Ratiometric electrochemiluminescence sensing and intracellular imaging of ClO- via resonance energy transfer.

Electrochemiluminescence resonance energy transfer (ECL-RET) is a versatile signal transduction strategy widely used in the fabrication of chem/biosensors. However, this technique has not yet been applied in visualized imaging analysis of intracellular species due to the insulating nature of the cell membrane. Here, we construct a ratiometric ECL-RET analytical method for hypochlorite ions (ClO-) by ECL luminophore, with a luminol derivative (L-012) as the donor and a fluorescence probe (fluorescein hydrazide) as the acceptor. L-012 can emit a strong blue ECL signal and fluorescein hydrazide has negligible absorbance and fluorescence signal in the absence of ClO-. Thus, the ECL-RET process is turned off at this time. In the presence of ClO-, however, the closed-loop hydrazide structure in fluorescein hydrazide is opened via specific recognition with ClO-, accompanied with intensified absorbance and fluorescence signal. Thanks to the spectral overlap between the ECL spectrum of L-012 and the absorption spectrum of fluorescein, the ECL-RET effect is gradually recovered with the addition of ClO-. Furthermore, the ECL-RET system has been successfully applied to image intracellular ClO-. Although the insulating nature of the cell itself can generate a shadow ECL pattern in the cellular region, extracellular ECL emission penetrates the cell membrane and excites intracellular fluorescein generated by the reactions between fluorescein hydrazide and ClO-. The cell imaging strategy via ECL-RET circumvents the blocking of the cell membrane and enables assays of intracellular species. The importance of the ECL-RET platform lies in calibrating the fluctuation from the external environment and improving the selectivity by using fluorescent probes. Therefore, this ratiometric ECL sensor has shown broad application prospects in the identification of targets in clinical diagnosis and environmental monitoring.

In Situ Ultrasound Irradiation for Regulating the Electrochemiluminescence Intensity and Layer.

Electrochemiluminescence (ECL) has manifested a surface-confined emitting character and a low light background occurring near the electrode surface. However, the luminescence intensity and emitting layer are limited by the slow mass diffusion rate and electrode fouling in a stationary electrolyte. To address this problem, we developed an in situ strategy to flexibly regulate the ECL intensity and layer thickness by introducing an ultrasound (US) probe to the ECL detector and microscope. Herein, we explored the ECL responses and the thickness of ECL layer (TEL) under US irradiation in different ECL routes and systems. ECL microscopy with an ultrasonic probe discovered that ultrasonic radiation enhanced the ECL intensity under the catalytic route, while an opposite trend was observed under the oxidative-reduction route. Simulation results demonstrated that US promoted the direct electrochemical oxidation of TPrA radicals by the electrode rather than oxidant Ru(bpy)33+, which made the TEL thinner than that in the catalytic route under the same US condition. In situ US boosted the ECL signal from 1.2 times to 4.7 times by improving the mass transport and weakening electrode fouling due to the cavitation role. It significantly enhanced the ECL intensity beyond the diffusion-controlled ECL reaction rate. In addition, a synergistic sonochemical luminescence is validated in the luminol system to enhance the whole luminescence because cavitation bubbles of US promoted the generation of reactive oxygen species. This in situ US strategy provides a new opportunity to understand ECL mechanisms and a new tool in regulating TEL to meet the needs of ECL imaging.

Serum Krebs von den Lungen-6 concentrations reflect severity of anti-melanoma differentiation-associated protein 5 antibody positive dermatomyositis associated interstitial lung disease.

OBJECTIVES: Rapidly progressive interstitial lung disease (RP-ILD) is a major complication of anti-melanoma differentiation-associated protein 5 antibody positive dermatomyositis (anti-MDA5+DM) with a high mortality rate. The aim of the study is to determine whether serum Krebs von den Lungen-6 (KL-6) could be a prognostic biomarker to predict RP-ILD and prognosis in anti-MDA5+DM patients. METHODS: A total of 21 anti-MDA5+DM patients with RP-ILD and 20 anti-MDA5+DM patients without RP-ILD were retrospectively included in this study. Serum KL-6 concentration (pg/mL) was measured using the latex agglutination test. RESULTS: Serum KL-6 level was higher in RP-ILD patients than those in non-RR-ILD patients (1195.61±872.93 vs. 452.6±465.51 pg/mL, p=0.002). The best cut-off value of KL-6 serum level was 500.9 pg/mL using ROC curve (AUC area = 0.7976, p=0.0011). KL-6 >500.9 pg/mL was an independent risk factor for RP-ILD using multivariate analysis (OR=56.38, 95% CI 5.51-577.504, p=0.001). Serum KL-6 concentrations were significantly higher in dead patients than those in the survivor group (1209.34±840.55 vs. 592.41±667.76, p=0.0033), and higher KL-6 concentration was also an independent risk factor for all-cause death after adjusting confounders (OR = 21.94, 95% CI 3.3-145.73, p=0.001). Anti-MDA5+DM patients with higher KL-6 level displayed a significantly decreased one-year survival rate, as compared with lower KL-6 level (36.36% vs. 89.47%, p=0.0008). CONCLUSIONS: The serum KL-6 levels reflect severity of lung injury and serve as a clinically useful biomarker in detection and monitoring RP-ILD progression in anti-MDA5+DM patients.

Effect of metal ions on lipopeptide secretion from Bacillus subtilis strain FJAT-4: Negative regulation by Ca2.

AIMS: This study aimed to investigate the effect of metal ions on lipopeptide production by Bacillus subtilis strain FJAT-4 and the mechanism of negative regulation by Ca2+ . METHODS AND RESULTS: The quantitative measurement of lipopeptides in response to K+ , Na+ , Mg2+ and Ca2+ addition was carried out by LC-MS. The contents of fengycin and surfactin varied within the range of 116.24-129.80 mg/L and 34.03-63.11 mg/L in the culture media containing K+ , Na+ and Mg2+ , while the levels were 0.86 and 0.63 mg/L in the media containing Ca2+ . Ca2+ at a high concentration (45 mM) did not adversely affect the growth of strain FJAT-4, but caused significant downregulation of lipopeptide synthesis-related gene expression, corresponding to a decrease in lipopeptide production. This inhibition by Ca2+ was further investigated by proteomic analysis. In total, 112 proteins were upregulated and 524 proteins were downregulated in the presence of additional Ca2+ (45 mM). Among these differentially expressed proteins (DEPs), 28 were related to phosphotransferase activity, and 42 were related to kinase activity. The proteomics results suggested that altered levels of three two-component signal-transduction systems (ResD/ResE, PhoP/PhoR and DegU/DegS) might be involved in the control of expression of the fen and srfA operons of FJAT-4 under high calcium stress. CONCLUSIONS: The Ca2+ at the high concentration (45 mM) triggers a decrease in lipopeptide production, which might be attributed to the regulation of three two-component signal-transduction systems ResD/ResE, PhoP/PhoR and DegU/DegS. SIGNIFICANCE AND IMPACT OF THE STUDY: The regulatory effect of calcium on the expression of genes encoding lipopeptide synthetases can be applied to optimize the production of lipopeptides.

Self-Assembled Micelles Improve the Oral Bioavailability of Dihydromyricetin and Anti-Acute Alcoholism Activity.

Dihydromyricetin (DMY) is highly effective in counteracting acute alcohol intoxication. However, its poor aqueous solubility and permeability lead to the low oral bioavailability and limit its clinic application. The aim of this work is to use Solutol®HS15 (HS 15) as surfactant to develop novel micelle to enhance the oral bioavailability of DMY by improving its solubility and permeability. The DMY-loaded Solutol®HS15 micelles (DMY-Ms) were prepared by the thin-film hydration method. The particle size of DMY-Ms was 13.97 ± 0.82 nm with an acceptable polydispersity index of 0.197 ± 0.015. Upon entrapped in micelles, the solubility of DMY in water was increased more than 25-fold. The DMY-Ms had better sustained release property than that of pure DMY. In single-pass intestinal perfusion models, the absorption rate constant (Ka) and permeability coefficient (Papp) of DMY-Ms were 5.5-fold and 3.0-fold than that of pure DMY, respectively. The relative bioavailability of the DMY-Ms (AUC0-∞) was 205% compared with that of pure DMY (AUC0-∞), indicating potential for clinical application. After administering DMY-Ms, there was much lower blood alcohol level and shorter duration of the loss of righting relax (LORR) in drunk animals compared with that treated by pure DMY. In addition, the oral administration of DMY-Ms greatly reduced oxidative stress, and significantly defended liver and gastric mucosa from alcoholic damages in mice with alcohol-induced tissue injury. Taken together, HS 15-based micelle system greatly improves the bioavailability of DMY and represents a promising strategy for the management of acute alcoholism. Graphical abstract.

Biocontrol of tomato bacterial wilt by the new strain Bacillus velezensis FJAT-46737 and its lipopeptides.

BACKGROUND: There is an urgent need to discover biocontrol agents to control bacterial wilt. This study reports on a new lipopeptide-producing biocontrol strain FJAT-46737 and explores its lipopeptidic compounds, and this study investigates the antagonistic effects of these compounds. RESULTS: Based on a whole genome sequence analysis, the new strain FJAT-46737 was identified as Bacillus velezensis, and seven gene clusters responsible for the synthesis of bioactive secondary metabolites in FJAT-46737 were predicted. The antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against three bacteria and three fungi. Pot experiments showed that the control efficiencies for tomato bacterial wilt of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was the secretion of lipopeptides consisting of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities against several pathogens (including Ralstonia solanacearum, Escherichia coli and Fusarium oxysporum) and fengycins were the major antibacterial components of the lipopeptides against R. solanacearum in vitro. Furthermore, the rich organic nitrogen sources (especially yeast extracts) in the media promoted the production of fengycin and surfactin by FJAT-46737. The secretion of these two lipopeptides was related to temperature fluctuations, with the fengycin content decreasing by 96.6% and the surfactins content increasing by 59.9% from 20 °C to 40 °C. The optimal temperature for lipopeptide production by FJAT-46737 varied between 20 °C and 25 °C. CONCLUSIONS: The B. velezensis strain FJAT-46737 and its secreted lipopeptides could be used as new sources of potential biocontrol agents against several plant pathogens, and especially the bacterial wilt pathogen R. solanacearum.

Strong inhibitory activities and action modes of lipopeptides on lipase.

Lipopeptides have been reported to exhibit anti-obesity effects. In this study, we obtained a Bacillus velezensis strain FJAT-52631 that could coproduce iturins, fengycins, and surfactins. Results showed that the FJAT-52631 crude lipopeptide, purified fengycin, iturin, and surfactin standards exhibited strong inhibition activities against lipase with dose-dependence manners (half maximal inhibitory concentration (IC50) = 0.011, 0.005, 0.056, and 0.005 mg/mL, respectively). Moreover, fengycin and surfactin had the comparable activities with orlistat, but iturin not. It was revealed that the inhibition mechanism and type of the lipopeptides were reversible and competitive. The quenching mechanism of lipase was static and only one binding site between lipase and lipopoeptide was inferred from the fluorescence analysis. The docking analysis displayed that fengycin and surfactin could directly interact with the active amino acid residues (Ser or Asp) of lipase, but not with iturin. Our work suggests that the B. velezensis lipopeptides would have great potential to act as lipase inhibitors.

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure.

Cry2Ab, a pore-forming toxin derived from Bacillus thuringiensis, is widely used as a bio-insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250-kDa prepore oligomer. Site-directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.

Bacterial community diversity associated with the severity of bacterial wilt disease in tomato fields in southeast China.

Tomato bacterial wilt caused by Ralstonia solanacearum is a devastating plant disease. The aims of this study were to investigate the relationship among soil nutrients, rhizobacterial community, and abundance of R. solanacearum, and to gather useful information for controlling the disease. Fifteen tomato rhizosphere soils were collected from three regions, encompassing five disease grades. Then, soil physicochemical properties and rhizobacterial communities were investigated. The content of soil organic carbon (SOC), total phosphorus (TP), total potassium (TK), and exchangeable calcium was significantly higher in the healthy plant rhizosphere soils than in diseased plant rhizosphere soils (P < 0.05). The healthy soils had a relatively higher abundance of Proteobacteria and a lower abundance of Acidobacteria than the diseased soils from the same region. Redundancy analysis demonstrated that R. solanacearum abundance was positively correlated with total nitrogen content and negatively correlated with soil pH, SOC, TP, TK, and exchangeable calcium. Ralstonia solanacearum abundance correlated positively with Chloroflexi, Acidobacteria, and Planctomycetes abundance but negatively with Nitrospirae, Bacteroidetes, and Proteobacteria abundance. These results suggested that improving soil pH, applying the amount of P and K fertilizers, and controlling the dosage of N fertilizer might be an effective approach in controlling bacterial wilt disease.

Modified xanthan gum for crystal violet uptake: kinetic, isotherm, and thermodynamic behaviors.

Modified xanthan gum (XG-AM-TTE) was employed as an adsorbent to study the adsorption behavior, thermodynamics and kinetics of crystal violet (CV) from an aqueous solution. Fourier transform infrared spectroscopy analysis indicates that the functional groups present in the adsorbent, such as carboxyl, ester and hydroxyl groups, are included on the external surface of the material, and these groups are potential active sites for interaction with CV. According to X-ray diffraction results, the structure of XG-AM-TTE after CV adsorption became more disordered, and the microstructure change is an indication of effective adsorption of CV to the surface, with CV becoming remarkably dispersed in the adsorbent according to the scanning electron microscopy observations. The adsorption kinetics and adsorption equilibrium were best described by the pseudo-second-order model and Freundlich isotherms, respectively. The thermodynamic parameters, as the Gibbs-free energy (ΔG), enthalpy (ΔH) and entropy (ΔS), indicated that the adsorption is a spontaneous, endothermic and entropy increase process. The maximum adsorption capacity of XG-AM-TTE was 183 ± 12 mg/g, suggesting that XG-AM-TTE is an efficient adsorbent.

Bacillus populi sp. nov. isolated from Populus euphratica rhizosphere soil of the Taklamakan desert.

A rod-shaped, endospore-forming, aerobic bacterium, designated FJAT-45347T, was isolated from rhizosphere soil collected from the Taklamakan desert in Xinjiang (PR China). Growth was observed at 15-35 °C (optimum 25 °C), in 0 % and 20.0 % NaCl (optimum 8.0 %) and at pH 7.5-12.0 (optimum 8.0), respectively. The cell-wall peptidoglycan contained meso-diaminopimelic acid and the isoprenoid quinone was MK-7. The main fatty acids were iso-C15 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analysis based on 16S rRNA gene sequences affiliated FJAT-45347T to the genus Bacillus, and it showed the highest sequence similarities to Bacillus clarkii DSM 8720T (96.1 %). The average nucleotide identity and in silico DNA-DNA hybridization values between FJAT-45347T and the most closely related species were 68.5 and 26.2 %, respectively, which were lower than the thresholds commonly used to define species (96 and 70 %, respectively), indicating that it represented a member of a different taxon. The DNA G+C content was 40.6 mol%. The phenotypic characters and taxono-genomics study revealed that FJAT-45347T represents a novel species of the genus Bacillus, for which the name Bacilluspopuli sp. nov. is proposed. The type strain is FJAT-45347T (=DSM 104632T=CCTCC AB 2016257T).

Invasive properties of Ralstonia solanacearum virulent and avirulent strains in tomato roots.

Ralstonia solanacearum causes bacterial wilt disease in a wide range of host plants, including tomato. To elucidate the invasion pattern of R. solanacearum, one avirulent strain, FJAT-1458, and one virulent strain, FJAT-91, were characterized according to their colonization and interaction with host in tomato roots. Both strains colonized in tomato roots. The highest colonization numbers were 1.66 × 108 cfu g-1 by FJAT-91 at 5 days (d) after inoculation and 1.09 × 108 cfu g-1 by FJAT-1458 at 6 d after inoculation. Infection with FJAT-91 caused tomato plant wilt with a disease index of 23.65% at 3 d after inoculation and 100% at 6 d after inoculation, and infection with FJAT-1458 did not cause plant wilt. Compared with FJAT-1458, infection with FJAT-91 reduced elongation of tomato roots, induced serious browning, and overflowed bacteria during the late stages of infection. Examination of cellular structure showed that infection with FJAT-1458 did not have obvious destructive effects on plant cells, while FJAT-91 induced a series of cytopathological changes, including swelling of mitochondria, degeneration of cytoplasm and nuclear heterochromatin, and collapse of host cells, which eventually resulted in the death of the host plant. The cytopathological changes appeared from the second to the fourth disease stages.

Bacillus wudalianchiensis sp. nov., isolated from grass soils of the Wudalianchi scenic area.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium, designated FJAT-27215T, was isolated from grass soil collected from Wudalianchi in the Heilongjiang Province of China. Growth was observed at 10-60 °C (optimum 30 °C), in 0 and 3.0 % NaCl (optimum 0 %) and at pH 5.0-10.0 (optimum 7.0), respectively. The cell-wall peptidoglycan contained meso-diaminopimelic acid and the isoprenoid quinone was MK7. The main fatty acids were iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0, and iso-C16 : 0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidyl ethanolamine. Phylogenetic analysis based on 16S rRNA gene sequences affiliated strain FJAT-27215T to the genus Bacillus. Strain FJAT-27215T showed high sequence similarities to Bacillus encimensis SGD-V-25T (98.6 %), Bacillus badius NBRC 15713T (98.6 %), Domibacillus indicus SD111T (96.9 %) and Bacillus thermotolerans SgZ-8T (96.5 %). The average nucleotide identity values between strain FJAT-27215T and the type strains of closely related species were much lower than the 96 % threshold value for delineation of genomic prokaryotic species. The in silico DNA-DNA hybridization values between strain FJAT-27215T and the most closely related strain B. encimensis SGD-V-25T showed a similarity of 22.4 % and lower than 70 %, indicating that they belong to different taxa. The phenotypic characters and taxono-genomics study revealed that strain FJAT-27215T represents a novel Bacillus species, for which the name Bacillus wudalianchiensis sp. nov. is proposed. The type strain is FJAT-27215T (=CCTCC AB 2015266T=DSM 100757T).

Bacillus praedii sp. nov., isolated from purplish paddy soil.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium, designated strain FJAT-25547T, was isolated from the purplish paddy soil collected from Linshan Township, Yanting Prefecture of Sichuan Province in PR China (31° 16' N 105° 27' E). Growth was achieved aerobically at temperatures between 15 and 40 °C (optimum 30 °C), with between 0 and 10.0 % NaCl (w/v) (optimum 4 %) and in the range of pH 5.0-12.0 (optimum pH 9.0). The cell-wall peptidoglycan contained meso-diaminopimelic acid, and the main isoprenoid quinone was MK-7. The major fatty acids were iso-C15 : 0 (55.4 %), anteiso-C15 : 0 (22.2 %), iso-C16 : 0 (5.1 %) and iso-C14 : 0 (6.5 %). The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain FJAT-25547T was a member of the genus Bacillus and was most closely related to Bacillus horneckiae DSM 23495T (97.7 % similarity), Bacillus eiseniae A1-2T (97.5 %), Bacillus mesophilum IITR-54T (97.2 %) and Bacillus kochii WCC 4582T (97.0 %). The average nucleotide identity value between strain FJAT-25547T and the type strain of the most closely related species, B. horneckiae DSM 23495T, was 77.7 %, less than the proposed cut-off value of 96.0 % for differentiating species within the genus. The in silico DNA-DNA hybridization value of strain FJAT-25547T with the most closely related species was 22.7 %, <70 %, again indicating they belong to different taxa. The DNA G+C content of strain FJAT-25547T was 39.1 mol%. This taxono-genomics study revealed that strain FJAT-25547T represents a novel species of the genus Bacillus for which the name Bacillus praedii sp. nov. (type strain FJAT-25547T=CCTCC AB 2015208T=DSM 101002T) is proposed.

Rapid differentiation of Ralstonia solanacearum avirulent and virulent strains by cell fractioning of an isolate using high performance liquid chromatography.

Ralstonia solanacearum is one of the most destructive plant bacterial pathogens worldwide. The population dynamics and genetic stability are important issues, especially when an avirulent strain is used for biocontrol. In this study, we developed a rapid method to differentiate the virulent and avirulent strains of R. solanacearum and to predict the biocontrol efficiency of an avirulent strain using high performance liquid chromatography (HPLC). Three chromatographic peaks P1, P2 and P3 were observed on the HPLC spectra among 68 avirulent and 28 virulent R. solanacearum strains. Based on the HPLC peaks, 96 strains total were assigned to three categories. For avirulent strains, the intense peak is P1, while for virulent strains, P3 is the majority. Based on the HLPC spectra of R. solanacearum strains, a chromatography titer index (CTI) was established as CTIi = Si/(S1+S2+S3) × 100% (i represents an individual HPLC peak; S1, S2 and S3 represent peak areas of P1, P2 and P3, respectively). The avirulent strains had high values of CTI1 ranging from 63.6 to 100.0%, while the virulent strains displayed high values of CTI3 ranging from 90.2 to 100.0%. Biological inoculation studies of 68 avirulent strains revealed that the biocontrol efficacy was the best when CTI1 = 100%. The purity and genetic stability of R. solanacearum strains were confirmed in the P1 fraction of avirulent strain FJAT-1957 and P3 fraction of virulent strain FJAT-1925 after 30 generations of consecutive subculture. These results confirmed that fractioning by HPLC and their deduced CTI can be used for rapid and efficient evaluation and prediction of an isolate of R. solanacearum. To the best of our knowledge, this is the first report that HPLC fractioning can be used for rapid differentiation of virulent and avirulent strains of R. solanacearum.

Bacillus loiseleuriae sp. nov., isolated from rhizosphere soil from a loiseleuria plant.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium, designated strain FJAT-27997T, was isolated from the rhizosphere soil of a Loiseleuria plant collected from Sichuan province in China. Growth was observed aerobically between 20 and 35 °C (optimum 30 °C), between 0 and 3.0 % (w/v) NaCl (optimum at 0 %) concentration and pH in the range 6.0-9.0 (optimum at pH 7.0). The cell-wall peptidoglycan contained meso-diaminopimelic acid and the major isoprenoid quinone was menaquinone MK-7. The main fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C14 : 0, C16 : 0 and C14 : 0. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analyses based on 16S rRNA gene sequences showed that isolate FJAT-27997T was a member of the genus Bacillus and was related most closely to Bacillus simplex DSM 1321T (97.95 % similarity), followed by Bacillushuizhouensis GSS03T (97.9 %). The average nucleotide identity value between strain FJAT-27997T and the most closely related species, B. simplex DSM 1321T, was 71.60 % (JSpecies), less than the previously proposed cut-off value of 96 % for differentiating species within the genus. The in silico DNA-DNA hybridization values between strain FJAT-27997T and its most closely related species were <70 %, again indicating they belong to different taxa. The main fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The novel strain could be differentiated from other known Bacillus species on the basis of several phenotypic characters and fatty acid profiles. This taxononomic/genomic study revealed that strain FJAT-27997T represents a novel Bacillus species, for which the name Bacillus loiseleuriae sp. nov. (type strain FJAT-27997T =CCTCC AB 2015285T=DSM 101776T) is proposed.

Effects of Dietary Glucose on Serum Estrogen Levels and Onset of Puberty in Gilts.

Metabolic signals and the state of energy reserves have been shown to play a crucial role in the regulation of reproductive function. This study was carried out to investigate the effects of dietary glucose levels on puberty onset in gilts. Weight-matched, landrace gilts (n = 36) 162±3 days old, weighing about 71.05±4.53 kg, were randomly assigned to 3 dietary treatment groups of 12 gilts each. The trial lasted until the onset of puberty. Gilts in each group were supplied with diets containing different levels of glucose as follows: i) starch group (SG) was free of glucose, contained 64% corn derived starch; ii) low-dose group (LDG) contained 19.2% glucose and 44.8% corn derived starch; iii) high-dose group (HDG) contained 30% glucose and 30% corn derived starch. Results indicated: i) The growth performance of gilts were not affected by the addition of glucose, but the age of puberty onset was advanced significantly (p<0.05); ii) Compared with the SG, the concentration of insulin significantly increased before puberty in HDG (p<0.05); iii) There was no difference in serum progesterone (P) levels amongst the different feed groups, however, levels of estradiol (E2), luteinizing hormone, and follicle-stimulating hormone were significantly higher at puberty onset in HDG (p<0.05). Overall, our findings indicate that glucose supplementation significantly advances puberty onset, which can have practical purposes for commercial breeding.

Bacillus wuyishanensis sp. nov., isolated from rhizosphere soil of a medical plant, Prunella vulgaris.

A Gram-staining-positive, rod-shaped, endospore-forming, aerobic bacterium (FJAT-17212(T)) was isolated from the rhizosphere soil of a medical plant, Prunella vulgaris (common selfheal), on the Wuyishan mountain of China. Isolate FJAT-17212(T) grew at 10-50 °C (optimum 30 °C), pH 5-11 (optimum pH 7) and with 0-6% (w/v) NaCl (optimum 2%). Phylogenetic analyses based on 16S rRNA gene sequences showed that isolate FJAT-17212(T) was a member of the genus Bacillus and was most closely related to Bacillus galactosidilyticus DSM 15595(T) (97.3%). DNA-DNA relatedness between isolate FJAT-17212(T) and B. galactosidilyticus DSM 15595(T) was low (35.2% ± 2.3). The diagnostic diamino acid of the peptidoglycan of isolate FJAT-17212(T) was meso-diaminopimelic acid and the predominant isoprenoid quinone was MK-7 (80.8%). The major cellular fatty acids were iso-C15 : 0 (35.7%), anteiso-C15 : 0 (29.8%), iso-C14 : 0 (9.9%) and iso-C16 : 0 (9.9%) and the DNA G+C content was 39.8 mol%. Phenotypic, chemotaxonomic and genotypic properties clearly indicated that isolate FJAT-17212(T) represents a novel species within the genus Bacillus, for which the name Bacillus wuyishanensis sp. nov. is proposed. The type strain is FJAT-17212(T) ( = DSM 27848(T) = CGMCC 1.12709(T)).

A coronary artery disease-associated SNP rs6903956 contributed to asymptomatic hyperuricemia susceptibility in Han Chinese.

BACKGROUND: To investigate the association of a coronary artery disease (CAD) risk SNP rs6903956 with asymptomatic hyperuricemia (aHU) susceptibility in Han Chinese. METHODS: Two hundred and twenty one patients with aHU and 447 healthy controls were recruited for this study. SNP rs6903956 were genotyped using TaqMan probe. RESULTS: The overall genotype and allele frequency distribution of the rs6903956 showed significant difference between aHU cases and controls (p<0.001 for genotype and allele, respectively). AA genotype of rs6903956 was significantly associated with aHU (OR=8.672, 95% CI 2.811-26.753, p<0.001) in our Han Chinese aHU cohort. Multivariate logistic regression analysis indicated that rs6903956 might be an independent risk factor for aHU susceptibility (OR=10.642 [2.671-42.400], p=0.001 for codominant model and OR=9.205 [2.336-36.280], p=0.002 for recessive model) after adjustment for some well- known CAD risk factors including age, gender, body mass index, smoking, hypertension, diabetes mellitus, abnormal glycometabolism, lipid abnormality and alcohol intake. No significant genotype-specific difference in uric acid levels was observed in aHU patients and controls. CONCLUSIONS: Our findings are the first to establish a genetic link of a CAD-associated rs6903956 with aHU in a Han Chinese population, providing the genetic evidence to support the close relationship between hyperuricemia and CAD.

Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.

Bacillus thuringiensis (Bt) strain FJAT-12 was a novel Bt strain isolated by Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Science. In this study, a new cry2Ab gene was cloned from Bt strain FJAT-12 and named as cry2Ab30 by Bt delta-endotoxin Nomenclature Committee. The sequencing results showed there were two mutations in conservative sites which led to two amino acids modification. Homology modeling indicated that the two changes were located in ß-sheet of Domain II. A prokaryotic expression vector pET30a-cry2Ab30 was constructed and the expressed protein was analyzed by western blot using Cry2Ab antibody. The expression conditions including IPTG concentration, revolution and temperature were optimized to get the highest expression level by SDS-PAGE and BandScan. The bioassay results also showed that the Cry2Ab30 toxin had high insecticidal activity against Plutella xylostella and the LC50 value was 0.0103 µg.mL(-1). The two mutations in ß-sheet of Domain II might contribute to insecticidal activity of Cry2Ab30 toxin against Plutella xylostella.