The complete chloroplast genome sequences of six Hylotelephium species: Comparative genomic analysis and phylogenetic relationships.

To evaluate the phylogenetic relationships between Hylotelephium and Orostachys, and to provide important information for further studies, we analyzed the complete chloroplast genomes of six Hylotelephium species and compared the sequences to those of published chloroplast genomes of congeneric species and species of the closely related genus, Orostachys. The total chloroplast genome length of nineteen species, including the six Hylotelephium species analyzed in this study and the thirteen Hylotelephium and Orostachys species analyzed in previous studies, ranged from 150,369 bp (O. minuta) to 151,739 bp (H. spectabile). Their overall GC contents were almost identical (37.7-37.8%). The chloroplast genomes of the nineteen species contained 113 unique genes comprising 79 protein-coding genes (PCGs), 30 transfer RNA genes (tRNAs), and four ribosomal RNA genes (rRNAs). Among the annotated genes, fourteen genes contained one intron, and two genes contained two introns. The chloroplast genomes of the nineteen Hylotelephium and Orostachys species had identical structures. Additionally, the large single copy (LSC), inverted repeat (IR), and small single copy (SSC) junction regions were conserved in the Hylotelephium and Orostachys species. The nucleotide diversity between the Hylotelephium chloroplast genomes was extremely low in all regions, and only one region showed a high Pi value (>0.03). In all nineteen chloroplast genomes, six regions had a high Pi value (>0.03). The phylogenetic analysis showed that the genus delimitation could not be clearly observed even in this study because Hylotelephium formed a paraphyly with subsect. Orostachys of the genus Orostachys. Additionally, the data supported the taxonomic position of Sedum taqeutii, which was treated as a synonym for H. viridescens in previous studies, as an independent taxon.

Exploring the Potential of a Thermionic LaB6 Virtual Source Mode Electron Gun for a High Angular Current Density and a Narrow Energy Distribution.

To date, lanthanum hexaboride (LaB6) thermionic electron sources have not been able fully to capitalize on their inherent potential, resulting in an ambiguous position within the application area. Although they exhibit higher brightness compared with a tungsten filament source, they still fall short of the performance of Schottky electron sources. This study aims to explore the capabilities of the LaB6 electron source under different operating conditions to bridge the gap, ultimately to realize its untapped potential. Simulations in virtual source mode indicated enhanced beam brightness and a reduced beam half-angle with an increase the extraction voltage, promising up to tenfold times higher beam brightness compared with the crossover mode. The energy distribution measured using a prelens retarding field energy analyzer revealed an energy distribution of 0.55 eV and a high angular current density of 33 mA/sr in the virtual source mode. Therefore, the virtual source mode of LaB6 can provide a narrow energy distribution akin to that of a ZrO/W Schottky electron gun (1600 K) while having an angular current density over 2,000 times higher. In addition, the stability of the virtual source mode is ±0.022%, while that of the crossover mode is ±0.138%.

The complete chloroplast genome sequences of eight Orostachys species: Comparative analysis and assessment of phylogenetic relationships.

We analyzed the complete chloroplast genomes of eight Orostachys species and compared the sequences to those of published chloroplast genomes of the congeneric and closely related genera, Meterostachys and Hylotelephium. The total chloroplast genome length of thirteen species, including the eight species analyzed in this study and the five species analyzed in previous studies, ranged from 149,860 (M. sikokianus) to 151,707 bp (H. verticillatum). The overall GC contents of the genomes were almost identical (37.6 to 37.8%). The thirteen chloroplast genomes each contained 113 unique genes comprising 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Among the annotated genes, sixteen genes contained one or two introns. Although the genome structures of all Orostachys and Hylotelephium species were identical, Meterostachys differed in structure due to a relatively large gene block (trnS-GCU-trnS-GGA) inversion. The nucleotide diversity among the subsect. Orostachys chloroplast genomes was extremely low in all regions, and among the subsect. Appendiculatae, genus Orostachys, and all thirteen chloroplast genomes showed high values of Pi (>0.03) in one, five, or three regions. The phylogenetic analysis showed that Orostachys formed polyphyly, and subsect. Orostachys and Appendiculatae were clustered with Hylotelephium and Meterostachys, respectively, supporting the conclusion that each subsection should be considered as an independent genus. Furthermore, the data supported the taxonomic position of O. margaritifolia and O. iwarenge f. magnus, which were treated as synonyms for O. iwarenge in a previous study, as independent taxa. Our results suggested that O. ramosa and O. japonica f. polycephala were individual variations of O. malacophylla and O. japonica, respectively. The exact taxonomic position of O. latielliptica and the phylogenetic relationship among the three species, O. chongsunensis, O. malacophylla and O. ramosa, should be a topic of future study.

The complete chloroplast genome sequence of Orostachys minuta (Crassulaceae).

The chloroplast (cp) genome sequence is determined and analyzed for Orostachys minuta for the first time. The cp genome was 150,369 bp in length, containing a large single-copy (LSC) of 82,795 bp and a small single-copy (SSC) of 16,854 bp, which were separated by a pair of 25,360 bp inverted repeats (IRs). The overall G + C content of the O. minuta cp genome amounted to 37.7%. In total, 113 unique genes were annotated, consisting of 79 protein-coding genes (PCGs), 30 transfer RNAs (tRNAs), and four ribosomal RNAs (rRNAs). Among these genes, 18 contained one or two introns. A maximum-likelihood (ML) phylogenetic analysis based on 33 taxa showed that O. minuta formed a clade with O. japonica. This study will provide a baseline as well as valuable molecular phylogenomic information for various future studies to determine the taxonomic position and phylogenetic relationships of the genus Orostachys.

High-Performance Compact Pre-Lens Retarding Field Energy Analyzer for Energy Distribution Measurements of an Electron Gun.

The energy distribution of an electron gun is one of the most important characteristics determining the performance of electron beam-based instruments, such as electron microscopes and electron energy loss spectroscopes. For accurate measurements of the energy distribution, this study presents a novel retarding field energy analyzer (RFEA) with the feature of an additional integrated pre-lens, which enables an adjustment of beam trajectory into the analyzer. The advantages of this analyzer are its compact size and simple electrode configuration. According to trajectory simulation theories, the optimum condition arises when the incident electron beam inside the RFEA is focused on the center of a retarding electrode. Comparing I­V curves depending on whether the pre-lens working or not, it is confirmed that the use of the pre-lens dramatically improves the energy resolution and efficiency of the signal acquisition process. The pre-lens RFEA was applied to characterize a Schottky electron gun under various temperatures and extraction voltages as operational conditions. When the tip temperature was increased by 50 K, we were able to measure an energy distribution broadening of 13.8 meV with the proposed pre-lens RFEA. The relative standard deviation of energy distribution was 0.7% for each working condition.

Characterization of the chloroplast genome of Meterostachys sikokianus (Makino) Nakai (Crassulaceae) and its phylogenetic analysis.

Meterostachys is a monotypic genus of Crassulaceae, though its phylogenetic position remains unclear. Here, we report the complete chloroplast (cp) genome sequence of Meterostachys sikokianus using the Illumina high-throughput sequencing approach. The cp genome was 149,860 bp in length, containing a large single copy (LSC) of 82,293 bp and a small single copy (SSC) of 16,879 bp, which were separated by a pair of 25,344 bp inverted repeats (IRs). The overall GC content of the M. sikokianus cp genome was 37.6%. A total of 113 unique genes were annotated, consisting of 79 protein coding genes (PCGs), 30 transfer RNAs (tRNAs), and four ribosomal RNAs (rRNAs). Among these genes, eighteen contained one or two introns. A maximum-likelihood (ML) phylogenetic analysis based on 30 accessions of Crassulaceae showed that M. sikokianus was most closely related to Orostachys japonica and Orostachys fimbriata.

An access to highly enantioenriched cis-3,5-disubstituted γ-lactones from α-bromoacetate and silyl enol ether.

A novel synthetic strategy for highly enantioenriched cis-3,5-disubstituted γ-lactones has been developed by the AgOTf-promoted nucleophilic substitution of α-bromoacetates with silyl enol ethers and subsequent reductive lactonization. The utility of this synthetic method was further demonstrated through the concise stereodivergent synthesis of cis- and trans-2,4-disubstituted tetrahydrofurans.

Investigation of the Effect of Structural Properties of a Vertically Standing CNT Cold Cathode on Electron Beam Brightness and Resolution of Secondary Electron Images.

Carbon nanotube (CNT)-based cold cathodes are promising sources of field emission electrons for advanced electron devices, particularly for ultra-high-resolution imaging systems, due to their high brightness and low energy spread. While the electron field emission properties of single-tip CNT cathodes have been intensively studied in the last few decades, a systematic study of the influencing factors on the electron beam properties of CNT cold cathodes and the resolution of the secondary electron images has been overlooked in this field. Here, we have systematically investigated the effect of the structural properties of a CNT cold cathode on the electron beam properties and resolution of secondary electron microscope (SEM) images. The aspect ratio (geometric factor) and the diameter of the tip of a vertically standing CNT cold cathode significantly affect the electron beam properties, including the beam size and brightness, and consequently determine the resolution of the secondary electron images obtained by SEM systems equipped with a CNT cold cathode module. Theoretical simulation elucidated the dependence of the structural features of CNT cold cathodes and electron beam properties on the contribution of edge-emitted electrons to the total field emission current. Investigating the correlations between the structural properties of CNT cold cathodes, the properties of the emitted electron beams, and the resolution of the secondary electron images captured by SEM equipped with CNT cold cathode modules is highly important and informative as a basic model.

The KDM5 Inhibitor KDM5-C70 Induces Astrocyte Differentiation in Rat Neural Stem Cells.

Members of the lysine-specific histone demethylase 5 (KDM5/JARID1) family are known to play important roles in stem cell fate determination. Here, using the KDM5 inhibitor C70 (KDM5-C70), we demonstrated that the histone demethylase activity of the KDM5 enzyme is essential for the repression of astrocytic differentiation of neural stem cells (NSCs). KDM5-C70 treatment activated the glial fibrillary acidic protein (Gfap) gene by increasing the trimethylation of histone H3 lysine 4 in the promoter regions and subsequently induced astrocytogenesis in NSCs. In addition, treatment of NSCs with KDM5-C70 activated Janus kinase-signal transducer and activator of transcription (JAK-STAT3) signaling and increased the mRNA expression of transforming growth factor-beta 1 (Tgf-ß1). Our data provide evidence that KDM5 is a promising target for NSC fate modulation and suggest that epigenetic regulation is important for NSC fate determination.

Functional Group-Dependent Induction of Astrocytogenesis and Neurogenesis by Flavone Derivatives.

Neural stem cells (NSCs) differentiate into multiple cell types, including neurons, astrocytes, and oligodendrocytes, and provide an excellent platform to screen drugs against neurodegenerative diseases. Flavonoids exert a wide range of biological functions on several cell types and affect the fate of NSCs. In the present study, we investigated whether the structure-activity relationships of flavone derivatives influence NSC differentiation. As previously reported, we observed that PD98059 (2'-amino-3'-methoxy-flavone), compound 2 (3'-methoxy-flavone) induced astrocytogenesis. In the present study, we showed that compound 3 (2'-hydroxy-3'-methoxy-flavone), containing a 3'-methoxy group, and a non-bulky group at C2' and C4', induced astrocytogenesis through JAK-STAT3 signaling pathway. However, compound 1 and 7-12 without the methoxy group did not show such effects. Interestingly, the compounds 4 (2',3'-dimethoxyflavone), 5 (2'-N-phenylacetamido-3'-methoxy-flavone), and 6 (3',4'-dimethoxyflavone) containing only 3'-methoxy could not promote astrocytic differentiation, suggesting that both the methoxy groups at C3' and non-bulky group at C2' and C4' are required for the induction of astrocytogenesis. Notably, compound 6 promoted neuronal differentiation, whereas its 4'-demethoxylated analog, compound 2, repressed neurogenesis, suggesting an essential role of the methoxy group at C4' in neurogenesis. These findings revealed that subtle structural changes of flavone derivatives have pronounced effects on NSC differentiation and can guide to design and develop novel flavone chemicals targeting NSCs fate regulation.

Differential effects of MEK inhibitors on rat neural stem cell differentiation: Repressive roles of MEK2 in neurogenesis and induction of astrocytogenesis by PD98059.

Neural stem cells (NSCs) proliferate and differentiate into neurons and glia depending on the culture environment. However, the underlying mechanisms determining the fate of NSCs are not fully understood. Growth factors facilitate NSC proliferation through mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and MAPK activation, and NSCs differentiate into neurons, astrocytes, or oligodendrocytes when mitogens are withdrawn from the culture media. Here, we aimed to identify the effects and roles of MEK signaling on the determination of NSC fate. MEK inhibitors, U0126, SL327, and PD98059, had differential effects on NSC differentiation. U0126 and SL327, which are known to inhibit MEK1 and MEK2, induced neuronal differentiation, whereas PD98059, which is reported to preferentially inhibit MEK1 at higher concentrations, increased astrocytogenesis. Knockdown of MEK2 using small interfering RNA increased neurogenesis and over-expression of wild type (WT) MEK2 inhibited neurogenesis, suggesting a repressive role of MEK2 in neuronal differentiation. The chemical structure of PD98059 appears to be important for induction of astrocytogenesis because not only PD98059 (2'-amino-3'-methoxyflavone) but also its chemical structural mimetic, 3'-methoxyflavone, enhanced astrocytogenesis. Therefore, in our study, we suggest that MEK inhibitors have distinct functions in determining NSC fate. Inhibition of MEK2 is important for induction of neurogenesis in NSCs. U0126 and SL327 increase neurogenesis through MEK2 inhibition, whereas PD98059 induced astrocytogenesis in NSCs, which is mediated by the chemical structure, particularly the 3'-methoxy group rather than its renowned MEK1 inhibition.

Characterization and classification of rat neural stem cells and differentiated cells by comparative metabolic and lipidomic profiling.

It is necessary to characterize and classify neural stem cells (NSCs) and differentiated cells (DCs) for potential use of NSC to treat neurodegenerative diseases. We therefore performed an analysis of NSCs and DCs using gas chromatography mass spectrometry (GC-MS) and direct infusion mass spectrometry (DI-MS) with elaborate multivariate statistical analysis for the characterization and classification of rat NSCs and DCs. GC-MS and DI-MS detected a total of 92 metabolites and lipids in NSCs and DCs, and the levels of 72 of them differed significantly between NSCs and DCs. The optimal model for partial least squares (PLS) discriminant analysis was constructed by applying 3 and 2 PLS components with a unit-variance scaling method for classifying NSCs and DCs based on the data obtained in the GC-MS and DI-MS analyses, respectively. The obtained results from PCA and PLS-DA suggest that creatinine, lactic acid, lysine, glutamine, glycine, pyroglutamic acid, PG 18:1/20:2, PS 18:0/20:2, PI 18:0/20:3, PC 16:0/20:4, PI 16:0/20:4, and PI 18:1/20:4 were the main contributors that provided distinct characteristics of NSCs and DCs. The results of this study suggest objective and complementary criteria for the characterization and classification of NSCs and DCs for potential clinical applications. Graphical abstract.

The histone demethylase KDM5A is required for the repression of astrocytogenesis and regulated by the translational machinery in neural progenitor cells.

Histone demethylases are known to play important roles in the determination of the fate of stem cells and in cancer progression. In this study, we show that the lysine 4 of histone H3 (H3K4), lysine-specific demethylase 5A (KDM5A) is essential for the repression of astrocyte differentiation in neural progenitor cells (NPCs), and its expression is regulated by translational machinery. Knockdown of KDM5A in NPCs increased astrocytogenesis, and conversely, KDM5A overexpression reduced the transcriptional activity of the Gfap promoter. Induction of astrocytogenesis by ciliary neurotrophic factor (CNTF) or small interfering RNA-induced knockdown of KDM5A decreased KDM5A recruitment to the Gfap promoter and increased H3K4 methylation. The transcript level of Kdm5a was high, whereas KDM5A protein level was low in CNTF induced astrocytes. During astroglial differentiation, translational activity indicated by the phosphorylation of eukaryotic translation initiation factor (eIF)4E was decreased. Treatment of NPCs with the cercosporamide, a MAPK-interacting kinases inhibitor, reduced eIF4E phosphorylation and KDM5A protein expression, increased GFAP levels, and enhanced astrocytogenesis. These data suggest that KDM5A is a key regulator that maintains NPCs in an undifferentiated state by repressing astrocytogenesis and that its expression is translationally controlled during astrocyte differentiation. Thus, KDM5A is a promising target for the modulation of NPC fate.-Kong, S.-Y., Kim, W., Lee, H.-R., Kim, H.-J. The histone demethylase KDM5A is required for the repression of astrocytogenesis and regulated by the translational machinery in neural progenitor cells.

Discovery of a Small Molecule that Enhances Astrocytogenesis by Activation of STAT3, SMAD1/5/8, and ERK1/2 via Induction of Cytokines in Neural Stem Cells.

Identification of small molecules that direct neural stem cells (NSCs) into specific cell types would be helpful to understand the molecular mechanisms involved in regulation of NSC fate, and facilitate the development of therapeutic applications. In the current study, we developed and screened small molecules that can modulate the fate of NSCs that are derived from rat fetal cortex. Among these compounds, compounds 5 and 6 successfully differentiated NSCs into astrocytes and neurons, respectively. Compound 5 induced astrocytogenesis by increasing expression of interleukin-6, bone morphogenetic protein 2 and leukemia inhibitory factor and through consequent phosphorylation of signal transducer and activator of transcription 3 and Sma- and Mad-related protein 1/5/8 in NSCs. In addition, compound 5 increased the expression of fibroblast growth factor (FGF) 2 and FGF8 which may regulate the branching and morphology of astrocytes. Taken together, our results suggest that these small molecules can serve as a useful tool to study cell fate determination in NSCs and be used as an inexpensive alternative to cytokines to study mechanisms of astrocytogenesis.

Kuwanon V inhibits proliferation, promotes cell survival and increases neurogenesis of neural stem cells.

Neural stem cells (NSCs) have the ability to proliferate and differentiate into neurons and glia. Regulation of NSC fate by small molecules is important for the generation of a certain type of cell. The identification of small molecules that can induce new neurons from NSCs could facilitate regenerative medicine and drug development for neurodegenerative diseases. In this study, we screened natural compounds to identify molecules that are effective on NSC cell fate determination. We found that Kuwanon V (KWV), which was isolated from the mulberry tree (Morus bombycis) root, increased neurogenesis in rat NSCs. In addition, during NSC differentiation, KWV increased cell survival and inhibited cell proliferation as shown by 5-bromo-2-deoxyuridine pulse experiments, Ki67 immunostaining and neurosphere forming assays. Interestingly, KWV enhanced neuronal differentiation and decreased NSC proliferation even in the presence of mitogens such as epidermal growth factor and fibroblast growth factor 2. KWV treatment of NSCs reduced the phosphorylation of extracellular signal-regulated kinase 1/2, increased mRNA expression levels of the cyclin-dependent kinase inhibitor p21, down-regulated Notch/Hairy expression levels and up-regulated microRNA miR-9, miR-29a and miR-181a. Taken together, our data suggest that KWV modulates NSC fate to induce neurogenesis, and it may be considered as a new drug candidate that can regenerate or protect neurons in neurodegenerative diseases.

Comparison of genetic epidemiology of vancomycin-resistant Enterococcus faecium isolates from humans and poultry.

This study was conducted to investigate the molecular characteristics and genetic relatedness of vancomycin-resistant Enterococcus faecium (VREF) isolates obtained from humans and poultry in Korea. A total of 147 VREF isolates from humans (71 clinical isolates) and poultry (76 isolates) in Korea were compared with respect to their antibiotic susceptibilities, organization of the Tn1546 transposon element, detection of virulence genes (esp and hyl), pulsed-field gel electrophoresis (PFGE) typing and multilocus sequence typing (MLST). All of the human and poultry isolates had the vanA gene and 11.3% (8/71) of the clinical isolates showed the VanB phenotype/vanA genotype. PCR mapping of the Tn1546 elements was different for isolates of the two groups: human isolates were classified into five transposon types, whereas all poultry isolates were identical to Tn1546 of E. faecium strain BM4147. The esp gene was detected in both human (93.0%, 66/71) and poultry (26.3%, 20/76) isolates, as was the hyl gene (human isolates: 80.3%, 57/71; poultry isolates: 26.3%, 20/76). Using MLST, the 71 human isolates could be divided into ten sequence types (STs) belonging to clonal complex (CC) 17 (except for one singleton). The 76 poultry isolates were categorized into 14 STs and 88.2% (67/76) of the poultry isolates belonged to CC26. PFGE typing of the human isolates demonstrated diverse PFGE profiles among the strains. However, the PFGE patterns of the poultry isolates were possibly related to the strains collected from individual farms. These data suggest that epidemic clonal groups of human and poultry VREFs in Korea have evolved through different evolutionary processes.

Evidence for clonal dissemination of the serotype K1 Klebsiella pneumoniae strain causing invasive liver abscesses in Korea.

Seventy-three liver abscess isolates of serotype K1 Klebsiella pneumoniae from a nationwide collection in Korea were genotypically characterized using pulsed-field gel electrophoresis and multilocus sequence typing. We found that serotype K1 K. pneumoniae strains that caused liver abscesses in Korea were genotypically related and that most were sequence type 23.