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Dendritic cells (DC) are highly specialized antigen-presenting cells with a unique ability to activate resting T lymphocytes and initiate primary immune responses. Angiotensin II (AII) is involved in key events of the inflammatory response. Because our previous work implicated an effect of AII on differentiation and function of murine and human DC, we investigated the impact of AII type 1 receptor (AT1) deficiency on the phenotypical and functional properties of mouse DC in vitro and in vivo. Bone marrow (BM) cells isolated from mice lacking AII subtype 1a receptor (AT1a), AII subtype 1b receptor (AT1b), or both receptor isoforms and control littermates [wild type (WT)] were cultured for 7 days in the presence of recombinant mouse granulocyte/macrophage colony-stimulating factor to generate myeloid DC in vitro. Generation of CD11c+ cells was less efficient in both AT1a- and AT1b-deficient BM cells than in WT BM cell cultures. Moreover, DC generated from AT1-deficient progenitors showed lower levels of expression of major histocompatibility complex II (MHC-II) and CD11c (p < 0.01) and a marked reduction in their allostimulatory activity (p < 0.01 or 0.001). Although AT1-deficient DC released comparable levels of interleukin (IL)-10 and IL-12p70 to WT DC, they produced significantly lower levels of tumor necrosis factor α (TNF-α) (p < 0.05). Remarkably, CD11c+ cells isolated from the spleen of AT1 knockout mice challenged with lipopolysaccharide in vivo up-regulated MHC-II, CD40, and CD80 as did WT, but released significantly lower levels of TNF-α (p < 0.01). These data provide clear evidence that AT1 controls differentiation and functionality of DC and thus may have a crucial impact on inflammatory processes where local angiotensinergic systems are known to be activated.
Angiotensin II (AII), the major bioactive peptide of the renin-angiotensin system (RAS), plays a critical role in controlling cardiovascular and renal homeostasis. AII acts through two main cell surface receptors, AT1 and AT2. Most of the recognized functions of AII involve AT1, which represents a critical pharmacological target in the treatment of cardiovascular disorders, such as hypertension, heart failure, and diabetic nephropathy. Humans possess a single AT1 (de Gasparo et al., 2000), whereas there are two AT1 isoforms described in rodents, AT1a and AT1b, which are products of separate genes (Agtr1a and Agtr1b, respectively) (Ito et al., 1995). These two isoforms share substantial DNA sequence homology, but are pharmacologically indistinguishable and differ in distribution and regulation among tissues and cell types. A growing body of evidence supports the notion that, in addition to its role in homeostasis of cardiovascular and renal systems, AII activates different inflammatory pathways. It increases vascular permeability, enhances the recruitment of leukocytes, and stimulates the production of inflammatory molecules such as reactive oxygen intermediates, adhesion molecules, growth factors, chemokines, and inflammatory cytokines (Fernandez-Castelo et al., 1987; Wolf et al., 1997; Sadoshima, 2000; Phillips and Kagiyama, 2002). In this way, acting mainly through the AT1, AII seems to contribute to the development of a great number of inflammatory diseases, such as cardiovascular diseases, immune-mediated glomerulonephritis, allograft rejection, and arthritis. Thus, independent of their hemodynamic benefits, the effectiveness of AT1 blockers has been attributed in part to inhibition of the proinflammatory actions of AII (Hisada et al., 1999; Nataraj et al., 1999; Dragun et al., 2005).
Dendritic cells (DC) are highly specialized antigen-presenting cells with a unique ability to activate resting T cells directing the differentiation of T CD4+ cells into different profiles (Banchereau and Steinman, 1998). DC progenitors from the BM enter the bloodstream and sparsely distribute in all tissues, where they differentiate into immature DC characterized by a high ability to capture and process antigens. Inflammatory cytokines and microbial products promote the maturation of DC, characterized by a high ability to present antigens to lymphocytes, triggering the adaptive immune response. Thus, they represent a crucial link between innate and adaptive immunity (Banchereau et al., 2000). We have reported that the blockade of AT1 by specific antagonists such as losartan or candesartan impaired differentiation and function of DC, supporting the existence of an autocrine/paracrine loop through which AII supports the normal development of DC. In the present study we investigated the impact of AII type 1 receptor deficiency on the differentiation and function of DC in vitro and in vivo. We report that the absence of AT1a and/or AT1b subtypes results in the generation of DC with major alterations in their phenotypical and functional properties.
Materials and Methods
RPMI medium 1640 with l-glutamine and fetal calf serum (FCS) was purchased from Invitrogen (Grand Island, NY). Recombinant mouse GM-CSF (rmGM-CSF) was purchased from R&D Systems (Minneapolis, MN). Lipopolysaccharide (LPS) from Escherichia coli 0111:B4, β-mercaptoethanol, BSA-FITC, and dextran-FITC (DXT-FITC) were purchased from Sigma-Aldrich (St Louis, MO). Carboxyl fluorescein succinimidyl ester (CFSE) was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK), and [3H]thymidine was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). Mouse anti-CD4 microbeads, mouse anti-CD11c microbeads (clone N418), Macs separation columns, and preseparation filters were from Miltenyi Biotec (Bergisch Gladbach, Germany). Collagenase D was from Roche Diagnostics (Mannheim, Germany). The following antibodies were purchased from BD Pharmingen (San Diego, CA): anti-CD 16/32 purified (2.4G2 clone), anti-CD4-FITC (L3T4) (RM4-5 clone), anti-Gr-1-PE (Ly-6G) (1A8 clone), anti-CD40-FITC (clone 3/23), anti-CD80-PE, anti-IA/IE-PE (clone M5/114.15.2), anti-Iab–FITC (AF6-120.1 C57BL/10J), and anti-CD11c-PE (clone HL-3).
Animals and Experimental Design.
Mice lacking AT1a or AT1b for AII were generated by using homologous recombination in embryonic stem cells as described previously. AT1a (−/−//+/+), and AT1b (+/+//−/−) single-knockout (KO) mice were used to generate the AT1a/AT1b double-knockout mice (DKO) (−/−//−/−) (Ito el al., 1995; Oliverio et al., 1998). To generate homozygous wild-type (WT) (+/+//+/+) and DKO animals, heterozygous offspring was intercrossed and bred on a 129 × C57BL/6 background at the animal facilities of the Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany, whereby experimental homozygous animals were generated by cross-breeding of heterozygous mice to ensure the most similar genetic background. Sets of males 3 to 6 months old were used. Experiments were performed in accordance with the American Physiological Society Guiding Principles for the Care and Use of Animals in Research and the guidelines of German federal law on the use of experimental animals (Animal Welfare Act) and were approved by the local authorities.
For the in vivo challenge with LPS, groups of four mice were injected intraperitoneally with either a sublethal dose of LPS (10 mg/kg) from Escherichia coli (serotype 0111:B4) or solubilized in 0.9% NaCl or 0.9% NaCl alone (control group). The dose of LPS was sufficient to elicit a significant response without the risk of endotoxic shock according to the literature (De Smedt et al., 1996; McCartney-Francis et al., 2004). Eighteen hours after injection the animals were killed by cervical dislocation and the spleens were removed.
Polymerase Chain Reaction for Genotyping.
Homozygosity in the three knockout genotypes used (AT1aKO, AT1bKO, and DKO) was proved by polymerase chain reaction after weaning and again after experiments were performed using specific primer pairs for each gene locus. Specific pairs of primers to detect AT1a have been described (Ito et al., 1995). For AT1b-specific polymerase chain reaction, two pairs of primers that amplified products for the AT1b WT (P1, 5′-CCA GGG CAA GAT TCA GAA GG-3′ and P2, 5′-CCA ACA AAG AGA CAT GAT C-3′) or knockout (P3, 5′-CCT GCG TGC AAT-3′ and P4, 5′-CCA TCT TGT TCA ATG-3′) allele were designed and used as described previously (Gembardt et al., 2008).
Generation of Mouse DC.
Mouse DC were generated as described previously with minor modifications (Lutz et al., 1999). In brief, after all muscle tissues were removed from the femurs, the bones were washed twice with PBS and transferred into a fresh dish with RPMI medium 1640. Both ends of the bones were cut with scissors in the dish, and then the marrow was flushed out by using 2 ml of RPMI medium 1640 with a syringe and 25-gauge needle. The tissue was suspended and passed through a 100-μm cell strainer (BD Biosciences, Franklin Lakes, NJ) to remove small pieces of bone and debris. Red cells were lysed with 0.45 M ammonium chloride, washed, and suspended at a concentration of 1 × 106 cells/ml in RPMI medium 1640 containing 10% heat-inactivated FBS, 100 IU/ml penicillin G, 100 g/ml streptomycin, 2 mM glutamine, and 5.5 × 10−5 M β-mercaptoethanol (Sigma-Aldrich), supplemented with 200 IU/ml rmGM-CSF (R&D Systems) and cultured for 7 days in 100-mm Petri dishes (5 × 106 cells/dish). Approximately 70% of the harvested cells expressed MHC class II and CD11c. DC were identified by flow cytometry as being positive for CD11c. At day 6, immature DC were further stimulated for 24 h with 100 ng/ml LPS to obtain mature DC.
Western Blot Analysis.
DC lysates were prepared with ice-cold lysis buffer, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes, which were blocked with 5% (w/v) skimmed milk in PBS, pH 7.4 and 0.05% (v/v) Tween 20 for 1 h at room temperature. Membranes were then incubated with rabbit polyclonal anti-AT1 antibody (N-10) (1:1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C. After incubation with peroxidase-linked secondary antibody (1:500) for 1 h at room temperature, immunoreactive proteins were visualized by ECL reagent and autoradiography film (Amersham Hyperfilm ECL; GE Healthcare). The same blots were stripped and reprobed with anti-β-actin antibody (1:1000) (Cell Signaling Technology, Danvers, MA). Quantification of the relative amount of protein was performed with ImageQuant Software (GE Healthcare) and normalized for the amount of β-actin in each experiment.
Purification of Splenic CD11c-Positive Cells.
Spleens were removed and digested with collagenase D (2.5 mg/ml; Roche Diagnostics) for 20 min at 37°C, then treated for 5 min with EDTA to disrupt T cell–DC complexes. The spleens were then suspended in RPMI medium 1640 with 10% FCS and softly passed through a 70-μm sterile cell strainer (BD Biosciences) to make a single cell suspension. Cells were washed twice with complete medium and stained directly with antibodies or further purified to isolate the CD11c+ splenocytes. Cells were sorted for positive selection with Mini-MACS by using the mouse CD11c cell isolation kit (clone N418) (Miltenyi Biotec). In brief, splenocytes were incubated with biotinylated N418 antibody and afterward with streptavidin microbeads. Cells were then positively enriched on a Mini-MACS column. Cells were centrifuged and washed twice with PBS containing 0.5% BSA and then suspended in 1% BSA/PBS. Cells were stained for 20 min at 4°C with the corresponding antibodies. More than 90% of these cells expressed CD11c.
Flow Cytometry.
Cells were harvested by pipetting with cold PBS. The cells were first blocked with anti-CD16/32 antibodies and then stained in phosphate-buffered saline (Invitrogen) at 4°C for 30 min with FITC- or PE-conjugated monoclonal antibodies [antigen-specific or control immunoglobulin G (IgG)]. Analyses of stained cells were performed on a FACS scan or FACSCalibur by using CellQuest software (BD Biosciences).
Allogeneic T Cell Proliferation.
Spleens were removed from BALB/c mice, and the tissue was disrupted to release a single cell suspension, which was then passed through a 70-μm sterile cell strainer. CD4+ cells were sorted by positive selection with Mini-MACS using a mouse CD4+ cell isolation kit (Miltenyi Biotec). Cells were centrifuged and washed twice with PBS containing 0.5% BSA, and then resuspended in culture medium. DC were washed with complete medium and irradiated (3000 rad from a 137-Cs source), and 2.5 × 104 cells were cocultured with 2.5 × 105 allogeneic splenic CD4+cells (BALB/c) for 96 h in U-bottom 96-well plates (Corning Life Sciences, Lowell, MA). Cells were pulsed with 1 μCi/well of [3H]thymidine (specific activity, 5 Ci/mM; DuPont, Wilmington, DE) for 18 h before harvesting on day 4 to measure proliferation. Plates were harvested with a Filter Mate (Canberra Industries, Meriden, CT), and incorporation of [3H]thymidine was determined by liquid scintillation spectroscopy using a TopCount device (Canberra Packard). Proliferation of allogeneic CD4+ splenocytes in response to the coculture with DC was also measured by CFSE dilution analysis by flow cytometry. Proliferation of splenocytes was calculated as the percentage of dividing cells.
Endocytosis Assay of BSA-FITC.
DC were incubated at 37°C in complete medium in the presence of BSA-FITC or DXT FITC (100 μg/ml) for 60 min. To determine unspecific binding a control at 4°C was incubated in parallel. Cells were washed three times with ice-cold 1% FCS/PBS and fixed with 1% paraformaldehyde. The BSA-FITC and DXT-FITC uptake was then evaluated by flow cytometry as described previously (Sallusto and Lanzavecchia, 1994).
Cytokine Assay.
TNF-α, IL-10, and IL-12p70 levels were measured in the culture supernatant by using a commercially available Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.
Statistical Analysis.
Results are expressed as the mean ± S.E.M. Data were analyzed by using single-factor analysis of variance (Prism 4.01; GraphPad Software Inc., San Diego, CA). If statistically significant effects were found, data were further analyzed by Dunnett's test. Statistical significance was assumed for p < 0.05.
AT1-Deficient BM Cells Give Rise to a Diminished Percentage of DC.
BM cells were obtained from mice lacking AT1a, AT1b, or both isoforms (AT1aKO, AT1bKO, and DKO, respectively) and control littermates (WT). We analyzed the percentage of positive cells for the lineage markers CD11b, CD3e, B220, GR-1, and TER-119 in the BM preparations and did not find any difference among the four genotypes (data not shown). After 7 days of culture in the presence of rmGM-CSF, the total number of cells recovered and the percentage of adherent cells were comparable among the genotypes (24 ± 3.0, 22 ± 4.3, 27 ± 3.7, and 26 ± 4.3% for WT, AT1a, AT1b, and DKO, respectively). DC were recognized for being positive for CD11c expression. Notably AT1aKO, AT1bKO, and DKO BM cells generated lower percentages of CD11c-positive cells compared with WT BM cells (p < 0.01; Fig. 1, A and B).
DC from AT1-Deficient BM Cells Express Low Levels of MHC-II and CD11c.
It is noteworthy that not only the percentage of CD11c+ cells was lower in cultures of AT1-deficient BM cells, but these AT1a KO- and DKO-deficient CD11c+ cells were also characterized by a lower mean fluorescence intensity of this marker (p < 0.01; Fig. 1C), suggesting that differentiation of DC from AT1-deficient progenitors is compromised under in vitro conditions. Supporting this assumption, the expression of MHC-II (IA/IE) molecules was also found to be lower in DC generated from AT1aKO, AT1bKO, and DKO BM cells as shown in representative histograms in Fig. 1D; CD40 and CD80 expression was comparable in the four genotypes analyzed.
Immature DC Generated In Vitro from AT1-Deficient Precursors Are Poor Stimulators of Allogeneic T Cells.
Immature DC are characterized for their high endocytic capacity. To evaluate the functional profile of AT1-deficient DC we first examined their ability to endocyte soluble markers such as DXT-FITC and BSA-FITC. We found no significant differences in the four genotypes analyzed (Fig. 2, A and B). Next, the antigen-presenting function of DC was evaluated by studying their ability to stimulate allogeneic T cell proliferation. We found that AT1a-, AT1b-, and double-deficient DC showed a markedly reduced allostimulatory capacity compared with wild-type DC (p < 0.01 or 0.001; Fig. 2C). Similar results were observed when the allostimulatory activity of DC was analyzed by flow cytometry using allogeneic splenocytes stained with CFSE (Fig. 2, D and E), indicating that the absence of AT1s in DC progenitors results in major defects in the function of immature DC.
LPS Induces Up-Regulation of AT1 Expression in WT DC.
Upon encountering inflammatory or microbial stimuli in peripheral tissues, DC become activated and undergo a number of physiological changes leading to their maturation. These changes enable DC to activate naive T cells and direct the differentiation of CD4+ T cells into different functional profiles. As expected, LPS induced up-regulation of MHC-II (IA/IE), CD80, and CD40 in WT BM-derived DC (Fig. 3A). It is noteworthy that this maturation of DC was associated with a marked increase in the expression of AT1 (p < 0.05; Fig. 3, B and C).
Mature DC Generated In Vitro from AT1-Deficient Precursors Remain Poor Stimulators of Allogeneic T Cells.
We then examined the role of AT1 in the phenotypical and functional maturation of DC triggered by LPS. Immature DC were stimulated for 24 h with 100 ng/ml LPS, and their phenotype was evaluated by flow cytometry. AT1a-, AT1b-, and double-deficient DC expressed significantly lower levels of IA/IE than WT immature DC (p < 0.01; Figs. 1D and 4A), whereas gene deficiencies had no effect on CD80 and CD40 expression (Fig. 4, B and C). LPS stimulation induced up-regulation of MHC-II (IA/IE), CD40, and CD80 in all genotypes. However, significant differences in the up-regulation were observed only for IA/IE, whereas DC from AT1bKO and DKO mice have been less responsive (Fig. 4, A–C).
We then studied the functional profile of mature AT1-deficient DC by analyzing their allostimulatory capacity. The proliferation rate of allogeneic T CD4+ cells was evaluated by using a [3H]thymidine incorporation assay (Fig. 4D). We observed that LPS-stimulated DC generated from AT1-deficient progenitors show a remarkably lower capacity to stimulate the proliferation of allogeneic T CD4+ lymphocytes compared with WT DC (p < 0.01), independent of whether they have been deficient in AT1a, AT1b, or double-knockout DC.
Effects of AT1 Deficiency on DC's Ability to Release IL-10, IL-12p70, and TNF-α.
An important property of DC is their capability to produce a great variety of cytokines. While IL-12 p70 is considered the key factor in the polarization of the adaptive immune response toward a T helper 1 profile, the production of IL-10 is usually associated with a tolerogenic profile, and TNF- α is a key inflammatory cytokine. We studied the pattern of secretion of IL-10, IL-12p70, and TNF-α in culture supernatants of immature and mature DC. AT1-deficient DC released IL-10 and IL-12p70 in a similar fashion to wild-type DC (Fig. 5, A and B). In contrast, immature DC generated from AT1-deficient BM cells produced significantly lower levels of the proinflammatory cytokine TNF-α (p < 0.05 or 0.01). However, under these in vitro conditions the significant reduction in TNF-α release vanished after LPS stimulation (Fig. 5C).
Spleen CD11c-Positive Cells Deficient in AT1 Release Lower Levels of TNF-α after In Vivo Challenge with LPS.
We first studied the phenotype of spleen DC isolated from AT1-deficient mice. We found no differences in the percentages of IA/IEhigh and CD11chigh double-positive cells or in the levels of expression of IA/IE and CD11c in the IA/IEhigh and CD11chigh constellation of double-positive spleen cells in the four genotypes analyzed (Fig. 6, A and B). Furthermore, purified CD11c+ spleen cells from AT1-deficient mice showed similar expression of IA/IE (WT: 1370 ± 25; AT1a KO: 1125 ± 109; AT1b KO: 1341 ± 71; DKO: 1349 ± 120), CD40 (WT: 72 ± 6; AT1a KO: 86 ± 11; AT1b KO: 71 ± 5; DKO: 61 ± 4), and CD80 (WT: 79 ± 11; AT1a KO: 84 ± 8; AT1b KO: 70 ± 10; DKO: 79 ± 10) (all numbers are mean fluorescence intensity; n ≥ 5) compared with the WT mice.
It is well known that LPS administration induces a systemic inflammatory response, which involves the massive production of inflammatory cytokines by splenic macrophages and DC. Our aim was to determine whether AT1 activation is necessary for the complete acquisition of a mature phenotype in spleen DC after administration of systemic LPS. WT, AT1aKO, AT1bKO, and DKO mice were challenged either with LPS or saline solution intraperitoneally. After 18 h, mice were sacrificed and spleens were removed to prepare a single cell suspension. Spleen cells were then analyzed for IA/IE and CD11c expression by FACS scan. CD11chigh spleen cells from DKO mice showed up-regulation of IA/IE in response to LPS challenge under these in vivo conditions as did WT. We then analyzed by FACS scan the phenotype of the CD11c+ spleen cells isolated from these mice. We found that WT and DKO CD11c+ spleen cells showed a comparable up-regulation of IA/IE, CD80, and CD40 in response to the systemic administration of LPS.
To further evaluate the CD11c+ spleen cells' response to systemic administration of LPS we studied the secretion of the proinflammatory cytokine TNF-α, a central mediator of the pathophysiological changes associated with endotoxemia. Purified CD11c+ spleen cells from mice challenged with either LPS or saline were cultured for 6 h, and TNF-α production was measured in the culture medium. Remarkably, AT1a and AT1b deficiency nearly abolished the increase in TNF-α release seen in CD11c+ spleen cells from wild-type mice challenged with LPS, suggesting that both isoforms play a critical role in the production of the key inflammatory cytokine TNF-α by CD11c+ spleen cells in response to LPS under in vivo conditions (Fig. 6C).
We investigated the impact of AII type 1 receptor deficiency in the phenotypical and functional properties of mouse DC in vitro and in vivo. Our present results provide clear evidence that AII type 1 receptor is required for the normal development and function of DC. Supporting our findings, other groups suggested that a functional RAS operates in DC. It has been previously demonstrated that human monocyte-derived DC express RAS components (Lapteva et al., 2001; Jurewicz et al., 2007). Lapteva et al. (2002) identified by DNA microarrays a subset of genes that were specifically regulated in human monocyte-derived DC by AII. Nie et al. (2009) observed that AII induces activation of extracellular signal-regulated kinases 1 and 2 in murine BM-derived DC. Furthermore, several lines of evidence suggest a role for the RAS in hematopoietic cell development. It has been demonstrated that there is a local RAS in the BM, present not only in the hematopoietic but also in the stromal compartment, which is thought to affect the growth and differentiation of hematopoietic colonies (Haznedaroğlu and Buyukasik, 1997; Strawn et al., 2004; Hubert et al., 2006).
We have previously examined the role of AII, AT1, and AT2 on the development of human and mice DC in an in vitro model using pharmacological antagonists of both receptors. We demonstrated that AII has a profound regulatory effect on the differentiation of human and mice DC, suggesting that AII supports the normal development of myeloid DC by interacting with AT1, the predominant class of AII receptors (Nahmod et al., 2003). In our previous study, we found that monocytes produce AII and express AT1 and AT2, and blockade of each of these receptors by specific antagonists induced opposite effects during the differentiation of DC. Blockade of AT1 results in the development of DC with very low levels of CD1a, HLA-DR, and CD40 expression and poor endocytic and allogeneic stimulatory activities, whereas blockade of AT2 results in DC with high levels of CD1a, CD40, CD80, and HLA-DR and high endocytic and allostimulatory activities. Results obtained from mice are similar to those in humans (Nahmod et al., 2003).
Those results obtained with AT1 antagonists are consistent with data shown here, because we found that DC differentiated from progenitors deficient in AII subtype 1 receptors express lower CD11c and IA/IE and have a severe defect in their allostimulatory capacity. Nevertheless, we found no differences in endocytic capacity or CD80 and CD40 expression in AT1-deficient DC compared with the WT DC. Differences in the observations made with pharmacological inhibitors versus knockout mice could be explained in different ways. The use of AT1 antagonist does not guarantee a complete inhibition of AT1 along the process of DC differentiation. On the other hand, AT1 blockers are suspected to interact with receptors other than AT1, such as peroxisome proliferator-activated receptor-γ (Schupp et al., 2004; Marshall el al., 2006; Ernsberger and Koletsky, 2007), thromboxane A2/prostaglandin H2 receptors (Liu et al., 1992), and formyl-Met-Leu-Phe receptor (Raiden et al., 1997). Thus, they might not be considered as the most appropriate tool to define the contribution of AT1 in DC physiology.
The model of in vitro DC differentiation with GM-CSF is one of the most common methods used to obtain murine myeloid DC. Here, we demonstrate in this in vitro model that AT1-deficient BM cells give rise to CD11c+ cells less efficiently than WT BM cells, suggesting that AII subtype receptor 1 is necessary for the normal development of DC. It is noteworthy that the absence of AT1a and/or AT1b results in DC expressing low levels of MHC class II molecules, whereas the absence of AT1a, but not AT1b, results in a marked reduction in the levels of expression of the DC marker CD11c. These results suggest that both receptors play a nonredundant function in the development of DC in vitro.
Although it is accepted that DC obtained through culture acquire different phenotypic and functional properties depending on the cytokine milieu used, it is believed that in vivo each tissue provides different microenvironments, which affect DC generation and maturation (Shortman and Liu, 2002). It is noteworthy that we found that AT1-deficient mice showed similar percentages of IA/IEhigh and CD11chigh double-positive cells isolated from spleens compared with the WT. These contrasting results between our in vitro and in vivo data might be related to the fact that during differentiation of DC in vivo the microenvironment is much more complex than the one generated artificially in a culture dish and might contain other factors able to promote DC development.
We also demonstrate here that AII type 1 receptors play a crucial role in DC's functionality, judging by the poor allostimulatory activity of AT1-deficient DC. The absence of AT1 in DC does not affect only the phenotype and function of immature but also mature DC. The main function of mature DC is to activate naive T cells. It is noteworthy that the results observed in allogeneic T cell proliferation assays show that the absence of either AT1a or AT1b isoform results in "mature" DC with a very low ability to act as antigen-presenting cells. These data clearly implicate, similar to our conclusion on CD11c expression pattern, that DC might require signals via both isoforms to be fully functional.
To our knowledge our study is the first to investigate the impact of AII type 1 receptor deficiency on the production of cytokines by DC. We investigated AT1-deficient DC's ability to produce IL-10, IL-12, and TNF-α in vitro. Immature and mature DC deficient in AT1 and WT-DC were able to release comparable amounts of IL-10 and IL-12p70 to the culture supernatant. This suggests that AT1 deficiency is not affecting DC ability to produce cytokines involved in the differentiation of T CD4+ cells into different functional profiles. In contrast, immature AT1-deficient DC produced lower levels of the proinflammatory cytokine TNF-α compared with WT DC.
However, the question arose whether the in vitro finding might have an impact under in vivo conditions, too. We thus examined the role of AT1 in the phenotypical and functional maturation of spleen DC triggered by LPS challenge in an in vivo model. We found that CD11chigh splenocytes from AT1-deficient mice produced significantly lower levels of the proinflammatory cytokine TNF-α, a central mediator of the pathophysiological changes associated with LPS challenge. These data clearly demonstrate that AT1 signaling is required for DC full response to LPS. This conclusion is further supported by the observation of other groups using AT1 antagonists or angiotensin-converting enzyme inhibitors, who postulated that AII enhances the LPS-induced production of cytokines in different cell types (Fukuzawa et al., 1997; Constantinescu et al., 1998; Miyoshi et al., 2003, 2008; Shimizu et al., 2004).
The fact that the AT1 is a key player for DC functionality after LPS stimulation is also supported by our molecular findings. We demonstrate for the first time that BM-derived murine DC express AT1 at the protein level and its expression is up-regulated in response to LPS. This novel finding supports the notion that AT1 may play a role in DC functionality during the inflammatory response. It shows that synthesis of RAS components is stimulated by inflammatory signals in distinct cell types or tissues (Brasier et al., 1996; Suzuki et al., 2003; Chan and Leung, 2007; Miyoshi et al., 2008; Sánchez-Lemus et al., 2009).
Our studies extend the importance of AII receptor subtype 1 to mediate physiological functions of the RAS in the immune system. Our results provide direct evidence that endogenously generated AII supports DC differentiation, maturation, and functionality through both AT1a and AT1b isoforms. The findings may also help to understand, at least partially, the beneficial effects of AT1 blockers in inflammatory and autoimmune diseases beyond antagonizing AII classical effects over blood pressure and fluid homeostasis.
In summary, we hypothesize that AII stimulates DC differentiation and function in an autocrine/paracrine manner by signaling via the AT1 to contribute to perpetuating inflammatory responses.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- dendritic cells
- angiotensin II
- AII type 1 receptor
- AT1a
- AII subtype 1a receptor
- AT1b
- AII subtype 1b receptor
- AII type 2 receptor
- knockout
- double KO
- bone marrow
- lipopolysaccharide
- renin-angiotensin system
- rmGM-CSF
- recombinant mouse granulocyte/macrophage colony-stimulating factor
- wild type
- TNF-α
- tumor necrosis factor α
- interleukin
- major histocompatibility complex II
- enzyme-linked immunosorbent assay
- phosphate-buffered saline
- bovine serum albumin
- fluorescein isothiocyanate
- dextran
- fetal calf serum
- carboxyl fluorescein succinimidyl ester
- phycoerythrin.
- Received September 17, 2009.
- Accepted May 28, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics


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Bread and Beer Exhibition
Date: Sun, 12 Jul, 2015
Time: 12:30 pm - 5:00 pm
Bread and Beer Exhibition
Featuring two Victorian Stotfold entrepreneurs. The Randall family ran their mill for over one hundred years and made extensive improvements. In the 1880s William Randall married into the Flitton family, who had business interests in Ashwell, Radwell and Stotfold. George Flitton founded a brewery near the Green. His prize beer, Stingo, is still remembered fondly by an older generation of Stotfolders.
Free Admission.

subset: commoncrawl


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2014 Forest Hills DriveMusic CD - 2014
From the critics
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2014 Forest Hills Drive is Cole's 3rd album and for sure his best so far. It came out in 2014 and it was highly rated by many people. It contains no features and went double platinum which was very impressive. It contains 13 songs. It was nominated for a Grammy and won an award at the BET awards. The most popular songs on Forest Hills are Apparently, Love Yourz, No Role Modelz and Wet Dreamz. This album is still very popular and is an album that took J. Cole to the next level. My favourite songs from this are A Tale of 2 Citiez, Fire Squad, No Role Modelz, Wet Dreamz and Love Yourz. If you are new to J. Cole this is an album you must hear and is a 5 out of 5.
- @sra1031 of the Teen Review Board at the Hamilton Public Library
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Tag: goals
Consistency is Key!
Do you suffer from procrastination like I do? Do you want to become more productive? I know I do, which is why Alex Altman's book Time is Money caught my eye on Amazon. It promised to: "Cure procrastination without willpower, Become more productive, Find your focus, and Get more done in less time." After I read […]
Posted: April 5th, 2017 under Life.
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Watch Out your Behind Hunter
A free online game for girls
Watch Out your Behind Hunter
Shoot out the lurkers or they will get you laid.
Tags: Fun - 1player - Flash - Shooting - Funny
Average Rating: 3.36 based on 11 ratings
- Fuzzy McFluffeinstein 2
One day in a quaint little pet store, a cat just suddenly brings havoc to all the rats his eyes laid on. This is the sequel to the bloody mouse mash.
- Food Memory 2
A classical memory game bedazzled by fine cuisine. Can you remember how table was laid?
- Making A Trukey Platter
It's thanksgiving day. The table has been laid - the candle-lit traditional dinner is going to be amazing! And you are going to make it even more special with your Turkey Platter!
- Gold Medal Cooker
In the crazy world of fine cuisine and exotic meals Matthew is knows as the Golden Medal Cooker. But the hectic job in the kitchen isn't for Matthew as he need more time to prepare meals and is soon going to be laid off. Help Matthew keep his title and his job!
- Game Day Chili Cooking
A big crowd is watching sports on TV in our restaurant and they have requested us to serve them mouth-watering snack. When it comes to serving a large crowd, the chef instantly chooses Game Day Chili. It is a perfect snack that you can prepare whenever you want to serve a large group of who come to your home to watch TV shows or a big crowd who gather in the stadium watch a football match or any other sports. The chef wants to show his cooking techniques to you. It is a wonderful chance for you to learn the recipe. So don't miss this opportunity.
- Watch Out
- Watch Out
Try to evade all the falling boulders!
- Sue Gardening
Dig a hole, sow plants, water and watch them growing!
- Flip Me Out
Test how fast you can watch Jack move.
- Car Coloring
Color the car and friends to watch them ride to the party.
- Deadly Dwarves
A fun shoot em up, watch out those little fellas will stab you in the back!
- Forest Brat
Catch all butterflies within time. But watch out for the bees!
- Great Cookie
Catch as many cookies as you can in 6 seconds and watch out for falling teacups!
- Rambo Bros.
Shoot the Mario characters and watch the blood spill.
- Car Can Racing
Race around the track against the other cars made of cans. Come in 1st. Watch out for those corners.
- Sunday Driver Remix
Drive down the street and take out the cop cars. Watch out for the helicopter!
- Field of Some Dreams
Catch as many baseballs as possible as theyre hit in your direction. Watch out for those holes!
- Jewel Hunter
Drive your car grabbing as many jewels as you can. Watch out for obstacles, especially other cars.
- Clowning Around
Help the clown to balance in tightrope with the aid of your mouse but watch out also for pies being thrown at you. Have fun!
- Boardn
You are skating through the city to catch up to a friend. Watch out for the obstacles. You must either go around them or jump over.
- 2d Army Swat
Shoot the drunk terrorists but watch out for them shooting back.
- Western Blitzkrieg
Aim and shoot in this sidescroller with nice graphics. Watch out for those landmines!
- Car Can Racing
Race around the track against the other cars made of cans. Come in 1st. Watch out for those corners.
- Ploop
Collect all the blue stone in the dungeon but watch out for the obstacles that may hurt you.


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anyone seen these? cute? or too cute?
sorry SCARVES
The triangular scarves are called pointus. The ribbon motif is called the Bolduc pattern.
Thanks Rose
Here is a thread that may be of interest:
THANKS! awesome
I have the fuschia bolduc one and it is so fun! After wearing the pointu, the "real" scarves feel like so much extra fabric.


text: Product Description:A spicy woody accord. Incense is dressed in head by spicy notes such as cardamom, cinnamon, pepper and pepper berries. The heart is accompanied by benjoin. Finally, at the bottom, the guaiac wood, sandalwood and ebony come surround to give roundness and warmth.Perfume family: woody, spicy, orientalTop notes: cardamom, cinnamon, black and pink pepperHeart notes: frankincense, benzoinBase notes: guaiac wood, sandalwood, ebony wood


text:
Plating Finishes | Black Chrome
Precision Plating offers the largest range of electroplated decorative finishes in Australia. This interactive selector details the name and some brief comments of most of the finishes we have.
The quality of the finish is also highly dependant on the surface of the metal or plastic you are plating on. Plating will not fill cracks in metals or cover large pitted surfaces. If you want a highly reflective finish, the surface must be highly polished before plating.
Black Chrome
An alternative to bright chrome, this finish is popular in the automotive markets. It is a durable coating normally plated over nickel and is available in high gloss, matt and brushed satin versions.


text:
Should MySpace carry a health warning?
A short while ago I wrote about Megan Meier who tragically committed suicide after becoming a victim of a MySpace hoax.
Now, a new MySpace hoax may have led to murder!
In Riverhead, New York, a 53 year old man, John White, is on trial for killing a friend of his son.
John White is charged with killing 17 year old Daniel Cicciaro on August 9, 2006, outside his Long Island home after a heated argument with Cicciaro and a group of his friends.
Cicciaro died later that night after allegedly being shot in the face by White who is charged with second-degree manslaughter.
With White being of African-American descent, claims that the incident may have been about racism and revenge have emerged.
White, however, claims that he was simply protecting his family from a white lynch mob and that his gun discharged purely by accident.
Prosecutors in the case disagree though.
They say that White should have called the police, instead of choosing to confront the boys himself, armed with a .32-calibre Beretta.
Riverhead police say that the incident was prompted by a hoax, issued from White's son's MySpace page.
Aaron White, 20, allegedly sent a message, threatening rape, to a teenage girl who was a good friend of Daniel Cicciaro.
On the evening that Cicciaro died, he was at a party with that same girl when they spotted Aaron White.
It is believed that Cicciaro then became angry, and White was asked to leave.
Aaron White, however, testified that after he left the party, Cicciaro and another man called him on his cell phone, saying to him, "Get back to this party you ni**er."
Aaron then says that he went home, only to receive more calls from Cicciaro and his friends, informing him they were going to come to his house to kill him.
At that point he awoke his father, who then grabbed his Beretta and waited for Cicciaro and the other boys to arrive.
After Cicciaro's death, it emerged that the rape threat that ignited the confrontation was false.
Under testimony, Aaron White's friend, Michael Longo, admitted that he had logged on to Aaron's MySpace page and had sent the threat to the girl as a joke.
Cicciaro's mother, Joanne, says that her son was simply trying to protect the girl, who was very much like a little sister to him.
She said, 'The thing is, that night, Daniel believed Aaron threatened to rape a girl that was like his little sister'.
John White's attorneys claim that racism fuelled the confrontation, and that White truly believed Cicciaro and his friends were going to kill his son.
Defense attorney Fred Brewington said, 'You had this group of white men who felt that they were going to defend this young white woman from a black man who said these things about her, and they felt justified to do so because of his race'.
In contrast, Cicciaro's father, also named Daniel, doesn't believe White's story, saying, 'He had 20 minutes to gather his thoughts, to call the police to defuse the situation, to find out why they were coming over, and he didn't take any of the precautionary steps'.
Joanne Cicciaro added, 'The Whites, none of them, ever called 9-1-1, even after he shot Daniel, but he did call their attorney'.
The jury are currently deliberating and have yet to return a verdict.


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1873 · Bording
by Smith, Joseph
Bording: C. G. Larsen / Trykt hos F. E., 1873. Early edition. Hardcover. LXXXI; 348pp. Sextodecimo [16 cm] 1/4 brown leather with black pebbled cloth covered boards, and a gilt stamped title and 6 double-ruled borders on the spine. Very good. The extremities are mildly rubbed, and the underlying boards are exposed at the corners. The threads are occasionally visible in the gutters of the pages, however the book is still very sturdy. The pages are darkened and very sporadically, lightly foxed. An early printing of the Doctrine and Covenants in Danish. Flake 2926. (Inventory #: 46008)


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By Ivan Gomez
To start off, let's talk about what you have been doing in the last couple of years. How is La Mega 87.7 treating you so far? Hey what's going on my friend! First off, thank you for reaching out for this interview! The past few years have been great. I have been super blessed to be busy with so much dj/production work. "La Mega" is cool; it is still at the beginning stages and is still growing. I would like to have my own show with full creative control; I know I can really change the radio for the good. Let's see what happens (laughs).
At what age did you decide you wanted to be a DJ? And why a Dj?
I started when I was twelve. I just fell in love with the party vibe and crowd control. I love being able to make people feel good on the dance floor and the air waves. It's all about having fun.
How did it all start for you?
My father used to let me tag along with him to all these Salsa concerts when I was a kid. I loved being there with him and just watching how the old heads lived life. It was such a different time back then. The love for music was so genuine; when the bands would take a break I could still hear music playing and still see people dancing. The energy was just unexplainable!, people jumping up and down, hands in the air and repeating whatever the person on the mic would say, someone was controlling hundreds of people all at once! I asked my father: who is that and he told me: that's the dj, I found where the dj was and that's the only place I wanted to be at every concert or festival; I no longer cared about the bands or what the old heads had going on! The rest is history!
What equipment did you use?
I started out with two Technic 1200 turntables, a Gemini mixer and a milk crate full of vinyl records, I would plug that mixer into the back of an old Magnavox boombox I had and just let loose. Today I use whatever the club has available (anything except numark products)!
Who were some of your favorite DJs growing up?
Danny Rivas from Lorain was the first dj I had ever seen playing live, he was the one at those concerts, the banner might have said Willie-D but the man with the control was Danny Rivas. Around my second or third year in I was able to see Dj Sipie from Cleveland play at a house party in South Lorain. I got to see him and Danny Rivas battle (it was great)! Other guys I looked up to was Kendo, Kaos, Jazzy Jeff, Jam Master Jay, Playero, Dj Laz and of course my mentor the late great Jinx Paul (may he rest in peace).
What advice would you have for DJs just starting out?
Man, I have to charge for that advice "lol". My only advice that I will give to the new guys is: Don't do this art form halfway! If you want to be a dj then RESPECT the art! Check your history, do your research, learn why we really do this, respect our craft! Do not undercut your fellow djs, do not and I repeat, do not DJ for free!, you wouldn't work at Ford for free so why would you dj For Free! Dj's if you are serious about it and need anything just reach out. We consult and manage djs all the time.
What's your opinion on the Latino Cleveland Dj scene? I always hear the same names… Or is it just me? Am I missing something?
All honesty some people will be offended by my following comments but hey, it is what it is, somebody has to be honest! I can say, there really isn't a Latino Dj Scene in Cleveland. You may hear the same names because 95% of these guys all low ball and undercut each other just so they can say that they are the dj at these bars or to hear their names on the radio (laughs).
Let's just keep it real, it's just a Dj Scene and a few guys who only cater to the Latin community by choice or because that is all they know. I personally refuse to be called a Latin or Latino dj, I am a DJ, I cater to every genre of music. I am what you may call an Open Format dj, I'm one of the very few who can merge all cultures and walks of life into one room and just provide a great vibe! This is something that took me many years to accomplish, one thing for sure there is no unity or loyalty among djs in Cleveland.
There are a few djs that understand the business aspect of the dj art form but then you have guys that just have zero respect for their fellow djs; you have the under cutter, the guy who just down talks his fellow djs to just get a booking at that bar, I say bar because there are no Latin clubs in Cleveland (just bars and restaurants). Then you have the guy with zero skills and talent, he just thinks he is a dj because he has folders of music and a laptop! That's not the case at all because everyone has folders of music on their laptop (that doesn't make them a dj).
What about the Dancing Clubs, do you think we have enough clubs or do we need more?
Cleveland has many clubs, could we use more? Yes, of course! Now for the Latin community that is more tricky, we have ZERO Latin clubs in Cleveland (we need one bad)!
The Latin community is very segregated in Cleveland, you have the salsa community and then you have everyone else, they do not mix at all, you might get a few people from each crowd to step out of their comfort zone but not enough to say Latinos united always.
What's your favorite track that you like to play?
I have so many (laughs); I guess it just comes down to what venue I am playing at or what crowd I'm playing for, every night I am playing for a completely different crowd at a different venue, I'm never playing the same set two nights in a row, the perks of being an open minded open format dj, you can have fun anywhere with any crowd (English or Spanish or both)!
I'm very critical to the main stream or commercial reggaeton, that ones we listen to on Radio or TV, and it's not because of the music, in fact, I love some reggaeton songs, but my problem is the lyrics, too much sex or too much sexual content, and remember, most of the Latina teenagers listen to this music, considering Latina teens continue to face the highest rates of teen pregnancies, what's your take on this? Remember I'm not talking about other cultures, I'm talking about Latinos in the Unites States.
Everyone is entitled to their own opinion, that's what is so great about our country, I totally understand what you mean, music has definitely changed over the years, well, let me take that back! Mainstream music has changed! You have to understand, the sexual content has always been in the music.
The only difference these days is the level of acceptance in the media has changed, people criticize reggaeton so much as if reggaeton was the first genre in Latin music to over express sexual content, please people, stop it! Calm down! Cálmate! Those same critics love Salsa, go to Salsa events and dance all night to Salsa, Salsa has a crazy amount of sexual content in its lyrics from the Fania days to today's Salsa movement.
The same with Latin Rock, Bachata, Merengue and Cumbia, the only thing that slows them down on down-talking those Latin genres is that they are held at more upscale venues and most of the time cater to an older crowd. I remember growing up and people talking down on Salsa and the lyrics of songs by artist such as Lalo Rodriquez, Frankie Ruiz, Eddie Santiago, Hector Lavoe. Today these guys are legends and their music is played at events and danced to by the same people who down talk reggaeton. It's just funny to me. Bachata was banned from radio when it first came out, now Romeo Santos and Prince Royce dominate radio and TV, now they don't talk about them anymore.
Turn your TV on to your favorite novela or turn your radio on right now, I guarantee you there is a Salsa, Merengue, Cumbia, Latin Rock or Bachata song playing right now that has crazy sexual content in the lyrics, nobody will say nothing about that though. Latin female teenagers aren't just listening to reggaeton, they love Romeo, Marc Anthony, Carlos Vives and Shakira. My thing is you can't judge our youth by the music they listen to on the radio or the novelas and videos they watch on TV; you have to remember there is some non-teenager in charge at all of these media outlets who is giving the ok for this content to be played on air or this sexual content to be advertised on billboards and shopping ads.
I believe if the parents raise their kids with good morals and understanding of everything that is going on in the world (the good and the bad) then your kids will make good choices, if you sit and just judge them or only single out what you as the parent feels is no good then the growing child will eventually become curious as to what else the parent has left out and why the parent was so judgmental and quick to single out only one thing. I'm a proud parent of a Latin teenager and I leave nothing out when explaining to him the good and the bad (in life, music, media and everything else). Nothing but good choices has come from this. Parents just need to communicate with their kids a little more instead of being so judgmental that's all.
You have to outline your definition of "judge"… Nowadays people use that word to silence opinions, criticisms or advice, we all on this together parents, family and society, and you right, most of the Latin music is full of sexual content, but, why should we think is ok? Again, mainstream music is just a reflection of our culture and has changed(?) So, how much has the scene changed since you started DJing?
Man! it has drastically changed, from the music to the equipment we use and the pay we get, some djs have been able to keep up with the times while others are still stuck in the past (musically, talent wise and technology). All in all we are still learning. It's endless!
What is the craziest thing that has happened to you as a DJ in 2016?
I'm so humble to have been blessed with a really great dj year but our dj community lost a great brother, friend, father and amazing dj: Jinx Paul. Losing Jinx has to be the craziest thing to happen to me last year. Miss you my brother!
What are your plans and goals in 2017?
Take more trips this year than I did last year and retire from the club scene as a dj. This year makes 25 years as a dj so I think it's the perfect time to walk away from the club scene, I will continue to manage and consult djs, I will also continue to dj private events (corporate, quinces and weddings). After this year I no longer want to dj in the clubs, unless I'm booked as an artist to perform my own records like: Diplo, Kaskade, Marshmello.
I will be stepping back into the promoter world again just like I used to back in the day. I see a lot of promoters just going about their events all wrong and treating their djs like they are not needed. The dj is key to all events! I'm going to change that again! They will respect the dj (especially if I'm managing them). So prepárate!
Other than Cleveland, what other cities or states have you performed in?
Man! The list is crazy! I have traveled all over the world to perform my dj sets (from Europe to South America to the Caribbean). In the US I have spun in most of the major cities from Miami to Los Angeles to Austin, Dallas, Phoenix, San Diego, Orlando, Connecticut, Kentucky, Chicago, Pittsburgh, Vegas and many other smaller cities, It's all about traveling as a dj! It's cool to dj in your city but why stop there!
Why not leave that 5 mile radius these local guys want to be kings of, I don't want to be king of Ohio! I want to be free with a nice bank account and own some land in a tropical island! So those fighting to be king of Cleveland can go ahead, it's all yours (laughs).
Do you follow a certain structure when creating DJ sets? Or just whatever!
No structure at all; I feel the vibe and just go from there! It's all about having fun with it and trying something new all the time!
Where can we hear your music?
You can find mixes on my site www.flacoflash.com (you can book me there too). I also have a mix-cloud page (mixcloud.com/djflacoflash)
Do you want to add anything else or have shout outs?
First off thanks to the higher power for allowing me to come this far! Much love and respect to my family (my #FINR circle).
Saludos a la gente mía de "Lo Máximo Productions" and "Porter Roc Ent." También saludos a todos los haters (I can't hear you all the way up here on this plane MMG) Dale!
Well my friend, thanks for this interview and your time!
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Hi Guys,
Please see below the coach details for Squires Gate game.
Tuesday 8th November
Chorley V Squires Gate
Depart from Duke Street at 18.00pm
Seats will be £10 for everyone.
To book your seats please call the club on 01257 230007
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text:
Question about Onkyo HTR500 Receiver
Will not switch to dolby digital mode
Posted by joesvolz on
You must use optical sound wire in order to reprduce dolby digital......You should have 2inputs on the back for this......plus the signal must be broadcasted in dolby digital.Such as HBO,SHOWTIME....they will tell you before the show comes on whether or not the movie or event is being broadcasted in dolby digital.Your unit will automatically change to dolbly digital once the show or event starts.Just make sure your input on video1,2,or3 is set to optical1 or 2.Just press the "Audio Sel" on the remote to change the audio source.But make sure the optical wire is connected first.....And just incase,make sure you are sending a dolby digital source to the unit from the sat reciever,cable box,playstation2.....make sure the dolby digital/optical option is on.You can find out what audio options you have in the menu screen.........or the redneck way.look on the back of the sat reciever/cablebox/playstaion2 ect...for a red laser light comming from these units.........i hope tht helps.
Posted on Apr 04, 2009
SOURCE: I have freestanding Series 8 dishwasher. Lately during the filling cycle water hammer is occurring. How can this be resolved
a 6ya Technician can help you resolve that issue over the phone in a minute or two.
Best thing about this new service is that you are never placed on hold and get to talk to real repair professionals here in the US.
click here to Talk to a Technician (only for users in the US for now) and get all the help you need.
Posted on Jan 02, 2017
Tips for a great answer:
Mar 07, 2013 | Audio Players & Recorders
Feb 09, 2012 | Audio Players & Recorders
Before using the remote control for the following
* Set the remote control mode selector to AUDIO
** There are exceptions in the operation mentioned
above. In such a case, follow each instruction
Dolby Pro Logic II*1
# Dolby Pro Logic II has a newly developed multichannel playback
format to decode all 2 channel sources - stereo source and Dolby
Surround encoded source - into a 5.1 channel.
Dolby Pro Logic II has two modes - Movie mode and Music
# Pro Logic II Movie (MOVIE)
Suitable for reproduction of Dolby Surround encoded sources
# Pro Logic II Music (MUSIC)
Suitable for reproduction of any 2 channel stereo music sources
# Dolby Digital*1
Used to reproduce multichannel soundtracks of the software
encoded with Dolby Digital
# DTS Digital Surround*2
Used to reproduce multichannel soundtracks of the software
encoded with DTS Digital Surround. DTS Digital Surround (DTS) is another discrete multichannel
digital audio format available on CD and DVD software
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Jul 22, 2007 | Yamaha RX-V3300 Receiver
May 14, 2014 | Onkyo HTR500 Receiver
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text:
|Preheat oven to 160C. Place lamb skin-side down on a work surface, scatter with pecorino and mint, season to taste, roll lengthways, tie at intervals with kitchen string and set aside. Heat oil in a casserole over medium heat, add carrot and onion, stir occasionally until golden (5-6 minutes), add stock and wine, bring to the simmer, add lamb, cover, transfer to oven and cook until lamb is fork-tender (2½-3 hours). Set aside to rest (5 minutes), remove string and thickly slice.
|Meanwhile, for sautéed artichokes, peel back tough outer leaves from artichokes and trim tops (discard). Remove choke with a teaspoon (discard), trim stems to 4cm lengths, and peel stems with a vegetable peeler until you reach the tender white part. Thinly slice artichokes, place in a bowl of acidulated water and set aside. Heat oil in a large frying pan over medium heat, add onion and stir occasionally until tender (6-7 minutes). Add artichoke and stock, cover with a round of baking paper, reduce heat to low and simmer until tender (15-20 minutes). Season to taste, scatter with mint and serve with slow-roasted lamb.


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Starting in the next couple of weeks, if you live north of Thornton Street, you may notice a two-man crew and bucket truck in your neighborhood. Work is scheduled to begin on the first phase of a yearlong process to install new LED fixtures on our older streetlight systems. The Relight Washington program is funded by the Transportation Improvement Board (TIB) and is being carried out by Puget Sound Energy (PSE). Aimed at improving street light infrastructure and lowering cities' street light bills across the state, Ferndale will benefit as each old fixture is converted to more energy efficient LEDs.
For more information about this project, including work schedules, locations, cost savings and benefits to LED lighting systems, please check out the project page at www.cityofferndale.org/LEDLights or call the Ferndale Public Works Department at (360) 384-4006.


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Há alguns meses, contamos a história de Bella, uma cadela da raça labrador que foi diagnosticada com câncer ósseo do tipo osteossarcoma em uma das patas dianteiras, que precisou ser amputada. Infelizmente, a doença acabou re-aparecendo nos pulmões da cadelinha.
Seu tutor, que é médico veterinário, viu que nada mais poderia ser feito por ela, então decidiu lhe proporcionar os melhores dias de sua vida, com várias viagens pelo país e atividades que enchiam a vida da cadela de alegria.
No último sábado (29), o tutor de Bella postou em sua conta no Instagram uma foto que a mostra de repouso. Na legenda da foto, Robert Kugler, tutor da labrador, contou que após um passeio de bicicleta, a respiração de Bella começou apresentar alguns tremores e espasmos, além de estar com dificuldade para conseguir respirar.
Ele então a levou até uma clínica, onde ela ficou no oxigênio e passou por um raio-x. "Os exames mostraram que os seus pulmões simplesmente não estavam mais conseguindo produzir oxigênio o suficiente….a hora está chegando," escreveu.
Well, #TeamBella…it's hard for me to tell you this, but today may have very well been our last full day in the beautiful outdoors. Bella started shivering today and her breathing became labored. There was actually a time after our bike ride that I looked into her eyes as I held her head in my lap and saw what they were telling me. I told her that I would be okay…that I'm ready if she is…and that I'll be holding her every step of the way. For a moment, I thought she may fall asleep right there in my arms. Yet, she held on..and we got up and chased some geese, and she went for another swim. Nonetheless, her breathing and shivering became worse and we zoomed up from the park in Denver to the FourSeasons Vet Hospital in Loveland. She's on oxygen right now and has been given a sedative to help. The X-rays showed that her lungs simply cannot produce enough oxygen…the time is near. We're doing our best to keep her comfortable through the night and set up a bridge crossing ceremony tomorrow at one of our new favorite outdoor spots we found here in Colorado. I'll be getting off my phone tonight and instead writing in a journal…possibly making another post in a few hours just to show her in her blanket and some comfort items that were given to her. Please don't ask for updates, judge any decisions, or even pray that she doesn't go. Just…know that it's okay that she is going. She has fulfilled her purpose. There are lessons to be learned from this timing. We are in a good place, with great people. I'll do my best to learn from this. It hurts…god it hurts…but it's going to happen…and that's okay. We cannot escape death, but we can embrace life. What you can send prayers and good energy for…is her comfort, her calm, my clarity, and my presence. She truly is the most perfect creature I've ever known…the spirit world is about to be that much more amazing, and I look forward to having the honor and gift of ushering her across that beautiful bridge. Now…it's time for me to observe every beautiful thing I Love about my best friend as I lay on the floor next to her…admiring the perfection of her existence. Much Love, Rob n Bella #GoBellaGo
Infelizmente, Bella faleceu no dia seguinte. Seu tutor fez uma postagem com um grande texto muito emocionado falando sobre a importância dela para ele e o quanto foi grato por todos esses anos em que ela viveu ao seu lado e alegrou os seus dias.
""Adeus meu amor. Obrigado por me amar…nunca me julgar…nunca desistir de mim… obrigado… por me amar com cada batida daquele lindo coração. Agora vá, torne-se um em meio a todos…dance com o vento, se flua com o oceano e aqueça os corações de todos aqueles que você tocou…seu amor agora eterno e imensurável," escreveu Robert.
Na postagem, ele ainda fez questão de homenagear seus parentes que já morreram e pediu para que eles recebessem bem a cadelinha e que fizessem carinhos na barriga dela.
Goodbye my Love. Thank you for never abandoning me…never judging me…never giving up on me… Thank you…for Loving me with every beat of that beautiful heart. Now go, become one with all…dance in the wind, flow in the ocean, and give warmth to the hearts of all you've touched…for your Love is now eternal and immeasurable. To my brother Mike, sister Charity and all my grandparents… give this girl some tummy rubs, would you? For all the spirits young and old who were brought to the other side far too soon due to some type of ailment or disease that took you before your time… welcome this girl as I'm sure she's greeting you with a wagging tail.
I wasn't sure if I would ever share this photo…as it's the last intimate moment that I spent with Bella. Yet…as I wondered this morning what will come of this social media following…what the purpose of sharing has been, and what it will now grow into after her passing…I remembered that there is such an opportunity to help others as they go through their own losses, or have pain the memories of their loss. As I'm struggling today with the fact that I didn't get to give Bella her picturesque waterside passing, I am reminded of our final goodbye. Instead of leaving her at the vet, I drove her to the crematorium and carried her from my vehicle myself. When it came time to leave…I couldn't. I couldn't just leave her there. My heart was crushed into a thousand pieces…my mind was spinning and my chest was full of anxiety. I felt it all happened too fast. I had so much time to say goodbye, but I hadn't really told her goodbye. I hadn't prepared her. I hand't done enough..and now I was just leaving her. How will I ever forgive myself for just leaving her there? Then…I caught myself. I knew what I needed to do. I went back to her perfectly beautiful body…kneeled down, placed my hand on her chest, and found my breath. Perhaps for merely even a minute or two…I breathed in…filling my lungs and my belly…and I breathed out. The world slowed down, along with my mind and heart. I told Bella that I was sending her all of my Love…and asked her to send me all of mine. I kissed her forehead one last time…and then remembered I could now kiss that beautiful nose of hers once again now that it was no longer in pain. I told her I Loved her…and left feeling fulfilled…and at peace. Perhaps this peace…has been what I've been searching for all along. Hopefully in my sharing this, you can remember to find your breath…and your breath can bring you your peace. Hang in there, #TeamRobandBella, we're still together in this thing called life…and what a beautiful thing it can be.


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Themes and Meanings
(Comprehensive Guide to Short Stories, Critical Edition)
Although Sonia Sanchez has worked in several genres, poetry is her primary craft; "After Saturday Nite Comes Sunday" might then best be considered an autobiographical prose poem, for her childhood years were spent in a New York tenement, where drug addiction was an everyday reality. In her essay "Ruminations/Reflections," she asserts that "the poet is a creator of social values." The story, without recourse to blunt didacticism, addresses from "a Black woman's view of the world" the struggle to avert the breakdown of the black family in the social context of white oppression.
The story's opening clarifies that context when Sandy is confronted by "that undersized/low expectation of niggahs/being able to save anything" at the bank. When she discovers that Winston is responsible for the missing money, she must confront not only his drug addiction but also the legacy of his frustrated dreams, "like some childhood disease," which have culminated in heroin fantasies from an early age. Those fantasies, based on the fear of failure and a sense of hopelessness, have led to drug-related crime, conviction, and prison—metaphors for being born black, for racism, and for exclusion. Having served his sentence, Winston remains criminally irresponsible and unemployed: He has confirmed the white stereotype of prejudice, which ignores the fact that he has grown up in a society that discriminated against him both legally and socially.
(The entire section is 545 words.)


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2004 Chevrolet Colorado Crew Cab - Consumer Review
Build Quality
Reliability Value
Build Quality
Reliability Value
Complaint free
By Chris on
Vehicle2004 Chevrolet Colorado 4dr Crew Cab Z71 LS 4WD SB (3.5L 5cyl 4A)
ReviewI've had this truck 2.5 yrs. and have put roughly 135,000 miles on it and thinking about a new 2007 replacement. Needless to say, with all the miles I've put on my truck, with no repairs needed, with the exception of two minor warranty repairs, this truck has been virtually trouble free. There is nothing wrong with the interior. It is what it is somewhat plain but extremely tough sensible hard plastic save for the cloth seats and well laid out. The exterior styling still impresses me as the best looking mid size crew cab on the road.
Best FeaturesThe automatic when manually shifted is great when going down slippery hills in the winter
Worst FeaturesThe wheel well trim and mudflaps on the Z71. I think it is different on the newer ones
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text:
ask the professor
Tanya Byron: My elderly husband refuses to give up driving
Q My husband and I are in our eighties, and are in good health for our age, but I don't think my husband should be driving a car. In the past five years he has had three accidents, all caused by his misjudgment. He is getting increasingly frail and has had difficulty walking for many years, which is why he is anxious to keep driving. However, his concentration is not what it was and I am afraid that he could cause death or injury to others.
The last accident caused our car and the one he crashed into to be written off. He insisted on buying another car, but could not get insurance so it was bought in my name and he was registered as…


text:
In stock
Santa's sleigh crashes and breaks in a snowstorm on Christmas Eve, and all of the presents are scattered across Adventure Bay. It's up to the PAW Patrol and your child to fix the sleigh, round up the reindeer, find the presents, find the magic star, and most of all, save Christmas for Adventure Bay.
- Please do not use double quotation marks (" ") in your personalization when entering book details. If necessary, only use single quotation marks (' ').
- Accents and other symbols cannot be printed.
- We automatically print the words "With Love From" on the title page, therefore please only enter your personalization requirements.
© 2016 Spin Master PAW Productions Inc.
All rights reserved. Nickelodeon is a trademark of Viacom International Inc.
Additional Information
|0 - 3, 4 - 7
|Book Size
|Large Softcover
|Product Category
|My Adventure Books
|Boys, Girls
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"A video game comic and blog that would have been awesome and relevant 10 years ago. Maybe." -Famous Website
Quite a lot of sparks have been flying in the community about the idea of having skippable combat in games. I'm amazed at just how much backlash it has created for the woman who brought the idea to the table as it digs at an important issue: battle fatigue.
We've all been there, right? If you've played games long enough, you have encountered sections in titles that drag on. This happens a lot in RPGs; you are invested in the story and want to see the progression but are tired of grinding dungeons. Maybe you have been playing and encountered an egregiously difficult battle. Up until that point, the game was sane but suddenly a boss decides to go aggro and relentlessly decorate his lawn with your body parts. Uh-oh! You didn't keep multiple save files and can't go back to grind out some levels to compensate? Have fun starting a new file.
Game developers are getting smarter and smarter at crafting the experience to be accessible. Certainly these sorts of issues appear less frequently and the easy answer is to say that it's up to a developer to ensure the combat is engaging and interesting throughout. Sure. But that is a very difficult line to walk and if the developer isn't spot on, then they will be alienating players that would have otherwise enjoyed their game.
I'm not a huge fan of nerfing; I personally love my games to have some bite. In multiplayer games this creates a whole other set of issues because competition is a critical component of what makes the game fun for some. But you know what? We solved this a long time ago with having difficulty levels and we've already seen games with story modes meant for players with this in mind. Hell, in the modern Tales games, you can even switch the difficulty whenever you want in the option screen.
It's funny because Pie and I are playing Earthbound right now and you know what? That game had a pretty elegant solution for battle fatigue: if you are more powerful than the encountered enemy, you auto-win the battle without ever seeing the battle screen. I thought that was a great idea then and at an age where game time is fleeting its especially great now.
The bottom line is this: Let the developers make smart difficulty modes and let the players choose what is right for them.


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Rose fends off Stenson with birdie on last to secure gold
Justin Rose claimed another gold for Great Britain as he saw off the Henrik Stenson after an exciting final-round duel in Rio.
Rose carded a closing 67 at Reserva de Marapendi to finish 16-under par and two shots ahead of Ryder Cup partner Stenson, with Matt Kuchar claiming bronze for America after a superb 63.
The outcome was only decided on the 72nd hole when Rose pitched to three feet to set up a decisive birdie and Stenson missed his attempt from long range, before the Swede also missed the return putt for par. That left the stage clear for Rose to tap in and seal victory before punching the air in delight.
"It feels absolutely incredible," Rose said. "I was on that last green, just sort of pinching myself and taking myself back to the quote that I had given about the Olympics all along - I hope my resume one day reads 'multiple major champion and Olympic gold medallist'.
"But let's just call it major champion and Olympic gold medallist, I'd be a very, very happy man.
"The whole week I've been so focused. I've been so into it. I've been so up for it. I've been just so determined to represent Team GB as best as I could, and it was just the most magical week, it really was."
Asked about his battle with Stenson after the pair won all three of their matches together at Gleneagles in 2014, Rose added: "I just said today that I had to out-Stenson Stenson. I knew I wasn't going to get much from him at all. Obviously the bogey at the last only came because he had to force the putt in. But he is unbelievable. He's relentless and a great player and I can't wait to be on the same team as him in the Ryder Cup."
Stenson had been hampered by a back problem, but refused to use it as an excuse.
"I had a bit of an issue, my thoracic spine locked up after 13 holes and put me out of rhythm a little, but I was still there at the end and it came down to whoever made birdie down the last," he said.
"I'm pleased with my performance. When you're in good position to try and win, you always kind of feel a little disappointed afterwards. But at the same time, there are some pretty good consolation prizes."
Irish Independent


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Hung Gar is known for its specific 12 Bridges (Sahp Yih Ji Kiu), or Kiu Sau.
However the concept of Kiu Sau is also common to many styles of Chinese martial arts.
The Bridges are a key characteristic of the Hung Gar style as evidenced by the right and left borders on every page of this website. The characters in these borders represent the Bridges.
To explain the Bridge concept I have put together a PDF document which has the content as outlined in the diagram below. To access the document click on the diagram, or see below.
Click the above diagram or click here for the document [PDF format] explaining the Bridge concept
If you need Acrobat Reader to access PDF files, click on this image to download


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Hogwarts Class of 2035!
I told you that 2017 would be full of big things! Sean and I couldn't be more thrilled to be welcoming our little lychee (codename: Foxlet) into the world this September!
Mischief managed.
Hogwarts Class of 2035!
I told you that 2017 would be full of big things! Sean and I couldn't be more thrilled to be welcoming our little lychee (codename: Foxlet) into the world this September!
Mischief managed.
It's 2017 and a full of year possibilities is ahead of us. I have a lot that I want to do this year, and I'm hoping that by jumping in full-force, I'll actually be able to say I've done a lot of it by this time next year!
I've openly admitted that I'm a starter of projects, but not all that great at finishing them. (Pretty sure it's smack-dab at the top of my "About" page, lol.) I know that makes me sound flaky, but it's just part of my personality that I've come to understand. I'm a passionate person, and I get excited easily. As such, I love jumping into things full force, and I love following my passions wherever they may lead. Unfortunately, what that sometimes means is that a new passion might steer me away from a previous one (see: Zumba, knitting, and my unfinished book sequel…). This ended up with me feeling guilty about leaving unfinished business behind (even if just temporarily.)
Worse than the guilt of knowing I've left loose threads dangling, however, is the fear of what people might say about it, say about me. Blogging and working for Yelp has definitely thickened my skin, but I can still be hyper-sensitive to criticism and critique. I'm also a Monica Gellar-level people-pleaser.
But, c'mon, I'm 28 years old! I can't live my life based on the fear of what others might say about me. And anyway, I'm growing up… kinda! While I'm pretty sure I'm just as passionate and excitable as ever, there are a lot of steady, firm, decidedly not-flaky parts of my life as well.
The average length of time I spent at all of my previous jobs was exactly 1 year. And then I found Yelp, and now I'm fast-approaching my 4-year anniversary! I'm a married lady with two fur-children, I'm older, and, hey, maybe even a tiny bit wiser.
I mentioned in a previous post that one of the things that I've been continuing to get more and more interested in is makeup. I've been getting more experimental and showcasing my looks on social media more often. I actually have received a few inquiries about whether I'd ever considered making tutorial videos, and the truth that I've actually considered it quite a bit. But, you know, the timing wasn't right, or I wasn't well-equipped, or it just seemed like too much work… and I didn't want to start another thing that would just peter out and disappear.
Alas, all of those things might still be true, but as part of my "resolution" (I kind of hate New Year's Resolutions, so I use that word with caution), I'm taking 2017 by the horns and testing the waters anyway! I went ahead and filmed my very first makeup tutorial video! It's kind of a mess — evidently putting me in front of a camera makes me unable to pronounce words anymore, and the amount of times I uttered the words "uh," "like," and "anyway" made me want to punch myself.
That said, if you're in any way interested in seeing what goes into an everyday glam look for me, you can check it out on my Youtube Channel:
Wow, you know it's been a minute since you've contributed to your blog when you have to remember the URL to get to the login page because it doesn't pop up in your browser history's autocomplete bar. To be honest, I don't think I even cognitively realized just how long it had really been, you know? I mean, with the loss of my friend, and then my grandmother, blogging just didn't seem "right" for a while. And then life just kept truckin' along, and before you know it, here we are! December. Crazy.
So the reality is, I don't even know where to begin with this post. It doesn't really feel productive to spend a ton of time covering what's been going on during last 6 months of my life because A) there's not really all that much to report, B) if you follow me on social media in some form or another, you probably already know a lot of it, and C) I am too lazy to try to remember it all. But, it would feel weird not at least touching on where I'm at in, like, my life, sooooooo… I'll try to make it brief.
Work: Yelp stuff is still going awesomely. I'm a little bit past 3.5 years at Yelp, and in July I was promoted to the role of Community Director. I am, it might go without saying, still loving my job, having a great time hosting events, working with local businesses, and championing my community. I also have the best #worksquad a girl could ask for. In August, I threw my 3rd huge Yelper Party event, and also attended CM Week at Yelp's HQ in San Francisco for the 3rd year.
Family: Even though 2016 has been the year of some seriously crappy stuff, it will still be remembered as an amazing year in the grand scheme of my life, since it started off with me gettin' hitched! Sean and my first year as newlyweds has been wonderful, and we haven't threatened to divorce each other even once.
My nieces are still awesome, and my family and I are just as weirdly close as ever. In early November, my sister, my parents, and I went to New York and my dreams came true because I finally got to see HAMILTON (in short: I cried like six…ty times, it was NOT overhyped despite how much I built it up in my head, it was everything and more):
And later in November (lol), the whole fam (minus Sean, who had to work), went to Orlando for a big family vaycay. Which meant I got to return to the Happiest Place on Earth (for Gretchen), Harry Potter World, and also, the Happiest Place on Earth (for Other People), Disneyworld:
My trip back to Diagon Alley went really well with my latest tattoo, too:
And, finally, my dogs are still my wonderful, weird, bearded children:
Does that pretty much cover all the bases? I think so, right? Eh, if there's anything I'm forgetting… I probably will never remember it. But! It should at least bring us up to the present, wherein I am actually trying to get back to updating this thing on the reg. But, for once, I'm not going to make any grand, sweeping declarations like, "I'm returning to my roots!" and "I'm getting back on the blog wagon!" because those will surely backfire and leave me eating my words. Instead, I'll just be honest and say this:
My life has gotten pretty great. I have a loving husband, a home of my own, furchildren, an awesome family, a job that I am legitimately still obsessed with, and the freedom to continue to dye my hair any color I want with impunity (it even recently got featured in a Buzzfeed listicle, so, y'know, #lifegoalscomplete). I'm also, obviously, extremely humble.
But I must admit, if anything has been missing over the past, I dunno, few months… a year… or two… it's that I've stopped writing.
I've basically stopped writing here on this blog, I've stopped writing creatively, and I think that I've been keeping myself busy enough that I haven't had to think about it. It's just so easy to put family, friends, work, holidays, trips, obsessively singing the soundtrack to Moana, binge-watching Gilmore Girls: A Year in the Life, and playing Final Fantasy XV nonstop for 3 straight days first. But I miss writing, I do, and I want to… stop… missing it. (Hmm, my skills may have already deteriorated.)
So, whether I start posting here regularly again, or whether I find the motivation to finally frickin' finish the sequel to Terra (I know, I know…), or whether I just find myself coming back to my words a little more often, I'd like to write more. And I'm going to try to do just that.
And with that said, hopefully, you'll see me back here in these parts very soon. Although, based on recent precedence, if I make it back for another post before… uh… April… I'll already have outdone myself. I do love setting that bar loooooow. 😉
This past Wednesday, July 27th, my beautiful grandmother passed away.
My PoPo (the Chinese term for maternal grandmother, and what my siblings and I all called her) was a strong and loving women, who raised a family of eight (8!!) children.
Those of you who have been reading this blog for a while may already know that my grandmother had not been in the greatest health for the past several years, so while nobody can say that her passing was a surprise, she is missed so deeply nonetheless. I'm grateful that my mother and her siblings were all there by her side until the very end.
PoPo leaves behind an amazing legacy in her children and children-in-law, 14 grandchildren (yeah, I have a lot of cousins), and even got to meet her first two great-grandchildren.
I savor memories of summers spent at her home in Virginia Beach, watching her sing Cantonese karaoke in the living room, letting our crazy gaggle of grandkids run amuck, cooking us seriously amazing and authentic Chinese dinners (which, of course, I never truly appreciated when I was young, but would kill for right now.)
She was an incredibly strong woman, and the love she held for us, for her entire family, was evident across age and culture and language. I think I mentioned back when I got married that almost every single member of my family on my mom's side was there. My aunts and uncles and cousins moved Heaven and Earth to change their flights, jump on buses, and outrun the storm in their cars — and they all made it here in time for my blizzard wedding.
That's the kind of family my PoPo raised. The kind that is there for each other, natural disaster or nah, because family really is the only thing.
我爱你, 婆婆. I love you, PoPo.
Whew! Yesterday wore me out. I was on my feet from morning til almost morning again, and I'm definitely feeling it. And yes, I realize there are many people in the world for whom this is a frequent occurrence, for my sedentary butt, it most definitely is not. On the bright side, I actually hit all of my fitness goals for once?
Also on the bright side, of course, are the reasons why I was out and about for the entire day. I met up with my wonderful Yelp DMV coworkers for lunch, after which we went to volunteer with Martha's Table at an elementary school in Anacostia.
It was a really neat setup: Martha's Table collected food donations which were then divvied up and set up in the school's auditorium like a mini grocery store. After school, the kids and their parents came through with a little shopping list and filled up their shopping bar with fresh produce and healthy foods. There was also a station where a chef prepared a dish using several of the items the kids would be "shopping" for. That way, they could taste it, take home the recipe, and get an idea for how to cook with the items they were receiving. Pretty neat, huh?
I was really glad that my coworker Kimberly set up this volunteering opportunity — it was particularly awesome since, as you all know, cooking and eating well is already a long-standing interest of mine!
After we packed everything up, it was time for the other exciting component of my day:
Seeing Pentatonix in concert!
My friend Lara and I went up to Merriweather Post Pavilion to watch the sensational acapella quintet (maybe you remember them from The Sing-Off?) delight us with their delicious vocal magic.
Sigh, I think I'm getting old, you guys. First my teeth start falling apart, then I'm the crotchety old grandma at the rock concert. Plus the very fact that I began this very post complaining about how tired I am. AND I already like knitting… oy. The evidence is really starting to stack up.
I don't know if this will make me seem even older, but I don't even care because my last piece of news is the amazing, spectacular, incredible, life-changing fact that…
In case you didn't already know, I am legitimately, inexorably, consumingly OBSESSED with the musical Hamilton. I watched the cast perform at the Grammys and it was just all over for me from that point on. Of course, my dreams of actually getting to see the show performed life seemed an ocean away, since tickets have been sold-out for, um, ever. Sure, you technically can purchase tickets…from "third party sellers" ::coughscalperscough:: for an outrageous upcharge. We're talking prices like $600 – $1300 for ONE ticket.
Yeeeeeah. I mean, I'm a bonafide musical theatre buff with a serious crush on Lin-Manuel Miranda's lyrical magnificence, but even I wasn't that thirsty. Until the day before yesterday, that is, when fate, providence, and fortune all came together to bless me with tickets, purchased at a regular price, in all their glory.
It went down like this: at 9:54 PM, I get a message on Facebook that Hamilton tickets just went on sale. Turns out, this new block of tickets were actually released at 9:00 PM, so already, I'm thinking they're probably all gone. That said, hell if I was throwing away my shot!
I scurried over to the Hamilton website, and clicked on literally the first available date (November 1st). I waited with baited breath. It transferred me to Ticketmaster, and the angels chorus began: 2 tickets. Front mezzanine. Row C.
Before I knew it, my fast-flying fingers had proceeded all the way to the checkout page and the tickets were mine!
Of course, it wasn't until after I had already purchased the tickets that I casually mentioned that I spent not a small amount of moolah on theatre tix. Heh. After all, even at regular price, Broadway tickets ain't cheap. Heh. #goodwifeaward
Luckily, my husband A) doesn't care about musicals and B) just wants me to be happy so the entire encounter went something like this:
Me: AUGUGHHGHEHRGHGHRHGEH!!!!!!!! ::flailing around the room::
Sean: What?!?! What happened?!?!?!
Me: I ::sob:: FINALLY ::sob:: GOT ::sob:: HAMILTON ::sob:: TICKETS!!!!!!
Sean: …yay?
Me: You don't understand! I got tickets! For regular price! From the regular box office! Not for like triple the cost from a third-party seller! Hamilton! HAMILTON!!!!!
Sean: …yay?
Me: You don't have to come with me.
Sean: YAY!
Anyway, so that's pretty much the best thing that's ever happened, ever, and come November I'mma tear UP in NYC!
Aaaaand there you have it! News that most certainly cannot be topped, so I might as well just peace out here. Today we've got an exciting trip to the vet on the books for Harry & Daxter, the wonder mutts, followed by a weekend of many adventures, so hopefully I'll have some fun stuff to report back on come Monday. Have a great weekend!


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Lately there has been increased demand for personal coaching. That said, for any coaching endeavor to be effective, whether it is business or executive or life, it is going to get personal. When I entered Coach U and began my two year professional training, students were to complete a program called Personal Foundation, created by Thomas […] Continue Reading
August 26, 2013
How is Your Personal Foundation?
June 19, 2013
Coaching – It's Personal
What does this photo have to do with goal setting, workplace productivity, and leadership skills? The other day I had dinner with my gal pal Dindy Yokel at her client's excellent new Chinese restaurant, Soi (think hole-in-the-wall China Grill). Over sticky black rice studded with shitake mushrooms and shrimp, baby bok choy and udon noodles with […] Continue Reading


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DECEMBER 1, 2011] – Mike
Hensley, executive vice president of gyro, the global ideas shop, received the
Business Marketing Association's most prestigious honor today—the 2011 G.D Crain
The G. D.
Crain Award is named in honor of the founder of Crain Communications and one of
the founders of the Business Marketing Association. Created in 1969, the
annual award recognizes a BMA member for career achievement in
business-to-business marketing and exceptional service to the advancement of
the Association at the national level.
The G.D.
Crain Award carries with it election to the BMA Hall of Fame and an honorarium
from the Crain Foundation.
"Mike has a
long history of success that only continues to grow through his great work here
at gyro," said Christoph Becker, ceo+cco, gyro. "He has been an essential part
of the creation our global ideas shop. Our daily mission is to ignite
something. Mike has been igniting the agency business for decades."
Rick Segal, president worldwide
& chief practice officer said, "On September 3, Mike and I inconspicuously
celebrated 30 years of professional partnership. All who have worked with Mike
over the years, or even more recently, know what a treasure he is to our
agency. Now, the whole industry is acknowledging this fact as well."
has been a creative director, account planner and account director during his
30 years in the business. He has helped to build service and creative teams to
meet the global needs of major brands including John Deere, USG, GE, Kodak, BP
and others.
at HSR Business to Business, which he co-founded with Segal, he established the
agency's digital practice in 1994 and helped launch the research and
brand-consulting group in 1998. Additionally, he developed many of the
processes, procedures and tools used to drive the agency's planning and
delivery systems, as well as client satisfaction assessment. HSR merged with
gryo in 2009.
has served on a number of industry boards including the AAF and the BMA, where
he served as national chairman in 2005. He has been a BMA member since 1981 and
has also served as chairman of the international board and president of the
Cincinnati chapter.
About gyro
We create ideas that ignite business decisions in a numb world. gyro is
an Advertising Age Top 50 global
agency deploying a 600-employee team of creative professionals in 17 offices in
nine countries throughout the world. Globally gyro works with Abbott, Audi,
FedEx, HP, John Deere, L'Oreal, USG and Virgin Atlantic. https://www.gyro.com
More information, please contact:
Kenneth Hein
Marketing Director, North America
ignite something…
d: 212-915-2482
m: 347-501-2591
e: email@example.com


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Justice Of The Peace in and around Bosque Farms, New Mexico.
Life's Moments are those times in life that we decide to make an unique acknowledgment of life changing occasions. Weddings are celebrations, weddings are events, yet above all weddings are a new beginning, a moment in time to be cherished. Having the right Minister is important.
For these minutes, we offer extensive Wedding Minister services, Weddings officiating, and wedding event solutions. From the very traditional to modern services, our weddings and also officiating solutions are made to show your desires, desires and also dreams. We can assist you to craft an event, spiritual or nonreligious that will be a treasured memory for many years to come.
Ministerial services and wedding event officiating in the Bosque Farms, New Mexico area is our specialty, however we could travel if the requirement occurs. We supply a total variety of Weddings plans; from elopement on the courthouse steps to the most fancy formal wedding you might want.
We also offer the finest in Justice of the Peace or secular weddings. These civil ceremonies provide their own kind of celebration, whether it be with family members or just a couple and a couple of witnesses, your wedding event is a special minute to be cherished.


text:
Kilpatrick Pal Gets 21 Years In Detroit Corruption Case
Bobby W. Ferguson, a construction company head who prosecutors say was the catalyst "at the center of a historic and unprecedented extortion scheme" along with Kilpatrick, was convicted in March of nine corruption-related charges. U.S. District Judge Nancy Edmunds imposed the sentence at a hearing in Detroit federal court.
Prosecutors said Ferguson, whose Ferguson Enterprises Inc. was heavily involved in...
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text:
I had the pleasure to meet a beautiful person in spirit and creativity last Fall at the Handmade Market and we've been fortunate to keep in touch and talk crafty pursuits over the last few months. Rovena of Handmades by Rovena is so witty! Her cards are stylish and smart and her creative science themed cards appeals to the nerd in all of us. We have a lot in common, both with science backgrounds and all!
As a result, we collaborated on a card together. What happens when a knitter and paper lover get together – well KNIT HAPPENS – that's what!
We debuted our joint card at a craft show yesterday (more on that later with photos or visit my facebook page for an early look) and the cards are now available on my shop online.
And there's more exciting things to come from the two of us! I can't wait to share!


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Last year at Father's Day I was suppose to be getting a wireless outdoor speaker for review. I made the mistake in telling my husband before I got the product. Well sometimes when companies contact you about doing a review, and they get your information, they do not always follow through. I have had this happen a few times.
Every since then my husband has had his mind set on getting a wireless out door speaker! Well, as going down the giveaway list of giveaways to enter I found out that Kristen from Our Ordinary Life, is giving one away! Want to know something awesome about this wireless speaker? It is also a planter holder HAHA so when I win, I mean if I win :p not only will hubs get his wireless outdoor speaker but I also get to plant pretty pink flowers in it LOL. Bet he didn't see that one coming!
Wanna chance to win it? I reckon I will not be greedy, and I will let yall in on the deets.
Giveaway: Audiovox Acoustic Research Wireless Indoor/Outdoor Speaker with Built In Planter – Audiovox Love.
Ends: April 23, 2011
Good luck to everyone who enters to win!


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As a well known and established franchise organisation with the majority territories already allocated, we do from time to time have franchise resale opportunities. After all, with over a quarter of a century's history some of our franchisees are now considering retirement or realising their investment. A resale can provide an exciting opportunity to buy a business which is already established.
Buying an existing business has its own unique advantages; not least that you are buying an established business with established trading patterns and customers. A resale can give you a flying start but does cost more. The cost will depend on the trading history and growth and profit performance of the business; a mature business driving significant profit to the bottom line can attract a premium price, which is what any business owner is ultimately striving for.
Please contact us for more details of current resale opportunities. OK, I'm interested. I'd like to request a brochure.