Impact of circadian nuclear receptor REV-ERBα on midbrain dopamine production and mood regulation.

The circadian nature of mood and its dysfunction in affective disorders is well recognized, but the underlying molecular mechanisms are still unclear. Here, we show that the circadian nuclear receptor REV-ERBα, which is associated with bipolar disorder, impacts midbrain dopamine production and mood-related behavior in mice. Genetic deletion of the Rev-erbα gene or pharmacological inhibition of REV-ERBα activity in the ventral midbrain induced mania-like behavior in association with a central hyperdopaminergic state. Also, REV-ERBα repressed tyrosine hydroxylase (TH) gene transcription via competition with nuclear receptor-related 1 protein (NURR1), another nuclear receptor crucial for dopaminergic neuronal function, thereby driving circadian TH expression through a target-dependent antagonistic mechanism. In conclusion, we identified a molecular connection between the circadian timing system and mood regulation, suggesting that REV-ERBα could be targeting in the treatment of circadian rhythm-related affective disorders.

An amygdala circuit that suppresses social engagement.

Innate social behaviours, such as mating and fighting, are fundamental to animal reproduction and survival1. However, social engagements can also put an individual at risk2. Little is known about the neural mechanisms that enable appropriate risk assessment and the suppression of hazardous social interactions. Here we identify the posteromedial nucleus of the cortical amygdala (COApm) as a locus required for the suppression of male mating when a female mouse is unhealthy. Using anatomical tracing, functional imaging and circuit-level epistatic analyses, we show that suppression of mating with an unhealthy female is mediated by the COApm projections onto the glutamatergic population of the medial amygdalar nucleus (MEA). We further show that the role of the COApm-to-MEA connection in regulating male mating behaviour relies on the neuromodulator thyrotropin-releasing hormone (TRH). TRH is expressed in the COApm, whereas the TRH receptor (TRHR) is found in the postsynaptic MEA glutamatergic neurons. Manipulating neural activity of TRH-expressing neurons in the COApm modulated male mating behaviour. In the MEA, activation of the TRHR pathway by ligand infusion inhibited mating even towards healthy female mice, whereas genetic ablation of TRHR facilitated mating with unhealthy individuals. In summary, we reveal a neural pathway that relies on the neuromodulator TRH to modulate social interactions according to the health status of the reciprocating individual. Individuals must balance the cost of social interactions relative to the benefit, as deficits in the ability to select healthy mates may lead to the spread of disease.

Dysregulation of Lipid Droplet Protein Expression in Adipose Tissues and Association with Metabolic Risk Factors in Adult Females with Obesity and Type 2 Diabetes.

BACKGROUND: Adipocyte dysregulation of lipid droplet (LD) metabolism caused by altered expression of LD proteins contributes to obesity-related metabolic diseases. OBJECTIVES: We aimed to investigate whether expression levels of PLIN1, CIDEA, and CIDEC were altered in adipose tissues of women with obesity and type 2 diabetes and whether their alterations were associated with metabolic risk factors. METHODS: Normal-weight (NW; 18.5 kg/m2 < BMI ≤ 25 kg/m2; n = 43), nondiabetic obese (OB; BMI > 30 kg/m2; n = 38), and diabetic obese (OB/DM; BMI > 30 kg/m2, fasting glucose ≥ 126 mg/dL, HbA1c ≥ 6.5%; n = 22) women were recruited. Metabolic parameters were measured, and expressions of PLIN1, CIDEA, CIDEC, and obesity-related genes were quantified in abdominal subcutaneous (SAT) and visceral adipose tissues (VAT). Effects of proinflammatory cytokines, endoplasmic reticulum (ER) stress inducers, and metabolic improvement agents on LD protein gene expressions were investigated in human adipocytes. RESULTS: PLIN1, CIDEA, and CIDEC expressions were lower in SAT and higher in VAT in OB subjects relative to NW subjects; however, they were suppressed in both fat depots in OB/DM subjects relative to OB (P < 0.05). Across the entire cohort, whereas VAT PLIN1 (r = 0.349) and CIDEC expressions (r = 0.282) were positively associated with BMI (P < 0.05), SAT PLIN1 (r = -0.390) and CIDEA expressions (r = -0.565) were inversely associated. After adjustment for BMI, some or all of the adipose LD protein gene expressions were negatively associated with fasting glucose (r = -0.259 or higher) and triglyceride levels (r = -0.284 or higher) and positively associated with UCP1 expression (r = 0.353 or higher) (P < 0.05). In adipocytes, LD protein gene expressions were 55-70% downregulated by increased proinflammatory cytokines and ER stress but 2-4-fold upregulated by the metabolic improvement agents exendin-4 and dapagliflozin (P < 0.05). CONCLUSIONS: The findings suggest that reduction of adipose LD protein expression is involved in the pathogenesis of metabolic disorders in women with obesity and type 2 diabetes and that increasing LD protein expression in adipocytes could control development of metabolic disorders.

Phosphorylation of LSD1 by PKCα is crucial for circadian rhythmicity and phase resetting.

The circadian clock is a self-sustaining oscillator that controls daily rhythms. For the proper circadian gene expression, dynamic changes in chromatin structure are important. Although chromatin modifiers have been shown to play a role in circadian gene expression, the in vivo role of circadian signal-modulated chromatin modifiers at an organism level remains to be elucidated. Here, we provide evidence that the lysine-specific demethylase 1 (LSD1) is phosphorylated by protein kinase Cα (PKCα) in a circadian manner and the phosphorylated LSD1 forms a complex with CLOCK:BMAL1 to facilitate E-box-mediated transcriptional activation. Knockin mice bearing phosphorylation-defective Lsd1(SA/SA) alleles exhibited altered circadian rhythms in locomotor behavior with attenuation of rhythmic expression of core clock genes and impaired phase resetting of circadian clock. These data demonstrate that LSD1 is a key component of the molecular circadian oscillator, which plays a pivotal role in rhythmicity and phase resetting of the circadian clock.

Endoplasmic reticulum stress increases LECT2 expression via ATF4.

Non-alcoholic fatty liver disease (NAFLD) is frequently associated with obesity, insulin resistance, and endoplasmic reticulum (ER) stress. Elevated circulating levels of the hepatokine leukocyte cell-derived chemotaxin-2 (LECT2) have also been noted in NAFLD; however, the mechanism underlying this association is unclear. To investigate a possible link between ER stress/unfolded protein response (UPR) signaling and LECT2 secretion, HepG2 cells were incubated with ER stress inducers with or without an ER stress-reducing chemical chaperone. Additionally, UPR pathway genes were knocked down and overexpressed, and a ChIP assay was performed. In diet-induced obese mice, hepatic expression of LECT2 and activating transcription factor 4 (ATF4) was measured. In HepG2 cells, LECT2 expression was increased by ER stressors, an effect blocked by the chemical chaperone. Among UPR pathway proteins, only knockdown of ATF4 suppressed ER stress-induced LECT2 expression, while overexpression of ATF4 enhanced LECT2 expression. The ChIP assay revealed that ATF4 binds to three putative binding sites on the LECT2 promoter and binding is promoted by an ER stress inducer. In steatotic livers of obese mice, LECT2 and ATF4 expression was concomitantly elevated. Our data indicate that activation of ER stress/UPR signaling induces LECT2 expression in steatotic liver; specifically, ATF4 appears to mediate upregulation of LECT2 transcription.

Hymenobacter taeanensis sp. nov., radiation resistant bacterium isolated from coastal sand dune.

An aerobic, Gram-stain-negative, non-motile, non-spore-forming, rod-shaped, and light pink-colored bacterial strain, designated TS19T, was isolated from a sand sample obtained from a coastal sand dune after exposure to 3 kGy of gamma radiation. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the isolate was a member of the genus Hymenobacter and was most closely related to H. wooponensis WM78T (98.3% similarity). Strain TS19T and H. wooponensis showed resistance to gamma radiation with D10 values (i.e., the dose required to reduce the bacterial population by tenfold) of 7.3 kGy and 3.5 kGy, respectively. The genome of strain TS19T consists of one contig with 4,879,662 bp and has a G + C content of 56.2%. The genome contains 3,955 protein coding sequences, 44 tRNAs, and 12 rRNAs. The predominant fatty acids of strain TS19T were iso-C15:0, summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B), summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), and C16:1 ω5c. The major polar lipids were phosphatidylethanolamine, and one unidentified aminophospholipid. The main respiratory quinone was menaquinone-7. Based on the phylogenetic, physiological, and chemotaxonomic characteristics, strain TS19T represents a novel species, for which the name Hymenobacter taeanensis sp. nov. is proposed. The type strain is TS19T (= KCTC 72897T = JCM 34023T).

Spirosoma taeanense sp. nov., a radiation resistant bacterium isolated from a coastal sand dune.

An aerobic, Gram-negative, non-motile, non-spore-forming, rod-shaped, and pale yellow-colored bacterial strain, designated TS118T, was isolated from a sand sample obtained from a coastal sand dune after exposure to 3 kGy of gamma radiation. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate was a member of the genus Spirosoma and most closely related to Spirosoma metallicum PR1014kT (95.1% similarity). The genome of strain TS118T is constituted by one chromosome (5,691,492 bp) and one plasmid (28,440 bp) and has a G + C content of 52.7%. The genome contains 4641 protein coding sequences (CDSs), 38 tRNAs, and 11 rRNAs. The predominant fatty acids of strain TS118T were C16:1 ω5c, iso-C15:0, C16:0, summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), and iso-C17:0 3-OH. The major polar lipids were phosphatidylethanolamine, an unidentified amino lipid and an unidentified aminophospholipid. The main respiratory quinone was menaquinone-7 (MK-7). The novel strain showed resistance to gamma radiation with a D10 value (i.e., the dose required to reduce the bacterial population by tenfold) of 4.3 kGy. Based on the phylogenetic, physiological, and chemotaxonomic characteristics, strain TS118T represents a novel species, for which the name Spirosoma taeanense sp. nov. is proposed. The type strain is TS118T (=KCTC 72898T =JCM 34024T).

Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion.

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.

DJ-1 Can Replace FGF-2 for Long-Term Culture of Human Pluripotent Stem Cells in Defined Media and Feeder-Free Condition.

Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b'a' Domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor.

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a') tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

Bacterial overexpression and purification of soluble recombinant human serum albumin using maltose-binding protein and protein disulphide isomerase.

Human serum albumin (HSA), the most abundant serum protein in healthy humans, plays important roles in many physiological processes and has wide clinical and research applications. Despite several efforts to obtain recombinant HSA (rHSA) from bacterial and eukaryotic expression systems, a low-cost and high-yield method for rHSA production is not available. The large molecular weight and high disulphide content hamper the expression and production of rHSA using bacterial hosts. Hence, a strategy that uses a fusion technique and engineered Escherichia coli strains was employed to improve the expression of soluble rHSA in the bacterial cytoplasm. The solubilities of the b'a' domain of human protein disulphide isomerase (PDIb'a')- and maltose-binding protein (MBP)-tagged rHSA expressed in Origami 2 at 18 °C were notably increased by up to 90.1% and 96%, respectively. A simple and efficient protocol for rHSA purification was established and approximately 9.46 mg rHSA was successfully obtained from a 500-mL culture at 97% purity. However, rHSA was mostly obtained in soluble oligomeric form. By introducing a simple refolding and size-exclusion chromatography step, monomeric rHSA was obtained at 34% yield. Native polyacrylamide gel electrophoresis confirmed the similarity in the molecular weights between E. coli-derived monomeric rHSA and commercial monomeric HSA.

Kisspeptin Neuron-Specific and Self-Sustained Calcium Oscillation in the Hypothalamic Arcuate Nucleus of Neonatal Mice: Regulatory Factors of its Synchronization.

INTRODUCTION: Synchronous and pulsatile neural activation of kisspeptin neurons in the arcuate nucleus (ARN) are important components of the gonadotropin-releasing hormone pulse generator, the final common pathway for central regulation of mammalian reproduction. However, whether ARN kisspeptin neurons can intrinsically generate self-sustained synchronous oscillations from the early neonatal period and how they are regulated remain unclear. OBJECTIVE: This study aimed to examine the endogenous rhythmicity of ARN kisspeptin neurons and its neural regulation using a neonatal organotypic slice culture model. METHODS: We monitored calcium (Ca2+) dynamics in real-time from individual ARN kisspeptin neurons in neonatal organotypic explant cultures of Kiss1-IRES-Cre mice transduced with genetically encoded Ca2+ indicators. Pharmacological approaches were employed to determine the regulations of kisspeptin neuron-specific Ca2+ oscillations. A chemogenetic approach was utilized to assess the contribution of ARN kisspeptin neurons to the population dynamics. RESULTS: ARN kisspeptin neurons in neonatal organotypic cultures exhibited a robust synchronized Ca2+ oscillation with a period of approximately 3 min. Kisspeptin neuron-specific Ca2+ oscillations were dependent on voltage-gated sodium channels and regulated by endoplasmic reticulum-dependent Ca2+ homeostasis. Chemogenetic inhibition of kisspeptin neurons abolished synchronous Ca2+ oscillations, but the autocrine actions of the neuropeptides were marginally effective. Finally, neonatal ARN kisspeptin neurons were regulated by N-methyl-D-aspartate and gamma-aminobutyric acid receptor-mediated neurotransmission. CONCLUSION: These data demonstrate that ARN kisspeptin neurons in organotypic cultures can generate synchronized and self-sustained Ca2+ oscillations. These oscillations controlled by multiple regulators within the ARN are a novel ultradian rhythm generator that is active during the early neonatal period.

Hymenobacter baengnokdamensis sp. nov., Isolated from the Soil of a Crater Lake in Korea.

An aerobic, Gram-stain-negative, non-motile, non-spore-forming, rod-shaped and pink-colored bacterial strain, designated BRD72T, was isolated from a crater lake (Baengnokdam) at the top of Mt. Hallasan in the Republic of Korea. Cells were catalase-positive and oxidase-negative. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the isolate was a member of the genus Hymenobacter and most closely related to Hymenobacter marinus KJ035T (96.2% similarity). The isolate was found to produce carotenoid pigment, but not flexirubin-type pigment. The predominant fatty acids of strain BRD72T were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c, 21.6%), iso-C15:0 (17.9%), anteiso-C15:0 (13.3%) and summed feature 4 (iso-C17:1 I and/or anteiso-C17:1 B, 11.3%). The major polar lipids were phosphatidylethanolamine, an unidentified amino lipid, and two unidentified aminophospholipids. The main respiratory quinone was menaquinone-7 (MK-7), and the main polyamine was homospermidine. The DNA G+C content was 59.8 mol%. Based on the phylogenetic, physiological, and chemotaxonomic characteristics, strain BRD72T represents a novel species, for which the name Hymenobacter baengnokdamensis sp. nov. is proposed. The type strain is BRD72T (= KCTC 72649T = JCM 33837T).

Comprehensive Genome-Wide Approaches to Activity-Dependent Translational Control in Neurons.

Activity-dependent regulation of gene expression is critical in experience-mediated changes in the brain. Although less appreciated than transcriptional control, translational control is a crucial regulatory step of activity-mediated gene expression in physiological and pathological conditions. In the first part of this review, we overview evidence demonstrating the importance of translational controls under the context of synaptic plasticity as well as learning and memory. Then, molecular mechanisms underlying the translational control, including post-translational modifications of translation factors, mTOR signaling pathway, and local translation, are explored. We also summarize how activity-dependent translational regulation is associated with neurodevelopmental and psychiatric disorders, such as autism spectrum disorder and depression. In the second part, we highlight how recent application of high-throughput sequencing techniques has added insight into genome-wide studies on translational regulation of neuronal genes. Sequencing-based strategies to identify molecular signatures of the active neuronal population responding to a specific stimulus are discussed. Overall, this review aims to highlight the implication of translational control for neuronal gene regulation and functions of the brain and to suggest prospects provided by the leading-edge techniques to study yet-unappreciated translational regulation in the nervous system.

Functional Elements Coatings on the Plasma Electrolytic Oxidation-Treated Ti-6Al-4V Alloy by Electrochemical Precipitation Method.

In this study, functional elements coatings on the plasma electrolytic oxidation (PEO)-treated Ti-6Al-4V alloy by electrochemical precipitation method were investigated. Ti-6Al-4V ELI disks were used as specimens for PEO and HA coating. The applied voltage and time were selected to be 280 V and 3 minutes for PEO treatment, respectively. The electrochemical precipitation on the Ti-6Al-4V alloy was carried out using cyclic voltammetry with cycle of 3, 5, 10, and 20 from -1.5 V to 0 V (vs. SCE electrode) in electrolyte containing Ca, P, Mg, Mn, Sr, Zn, and Si ions by cyclic voltammetry after PEO treatment. The morphology changes of the coatings on the PEO treated Ti-6Al-4V alloy surface were observed using FE-SEM and EDS. PEO surface has a uniformly distributed circular shape and a porous surface and the deposition of low cycles in electrolyte containing Mg, Mn, Sr, Zn, and Si-HA coated surfaces show uniform circular and granular structures. The precipitates of Mg, Mn, Sr, Zn, and Si-HA on the PEO treated surface showed a large number of circular particles as the number of deposition cycles increases with a mixture of rod-shaped particles and petal-shaped particles around pores. The precipitate nucleates around the pore and grows rapidly as the cycles increase.

Functional Elements Coatings on Ti-6Al-4V Alloy by Plasma Electrolytic Oxidation for Biomaterials.

In this study, functional elements coatings on Ti-6Al-4V alloy by plasma electrolytic oxidation for biomaterials were studied using various experimental techniques. For this study, Ti-6Al-4V ELI disk (grade 5, Timet Co. Ltd., Japan diameter; 10 mm) were used as the substrate for PEO treatment in the electrolyte containing Ca, P, Si, Zn, and Mn ions. The PEO treatment was performed using a pulsed DC power supply at 280 V for 3 min. The PEO-treated surface was observed with field-emission scanning electron microscope (FE-SEM), and energy dispersive X-ray spectrometry (EDS; Oxford ISIS 310, England), Image Analyzer software, and X-ray diffractometer (TF-XRD, X'pert Philips, Netherlands). The number of pores increases, as the Zn ion content increases. And the number of pores decrease, as the Mn ion content increases. The addition of Zn and Mn ions affects the number and size of pores and the area occupied by the pores. Mn and Zn are distributed around pores and in the pores. Anatase and rutile peaks appear and the HA peak shifted to the left in the case of Mn and Zn ion additions.

Hymenobacter defluvii sp. nov., isolated from wastewater of an acidic water neutralization facility.

A non-motile, pink-coloured and rod-shaped bacterium, designated strain POA9T, was isolated from a wastewater treatment facility, Republic of Korea. Cells were Gram-reaction-negative, aerobic, catalase-positive and oxidase-negative. The major fatty acids were C16 : 1ω5c, iso-C15 : 0, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and anteiso-C15 : 0. The strain contained MK-7 as the only isoprenoid quinone, phosphatidylethanolamine as the major polar lipid and sym-homospermidine as the major polyamine. The DNA G+C content was 57 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain POA9T forms a distinct evolutionary lineage within the radiation enclosing the members of the genus Hymenobacter, sharing the highest similarity with Hymenobacter actinosclerus CCUG 39621T (95.5 % sequence similarity) followed by Hymenobacterseoulensis 16F7GT (95.5 %), Hymenobactertibetensis XTM003T (95.4 %), Hymenobacterrutilus K2-33028T (95.4 %) and Hymenobacter psychrotolerans Tibet-IIU11T (94.9 %). A number of phenotypic characteristics distinguished strain POA9T from the related members of the genus Hymenobacter. On the basis of the evidence presented in this study, a novel species, Hymenobacter defluvii sp. nov., is proposed for strain POA9T (=KCTC 52270T=JCM 31658T).

Flagellimonas aquimarina sp. nov., and transfer of Spongiibacterium flavum Yoon and Oh 2012 and S. pacificum Gao et al. 2015 to the genus Flagellimonas Bae et al. 2007 as Flagellimonas flava comb. nov. and F. pacifica comb. nov., respectively.

A Gram-stain-negative, non-spore-forming, rod-shaped, aerobic and diffusible yellow-coloured bacterial strain, designated strain ECD12T was isolated from a seaweed, Ecklonia cava. The isolate required sea salts for growth. Catalase-positive and oxidase-negative. A phylogenetic tree based on 16S rRNA gene sequences showed that strain ECD12T formed an evolutionary lineage within the radiation enclosing the members of genera Spongiibacterium and Flagellimonas sharing the highest similarity to Flagellimonas eckloniae DOKDO007T (96.8 % 16S rRNA gene sequence similarity) followed by Spongiibacterium pacificum SW169T (96.4 %) and Spongiibacterium flavum DSM 22638T (96.1 %). The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G. The new isolate contained MK-6 as the only isoprenoid quinone and phosphatidylethanolamine, two unidentified amino lipids and two unidentified lipids as the major polar lipids. The genomic DNA G+C content is 39 mol%. A number of phenotypic characteristics such as the production of diffusible pigment distinguished strain ECD12T from the related species. On the basis of the evidence presented in this study, a novel species, Flagellimonas aquimarina sp. nov., is proposed for strain ECD12T (=KCTC 52351T=JCM 32292T). Based on the sequence similarity, phylogenetic relationship and common morphological, physiological and chemical characters among the members of the genera Spongiibacterium and Flagellimonas, it is recommended that the two genera are combined into a single genus. Thus, transfer of S. flavumYoon and Oh 2012 and S. pacificum Gao et al. 2015 to the genus FlagellimonasBae et al. 2007 as Flagellimonas flava comb. nov. and Flagellimonas pacifica comb. nov., respectively, is also proposed.

Pore Shape Changes and Apatite Formation on Zn and Si Ion-Doped HA Films of Ti-6Al-4V After Plasma Electrolytic Oxidation Treatment.

In this study, pore shape changes and apatite formation on zinc (Zn) and silicon (Si) ion-doped hydroxyapatite (HA) films of Ti-6Al-4V by plasma electrolytic oxidation (PEO) treatment has been investigated by several techniques. The PEO films and the Ti-6Al-4V surface after immersion in SBF were observed by X-ray spectroscopy, field-emission scanning electron microscopy, and energy dispersive X-ray spectroscopy. The number of pores decreased as Zn ion concentration increased from 5Zn to 10Zn. The maximum size of pores were increased from 5Zn to 20Zn concentration, whereas, the minimum size of pores decreased. The amount of bone-like apatite formation for the 5Zn/5Si sample was higher than those of other samples immersed in SBF.