Structurally diverse triterpenoids with antibacterial activities from Euphorbia humifusa.

An exploration of antibacterial components from the whole plant of Euphorbia humifusa led to the isolation of 14 new triterpenoids, euphohumifusoids A-N (1-7 and 9-15), as well as four known analogues (8 and 16-18). Their structures were elucidated by extensively analysis of the spectroscopic data and X-ray crystallography using Cu Kα radiation. Among them, euphohumifusoid A (1) bears an unique 6(7 â†’ 8)abeo scaffold originated from a D:C-friedo-oleanane skeleton for the first time, euphohumifusoids H and I (9 and 10) possess a rare α,ß-unsaturated-γ-lactone chain originated from 25,26,27-trinordammaranes, and euphohumifusoid L (13) is a highly modified 3,4-seco-25,26,27-trinorcycloartane. Notably, in antibacterial bioassay, compound 1 displayed excellent antibacterial activities against Bacillus cereus, Staphylococcus aureus, and S.epidermidis with MIC of 12.5, 25, and 25 µg/mL, comparable to the positive controls. Upon exposure to 1 and 2 MIC of 1, B.cereus underwent drastic morphological changes, resulting in complete disruption of the cells. Meanwhile, compound 1 also exhibited remarkable antibiofilm activity against B.cereus at 1 MIC and 2 MIC.

Paenibacillus mangrovi sp. nov., a novel endophytic bacterium isolated from bark of Kandelia candel.

A endospore-forming bacterium, designated strain KQZ6P-2T, was isolated from surface-sterilized bark of the mangrove plant Kandelia candel, collected from Maowei Sea Mangrove Nature Reserve in Guangxi Zhuang Autonomous Region, China. Strain KQZ6P-2T was able to grow at NaCl concentrations in the range of 0-3 % (w/v) with optimum growth at 0-1 % (w/v) NaCl. Growth occurred at 20-42 °C (optimal growth at 30-37 °C) and pH 5.5-6.5 (optimal growth at pH 6.5). The 16S rRNA gene sequence similarity between strain KQZ6P-2T and its closest phylogenetic neighbour Paenibacillus chibensis JCM 9905T was 98.2 %. Phylogenetic analyses using 16S rRNA gene sequences showed that strain KQZ6P-2T formed a distinct lineage with Paenibacillus chibensis JCM 9905T. The draft genome of strain KQZ6P-2T was 5 937 633 bp in size and its DNA G+C content was 47.2mol%. Comparative genome analysis revealed that the average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values among strain KQZ6P-2T and its related species were below the cut-off levels of 95, 70 and 95.5%, respec-tively. The cell-wall peptidoglycan of strain KQZ6P-2T contained meso-diaminopimelic acid as the diagnostic diamino acid. Major cellular fatty acids were anteiso-C15:0 and C16:0. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, four unidentified phospholipids, an unidentified aminolipid and five unidentified lipids. Based on phylogenetic, phenotypic and chemotaxonomic data, strain KQZ6P-2T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus mangrovi sp. nov. is proposed. The type strain is KQZ6P-2T (=MCCC 1K07172T =JCM 34931T).

Jiella avicenniae sp. nov., a novel endophytic bacterium isolated from bark of Avicennia marina.

A Gram-stain-negative and short rod-shaped strain CBK1P-4T, isolated from surface-sterilized bark of Avicennia marina was investigated by a polyphasic taxonomic approach to resolve its taxonomic position. Strain CBK1P-4T grew at 10-30 °C (optimum, 25 °C), pH 5.0-9.0 (optimum, pH 5.5) and in the presence of 0-9% (w/v) NaCl (optimum, 1-2%). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CBK1P-4T belonged to the genus Jiella and was most closely related to species of the genus Jiella (97.4-98.3%). The genome comparisons between strain CBK1P-4T and the closely related species indicated that average nucleotide identity and digital DNA-DNA hybridization values were below the recommended thresholds for assigning strains to the same species (95-96% and 70%, respectively). The cell wall peptidoglycan contained meso-diaminopimelic acid as diagnostic diamino acid. The principal fatty acids were C18:1ω7c and C19:0cycloω8c. The polar lipids were mainly comprised of phosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, two unidentified aminolipids, one unidentified phospholipid and one unidentified glycolipid. The dominant respiratory quinone was ubiquinone-10. The DNA G + C content of strain CBK1P-4T was 66.7%. Based on the phenotypic features, phylogenetic analysis as well as genome analysis, we conclude that strain CBK1P-4T represents a novel Jiella species, for which the name Jiella avicenniae sp. nov. is proposed. The type strain is CBK1P-4T (= CGMCC 1.18742T = JCM 34330T).

Stakelama flava sp. nov., a novel endophytic bacterium isolated from a branch of Kandelia candel.

A Gram-stain-negative, aerobic, motile, non-spore-forming, short-rod-shaped strain that did not produce diffusible pigment, designated CBK3Z-3T, was isolated from a branch of Kandelia candel, collected from the Beilun Estuary National Nature Reserve in Guangxi Zhang Autonomous Region, PR China, and investigated by a polyphasic approach to determine its taxonomic position. Strain CBK3Z-3T grew at pH 5.0-10.0 (optimum, pH 8.0), 20-37 °C (optimum, 25-30 °C) and with 0-10 % (w/v) NaCl (optimum, 2-3 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CBK3Z-3T was closely related to species of genus Stakelama and had the highest 16S rRNA gene sequence similarity of 98.7 % to Stakelama pacifica CGMCC 1.7294T. The DNA G+C content value of strain CBK3Z-3T was 62.6 mol%. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid and the polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid, an unidentified aminolipid and an unidentified lipid. The major fatty acids were C18 : 1 ω7c and C16 : 0. The average nucleotide identity, estimated digital DNA-DNA hybridization and average amino acid identity values between strain CBK3Z-3T and the type strain of Stakelama pacifica CGMCC 1.7294T were 80.4, 23.1 and 81.5 %, respectively. Based on the phylogenetic, phenotypic and chemotaxonomic data, strain CBK3Z-3T should be designated as a novel species of the genus Stakelama, for which the name Stakelama flava sp.nov. is proposed. The type strain is CBK3Z-3T (=JCM 34534T=CGMCC 1.18972T).

Description and genomic characterization of Jiella flava sp. nov., isolated from Acrostichum aureum.

Two endophytic bacteria, designated strains CQZ9-1T and MQZ9-1, were isolated from semi-mangrove plant Acrostichum aureum collected from Maowei Sea Mangrove Nature Reserve in Guangxi Zhuang Autonomous Region, PR China. The two strains possessed almost identical 16S rRNA gene sequences (99.7 %). The average nucleotide identity (ANI), average amino acid identity (AAI) and the digital DNA-DNA hybridization (dDDH) values between the two strains were 100 %, indicating that they represented the same species. The 16S rRNA gene sequence similarities between strains CQZ9-1T, MQZ9-1 and the most closely related type strains, Jiella mangrovi KSK16Y-1T, Jiella sonneratiae MQZ13P-4T and Jiella endophytica CBS 5Q-3T were 98.0-98.1, 97.3-97.4 and 97.3-97.4 %, respectively. The results of phylogenetic analyses based on 16S rRNA gene sequences and genome sequences indicated that CQZ9-1T and MQZ9-1 formed a distinct lineage with J. endophytica CBS5Q-3T, Jiella pacifica 40Bstr34T, J. mangrovi KSK16Y-1T, J. sonneratiae MQZ13P-4T, Jiella aquimaris JCM 30119T and J. aquimaris 22II-16-19i. The draft genomes of strains CQZ9-1T and MQZ9-1 were 4  162  933 bp and 4  164  266 bp in size, respectively, and their DNA G+C contents were both 63.8 %. Comparative genome analysis of the two strains and the type strains of related species revealed ANI, AAI and dDDH values below the cut-off levels of 95-96, 95.5 and 70 %, respectively. The ubiquinone detected in CQZ9-1T was Q-10. The major cellular fatty acid of strains CQZ9-1T and MQZ9-1 was found to be C18:1ω7c. Combined data from phenotypic, phylogenetic and chemotaxonomic studies indicated that CQZ9-1T and MQZ9-1 represent a novel species of the genus Jiella, for which the name Jiella flava sp. nov. is proposed. The type strain is CQZ9-1T (= CGMCC 1.18725T = JCM 34331T).

Nocardioides mangrovi sp. nov., a novel endophytic actinobacterium isolated from root of Kandelia candel.

A novel species of endophytic actinobacterium, designated strain GBK3QG-3T, was isolated from surface-sterilized root of Kandelia candel collected from Beilun Estuary, Guangxi Zhuang Autonomous Region, PR China. Cells of strain GBK3QG-3T were Gram-stain-positive, aerobic, non-spore-forming and rod-shaped. Growth of GBK3QG-3T was observed at 10-40 °C (optimum, 30 °C), pH 5.0-9.0 (optimum, pH 7.0) and in the presence of 0-3 % (w/v) NaCl (optimum, 0 %). The G+C content of genomic DNA was determined to be 72.2 mol%. On the basis of 16S rRNA gene sequence analysis, strain GBK3QG-3T was most related to the type strain of Nocardioides agrisoli CCTCC AB 2017058T (97.7 % 16S rRNA gene sequence similarity), followed by Nocardioides ginkgobilobae SYP-A7303T (97.0 %) and Nocardioides aquiterrae GW-9T (96.9 %). Average nucleotide identity and digital DNA-DNA hybridization values between strain GBK3QG-3T and some strains of the genus Nocardioides with publicly available genomes were below 84.3 and 30.0 %, respectively. The cell-wall peptidoglycan contained ll-diaminopimelic acid. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, an unknown phospholipid and an unknown lipid. The major menaquinone was identified as MK-8(H4), and the major fatty acids were identified as iso-C16 : 0, iso-C17 : 0 and anteiso-C17 : 0. Based on evidence collected from the phenotypic, genotypic and phylogenetic analyses, we propose a novel species named Nocardioides mangrovi sp. nov. The type strain is GBK3QG-3T (=CGMCC 4.7708T=JCM 34553T).

Jiella sonneratiae sp. nov., a novel endophytic bacterium isolated from bark of Sonneratia apetala.

A new endophytic bacterium, designated strain MQZ13P-4T was isolated from Sonneratia apetala collected from Maowei sea Mangrove Nature Reserve in Guangxi Zhuang Autonomous Region, PR China. The 16S rRNA gene sequence similarity between strain MQZ13P-4T and its closest phylogenetic neighbour Jiella endophytica CBS5Q-3T was 97.9 %. Phylogenetic analyses using 16S rRNA gene sequences and whole-genome sequences showed that strain MQZ13P-4T formed a distinct lineage with Jiella endophytica CBS5Q-3T, Jiella pacifica 40Bstr34T and Jiella aquimaris JCM 30119T. The draft genome of strain MQZ13P-4T was 5 153 243 bp in size and its DNA G+C content was 68.1 mol%. Comparative genome analysis revealed that the average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values among strain MQZ13P-4T and other related species were below the cut-off levels of 95, 70 and 95.5 %, respectively. The cell-wall peptidoglycan of strain MQZ13P-4T contained meso-diaminopimelic acid as the diagnostic diamino acid. The respiratory quinone was Q-10. The major cellular fatty acid was C18 : 1 ω7c. The polar lipids comprised phosphatidylcholine, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, two unidentified aminolipids and two unidentified lipids. Strain MQZ13P-4T had a typical chemical compositions of fatty acids, lipids, quinones and diagnostic diamino acid for Jiella species, but could be distinguished from known species of the genus Jiella. Based on polyphasic evidence, strain MQZ13P-4T represents novel species of the genus Jiella, for which the name Jiella sonneratiae sp. nov. is proposed. The type strain is MQZ13P-4T (=CGMCC 1.18727T=JCM 34333T).

Pseudooceanicola endophyticus sp. nov., a novel endophytic bacterium isolated from bark Aegiceras corniculatum.

A Gram-stain-negative, aerobic, short-rod-shaped bacterium, designated strain CBS1P-1T, was isolated from a surface-sterilized bark of Aegiceras corniculatum. Growth of strain CBS1P-1T was observed with between 0 and 12.0 % (w/v) NaCl (optimally with 5.0 %) and at between pH 6.0-9.0. It grew at temperatures between 25-37 °C (optimum, 30 °C). Chemotaxonomic analysis showed that ubiquinone-10 was the respiratory quinone. The lipids comprised diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid, an unidentified phospholipid and an unidentified aminolipid. The major fatty acids of strain CBS1P-1T were C18 : 1 ω7c, C16 : 0 and C19 : 0 cyclo ω8c. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain CBS1P-1T was most related to Pseudooceanicola antarcticus CGMCC 1.12662T with a sequence similarity of 96.5 %. The average nucleotide identity and digital DNA-DNA hybridization values between strain CBS1P-1T and P. antarcticus 1.12662T were 77.5 and 21.1 %, respectively. The G+C content of the genomic DNA was 67.3 mol%. Based on phylogenetic, chemotaxonomic and phenotypic data, strain CBS1P-1T is considered to represent a novel species of the genus Pseudooceanicola, for which the name Pseudooceanicola endophyticus is proposed. The type strain is CBS1P-1T (=KCTC 62836T=CGMCC 1.13743T).

Phycicoccus flavus sp. nov., a novel endophytic actinobacterium isolated from branch of Kandelia candel.

A Gram-stain-positive, aerobic, non-motile, non-endospore-forming and rod-shaped actinobacterium, designated strain CMS6Z-2T, was isolated from a surface-sterilized branch of Kandelia candel collected from the Maowei Sea, Guangxi Zhuang Autonomous Region, PR China. Strain CMS6Z-2T grew at 10-37 °C (optimum, 37 °C), pH 6.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 0-10.0 % (w/v) NaCl (optimum, 0-1.0 %). Strain CMS6Z-2T possessed meso-diaminopimelic acid as the diamino acid of the peptidoglycan and MK-8 (H4) as the predominant menaquinone. The major fatty acids were iso-C15 : 0, C16 : 0 and C18 : 1 ω9c. The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and two unknown phospholipids. The G+C content of the genomic DNA was 74.1 mol%. Comparative analysis of 16S rRNA genes showed that strain CMS6Z-2T should be assigned to the genus Phycicoccus and its closest relative was Phycicoccus endophyticus IP6SC6T with 98.3 % similarity. Phylogenetic analyses based on 16S rRNA gene sequence and phylogenomic analysis based on core proteomes alignment revealed that strain CMS6Z-2T belonged to the genus Phycicoccus and formed a robust cluster with Phycicoccus endophyticus IP6SC6T within the genus Phycicoccus. The average nucleotide identity value and estimated digital DNA-DNA hybridization value between strain CMS6Z-2T and the type strain of Phycicoccus endophyticus were 81.5 and 23.9 %, respectively. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain CMS6Z-2T represents a novel species of the genus Phycicoccus, for which the name Phycicoccus flavus sp. nov. is proposed. The type strain is CMS6Z-2T (=KCTC 49240T=CGMCC4.7549T).

Phycicoccus avicenniae sp. nov., a novel endophytic actinomycete isolated from a branch of Avicennia mariana.

A novel endophytic actinomycete, designated strain BSK3Z-2 T, was isolated from a surface-sterilised branch of Avicennia mariana from Shankou Mangrove Nature Reserve, Guangxi Zhuang Autonomous Region, China. Cells were observed to be Gram-stain positive, aerobic, asporogenous and rod-shaped. Strain BSK3Z-2 T was found to grow optimally at 30 °C, pH 7.0 and in the presence of 1% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BSK3Z-2 T belongs to the genus Phycicoccus and has high 16S rRNA gene sequence similarity of 98.1% with Phycicoccus endophyticus IP6SC6T. Phylogenetic analysis based on the genome of strain BSK3Z-2 T was performed by extracting and aligning 39 conserved proteins and 88 housekeeping genes, which further confirmed the phylogenetic assignment of strain BSK3Z-2 T. The draft genome of strain BSK3Z-2 T is 3.54 Mbp with a DNA G + C content of 73.8%. The average nucleotide identity (ANI), amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values between strain BSK3Z-2 T and species of genus Phycicoccus were 73.8-85.6%, 64.5-75.9% and 19.5-23.8%, respectively, which are below the standard cut-off values for bacterial species delineation. Strain BSK3Z-2 T contains MK-8(H4) as the dominant menaquinone. The cell wall peptidoglycan contains meso-diaminopimelic acid. The polar lipids profile of strain BSK3Z-2 T was found to contain diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The predominant fatty acids were identified as C15:0, C17:0, iso-C16:0 and C17:1ω8c. Comparing the phenotypic and phylogenetic features of the strain BSK3Z-2 T and related taxa, strain BSK3Z-2 T is concluded to represent a novel species of the genus Phycicoccus, for which the name Phycicoccus avicenniae sp. nov. is proposed. The type strain is BSK3Z-2 T (= CGMCC 1.18743 T = JCM 34335 T).

Ectobacillus aegiceratis sp. nov., a novel bacterium isolated from branch of Aegiceras corniculatum.

A Gram-stain-positive, non-motile, endospore-forming, rod-shaped and aerobic bacterium was isolated from surface-sterilized branch of Aegiceras corniculatum in Guangxi Zhuang Autonomous Region, China. The isolate, designated strain 165T, grew at 20-45 °C (optimum, 30 °C), pH 6.0-7.0 (optimum, 6.0) and with 0-3 % (w/v) NaCl (optimum, 1 %). The major respiratory quinone was MK-7 and the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid and an unidentified glycolipid. The major fatty acids were iso-C15:0, anteiso-C15:0 and iso-C16:0. On the basis of 16 S rRNA gene sequence and multiple genes of conserved core proteins analysis, strain 165T was a member of the genus Ectobacillus. Its closest phylogenetic neighbor was Ectobacillus panaciterrae Gsoil 1517T, with sequence similarity of 97.1 %. The average nucleotide identity value between strain 165T and type strain of Ectobacillus panaciterrae was 73.0 %. The estimated DDH value between strain 165T and type strain of Ectobacillus panaciterrae was 19.7 %. The genome of strain 165T was 3, 545, 051 bp long with a DNA G + C content of 38.2 % and encodes 3459 predicted proteins, 25 rRNAs, 87 tRNAs and 5 ncRNA. The genome of strain 165T comprised gene clusters of type 3 PKS, terpene, betalactone and lanthipeptide-class-ii for secondary metabolites. Phenotypic, chemotaxonomic and phylogenetic analyses supported the strain 165T as a representative of a novel species of the genus Ectobacillus, for which the name Ectobacillus aegiceratis sp. nov. is proposed, with strain 165T (= JCM 33,414T = CGMCC 1.13742T) as the type strain.

Brachybacterium halotolerans sp. nov., a halotolerant, endophytic actinomycete isolated from branch of Bruguiera gymnoirhiza.

A novel, aerobic, moderately halophilic Gram-positive actinomycete, strain MASK1Z-5T was isolated from a surface-sterilized branch of Bruguiera gymnoirhiza in Shankou Mangrove Nature Reserve, Guangxi, China. The taxonomic position of the strain was investigated using a polyphasic approach. Strain MASK1Z-5T tolerated up to 20% (w/v) NaCl (optimum 0-7%), and grew at pH 5.0-12.0 (optimum pH 7.0-8.0), 20-37 °C (optimum 30 °C). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MASK1Z-5T belonged to the genus Brachybacterium, and showed the highest 16S rRNA gene sequence similarity of 98.0% to B. endophyticum M1HQ-2T. The cell-wall peptidoglycan contained meso-diaminopimelic acid as diagnostic diamino acid. MK-7 was the predominant menaquinone. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, one unidentified glycolipid and three unidentified lipids. The major fatty acids were anteiso-C15:0, iso-C16:0 and anteiso-C17:0. The DNA G + C content was calculated to be 71.8 mol% based on the whole genome sequence. The estimated values of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) based on whole genome sequences between strain MASK1Z-5T and B. endophyticum M1HQ-2T were 81.8% and 25.0%, respectively. The phenotypic, chemotaxonomic and genotypic properties clearly indicated that strain MASK1Z-5T represents a novel species within the genus Brachybacterium, for which the name Brachybacterium halotolerans sp. nov. is proposed. The type strain is MASK1Z-5T (= CGMCC1.18660T = JCM 34339T).

Jiella mangrovi sp. nov., a novel endophytic bacterium isolated from leaf of Rhizophora stylosa.

A Gram-stain negative, non-motile, brilliant yellow and non-spore forming, coccoid- or short rod-shaped bacterium, designated strain KSK16Y-1T, was isolated from surface-sterilised leaf of Rhizophora stylosa collected from Shankou Mangrove Nature Reserve, Guangxi Zhuang Autonomous Region, China. Genome of strain KSK16Y-1T is 4.93 Mb with 68.1% DNA G + C content and encoded 4359 predicted proteins, 4 rRNAs, 45 tRNAs and 4 ncRNA. Comparative 16S rRNA gene sequence analysis showed that the strain KSK16Y-1T has 98.1%, 97.9% and 96.9% 16S rRNA gene similarities with Jiella aquimaris JCM 30119T, J. endophytica CBS5Q-3T and J. pacifica 40Bstr34T, respectively. Whole-genome comparisons between strain KSK16Y-1T and J. aquimaris 22II-16-19i, J. endophytica CBS5Q-3T, J. pacifica 40Bstr34T, using average nucleotide identity (ANI) values (< 82.0%) and digital DNA-DNA hybridization (dDDH) values (< 25.1%), confirmed low genome relatedness. Strain KSK16Y-1T grew at 20-30 °C (optimum, 30 °C), pH 6.0-11.0 (optimum, pH 6.0-7.0) and with 0-10% (w/v) NaCl (optimum, 0-2%). Cell wall contained meso-diaminopimelic acid and the major fatty acid was C18:1ω7c. The polar lipid profile consists of phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, one unknown phospholipid, one unknown aminolipid, one unknown aminophospholipid and four unidentified lipids. The predominant respiratory quinone is ubiquinone-10 (Q-10). The polyphasic characterization indicated that strain KSK16Y-1T represents a novel Jiella species. The name Jiella mangrovi sp. nov., type strain KSK16Y-1T (= CGMCC 1.18745T = JCM 34332T) is proposed.

Nocardioides acrostichi sp. nov., a novel endophytic actinobacterium isolated from leaf of Acrostichum aureum.

A short-rod-shaped, non-spore-forming endophytic actinobacterium, was isolated from a surface-sterilized leaf of Acrostichum aureum in Fangchenggang, Guangxi Zhuang Autonomous Region, China, designated strain CBS4Y-1T and examined by a polyphasic approach to determine its taxonomic position. This actinobacterium was Gram-staining-positive and aerobic. Substrate mycelia and aerial mycelia were not observed, and no diffusible pigments were observed on the media tested. Strain CBS4Y-1T grew optimally with 0-1.0% (w/v) NaCl at 30 °C, pH 7.0-8.0. Comparative analysis of 16S rRNA genes showed that strain CBS4Y-1T shared the highest 16S rRNA gene sequence similarities with Nocardioides marinus CL-DD14T (96.7%) and Nocardioides terrae BX5-10T (96.7%). Phylogenetic analyses based on 16S rRNA gene sequence and phylogenomic analysis based on core proteomes alignment revealed that strain CBS4Y-1T belonged to the genus Nocardioides and formed a distinct cluster within the genus Nocardioides. The DNA G + C content of strain CBS4Y-1T was 71.1 mol%. The cell-wall peptidoglycan contained LL-diaminopimelic acid and MK-8(H4) was the predominant menaquinone. Phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylinositol (PI) were detected in the polar lipid extracts. The major fatty acids were iso-C16:0, C18:1ω9c and iso-C17:0. On the basis of phylogenetic analysis, phenotypic and chemotaxonomic characteristics, strain CBS4Y-1T represents a novel species of the genus Nocardioides, for which the name Nocardioides acrostichi sp. nov. is proposed. The type strain is CBS4Y-1T (= KCTC 49238T = CGMCC 4.7548T).

Human platelet lysates for human cell propagation.

A pathogen-free and standardized xeno-free supplement of growth media is required for the ex vivo propagation of human cells used as advanced therapeutic medicinal products and for clinical translation in regenerative medicine and cell therapies. Human platelet lysate (HPL) made from therapeutic-grade platelet concentrate (PC) is increasingly regarded as being an efficient xeno-free alternative growth medium supplement to fetal bovine serum (FBS) for clinical-grade isolation and/or propagation of human cells. Most experimental studies establishing the superiority of HPL over FBS were conducted using mesenchymal stromal cells (MSCs) from bone marrow or adipose tissues. Data almost unanimously concur that MSCs expanded in a media supplemented with HPL have improved proliferation, shorter doubling times, and preserved clonogenicity, immunophenotype, in vitro trilineage differentiation capacity, and T-cell immunosuppressive activity. HPL can also be substituted for FBS when propagating MSCs from various other tissue sources, including Wharton jelly, the umbilical cord, amniotic fluid, dental pulp, periodontal ligaments, and apical papillae. Interestingly, HPL xeno-free supplementation is also proving successful for expanding human-differentiated cells, including chondrocytes, corneal endothelium and corneal epithelium cells, and tenocytes, for transplantation and tissue-engineering applications. In addition, the most recent developments suggest the possibility of successfully expanding immune cells such as macrophages, dendritic cells, and chimeric antigen receptor-T cells in HPL, further broadening its use as a growth medium supplement. Therefore, strong scientific rationale supports the use of HPL as a universal growth medium supplement for isolating and propagating therapeutic human cells for transplantation and tissue engineering. Efforts are underway to ensure optimal standardization and pathogen safety of HPL to secure its reliability for clinical-grade cell-therapy and regenerative medicine products and tissue engineering.

Nanofiltration of growth media supplemented with human platelet lysates for pathogen-safe xeno-free expansion of mesenchymal stromal cells.

BACKGROUND AIMS: Human platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow-derived MSCs. METHODS: Platelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated. RESULTS: Nanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages. CONCLUSIONS: Nanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.

Microbacterium excoecariae sp. nov., a novel endophytic actinobacterium isolated from bark of Excoecaria agallocha Linn.

A Gram-stain-positive, aerobic actinobacterium, designated strain CBS5P-1T, was isolated from bark of Excoecaria agallocha Linn collected from Guangxi Zhuang Autonomous Region, PR China. Cells were short rods. Colonies were light yellow, circular and had entire margins. Strain CBS5P-1T grew at 10-37 °C (optimum, 30 °C) and pH 6.0-12.0 (optimum, pH 7.0-8.0). Its nearest phylogenetic neighbour was Microbacterium amylolyticum DSM 24221T with 97.1 % 16S rRNA gene sequence similarity. The genomic DNA G+C content of strain CBS5P-1T was 71.8 mol%. Anteiso-C15  : 0, anteiso-C17 : 0, iso-C16 : 0 and C16:0 were predominant cellular fatty acids. Major menaquinones were MK-11 and MK-10. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid and an unidentified phospholipid. The combination of chemotaxonomic, phylogenetic and phenotypic data clearly distinguished strain CBS5P-1T from its phylogenetic neighbour. Accordingly, the name Microbacterium excoecariae sp. nov. is proposed to accommodate this new member of the genus Microbacterium. The type strain is CBS5P-1T (=KCTC 49239T=CGMCC 1.13862T).

Nakamurella flava sp. nov., a novel endophytic actinobacterium isolated from Mentha haplocalyx Briq.

A Gram-stain-positive, aerobic, coccus-shaped, non-spore-forming actinobacterium, designated strain N5BH11T, was isolated from a surface-sterilized sample of Mentha haplocalyx Briq. collected from Guizhou, PR China and tested by a polyphasic approach to determine its taxonomic position. Strain N5BH11T grew optimally at 30 °C, pH 6.0-7.0. Substrate mycelia and aerial mycelia were not formed, and no diffusible pigments were observed on the media tested. Phylogenetic analysis based on 16S rRNA gene sequence suggested that strain N5BH11T belonged to the genus Nakamurella and had the highest 16S rRNA gene sequence similarity to Nakamurella flavida DS-52T (98.1 %). The DNA G+C content of strain N5BH11T was 71.6 mol%. The average nucleotide identity values between strain N5BH11T and the type strains of Nakamurella panacisegetis, Nakamurella multipartita and Nakamurella lactea were 74.0, 76.5 and 73.6 %, respectively. The estimated DDH values between strain N5BH11T and the type strains of N. panacisegetis, N. multipartita and N. lactea were 20.3%, 21.4 and 20.2 %, respectively. The cell-wall peptidoglycan contained meso-diaminopimelic acid, and MK-8(H4) was the predominant menaquinone. The predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and unidentified phospholipids. The major fatty acids were iso-C15 : 0, anteiso-C15 : 0, C16 : 0 and C16 : 1ω7c. On the basis of the results of phylogenetic analysis and phenotypic and chemotaxonomic characteristics, strain N5BH11T represents a novel species of the genus Nakamurella, for which the name Nakamurella flava sp. nov. is proposed. The type strain is N5BH11T (=KCTC 49196T=CGMCC 4.7524T).

Comparison of three human platelet lysates used as supplements for in vitro expansion of corneal endothelium cells.

BACKGROUND: Human platelet lysates (HPLs) are emerging as the new gold standard supplement of growth media for ex vivo expansion of cells for transplant. However, variations do exist in the way how HPLs are prepared. In particular, uncertainties still exist regarding the type of HPL most suitable for corneal endothelium cells (CEC) expansion, especially as these cells have limited proliferative capacity. MATERIAL AND METHODS: Three distinct HPL preparations were produced, with or without calcium chloride/glass beads activation, and with or without heat treatment at 56°C for 30min. These HPLs were used to supplement basal D-MEM growth medium, each at a protein concentration equivalent to that of 10% fetal bovine serum (FBS; control). Impact on CEC (BCE C/D-1b cells) in vitro morphology, viability and capacity to express Zonula occludens-1 (ZO-1) tight junction marker was assessed by Western blotting. RESULTS: BCE C/D-1b cells grown in all HPL supplements exhibited four of essential characteristic properties: adhesion capacity, microscopic morphology and viability similar to that observed when using 10% FBS. In addition, Western blots analysis revealed an expression of the ZO-1 marker by BCE C/D-1b cells in all conditions of culture. CONCLUSION: CECs can expand ex vivo in a basal medium supplemented with the three HPLs without noticeable difference compared to FBS supplement. These data support further studies to evaluate the potential to use HPLs as a clinical-grade xeno-free supplement of CEC for corneal transplant.

Dissecting yield-associated loci in super hybrid rice by resequencing recombinant inbred lines and improving parental genome sequences.

The growing world population and shrinkage of arable land demand yield improvement of rice, one of the most important staple crops. To elucidate the genetic basis of yield and uncover its associated loci in rice, we resequenced the core recombinant inbred lines of Liang-You-Pei-Jiu, the widely cultivated super hybrid rice, and constructed a high-resolution linkage map. We detected 43 yield-associated quantitative trait loci, of which 20 are unique. Based on the high-density physical map, the genome sequences of paternal variety 93-11 and maternal cultivar PA64s of Liang-You-Pei-Jiu were significantly improved. The large recombinant inbred line population combined with plentiful high-quality single nucleotide polymorphisms and insertions/deletions between parental genomes allowed us to fine-map two quantitative trait loci, qSN8 and qSPB1, and to identify days to heading8 and lax panicle1 as candidate genes, respectively. The quantitative trait locus qSN8 was further confirmed to be days to heading8 by a complementation test. Our study provided an ideal platform for molecular breeding by targeting and dissecting yield-associated loci in rice.