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Brazilian Journal of Microbiology
Print version ISSN 1517-8382
Braz. J. Microbiol. vol.42 no.3 São Paulo July/Sept. 2011
Identification and biological activity of potential probiotic bacterium isolated from the stomach mucus of breast-fed lamb
H. Kiňová Sepová*; M. Dubničková; A. Bilková; M. Bukovský; L. Bezáková
Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic
The lactic acid bacterium E isolated from the stomach mucus of breast-fed lamb was identified by sequencing of 16S rDNA fragment and species-specific PCR as Lactobacillus reuteri. Its potential antimicrobial activity and ability to modulate immune system in vitro and in vivo was determined. The growth inhibition of potential pathogens decreased from Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica ser. Minnesota to Escherichia coli. The lowest inhibition activity was observed in the case of Candida albicans. The ability of L. reuteri E to modulate biological activities of human and mouse mononuclear cells was estimated in vitro and in vivo, respectively. The production of IL-1β by monocytes in vitro was significantly induced by L. reuteri E (relative activity 2.47). The ability to modulate biological activities of mononuclear cells by living L. reuteri E cells in vitro in comparison to disintegrated L. reuteri E cells in vivo differed. For example lysozyme activity in vitro was inhibited while in vivo was stimulated (relative activities 0.30 and 1.83, respectively). The peroxidase activity in vitro was stimulated while in vivo was inhibited (relative activities 1.53 and 0.17, respectively). Obtained results indicate that L. reuteri E is potential candidate to be used in probiotic preparations for animals and/or human.
Key words: Lactobacillus reuteri - mononuclear cells - PCR - antagonistic activity - phagocytosis
Lactobacillus spp. are the natural inhabitants of human and animal colon and vagina. Lactobacilli belong to the group of lactic acid bacteria that ferment saccharides to lactic acid. Specific strains of lactobacilli that are able to colonize body cavities seem to play an important role in maintaining a balanced microflora or preventing and lowering the incidence of recurrent urinary or digestive tract infections (18). Presence of Lactobacillus strains has positive effect on some digestive diseases, e.g. lactose intolerance, inflammatory bowel disease, constipation or post-antibiotic diarrhea (15).
Nowadays many researchers are looking for different probiotic bacteria that exhibit advantageous properties on human and/or animal health. These can be used in human or veterinary medicine in food supplements and/or probiotic preparations. According to FAO/WHO (5) guidelines, probiotic microorganisms must be exactly identified and characterize on strain level in several points: acid and bile salt resistance, ability to produce antimicrobial substances, adhere to mucus and modulate immune system.
The aim of this study was to identify and characterize bacterial isolate E derived from the stomach mucus of breast-fed lamb (Očová, Slovakia). According to microbiological and biochemical tests bacterium E showed several characteristics of lactic acid bacteria. For exact identification of isolate E, sequencing of fragment of 16S rDNA and PCR were used. To detect inhibition activity of isolate E against potential pathogens the streak line method on agar plates was performed. The ability of strain E to affect immune system of host was tested by in vitro and in vivo experiments.
Bacterial strains and growth conditions
Bacterium E was isolated from the stomach mucus of three weeks old breast-fed lamb (Očová, Slovakia). As the reference strains for lactobacilli identification Lactobacillus reuteri CCM 3625 and L. acidophilus CCM 4833 (Czech Collection of Microorganisms, Brno, Czech Republic) were used. Lactobacilli were cultivated in de Man-Sharpe-Rogosa (MRS) broth (Oxoid, UK) 18 h in anaerobic conditions at 37ºC. Antagonistic activity of strain E was tested against potential pathogens: Staphylococcus aureus CNCTC Mau 29/58 (Czech National Collection of Type Cultures, Prague, Czech Republic), Pseudomonas aeruginosa CNCTC Ps 79/70, Escherichia coli ATCC 11229 (American Type Culture Collection, Manassas, Virginia, USA), Salmonella enterica ser. Minnesota SK 99/39 (Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic) and Candida albicans CCM 8180. Strains of potential pathogens were cultivated in peptone water (peptone 10.0 g/L; sodium chloride 5.0 g/L; disodium hydrogen phosphate dodecahydrate 9.0 g/L; potassium dihydrogenphosphate 1.5 g/L) for 18 h at 37ºC. For testing immunomodulatory activity E. coli ATCC 11229, Micrococcus luteus ATCC 4698, Enterococcus faecalis CCM 2122 and Candida albicans CCM 8180 were used.
Identification of lactobacilli
Chromosomal DNA was isolated from 3 mL of an overnight culture of bacteria using DNA Easy® Tissue Kit (Qiagen, Germany), according to the manufacturer's protocol. For identification of new isolate E on the species level amplification of the part of 16S rDNA by PCR and sequencing of PCR product were used. The PCR was performed according to Heilig et al. (10). Sequence analysis was performed at the Faculty of Natural Sciences (Comenius University, Bratislava, Slovakia). The nucleotide sequence was processed in computer software Vector NTI and compared with known sequences in GenBank (www.ncbi.nlm.nih.gov/BLAST). Species-specific PCR for L. reuteri was performed according to Kwon et al. (14).
Electrophoresis of PCR products
The PCR products (8 µL) were separated in 1.5% agarose gel containing 1xTAE (18) in electric field of 80 V. DNA was stained by "GoldView" (SBS Genetech Co. Ltd., China) and visualised under UV light (λ = 254 nm).
Antagonistic activity
The turbidity of the broth cultures of strain E and potential pathogens was adjusted to equal that of 4 and 3 McFarland standard, respectively. 10 µL of Lactobacillus culture were seeded in line to the middle of Petri dish containing MRS agar. Cultures were cultivated for 24 h in anaerobic conditions at 37ºC. After incubation, lactobacilli were killed by exposure to chloroform gases for 2 h. Then the diluted cultures of potential pathogens were inoculated in volume 1 µL, in duplicate, perpendicular to the streak line of dead lactobacilli. Pathogens were incubated 24 h in aerobic conditions at 37ºC. After final incubation, zones of growth inhibition between lines of lactobacilli and pathogens were measured. Experiments were performed in twelve parallels. The degree of antagonistic activity was evaluated as following: 0 - 12 mm as low inhibition, 13 - 25 mm as intermediate inhibition and more than 25 mm as high inhibition according to Hütt et al. (12).
Isolation of human mononuclear cells and their pre-incubation with strain E
The human mononuclear cells (MOs) from 6 healthy independent volunteers (OHT-Derer's Hospital, Bratislava, Slovakia) were isolated and purified by HistoPaque-1077 (Sigma, USA) according to Böyum et al. (2). MOs used were suspended in RPMI medium (diluted in saline 1:4) supplemented with 10% fetal bovine serum to concentration 2x106 cells/mL. In the next step, they were cultivated 18 h (at 37ºC in 5% CO2 atmosphere) exposed to 3x102 CFU/mL and 6x102 CFU/mL of strain E suspension according to Bukovský et al. (3). Control samples contained untreated human MOs.
Administration of mouse model
For the experiments male C57 BL6 inbreeded mice (25-27 g) (Velaz, Czech Republic) were used. Bacterial suspensions of strain E (the dose of strain E was 3x105 CFU/kg of mouse) were administered intraperitoneally to mice daily during 5 days period. To animals in control group physiological saline was applied. Mice were weighted before and after experiment. All experiments were performed in accordance with the Proclamation of the Government of Slovak Republic in July 9, 2003, Law Nº 289/2003 in the Collection of Laws of the Slovak Republic, and in accordance to the local Ethical Committee of Faculty of Pharmacy (Comenius University, Bratislava, Slovakia). Mononuclear cells were isolated from mouse peritoneum using 5 mL of physiological saline.
Phagocytic activity and index
Phagocytic activity and index were determined microscopically. 100 µL of MOs (2x106 cells/mL) were incubated 1 h at 37ºC with 50 µL of heat inactivated E. faecalis (5x108 cells/mL) (3). The used ration strain/Wright's staining was performed according to the conventional methodology. Phagocytic activity was calculated as a percentage of the phagocyting mononuclear cells. Phagocytic index was determined as the average number of heat inactivated bacteria ingested per one active MO.
Microbicidal and metabolic activities
MOs, influenced in vivo and in vitro with strain E, were ultrasonically disintegrated (18 kHz, 10 s). The microbicidal activity of homogenate was tested on S. aureus, E. coli and C. albicans. 24 h cultures of S. aureus (grown on blood agar) and E. coli (grown on Endo agar) and 48 h culture of C. albicans (cultured on Sabouraud's agar) were washed twice in physiological saline and adjusted to OD540= 0.35 (all culture media were purchased from Imuna, Slovakia). Bacterial suspensions were resuspended to 1x105 cells/mL. The turbidity of C. albicans was adjusted to 1x104 cells/mL. Microbial cultures in different amounts (250 µL of E. coli, 100 µL of S. aureus, 100 µL of C. albicans) were incubated with 100 µL of crude homogenate of MOs. Incubation at 37ºC lasted 1 h. The number of surviving microbes was estimated after cultivation on appropriate media (bacteria - 24 h at 37ºC on blood agar or Endo agar, yeast 48 h at 25ºC on Sabouraud's agar). In control sample untreated MOs were used. Microbicidal activity was expressed as difference in number of CFU/mL between control and test samples.
The ultrasonically disintegrated monocytes (18 kHz, 10 s) were centrifuged (2500 g, 10 min, 4ºC). For lysozyme activity 150 µL of supernatant were mixed with 50 µL of the suspension of M. luteus (OD410 = 0.8) in phosphate buffer (pH 6.2, 0.07 mol/L KH2PO4 and 0.07 mol/L Na2HPO4.12H2O). After 20 min absorbance at 410 nm was measured (MR 5000, Dynatech, USA). For the determination of peroxidase activity 50 µL of the peroxidase substrate (benzene-1,2-diamine - 0.5 mg/mL, freshly diluted H2O2 - 10 µL/mL and 0.1 M sodium citrate tribasic dihydrate in distilled H2O, pH 5.0) was added to 150 µL monocyte supernatant. After incubation at 22ºC for 20 min reaction was stopped with H2SO4 (50 µL, 4 mol/L) and absorbance was measured at 490 nm (MR 5000, Dynatech, USA).
In vitro experiments were performed on human monocytes and repeated at least 4 times. In vivo experiments were carried out in two groups of 6 mice.
Influence on IL-1 production
Ability of bacterium E to induce production of IL-1 by phagocytes was determined spectrophotometrically (A450; MR 5000, Dynatech, USA). 2x106 phagocytes per mL were cultivated with strain E (6x102 CFU/mL) in total volume 50 µL in RPMI medium supplemented with 10% fetal bovine serum 18 h at 37ºC. Untreated monocytes were used as a control. The amount of IL-1β was determined by enzyme immunoassay method according to Immunotech's recommendations (Immunotech, France).
Statistical analysis
For calculations and statistical analysis OriginPro 7.5 was used. Data are expressed as mean values ± standard deviations or as relative activities (the control sample was considered as 1.00). The statistical comparison between control and samples was performed by Student's t-test. The value p < 0.05 was regarded as statistically significant.
Identification of strain E
Several bacteria were isolated from the stomach mucus of breast-fed lamb. Isolate E showed attributes typical for genus Lactobacillus by microbiological and biochemical tests (Bukovský et al. - data unpublished). For unambiguous identification the partial nucleotide sequence of the 16S rDNA was used. Amplification of this gene was performed using primer set Bact-0011f and Lab-0677r (10). Nucleotide sequence of cca 700 bp PCR product of strain E was used for the analysis of sequence similarity through BLAST (www.ncbi.nlm.nih.gov/BLAST). It showed a significant homology to the 16S rDNA of known Lactobacillus spp. The highest homology (99%) was achieved with strain L. reuteri LU3 (AY 735 406) (Figure 1). Identification of isolate E on species level was evaluated by species-specific PCR for L. reuteri according to Kwon et al. (14). Specific PCR product of 1100 bp length was observed. Finally the new isolate was identified as L. reuteri and named L. reuteri E.
Antagonistic activity of L. reuteri E
Antagonistic activity of L. reuteri E was tested against several potential pathogens. The streak line method on agar plates with few modifications was used (12). The highest inhibition activity was detected against S. aureus; the inhibition zone was 25.75 ± 4.90 mm from the line of lactobacilli. Other pathogens were inhibited weaker: P. aeruginosa (23.17 ± 3.42 mm), S. enterica ser. Minnesota (21.83 ± 3.47 mm), E. coli (20.75 ± 2.58 mm) and the lowest inhibition zone was observed in the case of C. albicans, 1.67 ± 1.61 mm (Figure 2).
The effect of L. reuteri E on human mononuclear cells in vitro
MOs were incubated with L. reuteri E overnight. After incubation the number of MOs was not significantly changed, in comparison to control sample (Table 1). The ability of L. reuteri E to affect the phagocytic activity and index of phagocytosis was estimated. Significant increase of both phagocytic activity and index was observed. The relative phagocytic activity was 1.09 and relative phagocytic index was 1.36. Microbicidal activity was presented as a number of killed CFU by MOs, that were pre-treated by L. reuteri E. Microbicidal activities of MO cells treated with L. reuteri E against E. coli and C. albicans were decreased, relative activities were 0.72 and 0.42, respectively. The lysozyme activity of induced MOs was significantly decreased (relative activity 0.30). Another protective metabolic activity of MOs against infection, peroxidase activity, was significantly stimulated by L. reuteri E (relative activity 1.53).
The phagocytes IL-1β production was significantly induced in vitro, relative activity was 2.47 (Table 1).
The effect of L. reuteri E on mouse macrophages in vivo
L. reuteri E exhibited no lethal activity after 5 days of intraperitoneal application to mice. The average weight of mice in control group was not significantly changed after 5 days (from 24.8 ± 0.9 g to 25.6 ± 1.8 g). The average weight of mice pretreated with L. reuteri E was significantly increased in comparison to control group (from 23.6 ± 1.5 g to 27.4 ± 0.1 g). Isolated peritoneal mouse macrophages were tested for several biological activities (Table 2). Pre-treatment with L. reuteri E increased phagocytic activity and phagocytic index of mouse phagocytes. Relative phagocytic activity was 1.13 and relative phagocytic index was 1.51. Microbicidal activities of macrophages isolated from pre-treated mice were not influenced. The lysozyme activity of effected macrophages isolated from pre-treated mice was significantly increased (relative activity 1.83). Peroxidase activity was significantly inhibited (relative activity 0.17) (Table 2).
For identification of new isolate E derived from the stomach mucus of lamb the nucleotide sequencing was performed. 665 bp nucleotide sequence of PCR product of isolate E showed highest homology (99%) with the 16S rDNA of L. reuteri LU3 (AY 735 406). Two nucleotides in obtained sequence were identified ambiguously (any of purines, A or G). In the reference sequence from GenBank there is G on both of these positions (Figure 1). Identification was confirmed by species-specific PCR (14), 1100 bp product was observed. This verifies the previous results, which recognized new isolate E as Lactobacillus reuteri.
The inhibition activity of L. reuteri E against potential pathogens decreased as follows: S. aureus > P. aeruginosa > S. enterica ser. Minnesota > E. coli > C. albicans (Figure 2). According to Ocaña et al. (15) S. aureus growth is inhibited by Lactobacillus strains, which produce hydrogen peroxide. This might be one of the reasons of high growth inhibition of S. aureus by L. reuteri E. Production of bacteriocins should be another explanation of this effect. Bacteriocins, the antimicrobial proteinaceous substances, are generally defined as substances that kill related bacteria (4). Bacteriocins produced by Gram-positive (G+) bacteria are active mostly against other G+ bacteria. Weaker inhibition activity of L. reuteri E was observed in the case of Gram-negatives (G-) than in S.aureus (G+). C. albicans that has diametrically different composition of cellular coating was inhibited the least.
Besides the exclusion of pathogens from the gut mucus lactobacilli can induce also host resistance to microbial pathogens by stimulation of non-specific immunity (6).
For evaluation of immunomodulatory potential of L. reuteri E in vitro and in vivo tests on mammalian cells were performed. The ability of induced MOs by L. reuteri E to phagocyte E. faecalis was comparable in both experiments (relative activity in vivo 1.13 and in vitro 1.09). Phagocytes release toxic agents, e.g., reactive oxygen intermediates and lytic enzymes, in various inflammatory reactions. Phagocytic activity results in the further recruitment of immunocompetent cells and the generation of inflammatory response. On the other hand microbicidal effect of MOs is limited to the production of cationic peptide molecules of different size and structure. During their interaction with bacteria they tend to find specific target in the bacterial cell membrane (9, 16). Modification of lipopolysaccharid (in G- bacterial membrane) or teichoic acid (in G+ bacterial membrane) often makes bacteria resistant to microbicidal activity of mononuclear cells (8, 16). The difference in bactericidal activity of modulated phagocyting cells against E. coli and S. aureus can be explained by the different mechanism of action of MOs on G- and G+ bacteria. Microbicidal activity of pre-treated human MO cells against E. coli and C. albicans in vitro was inhibited (Table 1). The obtained results in the case of C. albicans are in good agreement with data obtained by streak line method.
Results of metabolic activities in vitro (human MO cells) and in vivo (mouse macrophages) showed the opposite course. Lysozyme activity in vitro was reduced, while in vivo increased. Vice versa, the peroxidase activity of MOs was increased in vitro while in vivo was decreased. Explanation of differences in peroxidase and lysozyme activities in vivo and in vitro could be in different administration of lactobacilli to mononuclear cells. In in vitro experiment live lactobacilli were used while in in vivo test lactobacilli were disintegrated before administration. Alive lactic acid bacteria can produce several extracellular substances e.g. organic acids, H2O2, bacteriocins etc. (1) which can affect metabolic activities of immunocompetent cells. Moreover organic acids produced by lactobacilli can reduce pH of environment (in our experiment phosphate buffer, pH 6.2) and simultaneously influence activity of lysozyme. Optimal pH for lysozyme activity is 6.2.
Anyway, there are also other agents and biochemical systems, except H2O2 production, which can play a role in the modulation of the immune system of mouse.
Pro-inflammatory cytokines, including interleukin 1, play a pivotal, yet ambigous, role in inflammation. L. reuteri induced several proinflammatory and/or Th1 cytokines, such as IL-1, IL-2, and TNF, but not antiinflammatory or Th2 cytokines, such as IL-10. The authors are open to dispute, that outcome of probiotic treatment may depend on the immunologic state of the host and that changes in cytokine profile induced by probiotic strains may be site specific and dependent on the experimental system used (7).
Immunomodulating effects of probiotic bacteria may depend on the immunologic state of the host. It was further suggested that differences between specific strains of probiotic bacteria may exist. It was also observed that whole bacterial cells enhance immune cells proliferation and induce pro-inflammatory cytokines production, concretely tumor necrosis factor and interleukin 6. In contrast probiotic bacteria mediate suppression of lymphocyte proliferation and T cells cytokine production (13).
Isolauri et al. (13) attempted to compare the anti-proliferative effect of several probiotic bacterial strains in their nonviable forms. The suppressive effect of 10 µmol/L dexamethasone was compared with that of successful probiotics. Results indicated that some probiotic bacteria possess significant anti-inflammatory properties comparable to the therapeutic pharmaceutical agent. These findings further implicate the potential use of probiotic bacteria as immunomodulatory agents.
To estimate anti- or pro-inflammatory properties of L. reuteri E, bacterial cells were tested for ability to affect IL1β production of phagocytes in vitro (Table 1). There are several studies investigating the usage of probiotic cells components for cytokine production. Lactobacilli, bifidobacteria and E. coli Nissle 1917 can affect pro-and anti-inflammatory cytokines production in peripheral blood MOs (11).
Our results indicate that L. reuteri E isolated from the stomach mucus of lamb has auspicious probiotic properties, for example ability to inhibit the growth of several potential pathogens and modulate mammal's MOs activities in vitro as well as in vivo. Taken together, obtained data indicate that L. reuteri E is potential candidate for use in probiotic preparations for animals and/or human. However, additional experiments and testing are necessary.
This work was supported by grants of Slovak R&D Assistance Agency (APVV/0354/07) and Ministry of Education SR (VEGA 1/4290/07).
1. Annuk, H.; Shchepetova, J.; Kullisaar, T.; Songisepp, E.; Zilmer, M.; Mikelsaar, M. (2003): Characterization of intestinal lactobacilli as putative probiotic candidates. J. Appl. Microbiol. 94, p. 403-412. [ Links ]
2. Böyum, A. (1968): Isolation of leucocytes from human blood. Further observations. Methylcellulose dextran and ficoll as erythrocyte aggregating agents. Scand. J. Clin. Lab. Invest. Suppl. 97, p. 31-50. [ Links ]
3. Bukovský, M.; Mlynarčík, D.; Ondráčková, V. (1996). Immunomodulatory activity of amphiphilic antimicrobials on mouse macrophages. Int. J. Immunopharmacol. 18, p. 423-426. [ Links ]
4. Cleveland, J.; Montville, T.J.; Nes, I.F.; Chikindas, M.L. (2001). Bacteriocins: safe, natural antimicrobials for food preservation. Int. J. Food Microbiol. 71, p. 1-20. [ Links ]
5. FAO/WHO. 2002. Guidelines for the Evaluation of Probiotics in Food. Available at: ftp://ftp.fao.org/es/esn/food/wgreport2.pdf. Accessed 8 July 2010. [ Links ]
6. Foligne, B.; Nutten, S.; Grangette, C.; Dennin, V.; Goudercourt, D.; Poiret, S.; Dewulf, J.; Brassart, D.; Mercenier, A.; Pot, B. (2007). Correlation between in vivo and in vitro immunomodulation properties of lactic acid bacteria. World J. Gastroenterol. 13, p. 236-243. [ Links ]
7. Forsythe, P.; Inman, M. D.; Bienenstock, J. (2007). Oral treatment with live Lactobacillus reuteri inhibits the allergic airway response in mice. Am. J. Respir. Crit. Care Med. 175, p 561-569. [ Links ]
8. Gunn J.S. (2001). Bacterial modification of LPS and resistance to antimicrobial peptides. J. Endotox. Res. 7, p. 57-62. [ Links ]
9. Hancock, R.E.W.; Rozek, A. (2002). Role of membranes in the activities of antimicrobial cationic peptides. FEMS Microbiol. Lett. 206, p. 143-149. [ Links ]
10. Heilig, H.G.H.J.; Zoetendal, E.G.; Vaughan, E.E.; Marteau, P.; Akkermans, A.D.L.; de Vos, W.M. (2002). Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA. Appl. Environ. Microbiol. 68, p. 114-123. [ Links ]
11. Helwig U.; Lammers K.M.; Rizzello F.; Brigidi P.; Rohleder V.; Caramelli E.; Gionchetti P.; Schrezenmeir J.; Foelsch U.R.; Schreiber S.; Campieri M. (2006). Lactobacilli, bifidobacteria and E. coli Nissle induce pro-and anti-inflammatory cytokines in peripheral blood mononuclear cells. World J. Gastroenterol. 12, p. 5978-5986. [ Links ]
12. Hütt, P.; Shchepetova, J.; LUivukene, K.; Kullisaar, T.; Mikelsaar, M. (2006). Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero-and uropathogens. J. Appl. Microbiol. 100, p. 1324-1332. [ Links ]
13. Isolauri E.; Sütas Y.; Kankaanpää P.; Arvilommi H.; Salminen S. (2001) Probiotics: effects on immunity, Am. J. Clin. Nutr. 73, p. 2001, 444-450. [ Links ]
14. Kwon, H.S.; Yang, E.H.; Yeon, S.W.; Kang, B.H.; Kim, T.Y. (2004). Rapid identification of probiotic Lactobacillus species by multiplex PCR using species - specific primers based on the region extending from 16S rRNA. FEMS Microbiol. Lett. 239, p. 267-275. [ Links ]
15. Ocaña, V.S.; Pesce de Ruiz Holgado, A.A.; Nader-Macías, M.E. (1999). Growth inhibition of Staphylococcus aureus by H2O2-producing Lactobacillus paracasei subsp. paracasei isolated from human vagina. FEMS Immunol. Med. Microbiol. 23, p. 87-92. [ Links ]
16. Pesch A. (2002). How do bacteria resist human antimicrobial peptides? Trends Microbiol. 10, p. 179-185. [ Links ]
17. Tannock, G.W.; Tilsala-Timisjarvi, A.; Rodtong, S.; Ng, J.; Munro, K.; Alatossava, T. (1999). Identification of Lactobacillus isolates from the gastrointestinal tract, silage, and yoghurt by 16S - 23S rRNA intergenic spacer region sequence comparisons. Appl. Environ. Microbiol. 65, p. 4264-4267. [ Links ]
18. Versalovic, J.; Koeuth, T.; Lupski, J.R. (1991). Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucl. Acid Res. 19, p. 6823-6831. [ Links ]
19. Wilks, M.; Wiggins, R.; Whiley, A.; Hennessy, E.; Warwick, S.; Porter, H.; Corfield, A.; Millar, M. (2004). Identification and H2O2 production of vaginal lactobacilli from pregnant women at high risk of preterm birth rRNA through 23S and relation with outcome. J. Clin. Microbiol. 42, p. 713-717. [ Links ]
Submitted: February 25, 2010; Approved: March 14, 2011.
* Corresponding Author. Mailing address: Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Kalinciakova 8, 832 32 Bratislava, Slovak Republic.; Tel.: +421 2 50117313.; E-mail: firstname.lastname@example.com


text:
I was wondering why if I drink coffee always want to go to toilet? no matter what the time I drink it.?
8 Answers
- MarkLv 73 weeks ago
Côffėė is full of caffiene like any other
liquid will mak you feel like urinating all
over the place so that's the scoop on it !!
... ... ... ... ... ... ... ... ... ...
,,, ,,, ,,, ,,, ,,, ,,, ,,, ,,, ,,, ,,,
- Obi Wan KnievelLv 73 weeks ago
Because coffee has that effect of the body. That's why.
No really, it does. Caffeine is a stimulant, but it's also a mild diuretic (makes you pee) and laxative (makes you poop).
Alcohol is the same even though it's an opposite. Alcohol is a depressant, but it's also a diuretic and a laxative. That's why they say Schlitz gives you the Schitz.
- Anonymous3 weeks ago
It's a diuretic.
- 3 weeks ago
It happens to me too. Do you drink coffee often? I don't and I always assumed it was because my stomach is not used to it.
- How do you think about the answers? You can sign in to vote the answer.
- 3 weeks ago
Coffee is a laxative and a diuretic, this can affect different people in different ways... I'd say ditch the coffee.
- mrh-slosLv 73 weeks ago
Caffeine is a diuretic. If it is a problem try decaffeinated coffee.
- keerokLv 73 weeks ago
Sudden urge to pee?
Caffeine, the main ingredient of coffee is a diuretic, a drug that makes you pee. Ttada!
- 3 weeks ago
Coffee is a mild laxative, and its also a diuretic. So it naturally makes people need to go to the toilet

subset: commoncrawl


text:
Getting to the Farm
Most festivals are fairly straightforward when it comes to transportation. For Paradiso, you drive due east from Seattle, get in a line of cars, and get directed to your campsite. EDC is even simpler: Get to your hotel, hop in a shuttle to the Speedway, enter the festival grounds. Shambhala though is just a little different, especially if you're coming from the southern side of the Canadian border. Crossing the border can always be a hassle, so here are a few quick tips for anyone heading to the Farm from the United States.
DMNW Pro-Tip #1: Save the entirety of the route to Shambhala in "Offline Maps" in Google Maps, to ensure you can still find your way in areas with spotty cell service. You can find step-by-step instructions on that here.
Google Map your trip to Shambhala from Seattle, and the first result will likely tell you to head east to Spokane, and then head due north from there until you hit the border. That route will take you to the Nelway Border Crossing. Once you cross in Nelway, you'll be approximately 10-15 minutes away from Shambhala, continuing on until you see signs directing you to turn left into the festival and onto a narrow dirt road. Follow that dirt road for a few minutes and you'll soon find yourself in the line of cars leading into the campsite entryway.
Keep in mind, Nelway is a tiny crossing, manned by just a few border patrol guards with 1-2 lines for entry. It's also the most-used road into Shambhala, so be prepared to wait for a long time. The guards know there's a festival happening 12 miles north of the border, and as such will tear apart each and every car to ensure nothing illegal is making its way into Canada. It should go without saying, but do not attempt to bring any narcotics into the country. Also worth noting, the Nelway crossing is open from 8am-midnight.
To avoid the longer lines, you can (and honestly should) opt for the Paterson Crossing (denoted above), heading north right when you hit the Gorge, taking WA-283 north rather than continuing on I-90 east. Paterson is open 24 hours, and in terms of driving time will add approximately 45 minutes to your trip. Even with the added time though, you'll get less hassle at the border, with shorter lines and a larger staff of border guards to get you across in a timely manner. Head about 10 minutes north of that crossing, and you can go to Ferraro Foods in Rossland for affordable local groceries, before heading east to the Farm.
If you're headed to the Paterson Crossing, be sure you're following the correct signs — there's another, smaller border station that follows the Columbia River that's also going to be small, crowded, and difficult to get through.
DMNW Pro-Tip #2: Be sure to withdraw Canadian money before you skip town to avoid ATM fees at the festival; call ahead to your local branch to make sure they carry foreign money, as not every bank does (take out at least $200-300 for the weekend just to be on the safe side). The exchange rate right now is favorable to Americans, with $1 equaling $1.32 Canadian dollars.
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Consider helping out in updating the wiki for 3.8.0 - see Path of Exile Wiki:To-do list/3.8.0.
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|Resists Elemental Damage
Leaves Trail of Ice
Imperator Stantinus Bitterblade is a unique Blackguard Elite, located on the top floor of The Upper Sceptre of God. He is one of Dominus' champions. He uses Cyclone and deals Cold Damage, leaving behind a trail of ice that slows players who walk into it.
Map variants
A stronger version of Imperator Stantinus Bitterblade, named Oriath's Vigil, is featured on the Courtyard MapCourtyard MapMap Level: 75
Map Tier: 8
Guild Character: 4Travel to this Map by using it in the Templar Laboratory or a personal Map Device. Maps can only be used once., where he is part of a triple boss.


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This makes the reader dislike Mahtilde, especially compared to Veronica, as her life is reasonably good. A woman of the Makhzoom family with good connections was found guilty of theft. His birthday is observed as a national holiday. Sakhr, a chief of a tribe, had helped the Prophet greatly in the siege of Taaif, for which he was naturally obliged to him. Contoh Essay Gunawan Muhammad – nikeairhuarache. The Prophet of Islam Peace be upon him was the last messenger of God to the whole world.
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At age 40, he experienced visions 1. How to Find No Essay Scholarships – collegesindelaware. There is none like him. Through the next five years Khadijah became aware of how reliable and resourceful Muhammad was and proposed marriage to him when he was twenty-five.
Prophet Muhammad's justice and equality
Do not follow your own desires and deviate from the truth Allah loves those who do their duty. There are differing accounts of how the muhammads exemplary the commencement of the caliphate led to the Shia and Sunni essay. Germany totalitarian state essay – eossportsbets. The Prophet asked Qays to mount his qn with him.
When they did not, Umar broke in, resulting in Fatimah's essays being broken by being pressed between the door and the wall causing her to miscarry Muhsin which led to her eventual death.
Essay muhhammad hazrat muhammad as an exemplary judge After making the journey to Bethlehem, Mary and Joseph had to stay in a stable, because the inn was full. Amazing Impressions They say that you should never leave a book by its essay my first impression of college, and, third regarding people, its true. Some people were prepared to forgo their shares and remit the Prophet's ransom but he declined saying that he could make no distinctions.
The Prophet took upon himself the task of collecting firewood.
Allah does not like the man who considers himself superior to his companions. Disputes among tribes, an unjust economic order, plundering, intolerant attitudes between members of different religions, differences between the poor and the wealthy and many other injustices were the natural consequences of such discrimination.
essay on hazrat muhammad as an exemplary judge in english
His father Abdullah had died before he was born. Here I looked up and saw a cloud covering Assalam-o-Alaikum! Islamic Research Foundation International, Inc.
Despite all that I was excited for my dream to visit a judge court.


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Anyone Can Become Addicted to Drugs
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Uploaded on Feb 7, 2012
You might think that only some types of people can get addicted to drugs. The truth is, it can happen to anyone, whether you're young or old, rich or poor, male or female. This video from NIDA explains addiction in simple terms and offers a hotline to help you or a loved one find treatment. For more information, visit http://www.easyread.drugabuse.gov
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En México, marca 01800 911 2000
"If you can't explain it simply, you don't understand it well enough". Albert Einstein>
Fantastic video. Addiction simply explained. Thanks.
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Found 62 tutors in Gulfport, MS 39501 – page 1 of 7.
Gulfport 39501 · 0 miles from downtown · $35/hour · teaches Adobe Illustrator - Adobe
My passion for teaching centers around three main practices: I want students to learn, I want students to gain the study skills and learning strategies necessary to succeed in the future, and I want them to enjoy...certified
Gulfport 39501 · 0 miles from downtown · $50/hour · teaches Grammar - Proofreading -
My specialty is correcting improper English and constructing effective essays. I recently had the opportunity to teach English as a Second Language for a year in South Korea. Though their abilities are far below those...certified
Gulfport 39501 · 0 miles from downtown · $45/hour · teaches Desktop Publishing -
I am a retired attorney. While a student in law school, I outlined many of my courses and sold the outlines to other students; I also taught classes to other students while we were all studying for exams.certified
Gulfport 39501 · 0 miles from downtown · $35/hour · teaches Business - Career
I have fifteen years of adult education instruction in business and Microsoft office computer applications. My educational training consists of curriculum development for on-line and standard classroom coursework...certified
Gulfport 39501 · 0 miles from downtown · $450/hour · teaches Elementary Math - ESL/ESOL
I have an MEd from William Carey University, an EdD from the University of Southern Mississippi, and a degree in Education from Jefferson Davis Community College. I am a teacher with the Pike School District.certified
Gulfport 39507 · 4 miles from downtown · $30/hour · teaches ACT English - American
Bored and tired? That's what school is like sometimes, especially when you're stuck in a class you have zero interest in. Much of my high school career was filled with boring classes.certified
Gulfport 39507 · 4 miles from downtown · $50/hour · teaches Career Development - College
I have a Master's of Science degree in Counseling and have worked in a University Counseling Center. I have enjoyed tutoring others in English, social sciences and ACT/SAT Prep. I have also spent several years teaching...certified
Gulfport 39505 · 4 miles from downtown · $200/hour · teaches Architecture - Geometry
I am an architect with 20 years experience. I know AutoCAD and Microsoft. I am good at geometry. I graduated from Mississippi State University. I have never tutored, but believe I can help people learn drafting.certified
Gulfport 39507 · 4 miles from downtown · $25/hour · teaches ACT English - ACT Math - ACT
My name is Jordan, and I am from Gulfport, MS. I am 31 years old and have 10+ years of experience as a tutor. I have worked as a teacher in the public school system, and worked 5 years exclusively as a private tutor.certified
Gulfport 39507 · 4 miles from downtown · $50/hour · teaches Elementary (K-6th) -
I am currently working as a sixth grade math teacher in Gulfport, MS and I love every minute of it. My students and I strive to be the best in our roles as student and teacher. Everyday they are challenged...certified


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Rubbermaid Reveal Spray Mop
Product Features:
Save more and waste less with the Rubbermaid Reveal Spray Mop. The thick microfiber pad is washable and reusable so that you never have to run to the store for refills when you're getting ready to clean. Simply fill the bottle with hot water and two teaspoons of your…
Price as on: 2018-10-06 14:45:15


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|Vote for The Forward Look Network on the Mopar Top 100 Sites
From: mike myhrvold
Email: email@example.com
Date: January 11, 2002
In 1957, the "V-800" engine (dual quad 318) was standard in the Fury and available as an option in all Plymouth cars, even the least expensive. In 1958 the dual quad 318 was standard on the Fury and available only in the Fury. The Golden Commando 350 (dual quad 350)was optional in all Plymouth models. As for which engine is in the car, if it has a 4 barrel carb it is most likely the 301. If not it is probably a 277.


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Diy bracelets are mostly a summer vacation favorite pastime of many college girls. They love the delicate bracelets in braided style and have natural textures. These bracelets feature a sliding knot closure that is adjustable making it ideal to mix with everyday stack. Having simple braiding skills help in churning bracelets for your friends. You can have endless design combinations of diy bracelets if you have the required list of gold plated brass beads and colorful waxed cords.
Materials required
- Waxed cord of 1.5 yards, in 4 colors
- 15 round gold plated brass beads
- 15 mini star gold plated brass beads
- 3 spike charms gold plated
- Glue or lighter
- Scissors
- Clipboard or tape
- Divide into half the waxed- cord and cut. Fold one cut piece into two half pieces. Gather the two sides and tie into a knot.
- Make sure to leave extra inch of the loop. Leave 3 strands equally and snip off the fourth strand at the knots bottom.
- Tape onto a flat surface the loop. Start braiding the strands and begin beads braiding after an inch. On the outer right strand, thread a bead and push the bead, over the right strand in the middle.
- On the outer left strand, thread another bead. Push it to the braid base and crossover.
- Keep one finger at the braid base; hold the beads such that it is tight. Pursue the beads threading prior to each working strand is over.
- Normally, continue braiding, after adding few beads.
- Measure your wrist and stop braiding accordingly. Thread the bracelet end through the loop.
- Fold the cord back, make a sliding knot, create a zigzag, and over the zigzag, bend another inch and create a backward S.
- Tighten the bracelet and snip away excess cord, burn the ends carefully to seal, your diy bracelets are ready.
18 Farmhouse Garden Benches In Hardwood Ensure Longer Lifetime
Farmhouse garden benches are also referred to as rustic garden furniture or benches..
16 Diy Storage Boxes For Neat Houses
Are you looking for a prefect method to make diy storage box or..
16 Gorgeous Diy Plastic Spoon Crafts
Plastic spoons are used for summer barbecues and big events. Plastic spoons can..
DIY Refrigerator Magnets, A Beautiful Way To Spruce Your Appliance
Refrigerator magnets are easy and fun to make. In fact you can turn..


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[ eth-uh l-burt ]
/ ˈɛθ əlˌbɜrt /
a.d. 552?–616, king of Kent 560–616.
a male given name: from Old English words meaning "noble" and "bright."
Dictionary.com Unabridged Based on the Random House Unabridged Dictionary, © Random House, Inc. 2019
Examples from the Web for ethelbert
The other great figures of the period—Ethelbert and his Queen, and her chaplain—were also laid to rest in the church.Beautiful Britain|Gordon Home
Yet, whether I write or not, I know well that forgotten Ethelbert can never be.A King's Comrade|Charles Whistler
Ethelbert revived the reputation of his family, which had languished for some generations.
He also made a collection of the best laws of his predecessors, Ethelbert, Ine, and Offa.A Short Biographical Dictionary of English Literature|John W. Cousin
"Yes," continued Ethelbert, not at all understanding why a German professor should be contemptible in the eyes of an Oxford don.Barchester Towers|Anthony Trollope
British Dictionary definitions for ethelbert
thelbert (ˈæθəlˌbɜːt)
/ (ˈɛθəlˌbɜːt) /
Saint. ?552–616 ad, king of Kent (560–616): converted to Christianity by St Augustine; issued the earliest known code of English laws. Feast day: Feb 24 or 25
Collins English Dictionary - Complete & Unabridged 2012 Digital Edition © William Collins Sons & Co. Ltd. 1979, 1986 © HarperCollins Publishers 1998, 2000, 2003, 2005, 2006, 2007, 2009, 2012


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Posted by Momo on 3/7/2019, 5:14 am, in reply to "Game!"
there was a game!!! Those are three babies I would have picked! LOL!
I'd love for.... - PlumFairy 3/7/2019, 8:28 am
Will do!! n/t - Momo 3/7/2019, 3:50 pm
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Aftermarket Performance Parts and Accessories for the Aprilia Tuono V4 / R / 1100RR / 1100 Factory 2011, 2012, 2013, 2014, 2015, 2016 at Motostarz.ca
As part of our 2014 big-bore naked-bike comparison test, we ran all five contenders on our dyno. These charts show horsepower and torque for the Aprilia Tuono V4 R, BMW S 1000 R, Ducati Monster 1200 S.
M10 Bolt for Aprilia Tuono 1000 R 06-11 Titanium Bolt for Aprilia RSV4 15+ stainless steel bolt for Aprilia Tuono Factory 03-05 M10 Bolt for Aprilia Tuono 1000 R 06-11 M10 Bolt for Aprilia RSV 1000 R 04-10 Aprilia RSV-R 1000 MIVV sportline tintanium high-Up ohne db-Killer by Jacek Brzezinski. 0:13.. Aluminium and Stainless Steel – order online, supplied from stock, shipped worldwide..M6 Bolt for Yamaha R1 07-08 M10 Bolt for Aprilia Tuono 1000 R 06-11 Aprilia Tuono 1000 R (2006-2009) $149.99: buy +-customer reviews . M10 Bolt for Aprilia Tuono 1100RR 16+ M10 Bolt for aprilia rsv4 15+ Like its metric counterpart, the Honda is water-cooled, but the Shadow stands out from this testing crowd with its shaft drive.M10 Bolt for Aprilia Tuono 1000 R 06-11 M8 Bolt for Aprilia tuono factory 03-05 2014 aprilia tuono V4 Motorcycle Parts. Aprilia RSV4 RR is available in two attractive new color schemes on a black and grey base (Rivazza grey and Arrabbiata black) with red wheel rims.
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Titanium Bolt for Aprilia RSV4 15+ Set includes 4 caliper bolts. Shaves a few grams of weight off the front end. Rear titanium caliper bolts also available for most models. * Despite using 330mm rotors, the '19 RSV4 1100 Factory uses these early m10x55 bolts thanks to the thinner Brembo Stylema calipers. NOTE: As with any titanium fastener, an anti-seize thread lubricant is.Titanium Bolt for Aprilia RSV4 STD 09-10 Stainless Steel Bolt for Aprilia Tuono Factory 03-05 Aprilia Bolts Fasteners and Fixings for Fairings inluding Dzus Quick Release Fasteners. 10x m6 x 20mm Fairing Bolts Stainless Steel & Zinc Fairing Clips Our Price: 14.14 Inc VAT . 126 Bolt Universal stainless steel motorcycle frame bolts Kit FastenersM10 Bolt for Aprilia RSV4 STD 09-10 Upgrade your bike with our titanium front disc bolts. You'll benefit from a 45-50% weight saving over the standard steel fasteners and improve the look of your bike at the same time. precision manufactured from Grade 5 6AI-4AV titanium; Ideal for replacing/upgrading standard fasteners; Attractive polished.
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I've seen another 2019 Katana with the Samurai kit bolted on (£1000), but I was not too impressed with the aftermarket screen as it bolts over the old one and is only about an inch higher than the.
The Aprilia Tuono 125 is a 2 stroke, Sport bike with a Liquid cooled 124.80 ccm (7,58 cubic inches) Single cylinder type of engine. This engine then gets the power to the rear wheel with a Chain driven transmission. Wheels and Tyres. If you are looking for Aprilia Tuono 125 Tyres then you will need to look for the right sizes.
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Viciouscycleparts: 2017 Aprilia Tuono 1100rr Rear Rotor Back Brake Disc Abs Sensor & Ring. Model:Tuono 1100RR. This rear rotor is in good condition and shows normal signs of wear. It has a required thickness of 4.5mm and gauges at 4.9mm as shown in the pictures.
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There's nowhere where it feels ill at ease and so many places, from empty sinuous B-roads to bolt-upright motorway schleps. For the ultimate in speed, handling and tech Aprilia's Tuono V4 1100.
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iFixit was founded in 2003 by two college students. This forum is acclaimed in the tech community for its free step-by-step guides to working on everything from laptops to game systems to phones. It's crowdsourcing power at its best–everyday people post instructions and then the community updates and perfects them. The guides are a breeze to follow, even for beginners.
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With iFixit guides and a repair kit, your tech products don't have to be disposable. And you don't need to be beholden to pricey repairs from tech companies. Join the DIY repair revolution today.


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Ivy Mary Ricketts (Mary)
Ivy Mary Ricketts (Mary) Lifelong resident of Bearwood and Pembridge passed away peacefully on 30th July 2019 one day before her 90th birthday. Funeral service will be held at St Mary's Church, Pembridge on Friday 16th August 2019 at 2:30pm. Please feel free to wear something colourful, Family flowers only but donations can be made to the Special care baby unit, Hereford County Hospital. For further enquiries please contact A.W. Hughes and Son, Funeral Directors, Kington: 01544 370217 Presteigne: 01544 267778
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Sun | T.G.I.F.
Movies rated according to the following key: **** - excellent;*** - good; ** - fair; * - poor.
* 'ALONG CAME A SPIDER': See review on D1. R (violence, language). 1:43. **1/2
Bainbridge, Gig Harbor, Kitsap Mall, Poulsbo, South Sound
* 'BLOW': See review on D1. R (pervasive drug content and language, some violence and sexuality). 2:04. **1/2
Bainbridge, Kitsap Mall, Poulsbo, South Sound
* 'THE BROTHERS': Like "The Best Man" and "Waiting to Exhale," Gary Hardwick's directorial debut is a comedy-drama set in an mid-to-upscale urban background. It's about four pals (Morris Chestnut, Bill Bellamy, D.L. Hughley and Shemar Moore) and how they cope with the terrors of marriage, stormy relationships or, in one case, no relationship at all. R (strong sexual content and language). 1:43. **1/2
Kitsap Mall
* 'CHOCOLAT': Eating chocolate, some say, produces almost the same emotional effect as making love - and Lasse Hallstrom's lip-smacking "Chocolat" is a film that encourages such sensuous analogies. It's tantalizing, delectable and randy, a movie of melting eroticism and toothsome humor. PG-13 (a scene of sensuality and some violence). 1:58. ****
Gig Harbor, Plaza Twin, Silverdale
* 'CROUCHING TIGER, HIDDEN DRAGON': Ang Lee's Chinese period fantasy is one of the most ravishing movies of the year. PG-13. (martial arts violence and some sensuality). 1:59. ****
Kitsap Mall, Poulsbo, South Sound
* 'ENEMY AT THE GATES': Based on a book by William Craig, this Jean-Jacques Annaud film is both physically sumptuous and shockingly violent. Annaud gives us the hellish Battle of Stalingrad and a dramatic duel between the Russian and German armies' top sharpshooters: a sniper from the Urals named Vassili Zaitsev (Jude Law) and a crack German marksman and teacher named Major Konig (Ed Harris). R (graphic war violence and some sexuality). 2:11. ***
Kitsap Mall, Poulsbo
* 'EXIT WOUNDS': Steven Seagal is a tough-guy cop in the inner city who uncovers a ring of corrupt cops and, of course, commences to kick some serious butt. R.
Kitsap Mall, Poulsbo, South Sound
* 'HEARTBREAKERS': Sigourney Weaver and Jennifer Love Hewitt play mother-daughter grifters who con hapless bachelors Gene Hackman, Ray Liotta and Jason Lee. PG-13 (sex-related content, including dialogue). 2:03. **1/2
Bainbridge, Poulsbo, Silverdale, South Sound
* 'JUST VISITING': See review on D1. PG-13 (violence and crude humor). 1:28. **1/2
Kitsap Mall, Poulsbo, South Sound
* 'THE MEXICAN': Brad Pitt plays a mob errand boy ordered to retrieve a valuable antique revolver in Mexico, and Julia Roberts is his cranky honey, who makes good on her threat to move to Las Vegas if he makes the trip. Soon he's ducking bullets across the border and she's in the clutches of a hit man (James Gandolfini). The film never kicks into high gear, and instead drifts into siesta. R (violence, language). 2:03. **
South Sound
* 'POKEMON 3': If you've experienced anything PokÈmon you'll know what to expect from "PokÈmon 3": garish visuals and a convoluted plot that culminates in a fierce battle between good and evil. This Pokemon feature is preceded by a 22-minute short, "Pikachu and Pichu," featuring the ever-popular Pikachu and his two nephews, who get into trouble in the big city. G. 1:30. **
Bainbridge, Kitsap Mall, Poulsbo, South Sound
* 'POLLOCK': Ed Harris turns in a great performance as the alcoholic superstar painter Jackson Pollock, who was the king of the New York City abstract expressionists in the late 1940s. This is a fiercely concentrated work, totally committed, somberly truthful. R (language, brief sexuality). 2:03. ***1/2
Rose in Port Townsend
* 'SEE SPOT RUN': David Arquette stars as a mailman with a dog phobia, and his slapstick antics would do Jerry Lewis proud. However, this lowbrow comedy sinks under the weight of its endless pratfalls, toilet jokes and car-and-dog chases. Also stars Michael Clarke Duncan. PG (crude humor, language and comic violence). 1:34. No stars.
South Sound
* 'SOMEONE LIKE YOU': A TV talk-show booker (Ashley Judd) is torn between bad, sensitive men and good, macho ones (Greg Kinnear and Hugh Jackman). She becomes a popular, pseudonymous sex-advice columnist and is ordered to book herself by her boss (Ellen Barkin). PG-13 (sexual content including dialogue, and some language). 1:33. **
Bainbridge, Kitsap Mall, Poulsbo, South Sound
* 'SPY KIDS': Surprisingly sweet, this rollicking, gizmo-filled adventure is about a brother and sister (Alexa Vega and Daryl Sabara) who leap into the world of espionage to rescue their captured spy parents (Antonio Banderas and Carla Gugino). PG (action sequences). 1:30. ***
Bainbridge, Gig Harbor, Kitsap Mall, Poulsbo, South Sound
* 'TOMCATS': A bunch of buddies vow to stay bachelors, with the last one to remain single collecting a hefty tomcat pot. As one might expect, tasteless humor is abundant. With Jerry O'Connell, Shannon Elizabeth, Jake Busey. R (strong sexual content including dialogue, and language). 1:32. *1/2
Poulsbo, Silverdale, South Sound
* 'TRAFFIC': An extraordinary film about America's long-running war on drugs. Director Steven Soderbergh's canvas is vast, his story absorbing, and his cast deep and excellent as he interweaves three separate stories. R. 2:27. ****
Plaza Twin, Silverdale


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Events for November 27, 2018
Events Search and Views Navigation
11:00 am
Zumba Fitness
Zumba is a medium intensity dance style cardio workout and is led by certified instructor, Sandi Pas. 1st Class is Free! Otherwise it's $5 per person. No experience neccessary! All are welcome!Find out more »
2:00 pm
Meet in the Clubhouse by the book exchange every Tuesday, Thursday, and Sunday at 2 P.M. New and experienced players welcome.Find out more »
5:00 pm
Themed Potluck and Movie Night
Bring your favorite dish to share! Anything goes! Please sign up at the front desk. After dinner, stay for the 6:30 movie if you'd like! Movie TBD.Find out more »


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Evaluation of the jury system
Published by Admin in Reference Desk
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http://www.thelawbank.co.uk - A short video looking at the evaluation of the jury system. This is for use in preparation for the AQA series of AS exams in Law although can be used for most exam boards. For more information and more resources go to www.thelawbank.co.uk
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Lower House Payments With Less Expensive Home Insurance Rates
Get your house payments down without refinancing – Learn more.
Affordable Homeowners insurance is an aspect of home ownership that many do not always account for in projecting their budgets. It is also an area of tremendous mystery, as it is more complex and involved than basic auto insurance. Read this article to shed some light into this area of your life.
Be aware of what your home insurance actually covers. A regular home insurance policy doesn't cover things such as flood damage. This type of insurance needs to be purchased separately through the Federal Emergency Management Agency. Another thing to consider is earthquake damage. If this is something that you may experience, you will have to buy specialized coverage from a private home insurance company. Luckily, both of these types of coverage are relatively inexpensive.
You can reduce the premiums of your homeowners insurance in a couple of ways. The first step is to have a quality security system installed. This may lower your premiums every year by 5 percent. The insurance company won't automatically know you've installed a security system, however, so you must submit proof of this. You should also have smoke and fire alarms installed in every room in your house. Smoke alarms can reduce insurance premiums by 10 percent.
When buying a homeowner's insurance policy, what is excluded from coverage is sometimes more critical that what the policy does cover. Flood coverage, for example, is not typically covered in most homeowner's policies, but can be purchased as a separate policy. The same is true for other natural disasters such as earthquakes.
When you are dealing with homeowners insurance keep in mind that some insurance companies will actually lower your premium if you get your mortgage paid off. They will think that since you own the home outright you are more likely to take better care and pride in your home.
When you are going to purchase homeowners insurance there are certain things that you should look for in a policy. A good thing to have is guaranteed replacement value insurance. This means that no matter the cost your home will be rebuilt if a disaster were to happen. Most people think this is automatic, however, since home values increased it probably would cost more now than what you had originally paid for the home. This way you are covered.
Evaluate the needs of the homeowner's insurance when you have family since your household diminishes and your valuable material increases over time. Look over your policy again to check on coverage for different valuables. If you find that you do have limits, make sure that you get additional coverage for those items to cover them.
Affordable Homeowners Insurance
Earlier in this article, the complexity of homeowners insurance was alluded to. Now that you have read this piece, you should be more familiar with what homeowners insurance covers and how you should account for it in your life. Keep these tips in mind in the coming days, and you can make this a very successful area for yourself.
For more information on home insurance or to receive a quote visit http://www.InsuranceBrokersGroup.com or call: 800-459-6060


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Terry talks about River's history over the past almost 30 years and how that leading is directing us now to improve our building. The project is called Build@River and we are asking God to build our faith and our community.
More Messages from Terry Williams | Download Audio
Build@River Page
River Community Church
2130 N. Oliver
Wichita, KS 67208
Phone: (316) 652-9601
Email: email@example.com
9:30 a.m. and 11:00 a.m.Both services are identical and last about one hour.
Thursdays 7:30 p.m.Christian Challenge is River's College Ministry
Sunday 6:00 p.m.
For students 6th grade through 12th Grade.
Friday 7:00 p.m.
For post-college married couples and singles (ages 22-35).
View More…
© 2019 River Community Church.


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Nicola Sturgeon demands Scotland ready to GO after Brexit as UK too 'obsessed' with Brexit
SCOTLAND'S First Minister Nicola Sturgeon has continued her calls for a second Scottish independence referendum after calling the UK Government "obsessed" with Brexit, claiming that most Scottish people would choose independence over leaving the EU.
Ms Sturgeon made the comments as she reacted to an LBC poll which suggests that the majority Leave voters are happy to exit the EU if it means the UK going into recession.
Ms Sturgeon told reporters: "I don't think the obsession of the UK government with Brexit should be at the price of people being poorer and the country perhaps going into recession.
"The poll that I think is most interesting today is the one that shows that as people look at the prospect of a hard perhaps no deal Brexit then the majority of people in Scotland are actually saying they would choose the alternative of independence.
"A positive alternative that would put the future of our country into our own hands."
Ms Sturgeon was referring to a poll published by pro-Europe campaign group Best for Britain, which found that 52% of Scottish people would vote for independence after Brexit.
The poll asked 1022 in Scotland how they would vote if a referendum on Scotland's future was held after the UK leaves the European bloc.
Ms Sturgeon will announce her programme for government on Tuesday when MSPs return to the Scottish Parliament after the summer recess.
Ahead of revealing her proposals for the coming year, Ms Sturgeon said the UK's decision to leave the EU at the end of March 2019 "heightens the importance of everything else we are doing as a Government".
Scotland's First Minister said that the majority of Scottish people backed independence after Brexit
The First Minister added: "The people of Scotland did not vote to leave the European Union, but in this parliamentary year we are set to be taken out of the EU against our will - with continuing uncertainty around our future relationship with the world's largest trading block, which is around eight times larger than the UK market alone.
"In the face of this uncertainty, we have to intensify our focus on improving the wealth and the wellbeing of communities across Scotland while continuing to argue the case for a common sense approach to Brexit - for continued membership of the single market and customs union."
While Scots voted against independence in 2014, the country voted to stay in the EU two years later while the majority of voters across the UK backed Brexit.
Up to 55.3 per cent voted to remain in the UK compared to 44.7 per cent, while Scotland overwhelmingly backed Remain in the 2016 EU referendum by 62 per cent compared to the 38 per cent who voted for Brexit.


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Newly wed, Priyanka Chopra, is known for her mesmerizing Bollywood beauty. She's constantly showcasing Indian brands yet she has a soft spot for the Saudi brand, Honayda. The Internationally recognized Saudi designer has been featured on countless covers including Vogue Arabia and Harper's Bazaar.
From a young age, following in her historian mother's footsteps, Honayda Serafi was curious about art. As a result, she moved to Paris to study fashion and has been working as a designer since. She formed Honayda and now dresses many celebrity fans such as Priyanka, Angham, and Lupita.
The young duo shared their love-in-the-air moment at Joe Jones and Sophie Turner's wedding. The beauty modelled her shimmering lilac SS19 Honayda gown in the Northern French sunset. Doesn't get shinier than this!
Before the wedding festivities, she donned another shiny metallic high thigh strapless gown. The dress was a breathtaking pre-wedding party option.
But these were not the first time and certainly won't be the last time for the superstar to wear Honayda. She was spotted in an all-white pleated jumpsuit in Cannes. Rocking a feminine yet edgy look, perfectly representing the brand image of power and elegance.


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Digital transformation isn't easy. Too often, companies invest heavily in new technology or hire outside consultants without addressing internal, human barriers that stand in the way of effective change. In fact, 60 percent of pilots fail - even with technology in place - victims of this shortsightedness when it comes to considering the human elements required for change. This session will present lessons about managing the human side of digital transformation - because it's the people who make change possible.
Presented by:
Mario Montag, VP Chief Data & Analytics Officer, United Technologies


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Is Your Company's Strategy Aligned with Your Ownership Model?
When Vanguard founder John Bogle died last week, i … Continue reading
Why Family Businesses Need To Find The Right Level of Conflict
From the outside, the Alexander family seemed to h … Continue reading
How Does Ownership of a Company Affect its Ability to Compete – for Better or for Worse?
BanyanGlobal partner Josh Baron recently answered … Continue reading
Building Your Own Network When You're Part of a Family Business
Ashley Fina's grandfather was a natural networker. … Continue reading
The Common Traps of Working in Your Family's Business
"What's wrong — is the company going bankrupt? Are … Continue reading
Josh Baron has created 44 entries
Josh Baron
Josh Baron is a partner and co-founder of BanyanGlobal Family Business Advisors, and an adjunct professor at Columbia Business School.


text: Sponsored Post: Airports in Europe Did you know that every hour an average of 7.770 passengers travel through London Heathrow Airport? Or that at Brussels Airport Internet is more expensive than parking? Expedia published a little infographic with some facts about some selected european airports. I was very surprised by the distance some airports are away from the city centers.… Read more »


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Posted July 31, 2019
Last Sunday, July 28, more than 100 congregants, staff, members of clergy and their families joined together to fill the (thankfully, shaded) section 137 of Citi Field.
Rabbi Angela Buchdahl and Cantor Daniel Mutlu kicked off the day with a beautiful rendition of the national anthem, arranged by Music Director David Strickland, which you can watch here. (Cantor Cadrain is feeling great but wisely decided that singing in 90 degrees at 9 months pregnant was out!)
We wouldn't want to claim that Central clergy brought divine attention to the game, but the Mets undeniably triumphed over the Pirates, with an impressive 6-run first inning, including a 2-run homer that sent the big, red apple into the air within minutes of the first pitch. The end score was 8-7 with the Mets beating the Pirates for their fourth consecutive win.
In addition to an exciting game, Central guests received Mets baseball caps embroidered with the Central Synagogue logo.
Posted November 26, 2019
Posted November 26, 2019
Posted November 20, 2019
Posted November 15, 2019
Posted October 24, 2019


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Please read and accept before completing the order
General Conditions of Purchase
These terms and conditions d 'purchase governing the sale of products marketed by Ottica PT11 through the e-commerce site owned by www.occhialivistasole.it OTTICA PT11, Ottica PT11. All contracts entered into to purchase products through the website www.occhialivistaesole.it and according to the procedures described above, between the seller and the customer will be governed by these Terms of purchase.
Acceptance of Terms of purchase
Purchase transactions are governed, among other things, the provisions of Legislative Decree no. N. 206 of 6.9.2005.
By sending electronic confirmation of your purchase order, the customer declares to have read and accepted unconditionally all the terms and conditions of sale
The customer agrees and undertakes, once the purchase process online, to print and to keep the present general conditions, which has already seen and accepted as a necessary step in, and specifications of the product being purchased, and that in order to fully satisfy the conditions laid down in Articles. 52 and 53 of Legislative Decree no. 205/06.
And 'Any right of the Customer to damages or compensation, and any contractual or tort liability for direct or indirect damages to persons and / or property, caused by non-acceptance, even partial, of an order.
How to Buy
The customer may only purchase products in the electronic catalog of Ottica PT11 at the order and viewable to the address (URL) http://www.occhialivistaesole.it, as described in the relevant information sheets. The 'picture accompanying the description of a product may not be fully representative of its features but differ in color, size, products and accessories in the picture. Any purchase of accessories and products not listed in the catalog (products ordered on request) will be finalized through e-mail or telephone communication between the client and Ottica PT11 and will still be governed by these Terms of purchase.
Correct receipt of order is confirmed by Ottica PT11 by a reply by e-mail, sent to the email address supplied by the customer. The message will include all the data entered by the customer who agrees to verify the correctness and communicate any corrections, as described in this document.
Despite the constant updating of availability through a direct connection to the catalogs of suppliers, Optics PT11 reserves the right to accept orders received after checking the availability with your supplier for the product ordered. No responsibility or debit card will be charged in Optics PT11 in the event of non-acceptance.
In the case of non-acceptance, Ottica PT11 is committed to timely inform the Customer, in the same way.
All prices in this website are retail prices.
All glasses on sale on www.occhialivistaesole.it are new and come with tags. Come directly from the manufacturers and are free of defects.
All products are authentic and original 100%.
Every pair of glasses is shipped directly from Ottica PT11 with original case, cloth and certificate of authenticity.
On some products, the subject of promotions, Ottica PT11 reserves the right to accept orders by reducing the amount, notice and acceptance by the customer, failing which the order will be canceled.
Contact lens purchase
Purchase of contact lenses is addressed only to those who already use of contact lenses. Confirming the order so the buyer declares that what they are doing is not his first purchase and has no problems of tolerance in the use of contact lenses.
Methods of payment
Credit Card.Using Paypal, you can pay with major credit cards and prepaid cards.
Paypal. Using Paypal as method of payment, the order will be taken over immediately after e-mail confirmation of payment and processed normally in the time frame indicated in the order. If you have already registered to the PayPal service simply enter your e-mail address and password and follow the few simple instructions that will be provided.
Bank Transfer / Money Order. In case of payment by bank transfer or postal money order, the order will be taken over only after the receipt by the optics of the order PT11. The payment must be made no later than 5 working days. After this period, if no communication will be made by the customer, the order will be canceled without notice.
Bank details to make payment:
Beneficiary: Ottica PT11
Via Campitello 31
32040 San Nicolo 'Comelico (BL)
At: Unicredit Bank
Methods and costs of delivery
Ottica PT11 accept orders with international delivery.
For the shipment of products purchased, Ottica PT11 uses the Italian postal or courier Gls, Sda.
Delivery costs are calculated based on the weight of the goods and the geographical destination.
The published rates for the service for International shipments of goods outside of the European Community include the fee for the customs declaration in accordance with Ministerial Decree no. 060788, as amended, and any tariffs (customs duties, VAT, etc.) will be borne by the recipient.
The delivery of the goods shall be considered to street level. Upon delivery, the customer is required to check the conformity of the product delivered to him with the order made, the packaging is not damaged, or otherwise not altered, including the sealing materials. Any damage or the mismatch in the number of packages or particulars, must be immediately notified to the person making the delivery, signing "AGREE WITH RESERVE" on the delivery receipt. Failure to do so will make it impossible for any kind of recourse against the carrier and the application of calusole guarantee on shipping. Once signed the document delivery, the Customer may not oppose objection on the external characteristic of the delivered. Any problems concerning the physical, correspondence or completeness of the goods received must be reported in Ottica PT11 within 3 days from delivery, as provided herein.
For every order placed Ottica PT11 will issue an invoice sent, delivering only in pdf format to the e-mail address provided during registration. For the issuance of invoice, the information provided by the Customer. No change in the bill will be possible after the issuance thereof.
No responsibility can be attributed to Ottica PT11 in case of delay in the order or delivery of the order.
In the case of non-collection within 5 working days of the material due to repeated inability to deliver to the address specified by the Customer, the order will be automatically canceled and will be applied by the customer a fee of 20.00 € to compensate for the costs of storage and return of the products at the headquarters of Ottica PT11.
Right of withdrawal
Pursuant to art. D.Lgs. n.206/05, which regulates the selling, if the customer is not satisfied with your purchase may exercise the right of withdrawal by sending, within ten working days from the date of receipt of the goods, a written notice return receipt requested to the following addresses:
Ottica PT11
Via Campitello 31
32040 San Nicolo 'di Comelico (BL)
email: email@example.com
The products must be returned no later than the tenth business day following receipt of the product by direct delivery or shipment to the same address.
All costs of returning goods are the responsibility of the consumer. All items must be returned and received by Ottica PT11 intact and in the same condition in which they were received, in the original packaging and any manuals and / or handbooks that are part of the package, otherwise we reserve the right not make the repayment.
Reimbursement will be made as soon as possible, and within 30 days of notification of receipt of the request for reimbursement. The amount paid by the buyer will be deducted from the amount paid as a contribution towards the cost of shipment request and any costs incurred in returning.
All products sold by Ottica PT11 are covered by the manufacturer's standard warranty and 24-month warranty for defects of conformity, within the meaning of Dlg. 206/2005. To qualify for the warranty, the customer must necessarily keep the document sales tax.
In the event that, for whatever reason, will not be able to make its customers a product warranty (repaired or replaced), Ottica PT11 will proceed at its own discretion to refund the entire amount paid or to replace it with a product of equal or superior characteristics.
According to article 13 of Legislative Decree no. N. 196 of 30.6.2003 (Privacy Code), we inform the customer of the following.
The customer's personal data collected and processed by www.occhialivistaesole.it fall into the following categories:
1. identification data (company name, company address, tel., fax, email, tax data, etc.). 2. information concerning the business and commercial activity (orders, solvency, banking, accounting and tax data, etc.).
The personal information you provide will be used within the limits and to the achievement of the objectives relating to the contractual year. In particular, the treatment is aimed at the fulfillment thereon to carry out all the administrative, commercial, accounting and taxation. The provision of data and its processing are required in relation to the purposes related to the fulfillment of contractual and legal obligations. The provision is also compulsory for the completion of all activities of the undersigned required for the execution of contractual obligations. Any refusal to provide data for these purposes may prevent us from continuing the contractual relationship and obligations of the law. The conferment of data is optional for sending communications or advertising material that our company can also make through your e-mail address. At any time, you may object to such treatment by sending a request to the email address firstname.lastname@example.com. Any refusal to provide data for marketing purposes will not affect the smooth running of the contract year.
The data will be processed through the creation and management of a central repository, with paper, computer and data that is accessed by authorized personnel. Without prejudice to the notifications made in fulfillment of legal obligations and contract, all data collected and processed may be communicated in Italy and abroad transferred only for the purposes specified above:
1. service companies, data processing companies, accountant / tax and in general to all the bodies involved in audits and checks on the proper fulfillment of the above-stated purposes;
2. any third parties to our structure, with whom we have entered into specific agreements relating to the measures to be taken to guarantee the security of data entrusted to them, in any case, the data will be used exclusively for the pursuit of the above-stated purposes.
Remember that the customer can always exercise your rights under article 7 of the Privacy Code. The treatment is Ottica PT11
The customer, by sending electronic confirmation of your purchase order, acknowledges the foregoing disclosure and consent to the processing of personal data.
Any complaint must be sent to:
Ottica PT11
via Campitello 31
32040 San Nicolo 'di Comelico (BL)
email: email@example.com
Applicable Law
The contract of sale between the customer and Ottica PT11 is concluded in Italy and governed by Italian law. For the solution of civil and criminal litigation arising from the conclusion of this contract of sale at a distance, if the customer is a consumer, the territorial competence is that of the reference hole of his town of residence in all other cases, the local jurisdiction is the only forum of Belluno.
41. The conditions contained in this document may be modified by Ottica PT11, without notice, and will be valid from the date of publication on the website www.occhialivistaesole.it