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The oxidized bile acid 7-oxoLCA (7-oxolithocholic acid), formed primarily by gut micro-organisms, is reduced in human liver to CDCA (chenodeoxycholic acid) and, to a lesser extent, UDCA (ursodeoxycholic acid). The enzyme(s) responsible remained unknown. Using human liver microsomes, we observed enhanced 7-oxoLCA reduction in the presence of detergent. The reaction was dependent on NADPH and stimulated by glucose 6-phosphate, suggesting localization of the enzyme in the ER (endoplasmic reticulum) and dependence on NADPH-generating H6PDH (hexose-6-phosphate dehydrogenase). Using recombinant human 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), we demonstrate efficient conversion of 7-oxoLCA into CDCA and, to a lesser extent, UDCA. Unlike the reversible metabolism of glucocorticoids, 11β-HSD1 mediated solely 7-oxo reduction of 7-oxoLCA and its taurine and glycine conjugates. Furthermore, we investigated the interference of bile acids with 11β-HSD1-dependent interconversion of glucocorticoids. 7-OxoLCA and its conjugates preferentially inhibited cortisone reduction, and CDCA and its conjugates inhibited cortisol oxidation. Three-dimensional modelling provided an explanation for the binding mode and selectivity of the bile acids studied. The results reveal that 11β-HSD1 is responsible for 7-oxoLCA reduction in humans, providing a further link between hepatic glucocorticoid activation and bile acid metabolism. These findings also suggest the need for animal and clinical studies to explore whether inhibition of 11β-HSD1 to reduce cortisol levels would also lead to an accumulation of 7-oxoLCA, thereby potentially affecting bile acid-mediated functions.
- bile acid
- glucocorticoid
- 11β-hydroxysteroid dehydrogenase
- liver
- metabolism
- 7-oxolithocholic acid
Bile acids play an essential role in the processing and uptake of dietary lipids and fat-soluble vitamins, and in the elimination of cholesterol and toxic lipophilic compounds from the body. Impaired regulation of the composition and concentration of bile acids and bile salts has been associated with hepatobiliary and digestive diseases [1]. Thus it is important to identify the proteins involved in the maintenance of bile acid homoeostasis.
Bile acids are synthesized from cholesterol by CYP (cytochrome P450)-mediated oxidative type I biotransformation reactions. In addition to CYP enzymes, oxidoreductases and peroxisomal oxidases are involved in bile acid synthesis [2]. The major bile acids present in human bile are CDCA (chenodeoxycholic acid) (35–50%), its 12-hydroxylated derivative CA (cholic acid) (30–45%) and DCA (deoxycholic acid) (10–20%), the bacterial 7-deoxy metabolite of CA (for structures, see Figure 1) [3,4]. Several type II biotransformation reactions of bile acids occur in the liver. Unconjugated bile acids, either newly synthesized or reaching the liver after bacterial deconjugation via the enterohepatic circulation [5], are subjected to reamidation with taurine and glycine, and, to a lesser extent, by sulfation and glucuronidation. In some species, the liver catalyses the CYP-mediated rehydroxylation of the secondary bile acids DCA and LCA (lithocholic acid), which are formed from CA and CDCA through 7-dehydroxylation by bacterial enzymes in the colon during the enterohepatic circulation [5]. Furthermore, hepatic enzymes convert iso- or 3-oxo bile acids into the preferred 3α-hydroxy derivatives and 7-oxo bile acids into the 7α- and 7β-hydroxy forms [6]. Whereas several hydroxylating CYPs and conjugating liver enzymes have been identified and characterized, the enzyme(s) involved in the hepatic oxidoreduction of 7-oxo bile acids remained unknown.
In the human colon, several bacterial strains, including Escherichia coli, Bacteroides fragilis and Bacteroides intestinalis, express 7α-HSD (7α-hydroxysteroid dehydrogenase) enzymes that generate 7-oxoDCA (7-oxodeoxycholic acid) from CA, and 7-oxoLCA (7-oxolithocholic acid) from CDCA and UDCA (ursodeoxycholic acid) [7,8]. The gut microbiota also contains hydroxysteroid dehydrogenases that catalyse the epimerization of 7α- to 7β-hydroxy bile acids with the generation of a stable oxo-bile acid intermediate [9–11], thus contributing to the formation of the UDCA found in bile and faeces. The secondary bile acids DCA and LCA, as well as the 7-oxo bile acids 7-oxoDCA and 7-oxoLCA, are eliminated by the faeces. However, a substantial fraction of these secondary bile acids is not excreted, but reabsorbed in the distal intestine and transported back to the liver.
Early studies on the metabolism of radiolabelled 7-oxoLCA in rats with bile fistulas indicated the preferential formation of UDCA and lower amounts of CDCA and its metabolites [12]. Later, Fromm et al. [13] reported the preferential conversion of radiolabelled 7-oxoLCA into CDCA, with approximately 10% UDCA after a single hepatic passage following i.v. (intravenous) administration in humans. Similarly, 7-oxoLC-Gly (7-oxolithocholylglycine) and 7-oxoLC-Tau (7-oxolithocholyltaurine) were converted into the 7α-hydroxy epimer. After small intestinal infusion, 7-oxoLCA was metabolized primarily to CDCA as observed after i.v. injection. CDCA and UDCA were not metabolized by the liver, suggesting that UDCA is mainly produced by bacterial enzymes as a result of the epimerization of CDCA via the 7-oxoLCA intermediate [9,14,15]. Using human liver preparations Amuro et al. [16] provided evidence that 7-oxoLCA is primarily reduced to CDCA and lower amounts of UDCA by NADPH-dependent microsomal enzyme(s).
In order to identify this missing link, i.e. the source of hepatic reduction of bacterially derived 7-oxoLCA and formation of UDCA, we aimed in the present study to identify the hepatic 7-oxo bile acid reductase. Initially, human liver microsomes were used to characterize the 7-oxo bile acid reductase activity, which provided evidence for an ER (endoplasmic reticulum) luminal localization of the enzyme. Then, using recombinant enzyme, we demonstrated for the first time that 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1) catalyses the irreversible reduction of 7-oxoLCA.
11β-HSD1 functions in intact cells primarily as a reductase and converts inactive 11-oxoglucocorticoids into 11β-hydroxyglucocorticoids using co-substrate NADPH, which is provided by H6PDH (hexose-6-phosphate dehydrogenase) in the ER [17]. Owing to the adverse metabolic effects of elevated 11β-HSD1-dependent glucocorticoid activation in tissues such as liver, skeletal muscle and adipose, 11β-HSD1 has emerged as a promising therapeutic target to treat metabolic diseases, and there is considerable effort to develop therapeutic inhibitors (reviewed in [18–21]). However, regarding safety aspects of such inhibitors, it is necessary to identify other substrates of 11β-HSD1 [22], including bile acids as in the present study, and to understand the role of 11β-HSD1 in their metabolism.
Microsomal preparations and activity assays using human liver microsomes
Human liver microsomes (InVitro CYP H-class™ microsomes from a male donor, Celsis International) were thawed on ice and used immediately for activity assays. Microsomes, 0.2 mg per reaction, were incubated at 37 °C for 0–40 min in a total volume of 500 μl containing TS2 buffer (100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2, 250 mM sucrose and 20 mM Tris/HCl, pH 7.4), a final concentration of 500 μM NADPH, 1 μM substrate and vehicle or 5 μM 11β-HSD1 inhibitor as indicated. To assess the latency of 7-oxoLCA reduction and dependence on cofactor regeneration, microsomes were incubated in reaction mixture supplemented with the detergent Nonidet P40 (final concentration of 0.5%), G6P (glucose 6-phosphate) or G6S (glucose 6-sulfate) (final concentration of 1 mM), or the glucose-6-phosphate translocase inhibitor S3483 (obtained from Sanofi-Aventis; final concentration of 20 μM). Reactions were started by adding microsomes into freshly prepared reaction mixture and stopped by rapid freezing in solid CO2.
Rat liver microsomes and microsomes of HEK (human embryonic kidney)-293 cells transfected with human 11β-HSD1 or mock-transfected were prepared as described previously [23]. For immunoblotting, 50 μg of total microsomal proteins was separated by 12% PAGE, followed by transfer of proteins on to PVDF membranes. 11β-HSD1 was detected using primary anti-(human 11β-HSD1) antibody (Cayman Chemical).
Reduction of 7-oxoLCA by recombinant human 11β-HSD1
To assess 7-oxoLCA reductase activity in intact HEK-293 cells stably expressing recombinant human 11β-HSD1 or co-expressing 11β-HSD1 and H6PDH (AT6 and HHH7 clones respectively) [24], 20000 cells per well were distributed in 24-well plates and allowed to adhere for 16 h. Cells were then incubated with doubly charcoal-treated DMEM (Dulbecco's modified Eagle's medium), and the 7-oxidoreduction of 7-oxoLCA was measured at a final concentration of 1 μM after incubation for up to 24 h at 37 °C in a total volume of 1 ml. To determine the apparent Km of 11β-HSD1, frozen cell lysates were thawed, sonicated and immediately incubated for 10 min at 37 °C in a total volume of 500 μl containing 500 μM NADPH and 7-oxoLCA at concentrations between 62.5 nM and 4 μM. Reactions were terminated by freezing in solid CO2.
Impact of bile acids on the interconversion of glucocorticoids
The conversion of cortisone into cortisol using cell lysates was measured as described previously [25] (see the Supplementary Online Data at http://www.BiochemJ.org/bj/436/bj4360621add.htm). To assess the effect of 7-oxoLCA on the ratio of active to inactive glucocorticoids in intact cells at steady state, cells (30000 cells per well of a poly-L-lysine-coated 96-well plate, Becton-Dickinson) expressing 11β-HSD1 or co-expressing 11β-HSD1 and H6PDH were incubated for 24 h in a total volume of 40 μl of steroid-free DMEM in the presence of either 200 nM cortisone or cortisol and various concentrations of 7-oxoLCA.
Analysis of non-labelled steroids and bile acids by LC (liquid chromatography)–MS
Frozen samples from reactions using intact cells, cell lysates or microsomes were thawed, and a fixed amount of deuterated CDCA (0.5 nmol) or corticosterone (0.2 nmol) was added as an internal standard, followed by mixing and centrifugation at 3000 g for 5 min. Supernatants were loaded on to Oasis HBL SPE cartridges (pre-conditioned with 1 ml of methanol and 1 ml of water), followed by washing with 2 ml of water and elution with 2 ml of methanol. The solvent was evaporated and the residue was reconstituted in 100 μl of methanol.
7-OxoLCA and its metabolites were separated on an Atlantis T3 column (Waters) using an Agilent Technologies model 1200 liquid chromatograph (see the Supplementary Online Data). The liquid chromatograph was interfaced to an Agilent 6410 triple-quad mass spectrometer, operated in atmospheric pressure electrospray positive-ionization mode. Data acquisition was performed using MassHunter workstation software (version B.01.04).
Metabolites were identified by comparing their retention times and mass to charge ratios (m/z) with those of authentic standards. UDCA, 7-oxoLCA and CDCA were detected in the selected positive-ionization MS Scan2 mode (mass range, m/z 300–500). They were typically eluted at 5.2, 6.2 and 7.3 min, and were monitored at m/z 357.3, 373.2 and 357.3 (dehydrated bile acids) respectively. Quantitative determination of bile acids was performed by positive-ionization and MRM (Multiple Reaction Monitoring). Deuterated CDCA was used as internal standard (m/z 361.2).
Quantitative analysis of glucocorticoids was performed similarly by MRM. Cortisone (precursor and product ion at m/z 361 and 163) and cortisol (precursor and product ion at m/z 363 and 121) were eluted at 9.8 and 9.5 min respectively. Corticosterone (m/z 347.2) was used as internal standard and was eluted at 12.1 min.
Metabolites were quantified from calibration curves of the peak area ratio of the authentic standard and internal standard incubated in lysates of untransfected HEK-293 cells at a total protein concentration identical with that of the experimental setting and plotted against the concentration of authentic standards (normalization using internal standard).
Calculation of enzyme kinetic parameters
Enzyme kinetics was analysed by non-linear regression using four-parameter logistic curve fitting. For statistical comparisons, the ratio t-test in GraphPad Prism 5 software was used. Results (means±S.D.) were obtained from at least three independent experiments.
Molecular modelling of 11β-HSD1 with bile acids
Mouse 11β-HSD1 (PDB code 1Y5R) [26] was extracted from the PDB for use as a template to investigate the interactions of 7-oxo, 7α-hydroxy and 7β-hydroxy bile acids with 11β-HSD1. We used PDB code 1Y5R because it contains both corticosterone and NADP+, unlike other three-dimensional structures of 11β-HSD1 in the PDB. Human and mouse 11β-HSD1 have 79% sequence identity, which allows mouse 11β-HSD1 to be a good model for the interaction of bile acids with human 11β-HSD1.
To obtain 11β-HSD1 complexed with 7-oxoLCA, we superimposed the crystal structure of E. coli 7α-HSD [27] complexed with 7-oxoLC-Gly (PDB code 1FMC) with PDB code 1Y5R and extracted 7-oxoLC-Gly from PDB code 1FMC. Then we inserted 7-oxoLCA into 11β-HSD1. For conversion of the 7-oxo into either a 7α-hydroxy or a 7β-hydroxy, we used the Biopolymer option in Insight II. The glycine-conjugated bile acids also were constructed with Biopolymer. The final three-dimensional model of 11β-HSD1 with each bile acid was refined using Discover 3, which was run for 10000 iterations with a distant dependent dielectric constant of 2.
Reduction of 7-oxoLCA by human liver microsomes
Previous studies provided evidence for the existence of one or more hepatic enzymes catalysing the 7-oxo reduction of 7-oxoLCA to CDCA and/or UDCA [12,13,16,28,29]. These earlier studies suggested that the 7-oxo bile acid reductase is a microsomal enzyme preferentially using NADPH [16]. To identify this 7-oxo bile acid reductase, we first incubated human liver microsomes with 7-oxoLCA and studied the properties of the enzymatic reaction. After incubation for 40 min, approximately 70% of 7-oxoLCA was converted, mainly into CDCA and into approximately three times lower amounts of UDCA (Figure 2A). In addition, some minor products, including muricholic acids, were observed, but were not analysed further. The 7-oxoLCA reduction was approximately ten times more efficient in the presence of NADPH compared with NADH, and no 7-oxoLCA formation could be detected when microsomes were incubated with CDCA or UDCA and either NADP+ or NAD+ respectively, suggesting that the enzyme acts exclusively as a reductase.
Ketoconazole (5 μM) had no effect either on the amount of CDCA and UDCA formed or on their ratio, suggesting that CYPs play a minor role in the metabolism of 7-oxoLCA to its 7-hydroxylated forms. Experiments using the detergent Nonidet P40 suggested latency of the 7-oxo bile acid reductase; however, prolonged incubation with the detergent also seemed to inhibit the enzyme activity (results not shown).
In the ER lumen, NADPH is regenerated by H6PDH, which, under physiological conditions, is primarily dependent on G6P [30]. We therefore tested whether the 7-oxo bile acid reductase is stimulated in the presence of the hexose phosphate. In the presence of G6P, the 7-oxoLCA supplied was almost completely metabolized. Comparable stimulation was observed in the presence of G6S (Figure 2A), which is a specific substrate of the luminal H6PDH, but not the cytoplasmic G6PDH [30]. Furthermore, the glucose-6-phosphate translocase inhibitor S3483 abolished the G6P- and G6S-induced stimulation of 7-oxoLCA reduction (results not shown).
To our knowledge, the only currently known luminal oxidoreductase using NADPH is 11β-HSD1, which is a reversible enzyme and catalyses the interconversion of glucocorticoids and some other substrates, including 7-oxycholesterol, 7-oxydehydroepiandrosterone, 11-oxyprogesterone and 11-oxyandrogen metabolites [22,31–33]. An antibody raised against human 11β-HSD1 detected a single band at approximately 35 kDa and confirmed the high expression in human liver microsomes [34–36] (Figure 2B). A band at approximately 33 kDa was detected in rat liver microsomal preparations. The size difference can be explained by the presence of three glycosylation sites in human 11β-HSD1 and two in the rat enzyme [37]. The occurrence of three non-specific bands in rat liver microsomes and in HEK-293 microsomal preparations indicates some cross-reactivity of the antibody. The recombinant enzyme was constructed with a C-terminal FLAG epitope, resulting in a slightly slower migration of the protein in gel electrophoresis. Probing the blot with anti-FLAG antibody resulted in a single band at 35 kDa (results not shown).
To test whether 11β-HSD1 might catalyse the reduction of 7-oxoLCA, we used human liver microsomes and studied the effect of three structurally unrelated 11β-HSD1 inhibitors, i.e. glycyrrhetinic acid, T0504 (also known as Merck-544) and BNW16 [25]. All three inhibitors abolished the conversion of 7-oxoLCA into CDCA and UDCA (Figure 2A). Next, we compared the reduction of 7-oxoLCA and cortisone. The human liver microsomes (0.2 mg in a reaction volume of 500 μl) converted approximately 50 and 80% of 7-oxoLCA (1 μM) after 10 and 20 min respectively, and 7-oxoLCA was almost completely metabolized after 40 min (Figure 3). In comparison, in analogous experiments, 37, 56 and 67% of cortisone was converted, indicating a higher capacity to metabolize 7-oxoLCA compared with cortisone.
Reduction of 7-oxoLCA by recombinant 11β-HSD1
To verify that the reduction of 7-oxoLCA indeed is catalysed by 11β-HSD1, experiments in lysates of HEK-293 cells expressing the recombinant enzyme were performed. 11β-HSD1 efficiently catalysed the reduction of 7-oxoLCA with an apparent Km of 980±210 nM and a Vmax of 2.8±0.4 nmol·mg−1·h−1 as calculated by four-parametric non-linear regression (Figure 4A). Comparable values were obtained using the Hanes–Woolf equation (Figure 4B). No conversion of 7-oxoLCA was observed in untransfected HEK-293 control cells. The taurine- and glycine-conjugated 7-oxo bile acids, 7-oxoLC-Tau and 7-oxoLC-Gly, were similarly converted into the 7α-hydroxylated CDC-Tau (chenodeoxycholyltaurine) and CDC-Gly (chenodeoxycholylglycine) with minor amounts of UDC-Tau (ursodeoxycholyltaurine) and UDC-Gly (ursodeoxycholylglycine) respectively, demonstrating that 11β-HSD1 accepts both unconjugated and conjugated 7-oxoLCA as substrate.
Next, we studied the impact of H6PDH on 11β-HSD1-dependent reduction of 7-oxoLCA in intact HEK-293 cells stably expressing either human 11β-HSD1 alone or co-expressing 11β-HSD1 and H6PDH. Co-expression with H6PDH stimulated the 7-oxo reductase activity of 11β-HSD1 (Figure 5). In cells co-expressing 11β-HSD1 and H6PDH, the reaction was almost completed after 24 h, resulting in the formation of approximately 90% CDCA and 10% UDCA. In contrast, only approximately 50% of 7-oxoLCA was converted in cells expressing solely 11β-HSD1, and it took more than 48 h until the reaction was completed (results not shown). No oxidation of CDCA and UDCA was detected, independent of the cell line used, confirming the observation from human liver microsomes and showing that 11β-HSD1 catalyses the irreversible conversion of 7-oxoLCA into CDCA and lower amounts of UDCA.
Interference of bile acids with the metabolism of glucocorticoids by 11β-HSD1
Several bile acids, including CDCA and LCA, have been found in previous studies to inhibit 11β-HSD1 and 11β-HSD2 respectively [38–40]. We therefore compared the effect on 11β-HSD1 activities of 7-oxoLCA and its taurine- and glycine-conjugated forms with that of other relevant bile acids (Table 1). Whereas the 7α-hydroxylated CDCA and its conjugated derivatives CDC-Tau and CDC-Gly showed a more than 10-fold preference to inhibit the dehydrogenase over the reductase activity of 11β-HSD1, 7-oxoLCA displayed a slight preference to inhibit the reduction of cortisone, an effect that was more pronounced for the conjugated derivatives. The 7β-hydroxylated bile acids UDCA and UDC-Tau preferentially inhibited 11β-HSD1 dehydrogenase activity; however, they were approximately 10-fold less potent than the 7α-hydroxylated forms.
Impact of 7-oxoLCA on the ratio of cortisol to cortisone at steady state
Bile acids can reach high concentrations in the liver and may affect not only initial rates of conversion, but also steady-state ratios of cortisol to cortisone controlled by 11β-HSD1. We therefore determined the effect of 7-oxoLCA on the steady-state ratio of cortisol to cortisone in HEK-293 cells stably expressing 11β-HSD1 or co-expressing 11β-HSD1 and H6PDH. As shown in Figure 6(A), approximately 40% cortisol was produced in 11β-HSD1-expressing cells, whereas over 90% of initially supplied cortisone was converted into cortisol upon co-expression with H6PDH, in line with earlier observations [33]. A mirror image was obtained when cells were incubated initially with cortisol (Figure 6B). Co-incubation of the cells with the respective glucocorticoid and increasing concentrations of 7-oxoLCA resulted in diminished cortisol production when cortisone was supplied and enhanced cortisone formation when cortisol was supplied initially, thus reflecting a shift from the active to the inactive glucocorticoid at steady state in the presence of high concentrations of 7-oxoLCA.
Analysis of binding of bile acids to 11β-HSD1 by three-dimensional modelling
We used the crystal structure of 11β-HSD1 with corticosterone [26] (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/436/bj4360621add.htm) as a benchmark for comparison of the three-dimensional models of 11β-HSD1 with 7-oxoLCA, CDCA and UDCA (Figure 7). In the crystal structure of 11β-HSD1 in complex with corticosterone, the phenolic group of Tyr183 on 11β-HSD1 and C4 on NADP+ are 2.8 and 3.9 Å (1 Å=0.1 nm) respectively from the 11β-hydroxy group on corticosterone. The hydroxy group on Tyr183 is hydrogen-bonded with the 2′-hydroxy group on the nicotinamide ribose. The ∊-amino group on Lys187 is 3.2 Å from the 2′- and 3′-hydroxy groups on the nicotinamide ribose. This stabilizes the orientation of the nicotinamide ribose [26]. N∊ and Nη on the guanidinium group of Arg66 have an electrostatic interaction with the 2′-phosphate of NADP+. This electrostatic interaction is characteristic of NADP(H)-dependent SDRs (short-chain dehydrogenase/reductases), including 17β-HSD1 [41–43].
Although all three bile acid substrates have favourable interactions with 11β-HSD1, there are differences in some interactions that can explain differences in the observed binding and catalytic activity. In the three-dimensional model of 11β-HSD1 in complex with 7-oxoLCA, the phenolic group on Tyr183 on 11β-HSD1 and C-4 on the nicotinamide ring of NADPH are 2.9 and 3.5 Å respectively from the 7-oxo group on 7-oxoLCA. The ∊-amino group on Lys187 has two favourable contacts with the 2′- and 3′-ribose hydroxy groups on NADPH, and the hydroxy group on Tyr183 is 3.5 Å from the 2′-hydroxy group of the nicotinamide ribose. Furthermore, Arg66 and other residues in the N-terminal end of 11β-HSD1 have favourable contacts with the adenosine on NADPH. Together, this three-dimensional model indicates that 7-oxoLCA fits into 11β-HSD1 in an orientation that favours reduction of the 7-oxo group to an alcohol.
In the three-dimensional model of 11β-HSD1 in complex with CDCA, Tyr183 on 11β-HSD1 and C-4 on NADP+ are 2.9 and 3.4 Å respectively from the 7α-hydroxy group on CDCA. However, Lys187 has an asymmetric orientation to the ribose hydroxy groups. The ∊-amino group on Lys187 is 4.6 Å from 3′-hydroxy group, which is too far to form a hydrogen bond. Also, the hydroxy group on Tyr183 is 4.4 Å from the 2′-hydroxy group of the nicotinamide ribose. Thus the nicotinamide ribose lacks two stabilizing interactions that are present in 11β-HSD1 in complex with 7-oxoLCA. Arg66 and other residues in the N-terminal end of 11β-HSD1 have favourable contacts with the adenosine on NADP+.
In the three-dimensional model of 11β-HSD1 in complex with UDCA, Tyr183 on 11β-HSD1 and C-4 on NADP+ are 4.1 and 3.3 Å respectively from the 7β-hydroxy group on UDCA. Lys187 has an asymmetric orientation to the ribose hydroxy groups. The ∊-amino group on Lys187 is 4.5 Å from 3′-hydroxy group, which is too far to form a hydrogen bond. Arg66 and other residues in the Nterminal end of 11β-HSD1 have favourable contacts with the adenosine on NADP+ (see also the Supplementary Online Data).
We also constructed three-dimensional models of 11β-HSD1 in complex with the glycine conjugates of 7-oxoLCA, CDCA and UDCA, as shown in Supplementary Figure S2 at http://www.BiochemJ.org/bj/436/bj4360621add.htm. All three glycine–bile acid conjugates have stabilizing contacts with various backbone nitrogens or oxygens in 11β-HSD1. Examination of these three-dimensional models reveals an unexpected coulombic interaction between the glycine carbonyl group and Nη2 on Arg66, which also has a key electrostatic interaction with the 2′-ribose phosphate on NADP+. Thus Arg66 has two important stabilizing interactions in the complexes of 11β-HSD1 with glycine conjugates of bile acids.
Thus the three-dimensional models of 11β-HSD1 with bile acids (Figure 7) reveal that 7-oxoLCA has the most favourable interaction with the catalytic site in 11β-HSD1 and that glycine–bile acid conjugates also can fit into 11β-HSD1 in which they have a coulombic interaction with Arg66, a key residue in the stabilization of NADPH binding to 11β-HSD1.
By catalysing the biotransformation of gut bacteria-derived secondary bile acids, the liver plays a key role in damage and repair; damage being changes in the steroid nucleus by bacterial enzymes, and repair being rectification of these changes by the hepatocyte. Several bacterial strains express 7α-HSDs to yield 7-oxoLCA from CDCA and 7-oxoDCA from CA [7]. 7-OxoLCA can be metabolized further by reversible bacterial 7β-HSDs, or taken up actively via sodium-dependent transporter [SLC10A2 (solute carrier 10A2)] from the lumen of the ileal segment or passively in the colon. Although 7-oxoLCA is readily detectable in faeces and portal blood, it cannot be detected at substantial levels in bile and plasma [44,45], suggesting efficient hepatic metabolism.
In the present paper, we report the identification of 11β-HSD1 as a hepatic 7-oxoreductase, providing an explanation for the low circulating 7-oxoLCA concentrations. Human and rodent liver expresses high levels of 11β-HSD1 [34,35] (Figure 2B). Previous studies demonstrated that 11β-HSD1 purified from human or rodent liver catalyses the NADPH-dependent conversion of cortisone into cortisol and 11-dehydrocorticosterone to corticosterone respectively [36]. In addition, it was shown that 11β-HSD1 purified from rabbit and hamster liver accepts not only glucocorticoids as substrates, but also 7-oxycholesterol metabolites [46,47]. We now demonstrate that both human liver microsomes and recombinant human 11β-HSD1 expressed in HEK-293 cells catalysed the NADPH-dependent 7-oxo reduction of 7-oxoLCA to form preferentially the 7α-hydroxy bile acid CDCA and to a lesser extent the 7β-hydroxy isomer UDCA (10–20%). The ratio of CDCA to UDCA observed in our experiments with human liver microsomes as well as recombinant human enzyme is in line with earlier observations with human liver preparations and measurements in blood following i.v. administration [13,16]. Importantly, the 7-oxo reduction of 7-oxoLCA in liver microsomes was completely abolished by the 11β-HSD1 inhibitors glycyrrhetinic acid, T0504 and BNW16 (Figure 2A). Although we cannot exclude the existence of another enzyme that catalyses the 7-oxo reduction of 7-oxoLCA in the liver, it is highly unlikely that such an enzyme would be completely inhibited by all of the three structurally unrelated compounds. Thus the results provide strong evidence that 11β-HSD1 is the major enzyme catalysing the 7-oxo reduction of 7-oxoLCA in humans.
Analysis of the kinetic properties revealed that 11β-HSD1 efficiently catalyses 7-oxo reduction of 7-oxoLCA with approximately 2-fold lower affinity, but 2-fold higher Vmax compared with reduction of cortisone. The conversion of both 7-oxoLCA and cortisone was latent, dependent on H6PDH and stimulated to a similar extent by addition of G6P to the reaction mixture. 11β-HSD1 accepted the taurine- and glycine-conjugated forms as substrates, with catalytic efficiencies comparable with those for the free bile acids. This is consistent with predictions of the three-dimensional models of the three bile acids conjugated to glycine, which uncovered an unexpected interaction between the glycine carbonyl group and Nη2 on Arg66 on 11β-HSD1 (see Supplementary Figure S2). Arg66 has an important role in neutralizing the negative charge on the 2′-phosphate on NADP(H) in 11β-HSD1 and other SDRs that use NADP(H) as a cofactor [26,41–43]. Thus Arg66 has two key coulombic interactions in 11β-HSD1 complexed with the three glycine-conjugated bile acids.
Unlike other steroid and sterol substrates, 11β-HSD1 irreversibly catalyses the 7-oxo reduction of 7-oxoLCA, and the stereoselectivity for the bile acid metabolites formed is just opposite of that observed for the metabolites of 7-oxocholesterol [31,32], 7-oxodehydroepiandrosterone and 7-oxopregnenolone [33]. Neither CDCA nor UDCA, even upon prolonged incubation and at high concentrations, were converted into 7-oxoLCA, and there was also no isomerization of CDCA to UDCA or vice versa, as has been observed for 7α- and 7β-hydroxyepiandrosterone [48] and 7α- and 7β-hydroxydehydroepiandrosterone [33] respectively. Our results are in line with an earlier report on the metabolism of radiolabelled CDCA in rats with bile fistulas [12], where conversion of CDCA into trihydroxylated metabolites and minor amounts of UDCA, but no formation of 7-oxoLCA was observed. Furthermore, in humans, after a single hepatic passage following i.v. administration, neither CDCA nor UDCA was modified on the steroid ring.
The three-dimensional models indicate that only 7-oxoLCA has optimal binding of substrate and cofactor to Tyr183 and Lys187 (Figure 7), which is necessary for reduction of 7-oxoLCA to CDCA. In contrast, in the three-dimensional models of 11β-HSD1 with CDCA and UDCA, the ∊-amino group on the key catalytic residue Lys187 is too far from the 3′-ribose hydroxy group on NADP+ to form a stabilizing hydrogen bond required for catalytic activity. The distance between the 7β-hydroxy group on UDCA and the phenolic group on Tyr183 is 4.1 Å, which indicates weaker binding than found for the similar interaction between Tyr183 and either CDCA or 7-oxoLCA. This may explain the preference for CDCA as a product in the reduction of 7-oxoLCA by 11β-HSD1. Although only 7-oxoLCA was metabolized by 11β-HSD1, the three-dimensional models predicted binding of all three bile acids and their conjugates, and supported the more potent inhibitory effect of free and conjugated forms of CDCA compared with UDCA on cortisol oxidation (Table 1).
Alterations in the availability of bile acids, which reach high concentrations in the hepatocyte in cholestatic liver disease, may affect the hepatic activation of glucocorticoids. In intact HEK-293 cells expressing 11β-HSD1, but not H6PDH, CDCA and its conjugates preferentially inhibited 11β-HSD1 dehydrogenase activity and stimulated cortisone reduction. However, upon co-expression with H6PDH, which reflects the situation in hepatocytes, CDCA displayed weak inhibitory activity on 11β-HSD1. In contrast, 7-oxoLCA preferentially inhibited 11β-HSD1 reductase activity, and the presence of high concentrations of 7-oxoLCA stimulated cortisol oxidation and shifted the ratio of active to inactive glucocorticoids under steady-state conditions, probably by altering the ratio of NADPH to NADP+ in the ER lumen. It was shown previously that a ratio of NADPH to NADP+ greater than 10 is required for 11β-HSD1 to efficiently reduce cortisone [49]. The presence of high concentrations of 7-oxo bile acids, 7-oxo cholesterols or 7-oxo steroids may thus result in decreased ER luminal NADPH levels and lower concentrations of active glucocorticoids, thereby modulating redox signalling pathways and glucocorticoid-dependent adaptive responses.
Further research is needed to elucidate the physiological role for the rapid hepatic removal of 7-oxoLCA. Distinct effects of 7-oxoLCA, CDCA and UDCA on bile acid sensing receptors may affect the regulation of genes involved in lipid metabolism and inflammation. CDCA has been found to be a potent activator of the FXR (farnesoid X receptor)/RXR (retinoid X receptor) heterodimeric receptor [50,51]. In contrast, UDCA showed no or very little effect and 7-oxoLCA was a modest activator of human FXR, but did not activate mouse FXR. Thus, by converting the weak activator 7-oxoLCA into the more potent CDCA, 11β-HSD1 might play a role in modulating FXR activity. However, the amount of CDCA from de novo synthesis in the liver probably exceeds that from conversion of bacterially derived 7-oxoLCA, and the relative contribution remains to be determined. Also, there are currently no data available on potential effects of 7-oxoLCA on other nuclear receptors, including LXR (liver X receptor), VDR (vitamin D receptor) and PXR (pregnane X receptor).
Nevertheless, the results may be relevant regarding the current development of 11β-HSD1 inhibitors for treatment of metabolic diseases [19–21]. Inhibition of 11β-HSD1 is expected to abolish hepatic metabolism of 7-oxoLCA, thereby leading to elevated hepatic and circulating 7-oxoLCA levels, similar to the observed accumulation of 7-oxocholesterol following 11β-HSD1 inhibition in rats [32]. 11β-HSD1 inhibition is not expected to affect bacterially derived production of UDCA and its metabolism in the liver; however, as shown in Figure 5, some UDCA is formed in the 11β-HSD1-dependent reduction of 7-oxoLCA, and inhibition of the enzyme might lower the local availability of UDCA. Clearly, further studies in 11β-HSD1-knockout mice and pre-clinical and clinical studies using selective inhibitors are needed to elucidate the impact of 11β-HSD1 on bile acid composition and function.
Alex Odermatt had the responsibility for the overall planning and conduct of the work, performed inhibitor experiments, analysed data and wrote the paper. Thierry Da Cunha and Carlos Penno developed the LC–MS protocol, performed enzyme activity experiments and analysed data. Christian Reichert performed activity experiments. Min Dong and Armin Wolf assisted in the design of experiments with human liver microsomes and analysis of protein expression. Charlie Chandsawangbhuwana performed three-dimensional modelling and analysed data, and Michael Baker performed three-dimensional modelling, analysed data and wrote the paper. All authors read and approved the final paper.
This work was supported by the Swiss National Science Foundation [grant number 31003A-124912 to A.O.]. A.O. has a Chair in Molecular and Systems Toxicology by the Novartis Research Foundation.
We thank Dr Alan F. Hofmann (University of California, San Diego, San Diego, CA, U.S.A.) for providing some of the bile acids for this study, and also for critical comments on the paper. M. D. and A. W. are employees of Novartis AG, Basel, Switzerland. The employer did not influence the design and interpretation of the data of this study.
Abbreviations: CA, cholic acid; CDCA, chenodeoxycholic acid; CDC-Gly, chenodeoxycholylglycine; CDC-Tau, chenodeoxycholyltaurine; CYP, cytochrome P450; DCA, deoxycholic acid; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; FXR, farnesoid X receptor; G6P, glucose 6-phosphate; G6S, glucose 6-sulfate; HEK, human embryonic kidney; H6PDH, hexose-6-phosphate dehydrogenase; 7α-HSD, 7α-hydroxysteroid dehydrogenase; 11β-HSD, 11β-hydroxysteroid dehydrogenase; i.v., intravenous; LC, liquid chromatography; LCA, lithocholic acid; MRM, Multiple Reaction Monitoring; 7-oxoDCA, 7-oxodeoxycholic acid; 7-oxoLCA, 7-oxolithocholic acid; 7-oxoLC-Gly, 7-oxolithocholylglycine; 7-oxoLC-Tau, 7-oxolithocholyltaurine; SDR, short-chain dehydrogenase/reductase; UDCA, ursodeoxycholic acid; UDC-Gly, ursodeoxycholylglycine; UDC-Tau, ursodeoxycholyltaurine
- © The Authors Journal compilation © 2011 Biochemical Society


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text:
Ron Burgundy Helps the Dodge Durango 'Stay Classy'
Everyone's favorite vain 1970's anchorman, Ron Burgundy (portrayed by Will Ferrell), is hawking Dodge Durango SUVs in advance of his next movie.
The Daily Herald (of suburban Chicago) found out that Chrysler essentially got the bulk of the ads for free:
Francois got the talent to pitch a refurbished version of the Dodge Durango SUV for free. Paramount Pictures, he said, bartered work on the commercials by Will Ferrell, who plays Burgundy, in exchange for the promotion in the ads of an "Anchorman" sequel that is due out in December.
Ferrell ended up writing, and acting, in about 70 different commercials as the iconic Burgundy.
Here are two of the commercials:
This isn't the first time Burgundy has made an appearance outside of the movies. Burgundy made an appearance on Conan O'Brien's TBS show to announce the filming of Anchorman 2 last year.


text:
The Study of Infection and Cell Inflammation in Peritoneal Dialysate
The recruitment status of this study is unknown. The completion date has passed and the status has not been verified in more than two years.
Verified June 2005 by National Taiwan University Hospital.
Recruitment status was: Recruiting
Recruitment status was: Recruiting
National Taiwan University Hospital
Information provided by:
National Taiwan University Hospital
ClinicalTrials.gov Identifier:
First received: September 12, 2005
Last updated: November 23, 2005
Last verified: June 2005
- Full Text View
- Tabular View
- No Study Results Posted
- Disclaimer
- How to Read a Study Record
Detailed Description:
A progressive decline of plasma triggering receptor expressed on myeloid cells-1 (TREM-1) concentration indicates a favorable clinical evolution during the recovery phase of sepsis. The expression of TREM-1 in dialysate of peritoneal dialysis patients was not yet documented. We will collect the dialysate of peritonitis in peritoenal dialysis patients and analyze the time serial change.
|Peritonitis Kidney Failure
|Study Type:
|Study Design:
|Observational Model: Defined Population
Primary Purpose: Screening
Time Perspective: Cross-Sectional
Time Perspective: Prospective
Further study details as provided by National Taiwan University Hospital:
|Estimated Enrollment:
|Study Start Date:
|June 2005
|Estimated Study Completion Date:
|June 2009
The triggering receptor expressed on myeloid cells-1 (TREM-1) is a member of the immunoglobulin superfamily, and its expression is upregulated on phagocytic cells in the presence of bacteria or fungi (1). Several experiments by Bouchon and colleagues showed that TREM-1 mediates the acute inflammatory response to microbial products. Human tissues infected with bacteria are infiltrated with neutrophils and macrophages that express high levels of TREM-1. Conversely, TREM-1 is only weakly expressed in samples from patients with noninfectious inflammatory disorders. In addition, TREM-1 is shed from the membrane of activated phagocytes and can be found in a soluble form in body fluids. The presence of a soluble form of TREM-1 in samples of bronchoalveolar lavage fluid from mechanically ventilated patients has been shown to be a good indicator of infectious pneumonia. During sepsis, a progressive decline of plasma sTREM-1 concentration indicates a favorable clinical evolution during the recovery phase of sepsis. In addition, baseline sTREM-1 level may prove useful in predicting outcome of septic patients. We will collect the dialysate of peritonitis in peritoneal dialysis patients and analyze the time serials change.
|Ages Eligible for Study:
|18 Years to 80 Years (Adult, Senior)
|Sexes Eligible for Study:
|Accepts Healthy Volunteers:
Inclusion Criteria:
peritoneal dialysis patients, dialysis period for more than 3 months.
Exclusion Criteria:
age younger than 18 years old, Pregnant or lactating women. (Pre-menopause women, capable of bearing children will undergo pregnancy test)
Contacts and Locations
Choosing to participate in a study is an important personal decision. Talk with your doctor and family members or friends about deciding to join a study. To learn more about this study, you or your doctor may contact the study research staff using the Contacts provided below. For general information, see Learn About Clinical Studies.
Please refer to this study by its ClinicalTrials.gov identifier: NCT00173485
Please refer to this study by its ClinicalTrials.gov identifier: NCT00173485
|Contact: Vin-cent Wu, MD
|National Taiwan University Hospital
|Taipei, Taiwan
|Contact: Vincent Wu, MD 886-2-2356-2000 email@example.com
Sponsors and Collaborators
National Taiwan University Hospital
|Study Chair:
|Kwan-Dun Wu, MD, PhD
|National Taiwan University
More Information
Keywords provided by National Taiwan University Hospital:
Additional relevant MeSH terms:
ClinicalTrials.gov processed this record on August 21, 2017
Bouchon A, Dietrich J, Colonna M. Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes. J Immunol. 2000 May 15;164(10):4991-5.
Bouchon A, Facchetti F, Weigand MA, Colonna M. TREM-1 amplifies inflammation and is a crucial mediator of septic shock. Nature. 2001 Apr 26;410(6832):1103-7.
|ClinicalTrials.gov Identifier:
|NCT00173485 History of Changes
|Other Study ID Numbers:
|Study First Received:
|September 12, 2005
|Last Updated:
|November 23, 2005
Keywords provided by National Taiwan University Hospital:
peritonitis, peritoneal dialysis, dialysate
Additional relevant MeSH terms:
Renal Insufficiency
Kidney Diseases
Urologic Diseases
Intraabdominal Infections
Peritoneal Diseases
Digestive System Diseases
Dialysis Solutions
Pharmaceutical Solutions
ClinicalTrials.gov processed this record on August 21, 2017


text:
Learn more about GOODWYN SCHOOL LIMITED. Check the company's details for free and view the Companies House information, company documents and list of directors.
Know about every new company document that is added!
Take advantage of our watch this company feature free of charge and have notifications sent directly to your inbox. Whenever a new document appears on this website, or if their particulars change, you will be immediately informed about such changes. To monitor these changes, all you need to do is click on watch this company.
|Fixed Assets
|£ 115,591
|Tangible Fixed Assets
|£ 115,591
|Current Assets
|£ 996,234
|£ 454,499
|Shareholder Funds
|£ 895,872
|Profit Loss Account Reserve
|£ 895,372
|Called Up Share Capital
|£ 500
|Net Assets Liabilities Including Pension Asset Liability
|£ 895,872
|Net Current Assets Liabilities
|£ 780,281
|Creditors Due Within One Year
|£ 215,953
|Cash Bank In Hand
|£ 541,435
|Stocks Inventory
|£ 300
|Par Value Share
|Ordinary Shares of £1 each
|Number Shares Allotted
|£ 500
Information about the Private Limited Company GOODWYN SCHOOL LIMITED has been prepared for information purposes only. It is not intended to be nor does it constitute legal advice. This is public information provided by the official company register.


text:
Brazil, like Canada, is getting fatter by the year.
Obesity has reached such epic proportions that Brazil's new soccer stadiums include more than double the number of extra-wide seats required by World Cup authorities to accommodate both people with disabilities and heavyweight fans. On Brazil's famous beaches, adipose folds have multiplied as fast as the pizza joints and Burger Kings that were virtually non-existent two decades ago.
Nevertheless, Brazil has a plan to reverse obesity – and it's winning raves from North America's toughest nutrition critics, including author Michael Pollan, food-industry watchdog Marion Nestle and Canadian bariatric expert Yoni Freedhoff.
Unlike food guides in Canada and the United States, it does not include arranging food groups in pie or pyramid shapes, adding up recommended servings listed in grams, or colour-coding nutrient groups that correspond to sectors of the agricultural industry – dairy, meat and grain. Nor does it require people to measure servings of pasta by the half-cup, or carve up steak into helpings the size of a deck of cards.
Instead, Brazil's proposed guide (in public consultation until May) emphasizes meals, not nutrients. It urges people to be critical of food-industry advertising, and introduces healthy eating as a lifestyle choice that involves learning how to cook from scratch and taking the time to sit down and eat with others.
The concept is so simple, experts say, that it just might work.
Pollan (known for the phrases, "Eat food. Not too much. Mostly plants.") called the new dietary guidelines "radical" in a recent tweet. The highly influential Nestle, who blogs at FoodPolitics.com, praised Brazil for its "sensible, unambiguous" approach.
And Freedhoff, Canada's own healthy-eating guru, described Brazil's food guide, released in February, as "refreshingly free of industry bias" and "exactly what we need to be doing as a society."
How people eat is as important as what they eat, said Jean-Claude Moubarac, a Montreal-based postdoctoral scholar of public health and nutrition who spent two years as a member of the University of Sao Paulo team responsible to the Brazilian ministry of health for developing the new guidelines.
Research has shown that when people sit down at mealtimes with friends and family, "they tend to eat less" than when they're eating alone or on the run, Moubarac explained.
Brazil's new food guide contains no information on calories or how to limit serving sizes to reduce body mass index. Instead, it tells Brazilians what to leave off the dinner plate. The guide recommends that people limit or avoid ready-to-eat products such as snack foods, candy, sweetened beverages, instant soups and microwaveable meals.
The warning to be wary of food-industry advertising is a world's first for government-issued dietary guidelines, Moubarac said. Compared with U.S. and Canadian guidelines, which don't address the impact of the food industry on dietary habits, "It's a huge thing that a ministry of health is saying, 'be critical of commercial advertising,'" he said.
Canada's Food Guide has not succeeded in shrinking waistlines in this country, where 18 per cent are now obese – a rate that has tripled since 1985.
Canada's guidelines are based on recommended daily intake of specific nutrients derived from food groups. But the guidelines are not as evidence-based as they seem, according to Freedhoff, director of the Bariatric Medical Institute in Ottawa. For the most part, "RDIs are theoretical," he said.
The nutrient-based approach has enabled the food industry to market products such as sugary cereals and oversalted soups as part of a healthy diet, based on added minerals or protein advertised on the box, Freedhoff explained. "This focus on nutrients has steered people towards nonsensical patterns," he said.
Added vitamins or not, heavily processed foods are loaded with excess sugar, fat and salt, which work against the biological mechanisms that let us know when we've eaten enough, studies have shown. Meals made from scratch are more likely to trigger feelings of satiety, Freedhoff said. "It is very challenging to consume very large quantities from fresh foods – you're full."
Cooking a healthy meal can be as simple as frying an egg, tossing a salad or making a sandwich, he added.
The trouble is that a growing number of people – Canadians and Brazilians – have either gotten out of the habit of making their own food, or never learned in the first place.
In Brazil, many grow up with a team of maids or grandmas who spend hours in the kitchen peeling and chopping meat and vegetables for almoco, the midday meal. A typical almoco consists of a dozen plates of food laid out on the table for the family to enjoy on a two-hour break from work or school.
But Brazil's booming economy has resulted in longer working hours – which cut into almoco time – as well as job opportunities that lure workers far from home.
Young Brazilians who didn't learn to cook from their parents are easily seduced by the convenience of fast food, Moubarac said. Focus groups conducted by the researchers who created Brazil's new guidelines identified university students as the group most likely to say they did not like, or know how, to cook.
And now, many in Brazil have disposable income for the first time, thanks to economic and social reforms that pulled 20 million Brazilians out of poverty between 2003 and 2009. This means new freezers and microwaves to store and reheat food, and money for fast food.
Transnational food companies have seized the market opportunity, including McDonald's (which has nearly 700 restaurants in Brazil), KFC and Burger King, which was acquired in 2010 by a Brazilian-owned global investment firm.
For lower-income Brazilians, eating a Big Mac holds far more social cachet than cooking rice and beans from the corner market. "It's a way to climb the social hierarchy," Moubarac said.
Escalating obesity has been the result. Nearly half of Brazilians are overweight – a proportion that has more than doubled since 1990. More than 15 per cent are obese, and in a decade or so, Brazil is predicted to reach the current U.S. obesity level of 35 per cent.
Moubarac acknowledged that dietary guidelines alone can't counteract international market forces and major socioeconomic shifts that have added to Brazil's girth. The new food guide is the first step in a larger plan that will include community-based programs designed to help lower-income families learn about healthy portion sizes and how to cook homemade meals with moderate amounts of sugar, fat and salt, he said.
He added that Brazilians who lack the time or inclination to cook can still make healthy choices by frequenting Brazil's ubiquitous "por quilo" (by the kilogram) buffet-style restaurants, which offer a wide variety of freshly prepared meat and vegetarian dishes sold by weight.
Canada should borrow pages from Brazil's new book, Freedhoff said.
Instead of focusing on abstract nutrition concepts, health authorities should support and encourage Canadians to cook simple, healthy, everyday meals.
Initiatives could include reintroducing home-economics classes for boys and girls in primary school and offering after-school programs that help parents learn how to cook, Freedhoff said.
In the meantime, he said, Brazil is a trailblazer: "These are guidelines that countries around the world should encourage their citizens to adopt."
10 key points in Brazil's proposed guidelines
The authors of Brazil's proposed dietary guidelines boiled down the 87-page document into 10 basic steps:
1. Prepare meals using fresh and staple foods.
2. Use oils, fats, sugar and salt in moderation.
3. Limit consumption of ready-to-eat food and drink products.
4. Eat at regular mealtimes and pay attention to your food instead of multitasking. Find a comfortable place to eat. Avoid all-you-can-eat buffets and noisy, stressful environments.
5. Eat with others whenever possible.
6. Buy food in shops and markets that offer a variety of fresh foods. Avoid those that sell mainly ready-to-eat products.
7. Develop, practise, share and enjoy your skills in food preparation and cooking.
8. Decide as a family to share cooking responsibilities and dedicate enough time for healthy meals.
9. When you eat out, choose restaurants that serve freshly made dishes. Avoid fast-food chains.
10. Be critical of food-industry advertising.
Source: Guia Alimentar Para a Populacao Brasileira (2014)Report Typo/Error
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text:
ASKING PRICE: $1.2-million
AGENTS: Jill and Bill Parlee (Chestnut Park Real Estate Ltd. Brokerage)
Alison Smith's passion for art drove her out of her native Montreal to Toronto to set up shop as an art dealer five years ago. She chose Dundas Street West near Ossington as the location of her eponymous Alison Smith Gallery, wanting to take advantage of an area of the city that scenesters have dubbed the new Queen Street West for its proliferation of new bars, restaurants, live music venues and cutting-edge galleries. "We bought the building because of the vibrant neighbourhood," said Ms. Smith, whose intention was to convert an early 20th-Century structure into a blended commercial and residential space for herself, her husband - author Larry Gaudet - and their two young sons. She says that she was particularly drawn to the property because it had excellent bones - sturdy brick, high-ceilings, a very dry basement, a huge garage and a courtyard. The building had gone through several renovations that had cut up the space, especially on the main floor, into a warren of small rooms, so an extensive renovation was necessary to give the building back "its original sense of high-street dignity and elegance," said Ms. Smith.
To convert an old building into a contemporary work-live space, Ms. Smith hired the award-winning Toronto architecture firm superkül. "The direction was to give the building back its unstudied elegance and the dignity of its years, while making it habitable for a young family with diverse modern needs: a place to store and hang plenty of art, but also hockey equipment for the boys. It also had to have plenty of running room for a tabby cat." The end result is a 3,000-square-foot shoebox of a building that has undergone a complete makeover without losing its original flavour. The façade was completely refashioned with grey concrete board surrounding a teak window frame and door at the entrance level, with a large silver window box jutting out at an angle from the upper floor. Inside, the main floor renovation was essentially a complete gutting, turning a cluster of tight rooms into a wide-open gallery space warmed by maple floors and an art collection featuring both historical (Goya) and contemporary, colour-saturated (Goran Lucic) works.
The courtyard squeezed between the building and the garage, and viewed from behind glass on the main floor, is this property's best feature, by far. Intimate in scale, it represents a private retreat within the bustle of the city beyond the building's threshold. A huge window at the rear of the main floor space looks over the courtyard. A black steel staircase descends to the second floor. "It feels very downtown and urban, but also peaceful," said Ms. Smith, who is now moving to give her growing children - and her stable of artists - more room to grow.
Editor's Note: The asking price has been dropped from $1.5-million to $1.2-million. This article has been updated.
Report Typo/Error
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text:
US GOVERNMENT researchers who purposely infected subjects with sexually transmitted diseases in Guatemala in the 1940s had conducted similar experiments a few years earlier in Indiana but had obtained permission, investigators said.
The contrast between how the US Public Health Service scientists experimented with Americans and Guatemalans showed that researchers knew their conduct was unethical, members of the Presidential Commission for the Study of Bioethical Issues, which is investigating the experiment, said this week.
The commission found that 1300 people were exposed to the sexually transmitted diseases syphilis, gonorrhea and chancroid and that at least 83 people had died.
In one case, a woman who was infected with syphilis was clearly dying from the disease, but instead of treating her, the researchers poured gonorrhea-infected pus into her eyes and other orifices and infected her again with syphilis. She died six months later.
The ultimate goal of the Guatemalan research was to discover if using penicillin after sex would protect against syphilis, gonorrhea and chancroid.
The experiments included infecting prisoners by bringing them prostitutes who were either already carrying the diseases or were purposely infected by the researchers.
In the studies conducted in Indiana, researchers exposed 241 prison inmates to gonorrhea in 1943 and 1944. But there, the researchers explained the experiments in advance and used only prisoners who volunteered.
In contrast, many of the same researchers who began experimenting on Guatemalans a few years later actively hid what they were doing and never tried to obtain permission.
Washington Post


text:
Dear Christopher Dorner,
I understand your feelings of hurt and pain. I make this plea to you to
stop spreading the pain, the hurt, and the fear. Please stop. Don't take
any more lives.
Christopher, your mother is distraught and deeply concerned for your safety.
There are many good and credible people in Los Angeles who will help you.
Danny J. Bakewell Sr. (The Los Angeles Sentinel's Executive Publisher/CEO) ,
Bishop Noel Jones, Bishop Kenneth Ulmer and Rev. Charles Singleton are all
individuals I know personally. I promise that they will gladly receive you.
I will do whatever I can do for you without hesitation. Please contact me
through Danny Bakewell at the L.A. Sentinel at 323-299-3800
Reverend Jesse L. Jackson Sr.
Coalition–Keeping Hope Alive


text:
link. Priced applications will be available in the Android Market by the middle of next week for US customers. Later this quarter, developers in Germany, Austria, Netherlands, France, and Spain will be allowed to offer priced applications with additional countries expected to be announced by the end of the first quarter. As you might have expected, payments and billings will be handled by Google Checkout. Also announced today was availability of free apps in the Android Market for users in Austrailia starting this Sunday.You have a great idea for an app for the Android Market, but you want to get paid for it. Well, the Android Market is now accepting priced applications from US and UK developers. Developers are asked to visit the publisher web site and upload their application along with pricing for end users. You can get to that page by clicking on this
source: Android Developers
It's coming!
0 d | 0 0 0Galaxy Note 8 to be announced with Infinity Display | here's what we expect
Android Market now accepting priced applications in US and UK
1 Comments
posted on 20 Oct 2010, 01:42 0
1. cool_water2008 (Posts: 5; Member since: 23 Dec 2008)
NOOOOOOOOOOOOOOOOOOOOO. Why the hell are they putting the costs for the market place. Does anyone know the price
Android Market now accepting priced applications in US and UK


text:
Type of Document Dissertation Author Tarifa, Todd Anthony Author's Email Address email@example.com URN etd-11162006-154047 Title Level of Youth Voice in the Decision-Making Process within the 4-H Youth Development Program as Perceived by State 4-H Program Leaders, State 4-H Youth Development Specialists, and 4-H Agents/Educators Degree Doctor of Philosophy (Ph.D.) Department Human Resource Education Workforce Development Advisory Committee
Advisor Name Title Krisanna Machtmes Committee Chair Gerri Johnson Committee Member Janet E Fox Committee Member Michael F Burnett Committee Member James Harvey Moore Dean's Representative Keywords
- Youth Voice
- Youth Development
- Youth
Date of Defense 2006-11-13 Availability unrestricted AbstractThis study aimed at determining the level of inclusiveness of youth voice in the decision-making process in the 4-H youth development program. It described the 4-H program's position on youth voice among State 4-H Program Leaders, 4-H State Youth Development Specialists, and 4-H Youth Agents/Educators. The information gathered was utilized in identifying the level of inclusiveness of youth voice in the decision-making process to better understand how to suit youth's needs, identify promising practices, and barriers.
Three underlying constructs were identified that represented State 4-H Program Leader's, 4-H Youth Development Specialist's, and 4-H Agent's/Educator's personal views on youth voice in the decision-making process. They are as follows: Adult Initiated, Youth Involved; Youth Led, Adult Supported; and Youth Involved, Adult Decision-Making.
Findings indicated that as an organization, 4-H, has to gather emerging research on youth voice and base trainings that address the primary issue of youth voice. Four-H needs to become more directional in its purpose and focus on trainings and issues that will aid youth development professionals in becoming true youth developers that address the needs of youth in our nation. Most interaction with youth within the 4-H program takes place at the county/parish level. It is evident that 4-H needs to include youth voice at the state level to ensure that youth needs are being met at the higher levels within the organization. While aware of emerging research on youth voice, 4-H state faculty, need to value the benefits of this new ideology.
When viewing youth voice inclusion from the standpoint of organizational culture, it was found that as a youth development program, 4-H has a double standard on including youth voice in the decision-making process. The 4-H youth development program can not include them as full partners and let them have little choice on how and when they participate. Four-H creates a sense of mistrust with youth, and this type of inconsistency will negatively affect youth voice on all levels. It will take time for the organizational culture to shift the paradigm and recognize and adjust the value of, and respect youth voice in the decision-making process.
Filename Size Approximate Download Time (Hours:Minutes:Seconds)
28.8 Modem 56K Modem ISDN (64 Kb) ISDN (128 Kb) Higher-speed Access TarifaDissertation.pdf 2.02 Mb 00:09:22 00:04:49 00:04:13 00:02:06 00:00:10


text:
Hey there, time traveller!
This article was published 11/12/2013 (1347 days ago), so information in it may no longer be current.
Call it breakfast, call it brunch -- and now you can even call it dinner if you have it at Bernstein's, where the breakfast menu is available all day, and later than it used to be. The hours have been extended to range from 8 a.m. to 7:30 p.m. on weekdays, until 2:30 p.m on Saturday, and from 9 a.m. to 2:30 p.m. on the once-closed Sunday.
That's only one of the changes at this durable (almost 30 years) deli-cum-butcher shop-cum-restaurant. It's still your typically bare-bones deli, but now son Aaron is a managing partner with his mother, Marla Bernstein, and they have been joined by new chef Beth Jacob, of the late and much lamented Fresh Cafe.
The top-notch deli foods are still available for eating there or taking home -- I'm crazy for the fresh salmon spread and the marinated herring -- but this time breakfast was what I had come for, and although there are still the traditional eggs with a choice of breakfast meat, hash browns and toast ($5.50 to $8.25), Jacob has added several interesting dishes, which are listed on a feature sheet.
I didn't get to her Jewish chicken waffles -- schnitzel bits with little pancakes, chipotle maple syrup and blueberry compote ($11.25) -- or the charcuterie hash of deli meats and hash browns with two eggs and toast ($8.75), or the quinoa breakfast cereal with maple syrup and dried cranberries ($5.50). But I did have what must be one of the city's best breakfast dishes, the glorious latke stacker of potato pancakes, topped by corned beef, a poached egg and swiss cheese, ($10.25, or $11.25 with toast).
Also new is a whole-wheat wrap with cream cheese, dill and scrambled eggs, paired with a latke and sour cream ($8.50). Of course there's still the classic platter of lox with a toasted bagel, cream cheese, capers, red onion, cucumber and tomato ($11), but my favourite version is still the simplest -- a good, chewy bagel topped by a thick shmear of cream cheese and generous slices of silky, top-grade lox ($6.75). The Bernstein's omelette, with onions, corned beef, mushrooms, peppers and cheese, was always available ($8.50, hash browns $1 extra) but now it comes in a vegetarian version, and includes a latke or hashed browns and toast ($9.50).
Possibly some might not consider them breakfast on their own, but I would -- the excellent potato latkes, with apple sauce or sour cream ($5.75 for three, $2.25 for one). The crisp, skinny fries are delicious in the poutine with Bothwell curds ($6.50), which is even better with the addition of Montreal smoked meat ($8.50). I couldn't resist trying one lunch innovation which alone should earn Jacob a medal, i.e. the house-cured corned beef served warm in thick, hand-cut slices, on rye with hot mustard ($8.25). And the next time I'm there I'm determined to try the bison Reuben burger -- corned beef, bison patty, sauerkraut, swiss cheese and house-made Thousand Island dressing ($7.50).
Service is cheerful and willing but can be overwhelmed and confused at peak hours -- wise regulars know to come before or after the noon rush.
-- -- --
There have been some changes at Prairie Ink also, but in this case, not for the better. Sunday brunch, which had been mostly good, has been dropped, and now all breakfasts are only la carte, served from 9 a.m. to 11 a.m. Monday to Friday, to 11:30 a.m. on Saturday and 2 p.m. on Sunday.
An omelette was cold and thin, filled with crumbles of hard bacon, mushrooms that were black with age and (representing the promised mixed cheese) two wafer-thin wee squares of cold cheddar ($11 with toast). Eggs Benedict came on a knife-defying hard muffin ($12). Worse, the thick slab of back bacon had a greyish tint, and was as dry and difficult to cut as the muffin -- that (our server explained, as though that made it acceptable) was because it was cut thick. And when we pointed out that some of the back bacon slices (with a dish of poached eggs and fried potatoes, $8.75) were pink, moist and tender, while others on the same plate were as poor as those with the Benedict, the server had no comment at all. They were left uneaten, there was no apology, and no adjustment to the bill.
Only huevos rancheros were acceptable -- two eggs with chorizo, salsa, corn and beans and a cheese quesadilla ($12).
But if your usual breakfast runs only to some kind of baked goods, you'd be in luck with the delicious individually baked cinnamon bun with caramel sauce, or the decent, if not very airy croissant.
If breakfast was mostly a bust, another of Prairie Ink's innovations -- High Tea -- was a winner, served on Tuesdays only (reservations essential) from 2 p.m. to 4 p.m. for a flat $16.50, all of it good, and good value, although some might object to paying an obligatory 15 per cent tip.
For the tea the inner section of the restaurant is set aside, with crisp white linens and pretty Royal Dalton tea cups on the tables. The set menu is changed each week; in our case there were appetizers of hummus-topped naan, cucumber cups with feta, tomato and olive relish, and roma tomatoes filled with a mixture of bacon, cheddar and panko crumbs; three tea sandwiches (salmon, egg and mixture of pureed peas with prosciutto), and a plate of fresh fruits and two tiny portions of cheese.
But for sweet teeth, the best was yet to come, in the pastries, for which Prairie Ink is justly noted -- a square of chocolate sacher torte, a puff filled with praline cream and cake layers with pistachio-flavoured filling, all perfectly lovely (although I did miss not having a scone). Less satisfying was the tea itself (13 varieties), which wasn't loose leaf, but had been tied into bags by the restaurant, and which didn't taste in the least like the Earl Grey we had ordered.
To see the location of this restaurant as well as others reviewed in the Winnipeg Free Press, please see the map below or click here.
Restaurants marked with a red flag were rated between 0.5 to 2.5 stars; yellow flags mark those rated between 2.5 to 4 stars; and green flags mark those rated rated 4.5 to 5 stars. Locations marked with a yellow dot were not assigned a star rating.


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But the struggle continued.
In March 1965, deputies and troopers attacked voting rights activists with clubs and tear gas, on a march from the Alabama town of Selma to Montgomery.
The events helped to pave the way for the Voting Rights Act of 1965, which enabled far wider registration of black voters.


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Police intercept North Huntingdon man's growth hormone package
Editor's note: Charges of possession of a controlled substance filed against Frank Joseph Lodovico Jr., 38, of Haflinger Drive in North Huntingdon were dismissed.
A North Huntingdon man landed behind bars when police said they intercepted a parcel of human-growth hormones that he is accused of ordering from Slovakia.
Frank Joseph Lodovico Jr., 37, of Haflinger Drive, is accused of ordering numerous vials of schedule III testosterone from Bratislava, Slovakia. He is charged with possession of a controlled substance and illegally obtaining a controlled substance.
Lodovico was held in Westmoreland County Prison on Nov. 14. He was released the next day on $10,000 monetary bail.
A phone number is not listed for Lodovico. Court records do not indicate if he has an attorney.
Customs and Border Protection officers spotted a suspicious parcel at a shipping facility in Philadelphia on Nov. 13, according to court documents.
Officers opened the parcel and conducted a search of the contents.
Inside the package, officers logged four vials of testosterone, six vials of testosterone enanthate, two vials of methenolone enanthate, one vial of boldenone undecylenate, tablets of oxymetholone, tablets of mesterolone and tablets of anastrozole.
According to the U.S. Drug Enforcement Administration, all except anastrozole are schedule III non-narcotic drugs. Anastrozole is listed as a U.S. prescription drug used to counteract effects of steroid use.
Methenolone enanthate is the same steroid for which baseball players Alex Rodriguez and Barry Bonds were accused of testing positive in the early 2000s.
Under the state's Controlled Substance, Drug, Device and Cosmetic Act, possession of schedule III substances is illegal if someone is not a registered practitioner or licensed by the state board or did not obtain it directly from a practitioner.
Officers shipped the suspect package to the Homeland Security Investigations office in Pittsburgh in anticipation of a controlled delivery, according to court documents.
State police obtained a search warrant for the Haflinger Drive home where the package was intended to go.
The package was delivered around 5:45 p.m. Nov. 14. An occupant accepted the package, according to court documents.
Officers executed a search warrant and discovered "additional steroid-type substances," according to court documents.
A preliminary hearing before District Judge Douglas Weimer has been set for Feb. 12.
Amanda Dolasinski is a staff writer for Trib Total Media. She can be reached at 412-856-7400, ext. 8626.


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Children's rights have been celebrated in Myanmar for the first time in 20 years.
The occasion was to mark the anniversary of the signing of the United Nations Convention on the Rights of the Child.
The international convention, signed at the UN General Assembly in 1989 and ratified by the Myanmar government in 1991 has never been officially marked in this country.
The celebration was organised with the help of the Myanmar National Human Rights Commission and attended by the chief minister of Yangon Region and representatives of UNICEF, as well as children and teachers.
Chief Minister U Myint Swe said Myanmar had assumed its responsibilities by adopting the convention, which built on the country's 1993 law on child protection. The convention's 51 clauses are designed to protect children's rights and meet their basic needs.
"Children are a vital human resource for the future of the state. We need to pay serious attention to promoting the lives of children and protecting them from violation of their rights," said U Myint Swe. "We expect this anniversary to be celebrated throughout the country next year."
U Win Mra, chairman of the Myanmar National Human Rights Commission, said the convention on children's rights was one of the three core human rights convention ratified by Myanmar. He urged all concerned to protect, nurture, promote and care for children, as well as to protect their rights.
"The convention has been observed in past years on an individual basis, but this year the Myanmar National Human Rights Commission has marshalled all the resources under one banner to celebrate this occasion," said U Win Mra.
U Myint Swe said the Myanmar government had set up committees from the national level down to the township level to carry out child protection activities.
UNICEF representative Mr Bertrand Bainvel said many challenges to children's rights persisted throughout the country, including violence and lack of access to basic needs and services.
"We request all of you to create a better life for us. We don't want to live under conditions that are insecure," said Ma Thin Me Me Zaw, a middle school student from Yangon's Shwe Pyi Thar township who attended the ceremony.


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Private businesses with fewer than 50 employees added 58,000 jobs in April, almost half (49%) of the total increase of 119,000 jobs, according to the ADP National Employment Report released this morning.
The economy needs to add 125,000 jobs a month to keep up with population growth, and analysts had been expecting the closely watched ADP report to be much higher. The Bureau of Labor Statistics will report on public and private employment and the official unemployment rate on Friday. Check the Handling Hard Times blog for the scoop.
ADP breaks out the jobs growth by size of company:
- Firms with fewer than 50 employees, +58,000 jobs
- Businesses with 50 to 499 employees, +57,000 jobs
- Companies with 500-plus employees, +4,000
"While April's increase was the 27th consecutive monthly advance, it nonetheless reflected a deceleration in the recent pace of hiring. This deceleration seems consistent with other data including a disappointingly weak report on first-quarter gross domestic product, a recent back-up in initial unemployment claims and last month's relatively weak reading on establishment employment reported by the Bureau of Labor Statistics."
Other reports that specialize in small-business employment agree that small businesses are adding jobs.
Intuit Inc., which makes QuickBooks and Turbo Tax, says small business jobs increased approximately 40,000 in April for an annual growth rate of 2.5%. However, hours that the average employee worked declined.
Intuit's report is based on businesses with fewer than 20 employees.
SurePayroll, whose average client has seven employees, said its payrolls reflect a decline in hiring during April both nationwide and in Los Angeles. Nationwide hiring was down 0.1% March to April and down 0.4% in Los Angeles.
The average paycheck size also declined in April, according to SurePayroll.
Other business stories…
- 11 worst businesses to start in 2012
- 11 hottest businesses to start in 2012
- Crocs dangerous for kids, lawsuit claims
- 10 brands youngsters like most
- Sexy photos anger O.C. fitness club members
- 8 foods Americans like best
- 11 highest profit businesses to start
- Calif. lost most jobs, firms in recession
- Local landscapes win beautification awards
- 27% of California firms 'a sinking ship'
- 10 consumer products that beat the recession
- Report: 99.7% of O.C. residents pay no use tax
- Poll: How much money does government waste?
- O.C. chip maker gets $4.5 million, new CEO
- 11 businesses that have no future
- O.C. manufacturer turns 50
- 10 industries that are booming
- 10 companies with worst customer satisfaction
- Facebook most hated social media site
- Top 10 franchises that sell for under $50,000
- Crevier BMW sold to Penske Automotive Group
- Amazon terminates deal with Calif. affiliates
- O.C. manufacturer moves, adds 270 jobs in D.C.
- Top 5 things Americans won't do without
- Top branding expert makes name for her own line
- OC. manufacturer to close, move to Texas


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Angels of Fur K-9 Rescue is a group of volunteer dog lovers. We dedicate our time and knowledge to save our loving dogs. Our mission is to rescue, provide sanctuary, and ultimately to re-home abandoned, stray and neglected dogs.
All of our dogs are housed in foster homes where they are provided with an enormous amount of love and any medical attention that may be required to insure that they are in good health before adoption. They are all spayed or neutered and brought up-to-date on shots before being placed in their new homes.
As we are all volunteers we have busy lives too, but as time permits us we will provide house-training and basic obedience while in our care.
We evaluate every dog for their temperment, ability, and personality. This helps us to place them in homes that are well suited to their needs and to the needs of their future owners.
If you are looking for a specific type of dog and do not see it on our website, please email us.
Tianna and I have a veterinary technician background and have both donated our time in the Local Spay and Neuter Clinics and have decided we want our own part in helping.
If you have any questions feel free to email us anytime at email@example.com
VISIT OUR WEBSITE FREQUENTLY @ www.angelsoffur.com
A dog is the only thing on earth that loves you more than he loves himself
- Josh Billings
Thank You!
Your message has been sent to ANGELS OF FUR K-9 RESCUE.
You'll receive a copy, too, at to help you keep track of which pets you've inquired about, and which shelters and rescues you've emailed.
NOTE: Some shelters have physical locations you can visit; some of these shelters may only have pets for a limited time, so please do not wait for a reply—just go visit the shelter! Other organizations are rescue groups run by busy volunteers who may take a while to reply. You can find information about the shelter or rescue group caring for this pet, and their adoption procedures, on the pet's details page on Adopt-a-Pet.com.


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Slouchy Newsboy Hat with Short Brim Visor
buy it now or visit pattern website
Unique Slouchy Newsboy hat with banded chunky ribbing and a soft visor. (see 4th picture for alternate as a regular newsboy! Pattern INCLUDED!)
This LOOM KNIT hat was created by Kristen Mangus of GoodKnitKisses to feature a unique slouch on a newsboy hat with rich texture to stand out. The Slouchy Newsboy was originally published in 2011 and has been a popular hallmark pattern of GoodKnit Kisses. (edit: a new NEEDLE knit version is available as of Feb 2016 see link below).
As usual Kristen walks you through techniques, with video links for more advanced steps, so that even beginners have been able to knit this pattern.
Loom Knit pattern but could be worked on needles. Visor is noted all right side and hat is in the round. Written for an adult and fits a range of sizes due to the stretchy. Can be made smaller on a different loom or less bulky yarn. No blocking needed.
Link to NEEDLE knit version
This pattern is meant to be worn with the slouchy part pushed back and patted down and the sides pulled down over the ears. If not worn that way, it has a very "stovetop pipe" look. Directions for snugger hat included as well as links to video tutorials at no additional charge (See 4rd photo above). This is warm hat but is light enough to not be restrictive and gets some air flow.
Size: Adult (alternates for baby-adult)
Size: Adult or Teen
Men love this in the non-slouchy version as I'm told from several happy clients! :-)


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Hey there, time traveller!
This article was published 30/4/2013 (1570 days ago), so information in it may no longer be current.
April 22 was Earth Day, an international call for action on the environment.
Hopefully, you already conserve water, refrain from idling your car, use online billing and energy-saving lights.
Here are some additional ways we can all 'reduce, re-use, recycle' right here in Westwood.
Reduce: Choose eco-friendly products. Vita Health at 3500 Portage is perfect for Earth-conscious shoppers, with everything from baby products and cosmetics to pet-care and cleaning supplies.
Even the lighting and refrigeration systems are eco-friendly, and if you forget your re-usable shopping bag, the plastic bag they'll give you is compostable.
Many other Westwood retailers also offer 'green' household products, which are better for our lakes and waterways including Sturgeon Creek. By the way, the annual 'Creek Clean Up' is set for Saturday May 11 at Woodhaven CC at noon.
Re-use: The garbage bin should be used only as a last resort — many things can be given new life with a little effort.
Newspapers for example, will be put to good use at the Winnipeg Pet Rescue Shelter at 3062 Portage Ave. They will also be glad to have your old towels and blankets. Items can be dropped off during regular hours, 11 a.m. to 7 p.m. Monday to Friday, 11 a.m. to 6 p.m. Saturday.
Gently used housewares and clothes? Value Village and Salvation Army have drop-off centres close by, while Big Brothers/Big Sisters and Canadian Diabetes will pick items up at your home.
And the Give-Away Weekend is coming up May 11 and 12.
All sorts of useful things can be placed near your curb with a 'free' sign; check the city's website or call 311 for details. (http://winnipeg.ca/waterandwaste/recycle/giveawayWeekend)
Recycle: Most Westwood homes actively recycle, but what about things you can't put in the blue bin?
Clean plastic bags (overwrap from paper towels, cereal or bread bags, etc.) are collected in October by students at École Robert Browning for the 'Bag Up Manitoba' campaign; just save your clean bags for Browning.
What about old household or electronics batteries? Westwood homeowners will soon receive a 'Battery Recycling Box' courtesy of Coun. Grant Nordman – he'll even come pick up the full box! (Westwood Library also has a drop-box for expired batteries).
Oh, and yard waste collection has been increased to every second week all summer long - stock up on paper bags!
Earth Day and every day, our environment is in our hands… thanks for doing your part.
Sharon Pfeifer is a community correspondent for Westwood.You can contact her at email@example.com


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Despite Kermit's insistence on never giving into Doc Hopper, The Muppets are highly exploitable. They're instantly recognizable and beloved, but with non-specific mouth movements that can be made to say almost anything. That means YouTube masher-uppers have had a blast dubbing song after song into the mouths of our feltish friends. Here are 5 examples:
Cookie Monster Sings Tom Waits – "God's Away On Business"
Cookie starts with the letter "C." So does "Christmas Card from a Hooker in Minneapolis."
Beaker Sings Rick Astley – "Never Gonna Give You Up"
You, sir, have been meaproll'd.
Bert and Ernie Perform M.O.P. – "Ante Up"
Why do they take baths together? Cause they're hardcore gangstas, bitch.
Rowlf The Dog Sings Tay Dizm (f/ Akon) – "Dreamgirl"
Is Miss Piggy your dream girl? I assume the answer is "hells yeah."
Kermit Sings Nine Inch Nails – "Hurt"
Because it's good to end on a lighter note.
Geoffrey Golden is the Editor in Chief of The Devastator, "The Quarterly Comedy Magazine For Humans," in comic book stores across the country.


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Items by Eric Barker
A controversial plan to dredge the Snake River west of Lewiston, Idaho, has been stalled by a judge – for now
The National Marine Fisheries Service wants to table for five to 10 years any serious debate on breaching four dams in the Snake River to save salmon.
If all works out, grizzly bears may be reintroduced into the Northern Rockies' Selway-Bitterroot Wilderness Area in as little as a year.
Both sides are surprised when dam-breaching supporters outnumber opponents at public meetings over breaching four Snake River dams to save salmon.
Protests and chainsaws in Idaho's controversial roadless Cove-Mallard area have both stopped since new Nez Perce National Forest Supervisor Bruce Bernhardt announced that six unsold timber sales planned for the area would remain unsold.


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Jump start your creativity with free projects, from crafts and gifts to fashion
and home decor
Free embroidery designs are just a click away
Each month, we introduce a specialty presser foot along with a corresponding project
Step-by-step guides.
Instructions on features and functions of your favorite machines.
Individual embroidery designs including designs from Disney, Warner Brothers and
Create a new account or log-in to update or view existing account.
Downloads, FAQs, Help and more.
Find Print Drivers & Utility Software for you machine
Download Manuals, Reference Guides, and other documents for your Product.
Contact us by Email or Phone
This set includes the arm for your machine type, 2" X 4" mono hoop, 2.5" X 4" pocket hoop, 3" X 4" pocket hoop, 5" X 4" holiday stocking hoop, 6" radius hat hoop for the side and back of hats, and 7" X 5" attaché case hoop.
For Use With: PR-600, PR600II, PR620, PR600IIUPG, Entrepreneur® PR650, Entrepreneur® Pro PR1000, Entrepreneur® Pro PR1000e, Entrepreneur® PR650e, Entrepreneur® PR655, PR-655C
Running out of Ink and Toner is a thing of the past with Brother Refresh
Running out of Ink and Toner is a thing of the past with Brother Refresh
Brother Introduces DesignNCut


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Product Spotlight
Featured Research
Quick Stats
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E-Cigs: The Next 'Big Thing'? Napster Founder Thinks So
Sean Parker among investors of NJOY's recent $75M round of funding
Sean Parker likes to get involved in "Next Big Things." He co-founded Napster, which changed the way we consume digital music; he founded Plaxo, an online Rolodex with viral tools that integrated with Outlook and quickly reached 20 million users; and he was the first to see the potential in Facebook and became its founding president when it was a few months old.
His new Next Big Thing – Electronic Cigarettes.
Parker has joined several investors in the recent $75 million funding of Scottsdate, Ariz.-based Electronic cigarette brand NJOY, money the company says it will use for marketing, clinical trials, research and development, and international expansion.
"As an entrepreneur and investor I'm drawn to disruptive companies in explosive new markets," Parker said. "The common thread between these companies is a missionary desire to leverage technology to change the world. I'm optimistic that the clever application of technology might someday obsolete the combustion cigarette and all the harm it causes. Solving this problem takes a world-class leadership team that brings together executives across a range of disciplines from electrical engineering, to chemistry, to consumer marketing."
And while he's investing in the NJOY brand specifically, Parker sees this as an investment in the e-cig segment overall. NJOY has also demonstrated a commitment to advancing research and scientific understanding of the electronic cigarette category and recently announced that Former Surgeon General Dr. Richard Carmona was appointed a member of NJOY's board and chair of its scientific advisory committee.
"We are excited to be an investor in this company and to participate in the massive growth of this segment," Parker added.
Right now, the electronic cigarette market is still in the Harvard Facebook stage – with a steadily growing group of consumers that is poised to explode once critical mass of retail availability, word-of-mouth promotion, and TV, print and digital marketing is reached. Indeed, shelf space for these products at my local drug and convenience stores – even the 100-square-foot c-store in our office building -- has quadrupled these past few months, which has led me to wonder if grocers have decided to stay out of the business or think it's a fad that will disappear.
People once thought Facebook was a fad as well.
Sean Parker believed in it…now virtually every grocer in the world has a Facebook page, which, incedentally, is a great way to engage your customers in a discussion as to whether e-cigs are right for your business... at least until the next "Next Big Thing" comes along.
So my question to grocers is: Are you in or are you out of the e-cig business?


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Fireballing during the Maine Higland Games
By Robert F. Bukaty, Special to the BDN
A fireball demonstration took place during the Maine Highland Games at the Topsham Fairground, Friday evening, August 16, 2013 in Topsham, Maine. The origins of fireballing in Scotland go back to ancient times when the Scots showed their fondness for celebrating various celestial events, pagan rites and clan victories with the lighting of large bonfires and swinging burning baskets on the bare hilltops of their rugged land, according to Dr. Bill Cunningham.
Posted Aug. 17, 2013, at 1:34 p.m.


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This weekend saw Trek World Racing's Justin Leov crowned New Zealand's Downhill National Champion after storming to victory in Nelson. Hopefully we'll see Justin Leov carrying this momentum into the World Cup in a few weeks time.
Here's the word from one of Justin Leov's latest sponsors, Redraven.
Redraven is super excited to announce Justin Leov's latest racing results from the New Zealand National Championships.
Justin stormed to victory in both the Super D and DH races in Fringed Hill, Brook Valley, Nelson. The downhill course is one of the hardest courses in New Zealand and drew a huge crowd ready to watch the country's top racers sprint their way to the finish line.
The Men's Elite DH race was extremely close. Justin Leov confirmed his liking and success history with Nelson race courses to win by just over a second. The Downhill National Championship win made it two titles for Leov for the weekend including his Super D gold from Friday night. Leov will now proudly wear his race jersey this year on the world stage with right sleeves constructed as a NZ flag, the internationally recognised downhill National Champions identification.
Redraven CEO, Jonathan Knight commented: "This was amazing news to wake up to. Justin has brought Speedview its first National title in Downhill mountain biking. Justin has proved just how amazing Speedview performs in all weather conditions even bone dry dirt and it also showcases just how much of an all-round goggle Speedview is. We now look forward to seeing Justin on the UCI World Cup stage proving again how awesome Speedview preforms. Bring the rain!!!"
Justin added: "I'm stoked after winning both races this weekend. Speedview is an integral part of my race kit. Having the ability to clear my vision in all weather conditions including dry, dusty trails allows me to focus on racing and winning. I'm really looking forward to the UCI World Cups come wet or dry!"
Redraven are currently looking for global MTB distributors – if interested give them a shout at firstname.lastname@example.com
Full results can be found here.
For more information on Speedview visit http://www.redravenracing.com


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To understand what it's like to have asthma, take in a deep breath and hold it; then without letting any air out, take another deep breath and hold that. Physically your brain wants to breathe in deeper but what your body needs is to exhale.

But a revolutionary new treatment, called bronchial thermoplasty (BT), provides a new treatment option for patients with severe asthma.

BT therapy uses mild heat energy to treat the airways of the lung, reducing the ability of airway muscle spasms to obstruct the airway, allowing patients to breathe more freely. It is designed for adults who struggle to control their asthma with standard asthma medication.

Until now, medication has been pretty much our only tool for fighting severe asthma. But some people don't respond well to the drugs we use. For those people, this technology may reduce life-threatening episodes of severe asthma.

In fact, studies conducted one year after BT treatment have shown:

79 percent of patients who were treated with BT saw significant improvements in their asthma-related quality of life.
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32 percent reduction in severe asthma attacks
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84 percent fewer emergency room visits for respiratory symptoms
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66 percent fewer lost days of work, school, and other daily activities due to asthma

\r\nAdditionally, data from a recently concluded five-year study has shown:

Effectiveness of BT is maintained out to five yearsA 48% average decrease over 5 years in severe exacerbation event rates
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A reduction in severe exacerbations requiring oral corticosteroids
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A 88% average decrease over five years in the rate of ER visits for respiratory symptoms
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Long-term patient safety is maintained

\r\nThe procedure is minimally invasive and begins when a physician inserts a small bronchoscope through the patient's mouth or nose and into the lungs. The tip of the catheter is expanded to contact the walls of the targeted airways. Controlled bursts of mild heat are then delivered. The procedure is performed under moderate sedation or light anesthesia, and the patient typically goes home the same day.

Treatment includes three outpatient visits, scheduled approximately three weeks apart; after all three procedures are performed, treatment is complete.

Patients who are 18 or over and who struggle with asthma that is not well controlled with medication may be candidates for treatment. For more information, patients should contact their allergist or primary care physician for a referral, or go to BTforAsthma.com to learn more.


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Bama bigs lead the way
Posted January 8, 2013
Barrett Jones, Chance Warmack, DJ Fluker, Cyrus Kouandjio, Anthony Steen. You may not have heard much about them because they are offensive linemen, but after watching Alabama blow up Notre Dame, these Bama bigs are a reason why the Tide rolled the Irish.
Jones won his 3rd national championship ring playing his 3rd different position on the offensive line. The senior played his final game with torn ligaments in his left foot and even gave his own quarterback a shove in the fourth quarter. But he and the four other offensive linemen protected their quarterback from Notre Dame and kept AJ McCarron's uniform clean.
The big fellas also created paths for Eddie Lacy and TJ Yeldon to run through. One particular play caught my eye. On Lacy's first touchdown run, right guard Anthony Steen brushed the defensive lineman on his way to his assignment, block Manti Te'o. Steen tripped before getting to the Notre Dame All-American but instead of staying grounded, Steen lifted himself to his knees quickly enough to get a shoulder on Te'o who couldn't reach Lacy much less tackle him. The result was a 20-yard opening drive touchdown.
Like their team, Alabama's offensive front was the best in college football. Some with much deeper knowledge of Bama football than me, are calling this the best offensive line in Crimson Tide history. They were certainly in tune during the National Championship game against one of the top statistical defenses of 2012.
This was Mack truck meeting Mitsubishi down in Miami.
Barrett Jones, Chance Warmack, DJ Fluker, Cyrus Kouandjio, Anthony Steen. They got much less attention during the national championship telecast than AJ McCarron's girlfriend, Katherine Webb. But from a football perspective, Bama doesn't score without them.


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Life Is A Game , Uneven Bars Is Serious !!! Sweatshirt
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Sweatshirt Life Is A Game , Uneven Bars Is Serious !!! is the all-time ultimate in comfort with its classic, relaxed fit. On the surface rich cotton with Life Is A Game , Uneven Bars Is Serious !!!, inside flannel for the softest sensation. Medium to heavy weight ensuring warmth and makes it great, weather as a throw-over in rainy and windy weather or to protect against the snow and ice in the winter.
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Sku: SR1BL0000033270202020636WHGP00000