Seasonal variations and sources of atmospheric EPFRs in a megacity in severe cold region: Implications for the influence of strong coal and biomass combustion.

Environmentally persistent free radicals (EPFRs) can pose exposure risks by inducing the generation of reactive oxygen species. As a new class of pollutants, EPFRs have been frequently detected in atmospheric particulate matters. In this study, the seasonal variations and sources of EPFRs in a severe cold region in Northeastern China were comprehensively investigated, especially for the high pollution events. The geomean concentration of EPFRs in the total suspended particle was 6.58 × 1013 spins/m3 and the mean level in winter was one order of magnitude higher than summer and autumn. The correlation network analysis showed that EPFRs had significantly positive correlation with carbon component, K+ and PAHs, indicating that EPFRs were primarily emitted from combustion and pyrolysis process. The source appointment by the Positive Matrix Factorization (PMF) model indicated that the dominant sources in the heating season were coal combustion (48.4%), vehicle emission (23.1%) and biomass burning (19.4%), while the top three sources in the non-heating season were others (41.4%), coal combustion (23.7%) and vehicle emissions (21.2%). It was found that the high EPFRs in cold season can be ascribed to the extensive use of fossil fuel for heating demand; while the high EPFRs occurred in early spring were caused by the large-scale opening combustion of biomass. In summary, this study provided important basic information for better understanding the pollution characteristics of EPFRs, which suggested that the implementation of energy transformation and straw utilization was benefit for the control of EPFRs in severe cold region.

Xanchryones I-N, Six Unusual Phloroglucinol-Amino Acid Hybrids from the Leaves of Xanthostemon chrysanthus.

Six new phloroglucinol derivatives, xanchryones I-N (1-6), were isolated from the leaves of Xanthostemon chrysanthus. Compounds 1-6 are unusual phloroglucinol-amino acid hybrids constructed through C2 -N and O-C1 ' bonds forming a peculiar oxazole ring. The structures and absolute configurations of compounds 1-6 were determined by MS, NMR, and single-crystal X-ray diffraction. Moreover, the anti-inflammatory and antibacterial activities of these compounds were evaluated.

Health Risk Assessment of Organophosphate Flame Retardants in Soil Across China Based on Monte Carlo Simulation.

Health risks from exposure to contaminants are generally estimated by evaluating concentrations of the contaminants in environmental matrixes. However, accurate health risk assessment is difficult because of uncertainties regarding exposures. This study aims to utilize data on the concentrations of organophosphate flame retardants (OPFRs) in surface soil across China coupled with Monte Carlo simulations to compensate for uncertainties in exposure to evaluate the health risks associated with contamination of soil with this class of flame retardants. Results revealed that concentrations of ∑OPFRs were 0.793-406 ng/g dry weight (dw) with an average of 23.2 ng/g dw. In terms of spatial distribution, higher OPFRs concentrations were found in economically developed regions. Although the values of health risk of OPFRs in soil across China were below the threshold, the high concentrations of OPFRs in soil in some regions should attract more attentions in future. Sensitivity analysis revealed that concentrations of OPFRs in soil, skin adherence factor, and exposure duration were the most sensitive parameters in health risk assessment. In summary, the study indicated that the national scale soil measurement could provide unique information on OPFRs exposure and health risk assessment, which was useful for the management of soil in China and for better understanding of the environmental fate of OPFRs in the global perspective.

Dyella amyloliquefaciens sp. nov., isolated from forest soil.

A Gram-stain-negative, aerobic, yellow-pigmented, rod-shaped and motile with single polar flagellum bacterial strain, designated DHC06T, was isolated from forest soil sampled at Dinghushan Biosphere Reserve, Guangdong Province, PR China. The strain grew at 4-37 °C (optimum, 28 °C), pH 4.5-8.0 (pH 6.0-7.5) and in the presence of 0-4.0 % (2.0 %, w/v) NaCl. In the 16S rRNA gene sequence phylogram, strain DHC06T formed a clade with Dyella solisilvae DHG54T and Dyella terrae KACC 12748T within the genus of Dyella. Strain DHC06T had 16S rRNA gene sequence similarities of 98.6, 98.3, 98.3 and 98.2 % to Dyella japonica DSM 16301T, Dyella terrae JS14-6T, Dyella soli KACC 12747T and Dyella solisilvae DHG54T, respectively. The distinctiveness of strain DHC06Tfrom all described Dyellaspecies was also supported by the results of phylogenomic analysis based on 92 single-copy gene sequences. The DDH values among strain DHC06T and closely related Dyella species were all lower than 70 %. Strain DHC06T contained Q-8 as the only respiratory quinone. Its main fatty acids were iso-C15 : 0, iso-C17 : 1 ω9c and summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c). The DNA G+C content of strain DHC06T was 64.6 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. On the basis of phenotypic, 16S rRNA gene sequence and genomic analyses and chemotaxonomic data, strain DHC06T represents a novel species of the genus Dyella, for which the name Dyella amyloliquefaciens sp. nov. (type strain DHC06T=GDMCC 1.1186T=LMG 30090T) is proposed.

Dyella dinghuensis sp. nov. and Dyella choica sp. nov., isolated from forest soil.

Two aerobic, Gram-stain-negative, non-motile, rod-shaped bacterial strains, designated as DHOA06T and 4 M-K27T, were isolated from soil samples collected from the forest of Dinghushan Biosphere Reserve, Guangdong Province, PR China (112° 31' E 23° 10' N). Strains DHOA06T and 4 M-K27T grew at pH 4.5-7.0 (optimum, pH 5.0-6.0) and pH 4.5-6.5 (pH 6.0), respectively. Both strains grew at 12-37 °C (optimum, 28 °C) and NaCl levels up to 1.0 % (optimum 0 %, w/v). Phylogenetic analysis based on both 16S rRNA gene sequences and the concatenated partial atpD, gyrB andlepA gene sequences showed that strains DHOA06T and 4 M-K27T formed two isolated clades with members of the genus Dyella, but they each occupied a distinctive position within the genus. Strains DHOA06T and 4 M-K27T showed the highest 16S rRNA gene sequence similarities to Dyellacaseinilytica DHOB09T (98.7 %) and Dyellaacidisoli 4M-Z03T (98.8 %), respectively. DNA-DNA hybridization values of strains DHOA06T/DHOB09T and 4 M-K27T/4M-Z03T were 27.4±2.4 % and 38.8±1.0 %, respectively. Ubiquinone-8 was the only respiratory quinone detected in both strains. Their major fatty acids consisted of iso-C15 : 0, iso-C16 : 0 and iso-C17 : 1ω9c, and strain DHOA06T had iso-C17 : 0 in addition. Their polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminophospholipid, one unidentified phospholipid and two unidentified aminolipids, and strain DHOA06T had phosphatidylmethylethanolamine and one unidentified lipid in addition. The DNA G+C contents of strains DHOA06T and 4 M-K27T were 59.1 and 61.7 mol%, respectively. Based on the above results, we propose that strains DHOA06T and 4 M-K27T represent two novel species of the genus Dyella, namely Dyelladinghuensis sp. nov. (type strain DHOA06T = KCTC 52129T=NBRC 111978T) and Dyellachoica sp. nov. (type strain 4 M-K27T=GDMCC 1.1189T=LMG 30267T).

Dyella monticola sp. nov. and Dyella psychrodurans sp. nov., isolated from monsoon evergreen broad-leaved forest soil of Dinghu Mountain, China.

Cells of bacterial strains 4 G-K06T and 4MSK11T, isolated from soil samples collected from monsoon evergreen broad-leaved forest of the Dinghushan Mountain (112° 31' E 23° 10' N), Guangdong Province, PR China, were Gram-stain-negative, aerobic, non-spore-forming, non-motile and rod-shaped. Strain 4 G-K06T grew at 10-37 °C, pH 3.5-7.5 and 0-3.5 % (w/v) NaCl; while 4MSK11T grew at 4-42 °C, pH 3.5-7.5 and 0-2.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed strain 4 G-K06T formed a clade with Dyellaflagellata 4 M-K16T, Dyella acidisoli 4M-Z03T, Dyellahumi DHG40T and Dyellanitratireducens DHG59T, while strain 4MSK11T formed a clade with Dyellacaseinilytica DHOB09T and Dyellamobilis DHON07T, both within the genus Dyella. The result of the partial atpD, gyrB and lepA gene sequence analysis supported the conclusion based on 16S rRNA gene sequence analysis, which showed that these two strains represent two novel species of Dyella. The average nucleotide identity and digital DNA-DNA hybridization value for the whole genomes were 75.0-79.0 and 20.3-22.6 % between strains 4 G-K06T, 4MSK11T and those described Dyella species with genome sequences; while the DNA-DNA hybridization rates between strains 4 G-K06T, 4MSK11T and closely related Dyella species (without genome sequence) were 29.5-41.8 %. The major cellular fatty acids of these two strains were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 1ω9c, while the major polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several unidentified phospholipids and aminophospholipids. The only ubiquinone of these two strains was ubiquinone-8. The DNA G+C contents of 4 G-K06T and 4MSK11T were 60.4 and 61.3 mol%, respectively. On the basis of the evidence presented here, strains 4 G-K06T and 4MSK11T represent two novel species of the genus Dyella, for which the names Dyella monticola sp. nov. (type strain 4 G-K06T=LMG 30268T=GDMCC 1.1188T) and Dyella psychrodurans sp. nov. (type strain 4MSK11T=KCTC 62280T=GDMCC 1.1185T) are proposed.

Dyella solisilvae sp. nov., isolated from mixed pine and broad-leaved forest soil.

A Gram-stain-negative, aerobic, motile, yellow-pigmented, rod-shaped with a single polar flagellum bacterial strain, designated strain DHG54T, was isolated from a forest soil sample of Dinghushan Biosphere Reserve, Guangdong Province, China. Strain DHG54T grew at 12-37 °C (optimum, 28 °C), pH 4.5-8.0 (optimum, pH 6.0-7.0) and in the presence of 0-3.0 % (w/v) NaCl (optimum, 0-1.5 %, w/v). Based on 16S rRNA gene sequence analysis, strain DHG54T formed a clade with the members of the genus Dyella and showed highest sequence similarities of 98.2 % to Dyella japonica DSM 16301T and Dyella terrae KACC 12748T. This was also supported by phylogenetic analysis based on the concatenated partial gyrB, lepA and recA housekeeping gene sequences. DNA-DNA hybridization results between strain DHG54T and closely related Dyella species were all lower than 70 %. Ubiquinone-8 was the only respiratory quinone, and iso-C15 : 0, iso-C17 : 0 and iso-C17 : 1 ω9c were major fatty acids. The DNA G+C content of strain DHG54T was 65.4 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. On the basis of the polyphasic characterization results presented here, strain DHG54T represents a novel species of the genus Dyella, for which the name Dyellasolisilvae sp. nov. (type strain DHG54T=GDMCC 1.1187T = LMG 30091T) is proposed.

Structural characterisation and discrimination of the aerial parts of Paris polyphylla var. yunnanensis and Paris polyphylla var. chinensis by UHPLC-QTOF-MS coupled with multivariate data analysis.

INTRODUCTION: As sources of Rhizoma Paridis are facing shortages, utilising the aerial parts of Paris polyphylla has emerged as a promising additional source. However, the components in the aerial parts still need to be explored, and it is difficult to distinguish the aerial parts of P. polyphylla Smith var. yunnanensis (PPY) and P. polyphylla var. chinensis (PPC), two varieties of P. polyphylla. OBJECTIVE: This study aimed to establish a comprehensive platform to characterise steroid saponins from the aerial parts of PPY and PPC and to discriminate these two varieties. METHODOLOGY: A dereplication approach and ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) analysis were used for the characterisation of steroidal saponins in the aerial parts of PPY and PPC. Multivariate statistical analysis was performed to differentiate these two varieties and screen discriminant variables. In addition, a genetic algorithm-optimised for support vector machines (GA-SVM) model was developed to predict P. polyphylla samples. The distribution of steroidal saponins in PPY and PPC was visualised by a heatmap. RESULTS: A total of 102 compounds were characterised from the aerial parts of PPY and PPC by dereplication. A clear separation of PPY and PPC was achieved, and 35 saponins were screened as marker compounds. The established GA-SVM model showed excellent prediction performance with a prediction accuracy of 100%. CONCLUSIONS: Many steroid saponins that have been reported in Rhizoma Paridis also exist in the aerial parts of P. polyphylla. Samples from the aerial parts of PPY and PPC could be discriminated using our platform.

Dyella halodurans sp. nov., isolated from lower subtropical forest soil.

A Gram-stain-negative, aerobic, non-endospore-forming, motile by a polar flagellum, rod-shaped bacterium, designated strain DHOG02T, which produced yellow-pigmented colonies, was isolated from a soil sample collected from the lower subtropical forest of the Dinghushan Biosphere Reserve, Guangdong Province, PR China. Strain DHOG02T grew at 12-37 °C, pH 4-9 and 0-4 % (w/v) NaCl, with optima at 28 °C, pH 6-7 and 0.5 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that this strain formed a clade with Dyella lipolytica DHOB07T and Dyella jejuensis JP1T, with sequence similarities of 98.0 and 97.4 %, respectively. The result of the concatenated partial gyrB, lepA and recA gene sequence analysis confirmed that strain DHOG02T belongs to the genus Dyella, but is distinct from all currently known species of the genus. The G+C content of the genomic DNA was 62 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid and phospholipid. Ubiquinone-8 was the only respiratory quinone detected, and iso-C15 : 0, iso-C17 : 1ω9c and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) were the major fatty acids, all of which supported the affiliation of strain DHOG02T to the genus Dyella. On the basis of the evidence presented here, strain DHOG02T represents a novel species of the genus Dyella, for which the name Dyella halodurans sp. nov. is proposed. The type strain is DHOG02T (=NBRC 111474T=CGMCC 1.15435T).

Dyella caseinilytica sp. nov., Dyella flava sp. nov. and Dyella mobilis sp. nov., isolated from forest soil.

Three strains, DHOB09T, DHOC52T and DHON07T, were isolated from the forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. They were all Gram-stain-negative, aerobic, rod-shaped cells. The ranges (optimum) for the temperature, pH and NaCl concentration for growth of DHOB09T, DHOC52T and DHON07T were 10-42 (25-28) °C, pH 5.5-9.0 (7.0-7.5) and 0-4.0 (0-0.5) % (w/v); 10-42 (28) °C, pH 4.0-7.0 (4.5-6.5) and 0-2.0 (0) % (w/v) and 10-37 (25-28) °C, pH 4.0-7.5 (5.5-6.0) and 0-2.5 (0)  % (w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that DHOB09T, DHOC52T and DHON07T formed a phyletic cluster with seven species of the genus Dyella within the major clade of Dyella with sequence similarities ranged from 96.9 to 98.6 %. This indicated that the three strains may represent three novel species of the genus Dyella. This result was also strongly supported by the concatenated analysis of partial gyrB, lepA and recA gene sequences. DNA-DNA hybridization between strains DHON07T and DHOB09T, as well as DHON07T and Dyella koreensis BB4T was much lower than 70 %. The G+C content of strains DHOB09T, DHOC52T and DHON07T were 59.4, 60.7 and 59.5 %, respectively. The major fatty acids of the three strains were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0 and the predominant respiratory lipoquinone was ubiquinone-8. All of the physiological, phylogenetic and chemotaxonomic data showed that strains DHOB09T, DHOC52T and DHON07T are distinctive from each other and from all species of the genus Dyellawith validly published names. Therefore, we suggest that they represent three novel species of the genus, for which the names Dyella caseinilytica sp. nov. (type strain DHOB09T=CGMCC 1.15434T=LMG 29202T), Dyella flava sp. nov. (type strain DHOC52T=NBRC 111979T=KCTC 52128T) and Dyella mobilis sp. nov. (type strain DHON07T=CGMCC 1.15400T=NBRC 111475T) are proposed.

Puia dinghuensis gen. nov., sp. nov., isolated from monsoon evergreen broad-leaved forest soil.

A Gram-stain-negative, rod-shaped, aerobic, non-motile bacterial strain, 4GSH07T, was originally isolated from the monsoon evergreen broad-leaved forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China (112° 31' E 23° 10' N). It grew optimally at 28-33 °C and pH 4.0. The 16S rRNA gene sequence analysis showed that strain 4GSH07T had the highest sequence similarity of 94.0 % to Parasegetibacter terrae JCM 19942T, and formed an independent lineage separable from other described genera of the family Chitinophagaceae. The main fatty acids (>5 %) were iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 0 3-OH, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and C15 : 0 2-OH. The organism contained MK-7 as the predominant respiratory quinone, and the total DNA G+C content was 50.3 mol%. The phenotypic, chemotaxonomic and phylogenetic data showed consistently that strain 4GSH07T represents a novel species of a novel genus of the family Chitinophagaceae, for which the name Puia dinghuensis gen. nov., sp. nov., is proposed, with 4GSH07T (=CGMCC 1.15448T=LMG 29214T) as the type strain.

Dyella lipolytica sp. nov., a lipolytic bacterium isolated from lower subtropical forest soil.

A Gram-stain-negative, aerobic, yellow-pigmented, non-spore-forming, non-motile, rod-shaped bacterium, designated strain DHOB07T, was isolated from a soil sample collected from the lower subtropical forest of the Dinghushan Biosphere Reserve, Guangdong Province, PR China (23° 10' N 112° 31' E). Strain DHOB07T grew at 10-37 °C, pH 4-7 and 0-0.5 % (w/v) NaCl, with an optimum at 28 °C, pH 5-5.5 and 0% (w/v) NaCl on R2A medium. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain formed a clade with Dyella jejuensis JP1T, Dyella nitratireducens DHG59T, Dyella koreensis BB4T, Dyella marensis CS5-B2Tand Dyellasoli JS12-10T, with sequence similarities of 98.9, 98.0, 97.9, 97.9 and 97.8 %, respectively. Multilocus sequence analysis based on the concatenated sequences of partial housekeeping genes gyrB, lepA and recA confirmed that strain DHOB07T belongs to thegenus Dyella but is distinct from all currently known species of the genus Dyella. The G+C content of the genomic DNA was 58.2 mol%. The DNA-DNA relatedness value between strain DHOB07T and D. jejuensis JP1T was 41.8 %. Iso-C16 : 0, iso-C15 : 0 and iso-C17 : 1ω9c were the major fatty acids, and ubiquinone-8 was the only respiratory quinone detected, all of which supported the affiliation of strain DHOB07T to the genus Dyella. On the basis of the polyphasic characterization results presented above, strain DHOB07T represents a novel species of the genus Dyella, for which the name Dyella lipolytica sp. nov. is proposed. The type strain is DHOB07T (=NBRC 111473T=KCTC 52132T).

Dyella acidisoli sp. nov., D. flagellata sp. nov. and D. nitratireducens sp. nov., isolated from forest soil.

Bacterial strains 4M-Z03T, 4M-K16T and DHG59T were isolated from forest soil samples collected from the Dinghushan Biosphere Reserve, Guangdong Province, PR China (112° 31' E 23° 10' N). The three strains grew well at 28 °C, pH 5.0-6.0 on R2A medium. On the basis of 16S rRNA gene sequence analysis, the three strains, together with Dyella humi DHG40T, formed a distinct phyletic clade within the genus Dyella, and the sequence similarities between any strains of the clade ranged from 97.8 to 98.5 %. Sequence analysis of concatenated partial gyrB, lepA and recA gene sequences also strongly suggested that the three strains represented three novel species of the genus Dyella. The respiratory lipoquinone of the three strains was ubiquinone-8, and their DNA G+C content was 58.2-59.0 mol%. The fatty acid profiles differed substantially among these three strains, although they had two common major fatty acids, iso-C15 : 0 and iso-C17 : 1ω9c. The DNA-DNA relatedness among the three strains and the type strains of the closest species of the genus Dyella examined was lower than 50 %. The results of genotypic and phenotypic characterization presented above demonstrate that the three strains examined represent three novel species of the genus Dyella, for which the names Dyella acidisoli sp. nov. (type strain 4M-Z03T=NBRC 111980T=KCTC 52131T), Dyella flagellata sp. nov. (type strain 4M-K16T=NBRC 111981T=KCTC 52130T) and Dyella nitratireducens sp. nov. (type strain DHG59T=NBRC 111472T=LMG 29201T=CGMCC 1.15439T) are proposed.

Aliidongia dinghuensis gen. nov., sp. nov., a poly-ß-hydroxybutyrate-producing bacterium isolated from Pinus massoniana forest soil.

A Gram-stain-negative, aerobic, motile, rod-shaped bacterium, designated 7M-Z19T, was isolated from a soil sample collected from a Pinus massoniana forest of Dinghushan Biosphere Reserve, Guangdong Province, PR China. Strain 7M-Z19T grew at pH 4.5-7.5 (optimum pH 6.0-6.5), 10 to 37 °C (optimum 28 °C) and NaCl concentration up to 2.0 % (optimum 0 %, w/v). iso-C17 : 0, C18 : 1ω7c and C19 : 0ω8c cyclo were the major fatty acids (>10 %) while ubiquinone-10 was the only respiratory quinone detected in 7M-Z19T. The polar lipids of the strain consisted of phosphatidylethanolamine, phosphatidyldimethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, six unidentified aminophospholipids, three unidentified phospholipids, six unidentified lipids and a glycolipid. The DNA G+C content was 65.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate formed a distinct lineage with Dongia mobilis and Dongia rigui within the family Rhodospirillaceae, but with a low sequence similarity of 92.7 and 92.0 %, respectively. On the basis of phylogenetic, phenotypic, physiological and chemotaxonomic distinctiveness, strain 7M-Z19T should be placed in the family Rhodospirillaceae as a representative of a novel genus and species, for which the name Aliidongia dinghuensis gen. nov., sp. nov., is proposed. The type strain of the type species is 7M-Z19T (=NBRC 112240T=KCTC 52134T=CGMCC 1.15725T).

Triterpenoids-Enriched Extract from the Aerial Parts of Salvia miltiorrhiza Regulates Macrophage Polarization and Ameliorates Insulin Resistance in High-Fat Fed Mice.

Adipose tissue inflammation and macrophage polarization are tightly associated with the development of obesity-associated insulin resistance. Our previous studies have demonstrated the triterpenoids-enriched extract from the aerial parts of Salvia miltiorrhiza (TTE) could significantly improve atherosclerosis in LDLR-/- mice. However, its molecular mechanisms of TTE ameliorating insulin resistance remain unclear. In the present study, obesity model with insulin resistance induced by feeding high-fat diet (HFD) was established. Dietary TTE attenuated hyperlipidemia, improved glucose intolerance in mice and mediated the activation of IRS-1/PI3K/Akt insulin signaling pathway. Meanwhile, dietary TTE also attenuated macrophage infiltrations into adipose tissue and modified the phenotype ratio of M1/M2 macrophages. Furthermore, our results showed that TTE regulated the polarization of macrophages partly via adenosine monophosphate-activated kinase (AMPK). Taken together, these findings suggested that TTE has a potential clinical utility in improving insulin resistance. Its mechanisms might be contributed to its beneficial effects on macrophage polarization via AMPK. Copyright © 2016 John Wiley & Sons, Ltd.

Construction of A New Random Short Hairpin RNA Library Based on microRNA Context.

Objective To build an efficient random short hairpin RNA(shRNA)library. Methods shRNA expression vector was constructed with enhanced green fluorescent protein(EGFP)in the upstream of shRNA,driven by pol Ⅱ promoter(CMV).After the constructs were transfected into cells,the proteins were collected.The inhibition efficiency of shRNA was determined by Western blot and dual luciferase reporter system.After the shRNA expression vector was constructed with EGFP in the upstream of shRNA,driven by pol Ⅱ promoter(CMV),shRNA was further embedded into microRNA(miRNA)context.The constructs were transfected into cells,and then the inhibition efficiency of shRNA against target genes was evaluated by quantificational real-time polymerase chain reaction.According to the result of quantificational real-time polymerase chain reaction,a new random shRNA library was constructed based on miRNA context. Results shRNA downstream of a large transcript was transcripted efficiently by pol Ⅱ promoter(CMV).The efficiency of shRNA interference on target gene was improved when shRNA was embedded into miRNA context.Thus,we constructed a new random shRNA library sized 1.8×107 based on miRNA context.Conclusion We successfully constructed a new large random shRNA library.

Dinghuibacter silviterrae gen. nov., sp. nov., isolated from forest soil.

A novel Gram-stain negative, non-motile, rod-shaped, aerobic bacterial strain, designated DHOA34T, was isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, China. Comparative 16S rRNA gene sequence analysis showed that it exhibited highest similarity with Flavisolibacter ginsengiterrae Gsoil 492T and Flavitalea populi HY-50RT, at 90.89 and 90.83%, respectively. In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, DHOA34T formed an independent lineage within the family Chitinophagaceae but was distinct from all recognized species and genera of the family. The major cellular fatty acids of DHOA34T included iso-C15:0, anteiso-C15:0, iso-C17:0 3-OH and summed feature 3 (C16:1ω6c and/or C16:1ω7c). The DNA G+C content was 51.6 mol% and the predominant quinone was menaquinone 7 (MK-7). Flexirubin pigments were produced. The phenotypic, chemotaxonomic and phylogenetic data demonstrate consistently that strain DHOA34T represents a novel species of a new genus in the family Chitinophagaceae, for which the name Dinghuibacter silviterrae gen. nov., sp. nov. is proposed. The type strain of Dinghuibacter silviterrae is DHOA34T (=CGMCC 1.15023T=KCTC 42632T).

Paraburkholderiapallidirosea sp. nov., isolated from a monsoon evergreen broad-leaved forest soil.

A Gram-stain-negative, rod-shaped, aerobic and motile bacterial strain, DHOK13T, was isolated from the forest soils of Dinghushan Biosphere Reserve, Guangdong Province, PR China (112° 31' E, 23° 10' N). It grew optimally at 28-33 °C and pH 7.0-7.5. The main fatty acids were C16 : 0, C17 : 0 cyclo, C19 : 0 cycloω8c, summed feature 2 (C12 : 0 aldehyde and/or unknown 10.9525) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The organism contained ubiquinone Q-8 as the predominant isoprenoid quinone. The total DNA G+C content of strain DHOK13T was 62.0 mol%. Phylogenetic analysis of the 16S rRNA gene, as well as the sequence of the partial housekeeping genes, gyrB and recA, showed consistently that strain DHOK13T formed an independent cluster with Paraburkholderia phenazinium LMG 2247T. DNA-DNA hybridization studies showed relatively low relatedness values (39 %) of strain DHOK13T with P. phenazinium LMG 2247T. The phenotypic, chemotaxonomic and phylogenetic data showed that strain DHOK13T represents a novel species of the genus Paraburkholderia for which the name Paraburkholderia pallidirosea sp. nov. is proposed. The type strain is DHOK13T (=KCTC 42626T=LMG 28846T).

Dyella humi sp. nov., isolated from forest soil.

A Gram-stain-negative, aerobic, motile, rod-shaped, yellow-pigmented bacterium, designated as DHG40T, was isolated from a soil sample collected from the forest of Dinghushan Biosphere Reserve, Guangdong Province, China. Strain DHG40T grew at pH 4.0-8.0 and 10-37 °C (optimum at pH 6.0-7.0 and 25-28 °C). NaCl inhibited growth at concentrations above 2.5 % (w/v). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate fell within the cluster of the genus Dyella. Strain DHG40T was closely related to Dyella ginsengisoli Gsoil 3046T (97.6 % 16S rRNA gene sequence similarity), Dyella marensis CS5-B2T (97.5 %), Dyella koreensis BB4T (97.4 %) and Dyella jejuensis JP1T (97.4 %). The DNA-DNA relatedness values between strain DHG40T and its phylogenetically closest relatives were all below 40 %. The DNA G+C content was 60.3 mol%. In addition, iso-C15 : 0, iso-C16 : 0, iso-C17 : 0 and iso-C17 : 1ω9c were the major fatty acids (>10 %) and ubiquinone-8 was the respiratory quinone. The major polar lipids were phosphatidylethanolamine, phosphatidylmethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, an unidentified aminolipid and an unidentified phospholipid. On the basis of phylogenetic, phenotypic, physiological and chemotaxonomic distinctiveness, strain DHG40T should be placed in the genus Dyella as a representative of a novel species, for which the name Dyella humi sp. nov. is proposed. The type strain is DHG40T (=KCTC 42629T=LMG 28842T).

Edaphobacter dinghuensis sp. nov., an acidobacterium isolated from lower subtropical forest soil.

An aerobic bacterium, designated DHF9T, was isolated from a soil sample collected from the lower subtropical forest of Dinghushan Biosphere Reserve, Guangdong Province, PR China. Cells were Gram-stain-negative, non-motile, short rods that multiplied by binary division. Strain DHF9T was an obligately acidophilic, mesophilic bacterium capable of growth at pH 3.5-5.5 (optimum pH 4.0) and at 10-33 °C (optimum 28-33 °C). Growth was inhibited at NaCl concentrations above 2.0 % (w/v). The major fatty acids were iso-C15 : 0, C16 : 0 and C16 : 1ω7c. The polar lipids consist of phosphatidylethanolamine, two unidentified aminolipids, two unidentified phospholipids, two unidentified polar lipids and an unidentified glycolipid. The DNA G+C content was 57.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain belongs to the genus Edaphobacter in subdivision 1 of the phylum Acidobacteria, with the highest 16S rRNA gene sequence similarity of 97.0 % to Edaphobacter modestus Jbg-1T. Based on phylogenetic, chemotaxonomic and physiological analyses, it is proposed that strain DHF9T represents a novel species of the genus Edaphobacter, named Edaphobacter dinghuensis sp. nov. The type strain is DHF9T ( = DSM 29920T = CGMCC 1.12997T).