Co-fermentation of glucose, xylose and l-arabinose from lignocellulosic biomass by an oleaginous yeast is anticipated as a method for biodiesel production. However, most yeasts ferment glucose first before consuming pentoses, due to glucose repression. This preferential utilization results in delayed fermentation time and lower productivity. Therefore, co-fermentation of lignocellulosic sugars could achieve cost-effective conversion of lignocellulosic biomass to microbial lipid. Comprehensive screening of oleaginous yeasts capable of simultaneously utilizing glucose, xylose, and l-arabinose was performed by measuring the concentration of sugars remaining in the medium and of lipids accumulated in the cells. We found that of 1189 strains tested, 12 had the ability to co-ferment the sugars. The basidiomycete yeast Pseudozyma hubeiensis IPM1-10, which had the highest sugars consumption rate of 94.1 %, was selected by culturing in a batch culture with the mixed-sugar medium. The strain showed (1) simultaneous utilization of all three sugars, and (2) high lipid-accumulating ability. This study suggests that P. hubeiensis IPM1-10 is a promising candidate for second-generation biodiesel production from hydrolysate of lignocellulosic biomass.
Oleaginous yeast; Fatty acids; Xylose; l-arabinose; Pseudozyma hubeiensis
Here, we report the draft genome sequence of Trichosporon domesticum JCM 9580, isolated from the house of a patient with summer-type hypersensitivity pneumonitis (SHP) in Japan. This genomic information will help elucidate the mechanisms of the development of SHP.
A 10-year-old castrated male mixed-breed dog exhibited vomiting, lethargy and anorexia.
An abdominal ultrasound examination revealed a focal dilation of the abdominal aorta. The
dog died 2 days after examination, and necropsy revealed a ruptured aneurysm at the
abdominal aorta between the kidneys. Histological examination revealed severe
granulomatous and necrotizing pan-arteritis with fungal organisms.
Graphium species was identified through DNA sequence analysis of the
PCR product from formalin-fixed paraffin-embedded samples. To our knowledge, this is the
first report of aortic aneurysm caused by Graphium species in a dog.
abdominal aorta; canine; Graphium species; mycotic aneurysm
The order Trichosporonales (Tremellomycotina, Basidiomycota) includes various species that have clinical, agricultural and biotechnological value. Thus, understanding why and how evolutionary diversification occurred within this order is extremely important. This study clarified the phylogenetic relationships among Tricosporonales species. To select genes suitable for phylogenetic analysis, we determined the draft genomes of 17 Trichosporonales species and extracted 30 protein-coding DNA sequences (CDSs) from genomic data. The CDS regions of Trichosporon asahii and T. faecale were identified by referring to mRNA sequence data since the intron positions of the respective genes differed from those of Cryptococcus neoformans (outgroup) and are not conserved within this order. A multiple alignment of the respective gene was first constructed using the CDSs of T. asahii, T. faecale and C. neoformans, and those of other species were added and aligned based on codons. The phylogenetic trees were constructed based on each gene and a concatenated alignment. Resolution of the maximum-likelihood trees estimated from the concatenated dataset based on both nucleotide (72,531) and amino acid (24,173) sequences were greater than in previous reports. In addition, we found that several genes, such as phosphatidylinositol 3-kinase TOR1 and glutamate synthase (NADH), had good resolution in this group (even when used alone). Our study proposes a set of genes suitable for constructing a phylogenetic tree with high resolution to examine evolutionary diversification in Trichosporonales. These can also be used for epidemiological and biogeographical studies, and may also serve as the basis for a comprehensive reclassification of pleomorphic fungi.
Cryptococcus neoformans causes life-threatening diseases mainly in immunosuppressed hosts such as AIDS patients; C. gattii causes disseminated infections even in healthy hosts. To identify the possible molecular mechanisms underlying this difference in virulence, we investigated the survival and histopathology of lung tissue in wild-type and CD4-depleted mice infected with C. neoformans H99 and C. gattii JP02 (the highly virulent strain isolated in Japan); we then compared dendritic cell (DC) cytokine release responses to different cell fractions from these two strains. JP02-infected mice exhibited shorter survival and fewer inflammatory cells in the lung than H99-infected control mice. Depletion of CD4-related cellular immunity reduced survival of H99-infected mice but had no effect on the survival or inflammatory cell infiltration in JP02-infected mice, suggesting that JP02 evades immune detection. To identify the molecule(s) conferring this difference, we measured cytokine production from murine DCs co-cultured with H99 and JP02 in vitro. The levels of inflammatory cytokines from DCs treated with intact JP02 cells, the extracted capsule, secreted extracellular polysaccharides, and purified glucuronoxylomannan (GXM) were markedly lower than those induced by intact H99 cells and corresponding H99 fractions. Structural analysis of GXM indicated that JP02 altered one of two O-acetyl groups detected in the H99 GXM. Deacetylated GXM lost the ability to induce inflammatory cytokine release from DCs, implicating these O-acetyl groups in immune recognition. We conclude that the highly virulent C. gattii processes a structural alteration in GXM that allows this pathogen to evade the immune response and therefore elimination.
Cryptococcus gattii; Cryptococcus neoformans; capsule; glucuronoxylomannan; structure; O-acetylation; innate immune responses; dendritic cells
Previous space research conducted during short-term flight experiments and long-term environmental monitoring on board orbiting space stations suggests that the relationship between humans and microbes is altered in the crewed habitat in space. Both human physiology and microbial communities adapt to spaceflight. Microbial monitoring is critical to crew safety in long-duration space habitation and the sustained operation of life support systems on space transit vehicles, space stations, and surface habitats. To address this critical need, space agencies including NASA (National Aeronautics and Space Administration), ESA (European Space Agency), and JAXA (Japan Aerospace Exploration Agency) are working together to develop and implement specific measures to monitor, control, and counteract biological contamination in closed-environment systems. In this review, the current status of microbial monitoring conducted in the International Space Station (ISS) as well as the results of recent microbial spaceflight experiments have been summarized and future perspectives are discussed.
International Space Station; microbial monitoring; on-site analysis
Starch is considered a potential feedstock for biofuel production, particularly in light of the large-scale landfilling of food waste and other starchy materials worldwide. Lipid accumulation by oleaginous yeast is a promising method for biodiesel production from starch. However, most oleaginous yeasts are grown on monosaccharides or oligosaccharides because they cannot directly utilize starch. We therefore investigated the starch-assimilation ability of 1,200 yeasts. We found that Cryptococcus terricola could be used for fuel production through consolidated bioprocessing. C. terricola JCM 24523 exhibited the highest lipid content of 61.96% on medium with 5% starch at 10 days. Fatty acid methyl ester analysis showed that this strain produced high proportions of C16:0 and C18 fatty acids when grown on starch, which are ideal for use in biodiesel. Considering the yield and cost, lipids derived from starch using C. terricola would be a promising alternative source for biodiesel production.
Hypersensitivity pneumonitis is defined as an allergic lung disease that occurs in response to inhalation of fungal antigens, bacterial antigens, chemicals, dusts, or animal proteins. The incidence of summer-type hypersensitivity pneumonitis is higher in the summer season, especially in Japan, due to the influence of the hot and humid environment and the common style of wood house or old concrete condominiums.
The present report describes a case of a middle-aged married couple who lived in the same house and who simultaneously suffered from summer-type hypersensitivity pneumonitis. This report analyzes these two cases in terms of environmental research and its microbiological, radiological, and pathological aspects. This case report is followed by a review of family occurrences of summer-type hypersensitivity pneumonitis from 22 studies with a total of 49 patients (including the two present cases) in Japan.
Summer-type hypersensitivity pneumonitis may be unrecognized and misdiagnosed as pneumonia or other respiratory diseases. A greater understanding of the clinical, pathologic, and environmental features of summer-type hypersensitivity pneumonitis might help improve diagnosis and delivery of appropriate management for this condition.
Familial summer-type hypersensitivity pneumonitis; Climate; Geography; Trichosporon species; Environmental factor
An understanding of the role of yeasts in the environment has been uncertain because estimates of population size and diversity have often been based on species identifications that were determined from a limited number of phenotypic characteristics. DNA-based species identification has now become widely used, allowing an accurate assessment of species in different habitats. However, there are still problems in classification because some genera are polyphyletic. Consequently, the identification of yeasts and measurement of their diversity at the genus level remains difficult, as does assignment of genera to higher taxonomic ranks.
A total of 1021 yeast strains was isolated from soil samples and plant materials collected from Japan’s subtropical Iriomote Island and the cool temperate Rishiri Island. Based on sequence analyses of the D1/D2 domain of the LSU rRNA gene, these 1021 strains were tentatively classified into 183 species, with apparent new species accounting for approximately half of the total species isolated (60 and 46, Iriomote and Rishiri, respectively). The yeast species composition was statistically different between the two sites with only 15 species in common. Rarefaction curves of respective sources/areas gave distinctive patterns when the threshold of sequence identity became broader, indicating that the yeast diversity was distinct at the different taxonomic levels compared.
Our isolation study of yeasts in Japan has enabled us to expand the inventory of species diversity because a large number of new species was observed in the sampling areas. Further, we propose use of a particular diversity threshold as an “indicator” to recognize species, genera and higher taxonomic ranks.
One of the major components of telomerase is the human telomerase reverse transcriptase (hTERT) as the catalytic protein. hTERT mRNA expression are reported to be associated with prognosis and tumor progression in several sarcomas. However, there is no clear understanding of the mechanisms of hTERT in human sarcomas. Recent studies have suggested that signals transmitted through p38 mitogen-activated protein kinase (MAPK) can increase or decrease hTERT transcription in human cells. The purpose of this study was to analyse the correlation between p38 MAPK and hTERT in sarcoma samples.
We investigated 36 soft tissue malignant fibrous histiocytomas (MFH), 24 liposarcomas (LS) and 9 bone MFH samples for hTERT and p38 MAPK expression. Quantitative detection of hTERT and p38 MAPK was performed by RT-PCR.
There was a significant positive correlation between the values of hTERT and p38 MAPK in all samples (r = 0.445, p = 0.0001), soft tissue MFH (r = 0.352, p = 0.0352), LS (r = 0.704, p = 0.0001) and bone MFH samples (r = 0.802, p = 0.0093). Patients who had a higher than average expression of p38 MAPK had a significantly worse prognosis than other patients (p = 0.0036).
p38 MAPK may play a role in up-regulation of hTERT, and therefore, p38 MAPK may be a useful marker in the assessment of hTERT and patients' prognosis in sarcomas.
p38 mitogen-activated protein kinase; human telomerase reverse transcriptase; malignant fibrous histiocytoma; liposarcoma
Atopic dermatitis of the head and neck (HNAD) is recognized as a separate condition. Malassezia, the predominant skin microbiota fungus, is considered to exacerbate atopic dermatitis (AD), especially HNAD. In the present study, we investigated the relationships between the levels of specific IgE antibodies, colonization frequency of eight predominant Malassezia species, and clinical severity in 61 patients with HNAD (26 mild, 24 moderate, and 11 severe cases). As clinical severity increased, the levels of specific IgE antibodies against eight Malassezia species also increased. Species diversity of the Malassezia microbiota in scale samples from patients was analyzed by nested PCR using species-specific primers. The clinical severity of HNAD was correlated with the total level of specific IgE antibodies against Malassezia species and the number of Malassezia species detected.
We report a patient in Japan infected with Cryptococcus gattii genotype VGIIa who had no recent history of travel to disease-endemic areas. This strain was identical to the Vancouver Island outbreak strain R265. Our results suggest that this virulent strain has spread to regions outside North America.
Cryptococcus gattii; central nervous system infection; pulmonary cryptococcosis; genotype; multilocus sequence typing; parasites; Japan; dispatch
Liposarcoma is categorized as a soft tissue sarcoma and most commonly appears in the lower extremities and rarely in the foot during adulthood. We present a very rare case report of a primary well-differentiated liposarcoma arising in the foot on a 60-year-old female. Marginal resection of the tumor with metatarsal ray amputation was eventually performed. The patient's postoperative course was uneventful without recurrence 5 years after the original operation. The authors review the literature and also report on the low incidence of this tumor arising in the foot.
The function of promyelocytic leukemia (PML) bodies is not well known but plays an important role in controlling cell proliferation, apoptosis and senescence. This study was undertaken to analyze the clinical significance of PML body expression in primary tumor samples from malignant fibrous histiocytoma (MFH) and liposarcoma patients.
We studied MFH and liposarcoma samples from 55 patients for PML bodies. Fluorescent immunostaining of PML bodies was performed in the paraffin-embedded tumor sections.
PML body immunostaining was identified in 63.9% of MFH and 63.2% of liposarcoma samples. PML body expression rates of all sarcoma cells were 1.5 ± 1.8% (range: 0–7.0) in MFH and 1.3 ± 1.4% (0–5.2) in liposarcoma samples. PML body expression (p = 0.0053) and a high rate of PML body expression (p = 0.0012) were significantly greater prognostic risk factors for death than the other clinical factors in MFH patients. All liposarcoma patients without expression of PML were disease free at the end of the study.
Our study suggests that the presence of PML bodies may indicate a poor prognosis for MFH and liposarcoma patients.
Forty-six strains of Malassezia spp. with atypical biochemical features were isolated from 366 fresh clinical isolates from human subjects and dogs. Isolates obtained in this study included 2 (4.7%) lipid-dependent M. pachydermatis isolates; 1 (2.4%) precipitate-producing and 6 (14.6%) non-polyethoxylated castor oil (Cremophor EL)-assimilating M. furfur isolates; and 37 (34.3%) M. slooffiae isolates that were esculin hydrolyzing, 17 (15.7%) that were non-tolerant of growth at 40°C, and 2 (1.9%) that assimilated polyethoxylated castor oil. Although their colony morphologies and sizes were characteristic on CHROMagar Malassezia medium (CHROM), all strains of M. furfur developed large pale pink and wrinkled colonies, and all strains of M. slooffiae developed small (<1 mm) pale pink colonies on CHROM. These atypical strains were distinguishable by the appearance of their colonies grown on CHROM. Three clinically important Malassezia species, M. globosa, M. restricta, and M. furfur, were correctly identified by their biochemical characteristics and colony morphologies. The results presented here indicate that our proposed identification system will be useful as a routine tool for the identification of clinically important Malassezia species in clinical laboratories.
Yeasts from caves have rarely been examined. We examined yeasts collected from bat guano samples from 20 bat-inhabited limestone and volcanic caves located in 11 prefectures in Japan. Of ∼700 yeast-like colonies, nine Trichosporon species were recovered from 15 caves. Two of these were known species, and the remaining seven are potentially novel species, based on molecular phylogenetic analyses. In addition to Trichosporon species, identifiable strains of eight ascomycetous yeasts and one basidiomycetous yeast were recovered at frequencies of 5 to 35%. Our findings suggest that Trichosporon spp. are the major yeast species in bat guano in Japan and that bat guano is a potentially rich source of previously undescribed yeast species.
Brachytherapy, interstitial tumor bed irradiation, following conservative surgery has been shown to provide excellent local control and limb preservation in patients with soft tissue sarcomas (STS), whereas little is known about the tolerance of peripheral nerves to brachytherapy. In particular, nerve tolerance to high-dose-rate (HDR) brachytherapy has never been properly evaluated. In this study, we examined the efficacy and radiation neurotoxicity of HDR brachytherapy in patients with STS in contact with neurovascular structures.
Between 1995 and 2000, seven patients with STS involving the neurovascular bundle were treated in our institute with limb-preserving surgery, followed by fractionated HDR brachytherapy. Pathological examination demonstrated that 6 patients had high-grade lesions with five cases of negative margins and one case with positive margins, and one patient had a low-grade lesion with a negative margin. Afterloading catheters placed within the tumor bed directly upon the preserved neurovascular structures were postoperatively loaded with Iridium-192 with a total dose of 50 Gy in 6 patients. One patient received 30 Gy of HDR brachytherapy combined with 20 Gy of adjuvant external beam radiation.
With a median follow-up of 4 years, the 5-year actuarial overall survival, disease-free survival, and local control rates were 83.3, 68.6, and 83.3%, respectively. None of the 7 patients developed HDR brachytherapy-induced peripheral neuropathy. Of 5 survivors, 3 evaluable patients had values of motor nerve conduction velocity of the preserved peripheral nerve in the normal range.
In this study, there were no practical and electrophysiological findings of neurotoxicity of HDR brachytherapy. Despite the small number of patients, our encouraging results are valuable for limb-preserving surgery of unmanageable STS involving critical neurovascular structures.
The lipophilic yeast Malassezia is an exacerbating factor in atopic dermatitis (AD) and colonizes the skin surface of patients with AD. With the goal of reducing the number of Malassezia cells, we investigated the antifungal activities of a therapeutic agent for AD, tacrolimus, and the azole agents itraconazole and ketoconazole against Malassezia species in vitro. We examined 125 strains of the 11 currently accepted Malassezia species by using the agar dilution method. All strains of the 11 Malassezia species were very susceptible to both azole agents, with MICs ranging from 0.016 to 0.25 μg/ml. Tacrolimus had antifungal activities against half of the strains, with MICs ranging from 16 to 32 μg/ml. Two of the major cutaneous floras, Malassezia globosa and Malassezia restricta, have several genotypes in the intergenic spacer region of the rRNA gene; the azole agents had slightly higher MICs for specific genotype strains of both microorganisms. A combination of azole agents and tacrolimus had a synergistic effect against Malassezia isolates, based on a fractional inhibitory index of 0.245 to 0.378. Our results provide the basis for testing these agents in future clinical trials to reduce the number of Malassezia cells colonizing the skin surface in patients with AD.
Summer-type hypersensitivity pneumonitis (SHP) is type III or IV allergies developed by repeated inhalation of arthroconidia of Trichosporon species. We identified 105 strains obtained from the homes of 36 SHP patients by analysis of the intergenic spacer (IGS) 1 region, which is located between the 26S and 5S rRNA genes; in addition, we analyzed the IGS genotypes of the strains. Serologically, Trichosporon species are classified as serotype I, II, III, or I-III. Of the 105 strains, 43 (41.1%), 53 (50.5%), and 9 (8.6%) strains were isolated as serotypes I, II, and III, respectively. Serotype I, II, and III strains were recovered from 19 (52.8%), 29 (80.6%), and 4 (11.1%) of the 36 houses of SHP patients, respectively. No serotype I-III strains were isolated from the houses. Of 43 serotype I strains, 42 (97.7%) were identified as Trichosporon dermatis, and the remaining one was T. terricola. Of 53 serotype II strains, 37 (69.8%) were identified as T. asahii, and the remaining serotype II isolates were T. aquatile (1.9%), T. coremiiforme (7.5%), T. faecale (1.9%), T. japonicum (15.1%), and T. ovoides (3.8%). There were nine serotype III strains comprised of T. montevideense (77.8%) and T. domesticum (22.2%). Intraspecies diversity was found only in T. asahii. This microorganism also causes opportunistic infections (trichosporonosis); seven genotypes of its IGS 1 region have been identified. While the strains of T. asahii obtained from Japanese patients with trichosporonosis were genotype I, the strains from the houses of SHP patients were genotype III. Based on our analysis, we conclude that the strains that play the most significant roles in the development of SHP are T. dermatis, T. asahii genotype 3, and T. montevideense, representing serotypes I, II, and III, respectively.
We have developed magnetite cationic liposomes (MCLs) and applied them to local hyperthermia as a mediator. MCLs have a positive charge and generate heat under an alternating magnetic field (AMF) by hysteresis loss. In this study, the effect of hyperthermia using MCLs was examined in an in vivo study of hamster osteosarcoma.
MCLs were injected into the osteosarcoma and then subjected to an AMF.
The tumor was heated at over 42°C, but other normal tissues were not heated as much. Complete regression was observed in 100% of the treated group hamsters, whereas no regression was observed in the control group hamsters. At day 12, the average tumor volume of the treated hamsters was about 1/1000 of that of the control hamsters. In the treated hamsters, no regrowth of osteosarcomas was observed over a period of 3 months after the complete regression.
These results suggest that this treatment is effective for osteosarcoma.
Lipophilic yeasts of the genus Malassezia are part of the normal cutaneous microflora and are considered one of the factors that trigger atopic dermatitis (AD). We isolated two strains of Malassezia from a healthy Japanese female. Analysis of the D1/D2 26S ribosomal DNA and internal transcribed spacer region sequences of the isolates suggested that they are new members of the genus Malassezia. We propose the name Malassezia japonica sp. nov. for the isolates. M. japonica is easily distinguished from the seven known lipophilic species by its ability to assimilate Tween 40 and Tween 60 and its inability to assimilate Tween 20 and Tween 80 and to grow at 40°C. Furthermore, by applying transparent dressings to the skin lesions of 36 patients with AD and the skin of 22 healthy subjects, M. japonica DNA was detected by a non-culture-based method consisting of nested PCR with M. japonica species-specific primers. M. japonica DNA was detected from 12 of the 36 patients (33.3%) and 3 of the 22 healthy subjects (13.6%). Although it is not known whether M. japonica plays a role in AD, this species was part of the microflora in both patients with AD and healthy subjects.
The lipophilic yeast Malassezia globosa is one of the major constituents of the mycoflora of the skin of patients with atopic dermatitis (AD). We compared the genotypes of M. globosa colonizing the skin surface of 32 AD patients and 20 healthy individuals for polymorphism of the intergenic spacer (IGS) 1 region of the rRNA gene. Sequence analysis demonstrated that M. globosa was divided into four major groups, which corresponded to the sources of the samples, on the phylogenetic tree. Of the four groups, two were from AD patients and one was from healthy subjects. The remaining group included samples from both AD patients and healthy subjects. In addition, the IGS 1 region of M. globosa contained short sequence repeats: (CT)n, and (GT)n. The number of sequence repeats also differed between the IGS 1 of M. globosa from AD patients and that from healthy subjects. These findings suggest that a specific genotype of M. globosa may play a significant role in AD, although M. globosa commonly colonizes both AD patients and healthy subjects.
We determined the sequence of the intergenic spacer (IGS) 1 region, which is located between the 26S and 5S rRNA genes, in 25 species of the genus Trichosporon. IGS 1 sequences varied in length from 195 to 719 bp. Comparative sequence analysis suggested that the divergence of IGS 1 sequences has been greater than that of the internal transcribed spacer regions. We also identified five genotypes of T. asahii, which is a major causative agent of deep-seated trichosporonosis, based on the IGS 1 sequences of 43 strains. Most of the isolates that originated in Japan were of genotype 1, whereas the American isolates were of genotype 3 or 5. Our results suggest that analysis of IGS regions provides a powerful method to distinguish between phylogenetically closely related species and that a geographic substructure may exist among T. asahii clinical isolates.