Vibrational microspectroscopy as a tool to unveil new chemotherapeutic strategies against osteosarcoma.

Over the years, osteosarcoma therapy has had a significative improvement with the use of a multidrug regime strategy, increasing the survival rates from less than 20 % to circa 70 %. Different types of development of new antineoplastic agents are critical to achieve irreversible damage to cancer cells, while preserving the integrity of their healthy counterparts. In the present study, complexes with two and three Pd(II) centres linked by the biogenic polyamines: spermine (Pd2SpmCl4) and spermidine (Pd3Spd2Cl6) were tested against non-malignant (osteoblasts, HOb) and cancer (osteosarcoma, MG-63) human cell lines. Either alone or in combination according to the EURAMOS-1 protocol, they were used versus cisplatin as a drug reference. By evaluating the cytotoxic effects of both therapeutic approaches (single and drug combination) in HOb and MG-63 cell lines, the selective anti-tumoral potential is assessed. To understand the different treatments at a molecular level, Synchrotron Radiation Fourier Transform Infrared and Raman microspectroscopies were applied. Principal component analysis and hierarchical cluster analysis are applied to the vibrational data, revealing the major metabolic changes caused by each drug, which were found to rely on DNA, lipids, and proteins, acting as biomarkers of drug-to-cell impact. The main changes were observed for the B-DNA native conformation to either Z-DNA (higher in the presence of polynuclear complexes) or A-DNA (preferably after cisplatin exposure). Additionally, a higher effect upon variation in proteins content was detected in drug combination when compared to single drug administration proving the efficacy of the EURAMOS-1 protocol with the new drugs tested.

Injectable and Self-Curing Single-Component Hydrogel for Stem Cell Encapsulation and In Vivo Bone Regeneration.

An ideal hydrogel for stem cell therapy would be injectable and efficiently promote stem cell proliferation and differentiation in body. Herein, an injectable, single-component hydrogel with hyaluronic acid (HA) modified with phenylboronic acid (PBA) and spermidine (SM) is introduced. The resulting HAps (HA-PBA-SM) hydrogel is based on the reversible crosslinking between the diol and the ionized PBA, which is stabilized by the SM. It has a shear-thinning property, enabling its injection through a syringe to form a stable hydrogel inside the body. In addition, HAps hydrogel undergoes a post-injection "self-curing," which stiffens the hydrogel over time. This property allows the HAps hydrogel to meet the physical requirements for stem cell therapy in rigid tissues, such as bone, while maintaining injectability. The hydrogel enabled favorable proliferation of human mesenchymal stem cells (hMSCs) and promoted their differentiation and mineralization. After the injection of hMSCs-containing HAps into a rat femoral defect model, efficient osteogenic differentiation of hMSCs and bone regeneration is observed. The study demonstrates that simple cationic modification of PBA-based hydrogel enabled efficient gelation with shear-thinning and self-curing properties, and it would be highly useful for stem cell therapy and in vivo bone regeneration.

Characterization of four novel bacterial species of the genus Sphingomonas, Sphingomonas anseongensis, Sphingomonas alba, Sphingomonas brevis and Sphingomonas hankyongi sp.nov., isolated from wet land.

Four novel bacterial strains, designated as RG327T, SE158T, RB56-2T and SE220T, were isolated from wet soil in the Republic of Korea. To determine their taxonomic positions, the strains were fully characterized. On the basis of genomic information (16S rRNA gene and draft genome sequences), all four isolates represent members of the genus Sphingomonas. The draft genomes of RG327T, SE158T, RB56-2T and SE220T consisted of circular chromosomes of 2 226 119, 2 507 338, 2 593 639 and 2 548 888 base pairs with DNA G+C contents of 64.6, 63.6, 63.0 and 63.1 %, respectively. All the isolates contained ubiquinone Q-10 as the predominant quinone compound and a fatty acid profile with C16 : 0, C17 : 1ω6c, C18 : 1 2-OH, summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and summed feature 8 (C18 : 1ω7c/C18 : 1ω6c) as the major fatty acids, supporting the affiliation of strains RG327T, SE158T, RB56-2T and SE220T to the genus Sphingomonas. The major identified polar lipids in all four novel isolates were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid and phosphatidylcholine. Moreover, the physiological, biochemical results and low level of DNA-DNA relatedness and average nucleotide identity values allowed the phenotypic and genotypic differentiation of RG327T, SE158T, RB56-2T and SE220T from other species of the genus Sphingomonas with validly published names and indicated that they represented novel species of the genus Sphingomonas, for which the names Sphingomonas anseongensis sp. nov. (RG327T = KACC 22409T = LMG 32497T), Sphingomonas alba sp. nov. (SE158T = KACC 224408T = LMG 324498T), Sphingomonas brevis (RB56-2T = KACC 22410T = LMG 32496T) and Sphingomonas hankyongi sp. nov., (SE220T = KACC 22406T = LMG 32499T) are proposed.

Structural Analysis of Spermidine Synthase from Kluyveromyces lactis.

Spermidine is a polyamine molecule that performs various cellular functions, such as DNA and RNA stabilization, autophagy modulation, and eIF5A formation, and is generated from putrescine by aminopropyltransferase spermidine synthase (SpdS). During synthesis, the aminopropyl moiety is donated from decarboxylated S-adenosylmethionine to form putrescine, with 5'-deoxy-5'-methylthioadenosine being produced as a byproduct. Although the molecular mechanism of SpdS function has been well-established, its structure-based evolutionary relationships remain to be fully understood. Moreover, only a few structural studies have been conducted on SpdS from fungal species. Here, we determined the crystal structure of an apo-form of SpdS from Kluyveromyces lactis (KlSpdS) at 1.9 Å resolution. Structural comparison with its homologs revealed a conformational change in the α6 helix linked to the gate-keeping loop, with approximately 40° outward rotation. This change caused the catalytic residue Asp170 to move outward, possibly due to the absence of a ligand in the active site. These findings improve our understanding of the structural diversity of SpdS and provide a missing link that expands our knowledge of the structural features of SpdS in fungal species.

Sphingomonas cremea sp. nov., isolated from ginseng soil.

A novel Gram-stain-negative, aerobic, rod-shaped, non-motile, cream-coloured strain (G124T) was isolated from ginseng soil collected in Yeongju, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain G124T belongs to a distinct lineage within the genus Sphingomonas (family Sphingomonadaceae, order Sphingomonadales and class Alphaproteobacteria). Strain G124T was closely related to Sphingomonas rhizophila THG-T61T (98.5 % 16S rRNA gene sequence similarity), Sphingomonas mesophila SYSUP0001T (98.3 %), Sphingomonas edaphi DAC4T (97.6 %) and Sphingomonas jaspsi TDMA-16T (97.6 %). The strain contained ubiquinone 10 as the major respiratory quinone. The major polar lipid profile of strain G124T comprised phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and sphingoglycolipids. The predominant cellular fatty acids of strain G124T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c; 33.4 %), summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c; 27.2 %) and C16 : 0 (18.3 %). The genome size of strain G124T was 2 549 305 bp. The genomic DNA G+C content is 62.0 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain G124T and other Sphingomonas species were in the range of 71.2-75.9 % and 18.7-19.9 %, respectively. Based on the polyphasic analysis such as biochemical, phylogenetic and chemotaxonomic characteristics, strain G124T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cremea sp. nov. is proposed. The type strain is G124T (=KACC 21691T=LMG 31729T).

Sphingomonas cannabina sp. nov., isolated from Cannabis sativa L. 'Cheungsam'.

A Gram-negative, aerobic, rod-shaped bacterium, designated DM2-R-LB4T was isolated from Cannabis sativa L. 'Cheungsam' in Andong, Republic of Korea. The strain DM2-R-LB4T grew at temperatures of 15-45 °C (optimum, 30-37 °C), pH of 5.5-9 (optimum, 8.0), and 0-2 % (w/v) NaCl concentration (optimum, 0%). Phylogenetic analyses based on the 16S rRNA gene sequences revealed that strain DM2-R-LB4T is related to species of the genus Sphingomonas, and shared 97.8 and 97.5% similarity to Sphingomonas kyenggiensis KCTC 42244T and Sphingomonas leidyi DSM 4733T, respectively. The DNA G+C content was 67.9 mol% and genome analysis of the strain DM2-R-LB4T revealed that the genome size was 4 386 171 bp and contained 4 009 predicted protein-coding genes. The average nucleotide identity (ANI) values between strain DM2-R-LB4T and S. kyenggiensis KCTC 42244T, and S. leidyi DSM 4733T was 76.8 and 76.7 %, respectively, while the values of digital DNA-DNA hybridization (dDDH) were 20.7 and 20.6 %, respectively. C14 : 0 2-OH, C16 : 0, and summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) were the major fatty acids (>10 %) in the strain DM2-R-LB4T. The polar lipids comprised diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), sphingoglycolipid (SGL), glycolipid (GL), phospholipid (PL), and two unidentified polar lipids (L1 and L2). Ubiquinone-10 (Q-10) was the only respiratory quinone. The polyamine pattern was found to contain homospermidine, putrescine, and spermidine. The results of phylogenetic anlayses, polyphasic studies, revealed that strain DM2-R-LB4T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cannabina sp. nov., is proposed. The type strain is DM2-R-LB4T (=KCTC 92075T = GDMCC 1.3018T).

The untapped potential of spermidine alkaloids: Sources, structures, bioactivities and syntheses.

Spermidine alkaloids are a kind of natural products possessing an aliphatic triamine structure with three or four methylene groups between two N-atoms. Spermidine alkaloids exist in plants, microorganisms, and marine organisms, which usually form amide structures with cinnamic acid or fatty acid derivatives. Their unique structures showed a wide range of biological activities such as neuroprotective, anti-aging, anti-cancer, antioxidant, anti-inflammatory, and antimicrobial. In order to better understand the research status of spermidine alkaloids and promote their applications in human health, this paper systematically reviewed the biological sources, structures, pharmacological actions, and synthetic processes of spermidine alkaloids over the past two decades. This will help to open up new pharmacological investigation fields and better drug design based on these spermidine alkaloids.

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement.

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl-putrescine or -spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin-labelled acetyl-spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10-mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10-inhibitor structure using X-ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.

Nucleosome destabilization by polyamines.

The roles and molecular interactions of polyamines (PAs) in the nucleus are not fully understood. Here their effect on nucleosome stability, a key regulatory factor in eukaryotic gene control, is reported, as measured in agarose embedded nuclei of H2B-GFP expressor HeLa cells. Nucleosome stability was assessed by quantitative microscopy [1,2] in situ, in close to native state of chromatin, preserving the nucleosome constrained topology of the genomic DNA. A robust destabilizing effect was observed in the millimolar concentration range in the case of spermine, spermidine as well as putrescine, which was strongly pH and salt concentration-dependent, and remained significant also at neutral pH. The integrity of genomic DNA was not affected by PA treatment, excluding DNA break-elicited topological relaxation as a factor in destabilization. The binding of PAs to DNA was demonstrated by the displacement of ethidium bromide, both from deproteinized nuclear halos and from plasmid DNA. The possibility that DNA methylation patterns may be influenced by PA levels is contemplated in the context of gene expression and DNA methylation correlations identified in the NCI-60 panel-based CellMiner database: methylated loci in subsets of high-ODC1 cell lines and the dependence of PER3 DNA methylation on PA metabolism.

Enhanced antibacterial, antioxidant and anticancer activity of caffeic acid by simple acid-base complexation with spermine/spermidine.

Caffeic acid (CA) is a naturally occurring plant-derived polyphenol possessing diverse biological properties. However, the poor water-solubility of CA restricts its widespread applications. On the other hand, biogenic amines such as spermine and spermidine are natural constituents in eukaryotes. In this work, we present water-soluble complexes of CA with spermine and spermidine by exploiting the acid-base interaction. Four different compositions have been prepared by varying the CA to amine ratios, whose chemical structures have been probed in detail using Fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) studies that have revealed the acid-base interaction between the constituent precursors. The obtained acid-base complexes at their native pH values have shown enhanced antibacterial and antioxidant activities than pristine CA. Further, the CA-polyamine complexes have shown high anticancer performances in the concentration range that is compatible with the normal cell lines.

Vineibacter terrae gen. nov., sp. nov., an ammonium-assimilating and nitrate-reducing bacterium isolated from vineyard soil.

A polyphasic taxonomic approach was used to characterize a Gram-stain-negative bacterium, designated strain CC-CFT640T, isolated from vineyard soil sampled in Taiwan. Cells of strain CC-CFT640T were aerobic, non-motile, nitrate-reducing rods. Test results were positive for catalase, oxidase and proteinase activities. Optimal growth occurred at 30 °Ð¡ and pH 7. Strain CC-CFT640T showed highest 16S rRNA gene sequence similarity to members of the genus Enhydrobacter (90.0 %, n=1) followed by Hypericibacter (89.4-90.0 %, n=2), Reyranella (88.8-89.8 %, n=5) and Nitrospirillum (89.2-89.4 %, n=2), and formed a distinct phyletic lineage distantly associated with the clade that predominately accommodated Reynerella species. The DNA G+C composition of the genome (2.1 Mb) was 67.9 mol%. Genes involved in the reduction of nitrate to nitrite, nitric oxide and nitrous oxide were found. In addition, genes encoding dissimilatory nitrate reduction to ammonia, ammonium transport and ammonium assimilation were also detected. Average nucleotide identity values were 73.3 % (n=1), 74.0-74.6 % (n=2), 67.5-68.3 % (n=2) when compared within the type strains of the genera Enhydrobacter, Reyranella and Niveispirillum, respectively. The dominant cellular fatty acids (>5 %) included C16 : 0, iso-C17 : 1 ω10c, C19 : 0 cyclo ω8c, C18 : 1 2-OH and C18 : 1 ω7c/C18 : 1 ω6c. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unidentified aminolipids, three unidentified phospholipids and an unidentified aminophospholipid. The major respiratory quinone was ubiquinone 10 and the major polyamine was spermidine. Based on its distinct phylogenetic, phenotypic and chemotaxonomic traits together with results of comparative 16S rRNA gene sequencing, digital DNA-DNA hybridization, average nucleotide identity and phylogenomic placement, strain CC-CFT640T is considered to represent a novel genus and species of the family Rhodospirillaceae, for which the name Vineibacter terrae gen. nov., sp. nov. is proposed. The type strain is CC-CFT640T (=BCRC 81219T=JCM 33507T).

The thiol-based reduction of Bi(V) and Sb(V) anti-leishmanial complexes.

Low molecular weight thiols including trypanothione and glutathione play an important function in the cellular growth, maintenance and reduction of oxidative stress in Leishmania species. In particular, parasite specific trypanothione has been established as a prime target for new anti-leishmania drugs. Previous studies into the interaction of the front-line Sb(V) based anti-leishmanial drug meglumine antimoniate with glutathione, have demonstrated that a reduction pathway may be responsible for its effective and selective nature. The new suite of organometallic complexes, of general formula [MAr3(O2CR)2] (M = Sb or Bi) have been shown to have potential as new selective drug candidates. However, their behaviour towards the critical thiols glutathione and trypanothione is still largely unknown. Using NMR spectroscopy and mass spectrometry we have examined the interaction of the analogous Sb(V) and Bi(V) organometallic complexes, [SbPh3(O2CCH2(C6H4CH3))2] S1 and [BiPh3(O2CCH2(C6H4CH3))2] B1, with the trifluoroacetate (TFA) salt of trypanothione and L-glutathione. In the presence of trypanothione or glutathione at the clinically relevant pH of 4-5 for Leishmania amastigotes, both complexes undergo facile and rapid reduction, with no discernible difference. However, at a higher pH (6-7), the complexes behave quite differently towards glutathione. The Bi(V) complex is again reduced rapidly but the Sb(V) complex undergoes slow reduction over 8 h (t1/2 = 54 min.) These results give the first insights into why the highly oxidising Bi(V) complexes display low selectivity in their cytotoxicity towards leishmanial and mammalian cells, while the Sb(V) complexes show good selectivity.

Comparative genomic analysis of the genus Novosphingobium and the description of two novel species Novosphingobium aerophilum sp. nov. and Novosphingobium jiangmenense sp. nov.

Members of the genus Novosphingobium are well known for their metabolically versatile and great application potential in pollution elimination. The three novel bacterial strains, designated 4Y4T, 4Y9, and 1Y9AT, were isolated from aquaculture water and characterized by using a polyphasic taxonomic approach. The 16S rRNA gene sequences phylogenetic analysis revealed that the three strains belonged to the genus Novosphingobium. The phylogenomic analysis indicated that the three strains formed two independent and robust branches distinct from all reference strains. The analyses of dDDH values and ANIs between the three strains and their relatives further demonstrated that the three strains represented two different novel genospecies. Comparative genomic analysis of the three isolates and 32 type strains of the genus Novosphingobium showed that the most important central metabolic pathways of these strains appeared to be similar, while specific and specialized metabolic pathways were flexible and variable among these strains. Chemotaxonomic characterization exhibited that the predominant cellular fatty acids were summed feature 8, summed feature 3, and C14:0 2OH; the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyldimethylethanolamine, phosphatidylglycerol, and sphingoglycolipid; the major respiratory quinone and polyamine were Q-10 and spermidine. The DNA G + C contents were 67.6 and 64.7 %. Based on the genotypic and phenotypic characteristics, strains 4Y4T and 1Y9AT are concluded to represent two novel species of the genus Novosphingobium, for which the names Novosphingobium aerophilum sp. nov. (type strain 4Y4T = GDMCC 1.1828 T = KACC 21946 T) and Novosphingobium jiangmenense sp. nov. (type strain 1Y9AT = GDMCC 1.1936 T = KACC 22085 T) are proposed.

X-ray Crystallographic Snapshots of Substrate Binding in the Active Site of Histone Deacetylase 10.

Histone deacetylase 10 (HDAC10) is a zinc-dependent polyamine deacetylase enriched in the cytosol of eukaryotic cells. The active site of HDAC10 contains catalytic residues conserved in other HDAC isozymes that function as lysine deacetylases: Y307 assists the zinc ion in polarizing the substrate carbonyl for nucleophilic attack, and the H136-H137 dyad serves general base-general acid functions. As an inducer of autophagy, HDAC10 is an attractive target for the design of selective inhibitors that may be useful in cancer chemotherapy. Because detailed structural information regarding the catalytic mechanism of HDAC10 may inform new approaches to inhibitor design, we now report X-ray crystal structures of HDAC10 in which reaction intermediates with substrates N8-acetylspermidine and N-acetylputrescine are trapped in the active site. The Y307F substitution prevents activation of the substrate carbonyl for nucleophilic attack by the zinc-bound water molecule, thereby enabling crystallographic isolation of intact enzyme-substrate complexes. The H137A substitution removes the catalytically obligatory general acid, thereby enabling crystallographic isolation of oxyanionic tetrahedral intermediates. Finally, the acetate complex with the wild-type enzyme represents a product complex after dissociation of the polyamine coproduct. Taken together, these structures provide snapshots of the reaction coordinate of acetylpolyamine hydrolysis and are consistent with a mechanism in which tandem histidine residues H136 and H137 serve as general base and general acid catalysts, respectively. The function of the histidine dyad in the HDAC10 mechanism appears to be similar to that in HDAC6, but not HDAC8 in which both functions are served by the second histidine of the tandem pair.

Aeromicrobium terrae sp. nov., isolated from a maize field.

A polyphasic taxonomic approach was used to characterize a Gram-stain-positive bacterium, designated strain CC-CFT486T, isolated from soil sampled in a maize field in Taiwan. Cells of strain CC-CFT486T were short rods, motile with polar flagella, catalase-positive and oxidase-positive. Optimal growth occurred at 30 °Ð¡, pH 8 and 1 % NaCl. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-CFT486T associated with Aeromicrobium panacisoli (97.0 % sequence identity), Aeromicrobium lacus (97.0 %), Aeromicrobium erythreum (96.8 %) and Aeromicrobium alkaliterrae (96.8 %), and lower sequence similarity values to other species. Average nucleotide identity (ANI) values were 70.6-77.8 % (n=11) compared within the type strains of the genus Aeromicrobium. Strain CC-CFT486T contained C16 : 0, C17 : 0, C17 : 1 ω8c and C18 : 1 ω9c as the predominant fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, two unidentified aminophospholipids and three unknown phospholipids. The cell wall peptidoglycan of strains CC-CFT486T contained ll-diaminopimelic acid (ll-DAP) and the major polyamine was spermidine. The DNA G+C content was 70.6 mol% and the predominant quinone was menaquinone 9 (MK-9). Based on its distinct phylogenetic, phenotypic and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence and ANI analyses, strain CC-CFT486T is proposed to represent a novel Aeromicrobium species, for which the name Aeromicrobium terrae sp. nov. (type strain CC-CFT486T=BCRC 81217T=JCM 33499T).

Sphingomonas lacunae sp. nov., isolated from a freshwater pond.

A novel bacterial strain, designated CSW-10T, isolated from a freshwater pond in Taiwan, was characterized using a polyphasic taxonomic approach. Cells were Gram-stain-negative, aerobic, non-motile, rod-shaped and formed yellow-coloured colonies. Optimal growth occurred at 30 °C, pH 7, and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain CSW-10T formed a phylogenetic lineage in the genus Sphingomonas. The 16S rRNA gene sequence similarity indicated that strain CSW-10T was most closely related to Sphingomonas fonticola TNR-2T (97.6%). Strain CSW-10T showed 69.8-70.7% average nucleotide identity and 19.0-23.0% digital DNA-DNA hybridization identity with the strains of other related Sphingomonas species. The major fatty acids of strain CSW-10T were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) and C17:1 ω6c. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylcholine, one uncharacterized sphingoglycolipid, five uncharacterized aminophospholipids, one uncharacterized phospholipid and one uncharacterized lipid. The predominant polyamines were homospermidine and spermidine. The major isoprenoid quinone was Q-10. Genomic DNA G+C content of strain CSW-10T was 62.0 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain CSW-10T should represent a novel species of the genus Sphingomonas, for which the name Sphingomonas lacunae sp. nov. is proposed. The type strain is CSW-10T (=BCRC 81190T =LMG 31340T).

Novosphingobium ovatum sp. nov., isolated from a freshwater mesocosm.

A bacterial strain, designated FSY-8T, was isolated from a freshwater mesocosm in Taiwan and characterized using the polyphasic taxonomy approach. Cells of strain FSY-8T were aerobic, Gram-stain-negative, rod-shaped, non-motile and formed yellow coloured colonies on Reasoner's 2A agar. Growth occurred at 20-40 °C (optimum, 30-37 °C) and pH 5-7 (optimum, pH 6) and in the presence of 0-0.5 % NaCl (optimum, 0 %, w/v). The major fatty acids (>10 %) of strain FSY-8T were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, sphingoglycolipid, diphosphatidylglycerol, an uncharacterized aminophospholipid, an uncharacterized glycolipid and an uncharacterized lipid. The major polyamine was spermidine. The major isoprenoid quinone was Q-10. The DNA G+C content was 64.8 mol %. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain FSY-8T formed a phylogenetic lineage in the genus Novosphingobium. Strain FSY-8T showed 71.6-77.2 % average nucleotide identity and 19.9-22.8 % digital DNA-DNA hybridization identity with the strains of other Novosphingobium species. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain FSY-8T should be classified in a novel species of the genus Novosphingobium, for which the name Novosphingobium ovatum sp. nov. is proposed. The type strain is FSY-8T (=BCRC 81051T=LMG 30053T=KCTC 52812T).

Cerasibacillus terrae sp. nov., isolated from maize field, and emended description of Cerasibacillus quisquiliarum Nakamura et al. 2004.

A polyphasic taxonomic approach was used to characterize a Gram-stain-positive bacterium, designated strain CC-CFT480T, isolated from soil sampled in a maize field in Taiwan, ROC. Cells of strain CC-CFT480T were rod-shaped, motile with polar flagella, catalase-positive and oxidase-positive. Optimal growth occurred at 30 °Ð¡, pH 8 and 3 % NaCl. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-CFT480T associated with Cerasibacillus quisquiliarum (97.2 % sequence identity), Virgibacillus soli (95.7 %), Virgibacillus carmonensis (95.4 %) and Virgibacillus byunsanensis (95.2 %), and lower sequence similarity values to other species. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain CC-CFT480T and C. quisquiliarum were 74.2 and 20.1 %, respectively. Strain CC-CFT480T contained iso-C15:0, C16:1 ω7c alcohol and iso-C17:1 ω10c as the predominant fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown aminophospholipids, one uncharacterized aminophospholipid and two unknown phospholipids. The major polyamine was spermidine. The DNA G+C content was 34.8 mol% and the predominant quinone was menaquinone 7 (MK-7). Based on its distinct phylogenetic, phenotypic and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence, ANI and dDDH analyses, strain CC-CFT480T is proposed to represent a novel Cerasibacillus species, for which the name Cerasibacillus terrae sp. nov. (type strain CC-CFT480T=BCRC 81216T=JCM 33498T).

ESI-Q-TOF-MS determination of polyamines and related enzyme activity for elucidating cellular polyamine metabolism.

We developed a new procedure for the comprehensive analysis of metabolites and enzymes involved in polyamine metabolism pathways. The procedure utilizes stable isotope-labeled polyamines and directly and precisely determines labeled products from enzymatic reactions by ESI-Q-TOF-MS. The activity of different enzymes could be determined in essentially the same manner by suitably adjusting the reaction conditions for each individual enzyme. We applied the procedure to extracts of regenerating rat liver and analyzed the changes in polyamine-metabolizing enzymes and polyamine contents during recovery from partial hepatectomy. A general outline of polyamine metabolism and information of polyamine dynamics were obtained. This kind of comprehensive information would be valuable in unifying detailed but fragmentary information obtained through conventional analyses focusing on one or a few enzymes and on a limited aspect of polyamine metabolic pathway.

Rhodovarius crocodyli sp. nov., isolated from a crocodile pond.

Bacterial strain CCP-6T, isolated from a freshwater pond in Taiwan, was characterized using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set (92 protein clusters) indicated that strain CCP-6T is affiliated with species in the genus Rhodovarius. Strain CCP-6T was most closely related to Rhodovarius lipocyclicus CCUG 44693T with a 98.9% 16S rRNA gene sequence similarity. Cells were Gram-stain-negative, aerobic, non-motile, rod-shaped and formed light pink-coloured colonies. Optimal growth occurred at 30 °C, pH 6 and in the absence of NaCl. The major fatty acids of strain CCP-6T were C18 : 1 ω7c, C16 : 0 and C19 : 0 cyclo ω8c. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidyldimethylethanolamine, phosphatidylmethylethanolamine, diphosphatidylglycerol, three unidentified aminophospholipids and an unidentified phospholipid. The predominant polyamine was spermidine. The major isoprenoid quinone was Q-10. The DNA G+C content of the genomic DNA was 69.3 mol%. Strain CCP-6T showed 85.8% average nucleotide identity and 14.5% digital DNA-DNA hybridization identity with Rhodovarius lipocyclicus CCUG 44693T. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain CCP-6T represents a novel species in the genus Rhodovarius, for which the name Rhodovarius crocodyli sp. nov. is proposed. The type strain is CCP-6T (=BCRC 81095T=LMG 30310T=KCTC 62188T).