Calprotectin consists of the Ca2+-binding proteins S100a8 and S100a9 that are induced in epithelial cells in response to tissue damage and infection. Both proteins are also secreted by activated innate immune cells and numerous studies demonstrate their crucial role in pathological conditions of acute and chronic inflammation.
Here, we established a conditional mouse model with simultaneous S100a8 and S100a9 transgene expression in hepatocytes (TgS100a8a9 hep ) under the control of doxycycline to unravel the role of epithelial-derived Calprotectin on tissue homeostasis and inflammation. TgS100a8a9 hep mice displayed a significant enrichment of neutrophils in peripheral blood and tissues with high blood content. Interestingly, Cxcl1 transcription was significantly induced in the liver of TgS100a8a9 hep mice and primary hepatocytes derived thereof as compared to Control mice, accompanied by an increase of Cxcl1 serum levels. However, expression of other chemokines with a known function in neutrophil mobilization from the bone marrow, e.g. Csf3 and Cxcl2, was not altered. Doxycycline treatment of TgS100a8a9 hep mice reduced Cxcl1 expression in the liver and resulted in normal numbers of neutrophils.
In summary, our data demonstrate for the first time that hepatocyte-specific S100a8 and S100a9 expression induces a systemic mobilization of neutrophils by a specific activation of Cxcl1 transcription in the liver.
S100 proteins are a family of small Ca2+-binding proteins which share a broad spectrum of functions including regulation of enzyme activity, Ca2+-homeostasis, and interaction with components of the cytoskeleton. Belonging to this family, S100A8 and S100A9 also known as migration inhibitory factor-related protein 8 (MRP8) and 14 (MRP14) are low molecular weight proteins of 8 kDa and 14 kDa, respectively, and were initially described as proteins expressed and released by infiltrating phagocytes[2, 3]. S100A8 and S100A9 form preferably heterodimers also known as Calprotectin (S100A8/A9) whereas monomers and homodimers could not be detected in isolated granulocytes and monocytes. Although expression of S100A8/A9 under physiologic conditions is restricted to cells of myeloid origin including circulating neutrophils, monocytes, and eosinophils[5, 6], strong induction of S100A8/A9 was detected in epithelial and endothelial cells under inflammatory and stress conditions. Furthermore, marked expression of S100A8/A9 was found in many tumors of epithelial origin including lung, breast, gastric, colorectal and prostate cancer. These data strongly imply important functions of S100A8/A9 under conditions of tissue damage and inflammation. Accordingly S100A8/A9 is strongly induced in a broad variety of different chronic inflammatory disorders including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, arthritis, and psoriasis. S100A8/A9 is able to support an inflammatory response via both intra- and extracellular mechanisms. In fact, intracellular S100A8/A9 has been shown to facilitate leukocyte trafficking through the transendothelial membrane via microtubule reorganization, upregulation of CD11b, as well as increase in the binding affinity of CD11b to the endothelial expressed integrin ICAM-1. Moreover S100A8/A9 is able to boost the inflammatory response by promotion of NADPH oxidase activity and production of ROS in phagocytes and epithelial cells. After secretion at the site of insult by epithelial and innate immune cells, S100A8/A9 directly acts as a chemoattractant for leukocytes, as demonstrated both in vitro and in vivo. Importantly, S100A8/A9 is known to amplify pro-inflammatory signals, as it is able to induce the production and release of many cytokines and chemokines by epithelial and innate immune cells, e.g. CXCL1, CXCL2, IL-6, and TNF. In this context S100A8/A9 was shown to promote pre-metastatic niche formation in the lungs of tumor-bearing mice[16, 17]. Moreover, S100A8/A9 can promote its own expression by a feed-forward loop.
In vivo studies highlighted a crucial role for S100a8/a9 in the promotion of inflammation and inflammation-induced carcinogenesis by using S100a9 -/- animals in models of antigen-induced arthritis and colon carcinogenesis, respectively. However these studies focused on the role of myeloid-derived S100a8/a9. In contrast, no causal link between epithelial-derived S100a8/a9 and the establishment of chronic inflammation and subsequent tumor-development has been demonstrated yet. Strong experimental evidence indicates that S100A8/A9 overexpression in hepatocytes is associated with liver carcinogenesis as epidemiological studies showed a profound correlation of both proteins with high grade hepatocellular carcinoma in humans. Moreover, our group demonstrated a strong increase in S100a8/a9 in the Mdr2 -/- model of inflammation-induced carcinogenesis in a NF-κB dependent manner. Interestingly, elevated S100a8/a9 levels protected hepatocellular carcinoma cell lines from TNF-mediated apoptosis and promoted the production of ROS. Moreover, liver injury induced by partial hepatectomy lead to induction of S100a8/a9 expression in hepatocytes, suggesting a role in liver regeneration. Hitherto, we do not know whether the impact on pathological conditions of liver injury and carcinogenesis is mediated by well-known functions of S100a8/a9 on the amplification of pro-inflammatory signals, including immune cell recruitment and activation[15, 23], because informative in vivo studies are still missing.
In contrast to other in vivo studies focusing on the function of heavily elevated systemic S100a8/a9 levels in settings of acute or chronic inflammation[18, 24], we have established a conditional mouse model under the control of doxycycline to specifically investigate the effects of local S100a8/a9 overexpression without inducing a pathologic situation. In the present study we show that the simultaneous expression of S100a8 and S100a9 transgenes in hepatocytes leads to a constant homeostatic mobilization of neutrophils from the bone marrow via local induction of Cxcl1 expression. Importantly, the enrichment of the neutrophils occurs without activation and therefore hints to a so far unknown mechanism by which epithelial-derived S100a8/a9 may increase the surveillance capacity of an organism by innate immune cells.
We established a new mouse line (TgS100a8a9 hep ) with conditional and hepatocyte-specific co-expression of S100a8 and S100a9 transgenes by using the Tetracyclin-inducible expression system (Tet-Off sytem) to investigate the role of epithelial-derived S100a8/a9 under conditions of tissue homeostasis as well as pathological conditions. TgS100a8a9 hep mice were generated by breeding of the hepatocyte-specific transactivator (tTA) mouse line TALap1 with the TetOa8a9 transgenic mouse line, containing the Myc/His-tagged S100a8 (tgS100a8) and S100a9 (tgS100a9) transgenes under the control of a tTA-dependent and bidirectional promoter (Figure1A). First, tissue-specific expression of tgS100a8 and tgS100a9 transcripts was confirmed by semi-quantitative PCR with cDNA from different tissues derived of TgS100a8a9 hep mice. Strong expression of both transgenes was detected in the liver, while minor tgS100a9 transcript levels were also found in brain and adipose tissue, in line with published data on the TALap1 transgene expression pattern (Figure1B). In contrast, back skin, lung, heart, thymus, spleen, kidney, stomach, lymph nodes, and bone marrow displayed no expression for both transgenes (Figure1B, Additional file1: Figure S1A, data not shown). Quantification of tgS100a8 and tgS100a9 transcripts in livers from six month-old TgS100a8a9 hep mice by qRT-PCR revealed a similar expression level compared to younger animals, demonstrating a persistent transgene expression over time (Additional file1: Figure S1B). To confirm conditional transgene expression, TgS100a8a9 hep mice were treated with or without doxycycline for three weeks, and abolished transgene expression upon doxycycline treatment was measured by qRT-PCR as compared to control-treated mice (Figure1C). Finally, expression of tgS100a8 and tgS100a9 proteins was found in TgS100a8a9 hep but not in Control mice by Western blot analysis, following enrichment from whole liver lysate (Figure1D). However, immunohistochemical (IHC) staining of liver tissue sections did not show a strong signal for tgS100a8 and tgS100a9 proteins in hepatocytes of TgS100a8a9 hep mice, whereas S100a8 and S100a9 positive immune cells were easily detectable (data not shown), suggesting that only minor amounts of transgenic proteins were produced. In line with this data, an S100a8/a9 heterodimer-specific ELISA revealed comparable amounts of the S100a8/a9 protein complex in serum samples of peripheral blood from Control and TgS100a8a9 hep mice (Figure1E). In summary, TgS100a8a9 hep mice were characterized by a conditional and tissue-specific transgene expression, which resulted in a low but persistent tgS100a8 and tgS100a9 protein level in the liver under conditions of normal tissue homeostasis.
Hepatocyte-specific and conditional S100a8 and S100a9 transgene expression in TgS100a8a9 hep mice. (A) Schematic representation of the transgene construct: the transcriptional transactivator (tTA) is expressed under the control of the hepatocyte-specific promoter Lap1 (TALap1), binds to and activates the tetracycline operator (TetO), which drives the simultaneous expression of S100a8-myc/his and S100a9-myc/his transgenes (TetOa8a9). Doxycycline interferes with the binding of tTA to the TetO and inhibits transgene expression. (B) Tissue-specific tgS100a8 and tgS100a9 expression was determined by semi-quantitative PCR in different tissues from TgS100a8a9 hep mice using specific primers for transgenic S100a8 and S100a9 transcripts. Hprt1 levels served as control for cDNA quantity and quality. (C) TgS100a8a9 hep mice were treated with (+dox) or without (-dox) doxycycline (10 μg/ml) for three weeks and transgene expression was assessed by qRT-PCR using liver cDNA. The mean value +SD of n=3 animals per group is depicted. (D) Whole liver lysates from a TgS100a8a9 hep mouse (dTg) and a single TetOa8a9 transgenic (sTg) control were purified with an anti-Myc-tag IP Kit and expression of transgenic proteins was monitored by Western blot analysis using a His-tag specific antibody. HeLa cells were transiently transfected with S100a8 and S100a9 expression vectors and whole cell lysate was used as a positive control (Co). (E) S100a8/a9 protein was quantified by a heterodimer-specific ELISA in serum of Control (n=7) and TgS100a8a9 hep mice (n=6); mean +SD.
The S100a8/a9 protein complex is associated with inflammation especially in the context of leukocyte chemoattraction and activation[14, 26]. To investigate the impact of transgene expression on normal liver homeostasis, we analyzed tissue sections from eight week-old Control and TgS100a8a9 hep mice. Staining with hematoxylin and eosin (HE) revealed no obvious difference in liver architecture between TgS100a8a9 hep and Control littermates (Figure2A). Lack of liver damage in response to tgS100a8 and tgS100a9 expression was further confirmed by similar AST and ALT levels in serum samples from Control and TgS100a8a9 hep mice (Additional file2: Figure S2A, B). In addition, immunohistochemical staining with anti-HNE antibodies revealed no sign of increased ROS levels in TgS100a8a9 hep as compared to Control livers (Additional file2: Figure S2C). Moreover, IHC staining for the monocyte-specific antigen F4/80 revealed a comparable number of Kupffer cells in both genotypes (Figure2A). In contrast, the staining for Gr1, a marker specific for granulocytes as well as monocytes and myeloid derived suppressor cells (MDSCs), displayed a significant increase in Gr1-positive immune cells in TgS100a8a9 hep mice as compared to Control mice (Figure2Aand B).
Histological characterization of TgS100a8a9 hep liver sections. (A) Tissue sections from Control and TgS100a8a9 hep mice were stained with hematoxylin and eosin (HE) or by IHC with specific antibodies for monocytes (F4/80) and granulocytes (Gr1). Representative images from at least n=5 mice are shown with red staining for F4/80 or Gr1, respectively, and counterstaining with hematoxylin. Bars represent 200 μm. Gr1-positive (B) and S100a9-positive (C) immune cells from five randomly taken pictures (magnification 50x) from Control (n=9) and TgS100a8a9 hep (n=10) were quantified. Box-plot shows median, 25 % and 75 % quartile (light grey box), 5 % and 95 % percentile (bars), students t-test, ***p≤0.001.
IHC staining of liver sections with specific antibodies for S100a8 and S100a9, respectively, well-characterized markers for granulocytes and circulating monocytes[3, 27], and subsequent quantification of S100a8 and S100a9 positive immune cells confirmed a significant increase of this subpopulation of immune cells (Figure2C, data not shown). Indeed, comparison of the relative amount of Gr1- and S100a9-positive cells in the liver of Control and TgS100a8a9 hep mice revealed a high correlation coefficient (R2=0.95). Moreover, qRT-PCR analysis with cDNA of liver samples and primers recognizing selectively endogenous but not transgenic transcripts confirmed increased S100a8 and S100a9 expression in TgS100a8a9 hep compared to Control mice (Additional file2: Figure S2D). In summary, our data supported a causal link between transgene expression and the accumulation of a distinct Gr1-positive immune cell subpopulation of the monocyte lineage in the liver of TgS100a8a9 hep mice.
To investigate whether the accumulation of this immune cell subpopulation increased with age or caused an alteration in liver architecture, liver sections of six month-old Control and TgS100a8a9 hep mice were stained with HE and for Gr1-positive cells by IHC staining. A substantial increase in Gr1-positive cells was still detectable in liver sections of six month-old TgS100a8a9 hep as compared to Control mice and no obvious difference was found when compared to younger TgS100a8a9 hep animals (Additional file3: Figure S3).
It is worth mentioning that liver sections of TgS100a8a9 hep mice exhibited an equal distribution of Gr1-positive cells, suggesting that these cells were not actively recruited to the liver parenchyma but rather accumulated in the liver sinusoids due to increased amounts in the circulating blood. Accordingly, different tissues from Control and TgS100a8a9 hep mice were analyzed for the expression of endogenous S100a8 and S100a9 transcripts by qRT-PCR, which was used as surrogate marker for granulocytes. Elevated expression of endogenous S100a8 transcript was detected in liver, brain, kidney, and heart from TgS100a8a9 hep (n=4) as compared to Control mice (n=3), whereas the expression of S100a8 was not altered in spleen, thymus, stomach, and adipose tissue (Figure3A). Similar results were measured for endogenous S100a9 transcript levels by qRT-PCR (Additional file4: Figure S4A), indicating a specific increase in the amount of S100a8/a9 positive immune cells in highly perfused tissues. In line with this assumption, an almost 2.5-fold enrichment of neutrophils, but not eosinophils, basophils, monocytes, or lymphocytes was found in peripheral blood counts of TgS100a8a9 hep as compared to Control mice (n=4, Figure3B, Additional file4: Figure S4B). Elevated levels of neutrophils were further confirmed by flow cytometric analysis using an antibody for Ly-6G, a specific marker for neutrophils[28, 29], in combination with antibodies for Cd11b and Gr1 to distinguish the neutrophil subpopulation (Cd11b+/Gr1+/Ly-6G+) from other cell types (Cd11b+/Gr1+/Ly-6G-) including monocytes and monocytic MDSCs[28, 30]. The total percentage of Gr1+/Ly-6G+ neutrophils within the Cd11b+ population was significantly increased in TgS100a8a9 hep as compared to Control mice (n=7, Figure3C), whereas the relative number of the Gr1+/Ly-6G- subpopulation was not affected by the transgene expression (Figure3D). To exclude the possibility that Gr1-specific antibodies interfered with binding of anti-Ly-6G to its dedicated epitope, staining for neutrophils using an antibody specific for Ly-6C instead of anti-Gr1 was performed on wild type blood cells. Comparable amount of neutrophils were found with both staining procedures (Additional file4: Figure S4C-E). In summary, our data demonstrate an age-independent systemic increase of neutrophils in the circulating blood of TgS100a8a9 hep mice, which resulted in the accumulation of these cells in peripheral tissues with a strong blood supply, but without obvious features of inflammation or signs of pathological condition.
Increased amount of neutrophils in peripheral tissue and blood samples. (A) Relative expression of endogenous S100a8 transcripts was determined by qRT-PCR using cDNA from different tissues of Control (n=3) and TgS100a8a9 hep (n=4) mice. Mean +SD, students t-test, *p≤0.05, **p≤0.01, n.s. not significant. (B) Leukocyte subpopulations in the peripheral blood from Control and TgS100a8a9 hep mice (n=4) were quantified and depicted as relative cell number. Box-plot shows median, 25 % and 75 % quartile (light and dark grey box), students t-test, **p≤0.01. (C-D) Peripheral blood from Control and TgS100a8a9 hep mice (n=7) was subjected to tri-color staining using antibodies against Cd11b (APC), Gr1 (PE), and Ly-6G (FITC). Graphs show the total percentage of Gr1+/Ly-6G+ cells (C) and Gr1+/Ly-6G- cells (D) from all Cd11b+ cells in peripheral blood. Box-plot shows median, 25 % and 75 % quartile (light grey box), students t-test, *p≤0.05, n.s. not significant.
Since TgS100a8a9 hep mice did not develop an obvious pathological phenotype with age, we investigated the activation state of the systemically enriched neutrophil subpopulation. Upregulation of Mac-1 (Cd11b/Cd18) and ectodomain shedding of L-Selectin (Cd62L), two frequently used activation markers for neutrophils, were determined by flow cytometry on peripheral blood neutrophils (Gr1+/Ly6G+ for Cd11b; Cd11b+/Ly6G+ for L-Selectin) derived from Control and TgS100a8a9 hep animals (n≥5). Importantly, no difference in the mean intensity was detectable for Cd11b and L-Selectin surface expression, while Cd11b upregulation and L-Selectin shedding was easily detectable upon TPA-stimulation of isolated neutrophils (Figure4A,B and Additional file5: Figure S5), suggesting an accelerated mobilization but no further activation of neutrophils in TgS100a8a9 hep mice.
Molecular markers of neutrophil activation in TgS100a8a9 hep mice. Blood neutrophils (Gr1+/Ly-6G+ or Cd11b+/Gr1+) from Control and TgS100a8a9 hep mice or isolated neutrophils treated with 10 μg/ml TPA for 5 min (Pos) were stained with specific antibodies for (A) Cd11b (APC) and (B) L-Selectin (FITC), or the respective isotype control antibody. The mean fluorescence intensities from Control (Cd11b n=9; L-Selectin n=5), TgS100a8a9 hep (Cd11b n=8; L-Selectin n=5) and Pos (n=3) samples were determined and depicted as means +SEM.
As neutrophils have a relatively short life span in peripheral blood, continuous renewal by mobilization from the bone marrow is mandatory. This homeostatic release from the bone marrow is tightly regulated by an equilibrium of retention and mobilization factors either keeping the neutrophils in the hematopoietic cords of the bone marrow or mediating the transition across the sinusoidal endothelium into the circulating blood. Recent studies demonstrated the importance of several chemokines such as Cxcl1, Cxcl2, and Csf3 (G-Csf) and the interplay with their cognate receptors Cxcr2 and Cxcr4 on the surface of neutrophils in the regulation of homeostatic neutrophil mobilization[33, 34]. Moreover, the factors Csf1 (M-Csf) and Csf2 (Gm-Csf) have been related to neutrophil mobilization under homeostasis as well. Although, several reports showed S100a8/a9-mediated chemoattraction of neutrophils[14, 16, 24, 26], a direct involvement of transgenic proteins in neutrophil mobilization in TgS100a8a9 hep mice was unlikely as no systemic difference in S100a8/a9 protein serum levels was detected (Figure1E). Consequently, we hypothesized that local expression of transgenic S100a8 and S100a9 in the liver of TgS100a8a9 hep mice induced the expression of one or more factors, which in turn were able to mediate neutrophil mobilization from the bone marrow. First, we analyzed cDNA from livers of Control and TgS100a8a9 hep mice by qRT-PCR for the expression of Csf1, Csf2, Csf3, Cxcl1, and Cxcl2. Transcripts of Csf2 and Csf3 were not detectable (data not shown), whereas the expression of Cxcl2 and Csf1 revealed no significant difference between TgS100a8a9 hep and Control livers (Figure5A). Interestingly, Cxcl1 transcripts were significantly upregulated in TgS100a8a9 hep as compared to Control mice (Figure5A).
Chemokine levels in liver, primary hepatocytes, and serum of Control and TgS100a8a9 hep mice. (A) Relative expression of Cxcl1, Cxcl2 and Csf1 was determined by qRT-PCR analysis on liver cDNA from Control (n=6) and TgS100a8a9 hep (n=9) mice and is depicted as mean +SD. (B) Primary hepatocytes were isolated from Control and TgS100a8a9 hep mice and cultivated in vitro for 24 hours. Relative expression of Cxcl1, Cxcl2, Csf1 was measured by qRT-PCR on cDNA from primary hepatocytes. Two biological replicates for each group were measured in triplicates and means are depicted +SD, students t-test, *p≤0.05. (C) Protein levels of Cxcl1, Cxcl2, and Csf3 in serum samples from Control and TgS100a8a9 hep (n≥4) mice were measured by ELISA. Mean +SD, students t-test, *p≤0.05.
To address the question whether hepatocytes are the source of increased Cxcl1 transcript levels in TgS100a8a9 hep mice, livers of Control and TgS100a8a9 hep mice (n=3 each) were perfused with collagenase and the cell suspension was cultivated in vitro for 24 hours to obtain primary hepatocytes. One aliquot of the cell suspension was directly taken after enzymatic digestion and a significant increase in Cxcl1 transcript levels in the liver of TgS100a8a9 hep mice was confirmed by qRT-PCR (Additional file6: Figure S6A). Subsequent qRT-PCR analysis with cDNA from primary hepatocytes, which were cultured in vitro for 24 hours revealed a strong expression in S100a8 and S100a9 transgenes in TgS100a8a9 hep hepatocytes and a significant increase in the expression of Cxcl1 in TgS100a8a9 hep as compared to Control hepatocytes, whereas Cxcl2 and Csf1 were not affected (Figure5B and Additional file6: Figure S6B). These data strongly suggest that hepatocytes are the main source of increased Cxcl1 transcription in the liver of TgS100a8a9 hep mice.
Finally, we analyzed serum and liver samples of Control and TgS100a8a9 hep mice by enzyme-linked absorbent immunoassays (ELISA) for Cxcl1, Cxcl2, and Csf3 protein levels. Substantial levels of Cxcl1 and Csf3 were measured in both groups, whereas only low amounts were detected for Cxcl2 (Figure5C). We found no major difference in Cxcl2 and Csf3 protein levels, but in accordance with the qRT-PCR data, Cxcl1 protein levels were significantly increased in TgS100a8a9 hep as compared to Control mice (Figure5C and Additional file7: Figure S7).
Control (Co) and TgS100a8a9 hep (Tg) animals were treated either with (Tg +dox, Co +dox) or without (Tg -dox, Co -dox) doxycycline-containing drinking water for three weeks to investigate, whether the observed increase in Cxcl1 levels and systemic enrichment of neutrophils critically depends on the presence of hepatocyte-specific tgS100a8 and tgS100a9 expression in adult mice. qRT-PCR analysis on livers from four animals per group, respectively, confirmed a significant increase in Cxcl1 transcript levels in Tg -dox mice as compared to Co -dox animals. While doxycycline treatment had no impact on Cxcl1 expression in Co +dox mice, the Cxcl1 transcript levels were reduced to basal levels in Tg +dox mice upon doxycycline treatment (Figure6A). In line with reduced Cxcl1 expression, we found a significant decreased in the amount of peripheral neutrophils in the blood of Tg +dox mice (Figure6B, Additional file8: Figure S8), as measured by FACS analysis and blood counts, accompanied by reduced neutrophil numbers in liver sections of Tg +dox as compared to Tg -dox littermates (Figure6C). In contrast, doxycycline treatment did neither affect neutrophil numbers nor the number of Cd11b+/Gr1+/Ly6G- cells in Co +dox mice (Figure6, data not shown). Absolute numbers of neutrophils in blood counts from Control and TgS100a8a9 hep mice with and without doxycycline treatment revealed similar results (Additional file8: Figure S8).
Measurement of neutrophil numbers and Cxcl1 expression in Control and TgS100a8a9 hep mice treated with doxycycline. Control (Co) and TgS100a8a9 hep (Tg) mice were treated with (Co +dox, Tg +dox) or without (Co -dox, Tg -dox) doxycycline (10 μg/ml) containing drinking water. (A) Relative expression of Cxcl1 was determined by qRT-PCR on liver cDNA. Mean +SD, students t-test, *p≤0.05, **p≤0.01, n.s. not significant. (B) Peripheral blood cells were stained with specific antibodies for Cd11b (APC), Gr1 (PE) and Ly-6G (FITC) followed by flow cytometry analysis. Box-plot shows the relative fold increase of Gr1+/Ly-6G+ population from all Cd11b+ cells in peripheral blood; Median, 25% and 75% quartile (light grey boxes), students t-test, *p≤0.05. (C) IHC staining with Gr1-specific antibodies was performed on liver sections and representative images (n=4 animals per group) are shown with red staining for positively stained cells and counterstaining with hematoxilin. Bars represent 200 μm.
Taken together our data confirmed that the increase in Cxcl1 expression, which is accompanied by systemic enrichment of neutrophils in TgS100a8a9 hep mice critically depends on conditional S100a8 and S100a9 transgene expression in the liver.
In order to unravel molecular mechanisms of transgene induce Cxcl1 expression, we prepared protein lysate from freshly isolated primary hepatocytes derived from Control and TgS100a8a9 hep liver. Western Blot analysis revealed similar expression and phosphorylation of p65, c-Jun, Stat3, and ERK1/2, but a substantial decrease in phospho-p38 protein levels was evident in TgS100a8a9 hep as compared to Control hepatocytes (Figure7A). Densitometric quantification of phosphorylated and total p38 protein levels confirmed a 50% reduced phosphorylation due to transgene expression (data not shown). To further confirm the role of phosphorylated p38 as a negative regulator for Cxcl1 expression, freshly isolated wild type primary hepatocytes were treated with the p38 MAPK-specific inhibitor SB203580 for 5 hr and qRT-PCR analyses revealed a significant increase of Cxcl1 transcript levels in hepatocytes with impaired p38 MAPK activity as compared to DMSO treated cells (Figure7B). In contrast, Cxcl2 and Csf1 transcript levels were significantly decreased upon p38 inhibition, which is in line with our previous observation that tgS100a8/a9 expression specifically induces Cxcl1 expression in hepatocytes from TgS100a8a9 hep mice (Figure5B). In summary, these data demonstrate that impaired p38 MAPK activity in the presence of tgS100a8/a9 expression triggers a specific induction of Cxcl1 expression in hepatocytes.
Involvement of signaling pathways in Cxcl1 upregulation in hepatocytes. (A) Primary hepatocytes were isolated from Control and TgS100a8a9 hep mice (n=2). Expression of p65, c-Jun, Stat3, p38, ERK1/2, and the corresponding phosphorylated forms was monitored by Western Blot analysis. (B) Primary hepatocytes were treated either with SB203580 or with (DMSO) for 5 h and relative expression of Cxcl1, Cxcl2, and Csf1 was determined by qRT-PCR; n=5, Mean +SD, students t-test, **p≤0.01.
A broad range of experimental studies demonstrate a direct link between S100a8/a9 protein expression and acute and chronic inflammation as well as inflammation-induced carcinogenesis. In fact, S100a8/a9 is involved in several steps of the inflammatory response including leukocyte differentiation and chemoattraction, transendothelial migration, activation of immune and epithelial cells, and the induction of pro-inflammatory mediators. Thereby S100a8/a9 might promote an inflammatory feed-forward loop, which facilitates the transition to a chronic inflammation. An established chronic inflammation strongly increases the likelihood of pathologic conditions such as tumor formation and is even considered as a hallmark of carcinogenesis. Indeed in vivo studies with S100a9 -/- animals highlighted a crucial role for S100a8/a9 in the promotion of inflammation and inflammation-induced carcinogenesis in models of antigen-induced arthritis and colon carcinogenesis, respectively[18, 19]. However these studies focused on the role of S100a8/a9 in the differentiation and function of myeloid-derived S100a8/a9. In contrast, no causal connection of epithelial-derived S100a8/a9 and the initiation and maintenance of a chronic inflammation and subsequent tumor-development have been demonstrated yet.
In order to investigate the role of epithelial-derived S100a8/a9 in liver homeostasis, we generated TgS100a8a9 hep mice with conditional and hepatocyte-specific S100a8 and S100a9 expression. The initial characterization revealed persistent transgene expression in liver. Furthermore the conditional tgS100a8/a9 expression in TgS100a8a9 hep mice was confirmed via doxycycline treatment. Additionally, transgenic protein was detected in purified whole liver lysates from TgS100a8a9 hep mice, whereas the serum levels of total S100a8/a9 were unchanged in TgS100a8a9 hep mice compared to Controls. In summary the TgS100a8a9 hep mice showed conditional and hepatocyte-specific expression of tgS100a8/a9 transcripts, which was not sufficient to reach a systemic increase of total S100a8/a9. Interestingly, all animal models, which were used so far to show S100a8/a9-dependent immune cell recruitment and activation accompanied by pathologic conditions were characterized by a strong S100a8/a9 overexpression[14, 16, 23, 24]. In contrast, the use of the TgS100a8a9 hep mouse model enabled for the first time a detailed analysis of physiological and pathophysiological alterations in settings of low and locally restricted S100a8/a9 induction in vivo.
S100a8/a9 has been shown to act as a potent chemoattractant and activator for monocytes and neutrophils[14, 21, 23]. Therefore, tgS100a8/a9 expression might be expected to induce pathologic changes affecting liver homeostasis. However, TgS100a8a9 hep mice showed neither obvious differences in liver architecture and liver damage nor changes in the number of resident Kupffer cells. Interestingly, TgS100a8a9 hep animals developed a significant enrichment of Gr1-positive immune cells in the liver as compared to Control mice. Further characterization showed that this immune cell subpopulation was positive for endogenous S100a8 and S100a9 proteins and transcripts, and importantly, aged TgS100a8a9 hep animals exhibited a comparable enrichment of this specific immune subpopulation, supporting the existence of a constitutive signal in TgS100a8a9 hep mice, which leads to a sustained increase of these cells. Of note, the Gr1-positive subpopulation did not infiltrate into the liver parenchyma but rather accumulated in the blood and therefore appeared in the sinusoids of the liver. Therefore we hypothesized a systemic accumulation of Gr1-positive cells in TgS100a8a9 hep mice. Indeed, qRT-PCR analysis revealed an elevation of endogenous S100a8/a9 transcripts, which was used as a surrogate marker for the affected subpopulation, also in other perfused tissues including brain, kidney, and heart. This further supported the hypothesis of a systemic enrichment in TgS100a8a9 hep mice, which was further confirmed by blood counts and flow cytometric analysis on peripheral blood samples. Although spleen is a highly perfused organ, it serves as marginating pool for granulocytes, ensuring rapid mobilization of innate immune cells upon infection and inflammation to facilitate an immediate inflammatory response. Consequently, the overall amount of S100a8/a9 is very high and the difference due to the enriched population in TgS100a8a9 hep mice was not detectable.
Neutrophils circulating in the blood stream under healthy conditions, serve as first defense line against pathogens, and represent key players in sterile inflammatory diseases by secreting a plethora of different pro-inflammatory mediators. Since the TgS100a8a9 hep mice developed no obvious pathological phenotype over time it was questioned whether the neutrophils were fully activated. Indeed, flow cytometric analysis on neutrophils of TgS100a8a9 hep mice revealed neither an increase in Cd11b expression nor ectodomain shedding of L-Selectin, which are well-known activation markers of neutrophils[31, 38]. Together, these data convincingly show that the enriched neutrophils in the TgS100a8a9 hep mice are not activated, and additional signals are missing to induce an acute and/or chronic inflammation.
Injection of high amounts of S100a8/a9 was shown to directly act as a chemoattractant for neutrophils and lead to tissue-specific recruitment but was also reported as an activator of neutrophils[14, 23]. Furthermore, a causal role for S100a8 in inducing myeloid cell migration has been demonstrated without further activation. However TgS100a8a9 hep mice showed a systemic enrichment of neutrophils without activation and site-specific recruitment. This argues for a so far unknown mechanism of S100a8/a9, which might be only visible under conditions when tgS100a8/a9 expression is locally restricted to the liver without potent systemic accumulation. Hence we assumed that a mechanism, which is implicated in regulation of circulating neutrophils under homeostasis, was affected in TgS100a8a9 hep mice resulting in elevated neutrophil levels. The number of circulating neutrophils is mainly regulated via a constant release from the bone marrow compartment. This homeostatic release is realized either by Csf3, which is able to interfere with the Cxcl12-Cxcr4 mediated neutrophil retention[33, 34, 38], or by Cxcl1 and Cxcl2, which directly act as chemoattractants for neutrophils via Cxcr2. Moreover, experimental evidence suggests that other factors e.g. Csf1 and Csf2 might be involved in neutrophil mobilization under homeostasis[40, 41]. Interestingly, only Cxcl1 was increased in the liver and serum of TgS100a8a9 hep compared to Control mice, whereas other factors were either not detectable or were not altered. Moreover, Cxcl1 was overexpressed in primary hepatocytes isolated from TgS100a8a9 hep mice, supporting that an increase in Cxcl1 expression is at least in part mediated by hepatocytes.
In line with our findings, recent reports demonstrated that administration of recombinant Cxcl1 in mice results in a three- to four-fold increase in the number of circulating neutrophils due to release from the bone marrow[34, 42]. Accordingly, transgenic mice overexpressing Cxcl1 in different tissues showed elevated numbers of circulating neutrophils. It is worth mentioning that also Cxcl1-induced neutrophils showed no signs of further activation in terms of Cd11b upregulation and L-Selectin shedding. On the other hand mice deficient for Cxcr2 displayed an abnormal retention of neutrophils in the bone marrow whereas the numbers of circulating neutrophils largely declined.
To finally prove the causal link between transgene expression and the Cxcl1 induction as well as the accumulation of neutrophils in TgS100a8a9 hep mice, the transgene expression was repressed by doxycycline treatment. Indeed, TgS100a8a9 hep mice with repressed transgene expression exhibited baseline Cxcl1 transcript levels in the liver, accompanied by a normal number of neutrophils in peripheral blood. These data supported the assumption that the expression of Cxcl1 transcripts is causally linked to tgS100a8/a9 expression in the liver and that the accelerated mobilization of neutrophils from bone marrow is mediated by the specific increased of Cxcl1 expression in the liver of TgS100a8a9 hep mice (Figure8).
Model of S100a8/a9-Cxcl1-mediated neutrophil enrichment in TgS100a8a9 hep mice. The S100a8/a9 protein complex is produced in the liver of TgS100a8a9 hep mice and induces a specific expression of the neutrophil chemoattractant Cxcl1 in hepatocytes, which in turn leads to an increased pool of Cxcl1 protein in the serum of transgenic mice. Consequently, Cxcl1 enhances the release of neutrophils from the bone marrow and increases the number of circulating non-activated neutrophils, which also appear in distant organs such as liver.
The expression of Cxcl1 is regulated by several transcription factors, including NF-κB, AP-1, and Stat3, which facilitate a rapid activation upon inflammatory stimulation. S100a8/a9 expressed by hepatocytes in TgS100a8a9 hep mice may be secreted in minor amounts and act in an autocrine manner via binding to Rage or Tlr4, both well established membrane receptors for extracellular S100a8/a9[19, 47]. Engagement of these receptors has been shown to induce relevant transcription factors for induced Cxcl1 transcription as well as their upstream signaling pathways, such as p38 and ERK1/2 MAPKs[48–50]. Western Blot analysis of primary hepatocytes revealed a substantial decrease in activation of the p38 MAPK pathway whereas other pathways were not altered. Inhibition of p38 activity was shown previously by our group and others in situations of intracellular S100a8/a9 expression[21, 51]. In contrast, extracellular S100a8/a9 rather lead to a positive regulation of p38[17, 51]. Together with the finding that S100a8/a9 serum levels are unchanged in TgS100a8a9 hep as compared to Control and increased Cxcl1 levels were observed in isolated hepatocytes our results point to an intracellular rather than an extracellular mode of action, how S100a8/a9 mediated Cxcl1 induction. Finally, inhibition of p38 MAPK lead to increased expression of Cxcl1 in primary hepatocytes by an as yet unknown mechanism indicating a role of p38 as a negative regulator for Cxcl1 expression under conditions without inflammatory stimuli.
TgS100a8a9 hep mice with conditional and hepatocyte-specific expression of S100a8 and S100a9 served as an useful tool to unravel a so far unknown function of calprotectin on the mobilization of neutrophils. Our new findings have a major impact on the current view, how calprotectin may influence acute and chronic inflammation under pathological conditions. Ichikawa et al. demonstrated in a model of colitis-associated carcinogenesis that myeloid-derived S100a8/a9 induces chemokine expression by tumor cells, including Cxcl1, which was associated with an accumulation of Cd11b+/Gr1+ immune cells. However, TgS100a8a9 hep mice revealed a neutrophil mobilization upon expression of S100a8/a9 in non-transformed epithelial cells (Figure8), and elucidated a complete new aspect of the role played by calprotectin in epithelial cells: S100a8/a9 may support an acute inflammatory response by increasing steady state levels of circulating neutrophils and thereby help to restore tissue homeostasis. However, under settings of sustained tissue damage the constitutive mobilization of neutrophils by a S100a8/a9-Cxcl1-dependent mechanism could enhance the risk for the development and maintenance of chronic inflammation.
Animals were maintained in a specific pathogen-free environment. All animal experiments were performed with age-matched male mice. The procedures for performing animal experiments were in accordance with the principles and guidelines of the Arbeitsgemeinschaft der Tierschutzbeauftragten in Baden-Württemberg and were approved by the German Regional Council in Karlsruhe.
TALap1 mice were described previously. In order to generate the TetOa8a 9 mouse line, a bidirectional Tet-inducible expression plasmid was cloned from the parental pBi-L (Clontech Laboratories) encoding S100a8-myc/his and S100a9-myc/his fusion constructs driven by Tet-responsive promoter. The linearized pBi-S100a8a9 construct was injected into blastocytes of C57B1/6 mice. Genomic integration was determined by Southern Blot analysis.
For the analysis TALap1 and TetOa8a9 single transgenic mice served as Control animals. For doxycycline treatment, 6 to 8 week-old animals received 10 μg/ml doxycycline 5% sucrose containing drinking water ad libitum for a period of 3 weeks. Primary mouse hepatocytes were isolated and cultured as described. Briefly, 8 week-old mice were anesthetized by intraperitoneal injection of 5 mg/100 mg body weight 10% ketamine hydrochloride and 1 mg/100 mg body weight 2% xylazine hydrochloride. After opening the abdominal cavity, the liver was perfused at 37°C with EGTA buffer (0.6% Glucose (w/v), 105 mM NaCl, 2.4 mM KCl, 1.2 mM KH2PO4, 26 mM Hepes, 490 μM L-Glutamine, 512 μM EGTA, 15% (v/v) amino acid solution, pH 8.3) via the portal vein for 5 min and subsequently for 5 min with Collagenase buffer (0.6% Glucose (w/v), 105 mM NaCl, 2.3 mM KCl, 1.2 mM KH2PO4, 25 mM Hepes, 490 μM L-Glutamine, 5.3 mM CaCl2, 12% (v/v) amino acid solution, 365 μg/ml collagenase type I-A, pH 8.3) until disintegration of the liver structure was observed. The liver capsule was removed, and the cell suspension was filtered through a 100 μm mesh. Cells were washed and, subsequently, viability of cells was determined by trypan blue staining. Two million living cells were seeded on collagen type I-coated 6 cm dishes and harvested after 24 hr. For in vitro inhibition of p38 MAPK, freshly isolated primary hepatocytes were treated with SB203580 (10 μM) or its solvent (DMSO) and harvested after 5 hr.
Preparation of whole cell extracts was done with radioimmunoprecipitation assay buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% (v/v) sodium deoxylcholate, 1% (v/v) NP-40, proteinase inhibitor cocktail), and purification of Myc-tagged transgenic S100a8/a9 was performed with mammalian cMyc-tag/Co-IP Kit (Pierce) according to the manufacturer's instructions. Proteins were separated on a 15% SDS-polyacrylamide gel in SDS-sample buffer, transferred to nitrocellulose membrane (Optitran BA-S83) and incubated with specific primary and HRP-labeled secondary antibodies (DakoCytomation). The blots were developed using the Classic E.O.S. developer (Agfa). Primary antibodies are listed in Additional file9 (Table S1).
ELISAs for Cxc11, Cxcl2, G-Csf (all from R&D Systems), and S100A8/A9 (Immundiagnostik AG) were performed according to the manufacturer's instructions.
IHC analysis on paraffin sections was performed according to the manufacturer's instructions (Vector Laboratories), and sections were counterstained with hematoxylin. Primary and secondary antibodies are listed in Additional file9 (Table S1). Stained tissue sections were visualized and photographed using a Leica DMLB microscope and Olympus XC50 camera at a magnitude of 100x. Representative pictures for each genotype are shown.
Total RNA extraction was performed according to the manufacturer`s instructions using peqGOLD RNAPure (PeqLab Biotechnology). RNA was reverse transcribed using RevertAid H Minus-MuLV RT (Fermentas) and oligo-dT primers, followed by standard qRT-PCR performed on a detection system (StepOnePlus™; Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed as described previously. Primers used for qRT-PCR analysis are listed in Additional file10 (Table S2). Target gene cycle of threshold values were normalized to the corresponding cycle of threshold values of Hprt using the change in cycle of threshold method.
For detection of immune subpopulations in peripheral blood, whole blood samples were mixed with EDTA (final 5 mM), subjected to erythrocyte lysis (ACK buffer, Lonza) and subsequently blockade of Fc-receptors (anti-Cd16/32, eBioscience). Staining was performed at 4°C for 30 min and cells were determined by flow cytometry analysis in FACSCalibur (BD Bioscience) using the software FlowJo (Tree Star, Inc.). Antibodies are listed in Additional file9 (Table S1). As positive control for Cd11b upregulation and L-Selectin shedding, peripheral blood cells were treated with 10 μg/ml TPA for 5 min and stained as described.
Digital images were elaborated using Photoshop CS3 (Adobe); the luminosity of brightest and dimmest pixel in each channel were adjusted to obtain the best visual reproduction, taking care to maintain linearity in the brightness scale. Error bars represent SD except were indicated. Statistical evaluation of obtained data was performed with SigmaPlot 11.0 (Systat Software Inc.).
Jochen Hess and Peter Angel contributed equally to this work.
We thank Angelika Krischke for excellent technical support and Julia Leibold for helpful discussion. This work was supported by the Cooperation in Cancer Research of the German Cancer Research Center and Israeli Ministry of Science, Culture and Sport (Ca-130 to PA, and JH, and Ca-147 to PA), the German Research Foundation (SFB Transregio 77, to SMar, AV, PA, and JH), the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Systems Biology (to PA and JH), the Federal Ministry of Science, Education and Art (MWK Excellence Cluster Initiative to PA and JH), the Foundation Tumorforschung Kopf-Hals e.V. (to JH), and the 'Virtual Liver' funding initiative of the German Federal Ministry of Education and Research (BMBF) (to SMan and UK).
LW, JN, TP, CB, AD, SMan, and SMar performed experiments; LW analyzed results, made figures, and wrote the paper; LW, AV, UK, JH, and PA designed the research. All authors read and approved the final version of this manuscript.


text:
D-CONSTRUCTION
D•Construction addresses the economic, social and political issues that arise as a result of the restructuring and gentrification of urban communities. Artists working in all media call into question the ongoing reconstruction of Detroit and other cities, its impact on a city's landscape, its citizens and their ways of seeing.
Artists in this exhibition: Victoria Bayagich, Matt Cain, Rowynn Dumont, Anne Fracassa, Thalya Hunter, Craig Maki, Jaime Pattison, Michelle Renee Perkins, Sarah Price, J. Gordon Rodwan, Nancy J. Rodwan, Mary Rousseaux, Toni-Lee Sagastiano, Gary Schwartz, Tim Tonachella, Svetlana Tsivinskaya
Victoria Bayagich's Construction series (Fig. 1) captures the idea of work and what that means in regard to the creation of comfortable, safe and secure environments. Her paintings focused on the M1 Rail construction isolate images of construction workers and the brightly colored, plastic warning pylons that are a feature of any construction site. The removal of background creates a moment of stillness upon which the viewer's eye can settle, while paradoxically showcasing the focused attention of the workers on the job at hand. It also places the work in an historical context that the viewer might not initially consider (https://www.victoriabayagich.com/home).
Jaime Pattison's works for this exhibit reflect history, explore the concept of decay and examine ideas of community and togetherness. Her mattress installation, Frame on Frame (Fig. 4), encompasses the spirit of our human connections. As Miss Pattison explains, "The coexistence of the mattresses and the coexistence of people are similar, the mattresses share the same general form and function, just as we do as human beings. By stripping the mattresses of their coverings in order to show them in their most basic form, I highlight our similarities as humans. When we reduce ourselves to our most basic form, we can find intimacy with one another."
Michelle Renee Perkins uses themes, materials and aesthetic considerations that evoke a prayer, chant or sacred dwelling. Her series of sculptures entitled Oshun's Den (Fig. 5) evoke memory, call forth history and radiate spirit. She states, "I'm attracted to the sweet waters, memories and dreams and the relationships between personal/ancestral narratives, black aesthetics, and black spirituality. Afrocentric spiritual practices create an opportunity for images and objects inhabit a space akin to primal or celestial intuitive state of being. A state of being home, space that manifests as the internal and external home we all desire."
J. Gordon Rodwan's photographic images, such as Danger (Fig. 6) juxtapose tradition with thought-provoking subjects. This image in particular encapsulates the struggle that often occurs when an urban setting is in the throes of change; while there are many positive aspects, there is often an underlying destructive effect that remains unexposed. Rodwan speaks of his work this way, "I seek arresting images with unique angles, details, shapes and shadows. I strive both to compose compelling pictures in unusual places and to expose unexpected aspects of traditional subjects."
(Fig. 1 Victoria Bayagich, Temple and Cass, 2016; Fig. 2 Rowynn Dumont, The Deterratorialization of Semiotic Space, 2004; Fig. 3. Anne Fracassa, Detroit Reclaimed IV, 2017; Fig. 4 Jaime Pattison, Frame on Frame, 2017; Fig. 5 Michelle Renee Perkins, Oshun's Den, 2018; Fig. 6 J. Gordon Rodwan, Danger)

redpajama_set_name: RedPajamaCommonCrawl


Harold Glazer, founder of H. Glazer Builder, understands the value of that view, maybe better than anyone else around. In the past six years, he has remodeled more than 20 homes in the North Ridge community, and two of this year's homes earned STAR awards from the Remodelors Council of the Home Builders Association of Raleigh, Wake County as top Triangle remodeling projects.
"This really is a unique area of the Triangle," says Glazer. "People built these homes in the mid-70s, and they're ready to update, upgrade and expand, but they realize they can't recreate what they have here anywhere else." Glazer points out that the value of this property in the Triangle real estate market is such that a homeowner can invest a few hundred thousand dollars in a project and end up with a million dollar property — and enjoy living in a showcase home while their investment just continues to grow.
That was the scenario for both of these STAR award-winning homes. Original owners wanted to open up boxy 70s floorplans, update kitchens with the latest bells and whistles, add space and just generally enjoy, through their homes, the rewards of a lifetime of hard work.
The Gold award winner for both best kitchen and best bath was a two-story Colonial on one-third of an acre overlooking the 13th green of the North Ridge Golf Course. The retired homeowners had looked around at alternatives and decided there wasn't anywhere else they wanted to live. They simply needed to make changes that would allow them to stay in their home as long as they could.
The kitchen was a dated 70s style (back when kitchens were still in denial about being the true heart of the home), and the homeowners wanted a space where they could be proud to entertain their friends and family. And the master suite, also true to its decade, lacked the luxe amenities that are de rigeur in today's upscale homes. In addition, the homeowners wanted a downstairs master that would be easily accessible to them in years to come.
Glazer gutted the existing kitchen of this home and replaced it with a state-of-the-art showplace, and he added a 1,200-foot wing to include a luxurious master suite and handsome paneled library/home office. He also added a new deck to take best advantage of the spectacular views this property afforded.
In the kitchen, Glazer replaced tired old oak cabinets with hand-glazed custom cherry cabinetry. These raised panel cabinets feature a host of up-to-the-minute features, like pullout spice racks and today's furniture-style detailing — fluting, carved corbels and deep crown molding. A decorative paneled hood surround was custom tailored of the cherry cabinetry. New counters are a creamy flecked granite, and the backsplash is imported tile, with a decorative hearth-like mosaic as a focal point behind the cooktop. The kitchen was reconfigured slightly for a better workflow. Old appliances were replaced with the latest sleek stainless steel, including a wine cooler integrated into the cabinetry. A raised counter, with barstool seating was added to the island for casual gathering and eating. And dated light fixtures were replaced with antique bronze pendants and recessed lighting.
The real challenge with this renovation, says Glazer, was the seamless integration of the existing home with the new wing. "The worst thing you can possibly do," he says, "is to just tack on an addition that looks just that — tacked on." Glazer was able to manage a transition that is virtually invisible from old space to new.
Oak floors were installed in the addition and finished to a perfect match with the medium shade of the old oak floors. A hallway serves as transition between old and new. Trim in this corridor was stained to match the woodwork of the rest of the home, so that standing at either end gives no clue that this space has not always been a part of this home.
First stop in the new addition is a cherry paneled home office. The homeowner wanted a true library-like office, convenient to the master bedroom and not just a converted spare bedroom. The same hand-rubbed cherry used in the kitchen was used to outfit this office with a built-in desk, flanking floor-to ceiling cabinets and a wall of bookshelves.
At the end of this corridor, is the pièce de résistance of this addition, the opulent master suite. Bathed in soft creamy colors and warm woods, this space exudes luxury and pampering. Glazer credits interior designer, Carmen Hunnicutt with pulling together the plush and luxurious ambience.
Custom cherry cabinetry was designed to create a lighted alcove for the bed, extending the length of the wall and incorporating nightstands for a very clean tailored look. The same cabinetry was used for a stepped back custom entertainment center for the opposite wall. An elegant arcing bay, with floor-to-ceiling windows creates a cozy seating area and takes full advantage of the home's spectacular views.
This master suite is not just a pretty face either. The entire addition was wired for high-tech control of lighting, sound system and security. And the enormous his-and-her closets are completely outfitted with built-ins.
The luxury extends into the spa-like master bath. The custom glazed cherry cabinetry continues through this space, with staggered-height counters for vanity and sink, topped with solid granite. Creamy 18-inch tile is set on the diagonal and more tile, with decorative accents, graces the walls, the garden tub surround and the oversized, glass-walled shower. A doorway from the master bath leads to a private deck — a hot tub retreat.
Glazer added another 500 square feet of deck at the back of the home with room for entertaining and dining al fresco.
Glazer's second award-winning North Ridge remodeling project was likewise for the home's original owners. This couple was especially dissatisfied with the boxy closed in rooms that made up their 3,300-square-foot contemporary ranch but loved their incredible long-range views of the golf course and exceptionally private lot. They wanted air and space and flow, and they wanted a space open to the kitchen where they could gather with children and grandchildren. Like Glazer's other award winning addition, this one included adding a home office as well as modernizing the kitchen.
On this project, Glazer completely gutted the existing home. Walls were torn out and moved and ceilings were raised. Floors were replaced and the old deck torn off. As often happens in renovations of this scale, surprises were in store for the remodelor. "You never know what you might find when you tear inside a wall," says Glazer. "Sometimes you discover it was not built to code and you have to make major repairs." Glazer credits an atmosphere of trust between remodelor and homeowner when it comes to successfully dealing with the inevitable uh-ohs of large-scale renovations.
And he is emphatic that the path to that trust is a history of communication, communication, communication. "It's a daily, ongoing process of keeping the homeowner absolutely informed about every aspect of the job," says Glazer. "This kind of careful hands-on management is absolutely critical to a successful remodeling job — really even more so than in new-home construction." On this project, all of the wiring in the entire house had to be replaced, a job not called for in the original plans.
But both remodelor and homeowner persevered and the end result is a spectacular new home, almost unrecognizable from its previous incarnation. Glazer reconfigured the room-after-room all-in-a-row house to one of open airy spaces that flow easily one into another. Beginning with the very first impression, the old cubical of a foyer was blasted out and turned on an angle. From here, the home radiates out, with this juxtaposition of angles creating a strong sense of movement and flow. Oak flooring, in a contemporary natural finish sweeps through the foyer into the rooms beyond.
Immediately ahead, the expansive living room (with its new raised ceilings) is separated by a massive built-in angled unit framing open glass shelving and displaying the homeowners' collection of glass sculpture. This truly dramatic architectural element sets the contemporary tone for the entire home, serving to define the living room and foyer spaces, without closing them in at all. Super-plush cream-colored carpeting soothes the living room with its eclectic mix of traditional and modern furnishings, artwork and family portraits. Again, Glazer credits the interior designer, Judy Pickett with pulling together the overall look that the new spaces made possible. Black granite and smooth ultra-wide white trim surround the fireplace for a clean, contemporary look. Multiple French doors open to the deck and the breathtaking views of the golf course.
Adjoining the wide-open spaces of the living room and foyer, the dining room, while still open to these rooms, is a more intimate space. Dark marbleized walls and lighted buffet alcove, appointed with sleek contemporary black and white cabinetry and mirrors, give this room a distinctive Art Deco flavor.
Fanning out to the left of the foyer, a bedroom wing terminates in the completely updated master suite. This spacious room takes full advantage of the natural setting with a bank of windows along one wall. A sitting area and large entertainment center make this an inviting space for quiet secluded lounging as well as sleeping.
Glazer created a new master bathroom for these homeowners that is all Hollywood glamour. Black and gold and curvaceous lines fill this space. Plush carpet, highlighted with a single dramatic swirl of black and cream sweeps into the room. Glossy cabinets float on the walls, with stylized rounded bottoms. And the huge walk-in closet is fit for a Hollywood starlet too.
Radiating out from the foyer in the opposite direction, are the newly updated kitchen and breakfast area and the added great room and play room. Here again, Glazer used juxtaposed angles to create movement and flow. The completely open kitchen features a huge pie-wedge island, whose curving face fans out to visually embrace the entire space.
The new kitchen was appointed with contemporary maple cabinets and black granite counters for a very clean modern look. An adjoining alcove is outfitted with matching cabinetry for desk space and an entertainment center.
Separating this very large and airy kitchen and casual dining space from the newly added great room is a huge aquarium, flanked on either side by wide openings. Much like the custom display case in the foyer, the aquarium ingeniously defines the spaces without sacrificing the open flow.
Architectural detailing like a deep tray ceiling with recessed lighting and a custom built-in entertainment center give this new room — a gold STAR award winner — both substance and interest. Tucked off the side of the new great room, is a sky blue playroom for the homeowner's beloved grandchildren. This space is set off just enough to obscure the typical plaything clutter, while still allowing little ones to be close to the center of everything.


With Washington's help, one tiny plane has become a popular alternative to high-tech combat aircraft—one that poor air forces can afford to fly.
Over the last decade, the Pentagon has gifted dozens of the single-engine Cessna Caravans—outfitted as transports, aerial spies and attack aircraft—to countries in Africa, the Middle East and Central Asia.
The propeller-driven aircraft guard borders, snoop on militants and attack terrorists in war zones from Iraq to Mauritania. They're easy to operate and cheap to keep in the air.
The Caravan—also known by the designation C-208—is much slower than a modern combat jet and lacks the endurance of a drone like the Predator. But it makes up for its lack of performance with sheer simplicity and reliability.
Warplanes that are "highly capable in terms of payload, flexibility of use and overall utility are in high demand," says Richard Jackson, who spent more than 20 years flying for the U.S. Air Force and the Coast Guard.
"I think it's fairly clear that these sorts of light aircraft provide a low-cost alternative that works for international partners," adds Dr. Brian Laslie, an Air Force historian.
Which is not to say the U.S. military doesn't also possess Caravans. The Air Force's super-secret 427th Special Operations Squadron operates a handful of the planes, probably for hauling cargo and personnel.
The slow-flying C-208 is an "inherently stable" platform for peering through video cameras and and launching missiles, according to Jackson, who still works as a professional pilot. The high-wing aircraft is perfectly suited "for mounting of sensors, weapons and additional fuel," Jackson explains.
Cessna and L-3 Communications work together to turn C-208s into recon and surveillance planes. In addition, ATK offers the "Combat Caravan," which adds Hellfire missiles.
To turn the Caravans into flying spies, contractors install a turret containing a night-vision camera in the plane's belly, plus monitors and other equipment in main cabin.
The AC-208 Combat Caravan combines a similar kit with two underwing missile launchers.
The result is a cross between a low-budget fighter and a Predator drone that happens to have a pilot aboard.
As a bonus, the Caravan is similar to the kinds of planes many air forces use to train pilots. In fact, Afghanistan and Iraq have both also flown their Caravans as basic trainers, in addition to other duties.
As a result, new flyers can quickly progress from practice flights to combat missions, Jackson notes. This saves precious time and money.
Baghdad, for one, is acutely aware of how long it can take to train pilots to fly more high-tech jets. For three years, the Iraqi air force has been getting ready to take delivery of 18 U.S.-made F-16s.
A basic C-208 aircraft costs around $2 million. High-performance fighter jets can cost 25 to 50 times as much. And even a Predator drone is at least twice as expensive.
Of course, the Cessna's price does go up when you add military gear like video cameras, extra radios and weapons. In 2009, Lebanon received a single Combat Caravan as part of a deal worth approximately $27 million, according to a Pentagon report.
But the American funds also paid for spare parts, training and other items. Four years later, the Pentagon's regional command for Africa sent two basic C-208 transports to Niger for some $11 million, which also included schooling on how to operate and fix the aircraft.
The cost and effort to convert a regular cargo-carrying Caravan into a spy plane or strike aircraft is also relatively low. The Pentagon is currently looking to help Lebanon convert another one of its C-208s into a Combat Caravan.
Just don't expect the Caravan to survive a major war. While ATK's AC-208s can deploy decoy flares to confuse incoming anti-aircraft missiles, recent video footage from the AlYaseri Media Channel shows an Iraqi Combat Caravan flying at night.
The aircraft's crew likely took advantage of the darkness to help them hide from Islamic State militants, who have managed to shoot down more than one low-flying Iraqi helicopter.
"I do believe this aircraft can only be flown in very permissive environments," Laslie notes.
Still, supplying the tiny Caravans to American allies "allows the U.S. to provide a security presence around the globe at a reduced cost to the American taxpayer," Laslie says. And this is probably why the flying branch is interested in securing a steady stream of them for the future.
In September, the Air Force Life Cycle Management Command put out a feeler for companies to regularly supply armed Caravans. "Historically, this acquisition has been awarded as a sole-source contract," the announcement explains.
Under a sole-source contract, the customer deals directly with just one vendor and avoids the time and effort it takes to run a competition every time it needs to re-up.
In August, the Air Force used this method to buy C-208s with camera turrets for Niger and Kenya, as well as spare equipment for another Caravan already in Mauritania.
And if current trends hold, lots more countries could eventually get the cheap and simple spy and strike planes, too.


text:
The synchronised swimming competitions at the 2017 Southeast Asian Games in Kuala Lumpur took place at National Aquatic Centre, Bukit Jalil, Kuala Lumpur.

The 2017 Games featured competitions in 5 events.

Solo Technical Routine

Solo Free Routine

Duet Technical Routine

Duet Free Routine

Team Free Routine

References

External links

R

redpajama_set_name: RedPajamaWikipedia


Help your family discover the Light of Christmas in a new way!
Every year, Christmas seems to arrive in the stores earlier than you expect. Beautiful Christmas displays appear mid-October, Christmas gift ideas fill store catalogues, and glittering ornaments are hung in the specially created Christmas-theme areas.
And that's fine... although I never seem ready for Christmas despite the early reminders and displays. But the Christmas proclaimed in the department stores and shopping centres is not the Christmas that's important to me. And it's not the legacy of Christmas that, by and large, I want for my family. It doesn't reflect the humble stable of the Saviour's birth or the gift of that first Christmas.
The store-bought Christmas is filled with music and joy and fun and feasting and unwrapping endless gifts. I want to help my family unwrap the real joy of Christmas. And I believe that families, like mine, can hear the Christmas story in a new way using creative family-focused devotions.
Focus on th Family has produced a beautiful Advent colouring and devotional book. It will help you focus on the anticipation, preparation and celebration of Jesus' birth.
The ongoing enthusiasm for colouring by all ages means the your whole family can be involved.
All the Colours of Christmas Advent Calendar.
This is just so much more than just a Christmas colouring activity for your kids.
Well... you'll find faith-filled activities and readings.. enough for 28 days of family devotions.
There are colouring pages and stickers, an Advent Calendar Christmas Tree to colour together and enjoy.
Yes... this is another colouring devotional for Christmas. This publication is by Nancy Guthrie. It includes bonus activities, discussion starters, and kid-friendly colouring pages- Christmas ornaments and decorations.
All is Bright colouring book by Nancy Guthrie provides rich Christmas content and kid-firnedly colouring pages for the whole family.
This Christmas resource takes you and your family on a journey that'll reconnect you with the miracle of Jesus birth.
There are gifts to make and give to friends, family and neighbours. Other faith-building activity suggestions are included: field trips, a Christmas movie makeover and other fun!
Simple kitchen-based mission ideas (cookies, nut baskets, Christmas morning mix) will get your kids up and serving others and discovering Jesus.
Do more that just read about the Christmas story; engage your whole family in faith-building activities. It starts with creating your own Nativity scene and builds into so much more.
Family ideas to focus on creating, caring and sharing with others.
Host a kid-friendly Christmas family night with the Christmas Movie Night Kit.
You'll find a read aloud picture book, activities, puzzles and games. The animated Story of Christmas dvd includes sing-along videos of Christmas carols and behind-the-scenes production footage.
Celebrate the real meaning of Christmas with this creative Christmas kit.
Explore the real meaning of Christmas with your family as you enjoy the animated Story of Christmas dvd. You'll discover more about the events surrounding the first Christmas and how this season can bring us hope, love, joy and peace.... Grab the popcorn and get started!
The birth of the Saviour on that first Christmas was news that was shared. This is a big part of the Advent Journey Colouring Posters, too, sharing the Christmas story with friends, family and the community through colouring.
The posters are big.. supersize. Four of the posters measure 3' x 2' and the other two are even bigger- 6' x 2'. So they are wonderful to colour together in groups. It also offers plenty of opportunities to talk and share together while colouring.
Using these posters requires some out-of-the-box thinking when it comes to using them. Sure, you can use them for children's ministry groups or teen groups. You can take them to nursing homes to colour in together. Use them following your Sunday services for your church family to colour and enjoy while having morning tea. Schools may wish to use them. Your family might invite friends over for a colouring evening. There is also an accompanying Advent Journey Devotional Guide. So start thinking now... how will you use these wonderful resources for sharing the Christmas story?
Oh.. and the finished posters make beautiful display posters, perfect decorations for large walls, hallways and even stage areas.
Use these super-sized posters to colour with others share the story of Christmas.
Use them with family, groups community.
Take time to explore the real meaning of Christmas with your family with these creative resources. Explore the events surrounding the first Christmas and learn how this season can bring us hope, love, joy and peace.
Putting The "Fun" Into "Funky Ways To Exercise"!
Exercise and fun. Two words that, for most people, will never go hand in hand.
The fact of the matter is that, as adults, the majority of us do not get our 150 minutes of cardio and two days of strength training every week. This sounds like an impossible task. We are just too busy now, the household chores take priority, the kids need their meals, and by the time everything is done, we are absolutely exhausted and need to get our rest in before it all begins again tomorrow!
And to top it off, we don't eat as well as we did 30 or 40 years ago. The pull of ready meals and takeaways beats the effort of making a meal from scratch. We just cannot cope with the amount of pressure being put upon us every day.
Doing any type of exercise, whether you are finally getting to the gym, or dancing at home, is fully enhanced by the addition of music. For the parents that are unable to get out of the house, put a song on that really gets the kids up and moving, and join in with them! Scientifically, they say that the type of music that you enjoy will really affect your workout for the better. So, if you like heavy metal, the speed of that type of music will get your heart pumping faster than Mozart's Requiem! Disco music, with its steady beats and upbeat rhythms, is far likelier to induce positive exercise than feeling like you can't go on. Dancing is one of the best forms of cardio, and you only have to look at the ballroom dancers on TV to see how fit they are!
There are many benefits to going walking with the family. Not only does walking along different gradients of land help to burn calories more efficiently, but the experience of being outdoors is a more positive one than running idly on the treadmill for 40 minutes and has been proven to release tension and stress. And by doing it before your evening meal, you are working up an appetite as well as improving your digestion.
If being at one with nature isn't your speed, there are many activities that you can participate in as a family. By scheduling family time, you are not only getting in the desired exercise, but you are bonding over the experience. That doesn't mean to say that you need to play an overly competitive sport.
There are many other fun activities that you can take part in, such as swimming or, for the uninitiated, Bubble Football -Holleyweb provide zorb-like football suits you can wear and get playing. If the exercise is fun, it won't even feel like exercise!
There are so many things you can do to inject fun into your exercise routines, and if you enjoy it, you will always want to come back to it.
It's always challenging for grandparents when their grandkids reach teenhood. After all, they can morph into mini adults who you might not see as often as before. And as for present buying, it can turn into a minefield. After all, it's challenging to know what's on-trend nowadays. But there are a few things you can go for which will definitely work for your teens. Therefore, here are some ideas of gifts that your teen grandchildren will love!
It's hard to go wrong when it comes to food with teenagers. After all, they tend to eat their parents out of house and home when they are in their teens! Although they do have an excuse; they are growing lads and girls. Therefore, if you want to ensure you make the right choice with a gift, you should consider going down the food angle. You could make them a hamper full of all their favorite sweets and chocolates. If they have a sweet tooth, they will absolutely love this present. Or you could even bake them some cookies or muffins. Nothing beats your grandparent's cooking so they will adore this present! And if your teen is a health nut, you could make healthy snacks for them instead. They will love the fact you are paying an interest in their life and will enjoy the gift.
Teens today are more interested in their appearance than ever before. Therefore, if you want to get the right gift for your teen grandchild, you should go down the grooming route. For girls, any form of body lotions or makeup would definitely be a winner. If you are unsure about which brands they use, go for a voucher they can use to buy some new makeup. And as for boys, you should opt for some shaving sets which are a must have once they have hit puberty! You can find out about the best ones on ManlyMatters, and other similar sites. Otherwise, some cologne would go down well with your teen grandchild. As we said before, they will love that it will help them to stand out!
You will find that your teen is always talking about some singer or band that they love. After all, music is a big part of their lives. Therefore, you should go for something music related for a gift that they will love. For one thing, you could always look to see if their favorite performer is on tour soon. You could put some money towards the ticket so that they can go see them live in concert. Or you might even choose to go for some music vouchers so that they can buy some tunes from Apple store. After all, they won't use a CD if you buy it from the store. Therefore, it's best to give them a voucher which they can use to download some new tunes!
And although it's a tricky one, clothes could be a good present idea. Just make sure you check their size and the stores they like to go in before making a purchase. It might be wise to go for a voucher instead so they can choose what they like to buy from their store!
How to start your morning well when you're not a morning person.
The bright sunshine in Australia is beautiful but intense for most of the day. It makes for a wonderful holiday destination, brilliant beach snapshots, and plenty of solar energy. But it's also really important to wear a hat, sunglasses and 30+ SPF sunscreen, frequently applied.
Although my grandmother was accustomed to cooler, softer English days, without exception, she would venture out for her daily walk despite the heat. Did it harm her? No.. she lived well into her 90s. In fact, current research would indicate that her daily walking habit probably contributed to her longevity.
We were designed to walk for miles and miles to hunt and gather our food. Yet, in modern society, we spend most of our time sitting down. That is not good for our bodies leading to a host of health problems. This is probably one of the most important reasons to make an effort to move around and go for a walk each day. Of course, that's easier said than done.
How can walking help me?
Research indicates that walking offers great health benefits, from helping you to lose weight and to de-stress to lowering your blood pressure and BMIs. And although it's seen as a convenient and easy 'keep fit' option, it can be difficult to develop a daily walking habit.
Most of us sit for our work, we eat sitting down, and to be honest all we want to do when we come home from work is plop down on the couch for a Netflix marathon until bedtime.
In other words, getting and staying in the habit of going for a walk each day can be a bit of a challenge. But that's exactly what we need to do. We need to get in the habit of going for that walk just like we're in the habit of brushing our teeth twice a day or taking out the trash on Tuesdays. Once it's a well ingrained habit, it won't be as much of a challenge to make sure we go for a walk each day.
Okay, how can I begin?
A great place to start is to find a walking route you enjoy. It helps to make it as easy as possible. Your favourite walk may offer beautiful vistas, but if it's a 30 minute drive there and back, you'll be less likely to do it every single day. Instead, save that walk for the weekends and come up with something convenient and pleasant for your everyday walking routine. If you can, find a route in your own neighbourhood so you can leave right from your front door. Just lace up your shoes and start walking.
Taking the same route every day helps form that habit. It's also encouraging to notice that you can walk the same loop faster or with less effort over time. It proves that you're making a difference and are getting stronger and increasingly more fit.
Listening to your favourite music, podcast or audio books is also helpful. It will make the time go by faster and give you something else to look forward to. You can even use your favourite media as a way to bribe yourself to go for your daily walk. Let's say you have a couple of podcasts you enjoy. Save them for your walks and only let yourself listen to them while you're walking. It's a great incentive to get out there even on days when you're not feeling it.
Last, but not least, consider walking with other people. Find a walking group in your area, or talk a friend or neighbour into becoming your walking buddy. Not only is it more fun to walk when you have someone to talk to, it also has some built in accountability. It's much harder to skip a walking workout when you know that other people are waiting for you and relying on you to join them. A growing number of walking groups are meeting at the local shopping centre. It's air conditioned and flat, making walking comfortable, fast and easy.
There is a growing number of apps that can encourage you and help develop your walking habit. Use these apps to map and measure your walks, set walking goals, earn rewards, and share your achievements with friends. Check out the readers choice awards for walking apps before downloading.
Why not take your smartphone with you and share your walk on social media? Develop your photography and Instagram skills with a quick online course and enjoy sharing amazing images from your walk.
Give these tips a try and see if they help you make walking a daily habit and an integral part of your health, your fitness, and your life. Download and print the free infographic below. Mount it on your fridge or noticeboard. Refer to it regularly for inspiration and encouragement.
*Choose a simple route for your first walk.
*Check with your friends and family and find a walking buddy.
*Choose your favourite music or podcast to listen to while walking.
*Do you wish you could find time to refuel your tank, improve your health, invest more into your family, or even just to slow down and breathe? Take the 14-Day Make Over My Mornings online course now!
*Find more ideas and inspiration in the articles below.
Want more out of life? Try journaling.


This is Little Elm's best kept secret. Union Park is a 787-acre master planned community with some of the most gorgeous landscapes found in Texas. Staying healthy and active is easy in this amenity-filled neighborhood. Discover nature along the walking trails or fish along the banks of the natural ponds. Between the community pool and the 35-acre park with greenbelts, you and your family will always find activities. And refuel at the food truck park.


NEW DELHI: Believe it or not, a leading hospital in the city has hired 36 doctors to operate ventilators, a task usually performed by OT technicians. According to sources, the hospital has failed to recruit the required number of technicians due to certain procedural hurdles.
Lok Nayak hospital, the biggest state-run healthcare centre in Delhi, was given 35 ventilators by the government in March following a series of TOI reports highlighting lack of the life support system in the institute.
But, most of these machines had been lying unused for past of three months since the hospital did not have the required manpower to run them. "OT technicians can be recruited through the Delhi Subordinate Services Selection Board (DSSB) only. We cannot hire them. So, we have appointed 36 junior residents (JRs) to do the job on a temporary basis," Dr J C Passey, medical director of Lok Nayak hospital, told TOI.
He added that the newly recruited were being trained under the anaesthesia division and all ventilators would be functional within five to six days.
Junior residents are fresh medical graduates. Senior doctors at the hospital, who did not want to be identified, said these medicos cannot fill up for OT technicians or anaesthetists. "The government should have ensured enough manpower before buying these machines," said a doctor. He added that Lok Nayak hospital was facing a shortage of anaesthetists and OT technicians, the staff who operate ventilators.
TOI had on December 10 last year reported how attendants of critically-ill patients at Lok Nayak hospital were being given ambu-bags to resuscitate them as there weren't enough ventilators.
One of the patients, Nizamuddin from Dadri in UP who had been kept on ambu-bag—or manual resuscitation device—post-surgery, died a day later. The incident appeared to have jolted the government into action. CM Arvind Kejriwal said new machines would be procured in 15-20 days.
Three months later, 125 ventilators find their way to Delhi government hospitals. Of them, 35 were given to Lok Nayak hospital. Sources said except Lok Nayak, the machines at other hospitals were fully functional.


A couple of years ago, the Sioux Falls Area Chamber of Commerce hosted a group of touring visitors from an out-of-state chamber.
We joked that it was a "chamber of commerce day," with sun shining and moderate temperatures that showcased the city at its best. The visitors raved about the experience.
Kristin Luna wasn't quite so lucky with the weather when she made her first trip to the city for five days in April.
It poured what felt like the entire time, Luna said. She ended up buying a winter coat — on sale, at least.
I've set the stage for you this way because it makes the experience Luna had all the more extraordinary.
Her trip was part of a strategy for marketing tourism using digital influencers. Luna, who has experience in journalism, is based in Tennessee and started a blog about her travels nine years ago.
She was hired through a marketing agency to put together a project about her first experience in Sioux Falls.
Many people didn't know she was coming as a paid blogger, she added, including several she met once she and her husband, Scott, arrived.
At Josiah's Coffeehouse & Cafe, an employee came up to her with three Post-it notes of recommended stops after learning about the project, she said.
At Jacky's Restaurant, a pair of youth pastors standing ahead of the Lunas in line randomly invited them to dine together.
Months after the visit, Luna easily recalled more of her favorite stops by name: Sanaa Abourezk and her restaurant, Stacy Newcomb-Weiland and her commitment "to historic preservation and the food scene." She raved about her afternoon at Strawbale Winery, her visit to the National Music Museum in Vermillion and a beer tasting with Fernson Brewing Co.
When she returned to Nashville, she wrote several pieces about the visit, including an entry for a "road trip journal" housed on the website for the South Dakota Tourism Department.
One her blog posts is titled "Eleven Reasons We Fell in Love With Sioux Falls."
"I was pleasantly charmed — make that dazzled — by South Dakota's largest city," she wrote, going on to extol the craft beer and coffee scenes, the parks, the Washington Pavilion and the entrepreneurial community.
If you are at all connected through social media, I suspect you've seen the piece. It was shared so much that it drove more traffic to her blog than anything else in its nine-year existence.
Let's give credit to the city and state tourism leaders who embraced the strategy of using digital influencers to help tell our story. Luna called it "very innovative" and "at the forefront" compared with other tourism departments.
South Dakota Tourism Secretary Jim Hagen called it a "mutually beneficial" relationship that gains online followers for Luna and the state.
Her road trip journals are part of a larger collection on the state's website. It's one of those pages that can become addicting as you read through all of the out-of-towners' experiences. I suspect that's probably the point.
When you think about how increasingly difficult it is to engage people digitally, it makes sense. Personal experiences paired with great-looking photos make for a more compelling marketing message than the typical pop-up or banner ad.
And just because these travel experiences were coordinated for marketing purposes, don't forget about the travelers who have come on their own and are sharing their experience digitally. That's why it's important to keep the city well-maintained and treat visitors with the welcoming attitude I know we embrace here.
As for Luna, not only did the cold and rain "not detract at all from the experience," but she also plans to return when it's nicer for more outdoor activities. Even more compelling was what she told me unexpectedly at the end of our conversation.
It's hard to ask for a better endorsement than that.
P.S. Feel free to share this column and amplify her message!


Bilocale Bellini is located in Oliveri. The air-conditioned accommodation is 36 km from Taormina. The apartment has a satellite TV and 1 bedroom. The kitchen comes with an oven and a fridge. If you would like to discover the area cycling is possible in the surroundings. Messina is 44 km from the apartment. The nearest airport is Reggio di Calabria Tito Minniti Airport 52 km from Bilocale Bellini.


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Music City taking NFL draft out in the open on Broadway
Posted 5:45 am, January 31, 2019, by AP
DANIEL SLIM/AFP/Getty Images
NASHVILLE, Tenn. — Music City will put its stamp on the NFL draft.
The main stage will be out in the open on Broadway among Nashville's honky-tonks. The league's fan festival is scheduled for across the river at the Tennessee Titans' stadium.
City officials on Wednesday announced details of the April 25-27 draft, which will kick off the NFL's 100th season.
The red carpet on the first night of the draft will be held at The Green at Riverfront Park. Draft selections will be introduced on the stage at the same spot where the Titans unveiled their new uniforms last April. NFL team tables will be located at Schermerhorn Symphony Center, and concerts will be included — this is Music City, after all — with artists announced later. Admission is free.
A pedestrian bridge links the main draft stage to the NFL Draft Experience at Nissan Stadium across the Cumberland River.
Peter O'Reilly, the NFL's senior vice president of events, says Nashville will showcase its music scene and downtown area with the largest footprint ever used by the NFL. The draft has been on the move since 2015, going to Chicago for two years, then to Philadelphia and last year to the Dallas area.
Topics: Nashville, nfl draft, Sports, Tennessee
A-plus NFL draft grade for Music City with record attendance
Country star, Titans coach talk about NFL draft in Nashville
Nashville gets its chance to step up for NFL draft
Former NFL defensive lineman says he needs kidney transplant
Linebacker Morgan retires after 9 NFL seasons with Titans
Nashville bachelorettes upset about NFL Draft invading their space
Peyton Manning adds another honor in TN Sports Hall of Fame
Nashville organizer apologizes for cherry tree uproar
Tennessee spikes bill allowing LGBTQ adoption refusals
Titans agree to terms with top draft pick Jeffery Simmons
Peyton Manning headlines Tennessee Sports Hall of Fame class
Grant Williams won't return to Vols for senior season
Titans take Ole Miss receiver A.J. Brown in the second round of the NFL Draft


25 YEARS PUTTING THE PATIENT FIRST!
Working on a quality management system is a commitment we continually overtake in order to assure progress and customer satisfaction.
Medicals International is proud to be a distributor of the world leader ArjoHuntleigh.


If you have suffered a work injury and your employer has sent you to a doctor of their choice, it is important for you to act quickly to reach out to a doctor who would be on your side. Your doctor and your diagnosis act as the foundation for your workers' compensation claim.
The right doctor can be the difference between fulfilment and denial of the payment of your medical bills and wage loss.
This is why you can't leave such an important decision to your employer. Employers and their lawyers would obviously try to avoid paying workers' compensation claims to prevent an increase in their insurance premiums, drawing attention to unsafe work environments, and workplace safety penalties.
In many states, employees have can choose their work injury doctor. Employers that tell their employees that they have to go to their doctor or else they don't get benefits. This is simply incorrect.
There is nothing more important than your physical health and you can't leave that to your employer. Your financial health is equally important. Most people can't afford to be stuck with huge medical bills and lost wages.
Without the right doctor, both physical and financial health could be at stake. However, before you choose a new doctor, ensure that changing your primary physician is in accordance with the workers' compensation rules in your state.
In many states, you have to see the doctor your employer chooses once. If you continue with that doctor, s/he becomes your treating physician. But you can choose your own work injury doctor after the first evaluation by your employer's doctor. If your employer or their insurer is pushing you to a doctor of their choice, you should seek the advice of a work injury attorney right away.
To consult the best workers compensation and personal injury doctors in Phoenix, Arizona, call Arizona Injury Medical Associates at 480-347-0941.


Our gorgeous carseat covers are as versatile as they are adorable! Use it is both a carseat cover, nursing cover, shopping basket cover and more! Comes with a plush handle cover as well!
Materials: 95% polyester & 5% spandex. We recommend washing on delicate cycle with mild detergent. Dry on low. Do not bleach. Iron as needed.


As fast-paced as the apocalypse can be, you need to hinder the fiery coals of a radically changing world and slow down for an emerging relationship.
(Season 8) Kids Incorporated is invited to a church's youth outreach, where they see a band using a familiar formula to attract the youth, and deal with a group of adults who think the church and rock-n-roll shouldn't mix.
Faith dawned on Tim slowly.
Jimmy is dying. He knows he's dying. He just... doesn't quite know how to deal with it.


There's actually more - you can check out the entire Vera Bradley Frill collection by visiting their website. They also have these new styles in select stores. Hmmm, can you guess where I am going to be tonight?
I think anyone who has ever referred to Vera Bradley bags as "granny bags" is going to eat their words when they see the new Frill collection. It's obvious they are targeting a younger crowd with these pieces. However, I think they will appeal to just about anyone who loves fashion (even 30-somethings like me).
What do you think about the new Vera Bradley Frill collection?
If anyone can direct me to where I can purchase the SOI tote in Pirouette I would love it!!!
The items you see listed here are current listings from eBay. If you see an item you like, you may want to contact the sellers directly with your question.


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Photo Credit | Virgin Records
Bastille, the UK's premier alternative rock band, is following up their platinum-winning debut, 'Bad Blood,' with 'All This Bad Blood,' a double-length special edition album featuring band favorites that didn't fit on the original album, as well as a few tracks offering a taste of Bastille's future.
Bastille was formed in 2010 by singer/songwriter Dan Smith, incorporating three of his peers and collaborators, drummer Chris Wood, percussionist Kyle Simmons, and guitarist Will Farquarson. Since their debut, the group has steadily climbed the charts, with their first full album, 'Bad Blood,' releasing as the #1 album in the UK last March.
One of the most unique features of Bastille's work is the sheer breadth of techniques they incorporate into their music. Alongside the melancholy vocals of Dan Smith, electronic music flourishes, violin accompaniments, a booming male choir, and the use of xylophones and other interesting percussion elements add an impressive level of texture to each song, with each track featuring new and exciting musical touches.
This breadth of techniques also led to one of my only complaints about the album, a pronounced feeling of dissonance that really kept the album from feeling like a cohesive whole. Some tracks evoke a feeling almost similar to Coldplay's 'Viva la Vida,' with a powerful male chorus and violin accompaniment, while others lean more towards electronica, sounding like a guitar-focused 'Radioactive' by Imagine Dragons. Each song, individually, features excellent ideas and a high degree of polish, but the number of styles represented makes it impossible to give any one group of people a definitive 'buy' recommendation.
I personally recommend starting with track 1, 'Pompeii,' because its superb male choir accompaniment, excellent use of percussion, and driving beat will offer a decent overview of Bastille's style. For fans of electronic rock, track 4, 'Overjoyed,' offers an excellent showcase of the band's electronic chops, as well as a beautiful piano accompaniment. If you want something closer to traditional alternative, try Track 2 off of Disc 2, 'The Silence,' which features an awesome bass accompaniment over textbook alt-rock drum riffs.
Overall, 'All This Bad Blood' was a pleasant experience, which is sure to offer at least one or two new songs to any fan of indie or alternative rock. I would, however, recommend making your song purchases à la carte, as there is a good chance that some of the musical styles represented may fall flat with individuals looking for a specific experience.
I give Bastille's 'All This Bad Blood' an eight out of 10


Thank you to each person who has been a part of Tekton Hospitality's team this year.
Putting our gratitude into action, it was our joy to order 44 turkeys and 36 hams, and pass them out to our employees.
*We don't recommend full-screen viewing due to the shakiness of the picture.