Background: Orexin plays a central role in the integration of sleep/wake states and feeding behaviors. Results: Orexin neurons were induced from pluripotent stem cells by supplementation of ManNAc. Conclusion: ManNAc induced switching of epigenetic factors from Sirt1/Ogt to Mgea5 at Hcrt gene locus. Significance: This study will be useful to investigate molecular mechanism in the orexin system and development of regenerative medicine.

We report here newly discovered O-linked-N-acetylglucosamine (O-GlcNAc) modification of histone H2A at Ser40 (H2AS40Gc). The mouse genome contains 18 H2A isoforms, of which 13 have Ser40 and the other five have Ala40. The combination of production of monoclonal antibody and mass spectrometric analyses with reverse-phase (RP)-high performance liquid chromatography (HPLC) fractionation indicated that the O-GlcNAcylation is specific to the Ser40 isoforms. The H2AS40Gc site is in the L1 loop structure where two H2A molecules interact in the nucleosome. Targets of H2AS40Gc are distributed genome-wide and are dramatically changed during the process of differentiation in mouse trophoblast stem cells. In addition to the mouse, H2AS40Gc was also detected in humans, macaques and cows, whereas non-mammalian species possessing only the Ala40 isoforms, such as silkworms, zebrafish and Xenopus showed no signal. Genome database surveys revealed that Ser40 isoforms of H2A emerged in Marsupialia and persisted thereafter in mammals. We propose that the emergence of H2A Ser40 and its O-GlcNAcylation linked a genetic event to genome-wide epigenetic events that correlate with the evolution of placental animals.

Genome-wide RNA interference (RNAi) with pooled and barcoded short-hairpin RNA (shRNA) libraries provides a powerful tool for identifying cellular components that are relevant to the modes/mechanisms of action (MoA) of bioactive compounds. shRNAs that affect cellular sensitivity to a given compound can be identified by deep sequencing of shRNA-specific barcodes. We used multiplex barcode sequencing technology by adding sample-specific index tags to PCR primers during sequence library preparation, enabling parallel analysis of multiple samples. An shRNA library screen with this system revealed that downregulation of ATP1A1, an α-subunit of Na+/K+ ATPase, conferred significant sensitivity to aurilide B, a natural marine product that induces mitochondria-mediated apoptosis. Combined treatment with ouabain which inhibits Na+/K+ ATPase by targeting α-subunits potentiated sensitivity to aurilide B, suggesting that ATP1A1 regulates mitochondria-mediated apoptosis. Our results indicate that multiplex sequencing facilitates the use of pooled shRNA library screening for the identification of combination drug therapy targets.

Phylogenetic analysis based on 16S rDNA sequences was performed on all type strains of the 14 validly described Methylobacterium species to ascertain the genealogic relationships among these species. The results showed that type strains of Methylobacterium were divided into two monophyletic groups whose members were distinct species with sequence similarity values greater than 97.0% between any two of the members in the same group. Only M. organophilum

Sixty strains of thermoacidophilic bacteria have been isolated from soil and water samples obtained from various acidic environments in Japan. An initial comparative sequence analysis of the hypervariable regions of the 16S rDNA revealed that all strains could be assigned to the Alicyclobacillus acidocaldarius- Alicyclobacillus genomic species 1 group, which could be further subdivided into three clusters (Clusters I-III). On the basis of phenotypic characteristics, chemotaxonomic profiles, and phylogenetic data of six selected strains, five strains were identified as either A. acidocaldarius or Alicyclobacillus genomic species 1; however, one strain (MIH 332) could not be determined to belong to either of these species. 16S rDNA sequence homology values between strain MIH 332 and the reference strains of A. acidocaldarius (ATCC 27009(T)) and Alicyclobacillus genomic species 1 (DSM 11984) were 98.8% and 99.1%, respectively, which were higher than the corresponding similarity between the reference strains (98.4%). On the other hand, DNA-DNA hybridization levels between strain MIH 332 and the reference strains were 39% and 44%, respectively, which were lower than the value between the reference strains (59% or 65%). However, the phenotype of strain MIH 332 was also similar to those of the reference strains, and a typical phenotype could not be found for the strain, thus indicating that the strain may be a new genomic species of A. acidocaldarius, for which the name Alicyclobacillus genomic species 2 is tentatively proposed. The results of this study suggest that A. acidocaldarius and its related species are widely distributed in acidic environments in Japan, with slight regional variations in morphological and genotypic characteristics.