Cultivation and biogeochemical analyses reveal insights into methanogenesis in deep subseafloor sediment at a biogenic gas hydrate site.

Gas hydrates deposited in subseafloor sediments are considered to primarily consist of biogenic methane. However, little evidence for the occurrence of living methanogens in subseafloor sediments has been provided. This study investigated viable methanogen diversity, population, physiology and potential activity in hydrate-bearing sediments (1-307 m below the seafloor) from the eastern Nankai Trough. Radiotracer experiments, the quantification of coenzyme F430 and molecular sequencing analysis indicated the occurrence of potential methanogenic activity and living methanogens in the sediments and the predominance of hydrogenotrophic methanogens followed by methylotrophic methanogens. Ten isolates and nine representative culture clones of hydrogenotrophic, methylotrophic and acetoclastic methanogens were obtained from the batch incubation of sediments and accounted for 0.5-76% of the total methanogenic sequences directly recovered from each sediment. The hydrogenotrophic methanogen isolates of Methanocalculus and Methanoculleus that dominated the sediment methanogen communities produced methane at temperatures from 4 to 55 °C, with an abrupt decline in the methane production rate at temperatures above 40 °C, which is consistent with the depth profiles of potential methanogenic activity in the Nankai Trough sediments in this and previous studies. Our results reveal the previously overlooked phylogenetic and metabolic diversity of living methanogens, including methylotrophic methanogenesis.

Long-term wheel-running prevents reduction of grip strength in type 2 diabetic rats.

Diabetic skeletal muscles show reduced contractile force and increased fatigability. Hands are a target for several diabetes-induced complications. Therefore, reduced handgrip strength often occurs as a consequence of diabetes. The aim of this study was to examine whether long-term exercise can prevent reduction of grip strength in type 2 diabetes mellitus (T2DM) model OLETF rats, and to explore the mechanisms underlying diabetes-induced grip strength reduction. Ten 5-week-old OLETF rats were used as experimental animals, and five non-diabetic LETO rats as controls of OLETF rats. Half OLETF rats performed daily voluntary wheel-running for 17 months (OLETF + EXE), and the rest of OLETF and LETO rats were sedentary. Grip strength was higher in OLETF + EXE and LETO groups than in OLETF group. OLETF group with hyperglycemia showed an increase in HbA1c, serum TNF-α, and muscle SERCA activity, but a decrease in circulating insulin. Each fiber area, total fiber area, and % total fiber area in type IIb fibers of extensor digitorum longus muscles were larger in OLETF + EXE and LETO groups than in OLETF group. There was a positive correlation between grip strength and the above three parameters concerning type IIb fiber area. Therefore, type IIb fiber atrophy may be the major direct cause of grip strength reduction in OLETF group, although there seems multiple etiological mechanisms. Long-term wheel-running may have blocked the diabetes-induced reduction of grip strength by preventing type IIb fiber atrophy. Regular exercise may be a potent modality for preventing not only the progression of diabetes but muscle dysfunction in T2DM patients.

Effects of whole-body vibration on bone properties in aged rats.

OBJECTIVE: This study aimed to explore optimal conditions of whole-body vibration (WBV) for improving bone properties in aged rats. METHODS: Eighty-week-old rats were divided into baseline control (BC), age-matched control (CON) and experimental groups, which underwent WBV (0.5 g) at various frequencies (15, 30, 45, 60 or 90 Hz) or WBV (45 Hz) with various magnitudes (0.3, 0.5, 0.7 or 1.0 g) for 7 weeks. After interventions, femur bone size, bone mechanical strength and circulating bone formation/resorption markers were measured, and trabecular bone microstructure (TBMS) and cortical bone geometry (CBG) of femurs were analyzed by micro-CT. RESULTS: Several TBMS parameters and trabecular bone mineral content were significantly lower in the 15 Hz WBV (0.5 g) group than in the CON group, suggesting damage to trabecular bone. On the other hand, although frequency/magnitude of WBV did not influence any CBG parameters, the 0.7 g and 1.0 g WBV (45 Hz) group showed an increase in tissue mineral density of cortical bone compared with the BC and CON groups, suggesting the possibility of improving cortical bone properties. CONCLUSION: Based on these findings, it should be noted that WBV conditions are carefully considered when applied to elderly people.

Methanogenic archaea use a bacteria-like methyltransferase system to demethoxylate aromatic compounds.

Methane-generating archaea drive the final step in anaerobic organic compound mineralization and dictate the carbon flow of Earth's diverse anoxic ecosystems in the absence of inorganic electron acceptors. Although such Archaea were presumed to be restricted to life on simple compounds like hydrogen (H2), acetate or methanol, an archaeon, Methermicoccus shengliensis, was recently found to convert methoxylated aromatic compounds to methane. Methoxylated aromatic compounds are important components of lignin and coal, and are present in most subsurface sediments. Despite the novelty of such a methoxydotrophic archaeon its metabolism has not yet been explored. In this study, transcriptomics and proteomics reveal that under methoxydotrophic growth M. shengliensis expresses an O-demethylation/methyltransferase system related to the one used by acetogenic bacteria. Enzymatic assays provide evidence for a two step-mechanisms in which the methyl-group from the methoxy compound is (1) transferred on cobalamin and (2) further transferred on the C1-carrier tetrahydromethanopterin, a mechanism distinct from conventional methanogenic methyl-transfer systems which use coenzyme M as final acceptor. We further hypothesize that this likely leads to an atypical use of the methanogenesis pathway that derives cellular energy from methyl transfer (Mtr) rather than electron transfer (F420H2 re-oxidation) as found for methylotrophic methanogenesis.

Correction: Possible cross-feeding pathway of facultative methylotroph Methyloceanibacter caenitepidi Gela4 on methanotroph Methylocaldum marinum S8.

[This corrects the article DOI: 10.1371/journal.pone.0213535.].

Koleobacter methoxysyntrophicus gen. nov., sp. nov., a novel anaerobic bacterium isolated from deep subsurface oil field and proposal of Koleobacteraceae fam. nov. and Koleobacterales ord. nov. within the class Clostridia of the phylum Firmicutes.

An anaerobic thermophilic, rod-shaped bacterium possessing a unique non-lipid sheathed-like structure enveloping a single-membraned cell, designated strain NRmbB1T was isolated from at the deep subsurface oil field located in Yamagata Prefecture, Japan. Growth occurred with 40-60°C (optimum, 55°C), 0-2% (2%), NaCl and pH 6.0-8.5 (8.0). Fermentative growth with various sugars was observed. Glucose-grown cells generated acetate, hydrogen, pyruvate and lactate as the main end products. Syntrophic growth occurred with glucose, pyruvate and 3,4,5-trimethoxybenzoate in the presence of an H2-scavenging partner, and growth on 3,4,5-trimethoxybenzoate was only observed under syntrophic condition. The predominant cellular fatty acids were C16:0, iso-C16:0, anteiso-C15:0, and iso-C14:0. Respiratory quinone was not detected. The genomic G+C content was 40.8mol%. Based on 16S rRNA gene phylogeny, strain NRmbB1T belongs to a distinct order-level clade in the class Clostridia of the phylum Firmicutes, sharing low similarity with other isolated organisms (i.e., 87.5% for top hit Moorella thermoacetica DSM 2955T). In total, chemotaxonomic, phylogenetic and genomic characterization revealed that strain NRmbB1T (=KCTC 25035T, =JCM 39120T) represents a novel species of a new genus. In addition, we also propose the associated family and order as Koleobacteraceae fam. nov and Koleobacterales ord. nov., respectively.

Unexpected diversity of acetate degraders in anaerobic membrane bioreactor treating organic solid waste revealed by high-sensitivity stable isotope probing.

In anaerobic membrane bioreactor (AnMBR) treating organic solid waste, acetate is one of the most important precursors to CH4. However, the identity and diversity of anaerobic acetate degraders are largely unknown, possibly due to their slow growth rates and low abundances. Here, we identified acetate-degrading microorganisms in the AnMBR sludges by high-sensitivity stable isotope probing. Degradation of the amended 13C-acetate coincided with production of 13CH4 and 13CO2 during the sludge incubation. High-throughput sequencing of RNA density fractions indicated that the aceticlastic and hydrogenotrophic methanogens, i.e., Methanosaeta sp. (acetate dissimilator) and Methanolinea sp. (acetate assimilator), incorporated 13C-acetate significantly. Remarkably, 22 bacterial species incorporating 13C-acetate were identified, whereas their majority was distantly related to the cultured representatives. Only two of them were the class Deltaproteobacteria-affiliated lineages with syntrophic volatile fatty acid oxidation activities. Phylogenetic tree analysis and population dynamics tracing revealed that novel species of the hydrolyzing and/or fermenting taxa, such as the phyla Bacteroidetes, Chloroflexi and Lentisphaerae, exhibited low relative abundances comparable to that of Methanolinea sp. (0.00011%) during the AnMBR operation, suggesting that these bacteria were involved in anaerobic acetate assimilation. Meanwhile, novel species of the phyla Firmicutes, Synergistetes and Caldiserica, the candidate phyla Aminicenantes and Atribacteria and the candidate division GOUTA4-related clade, as well as the known Deltaproteobacteria members, existed at relatively high abundances (0.00031%-0.31121%) in the reactor, suggesting that these bacterial species participated in anaerobic dissimilation of acetate, e.g., syntrophic acetate oxidation. The results of this study demonstrated the unexpected diversity and ecophysiological features of the anaerobic acetate degraders in the AnMBR treating organic solid waste.

Parasympathetic nervous activity is associated with oxytocin in multiparous, but not primiparous, women during the perinatal period.

Oxytocin (OXT) is thought to have antidepressant/anxiolytic effects in postpartum women. Primiparous women tend toward an attenuated lactation compared with multiparous women. However, so far, little is known about the relationship between OXT and autonomic nervous activity (ANA) in perinatal women and whether it may be different in primiparous and multiparous women. Therefore, the objective of this study was to answer this question by determining both ANA and salivary OXT levels in primiparous and multiparous perinatal women. In 18 primiparous and 18 multiparous women, who underwent a physical and physiological examination, ANA measurement by heart rate variability and saliva sampling were performed during the perinatal period. Saliva OXT concentration was determined by a highly sensitive ELISA. OXT release into saliva was obtained from multiplying saliva OXT concentration by saliva flow rate. In the postpartum period, multiparous women had higher parasympathetic nervous activity (PNA) and lower physical stress index (PSI) compared with primiparous women. Furthermore, multiparous postpartal women had higher OXT compared with primiparous or multiparous prepartal women. In addition, in multiparous perinatal women, OXT correlated positively with PNA, but negatively with PSI. These results suggest that after parturition, multiparous mothers may switch over to the "feed and breed" system more quickly due to increased OXT compared with primiparous mothers. Our findings support antidepressant/anxiolytic and anti-stress effects of OXT. In postpartal women exposed to synthetic OXT, ANA measurement may provide a clue to clarify the effects of exogenous OXT on postpartum psychiatric disorders.

Possible effects of whole body vibration on bone properties in growing rats.

OBJECTIVES: To examine the effects of whole body vibration (WBV) on bone properties in growing rats, and to explore the optimal conditions for enhancing bone properties. METHODS: Thirty-six 4-week-old male rats were divided into 1 control and 5 experimental groups. Each experimental group underwent WBV at 15, 30, 45, 60, and 90 Hz (0.5 g, 15 min/d, 5 d/wk) for 8 weeks. We measured bone size, muscle weight and bone mechanical strength of the right tibia. Trabecular bone mass and trabecular bone microstructure (TBMS) of the left tibia were analyzed by micro-computed tomography. Serum levels of bone formation/resorption markers were also measured. RESULTS: WBV at 45 Hz and 60 Hz tended to enhance trabecular bone mass and TBMS parameters. However, there was no difference in maximum load of tibias among all groups. Serum levels of bone resorption marker were significantly higher in the 45-Hz WBV group than in the control group. CONCLUSIONS: WBV at 45-60 Hz may offer a potent modality for increasing bone mass during the period of rapid growth. Further studies are needed to explore the optimal WBV conditions for increasing peak bone mass and TBMS parameters. WBV modality may be a potent strategy for primary prevention against osteoporosis.

Whole body vibration at low-frequency can increase trabecular thickness and width in adult rats.

OBJECTIVE: This study aimed to examine whether WBV becomes a possible modality for the primary prevention of osteoporosis by exploring WBV frequency that has positive effects on bone properties in adult rats. METHODS: Thirty-six 24-week-old rats were divided into one control and 5 experimental groups, which underwent WBV at various frequencies (15, 30, 45, 60 or 90 Hz), with a magnitude of 0.5 g, for 15 min/day, 5 days/week, for 8 weeks. Bone size, muscle weight and bone mechanical strength were measured at the end of experimental period. Bone mass, trabecular bone microarchitecture (TBMA) and cortical bone geometry were analyzed by micro-CT. Circulating bone formation/resorption markers were determined by ELISA. RESULTS: Body weight-corrected soleus weight in all experimental groups and body weight-corrected extensor digitorum longus muscle weight in the 15 and 30 Hz groups were significantly higher than those of the control group, respectively. Femur trabecular thickness and width were significantly higher in the 15 Hz group than in the control group. However, there was no difference in bone mechanical strength or bone formation/resorption markers among all groups. CONCLUSION: These results suggest that WBV at low-frequencies may become a potent modality for the primary prevention of osteoporosis in adults.

Possible cross-feeding pathway of facultative methylotroph Methyloceanibacter caenitepidi Gela4 on methanotroph Methylocaldum marinum S8.

Non-methanotrophic bacteria such as methylotrophs often coexist with methane-oxidizing bacteria (methanotrophs) by cross-feeding on methane-derived carbon. Methanol has long been considered a major compound that mediates cross-feeding of methane-derived carbon. Despite the potential importance of cross-feeding in the global carbon cycle, only a few studies have actually explored metabolic responses of a bacteria when cross-feeding on a methanotroph. Recently, we isolated a novel facultative methylotroph, Methyloceanibacter caenitepidi Gela4, which grows syntrophically with the methanotroph, Methylocaldum marinum S8. To assess the potential metabolic pathways in M. caenitepidi Gela4 co-cultured with M. marinum S8, we conducted genomic analyses of the two strains, as well as RNA-Seq and chemical analyses of M. caenitepidi Gela4, both in pure culture with methanol and in co-culture with methanotrophs. Genes involved in the serine pathway were downregulated in M. caenitepidi Gela4 under co-culture conditions, and methanol was below the detection limit (< 310 nM) in both pure culture of M. marinum S8 and co-culture. In contrast, genes involved in the tricarboxylic acid cycle, as well as acetyl-CoA synthetase, were upregulated in M. caenitepidi Gela4 under co-culture conditions. Notably, a pure culture of M. marinum S8 produced acetate (< 16 µM) during growth. These results suggested that an organic compound other than methanol, possibly acetate, might be the major carbon source for M. caenitepidi Gela4 cross-fed by M. marinum S8. Co-culture of M. caenitepidi Gela4 and M. marinum S8 may represent a model system to further study methanol-independent cross-feeding from methanotrophs to non-methanotrophic bacteria.

Microbial community structure in deep natural gas-bearing aquifers subjected to sulfate-containing fluid injection.

In the natural gas field located in central Japan, high concentrations of natural gases and iodide ions are dissolved in formation water and commercially produced in deep aquifers. In the iodine recovery process, the produced formation water is amended with sulfate, and this fluid is injected into gas-bearing aquifers, which may lead to infrastructure corrosion by hydrogen sulfide. In this study, we examined the microbial community in aquifers subjected to sulfate-containing fluid injection. Formation water samples were collected from production wells located at different distances from the injection wells. The chemical analysis showed that the injection fluid contained oxygen, nitrate, nitrite and sulfate, in contrast to the formation water, which had previously been shown to be depleted in these components. Sulfur isotopic analysis indicated that sulfate derived from the injection fluid was present in the sample collected from near the injection wells. Quantitative and sequencing analysis of dissimilatory sulfite reductase and 16S rRNA genes revealed that sulfate-reducing bacteria (SRB), sulfur-oxidizing bacteria, and anaerobic methanotrophic archaea (ANME) in the wells located near injection wells were more abundant than those in wells located far from the injection wells, suggesting that fluid injection stimulated these microorganisms through the addition of oxygen, nitrate, nitrite and sulfate to the methane-rich aquifers. The predominant taxa were assigned to the ANME-2 group, its sulfate-reducing partner SEEP-SRB1 cluster and sulfur-oxidizing Epsilonproteobacteria. These results provide important insights for future studies to support the development of natural gas and iodine resources in Japan.

High-fat/high-sucrose diet results in higher bone mass in aged rats.

Intake of high-fat/high-sucrose (HFS) diet or high fat diet influences bone metabolism in young rodents, but its effects on bone properties of aged rodents still remain unclear. This study aimed to examine the effects of HFS diet intake on trabecular bone architecture (TBA) and cortical bone geometry (CBG) in aged rats. Fifteen male Wistar rats over 1 year were randomly divided into two groups. One group was fed a standard laboratory diet (SLD) and the other group was fed a HFS diet for six months. The femur/tibia, obtained from both groups at the end of experimental period, were scanned by micro-computed tomography for TBA/CBG analyses. Serum biochemical analyses were also conducted. Body weight was significantly higher in the HFS group than in the SLD group. In both femur and tibia, the HFS group showed higher trabecular/cortical bone mass in reference to bone mineral content, volume bone mineral density and TBA/CBG parameters compared with the SLD group. In addition, serum calcium, inorganic phosphorus, total protein, triacylglycerol, HDL and TRACP-5b levels were significantly higher in the HFS group than in the SLD group. There were good correlations between body weight and bone parameters in the femur and tibia. These results suggest that HFS diet intake results in higher bone mass in aged rats. Such effects of HFS diet intake might have been induced by increased body weight.

Long-Term Intake of Green Tea Extract Causes Mal-Conformation of Trabecular Bone Microarchitecture in Growing Rats.

The purpose of this study was to examine the effects of green tea extract (GTE) intake on bone structural and physiological properties, such as bone mass, trabecular bone microarchitecture, cortical bone geometry, and bone mechanical strength, in growing rats. Four-week-old male Wistar rats were divided into the following four groups: standard diet feeding for 85 days (S-CON) or 170 days (L-CON), and GTE diet feeding for 85 days (S-GTE) or 170 days (L-GTE). At the end of the experiment, in addition to measurement of circulating bone formation/resorption markers, bone mass, trabecular bone microarchitecture, and cortical bone geometry were analyzed in the left femur, and bone mechanical strength of the right femur was measured. There was no difference in all bone parameters between the S-CON and S-GTE groups. On the other hand, the L-GTE group showed the decrease in some trabecular bone mass/microarchitecture parameters and no change in cortical bone mass/geometry parameters compared with the L-CON group, and consequently the reduction in bone weight corrected by body weight. There was no difference in bone formation/resorption markers and bone mechanical strength between the S-CON and S-GTE groups and also between the L-CON and L-GTE groups. However, serum leptin levels were significantly lower in the L-GTE group than in the L-CON group. Thus, the long-term GTE intake had negative effects on bone, especially trabecular bone loss and microarchitecture mal-conformation, in growing rats.

Origin of methane-rich natural gas at the West Pacific convergent plate boundary.

Methane emission from the geosphere is generally characterized by a radiocarbon-free signature and might preserve information on the deep carbon cycle on Earth. Here we report a clear relationship between the origin of methane-rich natural gases and the geodynamic setting of the West Pacific convergent plate boundary. Natural gases in the frontal arc basin (South Kanto gas fields, Northeast Japan) show a typical microbial signature with light carbon isotopes, high CH4/C2H6 and CH4/3He ratios. In the Akita-Niigata region - which corresponds to the slope stretching from the volcanic-arc to the back-arc -a thermogenic signature characterize the gases, with prevalence of heavy carbon isotopes, low CH4/C2H6 and CH4/3He ratios. Natural gases from mud volcanoes in South Taiwan at the collision zone show heavy carbon isotopes, middle CH4/C2H6 ratios and low CH4/3He ratios. On the other hand, those from the Tokara Islands situated on the volcanic front of Southwest Japan show the heaviest carbon isotopes, middle CH4/C2H6 ratios and the lowest CH4/3He ratios. The observed geochemical signatures of natural gases are clearly explained by a mixing of microbial, thermogenic and abiotic methane. An increasing contribution of abiotic methane towards more tectonically active regions of the plate boundary is suggested.

Petrothermobacter organivorans gen. nov., sp. nov., a thermophilic, strictly anaerobic bacterium of the phylum Deferribacteres isolated from a deep subsurface oil reservoir.

A novel thermophilic, anaerobic, chemoheterotrophic, acetate-oxidizing and iron(III)-, manganese(IV)-, nitrate- and sulfate-reducing bacterium, designated strain ANAT, was isolated from a deep subsurface oil field in Japan (Yabase oil field, Akita Pref.). Cells of strain ANAT were Gram-stain-negative, non-motile, non-spore forming and slightly curved or twisted rods (1.5-5.0 µm long and 0.6-0.7 µm wide). The isolate grew at 25-60 °C (optimum 55 °C) and pH 6.0-8.0 (optimum pH 7.0). The isolate was capable of reducing iron(III), manganese(IV), nitrate and sulfate as an electron acceptor. The isolate utilized a limited range of electron donors such as acetate, lactate, pyruvate and yeast extract for iron reduction. Strain ANAT also used pyruvate, fumarate, succinate, malate, yeast extract and peptone for fermentative growth. The major respiratory quinones were menaquinone-7(H8) and menaquinone-8. The strain contained C18 : 0, iso-C18 : 0 and C16 : 0 as the major cellular fatty acids. The G+C content of the genomic DNA was 34.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain ANAT was closely related to Calditerrivibrio nitroreducens in the phylum Deferribacteres with low sequence similarities (89.5 %), and formed a distinct clade within the family Deferribacteraceae. In addition, the isolate is the first sulfate-reducing member of the phylum Deferribacteres. Based on phenotypic, chemotaxonomic and phylogenetic properties, a novel genus and species, Petrothermobacter organivorans gen. nov., sp. nov., is proposed for the isolate (type strain=ANAT= NBRC 112621T=DSM 105015T).

Methane production from coal by a single methanogen.

Coal-bed methane is one of the largest unconventional natural gas resources. Although microbial activity may greatly contribute to coal-bed methane formation, it is unclear whether the complex aromatic organic compounds present in coal can be used for methanogenesis. We show that deep subsurface-derived Methermicoccus methanogens can produce methane from more than 30 types of methoxylated aromatic compounds (MACs) as well as from coals containing MACs. In contrast to known methanogenesis pathways involving one- and two-carbon compounds, this "methoxydotrophic" mode of methanogenesis couples O-demethylation, CO2 reduction, and possibly acetyl-coenzyme A metabolism. Because MACs derived from lignin may occur widely in subsurface sediments, methoxydotrophic methanogenesis would play an important role in the formation of natural gas not limited to coal-bed methane and in the global carbon cycle.

Methanomicrobium antiquum sp. nov., a hydrogenotrophic methanogen isolated from deep sedimentary aquifers in a natural gas field.

A mesophilic, hydrogenotrophic methanogen, designated strain MobHT, was isolated from sediments derived from deep sedimentary, natural-gas-bearing aquifers in Japan. Strain MobHT utilized H2/CO2 or formate, but not ethanol, 1-propanol, 2-propanol, 2-butanol or cyclopentanol, for growth and methane production. In addition, acetate and tungsten were required for growth. Yeast extract stimulated the growth, but was not required. The cells were weakly motile with multiple flagella, presented as a curved-rod-shaped (0.8×2.0 µm) and occurred singly or in pairs. Strain MobHT grew at 15-40 °C (optimum 35 °C) and at pH 5.9-7.9 (optimum pH 7.0-7.5). The sodium chloride range for growth was 0-5.8 % (optimum 2 %). The G+C content of the genomic DNA was 37.6 mol%. In the phylogenetic tree based on the 16S rRNA gene sequences, strain MobHT clustered together with Methanomicrobium mobile (95.4 % in sequence similarity), and formed a distinct clade from Methanolacinia petrolearia SEBR 4847T (95.6 %) and Methanolacinia paynteri G-2000T (95.4 %). The two species of the genus Methanolacinia utilized 2-propanol, whereas strain MobHT and Methanomicrobium mobile, the sole species of the genus Methanomicrobium, do not. Based on phenotypic and phylogenetic features, we propose a novel species for the isolate with the name, Methanomicrobiumantiquum sp. nov. The type strain is MobHT (=DSM 21220T=NBRC 104160T).

Changes in microbial communities associated with gas hydrates in subseafloor sediments from the Nankai Trough.

Little is known about the microbial distribution patterns in subseafloor sediments. This study examines microbial diversity and activities in sediments of the Nankai Trough, where biogenic gas hydrates are deposited. Illumina sequencing of 16S rRNA genes revealed that the prokaryotic community structure is correlated with hydrate occurrence and depth but not with the sedimentary facies. The bacterial phyla 'Atribacteria' lineage JS1 and Chloroflexi dominated in all samples, whereas lower taxonomic units of Chloroflexi accounted for community variation related to hydrate saturation. In archaeal communities, 'Bathyarchaeota' was significantly abundant in the hydrate-containing samples, whereas Marine Benthic Group-B dominated in the upper sediments without hydrates. mcrA gene sequences assigned to deeply branching groups and ANME-1 were detected only in hydrate-containing samples. A predominance of hydrogenotrophic methanogens, Methanomicrobiales and Methanobacteriales, over acetoclastic methanogens was found throughout the depth. Incubation tests on hydrate-containing samples with a stable isotope tracer showed anaerobic methane oxidation activities under both low- and seawater-like salinity conditions. These results indicate that the distribution patterns of microorganisms involved in carbon cycling changed with gas hydrate occurrence, possibly because of the previous hydrate dissociation followed by pore water salinity decrease in situ, as previously proposed by a geochemical study at the study site.

Femoral bone structure in Otsuka Long-Evans Tokushima Fatty rats.

OBJECTIVES: Type 2 diabetes mellitus (T2DM) increases fracture risk despite normal to high levels of bone mineral density. Bone quality is known to affect bone fragility in T2DM. The aim of this study was to clarify the trabecular bone microstructure and cortical bone geometry of the femur in T2DM model rats. METHODS: Five-week-old Otsuka Long-Evans Tokushima Fatty (OLETF; n = 5) and Long-Evans Tokushima Otsuka (LETO; n = 5) rats were used. At the age of 18 months, femurs were scanned with micro-computed tomography, and trabecular bone microstructure and cortical bone geometry were analyzed. RESULTS: Trabecular bone microstructure and cortical bone geometry deteriorated in the femur in OLETF rats. Compared with in LETO rats, in OLETF rats, bone volume fraction, trabecular number and connectivity density decreased, and trabecular space significantly increased. Moreover, in OLETF rats, cortical bone volume and section area decreased, and medullary volume significantly increased. CONCLUSIONS: Long-term T2DM leaded to deterioration in trabecular and cortical bone structure. Therefore, OLETF rats may serve as a useful animal model for investigating the relationship between T2DM and bone quality.