Brain-targeted delivery of neuroprotective survival gene minimizing hematopoietic cell contamination: implications for Parkinson's disease treatment.

BACKGROUND: Neurodegenerative diseases, including Parkinson's disease, Amyotropic Lateral Sclerosis (ALS) and Alzheimer's disease, present significant challenges for therapeutic development due to drug delivery restrictions and toxicity concerns. Prevailing strategies often employ adeno-associated viral (AAV) vectors to deliver neuroprotective survival genes directly into the central nervous system (CNS). However, these methods have been limited by triggering immunogenic responses and risk of tumorigenicity, resulting from overexpression of survival genes in peripheral blood mononuclear cells (PBMC), thereby increasing the risk of tumorigenicity in specific immune cells. Thus, by coding selectively suppressive microRNA (miRNA) target sequences in AAV genome, we designed CNS-targeted neuroprotective gene expression vector system without leakage to blood cells. METHODS: To minimize the potential for transgene contamination in the blood, we designed a CNS-specific AAV system. Our system utilized a self-complementary AAV (scAAV), encoding a quadruple repeated target sequence of the hematopoietic cell-specific miR142-3p at the 3' untranslated region (UTR). As a representative therapeutic survival gene for Parkinson's disease treatment, we integrated DX2, an antagonistic splice variant of the apoptotic gene AIMP2, known to be implicated in Parkinson's disease, into the vector. RESULTS: This configuration ensured that transgene expression was stringently localized to the CNS, even if the vector found its way into the blood cells. A single injection of scAAV-DX2 demonstrated marked improvement in behavior and motor activity in animal models of Parkinson's disease induced by either Rotenone or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Importantly, comprehensive preclinical data adhering to Good Laboratory Practice (GLP) standards revealed no adverse effects in the treated animals. CONCLUSIONS: Our CNS-specific vector system, which encodes a survival transgene DX2, signifies a promising avenue for safe gene therapy, avoiding unintended expression of survival gene in blood cells, applicable to various neurodegenerative diseases.

Repurposing of conformationally-restricted cyclopentane-based AKT-inhibitors leads to discovery of potential and more selective antileishmanial agents than miltefosine.

Conformational restriction was addressed towards the development of more selective and effective antileishmanial agents than currently used drugs for treatment of Leishmania donovani; the causative parasite of the fatal visceral leishmaniasis. Five types of cyclopentane-based conformationally restricted miltefosine analogs that were previously explored in literature as anticancer AKT-inhibitors were reprepared and repurposed as antileishmanial agents. Amongst, positions-1 and 2 cis-conformationally-restricted compound 1a and positions-2 and 3 trans-conformationally-restricted compound 3b were highly potent eliciting sub-micromolar IC50 values for inhibition of infection and inhibition of parasite number compared with the currently used miltefosine drug that showed low micromolar IC50 values for inhibition of infection and inhibition of parasite number. Compounds 1a and 3b eradicated the parasite without triggering host cells cytotoxicity over more than one log concentration interval which is a superior performance compared to miltefosine. In silico studies suggested that conformational restriction conserved the conformer capable of binding LdAKT-like kinase while it might be possible that it excludes other conformers mediating undesirable effects and/or toxicity of miltefosine. Together, this study presents compounds 1a and 3b as antileishmanial agents with superior performance over the currently used miltefosine drug.

Angiotensin I-Converting Enzyme (ACE) Inhibition and Molecular Docking Study of Meroterpenoids Isolated from Brown Alga, Sargassum macrocarpum.

Angiotensin I-converting enzyme (ACE) is an important blood pressure regulator. In this study, we aimed to investigate the ACE-inhibitory effects of meroterpenoids isolated from the brown alga, Sargassum macrocarpum, and the molecular mechanisms underlying ACE inhibition. Four fractions of S. macrocarpum were prepared using hexane, chloroform, ethyl acetate, and water as solvents and analyzed for their potential ACE-inhibitory effects. The chloroform fraction showed the strongest ACE-inhibitory effect, with an IC50 value of 0.18 mg/mL. Three meroterpenoids, sargachromenol, 7-methyl sargachromenol, and sargaquinoic acid, were isolated from the chloroform fraction. Meroterpenoids isolated from S. macrocarpum had IC50 values of 0.44, 0.37, and 0.14 mM. The molecular docking study revealed that the ACE-inhibitory effect of the isolated meroterpenoids was mainly attributed to Zn-ion, hydrogen bonds, pi-anion, and pi-alkyl interactions between the meroterpenoids and ACE. These results suggest that S. macrocarpum could be a potential raw material for manufacturing antihypertensive nutraceutical ingredients.

Fulvivirga lutea sp. nov., a marine bacterium isolated from seawater.

A strictly aerobic, Gram-stain-negative, gliding, rod-shaped bacteria, designated strain S481T, was isolated from a surface seawater sample collected at Gunsan marina, in the West Sea of the Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain S481T formed a monophyletic clade with members of the genus Fulvivirga, showing 93.7-95.8% sequence similarity to the type strains. Strain S481T has a single circular chromosome of 4.13 Mbp with a DNA G+C content of 37.3 mol%. The values of average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization between strain S481T and all genome-sequenced species of the genus Fulvivirga were below 71.2%, 68.6% and 18.9%, respectively, indicating lower values than the standard cut-offs for species delineation. Growth was observed at 20-42 °C (optimum, 37 °C), at pH 6-8 (optimum, pH 7) and with 0 - 6 % NaCl (optimum, 1-2 %). The major fatty acids (>10%) were iso-C15:0, iso-C15:1 G and C16:1ω5c. The respiratory quinone was MK-7. The major polar lipids were identified as phosphatidylethanolamine, three unidentified aminolipids and five unidentified lipids. Based on the results of phenotypic characterization, phylogenetic analysis and genome-based comparison, strain S481T represents a novel species in the genus Fulvivirga, for which we propose the name Fulvivirga lutea sp. nov. The type strain is S481T (=KCTC 82209T=JCM 34505T).

Isolation and Characterization of Efficient Active Compounds Using High-Performance Centrifugal Partition Chromatography (CPC) from Anti-Inflammatory Activity Fraction of Ecklonia maxima in South Africa.

Ecklonia maxima is a brown seaweed, which is abundantly distributed in South Africa. This study investigated an efficient approach using high-performance centrifugal partition chromatography (HPCPC), which has been successfully developed for the isolation and purification of phlorotannins, eckmaxol, and dieckol from the ethyl acetate fraction of E. maxima (EEM). We evaluated EEM for its inhibitory effect against lipopolysaccharide (LPS)-induced inflammatory responses in zebrafish embryos. The separation of eckmaxol and dieckol from samples of EEM using HPCPC was found to be of high purity and yield under an optimal solvent system composed of n-hexane:ethyl acetate:methanol:water (2:7:3:7, v/v/v/v). To evaluate the anti-inflammatory efficacy of EEM containing active compounds, zebrafish embryos exposed to LPS were compared with and without EEM treatment for nitric oxide (NO) production, reactive oxygen species (ROS) generation, and cell death two days after fertilization. These evaluations indicate that EEM alleviated inflammation by inhibiting cell death, ROS, and NO generation induced by LPS treatment. According to these results, eckmaxol and dieckol isolated from brown seaweed E. maxima could be considered effective anti-inflammatory agents as pharmaceutical and functional food ingredients.

Antihypertensive Effects of IGTGIPGIW Peptide Purified from Hippocampus abdominalis: p-eNOS and p-AKT Stimulation in EA.hy926 Cells and Lowering of Blood Pressure in SHR Model.

The aim of this study was to assess the potential hypertensive effects of the IGTGIPGIW peptide purified from Hippocampus abdominalis alcalase hydrolysate (HA) for application in the functional food industry. We investigated the antihypertensive effects of IGTGIPGIW in vitro by assessing nitric oxide production in EA.hy926 endothelial cells, which is a major factor affecting vasorelaxation. The potential vasorelaxation effect was evaluated using 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, a fluorescent stain. IGTGIPGIW significantly increased the expression of endothelial-derived relaxing factors, including endothelial nitric oxide synthase and protein kinase B, in EA.hy926 cells. Furthermore, oral administration of IGTGIPGIW significantly lowered the systolic blood pressure (183.60 ± 1.34 mmHg) and rapidly recovered the diastolic blood pressure (143.50 ± 5.55 mmHg) in the spontaneously hypertensive rat model in vivo. Our results demonstrate the antihypertensive activity of the IGTGIPGIW peptide purified from H. abdominalis and indicate its suitability for application in the functional food industry.

Discovery of Leishmania donovani topoisomerase IB selective inhibitors by targeting protein-protein interactions between the large and small subunits.

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 µM; LRL-TP-94: 2.9 µM; and LRL-TP-101: 35.3 µM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 µM in extracellular promastigote assay and 34.0, 53.7, and 11.4 µM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.

Ferrimonas lipolytica sp. nov., a facultatively anaerobic bacterium isolated from seawater.

A Gram-stain-negative, motile, facultatively anaerobic rod-shaped bacterium with a polar flagellum, designated strain S7T was isolated from seawater sample collected at Uljin marina, in the East Sea of the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain S7T was affiliated with members of genus Ferrimonas, showing the highest sequence similarities to the type strains Ferrimonas senticii P2S11T (95.7 %), Ferrimonas balearica PATT (95.7 %) and Ferrimonas pelagia CBA4601T (95.1 %). The genome was 4.13 Mbp with a DNA G+C content of 49.4 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between S7T and F. senticii P2S11T and F. balearica PATT yielded ANI values of 71.9 and 70.7 %, and dDDH values of 15.1 and 13.9 %, respectively. The genome of S7T was predicted to encode triacylglycerol lipase, phospholipase A1/A2 and lysophospholipase as well as esterase involved in lipolytic processes. Growth was observed at 8-31 °C (optimum 27 °C), at pH 7-9 (optimum pH 7), and with 1-6 % NaCl (optimum 2 %). The respiratory quinones were MK-7 and Q-7 and the major fatty acids (>10 %) were C16 : 0, C16 : 1ω9c, C17 : 1ω8c, and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major polar lipids were identified as phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids, and three unidentified lipids. On the basis of the results of this polyphasic analysis, it was determined that the strain represents a novel species of the genus Ferrimonas, for which the name Ferrimonas lipolytica sp. nov. is proposed. The type strain is S7T (=KCTC 72490T=JCM 33793T).

Shewanella maritima sp. nov., a facultative anaerobic marine bacterium isolated from seawater, and emended description of Shewanella intestini.

A Gram-stain-negative, motile, facultative anaerobic rod-shaped marine bacterium, designated strain D4-2T, was isolated from a sample of seawater collected at Dong-do marina, Dokdo Island, in the East Sea of the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain D4-2T was affiliated with members of genus Shewanella and closely related to Shewanella intestini XMDDZSB0408T (97.4%), followed by Shewanella gelidii RZB5-4T (96.7 %) and Shewanella inventionis KX27T (96.1 %). D4-2T has a single circular chromosome of 4.72 Mbp with a DNA G+C content of 44.5 mol%. Average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values between strain D4-2T and the previously mentioned type strains of species of the genus Shewanella were in range of 69-83.8 % and 20.5-21.7 %, respectively. Growth was observed at 10-36 °C (optimum 29-32 °C), at pH 6-9 (optimum pH 7), and with 1-6% NaCl (optimum 2%). The predominant fatty acids (>10 %) of D4-2T were iso-C15:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The respiratory quinones were Q-7, Q-8, MK-7 and MMK-7. Phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid, an unidentified aminolipid and four unidentified lipids were detected in D4-2T. On the basis of phenotypic, chemotaxonomic and molecular properties, D4-2T represents a novel species of the genus Shewanella, for which the name Shewanella maritima sp. nov. is proposed with D4-2T as the type strain (=KCTC 72040T=JCM 33294T).

In Vitro and in Vivo Activity of mTOR Kinase and PI3K Inhibitors Against Leishmania donovani and Trypanosoma brucei.

Kinetoplastid parasites, including Leishmania and Trypanosoma spp., are life threatening pathogens with a worldwide distribution. Next-generation therapeutics for treatment are needed as current treatments have limitations, such as toxicity and drug resistance. In this study, we examined the activities of established mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitors against these tropical diseases. High-throughput screening of a library of 1742 bioactive compounds against intracellular L. donovani was performed, and seven mTOR/PI3K inhibitors were identified. Dose-dilution assays revealed that these inhibitors had half maximal effective concentration (EC50) values ranging from 0.14 to 13.44 µM for L. donovani amastigotes and from 0.00005 to 8.16 µM for T. brucei. The results of a visceral leishmaniasis mouse model indicated that treatment with Torin2, dactolisib, or NVP-BGT226 resulted in reductions of 35%, 53%, and 54%, respectively, in the numbers of liver parasites. In an acute T. brucei mouse model using NVP-BGT226 parasite numbers were reduced to under the limits of detection by five consecutive days of treatment. Multiple sequence and structural alignment results indicated high similarities between mTOR and kinetoplastid TORs; the inhibitors are predicted to bind in a similar manner. Taken together, these results indicated that the TOR pathways of parasites have potential for the discovery of novel targets and new potent inhibitors.

Tamlana carrageenivorans sp. nov., a carrageenan-degrading bacterium isolated from seawater.

A Gram-stain-negative, aerobic, non-motile, rod-shaped, agarolytic and carrageenolytic bacterial strain, designated UJ94T, was isolated from seawater of Uljin in the Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain UJ94T shared sequence similarities of 98.4, 96.1 and 95.4 % with Tamlana agarivorans JW-26T, Tamlana sedimentorum KMM 9545T and Tamlana crocina HST1-43T, respectively. Growth of strain UJ94T was observed at 4-37 °C and pH 6.5-8.0 in the presence of 2-9 % (w/v) NaCl. The major fatty acids of strain UJ94T were iso-C15 : 0, summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and iso-C17 : 0 3-OH; MK-6 was the predominant menaquinone. Phosphatidylethanolamine, two unidentified aminolipids and five unidentified lipids were detected as major polar lipids. The whole circular genome comprised 4 116 543 bp and had a G+C content of 35.2 mol%. The ranges of average nucleotide identity and in silico DNA-DNA hybridization estimated by genome-to-genome distance were 90.6-74.2 % and 47.6-14.6 %, respectively, with the type strains of T. agarivorans and T. sedimentorum. The present polyphasic study, including phylogenetic, chemotaxonomic, biochemical and genomic data, suggested that strain UJ94T represents a novel species of the genus Tamlana, for which the name Tamlana carrageenivorans sp. nov. is proposed. The type strain is UJ94T (=KCTC 62451T=NBRC 113234T).

The Time Point-Specific Effect of Beta-Adrenergic Blockade in Attenuating High Fat Diet-Induced Obesity and Bone Loss.

We aimed to clarify the key factor determining the effect of beta blocker attenuating high fat diet- induced obesity and bone loss. Six-week-old C57BL/6 male mice were assigned to groups reflecting different relative onset of obesity and beta blocker administration, different diet (control vs. high fat), and treatment (vehicle vs. beta blocker: propranolol). Mice in Group 1 were fed a control diet (CON) or high fat diet (HIGH) with vehicle or propranolol for 12 weeks. Mice in Group 2 were fed a CON or HIGH without pharmaceutical treatment for the first 12 weeks, followed by another 12 weeks of treatment with vehicle or propranolol. Mice in Group 3 were fed a CON without pharmaceutical treatment for the first 12 weeks, followed by stratification into diet-based subgroups and another 12 weeks of treatment with vehicle or propranolol. Propranolol attenuated the HIGH-induced increase in body weight/fat mass in Group 1 mice and in Group 3 mice, but not in Group 2 mice. Propranolol mitigated HIGH-induced reduction in femoral trabecular bone mineral density and bone architecture deterioration in Group 1 mice but not in Group 2 mice. HIGH feeding in Group 3 did not compromise skeletal integrity. Taken together, propranolol attenuates HIGH-induced body weight increases while weight gain is in progress but not once obesity has already been established. HIGH feeding during the growth period results in compromised bone mass/architecture; which can be attenuated by propranolol administration during the growth period, but not by propranolol administration after obesity has already been established.

Marinobacterium aestuarii sp. nov., a benzene-degrading marine bacterium isolated from estuary sediment.

A Gram-stain-negative, aerobic, motile, flagellated rod-shaped bacterium, designated ST58-10T, was isolated from an estuarine sediment in the Republic of Korea. The strain was able to degrade benzene. Growth of strain ST58-10T was observed at 4-35 °C (optimum, 20-25 °C), pH 5-9 (optimum, pH 7-8) and 1-8 % NaCl (optimum, 3 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ST58-10T formed a phyletic lineage within the genus Marinobacterium of the family Oceanospirillaceae. Strain ST58-10T was most closely related to Marinobacterium profundum PAMC 27536T (99.6 %) and Marinobacterium rhizophilum CL-YJ9T (98.3 %), and to other members of the genus Marinobacterium(94.5-91.5 %). However, the mean value estimated by using the Genome-to-Genome Distance Calculator was 50.6±7.4 % with M. profundum PAMC 27536T and 30.9±2.8 with M. rhizophilum CL-YJ9T, respectively. An average nucleotide identity value was 89.0 % with M. profundum PAMC 27536T and 85.6 % with M. rhizophilum CL-YJ9T, respectively. The major fatty acids of strain ST58-10T were summed feature 3 (comprising C16 : 1ω7c/C16 : 1ω6c), summed feature 8 (comprising C18 : 1 ω7c/C18 : 1ω6c), C16 : 0 and C10 : 0 3-OH, and contained ubiquinone (Q-8) as the sole isoprenoid quinone. Phosphatidylethanolamine, phosphatidylglycerol, three unidentified aminolipids, an unidentified glycolipid and an unidentified lipid were detected as polar lipids. The DNA G+C content of strain ST58-10T was 58.78 mol%. On the basis of the phenotypic, chemotaxonomic and molecular properties, strain ST58-10T represents a novel species of the genus Marinobacterium, for which the name Marinobacterium aestuarii sp. nov. is proposed. The type strain is ST58-10T (=KCTC 52193T=NBRC 112103T).

Flavobacterium sediminis sp. nov., a starch-degrading bacterium isolated from tidal flat sediment.

A yellow-pigmented bacterium with the ability to degrade starch, designated MEBiC07310T, was isolated from tidal flat sediment collected in Taean County, Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain MEBiC07310T was affiliated with the genus Flavobacterium in the phylum Bacteroidetes and showed that the strain was most closely related to Flavobacterium haoranii LQY-7T (96.8 % similarity), followed by Flavobacterium indicum GPTSA 100-9T (95.2 %) and Flavobacterium urocaniciphilum YIT 12746T (94.6 %). Genome-based analysis of the average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) of strain MEBiC07310T compared with F. haoranii LQY-7T and F. indicum GPTSA 100-9T yielded ANI values of 77.0 and 73.3 % and DDH values of 18.0±2.7 and 16.1±3.6 %, respectively. The DNA G+C content of strain MEBiC07310T was 35.2 mol%. Cells of the strain were aerobic, Gram-stain-negative and rod-shaped, and negative for flexirubin-type pigments. Growth was observed at 17-43 °C (optimum 32 °C), at pH 5.0-8.0 (optimum pH 7.0) and with 0-3 % (w/v) NaCl (optimum 1 %). The major fatty acids (>10 %) of strain MEBiC07310T were iso-C15 : 0, iso-C17 : 0 3-OH, summed feature 1 (iso-C15 : 1 H and/or C13 : 0 3-OH) and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The major respiratory quinone was menaquinone MK-6. Based on its phenotypic and genotypic characteristics, strain MEBiC07310T should be classified as representing a novel species of the genus Flavobacterium, for which the name Flavobacterium sediminis sp. nov. is proposed. The type strain is MEBiC07310T (=KCTC 62132T=JCM 32291T).

Blocking ß1/ß2-Adrenergic Signaling Reduces Dietary Fat Absorption by Suppressing Expression of Pancreatic Lipase in High Fat-Fed Mice.

We investigated whether ß-adrenergic antagonists attenuates dietary fat absorption through the regulation of pancreatic lipase (PNLIP) expression in pancreatic acinar cells in the context of high fat diet feeding. Male six-week-old C57BL/6 mice were assigned into an ad libitum fed control diet (CON) and a high fat diet (HIGH). Within each diet group, subgroups of mice were treated with vehicle (VEH) or propranolol, a ß-adrenergic antagonist (BB). Over 12 weeks, body weight gain observed in HIGHVEH was mitigated in HIGHBB (+103% vs. +72%). Increase in fecal fat amount observed in HIGHVEH was further increased in HIGHBB. Increase in PNLIP expressions observed in HIGHVEH pancreatic tissues was abolished in HIGHBB. PNLIP expression in mouse primary pancreatic acinar cells and 266-6 cell lines increased with isoproterenol treatment, which was blocked by propranolol. Isoproterenol increased PNLIP expression in a cAMP/protein kinase A/ cyclic AMP response element binding protein (CREB)-dependent manner. CREB directly bound to the CRE on the mouse PNLIP promoter and transactivated PNLIP expression. These results suggest that sympathetic activation increases dietary fat absorption through the upregulation of PNLIP expression and that a ß-adrenergic antagonist attenuates obesity development partly through the downregulation of PNLIP expression and inhibition of dietary fat absorption in the context of high fat diet feeding.

Conditions Inducing Excessive O-GlcNAcylation Inhibit BMP2-Induced Osteogenic Differentiation of C2C12 Cells.

Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O-linked-ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O-GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N-acetylglucosamine concentrations or by O-GlcNAc transferase (OGT) overexpression. The effect of O-GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N-acetylglucosamine increased O-GlcNAcylation of Runx2 and the total levels of O-GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O-GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing ß-N-acetylglucosaminidase. Our findings suggest that excessive protein O-GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation.

Parasphingopyxis algicola sp. nov., isolated from a marine red alga Asparagopsis taxiformis and emended description of the genus Parasphingopyxis Uchida et al. 2012.

An aerobic, Gram-stain-negative, yellow-pigmented bacterium, designated strain ATAX6-5T, was isolated from a marine red alga, Asparagopsistaxiformis, in South Korea. Cells were non-motile rods showing catalase- and oxidase-positive reactions. Growth of strain ATAX6-5T was observed at 5-35 °C (optimum, 30 °C), at pH 6.0-9.5 (optimum, pH 7.0) and in the presence of 0-6.0 % (w/v) NaCl (optimum, 2 %). Ubiquinone-10 was detected as the sole isoprenoid quinone and C18 : 1ω7c, C16 : 0 and C17 : 1ω6c were identified as the major cellular fatty acids. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, phosphatidylcholine, an unknown phospholipid and four unknown glycolipids were detected as polar lipids. The G+C content of the genomic DNA was 60.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ATAX6-5T formed a tight phylogenic lineage with Parasphingopyxis lamellibrachiae JAMH 0132T with a 100 % bootstrap value. Strain ATAX6-5T was most closely related to P. lamellibrachiae JAMH 0132T with a 96.9 % 16S rRNA gene sequence similarity. Based on phenotypic, chemotaxonomic and molecular features, strain ATAX6-5T clearly represents a novel species of the genus Parasphingopyxis, for which the name Parasphingopyxis algicola sp. nov. is proposed. The type strain is ATAX6-5T (=KACC 18993T=JCM 31719T). An emended description of the genus Parasphingopyxis is also proposed.

Roseitalea porphyridii gen. nov., sp. nov., isolated from a red alga, and reclassification of Hoeflea suaedae Chung et al. 2013 as Pseudohoeflea suaedae gen. nov., comb. nov.

A Gram-staining-negative, strictly aerobic bacterial strain, designated MA7-20T, was isolated from a marine alga, Porphyridium marinum, in Korea. Cells showing oxidase-positive and catalase-positive activities were motile rods with bipolar flagella. Growth of strain MA7-20T was observed at 15-45 °C (optimum, 30-37 °C), at pH 6.0-10.5 (optimum, pH 7.0-8.0) and in the presence of 0-7 % (w/v) NaCl (optimum, 2-3 %). Strain MA7-20T contained summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c), 11-methyl C18 : 1ω7c and C18 : 0 as the major fatty acids and ubiquinone-10 as the sole isoprenoid quinone. The major polar lipids were phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidyl-N-methylethanolamine. The G+C content of the genomic DNA was 61.5 mol%. Strain MA7-20T was most closely related to Hoeflea suaedae YC6898T, Oricola cellulosilytica CC-AMH-0T and Nitratireductor basaltis J3T with 96.0, 95.8 and 95.8 % 16S rRNA gene sequence similarities, respectively, but the strain formed a distinct phylogenetic lineage from them within the family Phyllobacteriaceae with a low bootstrap value. H. suaedae also formed a clearly distinct phylogenetic lineage from other members of the genus Hoeflea and closely related genera. On the basis of phenotypic, chemotaxonomic and molecular properties, strain MA7-20T represents a novel species of a new genus of the family Phyllobacteriaceae, for which the name Roseitalea porphyridii gen. nov., sp. nov. is proposed. The type strain is MA7-20T (=KACC 18807T=JCM 31538T). In addition, H. suaedae is also reclassified as Pseudohoeflea suaedae gen. nov., comb. nov. (type strain YC6898T=KACC 14911T=NBRC 107700T).

Roseovarius confluentis sp. nov., isolated from estuary sediment.

A Gram-stain-negative and strictly aerobic bacterium, designated strain SAG6T, was isolated from estuary sediment in South Korea. Cells of strain SAG6T were found to be oxidase- and catalase-positive rods with gliding motility. Cell growth was observed at 15-40 °C (optimum 30 °C), at pH 6.5-10.0 (optimum pH 7.0) and in the presence of 0.5-13.0 % (w/v) NaCl (optimum 2.0 %). Ubiquinone-10 was the only detected respiratory quinone and summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c), C16 : 0, C18 : 1ω7c 11-methyl and C12 : 0 were the major fatty acids identified (>5 % of the total fatty acids). The polar lipids of strain SAG6T consisted of phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminolipid and two unidentified lipids. The G+C content of the genomic DNA was 66.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SAG6T formed a tight phyletic lineage within the genus Roseovarius. Strain SAG6T was most closely related to Roseovarius indicus B108T with 97.6 % 16S rRNA gene sequence similarity. Based on phenotypic, chemotaxonomic and molecular features, strain SAG6T clearly represents a novel species of the genus Roseovarius, for which the name Roseovarius confluentis sp. nov. is proposed. The type strain is SAG6T (=KACC 18598T=JCM 31541T).

TNFα Increases RANKL Expression via PGE2-Induced Activation of NFATc1.

Tumor necrosis factor α (TNFα) is known to upregulate the expression of receptor activator of NF-κB ligand (RANKL). We investigated the role of the calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathway in TNFα-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. TNFα-induced RANKL expression was blocked by the calcineurin/NFAT pathway inhibitors. TNFα increased NFAT transcriptional activity and subsequent RANKL promoter binding. Mutations in the NFAT-binding element (MT(N)) suppressed TNFα-induced RANKL promoter activity. TNFα increased prostaglandin E2 (PGE2) production, which in turn enhanced NFAT transcriptional activity and binding to the RANKL promoter. MT(N) suppressed PGE2-induced RANKL promoter activity. TNFα and PGE2 increased the expression of RANKL, NFAT cytoplasmic-1 (NFATc1), cAMP response element-binding protein (CREB), and cyclooxygenase 2 (COX2); which increment was suppressed by indomethacin, a COX inhibitor. Mutations in the CRE-like element blocked PGE2-induced RANKL promoter activity. PGE2 induced the binding of CREB to the RANKL promoter, whereas TNFα increased the binding of both CREB and NFATc1 to this promoter through a process blocked by indomethacin. The PGE2 receptor antagonists AH6809 and AH23848 blocked TNFα-induced expression of RANKL, NFATc1, and CREB; transcriptional activity of NFAT; and binding of NFATc1 or CREB to the RANKL promoter. These results suggest that TNFα-induced RANKL expression depends on PGE2 production and subsequent transcriptional activation/enhanced binding of NFATc1 and CREB to the RANKL promoter.