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Coral microbial flora has been attracting attention because of their potential to protect corals from environmental stresses or pathogens. Although coral-associated bacteria are considered to be acquired from seawater, little is known about the relationships between microbial composition in corals and its surrounding seawater. Here, we tested several methods to identify coral-associated bacteria in coral and its surrounding seawater to detect specific types of Ruegeria species, some of which exhibit growth inhibition activities against the coral pathogen Vibrio coralliilyticus. We first isolated coral-associated bacteria from the reef-building coral Galaxea fascicularis collected at Sesoko Island, Okinawa, Japan, via random colony picking, which showed the existence of varieties of bacteria including Ruegeria species. Using newly constructed primers for colony PCR, several Ruegeria species were successfully isolated from G. fascicularis and seawater. We further investigated the seawater microbiome in association with the distance from coral reefs. By seasonal sampling, it was suggested that the seawater microbiome is more affected by seasonality than the distance from coral reefs. These methods and results may contribute to investigating and understanding the relationships between the presence of corals and microbial diversity in seawater, in addition to the efficient isolation of specific bacterial species from coral or its surrounding seawater.

Coral symbionts are important members of the coral holobiont, and the coral bacterial flora is essential in host health maintenance and coral conservation. Coral symbionts are affected by various environmental factors, such as seawater temperature, pH, and salinity. However, the effect of communication between different coral species and water flow against coral bacterial flora on its community structure is not well understood. In this study, we designed an artificial rearing environment to examine the impact of environmental and biological factors on Acropora tenuis, one of the major coral species in Okinawa, and Montipora digitata, from their co-culture. We intervened with the water flow to reveal that the movement of the rearing environment alters the bacterial flora of A. tenuis. During the bacterial community reconstruction, the alpha diversity of the coral microbiota increased, suggesting the establishment of rare bacteria from the ocean. No differences in the bacterial composition between the control and water flow groups were observed under the rearing conditions. However, the structure of the bacterial flora was significantly different in the co-culture group. Comparison among each bacterial community succession strongly suggest that the difference was due to the suppressed transmission of bacteria from the ocean in the co-culture group. These results contribute to the understanding of communication between corals and shed light on the importance of regional differences and bacterial composition of coral flora.

Coral symbionts are important members of the coral holobiont, and coral bacterial flora are essential in host health maintenance and coral conservation. Coral symbionts are affected by various environmental factors, such as seawater temperature, pH, and salinity. Although physicochemical and chemical factors have been highlighted as possible causes of these effects, the effects of water flow and the co-culture of different species corals have not been elucidated. In this study, we designed an artificial rearing environment to examine the impact of environmental and biological factors on Acropora tenuis, one of the major coral species in Okinawa, and Montipora digitata, during their co-culture. We intervened with the water flow to reveal that the movement of the rearing environment alters the bacterial flora of A. tenuis. During the rearing under captive environment, the alpha diversity of the coral microbiota increased, suggesting the establishment of rare bacteria from the ocean. No differences in the bacterial composition between the control and water flow groups were observed under the rearing conditions. However, the structure of the bacterial flora was significantly different in the co-culture group. Comparison of bacterial community succession strongly suggested that the differences observed were due to the suppressed transmission of bacteria from the ocean in the co-culture group. These results enhance our understanding of interactions between corals and shed light on the importance of regional differences and bacterial composition of coral flora.

The bacterium “ Candidatus Hydrogenosomobacter endosymbioticus” is an intracellular symbiont of anaerobic scuticociliate GW7, which is associated with hydrogenosome together with methanogenic archaea. Here, we report a complete genome sequence of the symbiont consisting of 827 kbp. Knowing this sequence would contribute to the understanding of the metabolic interactions and evolution of the tripartite symbiosis.

Brown algae contain a polysaccharide-rich cell wall, mainly composed of alginate and fucoidan which have been extensively studied for their individual structure and bioactivities. Particularly, the cell wall of Cladosiphon okamuranus is rich in fucoidan rather than alginate. However, little is known about its arrangement or interlinking with other polysaccharides such as cellulose in the cell wall. To determine its structure in detail, the cell wall was sequentially fractionated into five fractions: hot water (HW), ammonium oxalate, hemicellulose-I (HC-I), HC-II, and cellulose. Almost 80% of the total cell wall recovered from alcohol insoluble residue in C. okamuranus consisted of HW and HC-I, which mainly contained fucoidan composed of fucose, glucuronic acid, and sulfate in molar ratios of 1.0:0.3:0.9 and 1.0:0.2:0.3, respectively. Methylation analysis revealed that fucoidan in HW and HC-I structurally differed in terms of content of sulfate, and sugar residue which was 1,4-linked xylose and 1,4-linked fucose. Small angle X-ray scattering measurements also showed distinct conformational differences between HW and HC-I. These structural heterogeneities of fucoidan may be related to their localization, and fucoidan in HC-I may be involved in reinforcing cell wall structure by cross-linking to cellulose.

Corals create an ecosystem, called a holobiont, with intracellular algae (zooxanthellae) and resident bacteria. Zooxanthellae and some bacteria play major roles in the physiological properties of the coral host. However, because of the difficulties in experimental verification of cross-organism interactions, the mechanisms underpinning these interactions are largely unknown. To address this, we here generated and then analyzed multi-omics datasets for corals, zooxanthellae, and bacteria collected at Okinawa, Japan, from November 2014 to September 2016. Using cross-organism co-expression analysis, we successfully characterized the host–alga relationship in the coral holobiont. Specifically, we observed that the coral host dominates the zooxanthellae. The multi-omics analysis also suggested that infection with coral-associated bacteria Endozoicomonas likely involves coral-like ephrin ligands, triggering an immune response of the coral host. This study highlights the potential of the multi-omics approach to elucidate coral–microbe interactions.

Fusarium head blight (FHB) of cereals is a severe disease caused by the Fusarium graminearum species complex. It leads to the accumulation of the mycotoxin deoxynivalenol (DON) in grains and other plant tissues and causes substantial economic losses throughout the world. DON is one of the most troublesome mycotoxins because it is a virulence factor to host plants, including wheat, and exhibits toxicity to plants and animals. To control both FHB and DON accumulation, a biological control approach using DON-degrading bacteria (DDBs) is promising. Here, we performed a disease control assay using an in vitro petri dish test composed of germinated wheat seeds inoculated with F. graminearum (Fg) and DDBs. Determination of both grown leaf lengths and hyphal lesion lengths as a measure of disease severity showed that the inoculation of seeds with the DDBs Devosia sp. strain NKJ1 and Nocardioides spp. strains SS3 or SS4 were protective against the leaf growth inhibition caused by Fg. Furthermore, it was as effective against DON accumulation. The inoculation with strains SS3 or SS4 also reduced the inhibitory effect on leaves treated with 10 &micro, g mL&minus, 1 DON solution (without Fg). These results indicate that the DDBs partially suppress the disease by degrading DON.

Manganites exhibit various types of electronic phenomena, and these electronic characteristics can be controlled by carrier doping. Herein, we report the dielectric and magnetic properties of electron-doped manganite Ca$_{1-x}$Sr$_x$Mn$_{0.85}$Sb$_{0.15}$O$_3$ ($x$ = 0, 0.1, 0.2, and 0.3). The temperature dependence of the real part of the dielectric constant exhibits a broad and large peak just below the kink temperature of magnetization and a sharp decrease at lower temperatures, accompanied by an anomaly of the imaginary part. Furthermore, isovalent Sr substitution enhances the temperature of the dielectric peak by more than 50 K. Interestingly, the dielectric peak exhibits a negative magnetic-field effect. For all measured samples, the low-temperature variation of the dielectric constant can be qualitatively explained based on the Maxwell-Wagner (MW) model that describes a system composed of grain boundaries and semiconducting grains. However, the observed peak and its negative magneto-capacitance effect at high temperatures cannot be reproduced by a combination of the MW model and magnetoresistance effect. The dielectric peak strongly indicates polaronic relaxation in the present system. These results suggest that polarons form clusters with a dipole ordering and magneto-electric coupling, which might be consistently understood by the charge-ordering scenario.

Okinawa is the southwesternmost part of Japan and represents the northern limit of coral reefs that are influenced by the Kuroshio warm current. We have been studying two coral reefs off Sesoko Island, in northern Okinawa, to evaluate coral reef environments since 2014. In this study, we applied a metagenomic approach to analyze the seasonal composition of picophytoplankton at these two reef sites (Sesoko Minami and Ishikawabaru). The two sites were selected for their differences: Sesoko Minami faces the open ocean and experiences relatively small land effects, while Ishikawabaru experiences relatively high land effects and is surrounded by barrier reefs. Seawater samples were collected from these two sites and filtered through a 1.6 μm pore filter followed by a 0.2 μm membrane filter. DNA was extracted from the 0.2 μm filters, and the V1-V2 region of the 16S rRNA gene (260 bp) was amplified before sequencing using next-generation sequencing techniques. Cyanobacteria or chloroplast sequences were selected for further metabarcording analyses. Interestingly, almost no differences were indicated between the two sites and more than 95% of the picophytoplankton at both sampling sites was composed of only 5 to 7 OTUs. The seasonal composition of picophytoplankton clearly changed; in summer (Jul 2014, May to Jul 2015, and May to Aug 2016) OTU 1 (Synechococcus sp. NC007513) was most abundant. OTU 4 (Prochlorococcus marinus NC009091) also reached its maximum abundance in summer (May to Aug 2016), while OTU 6 (Bathycoccous prasinos FN563099) increased in winter. OTU 8 (Micromonas commoda RCC299 FJ858267) was present at every sampling period and accounted for 5–20% of the total picophytoplankton contig numbers. Our results indicate that seasonal changes clearly influence fluctuations in picophytoplankton community compositions.

Study DesignControlled laboratory study.PurposeThis study aimed to evaluate the efficacy of platelet-rich plasma (PRP) stored at room temperature (RT), frozen, or after freeze-drying.Overview of LiteraturePRP enriches tissue repair and regeneration, and is a novel treatment option for musculoskeletal pathologies. However, whether biological activity is preserved during PRP storage remains uncertain.MethodsPRP was prepared from blood of 12 healthy human volunteers (200 mL/person) and stored using three methods: PRP was stored at RT with shaking, PRP was frozen and stored at −80℃, or PRP was freeze-dried and stored at RT. Platelet counts and growth factor content were examined immediately after preparation, as well as 2, 4, and 8 weeks after storage. Platelet activation rate was quantified by flow cytometry.ResultsPlatelet counts were impossible to determine in many RT samples after 2 weeks, but they remained at constant levels in frozen and freeze-dried samples, even after 8 weeks of storage. Flow cytometry showed approximately 80% activation of the platelets regardless of storage conditions. Almost no growth factors were detected in the RT samples after 8 weeks, while low but significant expression was detected in the frozen and freeze-dried PRP. Over time, the mean relative concentrations of various growth factors decreased significantly or disappeared in the RT group. In the frozen group, levels were maintained for 4 weeks, but decreased significantly by 8 weeks (p ConclusionsFreeze-drying enables PRP storage while maintaining bioactivity and efficacy for extended periods.

A number of anaerobic ciliates, unicellular eukaryotes, intracellularly possess methanogenic archaea and bacteria as symbiotic partners. Although this tripartite relationship is of interest in terms of the fact that each participant is from a different domain, the difficulty in culture and maintenance of those host species with symbiotic partners has disturbed both ecological and functional studies so far. In this study, we obtained a stable culture of a small anaerobic scuticociliate, strain GW7. By transmission electron microscopic observation and fluorescent in situ hybridization with domain-specific probes, we demonstrate that GW7 possesses both archaeal and bacterial endosymbionts in its cytoplasm. These endosymbionts are in dependently associated with hydrogenosomes, which are organelle producing hydrogen and ATP under anaerobic conditions. Clone library analyses targeting prokaryotic 16S rRNA genes, fluorescent in situ hybridization with endosymbiont-specific probes, and molecular phylogenetic analyses revealed the phylogenetic affiliations and intracellular localizations of these endosymbionts. The endosymbiotic archaeon is a methanogen belonging to the genus Methanoregula (order Methanomicrobiales); a member of this genus has previously been described as the endosymbiont of an anaerobic ciliate from the genus Metopus (class Armophorea), which is only distantly related to strain GW7 (class Oligohymenophorea). The endosymbiotic bacterium belongs to the family Holosporaceae of the class Alphaproteobacteria, which also comprises several endosymbionts of various aerobic ciliates. For this endosymbiotic bacterium, we propose a novel candidate genus and species, "Candidatus Hydrogenosomobacter endosymbioticus."IMPORTANCE Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates.

A number of anaerobic ciliates, unicellular eukaryotes, intracellularly possess methanogenic archaea and bacteria as symbiotic partners. Although this tripartite relationship is of interest in terms of the fact that each participant is from a different domain, the difficulty in culture and maintenance of those host species with symbiotic partners has disturbed both ecological and functional studies so far. In this study, we obtained a stable culture of a small anaerobic scuticociliate, strain GW7. By transmission electron microscopic observation and fluorescent in situ hybridization with domain-specific probes, we demonstrate that GW7 possesses both archaeal and bacterial endosymbionts in its cytoplasm. These endosymbionts are in dependently associated with hydrogenosomes, which are organelle producing hydrogen and ATP under anaerobic conditions. Clone library analyses targeting prokaryotic 16S rRNA genes, fluorescent in situ hybridization with endosymbiont-specific probes, and molecular phylogenetic analyses revealed the phylogenetic affiliations and intracellular localizations of these endosymbionts. The endosymbiotic archaeon is a methanogen belonging to the genus Methanoregula (order Methanomicrobiales); a member of this genus has previously been described as the endosymbiont of an anaerobic ciliate from the genus Metopus (class Armophorea), which is only distantly related to strain GW7 (class Oligohymenophorea). The endosymbiotic bacterium belongs to the family Holosporaceae of the class Alphaproteobacteria, which also comprises several endosymbionts of various aerobic ciliates. For this endosymbiotic bacterium, we propose a novel candidate genus and species, “Candidatus Hydrogenosomobacter endosymbioticus.” IMPORTANCE Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates.

Actinomycete Nocardioides sp. strain LS1, isolated from wheat leaf, is a bacterium that degrades and assimilates the mycotoxin deoxynivalenol (DON) as the carbon source., Actinomycete Nocardioides sp. strain LS1, isolated from wheat leaf, is a bacterium that degrades and assimilates the mycotoxin deoxynivalenol (DON) as the carbon source. This is the first study of the genome sequence of the DON-degrading genus Nocardioides, and it facilitates the study of genes encoding the DON-degrading pathway.

Gut bacteria of phytophagous and omnivorous marine invertebrates often possess alginate lyases (ALGs), which are key enzymes for utilizing macroalgae as carbon neutral biomass. We hypothesized that the exclusive feeding of a target alga to marine invertebrates would shift the gut bacterial diversity suitable for degrading the algal components. To test this hypothesis, we reared sea hare (Dolabella auricularia) and sea snail (Batillus cornutus) for two to four weeks with exclusive feeding of a brown alga (Ecklonia cava). Pyrosequencing analysis of the gut bacterial 16S rRNA genes revealed shifts in the gut microbiota after rearing, mainly due to a decrease in the variety of bacterial members. Significant increases in six and four 16S rRNA gene phylotypes were observed in the reared sea hares and sea snails, respectively, and some of them were phylogenetically close to known alginate-degrading bacteria. Clone library analysis of PL7 family ALG genes using newly designed degenerate primer sets detected a total of 50 ALG gene phylotypes based on 90% amino acid identity. The number of ALG gene phylotypes increased in the reared sea hare but decreased in reared sea snail samples, and no phylotype was shared between them. Out of the 50 phylotypes, 15 were detected only after the feeding procedure. Thus, controlled feeding strategy may be valid and useful for the efficient screening of genes suitable for target alga fermentation.

Bacterial xylose isomerases (XI) are promising resources for efficient biofuel production from xylose in lignocellulosic biomass. Here, we investigated xylose isomerase gene (xylA) diversity in three soil metagenomes differing in plant vegetation and geographical location, using an amplicon pyrosequencing approach and two newly-designed primer sets. A total of 158,555 reads from three metagenomic DNA replicates for each soil sample were classified into 1127 phylotypes, detected in triplicate and defined by 90% amino acid identity. The phylotype coverage was estimated to be within the range of 84.0-92.7%. The xylA gene phylotypes obtained were phylogenetically distributed across the two known xylA groups. They shared 49-100% identities with their closest-related XI sequences in GenBank. Phylotypes demonstrating

Background Context Platelet-rich plasma (PRP) accelerates bone union in vivo in a rodent model of spinal fusion surgery. However, PRP's effect on bone union after spinal surgery remains unclear. Purpose The objective of this study was to evaluate the efficacy of PRP after posterolateral lumbar fusion (PLF) surgery. Study Design/Setting Single-center prospective randomized controlled clinical trial with 2-year follow-up. Patient Sample The patient sample included a total 62 patients (31 patients in the PRP group or 31 patients in the control group). Outcome Measures The outcome measures included the bone fusion rate, the area of bone fusion mass, the duration of bone fusion, and the clinical score using the visual analog scale (VAS). Materials and Methods We randomized 62 patients who underwent one- or two-level instrumented PLF for lumbar degenerative spondylosis with instability to either the PRP (31 patients) or the control (31 patients) groups. Platelet-rich plasma-treated patients underwent surgery using an autograft bone chip (local bone), and PRP was prepared from patient blood samples immediately before surgery; patients from the control group underwent PLF without PRP treatment. We assessed platelet counts and growth factor concentrations in PRP prepared immediately before surgery. The duration of bone union, the postoperative bone fusion rate, and the area of fusion mass were assessed using plain radiography every 3 months after surgery and by computed tomography at 12 or 24 months. The duration of bone fusion and the clinical scores for low back pain, leg pain, and leg numbness before and 3, 6, 12, and 24 months after surgery were evaluated using VAS. Results Data from 50 patients with complete data were included. The bone union rate at the final follow-up was significantly higher in the PRP group (94%) than in the control group (74%) (p=.002). The area of fusion mass was significantly higher in the PRP group (572 mm 2 ) than in the control group (367 mm 2 ) (p=.02). The mean period necessary for union was 7.8 months in the PRP group and 9.8 months in the control group (p=.013). In the PRP, the platelet count was 7.7 times higher and the growth factor concentrations were 50 times higher than those found in plasma (p .05). Conclusions Patients treated with PRP showed a higher fusion rate, greater fusion mass, and more rapid bone union after spinal fusion surgery than patients not treated with PRP.

Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA ; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency ( k cat / K m ) of 6.4 mM −1 s −1 . The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat.

We investigated the chemical composition of hydrothermal fluids and deduced the origin of hydrothermal fluid methane in a sediment-associated Minami-Ensei (ME) hydrothermal field, the Okinawa Trough. The endmember hydrothermal fluid of the ME field contained isotopically 13 C-enriched methane (− 26.0 to − 24.7‰), which suggested a thermogenic origin. However, the observed methane-dominating hydrocarbon compositions (C 1 /C 2+ > 10 3 ) suggested that biogenic methane, not thermogenic hydrocarbons, is predominant. The conflict between these geochemical indications can be resolved by the hypothesis that the thermal reaction of sedimentary organic matter initially generates the primary source of hydrocarbons and the secondary thermal decomposition of hydrocarbons increases the C 1 /C 2+ ratio as high as that of biogenic methane. The low molecular hydrogen concentration (0.04 mmol/kg) in the ME fluid suggests that the thermal fluid–sediment interaction occurred in the recharge stage, not the discharge stage, of hydrothermal circulation. Less biogenic methane in the ME fluid implies a laterally shrunken hydrothermal fluid circulation that reduced biogenic methane incorporation at the recharge stage. A comparison of hydrothermal fluid chemistry and geological/geographical settings among the global sediment-associated hydrothermal systems also suggests representative patterns in the geochemical origins of hydrothermal fluid methane and their relevance to hydrothermal fluid circulation as a whole.

Triggering receptor expressed on myeloid cells-1 (TREM-1) was reported to play a key roll in amplification of production of inflammatory cytokines. TREM-1 is suggested to be a specific biomarker for sepsis for this reason, but the clinical significance of TREM-1 has not been elucidated. We investigated TREM-1 expression on the cell-surface, and plasma levels of soluble TREM-1 (sTREM-1) in patients with non-infectious systemic inflammatory response syndrome (SIRS) and sepsis admitted to the ICU.Thirty-five patients with SIRS and 21 patients with sepsis admitted to ICU were subjected to the study. TREM-1 expressions on the surfaces of monocytes and neutrophils were measured by flow cytometry. Plasma sTREM-1 level and serum interleukin (IL)-6 level were measured.Septic patients had decreased TREM-1 expression, clearly on neutrophils or to a lesser extent on monocyte compared to SIRS patients on ICU admission (neutrophils p0.001, monocyte p0.05). TREM-1 expression on neutrophils had a significant inverse correlation with serum IL-6 level (r=-0.64, p0.0001). Plasma sTREM-1 level in septic patients was significantly higher than that in SIRS patients (p0.05). Plasma sTREM-1 level positively correlated with severity score and non-survivors had increased plasma sTREM-1 level compared to survivors in all SIRS/sepsis patients (p0.05).Patients with sepsis had increased soluble TREM-1 and decreased TREM-1 expression on neutrophil compared to SIRS patients. sTREM-1 may be useful to evaluate disease severity and outcome of patients with SIRS or sepsis.

Activity-based screening techniques were applied to clone a gene encoding γ-hexachlorocyclohexane (γ-HCH) dehydrochlorinase with its flanking regions from a cosmid-based library of DNA that was extracted from a γ-HCH-added suspension of HCH-contaminated soil. A total of 11 cosmid clones showing the γ-HCH dehydrochlorinase activity were obtained through the screenings, indicating the validity of this approach. All the clones had a linA gene identical to known one, but its flanking regions showed some structural variations with known ones, suggesting high likelihood of genetic divergence in the linA-flanking regions.

Deoxynivalenol (DON) is a hazardous and globally prevalent mycotoxin in cereals. It commonly accumulates in the grain of wheat, barley and other small grain cereals affected by Fusarium head blight (caused by several Fusarium species). The concept of reducing DON in naturally contaminated grain of wheat or barley using a DON-degrading bacterium is promising but has not been accomplished. In this study, we isolated a novel DON-utilising actinomycete, Marmoricola sp. strain MIM116, from wheat heads through a novel isolation procedure including an in situ plant enrichment step. Strain MIM116 had background degradation activity, and the activity was enhanced twofold by the consumption of DON. Among Tween 20, Triton X-100 and Tween 80, we selected Tween 80 as a spreading agent of strain MIM116 because it promoted DON degradation and the growth of strain MIM116 in the presence of DON. The inoculation of MIM116 cell suspension plus 0.01% Tween 80 into 1,000 harvested kernels of wheat and barley resulted in a DON decrease from approximately 3 mg kg(-1) to less than 1 mg kg(-1) of dry kernels, even when cells had only basal levels of DON-degrading activity. To the best of our knowledge, this is the first report that describes (1) the isolation of a DON-degrading bacterium from wheat heads, (2) the effects of surfactants on the biodegradation of DON and (3) the decrease of DON levels in naturally contaminated wheat and barley grain using a DON-degrading bacterium.

“Super H 2 O-barrier film” with a water vapor transmission rate (WVTR) less than 1 mg/m 2 /day has been developed. The barrier layer is a single layer of amorphous SiCN grown by organic Cat-CVD (O-Cat-CVD) with a thickness of 100 nm. SiCN has been grown by using a gas mixture of monomethylsilane (MMS; Si (CH 3 )H 3 ), NH 3 and H 2 on polyethylene-naphthalate (PEN) film substrates. It has been found that the WVTR drastically depends on the W-filament temperature of O-Cat-CVD. The WVTR changed from 5 × 10 −1 to 1 × 10 −3 , corresponding to the W-filament temperature increase from 1100 to 1200 C. We have recently succeeded in developing the “super H 2 O-barrier film” by the coating of single layers of SiCN for both sides of the PEN film without using the widely used polymer/inorganic multilayer coating. The both-side coating has been found to be crucial to avoid the H 2 O penetration into PEN films and also to avoid the breakdown of the SiCN/PEN interface caused by the H 2 O accumulation at the interface.

We present 210Pb/226Ra and 87Sr/86Sr data of barite separates from mineralized hydrothermal sulfate-sulfide chimneys collected from two young and active back-arc basins (i.e., Okinawa Trough and southern Mariana Trough), to examine the age of chimney formation and the relationship between the fluid chemistry and the field occurrence. Precipitation ages of purified barites in the mineralized chimneys are 24.8 (±1.3)-73.9 (±13.6) years BP2005 for chimney #220 from the Hakurei site in the Izena Cauldron (middle Okinawa Trough), 14.1 (±0.6)-53.4 (±2.8) years BP2005 for chimney #562-R13 from the Yaeyama graben (southern Okinawa Trough), and 29.8 (±1.4)-39.2 (±2.9) years BP2005 for chimney #903-R7 from the Archaean site in the southern Mariana Trough. Therefore, the time required for hydrothermal fluid venting to form these chimneys is of the order of decades. 87Sr/86Sr values for barites from chimney #220 are ∼ 0.7094; these values are higher than those of the JADE site (0.7091) at the slope of the Izena Cauldron and ambient seawater (0.70918). Given the relatively high Ag and Sb contents in chimney #220 bulk samples, we suggest that interactions have occurred between the hydrothermal fluid and the sediment cover. In contrast, barites from the southern Mariana Trough have relatively low 87Sr/86Sr values (∼ 0.7046), implying that magmatic fluids play a significant role in mineralization in this sediment-starved setting.

gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.

The α-proteobacterial strain Sphingobium japonicum UT26 utilizes a highly chlorinated pesticide, γ-hexachlorocyclohexane (γ-HCH), as a sole source of carbon and energy, and haloalkane dehalogenase LinB catalyzes the second step of γ-HCH degradation in UT26. Functional complementation of a linB mutant of UT26, UT26DB, was performed by the exogenous plasmid isolation technique using HCH-contaminated soil, leading to our successful identification of a plasmid, pLB1, carrying the linB gene. Complete sequencing analysis of pLB1, with a size of 65,998 bp, revealed that it carries (i) 50 totally annotated coding sequences, (ii) an IS 6100 composite transposon containing two copies of linB , and (iii) potential genes for replication, maintenance, and conjugative transfer with low levels of similarity to other homologues. A minireplicon assay demonstrated that a 2-kb region containing the predicted repA gene and its upstream region of pLB1 functions as an autonomously replicating unit in UT26. Furthermore, pLB1 was conjugally transferred from UT26DB to other α-proteobacterial strains but not to any of the β- or γ-proteobacterial strains examined to date. These results suggest that this exogenously isolated novel plasmid contributes to the dissemination of at least some genes for γ-HCH degradation in the natural environment. To the best of our knowledge, this is the first detailed report of a plasmid involved in γ-HCH degradation.

Two haloalkane dehalogenases, LinBUT and LinBMI, each with 296 amino acid residues, exhibit only seven amino acid residue differences between them, but LinBMI’s catalytic performance towards β-hexachlorocyclohexane (β-HCH) is considerably higher than LinBUT’s. To elucidate the molecular basis governing this difference, intermediate mutants between LinBUT and LinBMI were constructed and kinetically characterized. The activities of LinBUT-based mutants gradually increased by cumulative mutations into LinBUT, and the effects of the individual amino acid substitutions depended on combination with other mutations. These results indicated that LinBUT’s β-HCH degradation activity can be enhanced in a stepwise manner by the accumulation of point mutations.

To investigate differences in helper T cell immune responses in cerebrospinal fluid (CSF) between neuromyelitis optica (NMO) and multiple sclerosis (MS), we measured CSF levels of interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-a and interferon-c at the time of relapse in 17 NMO patients and 21 MS patients using fluorescence-activated cell sorting. CSF IL-6 levels were significantly higher in NMO patients than in patients with MS (P = 0.001) and other neurological diseases (P = 0.001). The other cytokines tested were undetectable. Elevated CSF levels of IL-6 in only NMO supports the view of different pathophysiologies of NMO and MS. CSF IL-6 levels may be useful in the differential diagnosis of the two disorders.

The purpose of this study was to investigate the working accuracy of CAD/CAM by probing the surface of the master model abutment, and its effect on the fabricated surface of three materials (titanium, thermosetting resin, and porcelain) cut to make crown and bridge. The master model abutment was measured first, the tool path for the cutting tool to run was produced, and then numerical control fabrication was applied to each material cut to measure it by the Coordinate Measuring Machine. For detailed results, two additional steps were taken and it was concluded that one-way analysis of variance statistically contrasted each measurement to the master model measurement and scanning electron microscopy confirmed eachfabricated surface.1. Data on one-way analysis of varianceA. Titanium: smaller in the cervical area and larger in the other parts than the master model measurement.B. Thermosetting resin: all the measured areas were smaller than those of the model except for the height.C. Porcelain: smaller in the cervical area and larger in the other parts than those of the model.2. Surfaces confirmed by scanning electron microscopyA. Titanium:a. a tool trace on the surfaceB. Thermosetting resin:a. a tool trace on the surfaceb. a wave-like difference in the marginal form level of chamferc. an obtuse angle and chipping in the marginal angled aread. scratches by the diamond particles of the toolC. Porcelaina. a tool trace on the surfaceb. a rough cutting surfacec. an obtuse angle and chipping in the marginal angled area-clearer than that of the resinIn conclusion, the results proved the efficiency working accuracy of CAD/CAM by probing the surface of three kinds of material cuts. As the detailed data of each candidate for analysis, it was obvious that the fabrication of titanium is superior to the others and that it will be a great reference for future studies and dental treatment.

A rare case of idiopathic benign biliary stricture is reported. A 50-year-old man with liver dysfunction underwent ultrasonography, which revealed dilation of the intrahepatic bile ducts, and endoscopic retrograde cholangiopancreatography, which revealed a short, ring-like stenosis at the junction of the left and right hepatic ducts. Although a benign stricture was suspected, radiologic tests alone were insufficient to make a firm diagnosis. Therefore, a cholecystectomy and resection of the extrahepatic biliary tract were performed. Microscopic examination of the resected specimen demonstrated no evidence of malignancy. The final diagnosis was mild, localized, chronic cholangitis. The patient had not had previous biliary tract surgery, choledocholithiasis, nor did he have a congenital abnormality of the biliary tract, bile duct carcinoma, or pancreatic disease. Since there was no evidence of primary sclerosing cholangitis, the stricture was considered to be idiopathic.

The conventional instruments used in experiments for the soft X-ray region such as X-ray diffraction analysis are X-ray films or imaging plates. However, these instruments are not suitable for real time observation. In this paper, newly developed imaging devices will be presented, which have the capability to take X-ray images in real time with a high detection efficiency. Also, another capability, to take elementary particle tracking images, is described.

We studied the effect of the quality of platelet concentrate (PC) on the recovery rate of leukocyte removal filters in platelet transfusion.Apheresis PCs taken in our hospital were stored in two types of storage bags (polyvinylchloride bag and polyolefin bag) for 48 to 96h. PC quality was analysed using GMP140-positive rate and pH level as parameters. Under the storage conditions employed, PCs showed a wide distribution of parameters, with pH ranging from 5.82 to 7.39 and GMP140-positive rate from 6.8% to 96.3%. The PCs were filtered with a leukocyte removal filter and then platelet recovery rate was measured.A significant correlation was found between PC quality (pH level and GMP140-positive rate) and platelet recovery rate, with velves of Y=9.107X+18.618, r=0.345 (p

Multidrug resistance is a severe clinical problem in the chemotherapy of malignant disease. Acute megakaryoblastic leukemia (AMKL) is a rare form of childhood leukemia, and is often resistant to many anti-cancer chemotherapeutic drugs. Here we report the expression of the mdr-1/P-glycoprotein in a cell line, CMK, established from a patient with AMKL. Expression of mdr-1 mRNA in CMK11-5 cells, a well differentiated subline, was higher than in CMK6 cells, a poorly differentiated subline. The level of P-glycoprotein was also higher in CMK11-5 cells. The cytokines interferon-gamma (IFN-gamma), GM-CSF and IL-3, which were shown to induce megakaryocytic differentiation of CMK cells, enhanced the expression of the mdr-1 mRNA and levels of P-glycoprotein. These results imply that differentiated megakaryocytic cells may have higher levels of the P-glycoprotein expression, suggesting a possible normal physiological function of P-glycoprotein in mature megakaryocytes.

Establishing cooperative relationship is critical for departments associated with blood transfusion for massive intraoperative hemorrhage, because it is directly linked to saving lives. To achieve this aim, careful preparation and establishment of in-house rules based on the "Guidelines for Management of Critical Bleeding", depending on site-specific situations, are necessary. Blood transfusion departments should (1) share recognition with other departments in the hospital, (2) educate practitioners in blood transfusion, and (3) exchange information with blood centers. In addition, it should be recognized that regular simulations are indispensable for ensuring prompt response to emergency situations.

To search for new, fast, inorganic scintillators, we have developed a bench-top pulsed X-ray source for determining fluorescent lifetimes and wavelengths of compounds in crystal or powdered form. This source uses a light-excited X-ray tube which produces X-rays when light from a laser diode strikes its photocathode. The X-ray tube has a tungsten anode, a beryllium exit window, a 30 kV maximum tube bias, and a 50 /spl mu/A maximum average cathode current. The laser produces 3/spl times/10/sup 7/ photons at 650 nm per /spl sim/100 ps pulse, with up to 10/sup 7/ pulses/sec. The time spread for the laser diode, X-ray tube, and a microchannel plate photomultiplier tube is less than 120 ps fwhm. The mean X-ray energy at tube biases of 20, 25, and 30 kV is 9.4, 10.3, and 11.1 keV, respectively. We measured 140, 230, and 330 X-ray photons per laser diode pulse per steradian, at tube biases of 20, 25, and 30 kV, respectively. Background X-rays due to dark current occur at a rate of 1/spl times/10/sup 6/ and 3/spl times/10/sup 6/ photons/sec/steradian at biases of 25 and 30 kV, respectively. Data characterizing the X-ray output with an aluminum filter in the X-ray beam are also presented. >

The mycotoxin deoxynivalenol (DON), a secondary metabolite produced by species of the plant pathogen Fusarium, causes serious problems in cereal crop production because of its toxicity towards humans and livestock. A biological approach for the degradation of DON using a DON-degrading bacterium (DDB) appears to be promising, although information about DDBs is limited. We isolated 13 aerobic DDBs from a variety of environmental samples, including field soils and wheat leaves. Of these 13 strains, nine belonged to the Gram-positive genus Nocardioides and other four to the Gram-negative genus Devosia. The degradation phenotypes of the two Gram types were clearly different; all washed cells of the 13 strains degraded 100 μg mL(-1) DON to below the detection limit (0.5 μg mL(-1)), but the conditions inducing the DON-degrading activities differed between the two Gram types. The HPLC profiles of the DON metabolites were also distinct between the two genera, although all strains produced 3-epi-deoxynivalenol. The Gram-positive strains showed DON assimilation in media containing DON as a carbon source, whereas the Gram-negatives did not. Our results suggest that aerobic DDBs are distributed within at least two phylogenetically restricted genera, suggesting independent evolution of the DON-degradation mechanisms.

A γ-hexachlorocyclohexane (HCH)-degrading bacterium, Sphingomonas sp. MM-1, was isolated from soil contaminated with HCH isomers. Cultivation of MM-1 in the presence of γ-HCH led to the detection of five γ-HCH metabolites, γ-pentachlorocyclohexene, 2,5-dichloro-2,5-cyclohexadiene-1,4-diol, 2,5-dichlorohydroquinone, 1,2,4-trichlorobenzene, and 2,5-dichlorophenol, strongly suggesting that MM-1 has the lin genes for γ-HCH degradation originally identified in the well-studied γ-HCH-degrading strain Sphingobium japonicum UT26. Southern blot, PCR amplification, and sequencing analyses indicated that MM-1 has seven lin genes for the conversion of γ-HCH to β-ketoadipate (six structural genes, linA to linF, and one regulatory gene, linR). MM-1 carried four plasmids, of 200, 50, 40, and 30 kb. Southern blot analysis revealed that all seven lin genes were dispersed across three of the four plasmids, and that IS6100, often found close to the lin genes, was present on all four plasmids.

[1] Molecular hydrogen (H2) is one of the most important energy sources for subseafloor chemolithoautotrophic microbial ecosystems in the deep-sea hydrothermal environments. This study investigated stable isotope ratios of H2 in 20°–375°C hydrothermal fluids to evaluate usefulness of the isotope ratio as a tracer to explore the H2-metabolisms. Prior to the observation, we developed an improved analytical method for the determination of concentration and stable isotope ratio of H2. This method achieved a relatively high sensitivity with a detection limit of 1 nmol H2 within an analytical error of 10‰ in the δDH2 value. The δDH2 values in the high-temperature fluids were between −405‰ and −330‰, indicating the achievement of the hydrogen isotopic equilibrium between H2 and H2O at around the hydrothermal end-member temperature. In contrast, several low-temperature fluids showed apparently smaller δDH2 values than those in the high-temperature fluids in spite of a negligible δDH2 change due to fluid-seawater mixing, suggesting the possibility of δDH2 change in the low-temperature fluids and the surrounding environments. Since the δDH2 change in low-temperature environments is not well explained by the very sluggish abiotic thermal isotopic equilibrium between H2 and H2O, it could be associated with (micro)biological H2-consuming and/or H2-generating metabolisms that would strongly promote the isotopic equilibrium at low temperatures. Our first detection of the δDH2 variation in deep-sea hydrothermal systems presents the availability of the δDH2 value as a new tracer for microbes whose enzymes catalyze D/H exchange in H2., 論文

A 45-year-old woman, complaining of back pain and bloody stool was given a diagnosis of poorly differentiated adenocarcinoma of the rectum with disseminated carcinomatosis to bone marrow and disseminated intravascular coagulation syndrome (DIC). We started chemotherapy using mFOLFOX-6 with simultaneous DIC treatment. After we confirmed that DIC had improved following 2 courses of mFOLFOX-6, bevacizumab was added to mFOLFOX-6. Laboratory studies revealed a serum CEA level of 314.4ng/ml, which improved to 4.6ng/ml after a total of 6 courses of chemotherapy. Colonoscopy findings showed almost normal rectal mucosa after a total of 10 courses of chemotherapy. Outpatient treatment was started after 5 courses of chemotherapy, and was continuing according to schedule at 7 months from the onset of this disease.

gamma-Hexachlorocyclohexane (gamma-HCH, also called gamma-BHC and lindane) is a halogenated organic insecticide that causes serious environmental problems. The aerobic degradation pathway of gamma-HCH was extensively revealed in bacterial strain Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26. gamma-HCH is transformed to 2,5-dichlorohydroquinone through sequential reactions catalyzed by LinA, LinB, and LinC, and then 2,5-dichlorohydroquinone is further metabolized by LinD, LinE, LinF, LinGH, and LinJ to succinyl-CoA and acetyl-CoA, which are metabolized in the citrate/tricarboxylic acid cycle. In addition to these catalytic enzymes, a putative ABC-type transporter system encoded by linKLMN is also essential for the gamma-HCH utilization in UT26. Preliminary examination of the complete genome sequence of UT26 clearly demonstrated that lin genes for the gamma-HCH utilization are dispersed on three large circular replicons with sizes of 3.5 Mb, 682 kb, and 191 kb. Nearly identical lin genes were also found in other HCH-degrading bacterial strains, and it has been suggested that the distribution of lin genes is mainly mediated by insertion sequence IS6100 and plasmids. Recently, it was revealed that two dehalogenases, LinA and LinB, have variants with small number of amino acid differences, and they showed dramatic functional differences for the degradation of HCH isomers, indicating these enzymes are still evolving at high speed.