text:
Liliana Schaefer1, Andrea Babelova1, Eva Kiss2, Heinz-J. Hausser3, Martina Baliova1, Miroslava Krzyzankova1, Gunther Marsche4, Marian F. Young5, Daniel Mihalik1, Martin Götte6, Ernst Malle4, Roland M. Schaefer1 and Hermann-Josef Gröne2
1Department of Medicine D, Interdisciplinary Center of Clinical Research, Muenster, University of Muenster, Muenster, Germany. 2Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany. 3Division for Biochemistry of Joint and Connective Tissue Diseases, University of Ulm, Ulm, Germany. 4Institute of Molecular Biology and Biochemistry, Medical University Graz, Graz, Austria. 5Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland, USA. 6Department of Gynecology, Muenster University Hospital, Muenster, Germany.
Address correspondence to: Liliana Schaefer, Medizinische Klinik und Poliklinik D, Universitätsklinikum Muenster, Albert-Schweitzer-Strasse 33, 48149 Muenster, Germany. Phone: 49-251-834-7525; Fax: 49-251-86-9550; E-mail: firstname.lastname@example.com.
First published July 1, 2005Submitted: October 27, 2004; Accepted: May 24, 2005.
Biglycan, a small leucine-rich proteoglycan, is a ubiquitous ECM component; however, its biological role has not been elucidated in detail. Here we show that biglycan acts in macrophages as an endogenous ligand of TLR4 and TLR2, which mediate innate immunity, leading to rapid activation of p38, ERK, and NF-κB and thereby stimulating the expression of TNF-α and macrophage inflammatory protein–2 (MIP-2). In agreement, the stimulatory effects of biglycan are significantly reduced in TLR4-mutant (TLR4-M), TLR2–/–, and myeloid differentiation factor 88–/– (MyD88–/–) macrophages and completely abolished in TLR2–/–/TLR4-M macrophages. Biglycan-null mice have a considerable survival benefit in LPS- or zymosan-induced sepsis due to lower levels of circulating TNF-α and reduced infiltration of mononuclear cells in the lung, which cause less end-organ damage. Importantly, when stimulated by LPS-induced proinflammatory factors, macrophages themselves are able to synthesize biglycan. Thus, biglycan, upon release from the ECM or from macrophages, can boost inflammation by signaling through TLR4 and TLR2, thereby enhancing the synthesis of TNF-α and MIP-2. Our results provide evidence for what is, to our knowledge, a novel role of the matrix component biglycan as a signaling molecule and a crucial proinflammatory factor. These findings are potentially relevant for the development of new strategies in the treatment of sepsis.
Biglycan (BGN), a member of the family of small leucine-rich proteoglycans (SLRPs), consists of a core protein and a chondroitin/dermatan sulfate chain(s) (1). Although it is abundant in many tissues, its biological functions remain poorly understood. BGN is considered to have an organizing role in the assembly of the ECM. Targeted disruption of the BGN gene results in abnormal collagen fibril morphology (2) and an osteoporosis-like phenotype (3). BGN is also directly involved in cell signaling. By regulating the interaction of bone morphogenic protein-4 with its inhibitor chordin, it modulates signaling of this morphogen (4). Furthermore, BGN may affect signal transduction during cell growth and differentiation via induction of the cyclin-dependent kinase inhibitor p27KIP1 (5). BGN-induced activation of RhoA and Rac1 signaling increases migration of lung fibroblasts (6). Moreover, adenovirus-mediated gene transfer of BGN induced a fibroblastic response in the lung, indicating a role of BGN in fibrogenesis (7).
There is growing evidence that ECM molecules may convey proinflammatory signals (8–13). Previously, we reported increased expression of BGN in renal inflammation, associated with heavy infiltration of BGN-expressing macrophages (14). More recent data indicated that BGN expression is regulated by nitric oxide in monocyte-like mesangial cells (15). Based on its capacity to bind TGF-β (16) and TNF-α (17), BGN may be involved in regulating cytokine activity. BGN seems to be required for growth and differentiation of monocytic-lineage cells (18). Like other gene products involved in pathogen recognition (TLRs, resistance genes in plants, CD14, nucleotide-binding oligomerization domain proteins) (19, 20), the BGN core protein contains leucine-rich repeats that facilitate protein-protein interactions (1). Recently, binding of BGN to the scavenger receptor class A (SR-A) and the endocytic mannose receptor (21, 22), 2 pattern recognition receptors on macrophages (23), has been reported. Therefore, we investigated whether BGN is part of the innate immune system and a potential innate antigen analogous to pathogen-associated molecular patterns (PAMPs).
TLRs are seminal immune receptors that are capable of sensing PAMPs of organisms ranging from protozoa to bacteria, fungi, and viruses (24, 25). The inflammatory response of macrophages to LPS is mediated mainly by a receptor complex composed of TLR4, CD14, and myeloid differentiation protein–2 (MD-2) (24, 26–28), which is critically important in innate immune recognition of Gram-negative pathogens. TLR2 participates in the recognition of Gram-positive bacteria, peptidoglycan, and yeast zymosan (24). Although TLR4 and TLR2 are highly specific in their pathogen recognition and activation of signal transduction, they share a common signaling pathway (20, 24) involving an intracellular adapter protein called myeloid differentiation factor 88 (MyD88) (29), rapid phosphorylation of ERK (30) and p38 (31), and rapid downstream activation of NF-κB (32), which lead to the expression of TNF-α and macrophage inflammatory protein–2 (MIP-2) (20).
Here we demonstrate that biglycan-null (Bgn-null) mice have a considerable survival benefit in LPS- or zymosan-induced sepsis, due to lower levels of circulating TNF-α and reduced infiltration of mononuclear cells in the lungs. In macrophages, BGN induced signaling through TLR4 and TLR2, leading to rapid activation of p38, ERK, and NF-κB and finally to a pronounced expression of TNF-α and MIP-2. This study provides evidence that BGN is a secretory product of macrophages that can act as a "danger" motif analogous to PAMPs, exerting proinflammatory functions by signaling through TLR4 and TLR2.
Improved survival of Bgn-deficient mice in LPS-induced sepsis. To investigate the influence of BGN on systemic inflammation, sepsis was induced in female Bgn+/+, Bgn+/–, and Bgn–/– mice and in male Bgn+/0 and Bgn–/0 mice by i.p. injection of LPS. On average, 8-week-old Bgn+/0 mice died at 30 ± 8 hours after injection, while Bgn–/0 animals survived for 95 ± 19 hours (n = 6, P < 0.05) (Figure 1A). Ninety minutes after injection of LPS, plasma TNF-α levels were 6 times lower in Bgn–/0 mice than in WT animals, while Bgn–/0 and Bgn+/0 mice not treated with LPS had identical plasma levels of TNF-α (Figure 1B). Similarly, female Bgn+/+ mice died much earlier (20 ± 2 hours, n = 7) than Bgn–/– mice (127 ± 39 h, n = 5, P < 0.05), of which 2 animals even survived the treatment (Figure 1C). Again, improved survival was associated with lower plasma levels of TNF-α (Figure 1D). However, Bgn+/– mice had no survival benefit (23 ± 2 hours, n = 7, P > 0.05) (Figure 1C), and TNF-α plasma levels were not significantly different from those in Bgn+/+ mice (Figure 1D). Thus, a lack of BGN prolongs survival of septic animals, probably by tempering the inflammatory cytokine response to endotoxin.
Bgn deficiency improved survival and lowered plasma levels of TNF α in mice with LPS-induced sepsis. (A and C) Survival of male Bgn–/0 and Bgn+/0 mice (n = 6) (A) and female Bgn+/+ , Bgn+/– , and Bgn–/– mice (n = 7) (C) after a lethal dose of LPS. Two of the Bgn–/– mice survived. (B and D) Plasma levels of TNF-α in male control mice without LPS (Bgn+/0 and Bgn–/0, each group n = 3) and in Bgn+/0 (n = 5) and Bgn–/0 mice (n = 4) 90 minutes after a sublethal dose of LPS (B) and in female control Bgn+/+ mice without LPS and in Bgn+/+, Bgn+/–, and Bgn–/– mice (each group n = 3) 90 minutes after a sublethal dose of LPS (D). Data are given as means ± SD. *P < 0.05.
Increased expression of BGN attracts infiltrating cells to the lung parenchyma in sepsis. To test whether BGN expression increases during septic inflammation in the lung, a major target organ in sepsis, Bgn+/0 mice were injected with a lethal dose of LPS. BGN mRNA (1.7 ± 0.2 times control, n = 3, P < 0.05, 2 hours of sepsis) (Figure 2A) and protein (1.6 ± 0.3 times control, n = 4, P < 0.05, 8 hours of sepsis) (Figure 2B) were increased in septic lungs compared with lungs from healthy mice. Immunostaining (Figure 2, C and D) and in situ hybridization confirmed these results and revealed macrophages as a source of BGN protein (Figure 2E) and mRNA expression (Figure 2F) in septic lungs. We previously demonstrated an association of increased expression of BGN with enhanced infiltration of mononuclear cells (14). Therefore, absence of BGN might reduce inflammatory cell infiltration in septic lungs. Indeed, we found significantly fewer infiltrating mononuclear cells in the lungs of septic Bgn–/0 (Figure 2H) compared with Bgn+/0 mice (Figure 2G), as quantified by H&E staining and by F4/80 immunostaining (Table 1). The improved outcome of sepsis in Bgn–/0 mice cannot be attributed to a compensatory overexpression of decorin or fibromodulin in these animals (33), as immunostainings for SLRPs both in the lung and in the liver revealed no differences from those in WT mice (data not shown). Furthermore, type I collagen (Figure 2, I and J), fibronectin (Figure 2K), laminin (Figure 2L), and type VI collagen (not shown), all known to augment the adhesiveness of leukocytes (34, 35), were equally increased in septic lungs from Bgn+/0 and Bgn–/0 mice.
Increased expression of BGN in infiltrating cells of lung parenchyma in sepsis. (A and B) Northern blot of BGN mRNA normalized to α-tubulin (2 hours) (A) and Western blot of BGN core protein normalized to α-tubulin (8 hours) (B) from lungs of Bgn+/0 mice after a lethal dose of LPS versus PBS. (C and D) Immunostaining of BGN (red stain) in lungs from control (C) and septic Bgn+/0 mice (D) 8 hours after a lethal dose of LPS. (E and F) Double staining, marked by arrows, for BGN (blue) and macrophages (F4/80, red) (E) and in situ hybridization for BGN (F) in septic lungs from Bgn+/0 mice (8 hours). The lower right inset shows a magnified view of mononuclear cells expressing BGN (white arrowhead). The upper left inset represents the sense riboprobe. (G and H) Immunostaining of macrophages (F4/80, red), with numerous F4/80–positive macrophages (lower right insets), in septic lungs from Bgn+/0 (G) versus Bgn–/0 mice (H). (I–L) Immunostaining of type I collagen in lungs from Bgn+/0 (brown) (I) versus Bgn–/0 mice (J) and Western blots of fibronectin (K) and laminin (L) normalized to β-tubulin in lungs from Bgn+/0 versus Bgn–/0 mice 8 hours after a lethal dose of LPS. The lower right insets in C, D, F, G, and H represent higher magnifications. Scale bars in D, H, and J apply to panels in C, G, and I, respectively.
Quantification of mononuclear cells in septic lungs of Bgn+/0 and Bgn–/0 mice
LPS-triggered IL-1 β and IL-6 lead to BGN expression, and BGN induces TNF- α and MIP-2 in macrophages. Given that BGN was increased in LPS-septic lungs and macrophages acted as a source of BGN, we investigated whether BGN is produced by macrophages only during infection, and if so, what stimuli would trigger its synthesis. As the BGN promoter region contains an IL-6–responsive element (36), we hypothesized that LPS-induced proinflammatory cytokines may trigger BGN expression. LPS stimulation (1 hour) resulted in an equal increase of IL-6 (Figure 3A) and IL-1β (Figure 3B) levels in culture media from both Bgn+/0 and Bgn–/0 thioglycollate-elicited peritoneal macrophages. Unstimulated macrophages did not express BGN mRNA (Figure 3C) or protein (Figure 3D). However, upon stimulation with IL-1β or IL-6, WT macrophages expressed BGN mRNA (Figure 3C) and secreted BGN protein (Figure 3D).
LPS-stimulated macrophages secrete IL-6 and IL-1β, both of which induce the expression of BGN, which in turn stimulates TNF-α and MIP-2 mRNA and protein expression in macrophages. (A and B) ELISA for IL-6 (A) and IL-1β (B) in culture media from Bgn+/0 and Bgn–/0 macrophages left unstimulated or stimulated with 0.5 ng/ml LPS for 1 hour. (C and D) RT-PCR of BGN mRNA (C) and Western blot of BGN core protein (D) secreted into culture media from Bgn+/0 and Bgn–/0 macrophages 2 hours after stimulation with either IL-6 or IL-1β (both 10 ng/ml), normalized to GAPDH or β-tubulin, respectively. (E) Northern blots of TNF-α and MIP-2 mRNA (normalized to α-tubulin) in Bgn+/0 and Bgn–/0 macrophages after 6 hours of incubation with BGN (4 μg/ml). (F and G) Dose-dependent enhancement of TNF-α (F) and MIP-2 (G) concentrations in media from Bgn+/0 or Bgn–/0 macrophages cultured for 24 hours in the absence or presence of BGN (1 or 10 μg/ml). (H) Time-dependent enhancement of TNF-α concentrations in media from Bgn+/0 or Bgn–/0 macrophages cultured for 6 and 24 hours in the absence or presence of BGN (10 μg/ml). (I and J) ELISA for TNF-α (I) and MIP-2 (J) in media from Bgn+/0 or Bgn–/0 macrophages cultured for 6 hours in the absence or presence of LPS (0.5 ng/ml). Data are given as means ± SD from 3–7 animals. *P < 0.05 for macrophages with versus without BGN or LPS, respectively.
As the rise in circulating TNF-α was blunted in septic Bgn-deficient mice, we subsequently investigated whether BGN influences the expression of TNF-α and MIP-2. Incubation of peritoneal macrophages from Bgn+/0 and Bgn–/0 mice with BGN for 6 hours led to increased mRNA levels of TNF-α (Bgn+/0 plus BGN: 5.5 ± 0.6 times Bgn+/0; Bgn–/0 plus BGN: 3.3 ± 0.4 times Bgn–/0; n = 3, P < 0.05) and MIP-2 (Bgn+/0 plus BGN: 3.3 ± 0.6 times Bgn+/0; Bgn–/0 plus BGN: 1.9 ± 0.2 times Bgn–/0; n = 3, P < 0.05) (Figure 3E). When macrophages from Bgn+/0 and Bgn–/0 mice were cultured for 2–24 hours in the presence or absence of BGN, we found dose-dependent (Figure 3, F and G) and time-dependent increases of secreted (shown for TNF-α in Figure 3H) as well as cellular TNF-α and MIP-2 (TNF-α: 1.6 ± 0.4 times control [without BGN], n = 5, P < 0.05; MIP-2: 23 ± 11 times control [without BGN], n = 3, P < 0.05 after incubation of Bgn+/0 macrophages with 10 μg/ml BGN for 24 hours). BGN-mediated increases of TNF-α and MIP-2 expression and secretion (Figure 3, E–H) appeared to be slightly (though not significantly) lower in Bgn–/0 than in Bgn+/0 macrophages, possibly because of binding of the exogenously added BGN to additional binding sites that are partially occupied by endogenous BGN in Bgn+/0 macrophages. Importantly, though the LPS-mediated increases of TNF-α and MIP-2 expression and secretion (Figure 3, I and J) were significantly lower in Bgn–/0 than in Bgn+/0 macrophages, the differences were not as pronounced as in the in vivo situation (Figure 1B). Thus, it appears that in isolated macrophages LPS initially triggers IL-1β and IL-6 expression, which then leads to BGN expression with a subsequent induction of TNF-α and MIP-2. Under in vivo conditions, LPS effects may be further aggravated by liberation of BGN from the ECM (due to the activity of proteolytic enzymes in the course of sepsis), further enhancing the induction of TNF-α and MIP-2 expression in macrophages. Data confirming the specificity and requirement of the intact and soluble BGN molecule for induction of macrophages are shown in Supplemental Figure 1 (supplemental material available online with this article; doi:10.1172/JCI23755DS1).
BGN activates p38, ERK, and NF-κB in macrophages. Subsequently, potential intracellular signaling cascades by which BGN stimulates TNF-α and MIP-2 expression in macrophages were explored. Within 10 minutes of incubation, BGN enhanced phosphorylation of ERK (3.5 ± 0.6 times control without BGN, n = 3, P < 0.05) and p38 (2.4 ± 0.5 times control, n = 3, P < 0.05) in thioglycollate-stimulated Bgn+/0 macrophages, reaching a maximum after 30 minutes (p-ERK/ERK: 5.1 ± 0.4 times control without BGN; p-p38/p38: 3.5 ± 0.6 times control without BGN; each n = 3, P < 0.05) with a return to normal levels within 120 minutes (Figure 4A). Preincubation of macrophages for 1 hour with U0126, a specific inhibitor of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2), completely abolished ERK phosphorylation (Figure 4B) after 30 minutes. Furthermore, both U0126 and SB203580, an inhibitor of p38 phosphorylation, caused a significant reduction of BGN-induced secretion of TNF-α and MIP-2 into the culture media from BGN-stimulated macrophages after 6 hours (Figure 4, C and D). Finally, enhanced activation of NF-κB after 30 minutes of incubation with BGN was shown by electrophoretic mobility shift assay (EMSA) (Figure 4E). Taken together, these results show that the BGN-dependent induction of TNF-α and MIP-2 in macrophages involves the ERK, p38, and NF-κB pathways.
Activation of MAPKs and NF-κB by BGN in macrophages. (A) Phosphorylation of ERK and p38 in Bgn+/0 macrophages after 10–120 minutes of incubation with BGN (Western blots). (B) Effects of the MEK1/2 inhibitor (U0126) on the phosphorylation of ERK in Bgn+/0 macrophages 30 minutes after induction with BGN. (C and D) Reduced levels of TNF-α (C) and MIP-2 (D) in the presence of U0126 or SB203580 (an inhibitor of p38 MAPK) in culture media from Bgn+/0 macrophages 6 hours after induction with BGN (4 μg/ml). Data are given as means ± SD from 3–4 animals in each group. *P < 0.05 for macrophages with BGN and inhibitor versus macrophages with BGN and without inhibitor. (E) Electrophoretic mobility shift assay of nuclear extracts from Bgn+/0 macrophages cultured for 30 minutes with or without BGN. C1 to C3 represent negative controls: C1, without protein; C2 and C3, macrophages with 100 times free oligonucleotides and without (C2) or with BGN (C3).
BGN signals through TLR4 and TLR2 in macrophages. The effect of BGN on HEK293 cells, which functionally expressed human TLR2, -3, -4, -5, -7, -8, or -9 and an alkaline phosphatase gene under control of the IL-12p40 promoter, was tested. When 10 μg/ml BGN was used, only activation of TLR2 and TLR4 was observed (Figure 5A). A dose-response analysis showed that 10 μg/ml BGN stimulates TLR4 (Figure 5B) and TLR2 (Figure 5C) to the same extent that 10 ng/ml FSL-1 stimulates TLR2 or 1 ng/ml LPS-K12 stimulates TLR4.
Interaction of BGN with TLR4 and TLR2 in macrophages. (A) Screening for BGN-induced (10 μg/ml) activation of NF-κB in 293-TLR cells expressing only 1 type of TLR and alkaline phosphatase as reporter gene (gray bars). An HEK293 cell line expressing only the reporter gene was used as control (TLR–). As positive control, each cell line was induced with a specific ligand (black bars). Noninduced TLR clones were used as negative controls (white bars). TLR activation is shown as activity of the secreted alkaline phosphatase in arbitrary units. (B and C) Dose-response analysis of the effect of BGN (gray bars; 1× equals 10 μg/ml) on the activation of TLR4 (B) and TLR2 (C). "NI" indicates a noninduced TLR cell line. Black bars represent positive controls (1× equals 100 ng/ml of LPS-K12 or FSL-1). (D and E) Immunoprecipitation for BGN after incubation of Bgn–/0 or TLR4–/– (D) or TLR2–/– macrophages (E) with BGN (4 μg/ml) in the presence of a cross-linker, followed by Western blot for TLR4 (D, top panel) or TLR2 (E, top panel). The bottom panels represent Western blots for BGN core protein after chondroitinase ABC treatment. (F) Colloidal Coomassie G250–stained SDS-PAGE indicating bands obtained by immunoprecipitation and analyzed by ESI/MS/MS. (G–J) ESI/MS/MS of bands labeled in F as 1 (G and H) and 2 (I and J) recognized human BGN with a probability-based score of 221 (G) and with a sequence coverage of 13% (H) and mouse TLR4 with a probability-based score of 528 (I) and with a sequence coverage of 8% (J). MOWSE, molecular weight search.
Preincubation of macrophages from Bgn+/0 and Bgn–/0 mice with blocking anti-TLR4 antibodies significantly reduced the increase in TNF-α (Bgn+/0, 43% ± 13%; Bgn–/0, 76% ± 14%; percentage of inhibition calculated for the respective sample stimulated with BGN without blocking antibodies, n = 3, P < 0.05) and MIP-2 levels (Bgn+/0, 34% ± 6%; Bgn–/0, 55% ± 23%; percentage of inhibition, n = 3, P < 0.05) in culture media upon BGN stimulation after 6 hours.
Furthermore, incubation of Bgn–/0 macrophages with intact human BGN in the presence of a cross-linker and subsequent immunoprecipitation with anti-BGN antiserum showed that BGN coimmunoprecipitates with both TLR4 (Figure 5D) and TLR2 (Figure 5E). Electrospray ionization tandem mass spectrometry (ESI/MS/MS) analysis of 2 bands migrating at 42–44 kDa and a band at 96 kDa of the immunoprecipitate after digestion with chondroitinase ABC and separation by 1D gel electrophoresis (Figure 5F) identified human BGN with a probability-based score of 221 (Figure 5G) and a sequence coverage of 13% (Figure 5H) and mouse TLR4 with a probability-based score of 528 (Figure 5I) and a sequence coverage of 8% (Figure 5J). Individual ion scores greater than 46 indicate identity or extensive homology (P < 0.05). Other significant hits (Figure 5, G and I) represented mainly keratins and trypsin. Note that band 2 (Figure 5F) was at the limit of detection, which is probably why TLR2 was not detected by ESI/MS/MS.
By gel filtration we could demonstrate interaction of BGN with a high–molecular weight complex containing TLR4 and/or TLR2, MD-2, and CD14 as shown in Supplemental Figure 2.
Effects of BGN on phosphorylation of ERK and p38 were tested after 30 minutes in macrophages from WT (C57BL/6) mice, TLR2–/– mice, C3H/HeJ mice hyporesponsive to LPS because of a point mutation in the TLR4 gene (TLR4-M) (26, 37), TLR2–/–/TLR4-M mice, and MyD88–/– mice (shown for ERK in Figure 6A). Deficiency in either TLR2 or MyD88 as well as mutation in TLR4 markedly diminished BGN-mediated phosphorylation of ERK, while functional lack of both TLRs completely abolished phosphorylation (Figure 6B). These data provide evidence for differential signaling between BGN (4 μg/ml) and LPS (2 ng/ml), since LPS-mediated signaling was completely abolished in TLR4-M macrophages whereas BGN was still able to increase phosphorylation of ERK via TLR2 (Figure 6, A and B). Another difference was observed in MyD88–/– macrophages, where BGN-mediated phosphorylation of ERK was strongly reduced (Figure 6, A and B), while LPS could still stimulate ERK via a MyD88-independent pathway (Figure 6A; WT plus LPS: 5.4 ± 0.8 times control without LPS; MyD88–/– plus LPS: 3.8 ± 0.5 times control without LPS; each n = 3, P < 0.05). In agreement with this signaling pattern, BGN-induced increases in the levels of secreted TNF-α and MIP-2 were slightly reduced in TLR2–/–, markedly decreased in TLR4-M and MyD88–/–, and completely abrogated in TLR2–/–/TLR4-M macrophages (Figure 6, C and D). Finally, BGN-mediated activation of NF-κB was detected by EMSA in HEK293 cells transfected with human TLR4/MD-2/CD14 (Figure 6E). These data suggest that BGN is signaling in macrophages mainly through TLR4 in a MyD88-dependent manner, causing activation of NF-κB and MAPKs. Besides TLR4, TLR2 is also involved in BGN-mediated signaling in macrophages.
Absence of BGN-mediated stimulation of ERK, TNF-α, and MIP-2 in TLR2–/–/TLR4-M macrophages and reduced stimulation in macrophages from TLR2–/–, TLR4-M, and MyD88–/– mice. (A) Phosphorylation of ERK in C57BL/6, TLR2–/–, TLR4-M, TLR2–/–/TLR4-M, and MyD88–/– macrophages 30 minutes after incubation with BGN (4 μg/ml), LPS (2 ng/ml), or peptidoglycan (Pepti) (5 μg/ml) (Western blots). (B) Densitometric quantification of p-ERK/ERK ratio in macrophages indicated in A. Controls represent macrophages without BGN and are defined as 1 arbitrary unit. Data are given as means ± SD from 3 Western blots. (C and D) ELISA for TNF-α (C) and MIP-2 (D) in media from macrophages indicated in A after 6 hours of culture without or with BGN (4 μg/ml). Data are given as means ± SD from 7–8 animals in each group. Significant differences for TLR- or MyD88-deficient versus C57BL/6 macrophages incubated with BGN are indicated by an asterisk positioned over the respective bar: *P < 0.05. (E) BGN- or LPS-mediated (2 ng/ml) activation of NF-κB, detected after 30 minutes by EMSA in HEK-Blue-4 cells (transfected with human TLR4/MD-2/CD14 genes) versus 293/null cells. C1 (macrophages with 500 times free oligonucleotides) and C2 (without nuclear extract) represent negative controls.
To ensure that activation of macrophages by BGN was not attributable to contamination of the BGN preparation, we carefully ruled out the presence of LPS, other proinflammatory factors, and other TLR4 and TLR2 ligands (Supplemental Figure 3).
Since in TLR2–/–/TLR4-M macrophages an inflammatory response to BGN was completely abolished, the role of alternative receptors is very unlikely. However, as interactions of BGN with the SR-A type I (SR-AI) and the endocytic mannose receptor have been reported (21, 22), we excluded a role of these receptors in BGN signaling (Supplemental Figure 4).
Prolonged survival of Bgn-deficient mice in zymosan-induced sepsis but not in T cell–mediated lethal shock triggered by the superantigen staphylococcal enterotoxin B. To test whether TLR2-mediated activation of macrophages is relevant in vivo, zymosan-induced sepsis (38) was evaluated in Bgn–/0 and Bgn+/0 mice. Bgn–/0 mice revealed improved survival (Figure 7A), lower plasma levels of TNF-α (Figure 7B), and a reduced number of H&E- and F4/80–positive infiltrating cells in septic lungs (Table 1). Finally, we induced a lethal shock syndrome triggered by staphylococcal enterotoxin B (SEB) in Bgn–/0 and Bgn+/0 mice sensitized by D-galactosamine hydrochloride. The basic differences between this model and LPS- or zymosan-induced sepsis are that SEB triggers activation of T lymphocytes, not macrophages, and that this model does not involve TLRs (39). In this model, survival (Figure 7C), plasma levels of TNF-α (Figure 7D), and the number of infiltrating mononuclear cells in the lungs (Bgn+/0, 3.6 ± 0.5; Bgn–/0, 3.9 ± 0.9; Bgn+/0 plus SEB, 4.8 ± 1.5; Bgn–/0 plus SEB, 3.8 ± 0.5; H&E, n = 4, P > 0.05) did not differ between Bgn–/0 and Bgn+/0 mice. Thus, these data provide further evidence for a role of macrophages and TLR4 and TLR2 in BGN-associated inflammatory processes.
Survival and plasma levels of TNF-α in zymosan- or superantigen SEB–induced sepsis. Bgn deficiency improved survival (A) and lowered plasma levels of TNF-α (B) in mice with zymosan-induced sepsis but did not affect T cell–mediated lethal shock triggered in mice by the superantigen SEB (C and D). (A) Survival of male Bgn–/0 and Bgn+/0 (n = 5) mice after a lethal dose of zymosan A. (B) Plasma levels of TNF-α in Bgn+/0 and Bgn–/0 mice (n = 5) 5 hours after a lethal dose of zymosan A. (C and D) Survival (n = 6) (C) and plasma levels of TNF-α (n = 7) (D) 90 minutes after a lethal dose of SEB simultaneously injected with D-galactosamine hydrochloride in Bgn–/0 and Bgn+/0 mice. There were no differences in the plasma levels of TNF-α between zymosan- and SEB-free mice (data not shown). *P < 0.05.
In the present studies, we have identified a new role of BGN as a crucial proinflammatory factor. The most convincing evidence for the biological relevance of this finding is the observation that Bgn-null mice have a considerable survival benefit compared with WT animals, both in LPS-induced and in zymosan-induced sepsis, due to a mitigated inflammatory response that results in less end-organ damage. This survival benefit in Bgn-null mice is probably due to the fact that the initial sequence of inflammatory events, namely PAMP-triggered expression of proinflammatory factors such as IL-1β and IL-6, subsequent IL-1β/IL-6–induced expression of BGN in macrophages, and finally BGN-induced expression of TNF-α, is interrupted in the absence of BGN. The relevance of this interpretation of our data is underscored by the observation that the increased secretion of IL-1β and IL-6 as an initial response to LPS is indistinguishable in Bgn-deficient and WT macrophages, while the subsequent TNF-α production is significantly reduced in the Bgn-deficient macrophages because of the lack of costimulatory effects of BGN. Regulation of TNF-α by BGN has not been reported previously, although in lung fibroblasts TNF-α has been proven to induce the expression of BGN (40). Since the initial release of TNF-α is of critical importance for the outcome of sepsis, therapeutic strategies to neutralize this cytokine have been developed, but no clear-cut survival benefits have been achieved so far (41). This study demonstrates that in the early stages of sepsis, serum levels of TNF-α are considerably lower in Bgn-deficient than in WT mice.
The importance of BGN during sepsis is underlined by the fact that BGN overexpression could be observed in septic lungs, at both mRNA and protein levels. Early upregulation of BGN in pulmonary inflammation has already been reported (42, 43); however, its biological relevance remained unclear. We previously observed a striking concurrence of BGN overexpression and enhanced numbers of infiltrating cells in a model of renal inflammation (14). In the present study we confirmed this relationship in the lung and addressed the potential mechanism in vitro by showing that BGN, besides TNF-α, induces the expression of MIP-2, a potent chemoattractant of infiltrating cells (44). Enhanced numbers of macrophages were in fact triggered by BGN, since other ECM components, such as fibronectin, laminin, and types I and VI collagen, which could potentially increase monocyte sticking (34, 35), were upregulated to the same extent in lungs from septic Bgn+/0 and Bgn–/0 mice. BGN regulates lung fibroblast migration via stimulation of the small GTPases RhoA and Rac1 (6). Interestingly, both the Rho proteins and the p38 MAPK pathways have been shown to be important mediators of LPS-induced expression of IL-8, a human MIP-2 homolog, in human endothelial cells (45).
It is particularly noteworthy that macrophages, seminal cells of the innate immune system (24), are capable of producing BGN upon stimulation by proinflammatory factors. Previously, we have shown that macrophages do contain and secrete BGN in a model of renal inflammation (14). A recent study reported BGN in association with macrophages in human gingiva (46). In the present investigation, we found that unstimulated peritoneal macrophages do not express BGN. However, once activated by IL-6 and IL-1β, macrophages start to express BGN mRNA and secrete the proteoglycan into the culture medium. Although the presence of an IL-6–responsive element in the BGN promoter region has been reported (36), the IL-1β– and IL-6–triggered expression of BGN in macrophages is, to our knowledge, a novel finding. The fact that macrophages produce BGN only upon inflammatory stimulation underlines the biological importance of this proteoglycan in terms of its influence on inflammation. As only soluble BGN is active in stimulating macrophages, presumably because in the immobilized state the binding sites for macrophage receptors are inaccessible because of binding of BGN to other ECM components, we suggest that regulation is achieved via the amount of BGN liberated from immobilized pools by the activity of proteolytic enzymes in the course of sepsis, rather than via fine-tuning of BGN expression levels. Thus, only Bgn-null mice could be seen to have a survival benefit in sepsis, which suggests that, in heterozygous females, sufficiently high tissue content of BGN is present to support macrophage activation.
Our data showing that BGN interacts with TLR4 and to a lesser extent with TLR2, and the subsequent predominantly MyD88-dependent induction of ERK, p38, and NF-κB, significantly extend the knowledge of the biology of BGN for 2 reasons: (a) the data confirm that BGN acts as a signaling molecule, and (b) they firmly establish this proteoglycan as an integral part of the innate immune system. Although the involvement of BGN in various signaling processes has been addressed (4–6, 15, 33), this is, to our knowledge, the first report to provide evidence for BGN-mediated signal transduction in macrophages, starting from the initial receptors and describing the downstream signaling events and the inflammatory mediators subsequently generated. Various structural molecules have been reported to modulate an inflammatory response reaction (9–13, 47). Glycosaminoglycan (GAG) chains are well known to act as targets for bacterial, viral, and parasitic virulence factors for attachment, invasion, and immune evasion (48–50). In our study, only intact BGN and to a lesser extent the intact decorin molecule were able to deliver a proinflammatory signal to macrophages. Thus, BGN-mediated stimulation of macrophages is not due to a general effect of GAG chains but was shown to be in fact BGN-specific. Recent studies demonstrated that ECM components, previously only regarded as docking sites for microbial pathogens, might actively participate in immune defense as an integral part of the innate immune system (9, 11–13). Among these ECM molecules, fibronectin extra domain A (11) and hyaluronan-derived oligosaccharides (9) have been shown to activate TLR4. Our study provides evidence that the ECM component BGN is also actively involved in the innate immune response. In contrast to hyaluronan (9, 13), BGN strongly influences the effector phase of an immune reaction (e.g., increase in activity of macrophages).
BGN effectively uses the TLR signaling cascade by binding to both TLR4 and TLR2. Contaminations are unlikely to have caused the effects. Apart from the neutralizing activity of antibodies directed against BGN, which strongly implicates BGN as the active component, contaminatory products have been excluded by several assays as outlined in the supplemental material. Most importantly, our data show a clear survival benefit of Bgn-null mice in TLR4- and TLR2-dependent models of sepsis but not in SEB-mediated shock, where T lymphocytes and not macrophages are triggered in a TLR-independent way (39). Further evidence for BGN-specific TLR4- and TLR2-mediated proinflammatory effects is provided by the observation that BGN and LPS signal differently in TLR4-M and MyD88–/– macrophages. The fact that BGN-induced signaling in macrophages is mediated by 2 receptors important in the recognition of both Gram-negative (26–28, 37) and Gram-positive pathogens (24) emphasizes the key role of this proteoglycan within the innate immune system.
In summary, our results demonstrate the biological relevance of BGN in a prototypical innate immune process such as sepsis and identify a novel role of BGN as a crucial proinflammatory factor. Being ubiquitously present in the ECM, BGN can be liberated during inflammation by proteolytic enzymes, or alternatively be induced in macrophages by PAMP-triggered cytokines, to signal through the classical PAMP receptors TLR4 and TLR2. Subsequent expression of TNF-α and MIP-2 will cause recruitment of more macrophages, which also will start to produce BGN, creating a feed-forward cycle that is able to drive an inflammatory response forward in both an autocrine and a paracrine manner.
Mice. Female Bgn–/– and Bgn–/+ and male Bgn–/0, Bgn+/+, and Bgn+/0 mice (C57BL/6) have been previously described (3). Since the Bgn gene is located on the X chromosome, male animals have only 1 allele. TLR4-M mice with a TLR4 gene point mutation (C3H/HeJ) (26, 37) and TLR4 WT strains (C3H/HeN) were from Charles River Laboratories. TLR2–/– and TLR4–/– mice were kindly provided by M. Freudenberg (Max Planck Institute for Immunology, Freiburg, Germany), TLR4-M/TLR2–/– mice by C.J. Kirschning (Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Munich, Germany), and MyD88–/– mice by S. Akira (Osaka University, Osaka, Japan). Sepsis was induced by i.p. injection into 8-week-old mice of 200 μg/g body weight (μg/g b.wt.) or 45 μg/g b.wt. (sublethal dose) of LPS from Salmonella minnesota, trichloroacetic acid extracted from S. minnesota (Sigma-Aldrich), or ultrapure S. minnesota (InvivoGen). Zymosan A (Sigma-Aldrich) prepared in 0.9% NaCl as described previously (38) and endotoxin-tested was injected i.p. in a dose of 2 mg/g b.wt. (a lethal dose in 8-week-old Bgn+/0 mice). D-Galactosamine hydrochloride (4 mg/g b.wt. i.p.; Carl Roth GmbH) and the superantigen SEB (10 μg/g b.wt. in hind footpads; Sigma-Aldrich) were injected simultaneously (39) in 8-week-old Bgn–/0 and Bgn+/0 mice. Mean body weights were not different between groups. All animal work was done in accordance with the German Animal Protection Law and was approved by the Ethics Review Committee for laboratory animals of the District Government of Muenster, Germany.
Cell culture and stimulation. Macrophages were harvested by peritoneal lavage 5 days after injection of thioglycollate, and cultured in RPMI 1640 (Invitrogen Corp.) under serum-free conditions. Only adherent cells were used for primary cultures. Purity was controlled by flow cytometry and Giemsa staining. LPS, cell culture–tested, from S. minnesota and peptidoglycan from Staphylococcus aureus were both from Sigma-Aldrich. Ultrapure LPS from S. minnesota was from InvivoGen. To control responsiveness, cells from each isolation were stimulated with LPS (0.1 ng/ml) and/or peptidoglycan (5 μg/ml) for 2 hours and tested for secreted TNF-α. Intact BGN, BGN core protein, or BGN-derived GAG chains were used in concentrations of 1–10 μg/ml. If it is not indicated otherwise, experiments were done with 4 μg/ml of BGN. When required, macrophages were preincubated for 30 minutes with rat anti–mouse TLR4 blocking antibodies (40 μg/ml; Imgenex) recognizing the TLR4/MD-2 complex, or for 1 hour with 10 μM of MEK1/2 inhibitor (U0126; Cell Signaling Technology) or p38 MAPK inhibitor (SB203580; Sigma-Aldrich). Stimulation of macrophages was performed with recombinant mouse IL-1β (10 ng/ml; Sigma-Aldrich) or human IL-6 (10 ng/ml; R&D Systems).
HEK293 cells stably transfected with pNIFTY2-SEAP vectors containing human TLR4, MD-2, and CD14 genes (HEK-Blue-4) or human TLR2 and CD14 genes (TLR2/CD14) and 293/null (control) cells (all from InvivoGen) were cultured according to the manufacturer's instructions.
Northern blot analysis, RT-PCR, and in situ hybridization. Total RNA was extracted using TRIzol (Invitrogen Corp.). Membranes were hybridized with 32P-labeled cDNA probes for BGN (51), TNF-α (American Type Culture Collection), and MIP-2 (52). A mouse α-tubulin probe encompassing bp 365–965 (GenBank accession number M13446), generated by RT-PCR (53) with the primer pair 5′-AAGGAGGATGCTGCCAATAA-3′ and 5′-AGAGATCACCAATGCCTGCT-3′, was used to control for equal loading. Northern blots were performed and quantified as described previously (53). RT-PCR for BGN and GAPDH and in situ hybridization for BGN were described previously (51).
Western blotting and ELISA. Western and dot blots were performed and quantified as described previously (53). The following antibodies were used: rabbit anti–human TLR4 cross-reacting with mouse TLR4, and rabbit anti–β-tubulin (both from Santa Cruz Biotechnology Inc.); rabbit anti–mouse TLR2 and rabbit anti–human TLR2 (both from Imgenex Corp.); rabbit anti–mouse pan-laminin and rabbit anti–human fibronectin (both from Sigma-Aldrich); and rabbit antibodies against phosphorylated and total ERK (Thr202/204) and p38 (Thr180/Tyr182) MAPK (all from Cell Signaling Technology). BGN core protein from murine lungs was semipurified and quantified by dot blotting using LF-106, a rabbit anti–murine BGN antiserum kindly provided by L.W. Fisher (National Institute of Dental and Craniofacial Research) (54), as described previously (53). Dot blot results were confirmed by Western blotting and standardized to the content of β-tubulin in the samples before purification. TNF-α, MIP-2, IL-6, and IL-1β were measured in plasma, culture media, and homogenates of macrophages by mouse-specific ELISAs (R&D Systems). Results were normalized by protein content and cell number. All assays were performed in duplicate, and each experiment was carried out at least 3 times.
Activation of TLRs by BGN. BGN-induced stimulation via TLR2, -3, -4, -5, -7, -8, or -9 was tested in HEK293 cells expressing only 1 type of TLR and secreted alkaline phosphatase under the control of a modified human IL-12p40 promoter inducible by NF-κB, activator protein–1, and IFN-regulatory factors cloned into pNIFTY2-SEAP vectors. The amount of secreted alkaline phosphatase was determined using the SEAP Reporter Assay Kit (measurements were performed by TLR screening service of InvivoGen) and is given as arbitrary units. Screening was performed (in duplicate) by challenge of the 7 TLR clones with 10 μg/ml of BGN and comparison of the induced alkaline phosphatase activity to that of negative controls (respective noninduced TLR clones) and that of positive controls stimulated with the following ligands: FSL-1 (10 ng/ml) for TLR2, poly I:C (10 μg/ml) for TLR3, LPS-K12 (10 and 1 ng/ml) for TLR4, flagellin (100 ng/ml) for TLR5, R848 (1 μg/ml) for TLR7 and -8, and ODN 2006 (10 μg/ml) for TLR9 (InvivoGen). Dose-response analysis was performed (in triplicate) using BGN (10 μg/ml, 1 μg/ml, 0.1 μg/ml) on the respective TLR cell lines.
Immunoprecipitation and protein identification by ESI/MS/MS. Immunoprecipitation of human BGN was performed as described previously (55) with some modifications. Thioglycollate-elicited Bgn–/0, TLR2–/–, or TLR4–/– macrophages (107 cells) or HEK-Blue-4, TLR2/CD14, and 293/null cells were incubated with 8 μg of intact human BGN for 2 hours at 4°C and then with 1 mM 3,3′-dithiobis(sulfo-succinimidyl propionate) (DTSSP; Pierce Biotechnology Inc.) for cross-linking of BGN to binding proteins. Cells were harvested in radioimmunoprecipitation (RIPA) buffer, and a complex of normal rabbit serum bound to protein A-Sepharose (Sigma-Aldrich) was added for 6 hours to remove nonspecifically precipitated proteins. Then 10 μg of rabbit anti–human BGN (55) was added, and after 16 hours the immune complexes were precipitated with protein A-Sepharose. As control, a similar amount of protein A-Sepharose was incubated with the antibody but without cell lysate. Additional controls included samples incubated without cross-linker and/or without BGN. After washing (3 times in lysis buffer, twice in PBS), the material containing intact BGN was divided into 2 aliquots. One aliquot was treated with chondroitinase ABC. Bound proteins were eluted by boiling in the respective sample buffer (55). Some immunoprecipitations for BGN were performed using rabbit IgG TrueBlot (eBioscience) according to the manufacturer's suggestion. Samples were examined by Western blotting for the presence of TLR4 and BGN, using BGN core protein and homogenates of TLR2–/– and TLR4–/– macrophages, HEK-Blue-4, TLR2/CD14, and 293/null cells as positive or negative controls. After separation by 1D SDS gel electrophoresis, identification of human BGN and mouse TLR4 by proteomics service (MS-ESI-PROTID; Eurogentec) using ESI/MS/MS was performed in Bgn–/0 macrophages incubated with BGN, immunoprecipitated for BGN, and digested with chondroitinase ABC. Results were analyzed by a Mascot database search (Matrix Science Ltd.).
Morphology and immunohistochemistry. Formaldehyde- or zinc-fixed and paraffin-embedded lung tissue sections were stained for matrix antigens and mononuclear cells by immunoperoxidase, alkaline phosphatase, anti–alkaline phosphatase, or streptavidin/biotin–alkaline phosphatase techniques (51, 52). Primary antibodies included LF-113, a rabbit anti–murine decorin antiserum (54), LF-106 (1:500), rabbit anti–mouse fibromodulin (1:100) (33), rabbit anti–rat type I collagen (1:50; Quartett GmbH), rabbit anti–human fibronectin (1:200; Sigma-Aldrich), biotinylated goat anti–type VI collagen (1:20; Southern Biotechnology Associates Inc.), and, for populations of monocytes/macrophages, rat anti-mouse F4/80 (1:2,000; BD Biosciences — Pharmingen). Double-immunostaining for BGN (chicken anti–rat BGN) (51) and F4/80 was performed as described previously (56). Immunolabeled and H&E-stained mononuclear cells were counted by a blinded observer in 15 high-power fields (×400) per section and recorded as mean per high-power field.
Other procedures. Expression of human BGN and decorin in HEK293 cells and purification of the native proteoglycans were described previously (33, 57). Potential contamination of BGN preparations was ruled out as described in the supplemental material.
Nuclear extraction was performed with NucBuster Protein Extraction Kit (EMD Biosciences Inc.) and analyzed by EMSA. Briefly, 2.5 μg of nuclear protein was incubated with 40 kcpm of 32P-labeled oligonucleotide coding for the region of interest. Reactions were incubated for 45 minutes on ice. For oligonucleotide competition, 20 ng of specific oligonucleotide competitor was added prior to the probe. Oligonucleotide sequences for consensus or mutant DNA-binding domains were purchased from Santa Cruz Biotechnology Inc. (NF-κB/sc2505/sc2511).
Protein concentrations were determined using the BCA Protein Assay Reagent (Pierce Biotechnology Inc.).
Statistics. Data are given as means ± SD analyzed by 1-way ANOVA, with Dunnett's significance correction test (SPSS 13.0; SPSS Inc.). Differences were considered significant at a P value of less than 0.05.
View Supplemental data
This work has been supported by the Deutsche Forschungsgemeinschaft (SFB 492, B10 to L. Schaefer and R.M. Schaefer; SFB 405, B10 to H.-J. Gröne; and GO1392/1-1 to M. Götte), the University of Muenster (Interdisciplinary Center of Clinical Research, Muenster, Schae2/004/04 and IMF SC 11 04 07 to L. Schaefer and R.M. Schaefer), and the Austrian Science Fund (FWF, P17013-B05-MED to E. Malle). We are grateful to Monty Krieger (Massachusetts Institute of Technology, Cambridge, Massachusetts, USA) for providing control and CHO-SR-AI cells. We are grateful to B. Arnold (German Cancer Research Center), P.J. Nelson (University of Munich, Munich, Germany), and D. Vestweber (University of Muenster) for their critical reading of the manuscript. The technical support of Siegmund Budny, Martina Volz, and Claudia Schmidt is highly appreciated.
Nonstandard abbreviations used: BGN, biglycan; b.wt., body weight; EMSA, electrophoretic mobility shift assay; ESI/MS/MS, electrospray ionization tandem mass spectrometry; GAG, glycosaminoglycan; MD-2, myeloid differentiation protein–2; MEK1/2, mitogen-activated protein kinase kinase 1 and 2; MIP-2, macrophage inflammatory protein–2; MyD88, myeloid differentiation factor 88; PAMP, pathogen-associated molecular pattern; SEB, staphylococcal enterotoxin B; SLRP, small leucine-rich proteoglycan; SR-AI, scavenger receptor class A type I; TLR4-M, TLR4 mutant.
Conflict of interest: The authors have declared that no conflict of interest exists.


text:
The Darby Translation (DBY) | New International Version (NIV) |
6 And there was a day when the sons of God came to present themselves before Jehovah; and Satan came also among them. | 6 One day the angels came to present themselves before the LORD, and Satan also came with them. |
The Darby Translation is in the public domain. (The Darby Translation Bible Online) | Scripture quoted by permission. Quotations designated (NIV) are from THE HOLY BIBLE: NEW INTERNATIONAL VERSION®. NIV®. Copyright © 1973, 1978, 1984 by Biblica. All rights reserved worldwide. (New International Version Bible Online) |

subset: commoncrawl


text:
ASPB's Top Authors
Online Group
The Plant Cell publishes novel research of special significance in plant biology, especially in the areas of cellular biology, molecular biology, genetics, development, and evolution.
Now Featuring Teaching Tools
Established in 1926, Plant Physiology® is an international journal devoted to physiology, biochemistry, cellular and molecular biology, genetics, biophysics, and environmental biology of plants.
25,000th Article Classics
A boldly contemporary text, including molecular biology, cell biology, and plant physiology
Online ImagesOrder
A free online book reviewing aspects of the plant Arabidopsis thalianaNew Chapters!
Now FeaturingPLANTS ARE NOT ALONEOnline Group
Additional Publications & Resources:
1/20/2015Plant Biology 2015 – July26-30 – Minneapolis Convention Center – Minneapolis, Minnesota
1/12/2015Nominations open for ASPB Executive Committee positions and ASPB Awards
1/9/2015Plants are fascinating! On May 18, 2015, we will show the world why.
1/26/2015 » 1/30/2015Botanical Nomenclature Training Course
2/1/2015 » 2/6/20152015 Gordon Research Conference (GRC) on Plant Lipids: Structure, Metabolism, and Function
2/2/2015 » 2/5/2015ISF-BIDR Workshop on "Surviving the Extremes: Abiotic Stress Tolerance in Extremophile Plants
2/8/2015 » 2/13/2015Keystone Symposia - Plant Receptor Kinases: From Molecules to Environment
2/17/2015 » 2/22/2015Keystone Symposia - RNA Silencing in Plants
15501 Monona Drive Rockville, MD 20855-2768 USA Phone: 301-251-0560 Fax: 301-279-2996 email@example.com
© Copyright American Society of Plant Biologists 2014 (All Rights Reserved)


text:
Pfizer:No AstraZeneca antitrust issues
Comment on this storyLondon - Pfizer chief executive Ian Read said on Tuesday he did not believe there would be antitrust problems in taking over British drugmaker AstraZeneca.
"We don't see any substantial antitrust issues on this deal anywhere in the world," Read told a panel of British lawmakers.
The US drugmaker has fuelled a major political row with its $106 billion plan to acquire AstraZeneca, which has been rebuffed by the smaller British company.
The deal would be the largest foreign takeover of a British firm and is opposed by many scientists, trade unions and politicians who fear for British science jobs given Pfizer's track record of cost cutting after past acquisitions. - Reuters
Comment Guidelines
- Please read our comment guidelines.
- Login and register, if you haven' t already.
- Write your comment in the block below and click (Post As)
- Has a comment offended you? Hover your mouse over the comment and wait until a small triangle appears on the right-hand side. Click triangle () and select "Flag as inappropriate". Our moderators will take action if need be.


text:
Well, I guess that each one of us use Twitter, now a days. If you don't, join it right now, you might be missing a lot. Now, for those of you how have already joined Twitter and are using it everyday, trying to grow their followers, let me ask you one simple question –
"How many social media influencers follow you?"
If your answer is "NONE!", you are at the right place. Because today I am going to show you the exact thing I did to get top social media influencers including Aaron Lee, Ted Rubin, John Lee Dumas & Adam Connell to follow me on Twitter. And some of these are also in Forbes Top 50 Social Media Influencers Of 2013. But, why do they follow me? Because I'm so cool? Probably. But, I really don't know.


text:
CNBC this morning, Maria Bartiromasaid:Goldman Sachs initiates coverage of the oil servicesgroup with an overweight position. HAL and SLBsingled out.--------------------------------------------------------Appears fto be overall strrength in the drillers andservices sector this AM. PKD already up 1/4 today.
tail is my specialty. Not doing it too often can be very rewarding. Today, would be a good example. I intend to cover at 21 1/2 or, there abouts.
and my nasty ass in that oval office. I get those fucks working to make some cash for us stockholders.
I pity those Parker fools if I get my hands on em
Were havin big fun now!!!!!!!
PKD always leads the way and outpaces the competition. Parker always wins the battle of the down days, it's that bad a stock.
slugger down in the boardroom is the only thing going help this POS.
Can we assume the following news about theKazakhstan projects will benefit PKD? Sounds better than theCA attorney general Bill Lockyer whining about theprice of gas goingup.http://search.news.yahoo.com/search/news?p=%2Boil+%2Bprice&n=10
Thanks BlueDuck we all need positive reinforcement once in a while.
I may have offended. (but in jest only) Im sure hoping you guys are right.I'm gonna hide and watch,and see how the stick floats.good day to all.the apprenhesive kid....
BlueDuck2222...thanks for your clever posting, the prediction is a riddle: "for mid-August PKD to double". good luck to you also, LOL.


text:
King James Version (KJV) | New International Version (NIV) |
4 And the mixt multitude that was among them fell a lusting : and the children of Israel also wept again , and said , Who shall give us flesh to eat ? | 4 The rabble with them began to crave other food, and again the Israelites started wailing and said, "If only we had meat to eat! |
The King James Version is in the public domain. (The King James Bible Version Online) | Scripture quoted by permission. Quotations designated (NIV) are from THE HOLY BIBLE: NEW INTERNATIONAL VERSION®. NIV®. Copyright © 1973, 1978, 1984 by Biblica. All rights reserved worldwide. (New International Version Bible Online) |


text:
Just got a new grad job in neuro - what to expect? - page 3
I was offered and accepted a position on the neuroscience floor. It is at a teaching hospital and is a certified stroke center. I have never set foot through the door, so I don't know what to... Read More
0Aug 18, '11 by DeLanaHarvickWannabe, BSN, RNQuote from MochaNurseHi MochaNurse...Hi all,
Thanks for all the post I am a new grad RN, I just started on a neurosurgical unit in Albert Einstein Hospital in the Bronx on Tuesday and I feel like Im lost..Is that normal? It is alot of work that I pray to master one day soon.....I felt very overwhelmed when the day was done...there is so much to do for each patient and to learn..I feel like Im in school again getting ready to take my unit exam....Please help with any advice, It would be greatly appreciated..I feel like Im all alone? Has anyone else felt this way when they started their first nursing job..Do you guys have any tips that can save my job..My probation is about 3 months and I would love to pass it...I love nursing so I want to do my best!!!..Thanks in advance!!!
Mocha Nurse
Every nurse feels like this at first. A Neurosurg unit is challenging for any level of nurse, from novice to expert. But you will learn SO much. I think you will be fine. Just learn as much as you can, ask preceptors and nurse educators available to you for help...remember, they want you to stay on your unit also. If they lose you they are out time and money training you. So ask for help - it is in their best interest that you succeed! -
1Aug 18, '11 by MochaNurseQuote from DeLanaHarvickWannabeThank You so much for your response DeLanaHarvickWannabe I am very appreciative of your input. I have faith in GOD that I will be just fine. I will definitely take your advice and talk to my Preceptors and Nurse Educators. You are so right about the cost to train me as well as the time. Thanks so much Ill keep you posted as to how it goes from here on, I go back on the floor for 2 days starting tomorrow..I am so anxious and nervous but , I will say a prayer before i go.Hi MochaNurse...
Every nurse feels like this at first. A Neurosurg unit is challenging for any level of nurse, from novice to expert. But you will learn SO much. I think you will be fine. Just learn as much as you can, ask preceptors and nurse educators available to you for help...remember, they want you to stay on your unit also. If they lose you they are out time and money training you. So ask for help - it is in their best interest that you succeed!
Mocha Nurse..DeLanaHarvickWannabe likes this. -
1Oct 14, '11 by aNYTrini-n-FLRNQuote from DeLanaHarvickWannabeThank you for this post I start as a new grad on the neurology/neurosurgery unit in Jan at Johns Hopkins Hospital I would have never thought about Neuro until I did share time on the unit and learning all the different diagnosis that I could barely pronounce lol. I will do review with the literature you provided...First of all, congratulations!
The floor I work on is stroke overflow (stroke floor only has 19 beds) at a teaching hospital that is stroke certified, so we do receive quite a few neuro patients.
My first MedSurg clinical rotation, though, was on a floor that sounds like yours. (Same hospital - before it expanded and became really specialized - all neuro was lumped onto one floor). Oh my goodness, I learned SO MUCH! I personally think a neuro floor is a great choice for a new nurse, because you will perfect those skills you learned in school. The things you'll learn on that floor will help you in whatever field you enter in the future!
I'm not going to lie, it will be difficult. I think all new nursing jobs are, but neuro patients can be very stressful and the A & P involved is quite complex. But please do NOT give up...remember no matter what, that your AllNurses family is here for you!
As for what to study, I did a little research for you. (I am a huge dork who regularly spends $100 at a time on nursing books, even though I've been out of school for four years!)
These titles sound yummy! (Some are pricey, so shop around...all can be found on Amazon).
The Clinical Practice of Neurological and Neurosurgical Nursing (Clinical Practice of Neurological & Neurosurgical Nursing) - Joanne V. Hickey, 12/2008
Care of the Neurological Patient (Essential Clinical Skills for Nurses) - Helen Iggluden, 6/2006
Clinical Neuroanatomy Made Ridiculously Simple (3rd Edition) - Stephen Goldberg, 1/2007
Wish you the best!
My only concern, as a new grad starting out in a specialty does that hinder you if you decide to go to another unit such as cardio?DeLanaHarvickWannabe likes this. -
1Oct 18, '11 by xtxrnHickey's book is sort of the "Bible"...but there were errors re: pseudo seizures in the last edition I got (about 8 years ago). It gives good introductory info on a lot of neuro disorders, but for interest in specific diseases, you'll probably want to look for books on those disorders on their own.... it can get pricey, but there is SO much to learn.
But Hickey is a good intro bookaNYTrini-n-FLRN likes this. -
0Jun 19, '12 by alannah_smthI am a new grad RN starting on a Neuro floor in less than a month! I am so nervous. Neuro wasn't necessarily my first choice but I was really just happy to have a job at the hospital of my choice.
This thread was great to read. I'm getting some ideas of what I need to brush up on and some important tips that will hopefully make orientation easier.
Thanks everyone! Anymore new grad advice would be appreciated!


text:
Unbiased Reviews by Real People
I'm a geographer and cartographer by trade. I design maps using GIS (geographic information systems). My software of choice is ArcGIS 10, which I've used since 1991.
In addition to being a wife and homemaker, parenting and shlepping two active teens, I have a part-time job making maps that keeps me pretty busy.
USA State Graphics


text:
Housing Options
Centennial Hall
Centrally located on campus, Centennial Hall has the capacity to house over 650 students in air-conditioned double rooms with private bathrooms. This hall is co-ed throughout the building and is for first-year students only. Centennial shares a courtyard with Kohl Hall and is steps away from Carillon Place dining center which hosts all-you-care-to-eat meal plans.
Conklin North is home to nearly 250 students. BGSU sororities will occupy a large portion of this building untill fall 2016 while the new Greek Village is being built. Conklin North is near the middle of campus, has air conditioning and is in close proximity to both Harshman and Kreischer Quadrangles, the Sundial Dining Center and Jerome Library.
Falcon Heights
Falcon Heights has the capacity to house nearly 650 upperclass (and transfers) in air-conditioned double- and single-room suites with private bathrooms. Typically 4 students share a suite. This building is located near many math and science buildings and is a short walk to the Student Union. Many residents dine at The Oaks dining center which hosts all-you-care-to-eat meal plans. Returning students only.
Founders Hall houses nearly 600 residents and is the home of the Honors Learning Community (HLC). Freshmen are eligible to live in Founders if they are part of the HLC. All other spaces are for returning and transfer students. Founders offers six-person and two-person suites with air-conditioned double and single rooms, as well as private bathrooms. Gender Neutral housing is also available in Founders on a limited basis. The hall features an Outtakes Quick Cuisine location and is close to the Student Union, many academic buildings and the heart of campus.
Harshman Quadrangle currently has two halls open: Anderson and Bromfield. It's a short walk to the Sundial Food Court across the Alumni Mall, a large courtyard area accompanied by outdoor basketball courts. The Rec. Center, intramural fields and athletic complexes are nearby as well as many local businesses. All students are eligible to live in Harshman.
Kohl Hall
Kohl Hall houses over 300 students in double occupancy rooms. Kohl is centrally located on campus and is right next door to the Carillon Dining Center. Kohl hosts several learning communities including, Chapman Learning Community, the Global Village, La Comunidad, and La Maison Française (starting Fall 2014). Kohl Hall residents are encouraged to participate in one of the communities.
Kreischer Quadrangle
Kreischer Quadrangle consists of four residence halls: Ashley, Batchelder, Compton, and Darrow. Kreischer features the Sundial Food Court along with the Alumni Mall, a large courtyard area accompanied by outdoor basketball courts. The Rec. Center, intramural fields and athletic complexes are nearby. The Arts Village learning community is housed in Kreischer Compton and the Music Community is located in Batchelder. All students are eligible to live in Kreischer.
McDonald has a new entrance, front desk and lounge area and updated rooms including new carpet, paint, lighting and furniture. McDonald's courtyard features The Oaks dining center which host all-you-care-to-eat style of dining. Members of the ARMY ROTC, the Aviation Community, the Educators in Context and Community (ECCO), the Construction Management Theme Community, and the SEARCH Community also live in McDonald. It is close to the Student Union and many academic buildings. All students are eligible to live here.
Offenhauer Towers house over 800 students in double and single rooms
with air conditioning. The rooms are some of the largest on campus.
Offenhauer features Outtakes Quick Cuisine in the lobby. Members of
the Natural
and Health Science Residential Community live in Offenhauer
West. The Offenhauer Towers are the two tallest buildings on campus at
ten and eleven stories. Prioritiy is given to returning students but
freshmen are welcome.
Fraternity and Sorority Residences
New Greek housing will be opening on campus for the fall 2016 semester. Many Greek chapter will be living in groups within the residence halls while constrution of the Greek Village is taking place. Fraternities are living in Falcon Heights and sororities are living in Conklin North. Some small group living units will continue to house addtional chapters away from central campus.
Contact Us
470 Math Science Bldg.
Bowling Green, OH 43403
Phone: 419-372-2011
Fax: 419-372-0477


text:
GLENTHOMPSON Dunkeld got off to a great start to surprise Lismore Derrinallum.
This resulted in the Demons having to play catch-up all game, only to be defeated by the Rams.
In the six goal end result, the Rams had best players in wing defender Jess McMahon and a high percentage goal haul by Jess Fishburn sealed Lismore Derrinallum's fate.
Full story in The Spectator on Thursday, July 24, 2014, or subscribe to our epaper at top of page. Only $2 per week.


text:
Holman Christian Standard (CSB) | New International Version (NIV) |
17 The word of the Lord came to me: | 17 The word of the LORD came to me: |
Holman Christian Standard Bible ® Copyright © 2003, 2002, 2000, 1999 by Holman Bible Publishers. Used by permission. All rights reserved. (Holman Christian Standard Bible Online) | Scripture quoted by permission. Quotations designated (NIV) are from THE HOLY BIBLE: NEW INTERNATIONAL VERSION®. NIV®. Copyright © 1973, 1978, 1984 by Biblica. All rights reserved worldwide. (New International Version Bible Online) |


text: Spurs' Tim Duncan sues financial advisor for more than... Spurs' Tim Duncan sues financial advisor for more than... Space Jam Inspired Art Exhibition Hits NYC Space Jam Inspired Art Exhibition Hits NYC SMU notified of allegations by NCAA SMU notified of allegations by NCAA Chargers WR Keenan Allen potentially out for rest of th... Chargers WR Keenan Allen potentially out for rest of th... Jon Lester featured on this week's Sports Illustrated c... Jon Lester featured on this week's Sports Illustrated c... Marcus Mariota shines on Letterman Marcus Mariota shines on Letterman Bobby Petrino's unique bonus Bobby Petrino's unique bonus Close Tweet0 Share0 Comment0 Share0 Share0 Stumble It0 Print0 More SI Wire Report: MLB owners approve replay funding by Scooby Axson Email Posted: Thu Nov. 14, 2013 MLB managers would get two chances each game to challenge disputed calls. (AP Photo/Michael Dwyer)Major League Baseball owners approved funding for replay expansion for the 2014 season, reports Jerry Crasnick of ESPN.com.The Major League Baseball Players Association and the World Umpires Association still must approve the plan.Rob Manfred, MLB's chief operating officer, said present and former umpires likely would make the decisions on contested calls after reviewing video at MLB headquarters in New York.The current system calls for replay only for boundary calls involving home runs.Managers will now get two challenges that can be used at any point in the game. If a certain play is challenged and overturned, the manager gets to keep that challenge. Managers cannot argue reviewable calls, but if a manager enters the field of play to argue a play, he must challenge.CORCORAN: MVP Awards preview: McCutchen vs. Goldschmidt, Trout vs. Cabrera The new plan will allow an appeal of most disputed plays. A review of any disputed play will have to be initiated by a manager when he informs an umpire that he wants to challenge a play. Promoted Stories Comments


text:
Believe it or not, this was created from a photo I took of a hair dryer diffuser =D I get bored sitting still…so when I looked up I whipped out my Canon PowerShot SX210 IS and took a few shots then add texturelayer and effects in Picnik. This is now on exhibit at LC Art Gallery and Urban Nervan wants to buy it and hang it in the Salon LOL!
398 Views, 5 Features and Sojie 18 Nomination as August 24, 2012 and one SoJie 18 Award – Thank you all!
Featured on September 5, 2012!
Featured in the ARTISTS ON FACEBOOK Group on August 24, 2012!
Featured in THE GROUP-GALLERY OF ART & PHOTOGRAPHY on August 24, 2012!
Featured in THE BEST OF REDBUBBLE Group on August 23, 2012!
Nominated for SoJie 18 Abstracts Exhibit on August 18, 2012!
Featured in the "Superbly Visual Group" on 3/19/2011!
Featured in the "Creation Group" on 3/22/11!
abstract, photo manipulations, deez, dl honeycutt, sojie 18 nominee
Commercial Artist & Freelance Photographer
Wedding & Event Photography
Redbubble Host of 6 Groups
Greenville, South Carolina USA
E-Mail: email@example.com
Congratulations from your hosts at Superbly Visual!
Thanks Colin =D
– DeeZ (D L Honeycutt)
Thanks Rene! =D
– DeeZ (D L Honeycutt)
Congratulations from your hosts at Creation!
Wow, thanks Colin! =D The girls at Urban Nervana will be thrilled…I'm headed that way in about an hour LOL!
– DeeZ (D L Honeycutt)
wow! hair dryer? I will be very respectful for ordinary things now. great shot and congrats ;)
Yes, my Mom always said I have an over-active imagination…so she laughed out loud when she saw this one and grinned. Thanks so much for the great comment!
– DeeZ (D L Honeycutt)
WOW!!!! Stunning work!!!!
– DeeZ (D L Honeycutt)
Yes, you can create interesting abstracts from ordinary objects, I do this too. Must be the incessant photographer in us (LOL)l
You're right Mary! Thanks so much!
– DeeZ (D L Honeycutt)
This work has been nominated to show in the
August 2012 Juried Invitational of Solo Exhibition,
SoJIE 18 – 'Abstracts'.
Click and follow Instructions for entering, now….
Wow, what a truly nice honor!
– DeeZ (D L Honeycutt)
Love this, what gorgeous colour
Thank you! =D
– DeeZ (D L Honeycutt)
Incredible!! :)
Thank you! =D
– DeeZ (D L Honeycutt)
WOW…..Super Cool abstract D……:)))))
Thanks T! =D
– DeeZ (D L Honeycutt)


text:
| Email | Full Article (TrendHunter.com/id/55912) |
Miss Plastic Hungary is a beauty pageant that celebrates plastic surgery by honoring the contestant with the best cosmetic enhancements—-those that, according to the organizers, display a "perfect harmony of body and soulâ€â€"-with cash and prizes.
The gallery and video above show contestants from this year's Miss Plastic Hungary contest. There were breast implants aplenty, but also rhinoplasties and even surgically-enhanced toes.


text:
More Recipe Collections
Mary's desserts and other treats
(9 Recipes) Created by MWEIMER
Recipes in this Collection
Very Good 4.2/5
(95 ratings)
mmm...mmm... good Submitted by LEPRECHAUN0610 Carbs: 15.9g | Fat: 0.3g | Fiber: 2.4g | Protein: 2.8g | Calories: 71.1
Delicious, low-cal drink I found on a forum for moments when you want chocolate. I will be drinking this even after I reach my goal. It is that good. Submitted by SERMAGRA Carbs: 14.3g | Fat: 0.4g | Fiber: 0.7g | Protein: 5.1g | Calories: 65.6
Very Good 4.5/5
(21 ratings)
Sugar Free Pumpkin Custard Submitted by RELOUDEN Carbs: 12g | Fat: 1.9g | Fiber: 2.1g | Protein: 4.3g | Calories: 63.3
What a sweet warm treat for a cold afternoon! This dish is like having a piece of apple pie but without the large amount of fat and carbs from the dough. The oats inside will trick your mind into thinking that you did just eat pie! Submitted by CHEF_MEG Carbs: 22.1g | Fat: 4.3g | Fiber: 3.3g | Protein: 0.6g | Calories: 119.8
Cookbook creator says: Probably could use Soy milk instead of powdered milk & water. Submitted by JESSICAB1221 Carbs: 28.4g | Fat: 0.3g | Fiber: 2.5g | Protein: 5.7g | Calories: 133.2
Cookbook creator says: I really don't think this can be considered sugar free, because it uses molasses & honey. Maybe you could use Splenda? Submitted by SAMANTHA_SP Carbs: 26.6g | Fat: 6.5g | Fiber: 1.4g | Protein: 1.7g | Calories: 166.4
Cupcakes are all the rage these days, and for good reason. These diminutive desserts are automatically portion controlled, and theyre just darn cute! Submitted by SPCOOKBOOK Carbs: 27.9g | Fat: 4.9g | Fiber: 2.4g | Protein: 2.6g | Calories: 145.7
Cookbook creator says: Don't use dates Submitted by CHEF_MEG Carbs: 23.1g | Fat: 4.8g | Fiber: 2.7g | Protein: 3.6g | Calories: 144.7
(no ratings)
This low calorie, low fat bread is packed with zucchini and flavor! Submitted by MWEIMER Carbs: 29.6g | Fat: 3.4g | Fiber: 4.4g | Protein: 5.1g | Calories: 160.6


text:
Try me ..... you will see......
These are some of my friends
Come to me and i will show you all my experiense
More adult webcams in this website :
erotic webcam films
sex live in realplayer
freechat girls cam
uk strip webcams
nn girls com cam
midget women xxx cam
philippine naked women pictures cam
ree live teaser chat
webcamsex totaly free
free live camxxx
filipino anal sex cam
old men cams
amature free webcams
Oh my Jenny Wren my baby I never meant to--I never--never-mm-m-meant-t-t-tt-to h-h-hurt-t-t m-m-my-y-y ba-a-by and she broke completely she sat crushed shakins as the tears came and the sobs rose to hysteria. We get this signal working efore we run into any trouble. i hope you don't mind me going off by myself for a little white to work this out. The dress fits perfectly over her backside tightly fitting into her back with thn shoulder straps she takes her wide deep red belt and wraps it around hertiny waist and fastens the large buckle. Hell . T's their money. Bob said They just loaned it to me for a while still bent over I ask Bob will you please take my skirt off. As he pulled down my skirt an I step out of it I stay bent over with my legs spread. He is the most handsome man you've ever seen in your life and . Our breath catches as he smiles at you and bows what are you? you ask trying to keep controlof yourself. My butt hurts fro. all the sitting! This is worse than a night with That's a good girl Shiloh. dia smiled her eyes turning soft. With both hands I stroked him delighting in the feel of his thick cock in my hands reveling in theadrenaline rush our affair was providing we're twenty four years old now and nobody can tell us apart not even our husands. Just me the city attorney the city clerk and the Chief of olice. i'd like David Dell and a cop Sally Monk to. Be included in the city's squad at the ceremony. In a concerned manner Jasn states You don't have to do it. an exacerbated Rebecca responds You don't really listen do you! I want todo it I am just nervous. Sam reached in andpulled a slip of paper then handed it to me i looked to her with a quesbion in my eyes and asked. 'Yes . Very man hopes for the same come lets go' she said makink a point to enquire about her he went to his room.


text:
Two dangerous welterweights were hoping to either contend or return to contention last night (March 3, 2012) as Pride and Sengoku veteran Kazuo Misaki took on former Strikeforce title contender Paul Daley in what was expected to be an action-packed showdown on the Strikeforce: "Tate vs. Rousey" main card.
While the powerful Daley, having earned his "Semtex" nickname for his explosive style and knockout power, was expecting a cakewalk against an aging Japanese veteran, he perhaps bit off more than he could chew.
read more
Paul Daley made his return to the United States against Kazuo Misaki in a welterweight battle on the Strikeforce: Tate vs. Rousey fight card. Daley showed his improving wrestling skills, but unfortunately couldn't get his striking g... Read Post
The Strikeforce: "Tate vs. Rousey" main card continued rolling along with a welterweight war pitting Paul Daley (who made weight without incident) against former Pride Grand Prix winner Kazuo Misaki. Daley's goals coming into the ni... Read Post
An established welterweight will take on an established middleweight who's dropping a division this Saturday night (March 3, 2012) as British bomber Paul Daley takes on Sengoku and Pride veteran Kazuo Misaki on the main card of Stri... Read Post


text:
Foods I Want to Try.
(19 Recipes) Created by P3ALEMANBRENDA
Recipes in this Collection
Very Good 4.1/5
(22 ratings)
Quick and easy and you will think it came from a high quality restaurant. Submitted by LESS_M Carbs: 13.3g | Fat: 7.6g | Fiber: 1.9g | Protein: 23.7g | Calories: 212.2
(no ratings)
Submitted by KATEINDENVER Carbs: 59.5g | Fat: 20.8g | Fiber: 8.1g | Protein: 8.3g | Calories: 385.3
These pumpkin muffins are so simple to make. They will fill your house with a sweet aroma, too! Submitted by LORILARSENRN Carbs: 24.4g | Fat: 0.4g | Fiber: 2g | Protein: 2.9g | Calories: 113.8
Quick, thick, delicious, satisfying drink chock full of vitamins. When made with homemade almond milk and raw honey it is suitable for raw diets. Submitted by AUNTIEANDIE Carbs: 90.3g | Fat: 4.7g | Fiber: 10.8g | Protein: 4g | Calories: 386.1
These cookies get a boost of nutrition from dried cherries, blueberries and cranberries. Submitted by PHULLER Carbs: 16.6g | Fat: 2.1g | Fiber: 1g | Protein: 1.2g | Calories: 76.5
In the morning, throw all the ingredients for this simple recipe in the slow cooker. A hearty and healthy dinner will be waiting for you when you get home! Submitted by CHEF_MEG Carbs: 7.9g | Fat: 3.7g | Fiber: 1.8g | Protein: 26.8g | Calories: 176.8
Sweet and creamy, peanut butter doesn't always have to be served with jelly. Carbs: 30.3g | Fat: 9.4g | Fiber: 1.7g | Protein: 9.7g | Calories: 229.9
Very Good 4.6/5
(27 ratings)
This recipe is for 1 person but can be multiplied to easily make dinner for as many as you'd like. Submitted by AMITY_PALMER Carbs: 30.8g | Fat: 18.9g | Fiber: 7g | Protein: 28.8g | Calories: 392
In 15 minutes, this easy recipe will take the chill off a cold evening. Carbs: 32.3g | Fat: 13.3g | Fiber: 10.1g | Protein: 31.3g | Calories: 370.8
The same great taste of the Italian favorite, but this one only has five grams of fat! Submitted by CONNIE563 Carbs: 13.6g | Fat: 5.4g | Fiber: 0.9g | Protein: 17g | Calories: 173.3
The perfect fall dessert. I always have at least one person ask me for the recipe! Submitted by ANDREWMOM Carbs: 28.5g | Fat: 1.7g | Fiber: 1.4g | Protein: 2.8g | Calories: 131.6
A quick and delicious smoothie to satisfy that sweet tooth in a healthy way! Submitted by RAEBRALOP Carbs: 25.7g | Fat: 1.3g | Fiber: 2.3g | Protein: 5.3g | Calories: 130.3
Banana and oatmeal are a perfect pair in these not-too-sweet treats. Carbs: 12.2g | Fat: 2.5g | Fiber: 0.5g | Protein: 1.1g | Calories: 66.8
Moist and not too sweet, these banana-chocolate muffins are tasty for breakfast, dessert, or a snack. Submitted by TRUTHISBEAUTY Carbs: 20.5g | Fat: 2.5g | Fiber: 1.8g | Protein: 1.7g | Calories: 102.4
Good 3.8/5
(69 ratings)
Sweet treat for dessert, or at any time of the day. Carbs: 21.2g | Fat: 3.2g | Fiber: 2.5g | Protein: 3.6g | Calories: 123.5
This recipe has been marked private.
Good 3.9/5
(106 ratings)
A quick stir fry that packs a spicy punch. Submitted by CHRISSY_8106 Carbs: 20.9g | Fat: 9.1g | Fiber: 2.2g | Protein: 13.4g | Calories: 211.7
Very Good 4.0/5
(107 ratings)
This is so easy, you'll be ready to eat in no time! Carbs: 24.7g | Fat: 27.7g | Fiber: 1.7g | Protein: 24.6g | Calories: 457.9
Very fiber-full recipe!! Submitted by TONIBALOGNA_11 Carbs: 12.2g | Fat: 5.2g | Fiber: 3.3g | Protein: 8.5g | Calories: 127.9


text:
Domain Report
This is a report about clasificados.elonce.com. according to this report,The linksin number is 215 ,the more linksin number,the higher seo ranks it may be. the alexa rank of clasificados.elonce.com is #21,115. the lower alexa rank, the higher value of this site is considered to be. The IP address of this site is 126.96.36.199, and it servers in the United States. City: Houston Continent Code: NA Postal Code: 77002 Host Target: 126.96.36.199-static.reverse.softlayer.com . We considered the estimated worth of this site is $469,004 .
The Domains Hosted On IP 188.8.131.52


text:
Who Will Google Buy Next?
Re:Australia maps? (306 comments)
I can think of some much better reasons...
Search engine... already got one.
Classifieds... ebay is slamming trading post from what I can see. Google engineers could program a trading post in a few hours... with everything free and listed int he google search engine... would crash the business plan of Trading post anyway.
Directory listings... would be handy to have... for yellow pages could just ask businesses to list for free... who wouldn't take that up!
Put a bit of effort in and you have smashed half the value of yellow pages to businesses without decreasing yellow pages costs. goodbye profits!
mapping... just need the maps... street-directory.com.au has better maps anyway.
citysearch... does that make money?
Why would google go and buy something that will probably go broke in a couple of years, or at least loose 90% of its profits!


text:
Investors Build New Marketing Tools For The Visual Web
Navigating the web these days is increasingly a case of what you see is what you get. Sites like Pinterest and Tumblr, and apps like Instagram, Vine, and Snapchat are replacing text updates with images and video as the primary mode of communication — and marketers and investors are taking notice. Read More
Chute Makes It Easy For Brands To Ask Fans For Permission To Use Their Photos
Chute is a startup aiming to become a "complete visual platform" for brands and publishers, initially by helping customers collect and publish user images and videos. Today it's taking an important step in that direction by adding tools for companies to actually get user permission to republish their content. There are companies, including Chute's customers, who… Read More
Chute Brings Its Photo Aggregation Tools Into Real-World Locations With Chute Live
Chute is a Y Combinator-backed startup that helps publishers and brands incorporate photos and other media (sometimes submitted directly by users, sometimes aggregated from social networking sites) onto their own websites. Now it's offering similar functionality for real-world locations, thanks to a new feature called Chute Live. For example, according to co-founder Ranvir Gujral… Read More
YC-Backed Chute Nabs $2.7M From Salesforce & More To Become The Twilio For Media Content
Chute knows you're tired of hearing about yet another photo-sharing or photo-syncing app. So, although it dwells in the photo sphere, thankfully Chute is taking a different approach. The recent Y Combinator grad set out to become the go-to service that app developers and content producers turn to for managing and enhancing photos in their app or on their website. You can think of Chute as… Read More
Y Combinator-Backed Chute Launches A Twilio For Photos
We've heard a lot about photo-sharing apps over the last year or two, and although the launch of a new social photo app may result in eye-rolling by some, images are becoming an ever-more important medium for communication. As our lives, products, and communications become increasingly visual, content producers, app developers, and site owners alike all have to ramp up their services to… Read More


text:
Get the insider tips from our Concierge and enjoy Shanghai!
We suggest that you can contact with our concerge in advance so they can book a cab for you. In addition, subway is an alternative option and it is much more efficiency.
Shanghai has a long term Summer & Winter season, it often rains and really clammy in winter.
The attentive staffs at the Grand Kempinski Hotel Shanghai cater to the needs of guests by providing an extensive range of services and facilities:
- Car rental
- Courier services
- Limousine service
- Luggage assistance
- Newspapers


text:
I love this season -- autumn reminds me of fresh notebooks, brand new pencils, falling red and golden leaves, and a sense of new possibilities. And, the planetary action in the past few weeks has deepened the message of new starts and clarity of purpose. It's time to connect to your long term highest path and take action. We feel this in both our personal lives and in the larger world around us. We are drawn to take a stand because the pressure is intense.
On the other hand, because of the intensity, we need to integrate self care in a more nourishing way than ever before. Even if you consciously take good care of yourself you might be finding the usual regime is simply not enough. I know I'm having to be way more diligent and daily than ever before.
Recently I spent a week studying restorative yoga, the yoga of deep rest. I have taken a few restorative classes over the years, and of course we rest in savasana at the end of every yoga class, but it is quite different to rest for 2 or 3 minutes at the end of a class and to fully support your body to be able rest for 20 minutes or longer in each pose. It's amazing how even a comfortable position becomes annoying in longer holds if any part of your body is touching something hard, or if light is in your eyes, or noises, or ... We are all a 'Princess and the Pea' waiting to happen!
So restorative yoga is like a science of swaddling yourself to be able to fully rest the nervous system without any distractions.
The four principles we cultivate in restorative yoga are:
Unlike all the vigorous, sweaty power yoga that is popular, in restorative yoga we use many props such as blankets, eye pillows, towels and bolsters to create an absolutely supported, cozy, warm, position for the body to lay or recline or even invert gently or forward fold slightly. Judith Hanson Lasater, one of the original Iyengar teachers (blessings to BKS Iyengar who passed from our physical world recently), says that sleep and rest are two completely different things, and the body needs both.
I was astonished by how much my body and spirit began to love the restorative poses we lazed around in all day at the workshop. I'm not normally a 'napper' but this is different. It's a deep meditative state attained in about 20 minutes.
Here is a basic savasana (resting) pose you can do easily by yourself.
1. Preparation:
a. Find a quiet place in your home and let your household know you don't want to be disturbed. This may be a big step for many!
b. Assemble your props: A large bolster or pillow, a very soft pillow or small soft blanket, an eye pillow or washcloth, and a soft and cozy blanket.
c. Set an alarm set for 20 minutes that will ring gently.
2. Lay down on your yoga mat (or a thick blanket on the floor). If necessary, it can even be your bed.
3. Place the pillow under your head so the edge comes just under your shoulders. Draw some thickness of the pillow a little more under your head so your chin is slightly tucked toward your chest yet the neck supported and elongated. You can do this by folding a small blanket or towel under your head if needed.
4. Place the bolster or large pillow under your knees.
5. Wrap the blanket around you.
6. Place the folder washcloth or eyepad on your eyes lightly.
As you lay there, draw your gaze inside of yourself, feeling simply how you feel in that moment without trying to change anything. It can help to count how long your inhale is and then match the exhale to the same length. If your mind wanders come back to the breath, and even to the counting if that helps. It will normally take at least 10 to 15 minutes to begin to calm down enough to reach a very deep resting state where the magic happens. You will know when you reach it. It feels heavy, deep, and very peaceful. It is almost like sleeping but it isn't. You maintain some consciousness as if you're under the ocean.
Share a free sample Dharma and Karma Charts. Astrology for the soul! Try your free Dharma Chart Karma Chart today!
Even if you are in this ultimate state only for a few minutes you will receive the healing effects. In the beginning you might fall asleep, or you might not attain the full deep peaceful sense. In fact your mind may race with anxiety not knowing what to do with the permission to really rest. If that happens, be patient with yourself. Modern life often asks us to be revved up all of the time, striving and accomplishing. We are habituated to stress and a constant high adrenal, fight/flight vibration.
By resting deeply we can create a foundation that will allow us to take the vibrant focused action we are called to ... yet from a grounded and well rested core.
with Love & Gratitude,
Marjorie Orr has an M.A. (Hons) in English Literature and Philosophy from Glasgow University, and was a current affairs journalist, award-winning BBC TV documentary producer and then a psychotherapist. She has a worldwide following for her astrology... read more
Drifting back into old and stress-producing behavior patterns is almost always a potential hazard when the Moon passes... read more »
Help Arrives! This week begins with romance, as the love planet, Venus, enters the most sensitive sign of the zodiac,... read more »


text:
Fed up with being carried around, he wants to go for a walk …
great work Bleee!
now that is an awesome idea X)…
I concur…this IS an awesome idea…and very well composed! ;D
you are awesome, the colours, shadows and hi-lites all sooo good
fantastic tee!
woah .. thanks everyone!
Great tee…love the shadow!
Thanks, that shadow was the hardest bit .. i did it three times!
Funny t-shirt! Congrats…


text:
You're leaving Wedding Paper Divas to see the Deal of the Day at our parent site, Tiny Prints.
You won't be able to dismiss this by usual means (escape or click button), but you can close it programatically based on user choices or actions.
"This is such a simple, effective and beautiful place card. You can also personalize it to add a little saying or something to make it you."


text:
Mon, May 21, 12 at 3:13
The time to repair the roof is when the sun is shining.
John F. Kennedy |
Follow-Up Postings:
I agree, but when we had a leak around our chimney cap last November and got an ugly stain on the ceiling, we couldn't wait for sunshine! What a bummer! A new roof doesn't come cheap, even on a small house, but it had to be done. Now, six months later, the ceiling and stain are going to be repaired. Of course, the whole ceiling has to be repainted, just one little section would stand out like a sore thumb! |
- Posted by anneliese_32 6 (My Page) on Mon, May 21, 12 at 18:30
Had a similar problem, it took one (1) roofing nail to repair, a 3 x 3" spot on the ceiling and a whole bedroom ceiling to paint. Somehow the whole thing was somewhat disproportioned. |
- Posted by west_gardener (My Page) on Mon, May 21, 12 at 19:15
I was so surpised to see JFK talking about repairing roofs. Who knew, not me. I guess it comes along with something like, make hay while the sun shines or get a loan when you don't need it, because when you need it, you can't get a loan. |
Please Note: Only registered members are able to post messages to this forum. If you are a member, please log in. If you aren't yet a member, join now!
Return to the The Garden Party Forum
Information about Posting
- You must be logged in to post a message. Once you are logged in, a posting window will appear at the bottom of the messages. If you are not a member, please register for an account.
- Please review our Rules of Play before posting.
- Posting is a two-step process. Once you have composed your message, you will be taken to the preview page. You will then have a chance to review your post, make changes and upload photos.
- After posting your message, you may need to refresh the forum page in order to see it.
- Before posting copyrighted material, please read about Copyright and Fair Use.
- We have a strict no-advertising policy!
- If you would like to practice posting or uploading photos, please visit our Test forum.
- If you need assistance, please Contact Us and we will be happy to help.
Learn more about in-text links on this page here


text:
I am
I am only a translator.
From the pictures in my head,
the language of monsters,
I am transfigurating words.
I am only a woman.
The visions impregnate my mind,
and I give birth to worlds to be described.
I am only a worker.
My true executives live
in the most luxurious tunnels of Hell.
I follow the instructions
through well-honed instinct
to produce them anew.
I am only a human,
using the peculiar art of speech
trying to communicate ideas
higher than humanity.
Naked Trees
The trees are stretching their spines
trying to reach and claw the sky -
dig into blue flesh for an answer why -
extending greedy and curious vines
into the air:
into the lair
of the howling, swirling dead -
and inside your lonely head.
Throbbing lines on my body -
wet kisses, vicious bites -
the fatal lust of a woman
in these voluptuous rites
bursts forth, like a fountain
of my boiling blood -
she engulfs me and drowns me
in her scrumptious flood.
Silence Is The Song Of The Dead
has its voices:
sparrow-tongued love songs,
solemn sighs of sycamores,
traces of whispering memories,
where we lie.
shadows play, black and green,
leaf and stone, wood and clay,
in peace's garden - war has left us
here to wait.
among roots deep, where streams of rain
caress the bones, a wakeful sleep
awaits you, with your friends and foes -
silence calls.


text:
Friday, June 10, 2011 - 8:29pm
BATON ROUGE, La (NBC33) - — Exxon employees had an opportunity to help out the environment and those less fortunate at the same time.
The oil giant teamed up with the Capital Area Corporate Recycling Council to collect any and all personal electronics from Exxon employees.
Alon with being able to help out the planet, it also allows CACRC to assist those who need it the most. Exxon collected more than a ton of personal electronics from their employees during today's event.


text:
- Shopping Bag ( 0 items )
From the Publisher
"This is a great (and unique) book for anyone who has received a diagnosis of borderline personality disorder (BPD), written by two experts who study and treat BPD. The features of BPD are described in very understandable terms, and there are clear suggestions for coping with these features and problems, as well as for understanding their impact. I highly recommend this book for those receiving the diagnosis, as well as for those with family members or friends with BPD."
—Timothy J. Trull, PhD, curators' professor of psychological sciences at the University of Missouri


text:
Your basket is empty.
There's a problem previewing your basket right now.
Check your Internet connection and go to your cart, or try again.
These silk teddy bear-sized Harajuku panties are the pawfect addition to a fashion-forward Harajuku outfit for your teddy bear. The Harajuku Hugs™ panties are inspired by Gwen Stefani's clothing & fashion and part of the Harajuku Hugs™ collection. They feature Harajuku Hugs™ Panda graphic and black lace trim.
Harajuku Hugs (hara-ju-koo-hugs)n: a magical and inspiring world of fashion where G and the Harajuku Girls (Love, Angle, Music and Baby) "embrace" the super cute styles found on fashion-loving girls throughout the Harajuku district of Tokyo.


text:
Re: [jira] [Updated] (FLUME-2333) HTTP source handler doesn't allow for responses
Ok, I¡¯ll update the ticket without the 3rd point.
I understand your concern though, and I¡¯ve actually heard it before when this source was first designed. I¡¯ll create a different ticket for that. Thank you! Gabriel From: Jeremy Karlson <[EMAIL PROTECTED]> Reply-To: <[EMAIL PROTECTED]> Date: Friday, February 28, 2014 at 11:00 AM To: <[EMAIL PROTECTED]> Subject: Re: [jira] [Updated] (FLUME-2333) HTTP source handler doesn't allow for responses I am not a committer, but I have no problem with additional changes to this report or patch item. It's true that the HTTP handler is limited in its openness for extension and if we can make a few changes to make it better, I'd be happy to look at any changes you make. The only thing that I'm uncertain on is point 3. It sounds like the plan is to build a simple servlet routing mechanism? My gut feeling is that Flume isn't really intended to be be a servlet container. If things are that complex, maybe a better choice is to run a standalone Tomcat or Jetty out front instead? Or alternatively, can the same thing be accomplished reasonably by running each handler on a separate port (the way it would be done now)? Point 3 sounds like a fair bit of work and I'd hate to see you do that and have it rejected. Maybe we should wait for some committers to comment before you start down that path? On Fri, Feb 28, 2014 at 8:20 AM, Gabriel Commeau <[EMAIL PROTECTED]>wrote: |


text:
Father/Son team; Don & Brandon DeHass. We are glove doctors that specialize in changing the lace color of baseball gloves. In addition to relace, we also enjoy complex repairs, tightening, glove break ins, and conditioning. With 30 years of experience; we understand that gloves are personal, and should last a lifetime. Unparalleled service, competitive prices, and superior craftsmanship are why loyal customers won't go anywhere else.
Within the greater Columbus, Ohio area, I have received mitts at many predetermined meeting places. If outside of Central Ohio, simply send your glove(s), desired service, payment, and a prepaid return shipping label.
24 HOUR GUARANTEE; simply request, 'URGENT', and your mitt will return ship within 24hrs after it's in my possession, or the service is FREE.
Don & Brandon DeHass
8708 Belworth Square
New Albany, Ohio 43054
Call or Text: (330) 231-7133
Email: email@example.com
Twitter: @DHass22 & @Elite_Brandon41
Price List:
(All gloves receive FREE tightening & conditioning.)