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Antimicrobials, Resistance and Chemotherapy
Natural compounds as antiquorum sensing agents View all 4 Articles
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Front. Microbiol., 13 May 2015 | https://doi.org/10.3389/fmicb.2015.00420
Sub-MICs of Mentha piperita essential oil and menthol inhibits AHL mediated quorum sensing and biofilm of Gram-negative bacteria
Fohad M. Husain1,2*, Iqbal Ahmad1*, Mohammad S. Khan1, Ejaz Ahmad3, Qudisa Tahseen4, Mohd Shahnawaz Khan5 and Nasser A. Alshabib2
1Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, India
2Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia
3School of Pharmaceutical Sciences, Sao Paulo State University, Araraquara, Brazil
4Department of Zoology, Aligarh Muslim University, Aligarh, India
5Department of Biochemistry, Protein Research Chair, College of Science, King Saud University, Riyadh, Saudi Arabia
Bacterial quorum sensing (QS) is a density dependent communication system that regulates the expression of certain genes including production of virulence factors in many pathogens. Bioactive plant extract/compounds inhibiting QS regulated gene expression may be a potential candidate as antipathogenic drug. In this study anti-QS activity of peppermint (Mentha piperita) oil was first tested using the Chromobacterium violaceum CVO26 biosensor. Further, the findings of the present investigation revealed that peppermint oil (PMO) at sub-Minimum Inhibitory Concentrations (sub-MICs) strongly interfered with acyl homoserine lactone (AHL) regulated virulence factors and biofilm formation in Pseudomonas aeruginosa and Aeromonas hydrophila. The result of molecular docking analysis attributed the QS inhibitory activity exhibited by PMO to menthol. Assessment of ability of menthol to interfere with QS systems of various Gram-negative pathogens comprising diverse AHL molecules revealed that it reduced the AHL dependent production of violacein, virulence factors, and biofilm formation indicating broad-spectrum anti-QS activity. Using two Escherichia coli biosensors, MG4/pKDT17 and pEAL08-2, we also confirmed that menthol inhibited both the las and pqs QS systems. Further, findings of the in vivo studies with menthol on nematode model Caenorhabditis elegans showed significantly enhanced survival of the nematode. Our data identified menthol as a novel broad spectrum QS inhibitor.
Emergence and spread of antibiotic resistance among pathogenic bacteria represents a major obstacle in treatment of infectious diseases. About 80% of the infections caused by microorganisms are biofilm based (Davies, 2003). Biofilm architecture consists of structured and aggregated communities of bacteria encased in a self-secreted exopolymeric substance (EPS; Costerton et al., 1995). Several studies have revealed that bacteria have developed resistance because of the prolonged treatment with conventional antibiotics possessing a broad-range efficacy via toxic or growth-inhibitory effects on target organisms rendering the traditional antibiotic treatment virtually ineffective (Xavier and Bassler, 2003). It has been found that bacteria living in the biofilm mode of growth are resistant to antibiotic up to 1000 times more than their planktonic counterparts (Caraher et al., 2007). Therefore, there is an increased demand for developing alternative strategies to the conventional antibiotic therapy (Zeng et al., 2008). Bacterial quorum sensing (QS) has been identified as a promising anti-infective drug target (Hentzer et al., 2002). It would be possible to repress the expression of QS regulated phenotypes through which the development of biofilm and virulence are being accomplished in many Gram-negative bacterial pathogens; such interference is expected to be useful in the treatment of bacterial infections (Hentzer and Givskov, 2003; March and Bentley, 2004). The QS mechanism enables bacteria to detect their population density through the production, release, and perception of small diffusible molecules called autoinducers and to coordinate gene expression accordingly (Williams et al., 2007; Rumbaugh et al., 2012).A wide array of functions in bacteria ranging from bacterial cell motility to complex behaviors such as biofilm formation and production of virulence factors are regulated by QS in pathogenic bacteria (Atkinson et al., 2006; Rumbaugh and Armstrong, 2014). Several Gram-negative pathogens employ N-acyl homoserine lactones (AHLs)-mediated QS systems to coordinate and synchronize specific gene expression of particular phenotypic features between the individual cells (Fuqua et al., 1994).
In Pseudomonas aeruginosa, biofilm formation and the production of various virulence factors are regulated via the action of a hierarchical quorum-sensing system mediated by the two chemically different classes of signal molecules, the N-acylhomoserine lactones and the 4-quinolones which comprise of more than 50 compounds and includes the most active signal molecule 2-heptyl-3-hydroxy-4-quinolone which is popularly known as the pseudomonas quinolone signal (PQS; Häussler and Becker, 2008; Heeb et al., 2011).
Quorum sensing plays an important role during the initial event of infection for the common opportunistic Gram-negative human pathogen P. aeruginosa, which is associated with nosocomial and wound infections, immunocompromised (Obritsch et al., 2005; LaSarre and Federle, 2013) and the genetically inherited disease cystic fibrosis (Fothergill et al., 2007). Therefore, compounds that interfere with the QS system to attenuate bacterial pathogenicity are termed as anti-QS compounds. Such compounds neither kill the bacteria nor stop their growth and are less expected to develop resistance toward antibiotics.
Various compounds both synthetic and natural from algae, fungi, and other organisms have been reported with quorum sensing inhibitory (QSI) activity, but they have little or no therapeutic value due to their unstable nature or toxicity associated with them (Rasmussen and Givskov, 2006). Therefore efforts have been directed in search of safe and effective QSI compounds from natural products particularly from medicinal plants, edible vegetables, spices and fruits, marine sponges and seaweeds (Zahin et al., 2010; Husain and Ahmad, 2013; Kalia, 2013).
Previously, we had screened 21 commonly available essential oils for their anti-QS activity using biosensor strains, Chromobacterium violaceum CV12472 and CVO26. The anti-QS activity of four essential oils namely cinnamon, lavender, clove, and peppermint was detected. Interestingly, most promising anti-QS activity against both C. violaceum pigment production and swarming motility of P. aeruginosa was demonstrated by clove oil at sub-Minimum Inhibitory Concentration (sub-MIC; Khan et al., 2009). Similarly, inhibition of QS signals by oils of rose, geranium, rosemary, and lavender has also been reported (Szabo et al., 2010; Yap et al., 2014).
Considering the encouraging results of the primary screening on peppermint oil (PMO; Hi-media, Mumbai, India), we have investigated its anti-pathogenic potential and its major phytoconstituent menthol (Hi-media, Mumbai, India), in three test organisms (C. violaceum, P. aeruginosa, and Aeromonas hydrophila). As above test strains use one or more different types of autoindiceer molecules activity all three organism may provide a strong basis for selection of broad spectrum anti-QS compounds. Both PMO and its major constituent menthol were studied for their efficacy in vitro. Further efficacy of major active component, Menthol was evaluated in Caenorhabditis elegans model to uncover the therapeutic potential of menthol as effective QS inhibitor.
Bacterial Strains and Growth Conditions
Bacterial strains used in this study were Chromobacterium violaceum CV026 (a mini-Tn5 mutant of C. violaceum 31532 that cannot synthesize its own AHL but responds to exogenous C4 and C6 AHLs), P. aeruginosa PAO1 (C4 and 3-oxo-C12 HSL producer, McLean et al., 2004), Escherichia coli pEAL08-2 (E. coli DH5α harboring plasmid pEAL08-2, Cugini et al., 2007), and E. coli MG4/pKDT17 (E. coli DH5α harboring plasmid pMG4/pKDT, Pearson et al., 1994). All the bacterial strains were grown in Luria-Bertani (LB) medium at 30°C for 24 h. When required, the medium for C. violaceum CV026 was supplemented with hexanoyl homoserine lactone (C6-HSL; Sigma–Aldrich, St Louis, MO, USA).
Determination of Minimum Inhibitory Concentration
Minimum Inhibitory Concentration of the test agents were determined against C. violaceum CVO26, P. aeruginosa PAO1, and A. hydrophila WAF38 by broth macrodilution method (Clinical and Laboratory Standards Institute [CLSI], 2007). Sub-MICs were selected for the assessment of anti-virulence and anti-biofilm activity in the above test strains.
Quantitative Estimation of Violacein
Extent of violacein production by C. violaceum (CVO26) in the presence and absence of Sub-MICs of test agents was studied by extracting violacein and quantifying photometrically using method of Blosser and Gray (2000) with little modifications (Packiavathy et al., 2012). Briefly, 1-ml culture from each flask was centrifuged at 13000 rev/min for 10 min to precipitate the insoluble violacein. The culture supernatant was discarded and 1 ml of DMSO was added to the pellet. The solution was vortexes vigorously for 30 s to completely solubilize violacein and centrifuged at 13000 rev/min for 10 min to remove the cells. Two hundred microlitres of the violacein-containing supernatants were added to 96-well flat bottomed microplates (Polylab, India), four wells per each solution, and the absorbance was read with a microplate reader (Thermo Scientific Multiskan Ex, India) at a wavelength of 585 nm. Reduction in the production of pigment in the presence of test agents was measured in terms of % inhibition as, [(OD of control – OD of treated)/OD of control] × 100.
Effect on Virulence Factor Production
Effect of sub-MICs of PMO and menthol on virulence factors of P. aeruginosa and A. hyrophila such as LasB elastase, protease, pyocyanin, chitinase, swarming motility, EPS extraction, and quantification was assessed as described previously (Husain et al., 2013).
Assay for Biofilm Inhibition
The effect of PMO and menthol on biofilm formation was measured using the microtitre plate assay (O'Toole and Kolter, 1998). Briefly, 1% overnight cultures (0.4 OD at 600 nm) of test pathogens were added into 1 mL of fresh LB medium in the presence and the absence of sub-MICs of test agents. Bacteria were allowed to adhere and grow without agitation for 24 h at 30°C. After incubation, microtitre plate was emptied by removing the media along with free-floating planktonic cells and the wells were gently rinsed twice with sterile water. The surface-attached cells (biofilm) were stained with 200 μL of 0.1% crystal violet (CV; Hi-media, Mumbai, India) solution. After 15 min, CV solution was discarded completely and wells were filled with 200 μL of 95% ethanol to solubilize CV from the stained cells. The biofilm biomass was then quantified by measuring the absorbance at OD 470 nm in a microplate reader (Thermo Scientific Multiskan Ex, India).
Biofilms were grown on glass coverslips, in the treated and untreated cultures of PAO1. After 24 h incubation, the cover slips were rinsed with distilled water to remove planktonic cells and processed for scanning electron microscopy (SEM) examination as described by Husain et al. (2013).
Caenorhabditis elegans Survival Assay
The method described by Musthafa et al. (2012) was adopted to study the in vivo efficiency of sub-MIC of menthol in Caenorhabditis elegans here nematode model. Briefly, the young adult nematodes were infected with PAO1 in the 24-well microtitre plate and incubated at 25°C for 12 h. After incubation, C. elegans from the wells were washed thrice with M9 buffer to remove surface-bound bacteria. Around 10 infected worms were transferred to the wells of microtitre plate containing 10% LB broth in M9 buffer and incubated at 25°C with or without 800 μg/mL menthol treatment. The plate was scored for live and dead worms every 12 h for 4 days. To assess the toxicity if any of the oil, C. elegans with menthol was maintained.
GC–MS Analysis
The compositions of the PMO was analyzed by Perkin Elmer GC Autosystem XL and Turbomass with EI source using PE-Wax column (30 m × 0.25 mm i.d., film thickness 0.25 mm), carrier gas was helium with column head pressure 7 psi connected to data station. Temperature programming: 4 min at 60°C then using at 4°C min-1 to 200°C with hold time of 21 min, at 200°C, split ratio 1:50. The components were identified by comparing their retention times to those of authentic samples, as well as by comparing their mass spectra with those of Wiley 8 and NIST 05 Libraries described by Masada (1976). Quantitative data were obtained by the peak normalization technique using integrated FID response.
Molecular Docking Analysis
The protein data bank (PDB) structure 2UV0 of LasR was downloaded from Brookhaven Protein Databank for molecular docking of phytoconstituents obtained from Mentha piperita essential oil to natural autoinducer AHL binding site of LasR. The residues falling within 5 Å of the binding site were extracted and combined to define the binding site residues. From Pubchem database the SDF format for 3D structures of all the phytoconstituents Limonene (I); Menthone (II); Isomenthone (III); 1-Hydroxyoctane (IV); Isopulegol (V); Menthyl acetate (VI); Neoisomenthol (VII); 2-Isopropyl-5-methylcyclohexanol (VIII); Menthol (IX); Lavandulol (X); Piperitone (XI) as well as the natural autoinducer 3-oxo-C12-HSL or AHL (0) with their chemical identifier (CID) 22311; 26447; 6986; 957; 24585; 27867; 19244; 1254; 165675; 5464156; 6987; and 3246941, respectively, were downloaded. The whole LasR molecule was defined as the binding site residues. Molecular docking simulation of the phytoconstituents (inhibitors) to LasR was performed with Autodock Vina program (Trott and Olson, 2010). Autodock uses Lamarkian genetic algorithm to calculate the possible conformations of the ligand that binds to the protein. Here the flexible ligands were set to free in the binding clefts of LasR to dock in the most feasible way. The grid centers of 24.327, 11.9665, and 77.3765 with the grid sizes of 40.368, 46.223, and 56.579 for X, Y, and Z axes, respectively, were used for covering all the binding site residues. For docking simulations the exhaustiveness was set to 8. Best docked structures based on the binding energy scores (ΔG) were chosen for further analyses. The hydrogen bonding and hydrophobic interactions between ligand and protein were calculated by Accelrys DS Visualizer 2.0 (Accelrys Software Inc., 2012) while the Figure 2 were generated by PyMol version 0.99 (DeLano, 2002) and Accelrys DS Visualizer 2.0 (Accelrys Software Inc., 2012). To validate the LasR-inhibitor docking experiments, we have compared our docking results with the structures already available at the PDB where the the natural autoinducer AHL has been co-crystallized with LasR, as for example, 2UV0 (BottomLey et al., 2007). Here, all the ligands from the crystal structure have been removed and the AHL was re-docked.
The binding constants (Kb) for protein-ligand interactions were calculated from the obtained free energy changes of docking by using the following equation:
ΔG=−RTlnKb
where R is the gas constant (1.987 cal.mol-1.K-1) and T is 298 K (Ahmad et al., 2011).
Analysis of lasB and pqsA Transcriptional Activity in E. coli
Measurements of β-galactosidase luminescence in E. coli MG4/pKDT17 and E. coli pEAL08-2 was done by the adopting the method described by Pearson et al. (1994) and Cugini et al. (2007).
All experiments were performed in triplicates and the data obtained from experiments were presented as mean values and the difference between control and test were analyzed using student's t-test.
Minimum inhibitory concentration of PMO was determined to select the sub-MICs to study the effect on growth and inhibition of QS regulated functions. MIC of oil was found to be 0.6% v/v against C. violaceum CVO26, 6.4% v/v against P. aeruginosa PAO1, and 1.6% v/v against A. hydrophila WAF38. The MIC of menthol was found to be 1 mg/mL, 800 μg/mL, and 400 μg/mL against PAO1, CVO26, and WAF 38, respectively.
Effect of Peppermint Oil on Violacein Production
Anti-QS property of M. piperita (peppermint) oil was firstly assessed for pigment inhibition in CVO26. The result of the violacein quantification assay is depicted in Figure 1. The sub-MICs of PMO exhibited concentration-dependent violacein inhibitory activity at all tested concentrations and maximum reduction of 83.3% was recorded. Viable plate count performed on MHA plates at 24 h incubation showed no significant difference in the number of colony-forming units (CFUs) between untreated C. violaceum CVO26 and C. violaceum CVO26 treated with sub-MICs of the oil (Figure 1).
FIGURE 1. Quantitative assessment of violacein inhibition in CVO26 by sub-MICs of peppermint oil (PMO). All of the data are presented as mean ± SD. Significance at ∗p ≤ 0.05, significance at ∗∗p ≤ 0.005, significance at ∗∗∗p ≤ 0.001.
Effect OF PMO on Production of Virulence Factors and Biofilm Formation
The effect of sub-MICs of PMO in reducing the production of QS-dependent LasB Elastase, protease and chitinase in PAO1 is presented in Table 1. Significant decrease in LasB elastase activity was observed in the culture supernatant of PAO1 treated with sub-MICs of PMO. A minimum of 30% inhibition was observed when PAO1 was cultured with oil at a concentration of 0.37% v/v and maximum of 80% inhibition was observed at 3% v/v oil concentration. The impact of PMO in inhibiting the QS-dependent protease activity of PAO1 was also determined and results of the obtained showed 26.6, 48.1, 70.4, and 76% decrease in total protease production when treated with 0.37, 0.75, 1.5, and 3% v/v oil concentrations, respectively. Treatment of PAO1 with sub-MICs of PMO showed significantly reduced chitinase activity, the oil (0.3–3% v/v) demonstrated inhibition in chitinase production to the level of 21–78% (Table 1).
TABLE 1. Effect of sub-MICs of peppermint oil (PMO) on inhibition of quorum sensing regulated virulence factors in Pseudomonas aeruginosa PAO1.
To examine the efficiency of the oil against pyocyanin production, the PAO1 cells were cultivated in the presence and absence of PMO. As a result, a significant decrease in pyocyanin production was observed, up to the level of 52.4–85.2%, respectively, in PAO1, at concentrations ranging from 0.3 to 3% v/v as compared to the control. The effect of PMO on swarming motility of PAO1 was also examined. The oil effectively inhibited QS dependent swarming migration in a dose dependent manner in P. aeruginosa PAO1. The maximum reduction of 81.3% in swarming migration was recorded at highest tested concentration (3% v/v) followed by 70.6, 61.3, and 50.6% at 1.5, 0.75, and 0.37% v/v oil concentration, respectively, (Table 1).
The spectrometric analysis of EPS extracted from oil treated and untreated cultures of PAO1 revealed that the production of EPS decreased with increasing concentration of PMO. The test oil at 3% v/v concentration exhibited 76% decrease in EPS production in P. aeruginosa PAO1. Anti-biofilm activity of the test oil shows biofilm inhibition of the pathogen. Addition of 0.37, 0.75, 1.5, and 3% v/v concentration of oil led to a dose dependent reduction in biofilm formation in the order of 42.7, 61, 73, and 84%, respectively, (Table 1).
Similarly, the oil of peppermint (0.1–0.8% v/v) effectively interfered with the QS system of A. hydrophila WAF38 by significantly reducing the total protease activity to the level of 24.5–71% (p ≤ 0.005) and EPS production by 39–77.9%. Maximum decrease (74.8%) in biofilm formation at 0.8% v/v concentration of the oil was observed as depicted in Table 2.
TABLE 2. Effect of sub-MICs of PMO on inhibition of quorum sensing regulated virulence factors in Aeromonas hydrophila WAF-38.
Effect of Peppermint Oil on las System
The addition of PMO decreased significant β-galactosidase activity in E. coli MG4/pKDT17 by up to 41.9 and 54.5% at 1.5 and 3% v/v (Figure 5), demonstrating that PMO reduces AHL levels significantly to inhibit las-controlled transcription.
GC-MS Analysis of Peppermint Oil
Major ingredient of PMO as revealed by GC-MS analysis was menthol (36.87%), and other constituents identified were menthone (16.44%), neoisomenthol (11.33%), isomenthone (10.47%), menthyl acetate (7.47%), 2-isopropyl-5-methylcyclohexanol (2.74%), piperitone (2.17%), and limonene (0.53%) as given in Supplementary Table S1 and depicted in Supplementary Figure S1.
For the better understanding of LasR-phytoconstituents binding the complementary applications of molecular docking of compounds on LasR has been performed with Autodock simulation analyses. The best energy ranked results are shown in Figure 2 and are summarized in Table 3. As a reference model, the natural autoinducer AHL was docked in ligand binding domain of LasR. We found that L36, L40, Y47, A50, Y56, W60, Y64, D73, T75, V76, C79, W88, Y93, F101, A105, L110, L125, G126, A127, and S129 of LasR were interacted with AHL. Out of these, only Y56, W60, D73, and T75 formed hydrogen bonds whereas others were bound with hydrophobic interactions. In this way, the best scored docked AHL molecule occupied almost in the identical pattern as in the ligand-complexed crystal structure. This docked structure was further evaluated by superimposing with the LasR-AHL bound crystal structure and was found to be quite significant. From the above interacted residues Y47, A50, Y56, W60, Y64, D73, V76, and S129 are the main residues responsible for the activation of LasR. In case of all the 11 phytocompounds, most of the residues involved in the interactions are common with the natural ligand AHL (Table 3). Flexible ligands were set to free in the LasR to dock in the most feasible orientation according to the least free energy change (ΔG) and the interactions between the LasR and the inhibitors were exclusively hydrophobic in nature as reflected by several non-polar residues at binding site. These residues were in the proximity distance of 5 Å of the bound ligands. The essential oil constituents were docked in the active site with binding affinities ranging from -5.1 to -7.9 kcal/mol as compared to the binding affinity of AHL (-8.7 kcal/mol) and from these values the obtained binding constants (Kb) were 5.50 × 103 to 6.22 × 105 as compared to the AHL (2.39 × 106 M-1; Table 3). All the non-cyclic aliphatic compounds showed the lowest affinity while limonene was the weakest interacting cyclic entity. Here, most of the residues of LasR interacted hydrophobically with all the 11 tested compounds. Highest affinities are shown by menthol followed by isopulegol and 1-Hydroxyoctane as given in Table 3.
FIGURE 2. (A) Inhibitors obtained from the essential oil of Mentha piperita as well as the natural autoinducer 3-oxo-C12-HSL acyl homoserine lactone (AHL) are docked in the hydrophobic pocket of LasR. (B) Residues involved in the interaction of LasR-inhibitors as revealed from molecular docking 0, 3-oxo-C12-HSL; I, Limonene; II, Menthone; III, Isomenthone; IV, 1-Hydroxyoctane; V, Isopulegol; VI, Menthyl acetate; VII, Neoisomenthol; VIII, 2-Isopropyl-5-methylcyclohexanol; IX, Menthol; X, Lavandulol; XI, Piperitone.
TABLE 3. Molecular docking results of LasR-inhibitor interactions.
Effect of Menthol on Violacein Production
Menthol exhibited a concentration dependent decrease in QS regulated violacein production. Maximum reduction of 85% was recorded at 400 μg/mLconcentration while lowest of 26% decrease over control was observed at 50 μg/mLmenthol concentration (Figure 3).
FIGURE 3. Quantitative assessment of violacein inhibition in CVO26 by sub-MICs of menthol. All of the data are presented as mean ± SD. Significance at ∗p ≤ 0.05, significance at ∗∗p ≤ 0.005, significance at ∗∗∗p ≤ 0.001.
Effect of Menthol on Virulence Factor Production
Effect of menthol on QS regulated virulence factors of P. aeruginosa PAO1 revealed a concentration dependent decrease in all the functions. Highest reduction in all the virulence factors was observed at 800 μg/mLin PAO1. Decrease in total protease activity was highest (84.2%) followed by pyocyanin production (83.5%), elastase activity (78.7%), swarming motility (78%), EPS production (57.7%), and least in chitinase activity (54.6%) as presented in Table 4.
TABLE 4. Effect of sub-MICs of menthol on inhibition of quorum sensing regulated virulence factors in P. aeruginosa PAO1.
In A. hydrophila WAF38, menthol inhibited total protease significantly (52.5%) at 200 μg mL-1 while at lower concentrations reduction observed was not statistically significant. EPS produced by untreated A. hydrophila WAF38 was lowered significantly (58.3–66.6%) at sub-MICs (50–200 μg/mL) as depicted in Table 5.
TABLE 5. Effect of sub-MICs of menthol on inhibition of quorum sensing regulated virulence factors in A. hydrophila WAF-38.
Effect of Menthol on Biofilm Formation
A significant decrease in biofilm formation was observed in test bacterial strains when grown in the presence of menthol. Highest reduction (69.4%) in biofilm formation was observed at 800 μg/mLconcentration followed by 54 and 43.2% reduction at 400 and 200 μg/mL, respectively. To ascertain the structures visualized by light microscopy that exhibited antibiofilm activity, we used SEM to elucidate the potential of menthol against biofilm formation (Figure 4). The results of electron microscopic analysis revealed that the control slides showed well developed dense biofilm growth of PAO1, whereas, PAO1 treated with the menthol developed poor biofilm growth compared to that of the control sample (Figure 4). In A. hydrophila WAF38, biofilm formation was also reduced considerably ranging from 27.9–80% over untreated control at sub-MICs of menthol tested (Table 5).
FIGURE 4. Scanning electron microscopic (SEM) images for inhibition of biofilm of Pseudomonas aeruginosa PA01 at sub-MICs. (A) Control; (B) PMO (3% v/v); (C) Menthol (800 μg mL-1); (D) Aeromonas hydrophila WAF38 control; (E) PMO (0.8% v/v); (F) menthol (200 μg mL-1).
FIGURE 5. Effect of PMO on las systems. β-Galactosidase activity was measured in the Escherichia coli MG4/pKDT17 with and without sub-MICs of PMO. All of the data are presented as mean ± SD. Significance at ∗p ≤ 0.05.
Effect of Menthol on las and pqs Systems
To exclude the influence of other QS systems in P. aeruginosa, we used E. coli MG4/pKDT17 that produces LasR and contains the lasB promoter fused to lacZ. The addition of menthol decreased β-galactosidase luminescence in E. coli MG4/pKDT17 up to 60% at 800 μg/mL (Figure 6A), which shows that menthol directly inhibits las-controlled transcription.
FIGURE 6. Effect of menthol on las and pqs systems. (A) β-Galactosidase activity was measured in the E. coli MG4/pKDT17 with and without sub-MICs of menthol. (B) β-Galactosidase activity was measured in the E. coli pEAL08-2 with and without menthol. All of the data are presented as mean ± SD. Significance at ∗p ≤ 0.05, significance at ∗∗p ≤ 0.005.
Pyocyanin production which is mainly regulated by the pqs system, therefore a heterologous strain E. coli pEAL08-2 that produces PqsR and contains the pqsA promoter fused to lacZ was used to determine whether the inhibition of pyocyanin production was directly due to the effects of pqs system (Cugini et al., 2007). The addition of menthol reduced the β-galactosidase luminescence in E. coli pEAL08-2 up to 55% at 800 μg/mL (Figure 6B), which proved that menthol directly inhibits PQS-stimulated transcription.
In Vivo Assessment with C. elegans
The anti-infection potential of the sub-MIC of menthol was assessed using a liquid killing assay of C. elegans animal model by PAO1 in a 24-well microtitre plate. Complete mortality of the P. aeruginosa PAO1 preinfected C. elegans was observed within 72 h. However, C. elegans preinfected with PAO1 further treated with menthol (800 μg/mL) displayed enhanced survival upto 58% (Figure 7). However, menthol alone demonstrated no significant mortality of C. elegans at tested concentrations.
FIGURE 7. Anti-infection potential of sub-MIC of menthol (800 μg/mL) in increasing the survival of Caenorhabditis elegans preinfected with P. aeruginosa PAO1. Means values of triplicate independent experiments and SDs are shown.
In the present investigation, we showed the QS inhibitory potential of PMO and menthol based on its ability to inhibit AHL-dependent violacein production in C. violaceum and virulence factors such as elastase, protease, pyocyanin, EPS production, and biofilm formation in PAO1. It has previously been confirmed that in C. violaceum, the CviIR-dependent QS system coordinates the production of violacein pigment, and the compound, which has the ability to inhibit the violacein production without any antibacterial activity, is considered to be the promising QS inhibitor (Choo et al., 2006; Zhu and Sun, 2008). In this study, a dose dependent decrease of AHL mediated violacein production was recorded in C. violaceum CVO26. The concentration dependent response of anti-QS potential as found in our study is also supported by the findings on clove oil (Khan et al., 2009), marjoram (Kerekes et al., 2013), and three isothiocyanates (Borges et al., 2014) that demonstrated reduction in AHL dependent violacein production in C. violaceum CVO26 without inhibition of growth.
The LasIR-encoded protease and elastase play a key role in the pathogenesis of PAO1 (Kessler et al., 1993). These enzymes degrade the structural components of the infected tissue and enhance the growth and invasiveness of the organism. In this present investigation, the oil of peppermint and menthol demonstrated a concentration-dependent inhibition of virulence factor production such as total protease, elastase in PAO1, as shown in Table 1. This result is in agreement with the study of Adonizio et al. (2008), who demonstrated significant inhibition of LasB activity by medicinal plants from Florida. In addition to this recently, flavanones (Vandeputte et al., 2011), Lagerstroemia speciosa fruit extract (Singh et al., 2012) and Sclerocarya birrea bark extract (Sarkar et al., 2014) have shown to inhibit elastase activity to substantial levels. Our data shows that menthol decreases both the elastase activity of PAO1 and the transcriptional activation of lasB in E. coli, which indicates that menthol inhibits the las system. Pyocyanin metabolite causes severe toxic effects by damaging the neutrophil-mediated host defense in patients with cystic fibrosis (Fothergill et al., 2007). Similarly, menthol reduced both the pyocyanin production of PAO1 and the transcriptional activation of pqsA in E. coli, which indicates that menthol inhibits the pqs system. Considering the las and pqs systems regulate the expression of numerous virulence-related genes, using menthol to inhibit these two systems would significantly decrease P. aeruginosa virulence. Our results are in accordance with the results of a recent report wherein Vandeputte et al. (2011) and Zhou et al. (2013) demonstrated that eugenol and flavanones (i.e., naringenin, eriodictyol, and taxifolin) reduced the production of pyocyanin significantly. QS-regulated flagellar and pili dependent swarming motility is considered as one of the virulence factors because of its involvement in biofilm formation (Niu and Gilbert, 2004). In the present study, the oil of pepermint and menthol reduced the swarming migration of target organism. Our findings are in agreement with the results of Khan et al. (2009) in which sub-MICs of clove oil decreased the swarming behavior in PAO1 by 78%. Recently, studies on Capparis spinosa, Cuminum cyminum, and S. birrea (Packiavathy et al., 2011, 2012; Sarkar et al., 2014) have shown significant reduction in swarming motility of P. aeruginosa PAO1.
Exopolymeric substance plays a defining role in maintaining the biofilm architecture. Therefore, decrease in production of EPS will render the biofilm structure weak and susceptible (Bomchil et al., 2003). The observed results in the present study indicated the potential PMO to inhibit EPS production in PAO1 at sub-MIC values. This is probably the first report on the inhibition of EPS by M. piperita oil in P. aeruginosa PAO1. Formation of biofilm by PAO1 is QS regulated and this mode of growth confers increased drug resistance and infection caused is quite severe in patients suffering from cystic fibrosis. AHL mediated cell–cell signaling plays a crucial role in the initiation and maturation of biofilms in PAO1 (Rasmussen et al., 2005). The results of our present investigation revealed the biofilm inhibiting potential of PMO and its major phytoconstituent menthol against PAO1 biofilms in a concentration dependent manner, as shown in Figure 3. Recent studies on herbal extract of M. piperita have demonstrated 57% reduction in adhesion property of P. aeruginosa (Sandasi et al., 2011); our findings on the biofilm inhibitory property of M. piperita oil finds support from above work. Further, the results obtained are comparable to the decrease in biofilm formation reported in studies for methyl eugenol (Packiavathy et al., 2012), marjoram (Kerekes et al., 2013), and Rosa rugosa tea extract (Zhang et al., 2014).
The mortality of the nematode by PAO1 is caused by the cyanide asphyxiation and paralysis (Gallagher and Manoil, 2001). We demonstrated an enhanced survival of pre-infected nematode model maintained with menthol (800 μg/mL) as depicted in the Figure 7. The data obtained clearly indicates that the menthol interferes with the QS of PAO1 leading to reduced mortality of the C. elegans. The findings are similar to the previous observations of Husain et al. (2013), on enhanced survival of the nematode (C. elegans) after treatment with sub-MICs of clove oil.
In many pathogens, virulence potential and biofilm formation is under the regulation of QS. Therefore, instead of bactericidal or bacteriostatic strategies, attenuation of virulence via QS inhibition approach could be more effective strategy to combat bacterial infection caused by drug resistant bacteria.. In the present study oil of M. piperita and menthol showed QS inhibitory properties, resulting in decreased biofilm formation capabilities and the attenuation of P. aeruginosa virulence in vitro and in vivo. Findings of this study also suggest that essential oil of M. piperita and menthol can inhibit QS, biofilm and its related virulence processes in pathogenic bacteria, and can be exploited by the pharmaceutical industry for the development of new safe broad spectrum antibiofilm and anti-QS drugs with reduced toxicity and antibiotic resistance.
The authors extend their appreciation to the Deanship of Scientific Research at KSU for funding this work through research group project number RGP-215.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fmicb.2015.00420/abstract
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Keywords: anti-quorum sensing activity, peppermint oil, menthol, biofilm, molecular docking, C. elegans
Citation: Husain FM, Ahmad I, Khan MS, Ahmad E, Tahseen Q, Khan MS and Alshabib NA (2015) Sub-MICs of Mentha piperita essential oil and menthol inhibits AHL mediated quorum sensing and biofilm of Gram-negative bacteria. Front. Microbiol. 6:420. doi: 10.3389/fmicb.2015.00420
Received: 11 February 2015; Accepted: 21 April 2015;
Filomena Nazzaro, Consiglio Nazionale delle Ricerche – Istituto di Scienze dell'Alimentazione, Italy
Efstathios D. Giaouris, University of the Aegean, Greece
Taoufik Ghrairi, Faculty of Medicine Ibn El Jazzar of Sousse, Tunisia
Copyright © 2015 Husain, Ahmad, Khan, Ahmad, Tahseen, Khan and Alshabib. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Iqbal Ahmad, Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh 202002-UP, India, ahmadiqbal8@yahoo.co.in; Fohad M. Husain, Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, Riyadh 11451, Saudi Arabia, fahadamu@gmail.com, fhussain@ksu.edu.sa


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text:
George Regas
The Rescue (Horsell Common album)
Warriors (novel series)
List of Chip 'n Dale Rescue Rangers episodes
Scouts to the Rescue
George Regas (November 9, 1890 – December 13, 1940) was a Greek-born American actor.
Born in Sparta, Greece, he was the brother of actor Pedro Regas. He was a stage actor in Athens before coming to the U.S. In New York he played Romeo in a Grecian version of Romeo and Juliet.
In 1921 Regas acted in his first motion picture: The Love Light with Mary Pickford. This film was produced by Pickford's production company. He would go on to create character roles in over one hundred films. His rugged looks and Mediterranean complexion allowed him to play a wide variety of nationalities in action and adventure films. He was married to actress Reine Davies, the sister of Marion Davies. Starred as Mateo in The Adventures of Sherlock Holmes (1939).
Regas died after an operation for a throat infection on December 13, 1940 in Hollywood, California, at age fifty. His last film role was Sergeant Gonzales in The Mark of Zorro. He is entombed at Hollywood Forever Cemetery alongside his brother.
This page contains text from Wikipedia, the Free Encyclopedia - https://wn.com/George_Regas
The Rescue may refer to:
The Rescue (painting), an 1855 painting by John Everett Millais
The Rescue (statue), a marble sculpture group (1837–50) by Horatio Greenough
The Rescue (1917 film), a silent drama starring Lon Chaney, Sr.
The Rescue (1929 film), a romantic adventure by Herbert Brenon, based on Joseph Conrad's novel (see below)
The Rescue (1971 film), a Shaw Brothers film
The Rescue (1988 film), a film directed by Ferdinand Fairfax
"The Rescue", seventh episode of the 1964 Doctor Who serial The Daleks
The Rescue (Doctor Who), a serial from the second season of Doctor Who
"The Rescue" (Dynasty 1984), a 1984 episode of Dynasty
"The Rescue" (The O.C.), an episode of The O.C.
The Rescue (Conrad novel), a 1920 novel by Joseph Conrad
The Rescue (Sparks novel), a 2000 novel by Nicholas Sparks
The Rescue, a 2006 novel by Gordon Korman
The Rescue, a manga based on Erin Hunter's novel series Warriors
This page contains text from Wikipedia, the Free Encyclopedia - https://wn.com/The_Rescue
The Rescue is the first LP of Australian band Horsell Common. The album was released on 29 September 2007. The first single released from the album was "Good From Afar" which peaked in the Australian Independent Records Charts at #5.
The album entered the Australian Independent Records Charts at #6.
"Good from Afar" - 3:17
"Bruise Easy" - 3:00
"Automation" - 5:22
"Help is on Its Way" - 3:40
"Sing the News" - 3:27
"Annie, If You're Listening" (featuring Lisa Gammaldi of Capeside) - 3:39
"It's OK" - 3:33
"I'm Dead" (featuring Stephen Christian of Anberlin) - 5:02
"The Sound of Breaking Records" - 2:48
"Gone For the Summer" - 3:01
"Surgery" - 5:41
Mark Stewart - vocals, guitar
Luke Cripps - bass guitar
Leigh Pengelly - drums
Stephen Christian - guest vocals on "I'm Dead"
Lisa Gammaldi - guest vocals on "Annie, If You're Listening"
Stephen Haigler - producer
↑ "Horsell Common bring Good[s] From Afar". http://www.fasterlouder.com.au/. 9 August 2007.
This page contains text from Wikipedia, the Free Encyclopedia - https://wn.com/The_Rescue_(Horsell_Common_album)
Warriors is a series of juvenile fantasy novels published by HarperCollins; it is written by authors Kate Cary, Cherith Baldry, Tui Sutherland, with the plot developed by editor Victoria Holmes, who collectively use the pseudonym Erin Hunter. The series follows the adventures of four Clans of wild cats—ThunderClan, ShadowClan, WindClan, and RiverClan—in their forest and lake homes. SkyClan, the long-forgotten fifth Clan of the forest, is later introduced in the stand-alone novel Firestar's Quest. It receives additional focus in the novel SkyClan's Destiny, the manga trilogy SkyClan and the Stranger, and the 2013 novella Cloudstar's Journey. A few cats in the series are
There are currently six sub-series, each containing six books. The first, Warriors (later re-titled as Warriors: The Prophecies Begin), was published from 2003 to 2004. Warriors: The New Prophecy, published from 2005 to 2006, follows the first sub-series, chronicling the Clans as they move to a new home. The third story arc, Warriors: Power of Three, was published from 2007 to 2009. The fourth sub-series, Warriors: Omen of the Stars, was published from 2009 to 2012 and continued where the third story arc left off. The fifth sub-series is Warriors: Dawn of the Clans, and the first book, The Sun Trail, was released 5 March 2013. The sub-series acts as a prequel series, detailing the formation of the Clans. A sixth sub-series with the working title Warriors: A Vision of Shadows has been announced, with Kate Cary posting a working character list to her blog site asking fans to check for inconsistencies. The series appears to focus on Bramblestar's kits, Alderkit and Sparkkit.
This page contains text from Wikipedia, the Free Encyclopedia - https://wn.com/Warriors_(novel_series)
Chip 'n Dale Rescue Rangers is an American animated television series produced by Walt Disney Television Animation. Created by Tad Stones and Alan Zaslove, it featured the established Disney characters Chip 'n Dale in a new setting. The series premiered on The Disney Channel on March 4, 1989, having aired the first produced episode, "Catteries Not Included", as a special preview on August 27, 1988.
A two-hour movie presentation of "Rescue Rangers: To The Rescue" began airing in syndication the weekend of September 29, 1989. While not produced first, the movie told the story of how the Rescue Rangers met.
The series then began airing in weekday syndication. From that point forward, the movie was broken up into five standard-length episodes that were considered part of season 2. In 1990, it became part of the syndicated programing block The Disney Afternoon. The final episode aired on November 19, 1990. As a part of the Disney Afternoon lineup, reruns of the show were aired until September 3, 1993.
This page contains text from Wikipedia, the Free Encyclopedia - https://wn.com/List_of_Chip_'n_Dale_Rescue_Rangers_episodes
Scouts to the Rescue (1939) is a Universal film serial.
A troop of boy scouts use a treasure map to find a stash of counterfeit notes and a lost tribe with a secret Radium deposit.
Jackie Cooper as Bruce Scott, leader of the Scout Troop
David Durand as Rip Rawson
Bill Cody, Jr. as Skeets Scanlon
Vondell Darr as Mary Scanlon
William Ruhl as Hal Marvin, a G-Man on the trail of the counterfeiters
Sidney Miller as Hermie, a boy scout
Ivan Miller as Turk Mortensen
Edwin Stanley as Pat Scanlon
Ralph Dunn as Pug O'Toole, a gangster
George Regas as Lukolu, high priest of the lost tribe
Jack Mulhall as Scoutmaster Hale
Jason Robards, Sr. as Doc, a gangster
Dick Botiller as Leeka, Leader of Warriors
Victor Adams as Hurst, a heavy
Max Wagner as Joe, a henchman
Death Rides the Air
Avalanche of Doom
Trapped by the Indians
River of Doom
Descending Doom
Ghost Town Menace
Destroyed by Dynamite
Thundering Hoofs [sic]
The Fire God Strikes
The Battle at Ghost Town
Hurtling Through Space
This page contains text from Wikipedia, the Free Encyclopedia - https://wn.com/Scouts_to_the_Rescue
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The Rescue, Keeping Secrets
The Rescue, American HI-FI
The Rescue, Adam Cappa
The Rescue, Triple Fast Action
The Rescue, Fast Forward
The Rescue, Codeseven
The Rescue, Tyler Ward
The Rescue, On The Last Day
The Rescue, Birth Control
The Rescue, Kutless
The Rescue, Search The City
The Rescue, Futures
I lay this out on the table
Only if you catch me
Call me out on this fable
Just to hear my story
I will look you in the eye
Say those words that make you melt inside
History repeats again just like me
This will never end I will never end
I'll be as sketchy as this pen and paper
Sending you to war without a weapon
Using my ability to get your sympathy
The only thing this is, its apathy
You've seen this so many times yet you can't walk away
Lights out you think you know me
So delicately I wanted more
So I lied and betrayed you
This must be love
How could you miss the doubtful look in my eyes?
When I told you that this felt right
I will kiss your lips again
With the passion that you only see in movies
I'll set you up to take this fall
Make you think that I am perfect I'm so perfect
I own the blueprints to your heart
And just like mines on a battlefield
I'll use these phone calls so strategically
Placing them perfectly
the situation arose where the 89-year-old had to be revived," continued Bockhop. Two paramedics and two doctors were on site to accompany the vaccination team ... "At the moment the only connection that is visible to us is the temporal connection," emphasized Bockhop ... Due to data protection, the district cannot provide any information about previous illnesses or other health problems, Bockhop continued....
After condemning the pro-Trump invasion of the Capitol, the incoming Biden administration invited Carlos Vecchio – a coup leader charged in the 2014 torching of the Venezuelan Attorney General's office – to its inaugural ceremony ... Who would be attending incoming President Joe Biden's inauguration on January 20, scheduled to take place exactly two weeks following the Capitol riot?....
Russian opposition leader Alexei Navalny's defiant pledge to return to Moscow from Germany after surviving a poisoning attack and subsequent arrest has put strongman Vladimir Putin and the Kremlin in another bind ... He did the same to Yegor Zhukov in 2019 ... (2)Narrow EscapeMost Americans do not realize how close the United States came to an authoritarian regime on Jan ... Mussolini to the Present....
Taking to Twitter, the environmental activist, 17, shared a picture of the outgoing president, 74, stepping into helicopter Marine One and turning to raise his fist ... At the time, the entrepreneur had already begun to accuse Joe Biden of committing election fraud ... The Swedish activist is more likely to get along with Joe Biden(Picture ... The new president has already set out plans for the US to rejoin the Paris climate accords....
Mali : UNTM –gouvernement : Reprise demain des négociations
Edit Mali Actualités 06 Jan 2021
Les discussions achoppent sur nombreux points de revendications dont la satisfaction pourrait lourdement peser sur le budget de l'Etat fortement impacté par la crise sécuritaire et la Covid 19 ... «Tout le problème est là » ... Autres revendications sur la table des négociations ... Les grèves ont paralysé divers secteurs stratégiques notamment l'éducation, la santé, et les régies financières. Georges François Traoré. Source ....
COVID-19 : des renforts déployés à Fort St. James
Edit Radio Canada 10 Dec 2020
Au moins 60 personnes ont reçu un diagnostic de COVID-19 dans cette communauté d'environ 1500 personnes située à quelque 160 km au nord-ouest de Prince George ... Les patients sont envoyés dans les hôpitaux de Prince George et de Vanderhoof, et la communauté travaille avec la régie de la santé du Nord et l'autorité sanitaire des Premières Nations de la Colombie-Britannique, indique la chef Aileen Prince ... ....
Radio France et France 5 bouleversent leurs grilles pour rendre hommage à Daniel Cordier
Le documentaire de 90 minutes, écrit par Régis Debray et Bernard George et diffusé pour la première fois en 2010, mêle le suspense du récit à l'authenticité du témoignage ... Dans un aller- retour entre passé et présent, le grand résistant partage avec Régis Debray son expérience de Français Libre sur les lieux mêmes de son engagement....
Daniel Cordier, secrétaire de Jean Moulin
Il a fait don de centaines d'oeuvres au Musée Georges-Pompidou ... A sa mort, Hubert Germain sera ainsi inhumé dans la crypte du Mont Valérien dans le dernier caveau vide, entre George Brière, matelot au 1er régiment de fusiliers-marins, tué dans les Vosges en novembre 1944, et Alfred Touny ("Colonel Guérin") fusillé en avril 1944, également Compagnon de la Libération....
Daniel Cordier, secrétaire de Jean Moulin AFP•20/11/2020 à 17:02
Il a fait don de centaines d'oeuvres au Musée Georges-Pompidou.  ... A sa mort, Hubert Germain sera ainsi inhumé dans la crypte du Mont Valérien dans le dernier caveau vide, entre George Brière, matelot au 1er régiment de fusiliers-marins, tué dans les Vosges en novembre 1944, et Alfred Touny ("Colonel Guérin") fusillé en avril 1944, également Compagnon de la Libération....
La Régie inflige une sanction de 40 000 $ à Westcoast Energy Inc. English
Edit Canada Newswire 13 Nov 2020
L'enquête visant l'application de la réglementation que la Régie a menée à la suite de la rupture de pipeline survenue en 2018 près de Prince George, en Colombie-Britannique, a révélé que la société n'avait pas suivi son programme de gestion de l'intégrité. CALGARY, AB, le /CNW/ - La Régie de l'énergie du Canada a dévoilé aujourd'hui tous les détails de la sanction administrative pécuniaire de 40 000 $ infligée à Westcoast Energy Inc....
Communiqué - La Régie inflige une sanction de 40 000 $ à Westcoast Energy Inc. (National Energy Board - Government of Canada)
Edit Public Technologies 12 Nov 2020
L'enquête visant l'application de la réglementation que la Régie a menée à la suite de la rupture de pipeline survenue en 2018 près de Prince George, en Colombie-Britannique, a révélé que la société n'avait pas suivi son programme de gestion de l'intégrité ... Le 12 novembre 2020 - Calgary (Alberta) - Régie de l'énergie du Canada ... La Régie a dépêché du personnel sur les lieux et a participé à l'intervention d'urgence....
Nicolas Dupont-Aignan cite George Orwell pour critiquer le couvre feu... avec une fausse citation
Parmi eux, Nicolas Dupont Aignan, qui a cité le célèbre auteur de science fiction George Orwell pour critiquer cette décision ... marcher dans les rues, se distraire, chanter, danser...", George Orwell, 1984" ... Le livre de George Orwell, publié en 1949, imagine une Grande-Bretagne régie par un régime autoritaire, où la liberté d'expression n'est plus et où les citoyens sont surveillés de près....
Une trop bruyante certitude
Edit Les Affaires 14 Oct 2020
BILLET. Joyce Echaquan, George Floyd, Regis Korchinski-Paquet, Breonna Taylor Les drames se suivent et se ressemblent. Il y a tellement de victimes de discrimination qu'il n'y aurait .......
Rieux-Minervois. Le livre "81e Régiment d'infanterie", offert à deux anciens combattants
Edit La Dépêche 30 Sep 2020
... présence de l'auteur, le livre à René Pech, 95 ans, membre de ce régiment à 17 ans, ayant menti sur son âge au commandant Henri Bousquet, et à Georges Tarbouriech, 97 ans, membre actif de l'association....
Rapport des commissaires aux comptes sur la réduction de capital dans le cadre d'un programme de rachat d'actions et par voie d'annulation d'actions ordinaires
Edit Public Technologies 30 Aug 2020
DocuSign Envelope ID. F45F3671-5EFA-408D-A3D4-A56587F07D26. KPMG Audit. Audit et Conseil Union. Tour EQHO. 17 bis, rue Joseph de Maistre. 2 Avenue Gambetta. 78576 Paris Cedex 18. CS 60055. France. 92066 Paris la Défense Cedex. France. AB Science S.A ... 3, avenue George V - 75008 Paris ... reg appx 0 ... 3, avenue George V - 75008 Paris ... AB Science SA published this content on 31 August 2020 and is solely responsible for the information contained therein....
Rapport des commissaires aux comptes sur l'autorisation d'attribution d'options de souscription d'actions
DocuSign Envelope ID. F45F3671-5EFA-408D-A3D4-A56587F07D26. KPMG Audit. Audit et Conseil Union. Tour EQHO. 17 bis, rue Joseph de Maistre. 2 Avenue Gambetta. 78576 Paris Cedex 18. CS 60055. France. 92066 Paris la Défense Cedex. France. AB Science S.A ... 3, avenue George V - 75008 Paris ... reg appx 0 ... 3, avenue George V - 75008 Paris ... 3 ... AB Science SA published this content on 31 August 2020 and is solely responsible for the information contained therein....
Attestation des commissaires aux comptes sur les informations communiquées dans le cadre de l'article L. 225-115 4° du code de commerce relatif au montant global des rémunérations versées aux personnes les mieux rémunérées pour l'exercice clos le 31 décembre 2019
DocuSign Envelope ID. F45F3671-5EFA-408D-A3D4-A56587F07D26. KPMG Audit. Audit et Conseil Union. Tour EQHO. 17bis Rue Joseph de Maistre. 2 Avenue Gambetta. 75876 Paris Cedex 18. CS 60055. France. 92066 Paris la Défense Cedex. France. AB Science S.A ... 3, avenue Georges V - 75008 Paris ... reg ... Tour EQHO ... 3, avenue Georges V - 75008 Paris ... AB Science SA published this content on 31 August 2020 and is solely responsible for the information contained therein....

redpajama_set_name: RedPajamaCommonCrawl


From the bright lights of a city street to the soft hues of a mountain trail, running can feel like a form of art. Start your masterpiece with Stance's Women's Crew, a sock that blends art and science with a beautiful balance. From our Fusion Run Collection comes a performance running style with 200-needle count construction for a lightweight feel, reinforced with flat-linked heel and toe stitching that outlasts your longest mile. With blocked cushioning and an engineered cuff that never loses its grip, fast will be your new favorite color with Stance's Crew.


text:
DIRECTIONS = 'N', 'E', 'S', 'W'
NORTH, EAST, SOUTH, WEST = DIRECTIONS

class Player(object):
    def __init__(self, location, name='Player'):
        assert isinstance(location, Location)
        self.location = location
        self.name = name

class Location(object):
    def __init__(self, name, description=""):
        self.name = name
        self.description = description
        self.exits = dict()

    def __str__(self):
        return self.name

    def add_direction(self, direction, other_location):
        assert direction in DIRECTIONS
        self.exits[direction] = other_location

    def describe(self):
        out = ''
        out += "Current location: %s\n%s\n" % (self.name, self.description)
        for direction, location in self.exits.items():
            out += "\t%s (%s)\n" % (location, direction)
        return out
sample_universe = """
:Garage
You are in the garage. There are no cars here currently.
E:Bedroom
W:Kitchen

:Kitchen
The kitchen is immaculate. You suspect that nobody has ever actually prepared any food here.
E:Garage

"""

def test_location():
    startroot = Location('Start room')
    kitchen = Location('Kitchen')
    startroot.add_direction(NORTH, kitchen)

def test_player():
    lobby = Location('Lobby')
    john = Player(lobby, 'John')

def _load_universe(content):
    location = first_location = None
    locations = {}

    for line in content:
        line = line.strip()
        if not line or line.startswith('#'):
            continue

        if line.startswith(':'):
            location = Location(line[1:])
            locations[line[1:]] = location
            if not first_location:
                first_location = location
        elif location is not None and not location.description:
            location.description = line
        else:
            direction, destination = line.split(':', 1)
            location.add_direction(direction, destination)

    for location in locations.values():
       for direction, destination in location.exits.items():
           try:
               location.add_direction(direction, locations[destination])
           except KeyError:
               raise SystemError("Your universe file sucks! %s" % destination)

    return locations, first_location

def _create_universe():
    start = Location('Start room')
    kitchen = Location('Kitchen')
    garage = Location('Garage')
    start.add_direction(NORTH, kitchen)
    start.add_direction(WEST, garage)
    hobby_room = Location('Hobby room')
    garage.add_direction(EAST, hobby_room)

    return start

def play(gamefile):
    #start_room = _create_universe()
    locations, start_room = _load_universe(file(gamefile))
    player_name = raw_input('Player name?: ') or 'No name'
    player = Player(start_room, player_name)
    while True:
        print player.location.describe()

        if not player.location.exits:
            print "No more exits! GAME OVER!"
            break

        next_direction = raw_input('Where to next? ').upper()
        while next_direction not in player.location.exits.keys():
            next_direction = raw_input('Where to next? (%s) ' %\
            ', '.join(player.location.exits.keys())).upper()
        player.location = player.location.exits[next_direction]

if __name__ == '__main__':
    import sys
    if sys.argv[1] == 'test':
        test_location()
        test_player()
        sys.exit(0)

    try:
        play(sys.argv[1])
    except KeyInterupt:
        pass

redpajama_set_name: RedPajamaGithub


This is a placeholder page for Gross Adam, which means this person is not currently on this site. We do suggest using the tools below to find Gross Adam.
You are visiting the placeholder page for Gross Adam. This page is here because someone used our placeholder utility to look for Gross Adam. We created this page automatically in hopes Gross Adam would find it. If you are not Gross Adam, but are an alumni of Larkin High School, register on this site for free now.


Considering putting your name forward as a candidate for a political party is an interesting process.
First you have to ask yourself, honestly, why do you want to do it? The answer will be different for many people. They may crave recognition, it could be ego, it could be the opportunity to make a difference, or it might be the pursuit of an attractive salary package and a lucrative pension.
Your motivation will help answer the question about what you are prepared to sacrifice to run and win. The life of a politician is not easy on families and relationships. Politicians spend a lot of time on the road away from their spouses and children. Even when they are at home the cell phone, e-mail, messenger and meetings drone on. Finding a balance for you family against the needs of your constituents, your party, the media and the legislature can be very difficult.
I have never lived the life of an elected official. However, I have been very close to it. One of the men that I worked for got married and raised two children over the period of his political career. In the end the career took a toll on his marriage. He and his wife raised two wonderful, respectful and successful children who have prospered academically and economically.
Sure, you can say family first - but as much as voters will not admit, they really do not care about you child's Christmas Concert or a Kindergarten Graduation. They expect to see you at the fireman's ball or the rally to save a the clinic. They will not forgive you for not attending and your political opponents will always use your unavailability as fodder.
The seat of government is in St. John's which means the vast majority of our MHA's have to live in the city when the House of Assembly is open. There are many days and evenings where they simply can not be at home.
These sacrifices are not always appreciated. Once you sign the contract to be the people's choice for a term, your first obligation is to them, or you will not get a second term.
Than there is the question of accomplishing your personal goals. If you are the type, and I am sure most are, that wants to make a difference - that feels passionately about issues - balancing that with the rigors of caucus solidarity is sure to be a challenge. Lets be honest, it is the glue that binds but how many PC MHA's see the writing on the wall because they chose to tow the party line instead of their personal convictions.
I am an old hand at organizing, running and participating in political campaigns for just about every conceivable election. The recipe for success and failure is not all that complicated. Fail to plan equates to planning to fail. Organizing, funding, communicating, speaking skills are based on 30 year of doing just that.
Can I accept the challenges of party solidarity, the sacrifices to my family life and the interplay of often childish petty politics that underscores the reality of how parties, caucuses, people and government work?
To me democracy begins at the local level, yet the reality is that government is a top down phenomena where a few party and government elites call all the shots.
I believe that public office is one of the highest aspirations that one can ever hope to achieve. Good politicians are builders, they look for solutions to the problems that we face. They see the big picture and are not anchored to the present.
I suppose it is like swimming, if I had never gotten in the water and tried, I would never have learned.
The road to hell is paved with good intentions. I have to ask myself a couple of questions: Can I make a difference to my community and my province?; is the timing right?; and most importantly - do I have enough faith in any team to jump on-board?
Labels: Contemplation, Politics, running for office.
Yes, it can be brutal having to be in St. John's up to 45 days a year, for up to four days in a row.
Beats being an unemployed Student Aid Information Officer. Go for it!! It is now or never!


Microsoft has released an update for windows to support Indian Rupee Symbol. This update is for Windows Vista, Windows Server 2008, Windows 7 and Windows Server 2008 R2.
This update is named as KB2496898 and before going for the update save all your works as it requires restart to take effect on your systems. Update KB2496898 is available for both 32 bit and 64bit systems.


Webster dictionary was developed by Noah Webster in the beginning of 19th century. On this website, you can find definition for extensive from the 1913 edition of Webster's Revised Unabridged Dictionary. Define extensive using one of the most comprehensive free online dictionaries on the web.
1. Having wide extent; of much superficial extent; expanded; large; broad; wide; comprehensive; as, an extensive farm; an extensive lake; an extensive sphere of operations; extensive benevolence; extensive greatness.


Manufacturers of Industrial Caravans and Mining Accommodation.
With 30 years experience in the design , manufacture and supply of industrial caravans throughout Australia.
Traymark Industrial Caravans are of robust construction and designed to last through the rigors of constant movement and are suited to adverse road conditions.
An Australian owned and run company Traymark has been a long time supplier of Mobile Lunchrooms,Toolrooms,Accommodation, Amenities, Mining Camps and various Special Purpose Caravans for many Governments departments and Commercial clients - great and small.
The materials and workmanship will be to the highest standard and meet all requirements as stated in the Australian Standards. From the chassis to the internal fitout these units are built to last .
The Traymark team has many years experience in the design and supply of Industrial vans . We can custom build to your requirements or we have many plans to choose from . Floor plans can include kitchens, bathrooms, office and sleeping facilities.
Our vans are constructed to suit site offices, lunch rooms, government departments, councils , the oil and gas industry and commercial ventures.


Please read the terms and conditions carefully before completing the entry, as they will form the basis of the contract between you and the ArtGemini Prize.
Decide on the number of works (maximum 6 entries) you wish to enter. You can submit your image now or at a later date if you are making new work. The contest is open to all artists worldwide with no age limit.
Open to all genres of 2D and 3D art eg painting, mixed media, drawing, ink, watercolour, calligraphy, original print, photography, digital art, sculpture and ceramic excluding installation and video.
Register by completing the online registration form.
Images of works must be in jpeg format. Recommended ideal size is 2MB, with minimum size 250KB, maximum 4MB. For best viewing, we recommend a maximum resolution of 300 dpi and pixel dimension of 1600 pixels or more wide. Remember to provide a high-quality image, as it may be used on the website and in promotional material.
You must include the title of your work, the year it was created, medium and size including frame (maximum size 150 x 150cm except Sculpture maximum size 50 x 50 x 50 cm). Limited edition print must declare the number of editions.
Any work submitted must be available for sale in the exhibition if shortlisted.
You must state an estimated sale price of the work, including VAT if you are registered, ie the price a gallery would normally charge. The price should include ArtGeminiPrize 50% sales commission which will be donated to selected charity. Sensible pricing is recommended.
An artist statement (max 100 words) is recommended and followed by a curriculum vitae (CV) if shortlisted.
You will need a credit/debit card ready to pay via Stripe. Use your full name for reference on your payment. Also a friend can pay on your behalf.
Please do not send your physical artwork to us unless you are notified that you are shortlisted.
Maximum 6 entries and entry fees are non-refundable.


Stan Polovets has served as Lead Member of the Advisory Board of L1 Energy since the formation of the company in 2013.
Prior to 2013, Mr. Polovets was the CEO of the Alfa-Access-Renova (AAR) Consortium, which managed $30 billion in oil and gas assets in Russia, Ukraine, Brazil, Vietnam, Venezuela and Iraq. AAR's main asset was TNK-BP, one of the world's 10 largest private oil companies. Mr. Polovets served as director and member of board committees of TNK-BP and its affiliated companies, including Slavneft and TNK Trading International. He represented TNK on its 2003 merger with BP and AAR on the $55 billion sale of TNK-BP to Rosneft in 2013.
Mr. Polovets is the Lead Non-Executive Director at Clal Industries, a major Israeli holding company with a diversified portfolio of investments in industrial, manufacturing, hi-tech, biotech and consumer sectors, and a lead director at Taavura, the leading logistics company in Israel. Mr. Polovets is a member of the New York University's President's Global Council, Council on Foreign Relations, and the Board of Overseers of Stanford University's Hoover Institution.


Looking at the series that helped revitalize the Transformers franchise, was an early CG cartoon and really did some great character work: Beast Wars!
A bootleg Pinhead shows up and says NO a lot. Meanwhile a family has an argument in their living room for a long time.
Nightwolf has computer problems while Kano hangs out in a pyramid. He might be thinking of some kind of scheme in there.


The amazing thing about cooking is, no matter who you are, it is for everybody. We are three completely different characters, but the love for food and cooking brings us together. We are looking at the world of food with very diverse approaches and meet in the center of this universe because cooking connects us.
Food is art. Without being a star chef – even with the simplest course, you can show beauty and create amazing meals. Food is a remembrance. It can be the taste of happy childhood, the reminder of a special person or evokes pleasant situations once again. It makes the past tangible in the present day. Food is individuality. With your eating habits, you can make a statement to the world; it is a channel to express your personality (because it is so closely connected to other topics).
You are Food. Your diet has a huge impact on how you can master the challenges of life. Good food provides you with the required energy to accomplish your tasks, not only with the help of nutrients. It gives you the opportunity of calming down and recharging your spirits. Food allows the world. While suffering from wanderlust, cooking can abduct to other countries. Exotic courses open new cultures when traveling to this country is too expensive. Food is the environment. With self-cooked courses, you can influence the market with the right of food origin and cultivation.
Food is a tradition. In the modern world, the knowledge about food preparation disappears. With this blog, I want to promote this skills and create awareness, therefore. Food is health. Cooking gives so much more than only a meal. The impact of a nourishing diet is extraordinary. With a home-cooked meal and natural ingredients, it is so easy to stay fit and healthy. Food is a pleasure. Enjoying a meal with all your senses allows quite different sensations. From own experience, I can tell that a self-made course always wins.
Food is everything you want it to be.


Lock in a great price for Grand Central Hotel Shanghai – rated 8.7 by recent guests!
I stay at this hotel at least one night every time I visit Shanghai. It's amazing.
Great location in the heart of tourist area. The room is clean, spacious and comfortable.
Excellent staff, friendly, helpful. Great location.
Helpful polite Staff. Everybody speak English. There's an exchange currency machine in the Lobby, which makes our lives easier. The lady who cleaned our room was so sweet! Rooms were large and very clean. Matress and pillows very comfortable. Breakfast was both Chinese and Western, with huge variety! Restaurant serves delicious soft steak! I'd definitely recommend this hotel to a friend! Hopefully we'll come back!
Very nice hotel with great lobby area, friendly staff and good clean rooms.
Great location, next to the shopping centers, delicious breakfast buffet . Nice and clean with very helpful staff.
The hotel has an older aesthetic. Room decor is all about old luxury with a lot of gold accents. Nonetheless, it's a very comfortable stay. The amenities in the room are complete, the bed was generally clean and comfortable. The location is excellent as it is near Nanjing Road which is accessible by many subway lines.
Room was spacious and service is fast. Free water daily. Bed super comfy and the location is prime. Lots of food options inside and around the hotel. Walking distance to the bund and other attractions and restaurants.
Good location, near to nanjing road and you can walk a few minutes to the bund.
One of our top picks in Shanghai.Grand Central Hotel Shanghai is located next to The Bund and East Nanjing Road Pedestrian Street. Its modern comforts include a large indoor pool and rooms with free internet access.
Grand Central Hotel Shanghai is a 3-minute walk from Shanghai's Metro subway line 1, 2 and 8. People's Square is 11 minutes' walk from the property. Lujiazui can be reached by metro within 25 minutes. It is a 20 minutes' drive from Hongqiao Airport and 40-minute ride from Pudong Airport. Shanghai Disneyland is a 46-minute drive away. The Bund is a 4-minute drive away.
Golden and brown hues characterize guest rooms, which are fitted with a flat-screen TV and work desk. A personal safe and spacious marble bathroom are also included.
Guests may exercise at the well-equipped fitness center or enjoy a game of billiards. The hotel also provides a business center and laundry services.
Bo Yuet Jin serves traditional Cantonese cuisine. You can also experience the authentic elegance of fine French cuisine here at the Central Grill on the 2nd floor with an extensive list of wines at your selection. Other dining options include buffet meals at the coffee shop and drinks at the lobby lounge.
When would you like to stay at Grand Central Hotel Shanghai?
Luxurious and modern, this spacious air-conditioned room comes with a flat-screen TV, personal safe and mini-bar. Private bathroom has a bathtub.
Luxurious and modern, this air-conditioned suite comes with a separate living room and an additional guest toilet. Includes a sofa set, flat-screen TV and a well-stocked minibar. Private bathroom has a bathtub.
Luxurious and modern, this air-conditioned room comes with a flat-screen TV, personal safe and minibar. Private bathroom has a bathtub.
Luxurious and modern, this spacious air-conditioned room comes with a flat-screen TV, a personal safe, a mini-bar and an iPod dock. Private bathroom has a bathtub.
For those who check in with third guest, an additional charge is required. The extra fee includes the access to executive lounge and 1 extra bed. More information, please contact the property in advance.
This twin/double room features a bathrobe, air conditioning and iPod docking station.
1. Prior reservation is recommended and subject to availability.
2. Offer is not valid in conjunction with other promotions.
This room offers breakfast for 1 guest.
Grand Central Hotel Shanghai is situated in the bustling Bund-East Nanjing Road Pedestrian Street commercial area in Shanghai. 3-minute walking to metro subway line 1, 2 , 8; 20-minute drive to Shanghai Hongqiao Airport, 45-minute drive to Shanghai Pudong International Airport. This unique and elegant five-star hotel has 353 rooms, offering a comprehensive range of luxurious amenities, including high-speed internet access, lavish bath amenities and convenient high-tech automation features. Whether visiting on business or escaping for a romantic holiday, the Grand Central Hotel Shanghai pampers guests with classic grandeur and dynamic service. Another highlight of Grand Central Hotel Shanghai is the Shanghai Grand Ballroom, a stunning, pillar-free room with an astonishing seven-metre high ceiling. World-class meeting facilities ensure that every event is a success. The Grand Central Hotel offers a choice of two fine hotels: enjoy authentic French cuisine at L'ile de France, or explore contemporary Cantonese delicacies at Bo Yuet Hin. The coffee shop and lobby lounge are also available for casual gatherings.
House Rules Grand Central Hotel Shanghai takes special requests – add in the next step!
Greatwall Peony Dragon Jin Grand Central Hotel Shanghai accepts these cards and reserves the right to temporarily hold an amount prior to arrival.
Grand Central Hotel Shanghai accepts these cards and reserves the right to temporarily hold an amount prior to arrival.
Children below 3.9 feet can enjoy 50% discount for breakfast; children above 3.9 feet dine with full price.
Very convenient location (closed to metro and closed to shopping place), excellent service and comfortable and clean hotel.
Room is very comfortable. Location is very near to Nanjing road and about 15 min walk to the bund.
There is no refrigerator in our room that we stayed in.
late check in , made me wait for two hours.
This hotel has an amazing location. The metro is less than 5 minute walk, one of the biggest shopping streets is in the parallel street and you can easily walk to the Bund and the people's square. Other tourist attractions are one or two stations away. The room was great and the breakfast very good.
That I couldn't stay longer.
WiFi connection was very poor.
Property is located in very convenient location and all staffs are very cooperative and helpful.
Very limited breakfast offer for a 5 star hotel. The healthy choice option was almost zero.
Have to order something to sit at the lobby area? That is news to me. The coffee was okay, but the cakes and especially the green dunplings were horrible, like 3 days old and expensive.
The location was good, and the room is big. The breakfast price is fair, comparing to other 5 star hotels.
Location were not bad. Staffs were very rude. Rooms were not clean.


Is modern painting as a serious art form dead? This was a topic of discussion on a LinkedIn web site recently and the answer is-- in some venues, yes. The good news is that out of these ashes, clear, meaningful art will arise.
Art in major galleries today is suffering like a drug and/or alcohol addict. It's addictive to get a lot of publicity for creating off-the-wall works. Sometimes these "addicts" have to hit rock bottom before climbing out of the abyss. Rock Bottom is definitely here.
In New York at MOMA (Museum of Modern Art) there is a show of disparate works, Alibis, by Sigmar Polke. He states " Art is Lies. Lies are art, Right?" He strove to have "no meaning" in his art. He succeeded. His works do not draw one in. One doesn't know where to look. One can easily breeze right by his massive amount of work.
In an odd way Polke's work relates to a show at the Asia Society in NY, "Golden Visions of Densatil" The Communists, attempting to destroy all art, literally obliterated the Densatil Tibetan Buddhist Monastery in their cultural revolution. Fortunately, photos of the Monastery before the destruction show the art. It was like looking into a home on the TV show "Hoarders". Chaos reigned with hundreds of pieces of art jammed together and stacked at least 10 feet high or higher in the monastery. Again, one does not know where to look. However, some of the art from Densatil was rescued and brought together for a show at the Asia Society. Here the works were separated, each one given a chance to breathe and be viewed separately. In this setting they are more meaningful and one is drawn to take a closer look.
This is where art is going. Rising from the ashes, look for art that is clear and meaningful. Even if one doesn't understand the work one should have a sense that the artist is trying to say SOMETHING.


"Middlebury College is on trial now. Its administration will either forthrightly defend liberal democratic norms, or it will capitulate. There is no middle ground. … These little Maoists studying at elite colleges and universities like Middlebury are on the fast track to move into the American ruling class. You see what they will do to dissenters. They must be resisted — and resisted strongly.


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text:
Q: POST request raises AssertionError I'm doing a POST request to an API, the code looks like this:
@gen.coroutine
def call():
    ...
    response = yield AsyncHTTPClient().fetch(
        HTTPRequest(
            url='https://api.mywebsite.com/v1/users',
            headers=headers,
            method='POST',
            body=json.dumps(body),
            validate_cert=False
        )
    )
    print response, response.body

if __name__ == "__main__":
    tornado.ioloop.IOLoop.current().run_sync(call)

The server responds first time with 201 Created and the second time with 200 OK.
But for that code I get this error for the first time. The second time works
Traceback (most recent call last):
  File "t.py", line 49, in <module>
    tornado.ioloop.IOLoop.current().run_sync(call)
  File "/usr/lib/python2.7/dist-packages/tornado/ioloop.py", line 389, in run_sync
    return future_cell[0].result()
  File "/usr/lib/python2.7/dist-packages/tornado/concurrent.py", line 129, in result
    raise_exc_info(self.__exc_info)
  File "/usr/lib/python2.7/dist-packages/tornado/stack_context.py", line 302, in wrapped
    ret = fn(*args, **kwargs)
  File "/usr/lib/python2.7/dist-packages/tornado/gen.py", line 574, in inner
    self.set_result(key, result)
  File "/usr/lib/python2.7/dist-packages/tornado/gen.py", line 500, in set_result
    self.run()
  File "/usr/lib/python2.7/dist-packages/tornado/gen.py", line 529, in run
    yielded = self.gen.throw(*exc_info)
  File "t.py", line 43, in call
    validate_cert=False
  File "/usr/lib/python2.7/dist-packages/tornado/gen.py", line 520, in run
    next = self.yield_point.get_result()
  File "/usr/lib/python2.7/dist-packages/tornado/gen.py", line 409, in get_result
    return self.runner.pop_result(self.key).result()
  File "/usr/lib/python2.7/dist-packages/tornado/concurrent.py", line 131, in result
    return super(TracebackFuture, self).result(timeout=timeout)
  File "/usr/lib/python2.7/dist-packages/concurrent/futures/_base.py", line 401, in result
    return self.__get_result()
  File "/usr/lib/python2.7/dist-packages/concurrent/futures/_base.py", line 360, in __get_result
    raise self._exception
AssertionError

A: Looks like you are using sync function HTTPRequest in future

from tornado.httpclient import AsyncHTTPClient
from tornado.httpclient import HTTPResponse

  def call():
        response = None
        http_client = AsyncHTTPClient()
        try:
            body = json.dumps(body)
            response: HTTPResponse = yield http_client.fetch('https://api.mywebsite.com/v1/users', method='POST', body=str(body), headers=headers, request_timeout=5)
        except Exception as e:
            print('get_request error:{0}'.format(e))

redpajama_set_name: RedPajamaStackExchange


We are open on Saturdays for busy families and accepting Medicaid and most major insurance plans. Our top priorities are always patient comfort, convenience, and great smiles.
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We welcome all travelers to experience our Fairfield Inn & Suites Branson hotel. Located just across the road from the Titanic Museum, our hotel in Branson, MO promises to satisfy each guests lodging needs with our premiere location, dedicated staff, and guest-oriented amenities. After a visit to the Titanic Museum, stop by the Hollywood Wax Museum, followed by some fun at the White Water Amusement Park. Experience Ballparks of America, just 5 minutes from our Branson hotel, and get some quality shopping done at Tanger Outlet Mall. Each morning is an opportunity to feast on our complimentary hot breakfast, with options for any breakfast preference. After a day exploring Branson, wind down in our indoor pool, and connect with family using our free wireless high speed Internet. At the Fairfield Inn & Suites Branson, you're our #1 priority. We promise you'll be satisfied, or we'll make it right. That's our commitment to you. Ask us about The Fairfield 100% Guarantee.


"SUPER SONS OF TOMORROW" finale!
"LEGACY" part five! Sovereign has come, and she's brought Olympus with her. The Great Darkness is growing, and it's ready to consume the Earth—and the Justice League is missing!
It's no fun for Jon Kent to be visiting Big City with his parents for the funeral of an old friend.
"LEGACY" finale! The truth about Sovereign is revealed—and her identity will shake the Justice League to its core!


text:
DHBVN to spend Rs 702 cr to boost power system
Chandigarh, July 28: The Dakshin Haryana Bijli Vitran Nigam (DHBVN) has chalked out a Rs 702 crore plan to renovate, augment and strengthen power distribution system in its area.
Stating this here today, a DHBVN spokesman said the Nigam had launched a scheme involving segregation of domestic and agriculture loads in villages at a cost of Rs 268 crore.
The scheme for segregation of domestic and agriculture load was an innovative scheme that could impact the day to day life in entire rural area, he said.
Implementation of the scheme would improve voltage level and quality of power supply to tube wells during pre-determined period and it would ensure uninterrupted power supply in domestic sector of villages.
Besides, the DHBVN planned to spend Rs 139 crore for rehabilitation of 100 overloaded and lengthier feeders of 11 kv by dividing each feeder into 2 or 3 feeders.
During the process of rehabilitation of the feeders, the complete system would be renovated and demand of additional distribution transformers would be met with.
To inject power in this strong distribution system, DHBVN has started construction of 37 additional substations and augmentation of ten existing substations of 33 KV level in rural area having target to complete with in a period of one year.
Under this plan costing Rs 37 crore plan, the capacity of transmission and distribution capacity would be augmented by 385 MVA, overcoming the problem of low voltage.
To improve power distribution system in villages, DHBVN was creating High Voltage Distribution System (HVDS) and Low Voltage Distribution System (LVDS) of power supply at a cost of Rs 258 crore.
Lord Krishna, Hanuman greatest diplomats of world: S Jaishankar