

BACKGROUND: Although the incidence of invasive aspergillosis in the intensive care unit (ICU) is scarce, it has emerged as major problems in critically ill patients. In this study, the incidence of pulmonary aspergillosis (PA) in ICU patients has evaluated and direct microscopy and culture has compared with nested polymerase chain reaction (PCR) and real-time PCR for detection of Aspergillus fumigatus and A. flavus in bronchoalveolar lavage (BAL) samples of the patients. METHODS: Thirty BAL samples obtained from ICU patients during a 16-month period were subjected to direct examinations on 20% potassium hydroxide (KOH) and culture on two culture media. Nested PCR targeting internal transcribed spacer ribosomal DNA and TaqMan real-time PCR assay targeting ß-tubulin gene were used for the detection of A. fumigatus and A. flavus. RESULTS: Of 30 patients, 60% were men and 40% were women. The diagnosis of invasive PA was probable in 1 (3%), possible in 11 (37%), and not IPA in 18 (60%). Nine samples were positive in nested PCR including seven samples by A. flavus and two by A. fumigatus specific primers. The lowest amount of DNA that TaqMan real-time PCR could detect was ≥40 copy numbers. Only one of the samples had a positive result of A. flavus real-time PCR with Ct value of 37.5. CONCLUSIONS: Although a significant number of specimens were positive in nested PCR, results of this study showed that establishment of a correlation between the conventional methods with nested PCR and real-time PCR needs more data confirmed by a prospective study with a larger sample group.

Use of mycological, nested PCR, and real-time PCR methods on BAL fluids for detection of Aspergillus fumigatus and A. flavus in solid organ transplant recipients.

Invasive aspergillosis continues to be a significant cause of morbidity and mortality in solid organ transplant (SOT) recipients. A reliable and early diagnostic method is needed to improve survival. In this study, four methods direct microscopy, culture, nested PCR on internal transcribed spacer region, and TaqMan real-time PCR targeted ß-tubulin gene were examined for the detection of Aspergillus fumigatus and A. flavus in sixty-four bronchoalveolar lavage (BAL) fluids that were obtained from SOT recipients. Direct examination with 20 % KOH (potassium hydroxide) and culture on mycological media were also performed. Of the 64 samples, seven (10.9 %) were positive in direct examination (five with septate hyphae and two with aseptate hyphae), and 15 (23 %) had positive culture including five A. flavus, four A. niger, two Penicillium spp., two Rhizopus spp., one Fusarium spp. and one mixed A. flavus/A. niger. Twenty five (39 %) samples had positive nested PCR with A. flavus and 6 (9.4 %) with A. fumigatus-specific primers. Only eight (12.5 %) had positive real-time PCR for A. flavus and nine (14 %) for A. fumigatus. The incidence of aspergillosis in these patients included proven invasive pulmonary aspergillosis (IPA) in two (3 %), probable IPA in 14 (22 %), possible IPA in 38 (59 %), and not IPA in 10 (16 %). A. flavus was the most common cause of pulmonary aspergillosis (PA) in the study. The results suggest that because nested PCR is too sensitive it may increase the number of false-positive results and is not recommended for BAL samples for diagnosis of PA. Although further studies with significant number of proved positive/negative standard BAL samples are necessary for better evaluation, the novel multiplex real-time PCR developed in the study could be promising as a valid diagnostic method for IPA.