Paenibacillus puerhi sp. nov., isolated from the rhizosphere soil of Pu-erh tea plants (Camellia sinensis var. assamica).

An aerobic, Gram-staining-positive, rod-shaped, endospore-forming and motile bacterial strain, designated SJY2T, was isolated from the rhizosphere soil of tea plants (Camellia sinensis var. assamica) collected in the organic tea garden of the Jingmai Pu-erh tea district in Pu'er city, Yunnan, southwest China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Paenibacillus. The closest phylogenetic relative was Paenibacillus filicis DSM 23916T (98.1% similarity). The major fatty acids (> 10% of the total fatty acids) were anteiso-C15:0 and isoC16:0. The major respiratory quinone was MK-7 and the major polar lipid was diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The peptidoglycan contained glutamic acid, serine, alanine and meso-diaminopimelic acid. Genome sequencing revealed a genome size of 6.71 Mbp and a G + C content of 53.1%. Pairwise determined whole genome average nucleotide identity (gANI) values and digital DNA-DNA hybridization (dDDH) values suggested that strain SJY2T represents a new species, for which we propose the name Paenibacillus puerhi sp. nov. with the type strain SJY2T (= CGMCC 1.17156T = KCTC 43242T).

Puerhibacterium puerhi gen. nov., sp. nov., a novel member of the family Promicromonosporaceae, isolated from Pu-erh tea pile-fermentation.

A Gram-staining positive aerobic bacterium, designated TLY-12T, was isolated from the Pu-erh tea pile-fermentation process in Pu'er city, Yunnan, China. Strain TLY-12T grew at 15-37 °C (optimum, 30 °C), pH 6.0-11.0 (optimum, pH 9.0) and 0-9.0% (w/v) NaCl (optimum, 3.0%). The major cellular fatty acids were anteiso-C15:0, C16:0 and iso-C16:0. The respiratory quinone were menaquinones MK-9 (H2) and MK-9 (H4). The polar lipids were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), phosphoglycolipid (PGL), glycolipid (GL) and an unidentified phospholipid (PL). The peptidoglycan contained glutamic acid, aspartic acid, alanine and lysine, with the last named being the diagnostic diamino acid. Whole-cell sugars of the isolate were ribose, galactose and glucose. Phylogenetic analyses of 16S rRNA gene showed that this strain belonged to the family Promicromonosporaceae, and was most closely related to Isoptericola cucumis DSM 101603 T, which gave sequence similarity of 97.9%. Genome sequencing revealed a genome size of 3.91 Mbp and a G + C content of 75.0%. Average nucleotide identity and digital DNA-DNA hybridization values were all below the species threshold of described Promicromonosporaceae species. Genome phylogenetic analysis showed that strain TLY-12T formed a separate evolutionary branch, and was parallel to other related genera of Promicromonosporaceae. Based on the phylogenetic, phenotypic, chemotaxonomic and genome pairwise data, strain TLY-12T is considered to represent a novel species in a new genus in the family Promicromonosporaceae, for which the name Puerhibacterium puerhi gen. nov, sp. nov. is proposed. The type strain is TLY-12T (= CGMCC 1.17157T = KCTC 49467T).

Comparison of volatile profiles and bioactive components of sun-dried Pu-erh tea leaves from ancient tea plants on Bulang Mountain measured by GC-MS and HPLC.

To explore the volatile profiles and the contents of ten bioactive components (polyphenols and caffeine) of sun-dried Pu-erh tea leaves from ancient tea plants on Bulang Mountain, 17 samples of three tea varieties were analyzed by headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) and high-performance liquid chromatography (HPLC). A total of 75 volatile components were tentatively identified. Laomaner (LME), Laobanzhang (LBZ), and other teas on Bulang Mountain (BL) contained 70, 53, and 71 volatile compounds, respectively. Among the volatile compounds, alcohols (30.2%-45.8%), hydrocarbons (13.7%-17.5%), and ketones (12.4%-23.4%) were qualitatively the most dominant volatile compounds in the different tea varieties. The average content of polyphenol was highest in LME (102.1 mg/g), followed by BL (98.7 mg/g) and LBZ (88.0 mg/g), while caffeine showed the opposite trend, 27.3 mg/g in LME, 33.5 mg/g in BL, and 38.1 mg/g in LBZ. Principal component analysis applied to both the volatile compounds and ten bioactive components showed a poor separation of samples according to varieties, while partial least squares-discriminant analysis (PLS-DA) showed satisfactory discrimination. Thirty-four volatile components and five bioactive compounds were selected as major discriminators (variable importance in projection (VIP) >1) among the tea varieties. These results suggest that chromatographic data combined with multivariate analysis could provide a useful technique to characterize and distinguish the sun-dried Pu-erh tea leaves from ancient tea varieties on Bulang Mountain.

Protective Effects of Pretreatment with Quercetin Against Lipopolysaccharide-Induced Apoptosis and the Inhibition of Osteoblast Differentiation via the MAPK and Wnt/ß-Catenin Pathways in MC3T3-E1 Cells.

BACKGROUND/AIMS: Quercetin, a flavonoid found in onions and other vegetables, has potential inhibitory effects on bone resorption in vivo and in vitro. In our previous study, we found that quercetin treatment reversed lipopolysaccharide (LPS)-induced inhibition of osteoblast differentiation through the mitogen-activated protein kinase (MAPK) pathway in MC3T3-E1 cells. In this study, we investigated the underlying mechanisms of pretreatment with quercetin on apoptosis and the inhibition of osteoblast differentiation in MC3T3-E1 cells induced by LPS. METHODS: MC3T3-E1 osteoblasts were treated with quercetin for 2 h; cells were then incubated with LPS in the presence of quercetin for the indicated times. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay, and cell apoptosis was evaluated using Hoechst 33258 staining. The mRNA expression levels of osteoblast-specific genes, Bax and caspase-3 were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of osteoblast-specific genes, caspase-3, Bax, cytochrome c, Bcl-2, Bcl-XL, phosphorylated MAPKs and Wnt/ß-catenin were measured using Western blot assays. The MAPK and Wnt/ß-catenin signalling pathways were blocked prior to pretreatment with quercetin. RESULTS: Pretreatment with quercetin significantly restored LPS-suppressed bone mineralization and the mRNA and protein expression levels of osteoblast-specific genes such as Osterix (OSX), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OCN) in a dose-dependent manner. Pretreatment with quercetin also inhibited osteoblast apoptosis, significantly restored the down-regulated expression of Bcl-2 and Bcl-XL and decreased the upregulated expression of caspase-3, Bax, and cytochrome c in MC3T3-E1 cells induced by LPS. Furthermore, pretreatment with quercetin not only decreased the abundance of phosphorylated p38 MAPK and increased the abundance of phosphorylated extracellular signal regulated kinase (ERK), but also triggered the Wnt/ß-catenin pathway through enhancing expression of Wnt3 and ß-catenin. Pretreatment with MAPK inhibitors or the Wnt/ß-catenin inhibitor XAV939 blocked the protective effects of quercetin against LPS-induced apoptosis and the inhibition of osteoblast differentiation. CONCLUSIONS: Our findings suggest that pretreatment with quercetin may be a potential drug for preventing abnormal human bone loss induced by LPS in bacteria-induced bone diseases.

Improvement of soybean transformation via Agrobacterium tumefaciens methods involving α-aminooxyacetic acid and sonication treatments enlightened by gene expression profile analysis.

KEY MESSAGE: Antagonists and sonication treatment relieved the structural barriers of Agrobacterium entering into cells; hindered signal perception and transmission; alleviated defense responses and increased cell susceptibility to Agrobacterium infection. Soybean gene expression analysis was performed to elucidate the general response of soybean plant to Agrobacterium at an early stage of infection. Agrobacterium infection stimulated the PAMPs-triggered immunity (BRI1, BAK1, BZR1, FLS2 and EFR) and effector-triggered immunity (RPM1, RPS2, RPS5, RIN4, and PBS1); up-regulated the transcript factors (WRKY25, WRKY29, MEKK1P, MKK4/5P and MYC2) in MAPK pathway; strengthened the biosynthesis of flavonoid and isoflavonoid in the second metabolism; finally led to a fierce defense response of soybean to Agrobacterium infection and thereby lower transformation efficiency. To overcome it, antagonist α-aminooxyacetic acid (AOA) and sonication treatment along with Agrobacterium infection were applied. This novel method dramatically decreased the expression of genes coding for F3'H, HCT, ß-glucosidase and IF7GT, etc., which are important for isoflavone biosynthesis or the interconversion of aglycones and glycon; genes coding for peroxidase, FLS2, PBS1 and transcription factor MYC2, etc., which are important components in plant-pathogen interaction; and genes coding for GPAT and α-L-fucosidase, which are important in polyesters formation in cell membrane and the degradation of fucose-containing glycoproteins and glycolipids on the external surface of cell membrane, respectively. This analysis implied that AOA and sonication treatment not only relieved the structural membrane barriers of Agrobacterium entering into cells, but also hindered the perception of 'invasion' signal on cell membrane and intercellular signal transmission, thus effectively alleviated the defense responses and increased the cell susceptibility to Agrobacterium infection. All these factors benefit the transformation process; other measures should also be further explored to improve soybean transformation.