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The Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene (pmoA) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated as INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription dataset of INp10 from biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help better understand ecological strategies of this possibly widespread marine-specific methanotrophic clade. Importance The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts such as bathymodiolin mussels Bathymodiolus japonicus and "Bathymodiolus" platifrons and a galatheoid crab Shinkaia crosnieri. Symbiotic methanotrophs have attracted significant attention, yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated it corresponds to a new species in the genus Methyloprofundus. This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus spp. suggests that the hydrothermal field is a promising locale to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus.

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A novel mesophilic, strictly anaerobic, chemolithoautotrophic sulphate-reducing bacterium, designated strain KT2T, was isolated from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Izu-Bonin Arc. Strain KT2T grew at 25–40 °C (optimum 35 °C) and pH 5.5–7.0 (optimum 6.6) in the presence of 25–45 g l−1 NaCl (optimum 30 g l−1). Growth occurred with molecular hydrogen as the electron donor and sulphate, thiosulphate, and sulphite as the electron acceptors. The isolate utilized CO2 as the sole carbon source for chemolithoautotrophic growth on H2. Glycerol, succinate, fumarate, malate, glutamate, or casamino acids could serve as an alternative electron donor in the presence of CO2. Malate, citrate, glutamate, and casamino acids were used as fermentative substrates for weak growth. The G+C content of genomic DNA was 46.1 %. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain KT2T is a member of the family Desulfobulbaceae , showing a sequence similarity of 94.3 % with Desulforhopalus singaporensis . Phylogenomic analysis based on concatenated 156 single-copy marker genes confirmed the same topology as the 16S rRNA gene phylogeny. The ANI and AAI values between strain KT2T and related genera of the family Desulfobulbaceae were 65.6–68.6 % and 53.1–62.9 %. Based on the genomic, molecular, and physiological characteristics, strain KT2T represents a novel genus and species within the family Desulfobulbaceae , for which the name Desulfomarina profundi gen. nov., sp. nov. is proposed, with KT2T (=JCM 34118T = DSM 111364T) as the type strain.

The scaly-foot snail (Chrysomallon squamiferum) inhabiting deep-sea hydrothermal vents in the Indian Ocean relies on its sulphur-oxidising gammaproteobacterial endosymbionts for nutrition and energy. In this study, we investigate the specificity, transmission mode, and stability of multiple scaly-foot snail populations dwelling in five vent fields with considerably disparate geological, physical and chemical environmental conditions. Results of population genomics analyses reveal an incongruent phylogeny between the endosymbiont and mitochondrial genomes of the scaly-foot snails in the five vent fields sampled, indicating that the hosts obtain endosymbionts via horizontal transmission in each generation. However, the genetic homogeneity of many symbiont populations implies that vertical transmission cannot be ruled out either. Fluorescence in situ hybridisation of ovarian tissue yields symbiont signals around the oocytes, suggesting that vertical transmission co-occurs with horizontal transmission. Results of in situ environmental measurements and gene expression analyses from in situ fixed samples show that the snail host buffers the differences in environmental conditions to provide the endosymbionts with a stable intracellular micro-environment, where the symbionts serve key metabolic functions and benefit from the host’s cushion. The mixed transmission mode, symbiont specificity at the species level, and stable intracellular environment provided by the host support the evolutionary, ecological, and physiological success of scaly-foot snail holobionts in different vents with unique environmental parameters.

BACKGROUND With the increase in endoscopic procedures, endoscopists are spending more time creating reports. Although medical reports have largely become electronic, most of the current reporting systems require manual operation. This study aimed to evaluate the efficacy of a novel endoscopic reporting system that uses voice recognition (VR) technology. METHODS We retrospectively reviewed consecutive patients who underwent esophagogastroduodenoscopy between September 2019 and March 2020 at a general hospital in Japan. The novel reporting system, used during endoscopic procedures, is equipped with VR and provides automatic responses by playing back recognized words. Differences in total time spent on the endoscopic procedure and report preparation between the manual entry (ME) and VR groups were evaluated using a propensity score matching method. RESULTS We enrolled 356 patients: 226 and 130 patients in the ME and VR groups, respectively. Propensity score matching created 101 matched pairs. After matching, the median report preparation time (311 vs. 383 s, P = 0.009) and median total time (765 vs. 842 s, P = 0.053) in the VR group were shorter than those in the ME group. The VR system independently shortened the total and report preparation times by 156 s (95% confidence interval, - 274 to - 37 s; P = 0.009) and 118 s (95% confidence interval, - 220 to - 15 s; P = 0.023), respectively, on multiple linear regression analysis. CONCLUSIONS The VR system could save the report preparation time and the total time. This novel system may improve the efficiency of endoscopy-related tasks.

A novel hydrogenotrophic methanogen, strain HHBT, was isolated from a deep-sea hydrothermal vent chimney sample collected from Beebe Vent Field at the Mid-Cayman Spreading Center, Caribbean Sea. The cells were non-motile regular to irregular cocci possessing several flagella. The novel isolate grew at 60–80 °C, pH 5.0–7.4 and with 1–4 % of NaCl (w/v). The isolate utilized H2/CO2 as the only substrates for growth and methane production. The results of phylogenetic analyses of both 16S rRNA and mcrA gene sequences and comparative genome analysis indicated that HHBT represented a member of the order Methanococcales , and was closely related to the members of the genera Methanothermococcus and Methanotorris . The most closely related species were Methanothermococcus okinawensis IH1T and Methanotorris igneus Kol 5T in comparison of 16S rRNA gene sequences (each with 93 % identity), and Methanotorris formicicus Mc-S-70T in the case of deduced amino acid sequence similarity of mcrA genes (92 % similarity). The ANI and AAI values between HHBT and the members of the genera Methanothermococcus and Methanotorris were 69–72 % and 66–70 %, respectively. Although many of the morphological and physiological characteristics were quite similar between HHBT and the species of the genera Methanothermococcus and Methanotorris , they were distinguishable by the differences in susceptibility to antibiotics, formate utilization, growth temperature and NaCl ranges. On the basis of these phenotypic, phylogenetic and genomic properties, we propose that strain HHBT represents a novel species, of a novel genus, Methanofervidicoccus abyssi gen. nov., sp. nov. The type strain is HHBT (=JCM 32161T=DSM 105918T).

South Chamorro Seamount (SCS) is a blueschist-bearing serpentinite mud volcano in the Mariana forearc. Previous scientific drilling conducted at SCS revealed highly alkaline, sulfate-rich formation fluids resulting from slab-derived fluid upwelling combined with serpentinization both beneath and within the seamount. In the present study, a time-series of ROV dives spanning 1000 days was conducted to collect discharging alkaline fluids from the cased Ocean Drilling Program (ODP) Hole 1200C (hereafter the CORK fluid). The CORK fluids were analyzed for chemical compositions (including dissolved gas) and microbial community composition/function. Compared to the ODP porewater, the CORK fluids were generally identical in concentration of major ions, with the exception of significant sulfate depletion and enrichment in sulfide, alkalinity, and methane. Microbiological analyses of the CORK fluids revealed little biomass and functional activity, despite habitable temperature conditions. The post-drilling sulfate depletion is likely attributable to sulfate reduction coupled with oxidation of methane (and hydrogen), probably triggered by the drilling and casing operations. Multiple lines of evidence suggest that abiotic organic synthesis associated with serpentinization is the most plausible source of the abundant methane in the CORK fluid. The SCS formation fluid regime presented here may represent the first example on Earth where abiotic syntheses are conspicuous with little biotic processes, despite a condition with sufficient bioavailable energy potentials and temperatures within the habitable range., Progress in Earth and Planetary Science, 5

The identification of microbial activity under extreme conditions is important to define potential boundaries of the habitable and uninhabitable zones of terrestrial and extraterrestrial living forms. The subseafloor regimes of serpentinite seamounts in the Mariana Forearc are among the most extreme environments for life on earth owing to the widespread presence of highly alkaline fluids with pH values greater than 12. The potential activity of sulfate-reducing microorganisms has been suggested within the South Chamorro serpentinite seamounts on the basis of depletion of sulfate and enrichment of dissolved sulfide in pore water. However, the vertical distribution of sulfate-reducing microorganisms and the origin of sulfate are still uncertain. To address these issues, we analyzed quadruple sulfur isotopes of sulfide minerals and pore water sulfate in the upper 56 m of sedimentary sequences at the summit of the S. Chamorro Seamount and those of dissolved sulfate in upwelling fluids collected as deep as 202 mbsf (meters below the seafloor) in a cased hole near the summit of the same seamount. The depth profiles of the concentrations and the δ 34 S and Δ 33 S ′ values of sulfide minerals and pore water sulfate indicate microbial sulfate reduction as deep as 30 mbsf. Further, apparent isotopic fractionations ( e 34 ) and exponents of mass dependent relationships ( λ 33 ) during sulfate reduction are estimated to be 62 ± 14‰ and 0.512 ± 0.002, respectively. The upwelling fluids show both the chlorine depletion relative to seawater and the negative δ 15 N values of ammonia (−4‰). Although these signatures point to dehydration of the subducting oceanic plate, the negative Δ 33 S ′ values of sulfate (−0.16‰ to −0.26‰ with analytical errors of ±0.01‰) are unlikely to originate from surrounding modern crusts. Instead, sulfate in the upwelling fluid likely possess non-mass-dependent (NMD) sulfur. Because NMD sulfur was produced primarily in the Archean atmosphere, our results suggest that the presence of recycled Archean crust that could be incorporated into the upper mantle through subduction of Archean oceanic crusts or from the NMD-bearing OIB seamounts located in the southern margin of the Pacific Plate.

We here report on specific chemical and microbial compositions observed at the Archaean hydrothermal site in the Southern Mariana backarc spreading center, which produces two remarkably different hydrothermal fluids. One was black smoker hydrothermal fluid at 340 °C containing a low concentration of methane with a relatively high carbon isotope ratio (δ13C of methane relative to VPDB), −7.8‰, indicating that methane originated from a magmatic source; thus, this is a fairly ordinary hydrothermal fluid for basalt-hosted hydrothermal activity. In contrast, the other fluid was clear smoker hydrothermal fluid at 117 °C containing a high concentration of methane with a very low carbon isotope ratio (−49.7‰). The host rock of the Archaean hydrothermal site is basalt, and therefore Fischer-Tropsch-type reactions resulting from serpentinization of mantle rocks are not feasible as a source for the “light” carbon in methane. Additionally, the carbon isotope ratio of carbon dioxide demonstrated that the sources of CO2 and CH4 were not organic materials. The remaining possibility, biogenic methane, was confirmed by cultivation of hyperthermophilic hydrogen-oxidizing methanogens from both rocks covering the vent of clear smoker hydrothermal fluids and in situ cultivation systems. Although the dominance and abundance of methanogens were very low in this ecosystem, the number was consistent with the relationship between hydrogen concentration and methanogen abundance (Takai et al., 2015). These results suggest that phase separation led to concentration of hydrogen and subsequent persistence of a hyperthermophilic subsurface lithoautotrophic microbial ecosystem in both serpentine-hosted and basalt-hosted hydrothermal systems. This type of ecosystem may occur in similar settings elsewhere on modern earth; in addition, similar communities may have existed in other types of deep-sea hydrothermal systems in the geological past.

Some deep-sea chemosynthetic invertebrates and their symbiotic bacteria can use molecular hydrogen (H2) as their energy source. However, how much the chemosynthetic holobiont (endosymbiont-host association) physiologically depends on H2 oxidation has not yet been determined. Here, we demonstrate that the Campylobacterota endosymbionts of the gastropod Alviniconcha marisindica in the Kairei and Edmond fields (kAlv and eAlv populations, respectively) of the Indian Ocean, utilize H2 in response to their physical and environmental H2 conditions, although the 16S rRNA gene sequence of both the endosymbionts shared 99.6% identity. A thermodynamic calculation using in situ H2 and hydrogen sulfide (H2S) concentrations indicated that chemosynthetic symbiosis could be supported by metabolic energy via H2 oxidation, particularly for the kAlv holobiont. Metabolic activity measurements showed that both the living individuals and the gill tissues consumed H2 and H2S at similar levels. Moreover, a combination of fluorescence in situ hybridization, quantitative transcript analyses, and enzymatic activity measurements showed that the kAlv endosymbiont expressed the genes and enzymes for both H2- and sulfur-oxidations. These results suggest that both H2 and H2S could serve as the primary energy sources for the kAlv holobiont. The eAlv holobiont had the ability to utilize H2, but the gene expression and enzyme activity for hydrogenases were much lower than for sulfur-oxidation enzymes. These results suggest that the energy acquisitions of A. marisindica holobionts are dependent on H2- and sulfur-oxidation in the H2-enriched Kairei field and that the mechanism of dual metabolism is controlled by the in situ H2 concentration.

Fluid chemistry and microbial community patterns in chimney habitats were investigated in two hydrothermal fields located at the Central Indian Ridge. Endmember hydrothermal fluid of the Solitaire field, located ~3 km away from the spreading center, was characterized by moderately high temperature (307°C), Cl depletion (489 mm), mildly acidic pH (≥4.40), and low metal concentrations (Fe ≤ 105 μm and Mn = 78 μm). Chloride depletion indicates that the subseafloor source fluid had undergone phase separation at temperatures higher than ~390°C while the metal depletion was likely attributable to fluid alteration occurring at a venting temperature of around 307°C. These different temperature conditions suggested from fluid chemistry might be associated with an off-spreading center location of the field that allows subseafloor fluid cooling prior to seafloor discharge. The microbial community in the chimney habitat seemed comparable to previously known patterns in typical basalt-hosted hydrothermal systems. Endmember hydrothermal fluid of the Dodo field, standing on center of the spreading axis, was characterized by high H2 concentration of 2.7 mm. The H2 enrichment was likely attributable to fresh basalt–fluid interaction, as suggested by the nondeformed sheet lava flow expansion around the vents. Thermodynamic calculation of the reducing pyrite–pyrrhotite–magnetite (PPM) redox buffer indeed reproduced the H2 enrichment. The quantitative cultivation test revealed that the microbial community associated with the hydrothermal fluid hosted abundant populations of (hyper)thermophilic hydrogenotrophic chemolithoautotrophs such as methanogens. The function of subseafloor hydrogenotrophic methanogenic populations dwelling around the H2-enriched hydrothermal fluid flows was also inferred from the 13C- and D-depleted signature of CH4 in the collected fluids. It was observed that the hydrothermal activity of the Dodo field had ceased until 2013.

The South Chamorro Seamount (13°47'N, 146°00'E) on the southeastern Mariana Forearc is one of a series of serpentine mud volcanoes in the area, and hosts an active fluid seepage area on its...

BackgroundThe deep-sea hydrothermal vent is one of the most ‘extreme’ environments in the marine realm. Few species are capable of inhabiting such ecosystems, despite extremely high productivity there supported by microbial chemosynthesis, leading to high biomass and low species richness. Although gastropod molluscs are one of the main constituents of megafaunal communities at vent ecosystems, most species belong to several typical families (e.g., Provannidae, Peltospiridae, Lepetodrilidae) specialised and adapted to life at vents.MethodsDuring recent surveys of Okinawa Trough hydrothermal vent systems, two snails atypical of vent ecosystems were unexpectedly found in newly discovered hydrothermally influenced areas. Shell and radular characteristics were used to identify the gastropods morphologically.ResultsOne species was a vetigastropod, the calliostomatidTristichotrochus ikukoae(Sakurai, 1994); and the other was a caenogastropod, the muricidAbyssotrophon soyoae(Okutani, 1959). Both gastropods were previously only known from regular non-chemosynthetic deep-sea and very rare—only two definitive published records exist forT. ikukoaeand three forA. soyoae. The radula formula ofTristichotrochus ikukoaeis accurately reported for the first time and based on that it is returned to genusOtukaia. For both species, barcode sequences of the cytochromecoxidase I (COI) gene were obtained and deposited for future references.DiscussionThese new records represent the second record of calliostomatids from vents (third from chemosynthetic ecosystems) and the third record of muricids from vents (tenth from chemosynthetic ecosystems), and extend the distribution of both species to the southwest. Neither family has been recorded at chemosynthetic ecosystems in the western Pacific. Both were from weakly diffuse flow areas not subject to high temperature venting but were nevertheless associated with typical vent-reliant taxa such asLamellibrachiatubeworms andBathymodiolusmussels. These new records show that these species are capable of tolerating environmental stress associated with weak hydrothermally influenced areas, despite not being vent endemic species, adding to the list of known vent/non-vent species intersections. This signifies that such weakly influenced areas may provide key habitats for them, and that such areas may play a role in the evolution of biological adaptations to ‘extreme’ chemosynthetic ecosystems.

Rich animal and microbial communities have been found at deep-sea hydrothermal vents. Although the biogeography of vent macrofauna is well understood, the corresponding knowledge about vent microbial biogeography is lacking. Here, we apply the multilocus sequence analysis (MLSA) to assess the genetic variation of 109 Sulfurimonas strains with greater than or equal to98% 16S rRNA gene sequence similarity, which were isolated from four different geographical regions (Okinawa Trough (OT), Mariana Volcanic Arc and Trough (MVAT), Central Indian Ridge (CIR) and Mid-Atlantic Ridge (MAR)). Sequence typing based on 11 protein-coding genes revealed high genetic variation, including some allele types that are widespread within regions, resulting in 102 nucleotide sequence types (STs). This genetic variation was predominantly due to mutation rather than recombination. Phylogenetic analysis of the 11 concatenated genes showed a clear geographical isolation corresponding to the hydrothermal regions they originated from, suggesting limited dispersal. Genetic differentiation among Sulfurimonas populations was primarily influenced by geographical distance rather than gas composition of vent fluid or habitat, although in situ environmental conditions of each microhabitat could not be examined. Nevertheless, Sulfurimonas may possess a higher dispersal capability compared with deep-sea hydrothermal vent thermophiles. This is the first report on MLSA of deep-sea hydrothermal vent Epsilonproteobacteria, which is indicative of allopatric speciation.

Hyperthermophilic hydrogenotrophic methanogens are considered to be one of the most predominant primary producers in hydrogen (H2)-abundant hydrothermal environments in the present-day ocean and throughout the history of the Earth. However, the nitrogen sources supporting the development of microbial communities in hydrothermal environments remain poorly understood. We have investigated, for the first time, methanogenic archaea commonly found in deep-sea hydrothermal environments to understand their physiological properties (growth kinetics, energetics, and metal requirements) and isotopic characteristics during the fixation of dinitrogen (N2), which is an abundant but less-bioavailable compound in hydrothermal fluids. Culture experiments showed that Methanocaldococcus strain (Mc 1–85N) (Topt = 85 °C) and Methanothermococcus strain (Mt 5–55N) (Topt = 55 °C) assimilated N2 and ammonium, but not nitrate. Previous phylogenetic studies have predicted that the Methanocaldococcus and Methanothermococcus lineages have nitrogenases, key enzymes for N2 fixation, with biochemically uncharacterised active site metal cofactors. We showed that Mt 5–55N required molybdenum for the nitrogenase to function, implying a molybdenum-bearing cofactor in the strain. Molybdenum also stimulated diazotrophic (i.e., N2-fixing) growth of Mc 1–85N, though further experiments are required to test whether the strain contains a molybdenum-dependent nitrogenase. Importantly, Mc 1–85N exhibited an apparently lower requirement of and higher tolerance to molybdenum and iron than Mt 5–55N. Furthermore, both strains produced more 15N-depleted biomass (−4‰ relative to N2) than that previously reported for diazotrophic photosynthetic prokaryotes. These results demonstrate that diazotrophic hyperthermophilic methanogens can be broadly distributed in seafloor and subseafloor hydrothermal environments, where the availability of transition metals is variable and where organic carbon, organic nitrogen, and ammonium are generally scarce. The emergence and function of diazotrophy, coupled with methanogenesis, in the early Earth is also consistent with the nitrogen isotopic records of 3.5 billion-year-old hydrothermal deposits.

Objective: This study evaluated the effects of ebastine on serum eosinophil cationic protein (ECP) levels in patients with bronchial asthma. Patients: Twenty patients with bronchial asthma (11 patients with atopic disease and nine with non-atopic disease) were enrolled in the study. Methods: In an open-label design, all patients received ebastine 10 mg/day for 4 weeks and serum ECP levels, peripheral blood eosinophil counts, morning peak expiratory flow rate (PEFR) and thresholds for airway hyper-responsiveness (Dmin in asthgraphy) were determined before and after treatment. Results: Serum ECP levels and peripheral blood eosinophil counts were significantly decreased. By disease type, no significant change was found in the non-atopic patients, while the serum ECP level was significantly (p < 0.001) decreased in the atopic patients. Furthermore, no significant change in Dmin was found, but PEFR was significantly (p < 0.019) increased in the atopic type. Conclusion: Ebastine not only inhibits type I allergic reactions, but may also inhibit airway inflammation by reducing serum ECP levels, particularly in patients with atopic bronchial asthma.

[1] The Integrated Ocean Drilling Program (IODP) Expedition 331 investigated the Iheya North hydrothermal field in the Okinawa Trough. Several post-drilling underwater vehicle investigations were conducted over 2 years to identify post-drilling changes in fluid discharge pattern, mineral deposition, and fluid chemistry. Drilling-induced high-temperature hydrothermal fluid vents were identified at deep holes not only near the naturally occurring NBC hydrothermal fluid vent (Site C0016) but also at the seafloor ∼450 m distal to the NBC vent (Site C0014), where no hydrothermal fluid discharge was observed prior to drilling. A chimney structure at Hole C0016A grew rapidly at the NBC mound crest, where only small chimneys had been found before drilling. A drilling-induced diffuse hydrothermal flow region spread at Site C0014, and this area was newly colonized by the galatheid crab. From a fluid chemistry perspective, the post-drilling hydrothermal fluids were enriched in Cl relative to seawater, although this fluid chemistry was not observed during the 12 years prior to drilling. The Cl-enriched fluid reservoir underlying the subseafloor impermeable layers, observed by IODP Expedition 331, is likely source for the Cl-enriched fluids discharging from the post-drilling vents. The drilling-induced physical disturbance of subseafloor hydrogeological structures would release such fluids to the seafloor. In turn, the rapid chimney growth at the NBC mound crest may also be attributed to highly turbulent fluid flow with the enlarged artificial vent of Hole C0016A, which can contribute to the retention of the fluid-seawater mixture for a sufficiently long period to precipitate sulfide/sulfate minerals on the seafloor.

A new hydrothermal vent site in the Southern Mariana Trough has been discovered using acoustic and magnetic surveys conducted by the Japan Agency for Marine-Earth Science and Technology's (JAMSTEC) autonomous underwater vehicle (AUV), Urashima . The high-resolution magnetic survey, part of a near-bottom geophysical mapping around a previously known hydrothermal vent site, the Pika site, during the YK09-08 cruise in June–July 2009, found that a clear magnetization low extends ∼500 m north from the Pika site. Acoustic signals, suggesting hydrothermal plumes, and 10 m-scale chimney-like topographic highs were detected within this low magnetization zone by a 120 kHz side-scan sonar and a 400 kHz multibeam echo sounder. In order to confirm the seafloor sources of the geophysical signals, seafloor observations were carried out using the deep-sea manned submersible Shinkai 6500 during the YK 10-10 cruise in August 2010. This discovered a new hydrothermal vent site (12°55.30′N, 143°38.89′E; at a depth of 2922 m), which we have named the Urashima site. This hydrothermal vent site covers an area of approximately 300 m×300 m and consists of black and clear smoker chimneys, brownish-colored shimmering chimneys, and inactive chimneys. All of the fluids sampled from the Urashima and Pika sites have chlorinity greater than local ambient seawater, suggesting subseafloor phase separation or leaching from rocks in the hydrothermal reaction zone. End-member compositions of the Urashima and Pika fluids suggest that fluids from two different sources feed the two sites, even though they are located on the same knoll and separated by only ∼500 m. We demonstrate that investigations on hydrothermal vent sites located in close proximity to one another can provide important insights into subseafloor hydrothermal fluid flow, and also that, while such hydrothermal sites are difficult to detect by conventional plume survey methods, high-resolution underwater geophysical surveys provide an effective means.

A series of piperazine ureas was designed, synthesized, and evaluated for their potential as novel orally available fatty acid amide hydrolase (FAAH) inhibitors that are therapeutically effective against pain. We carried out an optimization study of the lead compound 3 to improve its DMPK profile as well as in vitro potency. We identified the thiazole compound 60j with potent inhibitory activity, high brain permeability, and good bioavailability. Compound 60j showed a potent and dose-dependent anti-nociceptive effect in the acetic acid-induced writhing test in mice.

We report here the concurrence and interaction among forms of nitrogen metabolism in thermophilic microbial mat communities that developed in an ammonium-abundant subsurface geothermal stream. First, the physical and chemical conditions of the stream water at several representative microbial mat habitats (including upper, middle and downstream sites) were characterized. A thermodynamic calculation using these physical and chemical conditions predicted that nitrification consisting of ammonia and nitrite oxidations would provide one of the largest energy yields of chemolithotrophic metabolisms. Second, near-complete prokaryotic 16S rRNA gene clone analysis was conducted for representative microbial mat communities at the upper, middle and downstream sites. The results indicated a dynamic shift in the 16S rRNA gene phylotype composition through physical and chemical variations of the stream water. The predominant prokaryotic components varied from phylotypes related to hydrogeno (H 2 )- and thio (S)-trophic Aquificales , thermophilic methanotrophs and putative ammonia-oxidizing Archaea (AOA) located upstream (72 °C) to the phylotypes affiliated with putative AOA and nitrite-oxidizing bacteria (NOB) located at the middle and downstream sites (65 and 57 °C, respectively). In addition, the potential in situ metabolic activities of different forms of nitrogen metabolism were estimated through laboratory experiments using bulk microbial mat communities. Finally, the compositional and isotopic variation in nitrogen compounds was investigated in the stream water flowing over the microbial mats and in the interstitial water inside the mats. Although the stream water was characterized by a gradual decrease in the total ammonia concentration (ΣNH 3 : the sum of ammonia and ammonium concentrations) and a gradual increase in the total concentration of nitrite and nitrate (NO 2 − + NO 3 − ), the total inorganic nitrogen concentration (TIN: the sum of ΣNH 3 , NO 2 − and NO 3 − concentrations) was nearly constant (250 μM) throughout the stream. Based on the level of detectable dissolved molecular oxygen (O 2 ) of the stream water (⩾38 μM) along with metabolic measurements, it was predicted that nitrification by thermophilic AOA and NOB components in the microbial mats that were exposed to the stream water would constrain the concentrations and isotopic ratios of ΣNH 3 , NO 2 − and NO 3 − of the stream water. The δ 15 N value of ΣNH 3 increased from 0‰ to 7‰ with decreasing concentration, which was consistent with the previously reported isotopic fractionation for microbial ΣNH 3 oxidation. In contrast, the δ 15 N value of NO 2 − was 22‰ lighter than that of NO 3 − in the steam water at the same site, indicating an inverse isotopic fractionation for microbial NO 2 − oxidation. The variation in concentrations and δ 15 N values of ΣNH 3 , NO 2 − and NO 3 − was largely explained using a two-step nitrification model, and the apparent nitrogen isotopic fractionations of ΣNH 3 oxidation and NO 2 − oxidation were estimated to be 0.986 and 1.020, respectively. In the interstitial water within the microbial mats, the compositional and isotopic properties of TIN at the downstream site indicated potential denitrification by the anaerobic microbial components. The geochemically deduced transition of microbial nitrogen metabolism was substantiated through cultivation-independent microbiological analyses.

It has been suggested that iron is one of the most important energy sources for photosynthesis-independent microbial ecosystems in the ocean crust. Iron-metabolizing chemolithoautotrophs play a key role as primary producers, but little is known about their distribution and diversity and their ecological role as submarine iron-metabolizing chemolithotrophs, particularly the iron oxidizers. In this study, we investigated the microbial communities in several iron-dominated flocculent mats found in deep-sea hydrothermal fields in the Mariana Volcanic Arc and Trough and the Okinawa Trough by culture-independent molecular techniques and X-ray mineralogical analyses. The abundance and composition of the 16S rRNA gene phylotypes demonstrated the ubiquity of zetaproteobacterial phylotypes in iron-dominated mat communities affected by hydrothermal fluid input. Electron microscopy with energy-dispersive X-ray microanalysis and X-ray absorption fine structure (XAFS) analysis revealed the chemical and mineralogical signatures of biogenic Fe-(oxy)hydroxide species and the potential contribution of Zetaproteobacteria to the in situ generation. These results suggest that putative iron-oxidizing chemolithoautotrophs play a significant ecological role in producing iron-dominated flocculent mats and that they are important for iron and carbon cycles in deep-sea low-temperature hydrothermal environments., 論文

International audience; A novel strictly anaerobic, hyperthermophilic archaeon, designated strain CDGST, was isolated from a deep-sea hydrothermal vent in the Cayman Trough at 4964m water depth. The novel isolate is obligate anaerobe and grows chemoorganoheterotrophically with stimulation of growth by sulphur containing compounds. Its growth is optimal at 75 ◦C,pH6.0 and under a pressure of 50 MPa. It possesses the broadest hydrostatic pressure range for growth that has ever been described for a microorganism. Its genomic DNA G+ C content is 51.11 mol%. The novel isolate belongs to the genus Thermococcus. Phylogenetic analyses indicated that it is most closely related to Thermococcus barossii DSM17882T based on its 16S rRNA gene sequence, and to ‘Thermococcus onnurineus’ NA1 based on its whole genome sequence. The average nucleotide identity scores with these strains are 77.66% for T. barossii and 84.84% for ‘T. onnurineus’, respectively. Based on the draft whole genome sequence and phenotypic characteristics, strain CDGST is suggested to be separated into a novel species within the genus Thermococcus, with proposed name Thermococcus piezophilus (type strain CDGST = ATCC TSD-33T =UBOCC 3296T).

As part of a program to identify potent GPR40 agonists with drug-like properties suitable for clinical development, the incorporation of polar substituents was explored with the intention of decreasing the lipophilicity of our recently disclosed phenylpropanoic acid derivative 1. This incorporation would allow us to mitigate the cytotoxicity issues observed with compound 1 and enable us to move away from the multifunctional free fatty acid-like structure. Substitutions on the 2',6'-dimethylbiphenyl ring were initially undertaken, which revealed the feasibility of introducing polar functionalities at the biphenyl 4'-position. Further optimization of this position and the linker led to the discovery of several 4'-alkoxybiphenyl derivatives, which showed potent GPR40 agonist activities with the best balance in terms of improved cytotoxicity profiles and favorable pharmacokinetic properties. Among them, 3-{2-fluoro-4-[({4'-[(4-hydroxy-1,1-dioxidotetrahydro-2H-thiopyran-4-yl)methoxy]-2',6'-dimethylbiphenyl-3-yl}methyl)amino]phenyl}propanoic acid (35) exhibited a robust plasma glucose-lowering effect and insulinotropic action during an oral glucose tolerance test in rats with impaired glucose tolerance.

In 40 patients with moderate to severe persistent adult asthma who had been requiring more than 1200 micrograms/day of beclomethasone dipropionate (CAS 5534-09-8, BDP) inhalation, the present study evaluated the combined effects of pranlukast (CAS 103177-37-3, Onon) during highdose steroid inhalation (20 in the pranlukast group and 20 in the control group). In the pranlukast group, 450 mg/day of pranlukast was administered. The course of these patients was monitored for 6 weeks during which the dose of BDP was decreased to 1/2 of the initial dose 2 weeks later and to 1/4 of the initial dose additional 4 weeks later. The dose of BDP was also decreased in the control group. The concomitant use of pranlukast significantly improved asthmatic symptoms and respiratory functions in asthmatic patients receiving high-dose steroid inhalation. Furthermore, the concomitant use of pranlukast significantly inhibited worsening of asthmatic symptoms, respiratory functions and parameters, which are frequently observed when the dose of inhalation steroids is decreased.

An extremely thermophilic bacterium,Thermus thermophilus, possesses two glutamate dehydrogenase (GDH) genes,gdhAandgdhB, putatively forming an operon on the genome. To elucidate the functions of these genes, the gene products were purified and characterized. GdhA showed no GDH activity, while GdhB showed GDH activity for reductive amination 1.3-fold higher than that for oxidative deamination. When GdhA was co-expressed with His-tag-fused GdhB, GdhA was co-purified with His-tagged GdhB. Compared with GdhB alone, co-purified GdhA–GdhB had decreased reductive amination activity and increased oxidative deamination activity, resulting in a 3.1-fold preference for oxidative deamination over reductive amination. Addition of hydrophobic amino acids affected the GDH activity of the co-purified GdhA–GdhB hetero-complex. Among the amino acids, leucine had the largest effect on activity: addition of 1 mM leucine elevated the GDH activity of the co-purified GdhA–GdhB by 974 and 245 % for reductive amination and oxidative deamination, respectively, while GdhB alone did not show such marked activation by leucine. Kinetic analysis revealed that the elevation of GDH activity by leucine is attributable to the enhanced turnover number of GDH. In this hetero-oligomeric GDH system, GdhA and GdhB act as regulatory and catalytic subunits, respectively, and GdhA can modulate the activity of GdhB through hetero-complex formation, depending on the availability of hydrophobic amino acids. This study provides the first finding, to our knowledge, of a hetero-oligomeric GDH that can be regulated allosterically.

We investigated the diversity of fungal communities in 10 different deep-sea sediment samples by PCR-mediated internal transcribed spacer (ITS) regions of rRNA gene clone analysis. Although some of the amplified sequences were identified as common terrestrial fungal species, the majority were novel sequences that were not closely related to previously identified fungal ITS sequences in public databases. Phylogenetic analysis also suggested the novel phylogenetic affiliation of a group of predominant deep-sea phylotypes within the phylum Ascomycota. These phylotypes may be distributed widely in global deep-sea sediments. Another novel phylotype was placed in deep branches within the phylum Chytridiomycota with Rozella spp. as the closest related organisms. This study revealed the possible existence of previously unknown fungal components in various depths and sites of deep-sea sedimentary habitats. It is suggested that deep-sea environments are habitats hosting previously unexplored fungi, which may provide key insights into the early evolution of fungi and their ecological and physiological significance.

GPR40, one of the G protein-coupled receptors predominantly expressed in pancreatic β-cells, mediates enhancement of glucose-stimulated insulin secretion by free fatty acids. A potent and selective GPR40 agonist is theorized to be a safe and effective antidiabetic drug with little or no risk of hypoglycemia. Cyclization of the phenylpropanoic acid moiety of lead compound 1 produced fused phenylalkanoic acids with favorable in vitro agonist activities and pharmacokinetic profiles. Further optimization led to the discovery of dihydrobenzofuran derivative 9a ([(3S)-6-({2',6'-dimethyl-4'-[3-(methylsulfonyl)propoxy]biphenyl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetic acid hemi-hydrate, TAK-875) as a potent, selective, and orally bioavailable GPR40 agonist, with a pharmacokinetic profile enabling long-acting drug efficacy. Compound 9a showed potent plasma glucose-lowering action and insulinotropic action during an oral glucose tolerance test in female Wistar fatty rats with impaired glucose tolerance. Compound 9a is currently in clinical trials for the treatment of type 2 diabetes mellitus.

The potential for microbial nitrogen fixation in the anoxic methane seep sediments in a mud volcano, the number 8 Kumano Knoll, was characterized by molecular phylogenetic analyses. A total of 111 of the nifH (a gene coding a nitrogen fixation enzyme, Fe protein) clones were obtained from different depths of the core sediments, and the phylogenetic analysis of the clones indicated the genetic diversity of nifH genes. The predominant group detected (methane seep group 2), representing 74% of clonal abundance, was phylogenetically related to the nifH sequences obtained from the Methanosarcina species but was most closely related to the nifH sequences potentially derived from the anoxic methanotrophic archaea (ANME-2 archaea). The recovery of the nif gene clusters including the nifH sequences of the methane seep group 2 and the subsequent reverse transcription-PCR detection of the nifD and nifH genes strongly suggested that the genetic components of the gene clusters would be operative for the in situ assimilation of molecular nitrogen (N 2 ) by the host microorganisms. DNA-based quantitative PCR of the archaeal 16S rRNA gene, the group-specific mcrA (a gene encoding the methyl-coenzyme M reductase α subunit) gene, and the nifD and nifH genes demonstrated the similar distribution patterns of the archaeal 16S rRNA gene, the mcrA groups c-d and e, and the nifD and nifH genes through the core sediments. These results supported the idea that the anoxic methanotrophic archaea ANME-2c could be the microorganisms hosting the nif gene clusters and could play an important role in not only the in situ carbon (methane) cycle but also the nitrogen cycle in subseafloor sediments.

We have developed a technique for cultivation of chemolithoautotrophs under high hydrostatic pressures that is successfully applicable to various types of deep-sea chemolithoautotrophs, including methanogens. It is based on a glass-syringe-sealing liquid medium and gas mixture used in conjunction with a butyl rubber piston and a metallic needle stuck into butyl rubber. By using this technique, growth, survival, and methane production of a newly isolated, hyperthermophilic methanogen Methanopyrus kandleri strain 116 are characterized under high temperatures and hydrostatic pressures. Elevated hydrostatic pressures extend the temperature maximum for possible cell proliferation from 116°C at 0.4 MPa to 122°C at 20 MPa, providing the potential for growth even at 122°C under an in situ high pressure. In addition, piezophilic growth significantly affected stable carbon isotope fractionation of methanogenesis from CO 2 . Under conventional growth conditions, the isotope fractionation of methanogenesis by M. kandleri strain 116 was similar to values (−34‰ to−27‰) previously reported for other hydrogenotrophic methanogens. However, under high hydrostatic pressures, the isotope fractionation effect became much smaller (

A quantitative fluorogenic PCR method for detecting methanogenic and methanotrophic orders was established using a refined primer set for the methyl coenzyme M reductase subunit A gene (mcrA). The method developed was applied to several microbial communities in which diversity and abundance of methanogens or anaerobic methanotrophs (ANMEs) was identified by 16S rRNA gene clone analysis, and strong correlations between the copy numbers of mcrA with those of archaeal 16S rRNA genes in the communities were observed. The assay can be applied to detecting and assessing the abundance of methanogens and/or ANMEs in anoxic environments that could not be detected by 16S rRNA gene sequence analyses.

Sclerosing lipogranuloma (SLG) is a rare disease that occurs in the male genital region. We report here three cases of SLG, including two of Y-shaped lesions in the penile base forming an intrascrotal mass, as well as a patient with a mass in the epidydimal region. All three lesions histologically exhibited formation of multiple granulomas consisting of multinucleated giant cells and epithelioid cells, in the fibroadipose tissue or around the epidydimis, in association with eosinophil infiltration. Membranocystic changes were found within the cytoplasm of multinucleated cells. In the two patients with scrotal lesions, membranocystic changes were positive for diastase-PAS reaction and on Sudan black B staining in dewaxed sections. CD68 staining was strongly positive in multinucleated giant cells and epithelioid cells. Most of the lymphocytes infiltrating the lesions were T cells associated with some S-100-positive dendritic cells. T-cell mediated immune reaction appears to be important in the histogenesis of SLG. The histogenesis is generally considered to be a foreign-body reaction to degenerated or damaged fatty tissue or lipids, but no apparent causative factors can be identified in some reported cases. All three patients in the present study had a good clinical course after biopsy or surgical excision. We thus report three cases of SLG including an unusual case in the epidydimal region, with some considerations regarding the histogenesis of SLG.

Although monocyte infiltration is an important aspect of the host response to tumor growth, the mechanisms of recruitment and their impact on tumor growth are still unknown. The authors studied monocyte-endothelial interaction and the effect of chemokine CCL2 in orthotopic mouse pancreatic cancer.The rolling and adhesion of labeled monocytes in peritumoral and intratumoral areas were assessed by using an intravital microscope. Further, the effects of intratumoral injection or superfusion of CCL2 on in situ recruitment of monocytes and other immune cells and adhesion molecules were investigated.More monocytes were recruited in the peritumoral area than in the intratumoral area with increased vascular interaction, and the effect was more apparent by intratumoral CCL2 injection than superfusion. In both CCL2-treated groups infiltration of CD11b(+), CD68(+), and CD4(+) cells were increased, but the magnitude of increase was larger in intratumoral injection. Quantitative RT-PCR for the tumor tissue revealed that ICAM-1 expression was increased by the injection of CCL2.These results show intratumoral injection of CCL2 induces effective interaction between monocytes and endothelial cells in the peritumoral area of pancreatic cancer accompanied by the upregulation of ICAM-1 and may possibly become a tool for immunotherapy by promoting the infiltration of immune cells in cancers.

Hadal oceans at water depths below 6,000 m are the least-explored aquatic biosphere. The Challenger Deep, located in the western equatorial Pacific, with a water depth of ∼11 km, is the deepest ocean on Earth. Microbial communities associated with waters from the sea surface to the trench bottom (0 ∼10,257 m) in the Challenger Deep were analyzed, and unprecedented trench microbial communities were identified in the hadal waters (6,000 ∼10,257 m) that were distinct from the abyssal microbial communities. The potentially chemolithotrophic populations were less abundant in the hadal water than those in the upper abyssal waters. The emerging members of chemolithotrophic nitrifiers in the hadal water that likely adapt to the higher flux of electron donors were also different from those in the abyssal waters that adapt to the lower flux of electron donors. Species-level niche separation in most of the dominant taxa was also found between the hadal and abyssal microbial communities. Considering the geomorphology and the isolated hydrotopographical nature of the Mariana Trench, we hypothesized that the distinct hadal microbial ecosystem was driven by the endogenous recycling of organic matter in the hadal waters associated with the trench geomorphology.

To develop effective drugs for hypogonadism, sarcopenia, and cachexia, we designed, synthesized, and evaluated selective androgen receptor modulators (SARMs) that exhibit not only anabolic effects on organs such as muscles and the central nervous system (CNS) but also neutral or antagonistic effects on the prostate. Based on the information obtained from a docking model with androgen receptor (AR), we modified a hit compound A identified through high-throughput screening. Among the prepared compounds, 1-(4-cyano-1-naphthyl)-2,3-disubstituted pyrrolidine derivatives 17h, 17m, and 17j had highly potent AR agonistic activities in vitro and good tissue selectivity in vivo. These derivatives increased the weight of the levator ani muscle without influencing the prostate and seminal vesicle. In addition, these compounds induced sexual behavior in castrated rats, indicating that the compounds could also act as agonists on the CNS.