The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the ‘Thaumarchaeota’ and ‘Korarchaeota’. Here, we show the genome sequence of Candidatus ‘Caldiarchaeum subterraneum’ that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea.

Deep-sea hydrothermal activity results in diverse physical and chemical environments for the resident microbial communities. Using cultivation techniques and culture-independent molecular analyses, diverse lineages of archaea and bacteria have so far been observed from chimney structures, retrieved in situ colonization systems settled in or on the hydrothermal conduit, microbial mats, sediments, and chemosynthetic macrofaunal bodies (19,35,62). Especially in the domain Archaea, most of lineages derived from hydrothermal environments have not yet been cultivated, and little is known about their physiological and metabolic traits.

Microbial community structures in methane seep sediments in the Nankai Trough were analyzed by tag-sequencing analysis for the small subunit (SSU) rRNA gene using a newly developed primer set. The dominant members of Archaea were Deep-sea Hydrothermal Vent Euryarchaeotic Group 6 (DHVEG 6), Marine Group I (MGI) and Deep Sea Archaeal Group (DSAG), and those in Bacteria were Alpha-, Gamma-, Delta- and Epsilonproteobacteria, Chloroflexi, Bacteroidetes, Planctomycetes and Acidobacteria. Diversity and richness were examined by 8,709 and 7,690 tag-sequences from sediments at 5 and 25 cm below the seafloor (cmbsf), respectively. The estimated diversity and richness in the methane seep sediment are as high as those in soil and deep-sea hydrothermal environments, although the tag-sequences obtained in this study were not sufficient to show whole microbial diversity in this analysis. We also compared the diversity and richness of each taxon/division between the sediments from the two depths, and found that the diversity and richness of some taxa/divisions varied significantly along with the depth.

Uncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.

Subseafloor pelagic sediments with high concentrations of organic matter form habitats for diverse microorganisms. Here, we determined depth profiles of genes for SSU rRNA, mcrA, dsrA and amoA from just beneath the seafloor to 363.3 m below the seafloor (mbsf) using core samples obtained from the forearc basin off the Shimokita Peninsula. The molecular profiles were combined with data on lithostratigraphy, depositional age, sedimentation rate and pore-water chemistry. The SSU rRNA gene tag structure and diversity changed at around the sulfate-methane transition zone (SMTZ), whereas the profiles varied further with depth below the SMTZ, probably in connection with the variation in pore-water chemistry. The depth profiles of diversity and abundance of dsrA, a key gene for sulfate reduction, suggested the possible niche separations of sulfate-reducing populations, even below the SMTZ. The diversity and abundance patterns of mcrA, a key gene for methanogenesis/anaerobic methanotrophy, suggested a stratified distribution and separation of anaerobic methanotrophy and hydrogenotrophic or methylotrophic methanogensis below the SMTZ. This study provides novel insights into the relationships between the composition and function of microbial communities and the chemical environment in the nutrient-rich continental margin subseafloor sediments, which may result in niche separation and variability in subseafloor microbial populations.

Pancreatic cancer is one of the most difficult types of cancer to treat because of its high mortality rate due to chemotherapy resistance. We previously reported that combined treatment with gefitinib (GEF) and clarithromycin (CAM) results in enhanced cytotoxicity of GEF along with endoplasmic reticulum (ER) stress loading in non-small cell lung cancer cell lines. An epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) such as GEF induces autophagy in a pro-survival role, whereas CAM inhibits autophagy flux in various cell lines. Pronounced GEF-induced cytotoxicity therefore appears to depend on the efficacy of autophagy inhibition. In the present study, we compared the effect on autophagy inhibition among such macrolides as CAM, azithromycin (AZM), and EM900, a novel 12-membered non-antibiotic macrolide. We then assessed the enhanced GEF-induced cytotoxic effect on pancreatic cancer cell lines BxPC-3 and PANC-1. Autophagy flux analysis indicated that AZM is the most effective autophagy inhibitor of the three macrolides. CAM exhibits an inhibitory effect but less than AZM and EM900. Notably, the enhancing effect of GEF-induced cytotoxicity by combining macrolides correlated well with their efficient autophagy inhibition. However, this pronounced cytotoxicity was not due to upregulation of apoptosis induction, but was at least partially mediated through necroptosis. Our data suggest the possibility of using macrolides as ‘chemosensitizers’ for EGFR-TKI therapy in pancreatic cancer patients to enhance non-apoptotic tumor cell death induction.

In the cell cycle, the G1/S transition is controlled by the cyclin‐dependent kinase (CDK) 4/6‐cyclin D complex. Constitutive activation of CDK4/6 dysregulates G1/S transition, leading to oncogenic transformation. We found that 3 CDK4/6 inhibitors, abemaciclib, ribociclib, and palbociclib, exerted a cytocidal effect as well as a cytostatic effect at the G1 phase in cancer cell lines, including A549 human non–small cell lung cancer cells. Among these inhibitors, abemaciclib exhibited the most potent cytotoxic effect. The cell‐death phenotype induced by abemaciclib, which entailed formation of multiple cytoplasmic vacuoles, was not consistent with apoptosis or necroptosis. Abemaciclib blocked autophagic flux, resulting in accumulation of autophagosomes, however vacuole formation and cell death induced by abemaciclib were independent of autophagy. In addition, methuosis, a cell‐death phenotype characterized by vacuole formation induced by excessive macropinocytosis, was excluded because the vacuoles did not incorporate fluorescent dextran. Of note, both formation of vacuoles and induction of cell death in response to abemaciclib were inhibited by vacuolar‐type ATPase (V‐ATPase) inhibitors such as bafilomycin A1 and concanamycin A. Live‐cell imaging revealed that the abemaciclib‐induced vacuoles were derived from lysosomes that expanded following acidification. Transmission electron microscopy revealed that these vacuoles contained undigested debris and remnants of organelles. Cycloheximide chase assay revealed that lysosomal turnover was blocked by abemaciclib. Furthermore, mTORC1 inhibition along with partial lysosomal membrane permeabilization occurred after abemaciclib treatment. Together, these results indicate that, in cancer cells, abemaciclib induces a unique form of cell death accompanied by swollen and dysfunctional lysosomes.

A novel marine thermophilic and heterotrophic Anaerolineae bacterium in the phylum Chloroflexi, strain SW7T, was isolated from an in situ colonization system deployed in the main hydrothermal vent of the Taketomi submarine hot spring field located on the southern part of Yaeyama Archipelago, Japan. The microbial community associated with the hydrothermal vent was predominated by thermophilic heterotrophs such as Thermococcaceae and Anaerolineae, and the next dominant population was thermophilic sulfur oxidizers. Both aerobic and anaerobic hydrogenotrophs including methanogens were detected as minor populations. During the culture-dependent viable count analysis in this study, an Anaerolineae strain SW7T was isolated from an enrichment culture at a high dilution rate. Strain SW7T was an obligately anaerobic heterotroph that grew with fermentation and had non-motile thin rods 3.5–16.5 μm in length and 0.2 μm in width constituting multicellular filaments. Growth was observed between 37–65°C (optimum 60°C), pH 5.5–7.3 (optimum pH 6.0), and 0.5–3.5% (w/v) NaCl concentration (optimum 1.0%). Based on the physiological and phylogenetic features of a new isolate, we propose a new species representing a novel genus Thermomarinilinea: the type strain of Thermomarinilinea lacunofontalis sp. nov., is SW7T (=JCM15506T=KCTC5908T).