text: Altered Expression OF Fas Receptor on Alveolar Macrophages and Inflammatory Effects of Soluble Fas Ligand Following Blunt Chest TraumaSeitz, Daniel H.; Palmer, Annette; Niesler, Ulrike; Braumüller, Sonja T.; Bauknecht, Simon; Gebhard, Florian; Knöferl, Markus W.Shock: June 2011 - Volume 35 - Issue 6 - pp 610-617 doi: 10.1097/SHK.0b013e318213665d Basic Science Aspects Abstract Author Information Article Outline Blunt chest trauma impairs the outcome of multiply-injured patients. Lung contusion induces inflammatory alterations and Fas-dependent apoptosis of alveolar type 2 epithelial (AT2) cells has been described. The Fas/Fas ligand (FasL) system seems to exhibit a proinflammatory potential. We aimed to elucidate the involvement of the Fas/FasL system in the inflammatory response after lung contusion. Chest trauma was induced in male rats by a pressure wave. Soluble FasL concentrations were determined in bronchoalveolar lavage fluids and alveolar macrophage (AMΦ) supernatants. Alveolar macrophages and AT2 cells were isolated to determine the surface expression (FACS) of Fas/FasL, the mRNA expression (reverse transcriptase-polymerase chain reaction) of Fas, FasL, TNF-α, IL-6, and IL-10 and to measure the release of IL-6 and IL-10 after culture with or without stimulation with FasL. After chest trauma, FasL concentration was increased in bronchoalveolar lavage fluid, and AMΦ supernatants and Fas and FasL protein were downregulated on AMΦs and unchanged on AT2 cells. The mRNA expression of Fas was increased in AMΦs and AT2 cells and that of FasL only in AMΦs isolated after lung contusion. Fas ligand stimulation further enhanced IL-6 and suppressed IL-10 release in AMΦs after trauma. The results indicate that the Fas/FasL system is activated after chest trauma, and FasL is associated with the inflammatory response after lung contusion. The proinflammatory response of AMΦs is enhanced by FasL stimulation. Both AMΦs and AT2 cells seem to contribute to the mediator release after lung contusion. These results confirm the importance of the Fas/FasL system in the inflammatory response after chest trauma. Department of Trauma Surgery, Hand, Plastic and Reconstructive Surgery, University of Ulm, Ulm, Germany Received 3 Dec 2010; first review completed 15 Dec 2010; accepted in final form 7 Jan 2011 Address reprint requests to Markus W. Knöferl, MD, Department of Trauma Surgery, Hand, Plastic, and Reconstructive Surgery, University of Ulm, Steinhövelstr. 9,89075 Ulm, Germany. E-mail: firstname.lastname@example.com. This study was supported by the Deutsche Forschungsgemeinschaft (DFG SPP 1151: DFG KN 475/3-2, 4-1) and State of Baden Württemberg (Juniorprofessoren-Programm) to M.W.K. None of the authors have any financial interests or affiliations with commercial organizations whose products or services are related to the subject matter of this manuscript (no existing conflicts of interest). Part of this study was presented at the 8th World Congress on Trauma, Shock, Inflammation and Sepsis-TSIS 2010, March 9-13, 2010, Munich, Germany. ABSTRACT INTRODUCTION MATERIALS AND METHODS Animals Blunt chest trauma Collection of blood plasma samples Collection of bronchoalveolar lavage fluids and preparation... Preparation of AT2 cells Purification of AT2 cells Soluble FasL stimulation of AMΦs and AT2 cells in vitro Measurement of BAL and supernatant cytokine concentrations Flowcytometric (FACS) analysis of Fas or FasL protein expre... Annexin V and TUNEL stain of AT2 cell cultures Isolation of RNA Reverse transcriptase-polymerase chain reaction Statistics RESULTS FasL concentrations in BAL and AMΦ supernatants Apoptosis induction in AT2 cells by sFasL Counts of isolated AMΦs and AT2 cells Mediator release of AMΦs or AT2 cells isolated after sham p... Expression of Fas, FasL, IL-6, IL-10, and TNF-α mRNA in AMΦ... Flow cytometric analysis of AMΦs and AT2 cells for expressi... DISCUSSION REFERENCES Back to Top | Article Outline INTRODUCTION Blunt chest trauma is a very common injury (1). It occurs in isolation, but more frequently it is part of the injury pattern seen in combined injury (2, 3). In this regard, lung contusion plays an important role with respect to morbidity and mortality of multiply-injured patients. Mortality from polytrauma rises significantly when lung contusion is part of the injury pattern (4). Acute respiratory distress syndrome (ARDS) and respiratory failure are major causes of death in severely injured patients (1), and chest trauma is an important trigger for ARDS (5). Severe local and systemic inflammatory alterations after experimental lung contusion have been described by our laboratory (6, 7) and may contribute to the previously mentioned complications. In this context, we could show that apoptosis induction in alveolar type 2 epithelial (AT2) cells after blunt chest trauma was mainly mediated by the Fas/Fas ligand (FasL) system (8). Fas/FasL-mediated apoptosis of pulmonary epithelial cells is discussed as an important trigger for the induction of ARDS (9). In addition to the induction of apoptosis, the Fas/FasL system contributes to a septic inflammatory response. Perl et al. (10) found that the inflammatory response after acute lung injury induced by hemorrhagic shock followed by polymicrobial sepsis (cecal ligation and puncture) was markedly depressed by Fas silencing. Furthermore, stimulation of pulmonary epithelial cells with Fas-activating antibodies enhanced the release of inflammatory mediators, such as the chemoattractant IL-8 (11). The intratracheal application of Jo2 as a Fas-activating antibody induced a significant increase in neutrophil counts in the bronchoalveolar lavage of alveolar macrophage (AMΦ)-depleted mice (12). All of these observations postulate that the Fas/FasL system is involved in the pulmonary inflammatory response. Nevertheless, to the best of our knowledge, the contribution of the Fas/FasL system to pulmonary inflammation after a nonseptic insult such as blunt chest trauma remains still unclear. Because AT2 cells are located in the alveolar walls, they may be influenced by mediators present in the alveolar milieu. Therefore, this study was designed to determine if the Fas/FasL system is activated by chest trauma and if Fas ligation further enhances AMΦ or AT2 cell inflammatory response after lung contusion. Back to Top | Article Outline MATERIALS AND METHODS Animals Male CD rats (Charles River, Sulzfeld, Germany) with a body weight of 250 to 275 g were used. Each experiment consisted of at least five animals and up to 15 animals per group and time point. Animals were kept and treated as described previously (8). All methods including animal work as well as specimen collection were reviewed and approved by the institutional review board/ethics committee of the University of Ulm as well as by the Federal Animal Care and Use Committee (permit no. 780; Regierungspräsidium Tübingen, Germany) Back to Top | Article Outline Blunt chest trauma Rats were anesthetized with 4% sevoflurane (Sevorane; Abbott, Wiesbaden, Germany) in oxygen under a continuous flow of 2 L/min, fixed in supine position, and chest and abdomen were shaved. To induce the blunt chest trauma, a single blast wave, released by a pressure wave generator, was centered on the thorax as described previously (6, 13-15). Sham animals underwent the same procedures, but no blast wave was applied. Back to Top | Article Outline Collection of blood plasma samples At 6, 24, and 48 h after sham procedure or chest trauma, animals were killed by exsanguination in deep anesthesia. Blood samples were centrifuged; the supernatants were removed and stored at −80°C until further use. Back to Top | Article Outline Collection of bronchoalveolar lavage fluids and preparation of AMΦs Alveolar macrophages were isolated as described previously (8, 16). In brief, at 4, 6, 16, 24, 48, or 72 h after chest trauma, animals were killed, the trachea was cannulated, and the lungs were flushed once with 5 mL of cold phosphate-buffered saline (PBS) (Dulbecco's PBS; Gibco BRL, Grand Island, NY) to collect bronchoalveolar lavage fluid (BAL) for mediator analysis (BAL 1) and then another eight times with 10 mL PBS to obtain AMΦs (BAL 2). Bronchoalveolar lavage fluid was centrifuged, and the supernatants of BAL 1 were stored at −80°C for enzyme-linked immunosorbent assay (ELISA). The pellets of BAL 1 and 2 were combined, resuspended in Dulbecco's modified Eagle medium (DMEM; Gibco), counted, and transferred to 24-well culture plates (BD, Franklin Lakes, NJ). After incubation (60 min, 37°C, 5% CO2), cultures were washed with DMEM to remove nonadherent cells. Alveolar macrophages designated for RNA isolation were now ready for use. For determination of supernatant mediator levels, cells were cultured for another 24 h in DMEM + 0.5% fetal calf serum (FCS; Life Technologies, Rockville, Md). Supernatants were then removed, centrifuged, and stored at −80°C until further use. Back to Top | Article Outline Preparation of AT2 cells Alveolar type 2 epithelial cells were isolated as described previously (17, 18). In brief, the lungs were digested with elastase (Worthington Biochemical, Freehold, NJ) to obtain a cell suspension. The suspension was filtered successively through polyester meshes (500-, 200-, and 105-μm pore diameter; VWR International, Darmstadt, Germany) and plated on Petri dishes coated with rat-IgG (Sigma, Steinheim, Germany) to remove lung macrophages. Then AT2 cells were carefully collected by rinsing the Petri dishes three times with DMEM. After centrifugation, cells were resuspended in DMEM + 10% FCS and passed through a 100-μm cell strainer (BD Biosciences Discovery Labware, Bedford, Mass). Cells were counted, and viability was found to be higher than 95% by trypan blue exclusion. Back to Top | Article Outline Purification of AT2 cells For further purification of AT2 cells, neutrophils were removed by magnetic bead selection using the EasySep PE selection kit (StemCell Technologies, Vancouver, British Columbia, Canada) according to the manufacturer's recommendations. Briefly, cells were resuspended in selection buffer (PBS w/o Mg2+/Ca2+ + 1 mM EDTA [Sigma] + 2% FCS), and unspecific bindings were blocked with rat-IgG (Caltag Laboratories, Burlingame, Calif). Cells were then incubated with a mouse antigranulocyte antibody (BD Biosciences, Heidelberg, Germany), washed, and again resuspended in selection buffer. The cell suspension was incubated with EasySep PE Selection Cocktail and afterward with EasySep magnetic nanoparticles. For the magnetic separation, the cell suspension was diluted to a total volume of 2.5 mL by adding selection buffer, put in a FACS tube (BD), and placed in the magnet for 10 min. The supernatant, containing AT2 cells, was transferred to another FACS tube and centrifuged, and the pellet resuspended in 500 μL PBS. Cell counts and viability were determined by trypan blue. Back to Top | Article Outline Soluble FasL stimulation of AMΦs and AT2 cells in vitro Alveolar type 2 epithelial cells were isolated from naive animals. To determine apoptosis induction by soluble FasL (sFasL), AT2 cells were plated on eight-well chamber slides (Lab-Tek II; Nalge Nunc Int, Naperville, Ill) and stimulated with 50, 250, or 500 ng/mL sFasL (Kamiya Biomedical Company, Seattle, Wash) in DMEM + 5% FCS (18 h, 37°C, 5% CO2). Afterward, cells were fixed in 4% formaldehyde (J. T. Baker, Deventer, the Netherlands), washed, and stained with annexin V for apoptosis detection (see below). In addition, AMΦs or AT2 cells were isolated at 24 h after chest trauma or sham procedure and cultured for 24 h in DMEM + 0.5% FCS with or without 500 ng/mL sFasL (37°C, 5% CO2). Culture supernatants were then removed, centrifuged, and stored at −80°C until determination of mediator concentrations. Back to Top | Article Outline Measurement of BAL and supernatant cytokine concentrations The concentrations of IL-6, IL-10, MCP-1 (BD OptEIA ELISA Set; BD Pharmingen, San Diego, Calif), and MIP-2 (CytoSet; Biosource, Camarillo, Calif) in BAL, AMΦ, and AT2 cell culture supernatants were determined by ELISA according to the manufacturer's recommendations. The concentrations of sFasL in AMΦ supernatants were determined by direct ELISA as described previously (8). The absorbance of standards and samples was measured with a microplate reader (Tecan Austria GmbH, Gröding, Austria). Back to Top | Article Outline Flowcytometric (FACS) analysis of Fas or FasL protein expression on AMΦs and AT2 cells Alveolar macrophages and AT2 cells were isolated at 4 and 16 h after chest trauma or sham procedure. Cells were fixed and permeabilized using Leucoperm (AbD Serotec, Raleigh, NC). Isotype rabbit-IgG (Beckman-Coulter, Krefeld, Germany), anti-Fas (Santa Cruz Biotechnology, Santa Cruz, Calif), or anti-FasL (Santa Cruz) was added to both cell populations. Alveolar type 2 epithelial cells were additionally stained with anticytokeratin MNF Clone 116 (Dako, Carpinteria, Calif). Cell suspensions were incubated for 30 min at room temperature. Goat anti-rabbit-PE (Beckman-Coulter) was used as a detection antibody for Fas and FasL. Alveolar type 2 epithelial cells were additionally stained with goat anti-mouse SPRD (Spectral Red; Beckman-Coulter) as a secondary antibody for the MNF 116 stain. Cells were incubated for 25 min on ice and washed, and FACS analysis was performed on a BD FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software (BD Biosciences). The cells were gated according to their SSC versus FL1 characteristics (AMΦ) or their FSC versus SSC characteristics (AT2 cells), and 10,000 events were acquired. Back to Top | Article Outline Annexin V and TUNEL stain of AT2 cell cultures To detect apoptosis in AT2 cell cultures, cells were fixed as described above, and annexin V or TUNEL staining was performed as described before (8). Cells were evaluated by fluorescence microscopy (Axio Imager M1; Zeiss, Oberkochen, Germany). To quantify apoptosis, the total number of cells and the number of annexin V-positive cells in three representative high-power fields were counted by a researcher blinded to the groups. The percentage of apoptotic cells was calculated. Back to Top | Article Outline Isolation of RNA Total RNA from AMΦs and purified AT2 cells was isolated using the Absolutely RNA Miniprep Kit (Stratagene, Cedar Creek, Tex) following the manufacturer's instructions including the RNase-free DNase step to avoid contamination with genomic DNA. RNA samples were purified and concentrated using the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany). Back to Top | Article Outline Reverse transcriptase-polymerase chain reaction The relative quantities of mRNA for each gene of interest were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Reverse transcription was carried out in a 40-μL final volume from 2 μg of total RNA using the AffinityScript QPCR cDNA Synthesis Kit (Stratagene) according to the manufacturer's instructions. For the RT reaction, the samples were incubated for 15 min at 42°C followed by 5 min at 95°C. Samples were then diluted 1:5 with RNase-free water. Quantitative real-time PCR was performed on an Mx3000P QPCR System and software (MxPro; Stratagene) in combination with Brilliant SYBR Green QPCR Master Mix (Stratagene) and specific primers (Table 1). The thermal cycling program consisted of an initial denaturation step (95°C for 10 min) followed by 40 cycles of 15 s at 94°C, 30 s at 55°C, and 30 s at 72°C. A melting curve was generated by linear heating from 55°C to 95°C and showed the specificity by a single peak at the expected temperature. A negative control with no template and a No-RT control were included for each gene in each qPCR run. β2-Microglobulin was used as housekeeping gene. This gene was chosen for normalization because it showed the lowest variance of expression between sham and trauma in the particular cell population (data not shown). Results were expressed as the fold increase of trauma over sham animals. The logarithm (log2) of the ΔCT values was used for statistics as mentioned in the following section. Table 1 Back to Top | Article Outline Statistics Results are presented as mean ± SEM. A one-way ANOVA followed by the Student-Newman-Keuls test as a post hoc test for multiple comparisons was performed to determine significant differences between experimental means. Whenever more than one variable was tested, a two-way ANOVA was performed before the Student-Newman-Keuls test as mentioned above. P ≤ 0.05 was considered statistically significant. Back to Top | Article Outline RESULTS FasL concentrations in BAL and AMΦ supernatants Concentrations of sFasL were significantly increased in BAL collected at 6, 24, and 48 h after blunt chest trauma, compared with corresponding shams (Fig. 1A). Furthermore, FasL levels in BAL collected at 24 or 48 h after the insult were significantly higher than those in BAL sampled at 6 h. Alveolar macrophages isolated at 6 h after chest trauma released significantly higher levels of FasL than AMΦs from corresponding sham animals. This effect was lost at 24 and 48 h after the insult (Fig. 1B). Fig. 1 Back to Top | Article Outline Apoptosis induction in AT2 cells by sFasL Addition of sFasL (50, 250, 500 ng/mL) to AT2 cell cultures increased AT2 cell apoptosis in a dose-dependent manner when compared with unstimulated cells, as shown by annexin V stain (Fig. 1C). TUNEL stain revealed a tendency to increased counts of apoptotic AT2 cells after addition of sFasL (Fig. 1D). Back to Top | Article Outline Counts of isolated AMΦs and AT2 cells At 24, 48, and 72 h after chest trauma, significantly more AMΦs were isolated than at the same time points in sham (Fig. 2A). At 4 h after chest trauma, the counts of isolated AT2 cells were significantly higher than in sham (Fig. 2B). At the later time points, there was no further difference between trauma and sham AT2 cell counts. Fig. 2 Back to Top | Article Outline Mediator release of AMΦs or AT2 cells isolated after sham procedure or blunt chest trauma, cultured with or without stimulation with sFasL Alveolar macrophages showed a significantly increased release of IL-6, IL-10, and MCP-1 at 24 h after blunt chest trauma, when compared with corresponding sham (Fig. 3, A-C). Additional FasL stimulation (500 ng/mL) of AMΦs isolated after blunt chest trauma induced a further increase in the release of IL-6 compared with AMΦs cultured without stimulation (Fig. 3A). In supernatants of AMΦs isolated after chest trauma and stimulated with sFasL IL-10, release was significantly lower than in unstimulated AMΦs from traumatized animals (Fig. 3B). Alveolar type 2 epithelial cells isolated at 24 h after blunt chest trauma released significantly more IL-6, IL-10, and MCP-1 than the corresponding sham; additional stimulation with sFasL did not attenuate or further enhance the release of these mediators (Fig. 4, A-C). Fig. 3 Fig. 4 Back to Top | Article Outline Expression of Fas, FasL, IL-6, IL-10, and TNF-α mRNA in AMΦs and AT2 cells The mRNA expression of Fas in AMΦs isolated at 4 h after trauma was significantly downregulated when compared with sham or the 16-h time point (Fig. 5A). At 24 h after chest trauma, Fas mRNA expression in AMΦs was upregulated when compared with corresponding sham or the 48-h time point (Fig. 5A). Expression of FasL mRNA was markedly higher in AMΦs isolated at 4 h after chest trauma than in AMΦs from sham animals (Fig. 5B). In AT2 cells, Fas mRNA expression was significantly increased at 4 h after lung contusion, when compared with AT2 cells isolated at 4 h after sham procedure and compared with those isolated at 16 h after chest trauma (Fig. 5C). Alveolar type 2 epithelial cells isolated at 24 h after blunt chest trauma showed a significantly lower mRNA expression of FasL than AT2 cells from sham animals or AT2 cells isolated at 4, 16, or 48 h after chest trauma (Fig. 5D). Regarding the cytokines, blunt chest trauma induced an increased expression of TNF-α, IL-6, and IL-10 mRNA in AMΦs (Fig. 6, A-C). The peak of cytokine mRNA expression was at the 48-h time point. In AT2 cells, mRNA expression of TNF-α, IL-6, or IL-10 was detectable but did not change after blunt chest trauma (Table 2). Table 2 Fig. 5 Fig. 6 Back to Top | Article Outline Flow cytometric analysis of AMΦs and AT2 cells for expression of Fas and FasL Flow cytometric analysis revealed a significant decrease in Fas-positive (Fig. 7A) and FasL-positive (Fig. 7B) AMΦ cell counts at 4 or 16 h after lung contusion when compared with sham. The percentage of Fas-positive (Fig. 7C) or FasL-positive (Fig. 7D) AT2 cells did not change after blunt chest trauma. Nevertheless, in all the studied groups, the fraction of Fas-positive AT2 cells among the total of AT2 cells analyzed was about 50%. Fig. 7 Back to Top | Article Outline DISCUSSION The present study was designed to determine the contribution of the Fas/ FasL system to the inflammatory response after blunt chest trauma. Our results indicate that the Fas/FasL system not only induces apoptosis in alveolar epithelial cells but also represents an important factor in regulating the inflammatory response after lung contusion. Concentrations of sFasL were increased in both BAL and cell culture supernatants of unstimulated AMΦs isolated after blunt chest trauma (Fig. 1, A and B). These findings are supported by clinical observations of Matute-Bello et al. (19), who described significantly increased concentrations of sFasL in BAL fluids and LPS-stimulated BAL cell culture supernatants of patients with clinically defined ARDS. According to Matute-Bello et al. (19), increased levels of sFasL in the BAL at the onset of ARDS were associated with a poor outcome. These findings emphasize the relevance of Fas/FasL activation in lung inflammation and the impact of sFasL on the development of severe pulmonary complications, such as ARDS. Because the mentioned authors have shown that FasL has a proinflammatory potential in the context of lung injury or inflammation, this study was designed to further determine the release or expression of Fas and FasL after lung contusion and to analyze Fas/FasL effects on pulmonary immunological cells after blunt chest trauma. TUNEL stain revealed a tendency to increased apoptosis rates (Fig. 1D). Annexin V stain, however, indicates that incubation of untreated AT2 cells with different concentrations of sFasL induced apoptosis in a dose-dependent manner (Fig. 1C). The variance between these two apoptosis assays is explained by their different kinetics and sensitivity (20, 21). This observation stands in line with findings of Hagimoto et al. (11), who found a markedly increased apoptosis rate in human bronchiolar epithelial cells after incubation with a Fas-activating antibody (CH-11) or TNF-α. According to a report by Nakamura et al. (22), sFasL itself reduced the survival of human small airway epithelial cells but not normal human bronchial epithelial cells. Small airway epithelial cells are nonciliated primary lung epithelial cells that express cytokeratin and surfactant protein A and therefore are very similar to the alveolar epithelial cells used in our experiment. Interestingly, preincubation of small airway epithelial cells with TNF-α, IL-1β, or IFN-γ further enhanced apoptosis induction, showing that proinflammatory cytokines sensitize lung epithelium to Fas-induced death (22). To confirm these findings in vivo, these authors instilled a Fas-activating antibody (Jo2) in lungs of otherwise untreated mice. After 6 h of incubation, immunohistochemistry for activated caspase 3 indicated increased apoptosis induction in epithelia covering the alveolar spaces and in intraalveolar cells (22). Because sFasL was increased in BALs after blunt chest trauma in our experiments (Fig. 1A), these findings support the hypothesis that apoptosis induction after blunt chest trauma is mediated by the Fas/FasL system (8). According to our own previous findings, the counts of isolated AMΦs were significantly higher in traumatized animals than in sham at the later time points after the insult (23). In contrast to that, AT2 cell gain was significantly higher at 4 h after chest trauma than after sham procedure, but was not changed in the later time course. Interestingly, an increased apoptosis rate of AT2 cells as mentioned in a previous publication could not be reproduced by decreased AT2 cell gain during isolation (8). A reason for this discrepancy might be that early apoptotic cells were not detected by trypan blue stain and counted as viable. The release of the proinflammatory cytokine IL-6 by AMΦs isolated after chest trauma was further enhanced by additional stimulation with FasL, and the release of anti-inflammatory IL-10 by AMΦs isolated after chest trauma was suppressed by FasL stimulation in our experiment (Fig. 3, A and B). Those results indicate that FasL stimulation is associated with the enhancement of the inflammatory response, observed after blunt chest trauma. The fact that Fas activation induces a proinflammatory cytokine response is supported by a report by Park et al. (24) on Fas stimulation in human monocytes and monocyte-derived macrophages. The authors report that TNF-α and IL-8 release in naive monocytes significantly increased after 18 h of incubation with the Fas-activating antibody CH-11 as well as with sFasL. In contrast to our experiment, Park et al. (24) used only untreated cells, whereas we compared cells collected after sham injury or chest trauma. In another experiment by Perl et al. (25), acute lung injury was induced in Fas and FasL knockout mice and wild-type mice by cecal ligation and puncture, followed by hemorrhage. In vivo experiments revealed a marked decrease in proinflammatory cytokine release, such as IL-6 in lung tissue and bronchoalveolar lavage in both knockout mice examined. In contrast to Fas knockout, FasL knockout did not attenuate chemokine concentrations (KC and MIP-2) in lung tissue isolated early after lung injury in the same experiment. Findings of Perl et al. (25) and our data on distinct cell types indicate that other mechanisms besides Fas/FasL activation are involved in chemokine release after chest trauma or lung injury. It should be noted that, with regard to the study design, our data reveal only an association between Fas/FasL activation and posttraumatic immune response after blunt chest trauma. However, based on the remarkable results of this study, further experiments should focus on therapeutic effect of the modification in Fas/FasL activation by introducing knockout animals or the auspicious method of Fas silencing (10). Alveolar type 2 epithelial cells isolated after blunt chest trauma released significantly higher levels of IL-6, IL-10, and MCP-1 than AT2 cells from sham animals (Fig. 4). These data indicate that AT2 cells are activated by blunt chest trauma and contribute to posttraumatic alterations by the release of inflammatory mediators. Stimulation with sFasL did not further increase this cytokine release. In contrast to our results, human bronchiolar epithelial cells showed a significantly increased mediator release in response to stimulation with TNF-α or the Fas-activating antibody CH-11 (11). However, Hagimoto et al. (11) used naive bronchiolar epithelial cells, whereas we studied alveolar epithelial cells from rats subjected to chest trauma or sham procedure. Furthermore, the authors determined only the concentrations of the chemoattractant IL-8. Crestani et al. (26) detected that AT2 cells isolated from untreated rats released IL-6, and additional stimulation with murine IL-1β or TNF-α further augmented the IL-6 release. We have previously shown that IL-1β is increased in bronchoalveolar lavage after blunt chest trauma (8). Therefore, it is reasonable to suggest that mediators present in the alveoli after lung contusion may activate AT2 cells to increase their mediator secretion. The present findings support the hypothesis that AT2 cells serve as an important source of inflammatory mediators in the alveolar space and are associated with the regulation of the intraalveolar immune response and recruitment of circulating monocytes (26, 27). Alveolar macrophages and AT2 cells from sham animals expressed considerable amounts of Fas and FasL mRNA (Fig. 5). In AMΦs, Fas mRNA expression was upregulated late, and FasL mRNA expression was upregulated early after trauma (Fig. 5, A and B). The early increase of FasL mRNA in AMΦs is in line with the increased release of the corresponding sFasL protein by AMΦs isolated early after chest trauma (Fig. 1B). In AT2 cells, Fas mRNA expression was upregulated early after trauma, and FasL mRNA was downregulated late after the insult (Fig. 5, C and D). Comparable results were described by Nakamura et al. (22), who showed that Fas but not FasL mRNA was expressed in normal human bronchial epithelial cells and human small airway epithelial cells. In contrast to our experiment, however, Nakamura used a slightly different cell population. Our results indicate that AMΦs and AT2 cells express the Fas receptor and FasL constitutively. Chest trauma seems to increase the susceptibility of AMΦs and AT2 cells to Fas ligation in a time-dependent manner. IL-6 and IL-10 mRNA expression in AMΦs significantly increased after blunt chest trauma (Fig. 6). Vanderbilt et al. (28), however, found that unstimulated AMΦs did not express genes for these chemokines until they were cultured for at least 2 h, and the amount of mRNA expression depended on the time in cell culture. In our experiment, AMΦs were cultured for only 1 h until they were used for RNA isolation. As IL-6 and IL-10 mRNA expressions were barely detectable in sham animals, the activating effect of the cell culture conditions on AMΦs seems to be negligible. Thus, these data confirm our own previous findings indicating that AMΦs are activated by blunt chest trauma to release inflammatory mediators (23, 29) and to express the corresponding mRNA. Alveolar type 2 epithelial cells from sham animals showed a detectable mRNA expression of those cytokines; however, blunt chest trauma did not alter this expression (Table 2). The discrepancy between the unchanged mRNA expression for those cytokines and the increased mediator release might arise from the fact that AT2 cells were cultured for determination of mediator release, whereas the mRNA expression was determined in freshly isolated cells. Furthermore, because there is evidence in the literature that cytokines can be stored intracellularly (30, 31), another possible explanation is that preformed cytokines were present in the cells before release. Flow cytometric analysis of Fas and FasL expression on the surface of AMΦs revealed significantly decreased numbers of Fas- and FasL-positive AMΦs at 4 and 16 h after chest trauma when compared with sham. Nevertheless, a baseline surface expression of Fas and FasL was detectable in all animals (Fig. 7). Comparable results were described by Matute-Bello et al. (32), who detected a baseline expression of Fas on the surface of AMΦs from wild-type mice. Soluble FasL is known to be released by cleavage of the membrane-bound form of FasL by a metalloproteinase (33). As we observed an increase in FasL release by cultured AMΦs isolated early after blunt chest trauma when compared with sham (Fig. 1B), our data suggest that the observed decrease in membrane-bound FasL on AMΦs may be associated with an increased release of sFasL. FACS analysis revealed that 50% of AT2 cells expressed Fas on their surface and that the ratio did not change following blunt chest trauma. These data indicate that AT2 cells express the Fas receptor constitutively. Our results are in line with findings of Fine et al. (34), who immunohistochemically detected that Fas protein was expressed on the surface of a subpopulation of AT2 cells from untreated rats. An increased surface expression of Fas on human bronchiolar epithelial cells has been described in a report by Hagimoto et al. (11). In this study, the cells were stained for Fas expression and evaluated by FACS analysis. Interestingly, preincubation with TNF-α and IFN-γ further enhanced the expression of Fas in those cells (11). Although in our own previous experiments cytokine levels were significantly increased in bronchoalveolar lavage after blunt chest trauma (8), our results indicate that mediators detected in the alveoli of animals subjected to lung contusion do not enhance Fas receptor expression on AT2 cells. Furthermore, these findings are comparable to our previous observations that showed that the numbers of Fas-expressing AT2 cells in lung sections increased only late after lung contusion (8). However, the comparison between the studies is limited by the fact that Fas receptor expression was determined by different methods. Furthermore, AT2 cell apoptosis, which is known to occur after chest trauma (8), might explain the missing increase of Fas receptor-positive AT2 cells in the present study. Our findings suggest that the Fas/FasL system is activated after blunt chest trauma. Alveolar macrophages appear to contribute to the increased levels of sFasL in bronchoalveolar lavage by the release of this mediator. Furthermore, AMΦs were activated by sFasL in vitro. Because this mediator is increased in bronchoalveolar lavage after lung contusion, it has the potential to enhance the posttraumatic response of AMΦs in vivo. Interestingly, we could show that AT2 cells released higher levels of IL-6, IL-10, and MCP-1 after blunt chest trauma, but in contrast to AMΦ, cultured AT2 cells did not further enhance their mediator release after stimulation with sFasL in vitro. Taken together, our results indicate that the Fas/FasL system not only induces apoptosis in alveolar epithelial cells but is also associated with the regulation of the inflammatory response after lung contusion. Back to Top | Article Outline REFERENCES1. Scarborough K, Slone DS, Uribe P, Craun M, Bar-Or R, Bar-Or D: Reduced mortality at a community hospital trauma center: the impact of changing trauma level designation from II to I. Arch Surg 143(1):22-27, 2008. Cited Here... 2. Shorr RM, Crittenden M, Indeck M, Hartunian SL, Rodriguez A: Blunt thoracic trauma. Analysis of 515 patients. Ann Surg 206(2):200-205, 1987. Cited Here... 3. Pinilla JC: Acute respiratory failure in severe blunt chest trauma. J Trauma 22(3):221-226, 1982. Cited Here... 4. Gaillard M, Herve C, Mandin L, Raynaud P: Mortality prognostic factors in chest injury. J Trauma 30(1):93-96, 1990. Cited Here... 5. Guo-shou Z, Xiang-jun B, Cheng-ye Z: Analysis of high risk factors related to acute respiratory distress syndrome following severe thoracoabdominal injuries. Chin J Traumatol 10(5):275-278, 2007. Cited Here... 6. Knöferl MW, Liener UC, Seitz DH, Perl M, Brückner UB, Kinzl L, Gebhard F: Cardiopulmonary, histological, and inflammatory alterations after lung contusion in a novel mouse model of blunt chest trauma. Shock 19(6):519-525, 2003. Cited Here... 7. Perl M, Gebhard F, Brückner UB, Ayala A, Braumüller S, Büttner C, Kinzl L, Knöferl MW: Pulmonary contusion causes impairment of macrophage and lymphocyte immune functions and increases mortality associated with a subsequent septic challenge. Crit Care Med 33(6):1351-1358, 2005. Cited Here... 8. Seitz DH, Perl M, Mangold S, Neddermann A, Braumüller ST, Zhou S, Bachem MG, Huber-Lang MS, Knöferl MW: Pulmonary contusion induces alveolar type 2 epithelial cell apoptosis: role of alveolar macrophages and neutrophils. Shock 30(5):537-544, 2008. Cited Here... 9. Matute-Bello G, Martin TR: Science review: apoptosis in acute lung injury. Crit Care 7(5):355-358, 2003. Cited Here... 10. Perl M, Chung CS, Lomas-Neira J, Rachel TM, Biffl WL, Cioffi WG, Ayala A: Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis. Am J Pathol 167(6):1545-1559, 2005. Cited Here... 11. Hagimoto N, Kuwano K, Kawasaki M, Yoshimi M, Kaneko Y, Kunitake R, Maeyama T, Tanaka T, Hara N: Induction of interleukin-8 secretion and apoptosis in bronchiolar epithelial cells by Fas ligation. Am J Respir Cell Mol Biol 21(3):436-445, 1999. Cited Here... 12. Bem RA, Farnand AW, Wong V, Koski A, Rosenfeld ME, van RN, Frevert CW, Martin TR, Matute-Bello G: Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice. Am J Physiol Lung Cell Mol Physiol 295(2):L314-L325, 2008. Cited Here... 13. Irwin RJ, Lerner MR, Bealer JF, Brackett DJ, Tuggle DW: Cardiopulmonary physiology of primary blast injury. J Trauma 43(4):650-655, 1997. Cited Here... 14. Jaffin JH, McKinney L, Kinney RC, Cunningham JA, Moritz DM, Kraimer JM, Graeber GM, Moe JB, Salander JM, Harmon JW: A laboratory model for studying blast overpressure injury. J Trauma 27(4):349-356, 1987. Cited Here... 15. Liener UC, Knöferl MW, Sträter J, Barth TF, Pauser EM, Nüssler AK, Kinzl L, Brückner UB, Gebhard F: Induction of apoptosis following blunt chest trauma. Shock 20(6):511-516, 2003. Cited Here... 16. Huber-Lang M, Younkin EM, Sarma JV, Riedemann N, McGuire SR, Lu KT, Kunkel R, Younger JG, Zetoune FS, Ward PA: Generation of C5a by phagocytic cells. Am J Pathol 161(5):1849-1859, 2002. Cited Here... 17. Dobbs LG: Isolation and culture of alveolar type II cells. Am J Physiol 258(4 Pt 1):L134-L147, 1990. Cited Here... 18. Dobbs LG, Gonzalez R, Williams MC: An improved method for isolating type II cells in high yield and purity. Am Rev Respir Dis 134(1):141-145, 1986. Cited Here... 19. Matute-Bello G, Liles WC, Steinberg KP, Kiener PA, Mongovin S, Chi EY, Jonas M, Martin TR: Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS). J Immunol 163(4):2217-2225, 1999. Cited Here... 20. Zhao J, Schmid-Kotsas A, Gross HJ, Gruenert A, Bachem MG: Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells. Chin Med J (Engl) 116(12):1923-1929, 2003. Cited Here... 21. Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C: A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Methods 184(1):39-51, 1995. Cited Here... 22. Nakamura M, Matute-Bello G, Liles WC, Hayashi S, Kajikawa O, Lin SM, Frevert CW, Martin TR: Differential response of human lung epithelial cells to Fas-induced apoptosis. Am J Pathol 164(6):1949-1958, 2004. Cited Here... 23. Seitz DH, Niesler U, Palmer A, Sulger M, Braumüller ST, Perl M, Gebhard F, Knöferl MW: Blunt chest trauma induces mediator-dependent monocyte migration to the lung. Crit Care Med 38(9):1852-1859, 2010. Cited Here... 24. Park DR, Thomsen AR, Frevert CW, Pham U, Skerrett SJ, Kiener PA, Liles WC: Fas (CD95) induces proinflammatory cytokine responses by human monocytes and monocyte-derived macrophages. J Immunol 170(12):6209-6216, 2003. Cited Here... 25. Perl M, Chung CS, Perl U, Lomas-Neira J, de PM, Cioffi WG, Ayala A: Fas-induced pulmonary apoptosis and inflammation during indirect acute lung injury. Am J Respir Crit Care Med 176(6):591-601, 2007. Cited Here... 26. Crestani B, Cornillet P, Dehoux M, Rolland C, Guenounou M, Aubier M: Alveolar type II epithelial cells produce interleukin-6 in vitro and in vivo. Regulation by alveolar macrophage secretory products. J Clin Invest 94(2):731-740, 1994. Cited Here... 27. Thorley AJ, Ford PA, Giembycz MA, Goldstraw P, Young A, Tetley TD: Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages. J Immunol 178(1):463-473, 2007. Cited Here... 28. Vanderbilt JN, Mager EM, Allen L, Sawa T, Wiener-Kronish J, Gonzalez R, Dobbs LG: CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury. Am J Respir Cell Mol Biol 29(6):661-668, 2003. Cited Here... 29. Liener UC, Perl M, Huber-Lang MS, Seitz DH, Brückner UB, Gebhard F, Knöferl MW: Is the function of alveolar macrophages altered following blunt chest trauma? Langenbecks Arch Surg 396(2):251-259, 2010. Cited Here... 30. Spencer LA, Melo RC, Perez SA, Bafford SP, Dvorak AM, Weller PF: Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion. Proc Natl Acad Sci U S A 103(9):3333-3338, 2006. Cited Here... 31. Tsakiri N, Kimber I, Rothwell NJ, Pinteaux E: Mechanisms of interleukin-6 synthesis and release induced by interleukin-1 and cell depolarisation in neurones. Mol Cell Neurosci 37(1):110-118, 2008. Cited Here... 32. Matute-Bello G, Lee JS, Liles WC, Frevert CW, Mongovin S, Wong V, Ballman K, Sutlief S, Martin TR: Fas-mediated acute lung injury requires Fas expression on nonmyeloid cells of the lung. J Immunol 175(6):4069-4075, 2005. Cited Here... 33. Nagata S: Fas ligand-induced apoptosis. Annu Rev Genet 33:29-55, 1999. Cited Here... 34. Fine A, Anderson NL, Rothstein TL, Williams MC, Gochuico BR: Fas expression in pulmonary alveolar type II cells. Am J Physiol 273(1 Pt 1):L64-L71, 1997. Cited Here... Keywords: Lung contusion; inflammation; Fas/Fas ligand; alveolar macrophages; alveolar type 2 epithelial cells ©2011The Shock Society Article Outline ABSTRACT INTRODUCTION MATERIALS AND METHODS Animals Blunt chest trauma Collection of blood plasma samples Collection of bronchoalveolar lavage fluids and preparation... Preparation of AT2 cells Purification of AT2 cells Soluble FasL stimulation of AMΦs and AT2 cells in vitro Measurement of BAL and supernatant cytokine concentrations Flowcytometric (FACS) analysis of Fas or FasL protein expre... Annexin V and TUNEL stain of AT2 cell cultures Isolation of RNA Reverse transcriptase-polymerase chain reaction Statistics RESULTS FasL concentrations in BAL and AMΦ supernatants Apoptosis induction in AT2 cells by sFasL Counts of isolated AMΦs and AT2 cells Mediator release of AMΦs or AT2 cells isolated after sham p... Expression of Fas, FasL, IL-6, IL-10, and TNF-α mRNA in AMΦ... Flow cytometric analysis of AMΦs and AT2 cells for expressi... DISCUSSION REFERENCES Source Altered Expression OF Fas Receptor on Alveolar Macrophages and Inflammatory Effects of Soluble Fas Ligand Following Blunt Chest Trauma Shock35(6):610-617, June 2011. Full-Size Email + Favorites Export View in Gallery Email to a Colleague Colleague's E-mail is Invalid Your Name: (optional) Your Email: Colleague's Email: Separate multiple e-mails with a (;). Message: Thought you might appreciate this item(s) I saw at Shock. Send a copy to your email Your message has been successfully sent to your colleague. Some error has occurred while processing your request. Please try after some time. Email to a Colleague Colleague's E-mail is Invalid Your Name: (optional) Your Email: Colleague's Email: Separate multiple e-mails with a (;). Message: Thought you might appreciate this item(s) I saw at Shock. Send a copy to your email Your message has been successfully sent to your colleague. Some error has occurred while processing your request. Please try after some time. 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, Florida (PressExposure) June 18, 2009 -- For some detailers bad weather is another opportunity. The Wiper Pro.com has recently published an article outline how some auto detailers are still profiting, even in bad weather. While their competitors call the day off they are collecting profits other auto detailers have over looked.
This publication includes information about marketing strategies, ways to pull traffic and drive sales in addition to detailed information on exactly how some detailers are doing this.
Published as part of a larger series on Results Based Marketing for the Auto Detailer this article was written by The Wiper Pro co-founder Peter Ferrigan.
Copyright © 2009 Peter Ferrigan WiperPro.com

subset: commoncrawl


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Fez, Morocco
Merenid Tombs
Crumbling necropolis set high above Fez.
Looming above the northern walls of the Fez Medina are the skeletal remains of the Merenid Tombs. Built in the 14th century, the tombs were once the sumptuous resting places of the Merenids' finest, but after years at the mercy of conquests and looters, they lack most of their original decorative charm.
What the remaining tombs lack in aesthetics, though, the view makes up for.
Below, the whole of the Fez Medina stretches out to the East in a patchwork of minarets, laundry lines, satellites, and the occasional garbage fire. Carved into the hills are man-made caves that serve as dwellings for some of Fez's less fortunate. The amount of empty beer cans lying around is a good clue-in as to what the late-night atmosphere is like, and any local will tell you the tombs are a well-known mugging ground for "gauries" ("honkies" in colloquial Moroccan).
Directly below the tombs on either side of the cliff are two large graveyards that are used as grazing grounds for local shepherds. Unfortunately, the area is rife with trash, but there are also plenty of animal bones lying around to complement the spookiness.
Know Before You Go
Easily visible from from Casbah En Nouar, it's a 15 minute walk and next to Hotel Les Merenides.
- Lonely Planet: Merenid Tombs in Fez: http://www.lonelyplanet.com/morocco/the-mediterranean-coast-and-the-rif/fes/sights/archaeology/merenid-tombs


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null domain pointer - opening a gambit mesh
Your forum has been of a great help for me, ever since I started using fluent two weeks ago, however, I did this mesh that I have already checked on gambit for quality and it has a very good quality, but when I exported the mesh and tryed to open it on fluent it gave me this error:
warning: inappropriate zone type (interior) for one-sided face zone 26. Changing to wall.
Warning: inappropriate zone tyep (interior) for one-sided face zone 5. Changing to wall.
Cell centroid is xc -0.212418 yc -1.389475
Warning: no face with given nodes. Thread 2, cell 240465
Error: Build_Grid: grid error. Clearing partially read grid.
Error: Null domain pointer
Error Object : #f
if you can help me figure out what is going on there I would really appreciate it... the fluent path seems a long one right now..
|All times are GMT -4. The time now is 21:51.


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WWII US 1944 DATED Canteen/Cover/Belt/1ST Aid Case!
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WWII US 1944 DATED Canteen/Cover/Belt/1ST Aid Case!
WWII US 1944 DATED Canteen/Cover/Belt/1ST Aid Case! Up for auction a Vintage WWII Belt, Canteen & Accessory Kit!
Auction Includes: Stamped 1944 Canteen with fitted cup (stamped 1944) and cover (dated 1945) 1 st Aid Pouch 2 Utility Belts (1 dated 1943). All vintage WWII era! Check out the super-sized pics for a look at condition.
Check out our other auctions for MORE WWII GEARAnd $ave on COMBINED SHIPPING!
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COLORADO RESIDENTS MUST PAY 2.9% SALES TAX, EL PASO COUNTY, COLORADO RESIDENTS MUST PAY 3.9% SALES TAX, MONUMENT COLORADO RESIDENTS MUST PAY 6.9% SALES TAX. All other bidders are exempt from sales tax.
For merchandise not being mailed to the winning bidder, but rather picked up at our retail store (251 Front St. #4, Monument, CO 80132) t is NO charge.
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Surely you've heard by now what Hillary Clinton had to say about Bernie Sanders and health care reform in the 1990s:
And you've probably seen the photo or video of Sanders standing behind Clinton in a 1993 speech on the subject (above) as well as these thankful words from the same year:"I don't know where he was when I was trying to get health care in '93 and '94," a fired up @HillaryClinton says of Sanders.— Amy Chozick (@amychozick) March 12, 2016
The optics of this, admittedly, aren't flattering to Hillary and she should have been more careful with her wording of the criticism. But none of the pushback really answers her point: Just where was Bernie when the Clintons were trying to pass health care reform?Clinton today: "I don't know where he was when I was trying to get health care." Clinton in '93: pic.twitter.com/H4ETjeH4gU— Jennifer Epstein (@jeneps) March 12, 2016
Having listened to Sanders on the campaign trail for the past year or so, my guess is that he wanted single-payer and wouldn't compromise, therefore being not much help to the Clintons as they were fighting like hell for the Health Security Act well into 1994.
But it turns out that none of us have to guess, because Sanders himself wrote about it in his 1997 memoir Outsider in the House (later renamed and re-released in 2015 as Outsider in the White House):
So by his own admission, Bernie Sanders didn't support the Clinton health care initiative (which had a shot at passing) and instead backed a single-payer plan (which had zero chance at passing).
And now we know why Hillary Clinton said what she did the other day.
The White House needed every single Democratic (and democratic socialist) vote that they could get in order to have any shot at making health care reform a reality. Clearly Hillary was trying to woo Bernie's support, as she surely did with so many others in Congress, but it ultimately didn't work. In the end, Sanders wouldn't compromise, wouldn't support their plan, and ultimately wasn't much help to the Clintons.
But it doesn't stop there. In the same excerpt, he then blames the Health Security Act's failure solely on lobbying and ad spending by the insurance companies rather than a lack of votes for it in Congress! Did he really, seriously believe that there wouldn't have been a similar (or likely far worse) opposition effort if single-payer had a real shot at becoming law? Please.
He doesn't sound too bothered that 'Hillarycare' went up in flames, does he? It wasn't his thing, the insurance companies shot it down anyway, and that's that. But to his credit, perhaps he learned something...because he did end up supporting President Obama's similarly "complicated and compromised" Affordable Care Act in '09 and '10.
Whatever the case, it looks like we have an answer to the question of where Bernie Sanders was on health care reform in '93 and '94: Against the Clintons.
UPDATE: Just three hours after this was published, the Huffington Post has independently confirmed Hillary HQ's report.
But his presence at that speech -- like a gracious letter Clinton sent him in 1993 -- isn't proof that Sanders was a major ally of the Clinton health care effort. Sanders wasn't even endorsing the Clinton plan, unlike another member of Congress who attended the rally.
And an internal memo from the Bill Clinton administration shows that the White House was worried that Sanders might hold out. "Given his reputation for independence and his more combative style [he] may be of the more difficult members to get on board the Administration's proposal," the memo said.
In fact, Sanders opposed the proposal throughout the process, his campaign confirmed to The Huffington Post. As late as August 1994, while Democrats were making a last-ditch effort to pass Clinton's plan, Sanders held a press conference opposing it -- and touting single-payer, according to his campaign.
Sanders' commitment, or lack thereof, to the White House plan wasn't a cause of its failure, considering staunch resistance from the health care industry, Republicans and conservative Democrats. But Clinton clearly hasn't forgotten it.


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Caipirinhas and swimming shouldn´t mix
Arraial do Cabo Travel Blog› entry 5 of 99 › view all entries
Yesterday was excellent, the reason why people go on these sorts of trips.
We went out on a boat at 11am, a nice yacht but running the motor with the sails put away. There were unlimited drinks throughout the trip, including caipirinhas! Result! They took us all around the bays and then dropped us off at the first beach. We did some snorkelling and some people found a turtle swimming just off shore. I saw a Trumpet Fish, apparently, but yet to be confirmed.
Then back on the boat to go across to a beach on the other side with a big sand dune behind it, where we rented boards for 5 Reais and did sand boarding. The boat anchored fairly near to shore this time and so I was able to live the dream of diving off a yacht into the sea and swimming to the beach, and then swimming back later. We got two more caipirinhas in before arriving back in port. All for 25 Reais, or less than 10 pounds!
In the evening we got a bus to the nearby city of Cabo Frio to savour some nightlife, which is lacking in Arraial. Brilliant, there was a strip of bars and restaurants all along the side of a big canal. Water fronts and bars always make a great alliance!


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NEWINGTON — Management at the Mall at Fox Run, Pheasant Lane Mall, Mall at Rockingham Park and Mall of New Hampshire announced these properties will have extended holiday hours starting at 8:00 p.m. on Thursday, November 28, to greet shoppers and families eager to begin their tax-free holiday shopping for the 2013 season.
Families, friends and shoppers are welcome to join us at the Mall at Fox Run, Pheasant Lane Mall, Mall at Rockingham Park, and Mall of New Hampshire for special shopping events and activities during the Thanksgiving holiday weekend. Everyone can come and walk off those Thanksgiving calories at 8:00 p.m., and begin crossing names off their holiday lists as well as getting some great deals for themselves.
As many families enjoy entertainment traditions around Thanksgiving, whether it's sharing meals, going shopping, seeing a movie, or watching a game together, heading to the Mall at Fox Run, Pheasant Lane Mall, Mall at Rockingham Park and Mall of New Hampshire with family and friends is yet another perfect way to wrap up a holiday about gratitude and togetherness.
All four of the Simon New Hampshire malls are excited to meet their customers' different schedules and needs with the variety of retail and entertainment experiences they can enjoy at each property, plus extended opportunities for holiday 'doorbusters' and deals to be discovered.
Each Mall property is offering:
— Greeters. All Simon Malls will have greeters on Black Friday and some will have them during peak shopping times during the entire holiday season; the greeters are a great resource for shoppers to check in to get the latest and greatest information about the deals and promotions being offered that particular day.
— Gift Wrap stations benefiting various local charities are offered.
— Holiday Entertainment: Local holiday entertainment including carolers, choral groups, and other merry-makers will perform for holiday shoppers, adding to the festive holiday family environment.
— Santa and Holiday Pet Photos will be available to families to capture the moment. Times differ from mall to mall; check simon.com and/or each mall's Facebook page for ongoing updates.
— The American Express®/Simon Giftcard is a groundbreaking gift card available at Simon Malls across New England. The card comes in festive holiday designs, comes with no monthly fees or expiration date on funds, and is available in any denomination between $20 and $500. Follow Mall at Fox Run on Facebook: www.facebook.com/TheMallAtFoxRun or Twitter: @FoxRunMall


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Original paper
Antrodia neotropica sp. nov. (Polyporales, Basidiomycota): a new South American species of Antrodia s.s. from Brazil based on morphological, molecular and ecological data
Kaipper-Figueiró, Gesieli; Robledo, Gerardo Lucio; Reck, Mateus Arduvino; Góes-Neto, Aristóteles; Drechsler-Santos, Elisandro Ricardo
Nova Hedwigia Band 103 Heft 1-2 (2016), p. 125 - 143
published: Aug 1, 2016
DOI: 10.1127/nova_hedwigia/2016/0340
ArtNo. ESP050010301009, Price: 29.00 €
During the revision of the Antrodia species from the Atlantic Forest in southern Brazil, some specimens collected on dead branches of Baccharis (Asteraceae) were found to represent an undescribed species, Antrodia neotropica sp. nov. Morphologically, this new species is characterized by resupinate to effused-reflexed basidiomata with a cream to brown pore surface, a dimitic hyphal system and basidiospores that are cylindrical to subcylindrical, in dorsi-ventral view with an obovoid aspect, and from the side-view present a sigmoid ventral side. Molecular phylogeny inferred from nrITS and nrLSU sequence data confirmed that this species belongs to the Antrodia s.s. clade, closely related to European and North American species, A. serpens and A. heteromorpha. Its relationships within Antrodia s.s. as well as biological and morphological characters of Antrodia s.s. are discussed.


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Program of Study and Apply for Graduation
The Program of Study (PoS) form should be sent to the Grad School for approval when you think you are about a year from being finished with your degree requirements, or earlier if there is anything unusual about your plan (special permissions, transfer courses, etc). Use the Masters PoS form for either the MAS or the CAS program.
Everything on the PoS form is fairly self-explanatory. Under "degree" put either "Masters" or "Certificate" as appropriate. Under "major" put "Applied Statistics". Leave "track" blank. No foreign language is required. Leave the grades blank for classes you have not yet taken – just say when you plan to take them. If something in your plan changes after your PoS is submitted, we can file an amended PoS.
After filling in all remaining information on the form, save it to your computer, print it out and sign it. Send it to Brian Habing for signatures (scan-n-send to email@example.com or fax to 803-777-4048 is ok). He will add memos for exceptions if needed, and send it on to the Grad school for their approval.
Once the approved PoS is on file, when you are just beginning your last semester, you must apply for graduation using the "Application for degree" form. Please remember this application is due *very early* in the semester in which you hope to graduate. Do not apply for MAS graduation unless you have already passed both comprehensive exams !


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California Bucket List
Heading to California is a dream of many! However, not many people know what they will do when they get there. A bucket list is a list of items that you want to accomplish. There is never a set time given for the bucket list, however, most people want to accomplish those items ASAP. Here are a few items you will want to include in your California bucket list.
- Actually go to California – Before you can complete your bucket list, you have to go to Cali. However, make it a fun way to get there. Train? Plane? Car? Your choice! Maybe you can cross something off your bucket list.
- Walk under the Cypress Tree Tunnel – This has been something on my "must do" list for a while. Walking under that tunnel is going to be something I never forget, many say it is so beautiful you don't want to miss out on taking pictures.
- Watch the sunset – One item on my California bucket list is watching the sunset. The thing about California is there are many places to watch the sunset. One place I would love to watch it would be at Cliffs Natural Park. The sunset there is said to be like no other.
- Wine tasting in California – Cali is well known for its wine. However, my bucket list doesn't include making wine, it includes tasting the wine. I can't wait to grab a glass and taste the fruits of everyone's hard labor.
- Watch whales at the bay – Please, please, please put this on your California bucket list. Watching the whales on Monterey Bay will change your life forever. Many people say sightings of the humpback whales are frequent.
- Visit Hollywood – There is just something incredible about visiting the Hollywood sign in California. I have it on my bucket list to get a picture in front of that sign. As I'm sure many of you do as well!
- Eat the food – When in California, it is wise to try as much food as you can. This is exactly what I plan on doing as well!
As you can see, California is an amazing place. It is full of beaches, mountains, famous people, and landmarks that you can't forget anytime soon. Before you head out on your trip, make sure you get your car looked at.
I would love to hear your California bucket list. What do you plan on seeing while in this amazing state?


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With Father's Day coming up, I tend to think a bit more about my husband's role as a father to our kids. I've heard of some cases where the fathers have very little involvement with their kids, in comparison to the mothers. However, that is definitely not the case in our home. My husband is front and center in our family. And it's not just because "Abba" (Hebrew for father) is easier to say than "Eema" (Hebrew for mother).
He loves to make the kids laugh. He'll joke around with them, make silly faces, and sneak up and surprise them. He does different voices when he reads to them, which they also find funny. He isn't afraid to step out of his comfort zone in order to get a good laugh. I love hearing the uninhibited joy in their voices when they genuinely laugh at something he does. He loves to play and it shows. He plays board games with the boys or even invents a new game for them to play. He teaches them how to play video games, as well. He'll also take them to Chuck E. Cheese or the playground and become totally involved in whatever they are doing, even participating himself. He's also firm with them when they misbehave and makes sure to explain why their behavior earned consequences. He's fair and authoritative, but is not afraid to be the "bad cop" when it's warranted.
My husband is very much involved in their well-being. Not only does he take our daughter to speech therapy every week, but he's also involved with her physical therapy sessions. He also acts as advocate for both E and our daughter when it comes to anything regarding their hearing loss. He reads E's IEP from beginning to end, documenting all the details to make sure E is receiving the correct services. When M was experiencing a health issue that concerned him, he reached out to get some answers.
He cares deeply about their religious development. He involves them in discussions about Jewish topics, reads a children's version of the Torah to them every week, takes them to shul (if they are awake enough to join him), practices Hebrew with them, etc. I have a feeling he'll be helping them write their Bar Mitzvah speeches one of these days. He will do whatever it takes to keep Judaism positive and enriching for them. He recently came up with a game to help them learn Hebrew, so that it would also be positive and motivating for them. Shortly after playing the game on Shabbat, E wanted to participate in evening prayers.
I hope my husband knows how much I appreciate him both as a spouse and as a father. I don't just need Father's Day to tell him this, but his role in our family means so much more to me than anything that is printed in a card.
About Melissa A
Melissa A. and her husband have 2 young sons, E and M, and a new baby daughter. Melissa's son E has hearing loss and wears a cochlear implant. Melissa works as an administrative assistant for a non-profit and also runs a bullying prevention group and a book-related fan group, in addition to blogging for Empowering Parents. You can check out Melissa's personal blog here.


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K&L Gates has hired Addleshaw Goddard corporate crime chief Elizabeth Robertson into its equity partnership.
Robertson joined Addleshaws' fraud and regulatory practice in May 2007 from litigation boutique Peters & Peters. Hers was seen as a high level appointment intended to boost the firm's white collar crime expertise.
In 2010 Robertson represented the three Pakistani cricket players, Mohammad Amir, Mohammad Asif and Salman Butt, who were subsequently jailed for their part in a betting scam
Robertson left Addleshaws in December, sources close to the firm said, but it was not until this month that she secured a place in the equity partnership at K&L Gates.
It is understood that she will be the first partner in K&L Gates' white collar crime and criminal defence practice.
Calls for comment from K&L Gates were not returned.


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Carson Rau of Ventnor and Kristen Shema of Telford, Pa., were the overall winners of the 41st annual Harry Yates Memorial Swim on Saturday in Atlantic City.
Rau, a member of the Atlantic City Beach Patrol, won the race in 11 minutes, 56 seconds. Shema was third overall and the women's champion in 12:31.
Former A.C. lifeguard Mike Toy was second overall in 12:26, and Thomas Patterson was third among men and fourth overall in 12:32. Liz Casey was 10th overall and took second in the women's division in 13:48.
12 and under-Douglas Grisbaum 13:54; Erin Shema 16:14. 13-14-Michael Courter 12:53; Lainie Estlow 17:07. 15-19-Patrick Lavery 12:34; Sarabeth Henne 14:54. 25-29-Sergei Fedorov 16:41; 30-34-Dan Smith 16:20; 35-39-Thomas Patterson 12:32; 40-44-Nicholas Raben 16:25; 45-49-Doug Bergen 13:23; Liz Casey 13:48. 50-54-Brian Donnelly 14:41; 55-59-Mike Toy 12:26; Linda Hoke 15:14. 60-64-Jack Martin 13:07; Delia Perez 18:21; 65-69-Bill Gallagher 17:36.


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Data mining: comparing the empiric CFS to the Canadian ME/CFS case definition†
- †
We appreciate the financial assistance provided by the National Institute of Allergy and Infectious Diseases (grant numbers AI36295 and AI49720).
This article contrasts two case definitions for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We compared the empiric CFS case definition (Reeves et al., 2005) and the Canadian ME/CFS clinical case definition (Carruthers et al., 2003) with a sample of individuals with CFS versus those without. Data mining with decision trees was used to identify the best items to identify patients with CFS. Data mining is a statistical technique that was used to help determine which of the survey questions were most effective for accurately classifying cases. The empiric criteria identified about 79% of patients with CFS and the Canadian criteria identified 87% of patients. Items identified by the Canadian criteria had more construct validity. The implications of these findings are discussed. © 2011 Wiley Periodicals, Inc. J Clin Psychol 67:1–9, 2011.


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At the All-Star break, this award looked like Jose Reyes' to lose. The Mets shortstop was having a dominant year, and had piled up 5.2 fWAR in 80 games. In the 46 games after the break, he was worth just 1.0 fWAR, and tailed off in the race. Other candidates stepped up big time, and the award looked a lot more competitive at the end of the day. But when all the votes were tabulated, two candidates stood head and shoulders above the rest.
Midseason winner: Jose Reyes, New York Mets
1) Matt Kemp (88 points, seven first place votes)
2) Ryan Braun (81 points, two first place votes)
3) Joey Votto (50 points)
4) Justin Upton (45 points)
5) Roy Halladay (45 points)
6) Clayton Kershaw (43 points)
7) Troy Tulowitzki (38 points)
8) Jose Reyes (34 points)
9) Prince Fielder (21 points)
10) Cliff Lee (13 points)
11) Albert Pujols (10 points)
12) Andrew McCutchen (8 points)
13) Shane Victorino (8 points)
14) Ryan Howard (6 points)
15) Lance Berkman (5 points)
Matt Kemp is our winner, after his fantastic 2011 campaign. His 8.7 fWAR led the National League, nearly a full win more than runner up Ryan Braun's 7.8. Kemp finished one home run shy of going 40/40 on the year, he hit .324, he won both a gold glove and a silver slugger (not that I care much about those awards…), and it was an all-around fantastic year for the Dodgers center fielder. A year after hitting .249 with a .760 OPS, and rumors of him being shipped out of LA, Kemp absolutely exploded in 2011, increasing his batting average by 75 points and his OPS by more than 300. You want to talk about a superstar? Matt Kemp has become that guy for the Dodgers. Braun has also continued his path of rage as a Brewer, and 2011 was his best of five great seasons. He hit a career high .332, cracking the .300 mark for the fourth time in fifth seasons. He also cracked 30 homers for the fourth year, and 100 RBI for the fourth year. In a nice little note about Braun's career, his walk rate has increased and his strikeout rate has decreased in every year of his career. Braun is the face of the Brewers, and only Kemp was able to prevent him from winning the award this season. A 30/30 season with a .994 OPS is absolutely MVP worthy, though.
Joey Votto had another sneaky good season, a year after winning the NL MVP award. Votto wasn't as good this year, with his OPS dropping from 1.031 to .947, and his fWAR falling to 6.9 from 7.3, but when we're talking about a 6.9 WAR season as a decline, that's a pretty special player. Votto continues to walk at a monster rate, tallying 110 on the year in 2011. He also fell one home run short of 30 in 2011, which would be the second straight year at that mark. Votto couldn't carry his Reds to another NL Central title, but Justin Upton was able to carry his Diamondbacks to the NL West championship. Upton had the best season of his young career, with an .898 OPS, 31 bombs, and 21 steals. That OPS tied his career high, set in 2009. The scary part about Upton is that he's only 24, and getting even better.
I talked about Roy Halladay and Clayton Kershaw in the NL Cy Young voting on Tuesday, and the probable reason that Halladay finished a tad bit higher than Kershaw was probably because of Halladay's Phillies making the playoffs. Let's take a look at Rockies shortstop Troy Tulowitzki, who won both the silver slugger and gold glove this year (again, not that I care), was actually slightly less valuable in comparison to last year (6.5 fWAR to 6.3 fWAR), but he was still an amazing hitter, especially for a shortstop. Tulo homered 30 times, hit .302, OPSed .916 and played great defense for a Rockies team that struggled a lot in 2011. If his team finished higher than fourth in the NL West standings, you have to think that he would have finished higher in the MVP voting. Another NL shortstop, Jose Reyes, struggled in the second half while dealing with hamstring injuries. I talked about his year a little bit in the opening, but he still managed to lead the NL with a .337 batting average while OPSing a career high .877 and stealing 39 bases.
A pair of slugging NL Central first basemen received little support in our voting, with Prince Fielder finishing ninth, and Albert Pujols not even cracking the top ten. Fielder's 2011 wasn't as good as his awesome 2007 and 2009 seasons, but it was still damn good. 38 homers and a .981 OPS for a 27 year old free agent? Yeah, I'll take that. His value to Milwaukee was thwarted a little bit by his awful fielding and baserunning, and the presence of Braun in the lineup overshadowed him a little bit. As for Pujols, he was part of a loaded Cardinals lineup that would end up leading the team to the World Championship, but his .907 OPS was a career low. It was the first year of his career where he didn't hit .300 (.299) or drive in 100 runs (99). But regardless, he's still Albert Pujols, one of the best players in baseball. This year though, there were a few players better than him.
Cliff Lee of the Phillies got mentioned in the Cy Young balloting, but a pair of his teammates got some love in the back-end of the MVP voting: Shane Victorino and Ryan Howard. Howard is no stranger to MVP ballots, but 2011 was his worst season ever, with a career low .834 OPS. But hey, when that's your career low, you've got something going on. Howard gets a lot of love because of his high RBI total (116 this year, his six straight year in triple digits), but he's a terrible defender and baserunner. His teammate Victorino is neither, a fantastic baserunner and fielder who's bat matured greatly this year. His 19 stolen bases were his lowest since 2006, but his 17 homers were one off of a career high. And then there's his .846 OPS, a career high. Speaking of stud center fielders, Andrew McCutchen fell off in the second half, but still had a great year in relative anonymity in Pittsburgh. His batting average plummeted to a career low .259, but his rate stats remained consistent for an .820 OPS. McCutchen also went 20/20, with 23 homers and 23 steals on the season. The final man to receive votes on our ballots is NL comeback player of the year Lance Berkman. He had a .959 OPS a year after being left for dead with a season split between the Astros and Yankees. His 31 homers were his highest in a season since 2007. It was a great rebound season for Berkman.


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Digital, Startups & Tech Roundup — Issue #42
Who Knew A Volvo Ad Could Make You Re-Examine Your Life Priorities? — www.fastcocreate.com
"There is no point planning for a future, which, when you get to it, and it becomes the present, you won't be there." — Alan Watts
Google Developing Robust Mobile Analytics for YouTube — www.adweek.com
Snickers Turned Its Preroll Ads Into Wacky Little Video Games You Can Actually Play — www.adweek.com
From Snickers' point of view, skipping its YouTube preroll ads is just as bad skipping one of the Mars candy brand's chocolaty treats when you're hungry. So, to boost viewer engagement in the prerolls, Impact BBDO in Dubai just unwrapped "Pre-Video Videogames" — ads you can actually play that are also packed with lou
The mobile tactics that turn consumer off — www.marketingweek.com
Buying on mobile is set to rocket but consumers are becoming increasingly wary of retailers' tactics to push them into a sale or hand over data.
Amazon will now send your kids tech and science toys for $20 a month — www.theverge.com
Amazon is launching a new STEM Club subscription service for monthly, STEM-related toys. For $19.99 a month (plus tax), Amazon will deliver a "hand selected" educational toy that will theoretically…
Boom completes wind tunnel testing, paving the way for supersonic airplane construction — techcrunch.com
Supersonic airplane startup Boom has just completed a key step on the way to building a production supersonic passenger jet; the startup finished its wind..
Facebook Stories puts a Snapchat clone above the News Feed — techcrunch.com
Instagram put a dent in Snapchat by placing its own version of Snapchat Stories atop its feed, and now Facebook is doing the same. Today Facebook launched..
Instagram Confirms Live-Streaming Coming to All Users This Week — www.socialmediatoday.com
Instagram has confirmed that their new live-streaming tool is being rolled out to all users this week.
Tech News
Elon Musk says to expect "major" Tesla hardware revisions almost annually — techcrunch.com
Tesla CEO and founder Elon Musk has advice for prospective buyers hoping their vehicles will be future-proof: Shop elsewhere. Musk made the comment when..
Pipedrive, a CRM platform to help SMBs sell more, closes $17M Series B led by Atomico — techcrunch.com
Pipedrive, a CRM platform designed to help SMBs sell more, has closed $17 million in Series B funding. The round was led by Atomico, the global VC firm..
How to Write a Business Plan that Investors Will Love
You want to write a great business plan that investors will love, but you have no idea how. Luckily, the experts at Bizplan have given us some great tips!
Connected doorbell startup Ring raises $109 million from DFJ, Goldman Sachs, Qualcomm, others — venturebeat.com
From beds to fridges and beyond, the so-called Internet of Things took a giant leap forward in 2016, with consumers the world over encouraged to connect the most random of household items to the internet.


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What are people saying
about Rachel's Bake Lab?
We used Rachel's Bake Lab for a close friends baby shower. We ordered a red velvet and a blue funfetti cookie for each guest. Lets just say they were both a hit! I am not a big fan of red velvet and I even fell in love with them! Order was easy as 1-2-3, Rachel was super friendly and all the cookies were individually wrapped and fresh as can be! Thank you Rachel!
- Sean G.
Holy cow! Saying these are my favorite cookies would be an understatement. Not only do they have a great texture but the variety of flavors is refreshing. Rachel has to be using magic to make these and on top of that she's just the nicest person ever.
- John E.
I could not get this chocolate chip cookie in my mouth fast enough. The cookie is beautifully thick and the texture is both firm and cake-like simultaneously with the perfect amount of chocolate chips. Imagine a soft shortbread type cookie but in the classic flavor of chocolate chip. It is a perfectly delicious, classic cookie! I thoroughly enjoyed it!
-Michelle D.
Companies I've worked with...
Fantastic! I got my cookies hand delivered by the CEO! Delicious.
- David W.
Sent these to my brother for his birthday, and he called to thank me (which is by itself a 5 star review) and said they were the best cookies he's ever tasted!
- Emily M.
We are working with Rachel for weekly deliveries to our leasing office. She hand delivered our beautiful box of perfectly packaged cookies. We ordered the M&M spring time cookies this week and O-M-G please take them out of our office because I just want to stuff them all in my face!! They are the perfect texture, and aren't too sweet. Rachel has definitely perfected the cookie, which may or may not be a good thing for me lol.
-Julie M.
So so so good! I ordered the chocolate chip cookies and they were delicious. Love that they are individually packaged making them easy to share, though you certainly will want to keep them all for yourself! Thanks Rachel!
- Marisa C.
I got the chocolate peppermint cookies for Christmas and I can't decide whether to eat them all immediately or save them for the rest of my life. I am torn between keeping them forever and stuffing my face completely full right this minute. So good! Will definitely be a repeat customer!
- Kim B
I bought a mint green t-shirt and I love the way it fits! It's the perfect length and it's made from extremely comfortable material. Plus, it looks great on! I also ordered the strawberry shortcake cookies and they are a dream come true. I can't wait to order more!
- Heather Q.
Strawberry cheesecake cookies!... Are heaven in your mouth!! They were very soft and fresh when they found my place in New Jersey. Each cookie is individually wrapped, good for sharing and saving the rest in freezer. And needless to say, they go great with a cup of coffee and a good book after a long day wink emoticon Thank you, Rachel's Bake Lab!!
- Pelin A.


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ShellDockley wrote:I second the recommendation for Kintore Way. However I'm interested in why the nursery has to be rated as Outstanding by Ofsted?
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text: Beaded suns looped together, bracelet measures 6 1/2 inches, with clasp it is 7 1/4 inches long by 5/8 inches. Made with size 11 seed beads and bead netting. Pattern includes full instructions (step by step), easy to read diagrams, material list, bead guide and finished pictures of the piece. Instructions included to make your beaded bracelet shorter or longer. Purchase of this beaded bracelet pattern entitles you to use the instructions for personal use, does not entitle you to reproduce the pattern for resale or redistribution to others. Make and sell as many as you like, but state that the pattern is a design by ZaneyMay. BASIC SKILLS NECESSARY net beading SIZING / FINISHED MEASUREMENTS made to fit


text:
Mobile telecom company Fongo Inc. says it's making a bid to buy Wind Mobile's Canadian operations.
The Waterloo, Ont., company didn't say how much it will offer for Toronto-based Wind, which launched in 2009.
Fongo says it plans to retain the majority of Wind Mobile's employees but will rethink many aspects of the business.
The company also says that since February 2012 more than 250,000 Canadians have signed up for Fongo's free mobile phone service through a mobile app.
Wind Mobile, part of a wave of new wireless companies offering more cellphone competition in Canada, was launched by founder Tony Lacavera with financial backing from Egyptian telecom player Naguib Sawiris.
They are expected to make a bid to buy back the company from its current Dutch owner, VimpelCom, which has put Wind Mobile up for sale.
However, Dvai Ghose, an analyst with Canaccord Genuity, questioned the seriousness of Fongo's bid in a note to investors on Wednesday. Having never heard of the company before, he was left relying on the private company's description of itself from its website.
"Does Fongo really have the capital and expertise to acquire and build Wind Mobile?" Mr. Ghose asked, noting that the company was private and he had no access to its financial data but that aspects of the bid did not make sense. "While we are not taking the Fongo (news) release overly seriously at this time, the question remains is Wind for sale and are there real buyers?"
It was also not apparent that Wind executives were taking the Fongo bid seriously. Simon Lockie, Wind's chief regulatory officer, tweeted: "I hereby publicly offer to buy Fongo for $1 and half my sandwich. Hopefully the story gets picked up before I get too hungry to deliver."
With a file from The Globe and Mail staff


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History reveals itself when, in the 17th century, a Jesuit missionary ventures into the Canadian wilderness in search of converts - the defining moment of first contact between radically different worlds. What unfolds over the next several years is truly epic, constantly illuminating and surprising, sometimes comic, always entrancing, and ultimately all too human in its tragic grandeur.
Christophe has been in the New World only a year when his native guides abandon him to flee their Iroquois pursuers. A Huron warrior and elder named Bird soon takes him prisoner, along with a young Iroquois girl, Snow Falls, whose family he has just killed, and holds them captive in his massive village. Champlain's Iron People have only recently begun trading with the Huron, who mistrust them as well as this Crow who has now trespassed on to their land; and her people, of course, have become the Huron's greatest enemy. Putting both to death would resolve the issue, but Bird sees Christophe as a potential envoy to those in New France, and Snow Falls as a replacement for his two daughters who were murdered by the Iroquois.
The relationships between these three are reshaped again and again as life comes at them relentlessly: A dangerous trading mission, friendly exchanges with allied tribes, shocking victories and defeats in battle, and sicknesses the likes of which no one has ever witnessed.
The Orenda traces a story of blood and hope, suspicion and trust, hatred and love, that comes to a head when Jesuit and Huron join together against the stupendous wrath of the Iroquois, when everything that any of them has ever known or believed faces nothing less than annihilation. A saga nearly 400 years old, it is also timeless and eternal.
©2013 Joseph Boyden (P)2014 Recorded Books
"Thoughtful and interesting, if not always gripping"
I've read a few novels on the subject of the interactions between the French missionaries and the First Nations in North America during the 17th century, and this one is essential reading if you're interested in the subject. Boyden has clearly set out to immerse himself in both cultures and to try to give each an equal amount of respect. The missionaries are naive and arrogant but are also brave and have integrity in their spiritual beliefs. The native belief system and way of life is made fully comprehensible and possible for the reader to identify with yet Boyden doesn't sentimentalize the First Nations into New Age hippies - he pulls no punches in depicting their culture as patriarchal and militaristic. It's an amazing depiction of two worlds that feel intensely real and are trying to understand each other. And the plot never goes in the directions that you think it will.
I should also warn listeners of a sensitive disposition that the novel contains numerous detailed and intensely disturbing descriptions of the long, drawn-out tortures of prisoners that dominated the wars between the Huron and the Iroquois. It is the stuff of nightmares and while it's essential to the plot and themes, many listeners will find it hard to deal with.
Although I found the novel fascinating on an intellectual level, the characters and story sometimes left me cold and felt a little flat. The main problem is that although the two cultures are presented with superb complexity, the three protagonists are excessively good-hearted and admirable, to the extent that they feel rather cardboard when compared with the minor characters. This problem is exacerbated by the three readers, who are all competent but never exciting. This makes parts of the novel drag.
Overall though, this is essential reading for anyone with a strong stomach and an interest in the subject.
"Excellent, but very explicitly violent"
I learned a lot listening to this book. It gave me a better understanding of this period of history in eastern Canada, and also helped me appreciate the Huron's deep connection to the natural world. The book is VERY explicitly violent, which I did not like, but the culture of the Iroquois and Hurons was very violent and the book just presents that reality. The author does a great job of character development. Naration is excellent.
"One of the BEST books I've listened to so far!"
This story will capture you from it's very beginning and will hold you fascinated until the very end. Not only is it very entertaining but also enlightening. This book is a must read for all even though it is very violent and disturbing.
"Fantastic. Unbelievable."
Absolutely worth it. Harrowing, but beautifully narrated and a terrifying picture of early canada. A must read for anyone curious about early Canadian history and the relationship between the native Canadians and the Jesuit priests who landed there.
"A great realization of history but..."
I couldn't believe that people who lived to together in such a raw and intimate environment would be able to maintain the lack of understanding shown by both sides. The descriptions of violence fit the time and place, but overwhelmed the arc of the characters. The "tragedies" of the book where inevitable, but no one seemed to learn anything of value from them.
Probably. He writes well and his main characters are accessible and developed. I think it was a great effort but missed the most captivating opportunity.
Snow Fall's vengeful attack on Bird, while he slept ended with a wonderfully conceived consequence, and a powerful intertwining of their fates. Gosling's many applications of "magic" were also well thought out.
Not network. Perhaps a good director on a premium cable network could do it justice.
I'm fascinated by the world of the Iroquois and Huron. I'm eager to find good literature that "lives" with them before the appearance of the European blight.
Thank you Joseph Boyden for reading The Orenda yourself. You gave voice to the words that breathed life to your story that I could reach out and connect to -
"indigenous spirituality meeting western history"
Ceremonial real-life exposed.
The Huron leader's trusted friend is loyal and courageous, undaunted, a warrior.
The voices of the three main characters are extremely well differentiated.
The jesuit priest is so dogged in his missionary work, so disrespectful of the orenda, and his death is correspondingly marked with the respectfulness of his "sauvages".
Joseph Boyden brings to life that time in history (around the 16th century).
"Torture to listen to"
I dreaded listening to this book and admit that I gave up about halfway through. It is simply depressing and filled with graphic depictions of tortures, murders and general bad interpersonal relationships.
"Gripping in its realism"
Immersed in the world of colonialism and Huron-Iroquois warfare, the Orenda is vivid and intense in its telling of three paralel stories, offering rich point of views on all sides. Boyden is uncompromising when describing this at times violent and unforgiving period, while maintaining an immaculate sense of historical context. Not a light novel, it's simply fantastic in its realism as it immerses you in the great lakes region of the 16th century.
"this could be a great movie"
the narration of this book made me feel like I was watching a movie and reminded me of the movie The reverent. I could see this becoming a movie by some famous director like Martin Scorsese. The background took place in the north east of North America. I grew up in this area so it was interesting to hear the names of tribes we learned about in high school. a brutal story of imaginable hardships.
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text: I have a few inter-related issues:Why would one kernel boot a card that another kernel can't?Why would a card's disk geometry matter for boot?Who is a good manufacturer for getting hardware-identical cards in bulk? How can I probe the actual "disk geometry" of an sd card?I bought 100 Transcend SD cards a little while ago and duplicated them with an OpenEmbedded-based filesystem (linux-2.6.36). There were a few "bad" cards that I threw out, but the success rate was acceptable.In the next round of 40 SD cards I used a Linaro-based filesystem (linux-2.6.39) and had about a 50% failure rate when testing that the cards would boot, which is absurd. There kernel reports: [ 1.003204] mmcblk0: unknown partition tableHowever, the cards would mount and show files just fine.I reduplicated one of the non-booting cards with an OpenEmbedded filesystem and then it booted. Weird! After some investigation I found that using `gparted` (instead of `fdisk`) to create a new partition table and then `rsync`ing the contents of the original filesystem resulted in a booting Linaro card. Rinse and repeat and I ended up with 3 images which only vary by the disk geometry as reported by `fdisk -l`:50% -- 255 heads, 63 sectors/track, 974 cylinders 40% -- 2 heads, 4 sectors/track, 1957632 cylinders10% -- 247 heads, 62 sectors/track, 1022 cylinders 1 card still didn't bootI'm lost. Please advise.AJ ONeal Non-booting kernel message[ 0.923309] Waiting for root device /dev/mmcblk0p2...[ 0.957885] mmc0: host does not support reading read-only switch. assuming write-enable. [ 0.982025] mmc0: new high speed SDHC card at address b368[ 0.988494] mmcblk0: mmc0:b368 USD 7.46 GiB [ 0.993957] mmcblk0: detected capacity change from 0 to 8018460672[ 1.003204] mmcblk0: unknown partition table [ 1.036926] VFS: Cannot open root device "mmcblk0p2" or unknown-block(179,2)[ 1.044433] Please append a correct "root=" boot option; here are the available partitions: [ 1.053344] b300 7830528 mmcblk0 driver: mmcblk [ 1.058959] Kernel panic - not syncing: VFS: Unable to mount root fs on unknown-block(179,2)Booting kernel message[ 1.122070] mmc0: host does not support reading read-only switch. assuming write-enable. [ 1.146087] mmc0: new high speed SDHC card at address b368[ 1.152557] mmcblk0: mmc0:b368 USD 7.46 GiB [ 1.158020] mmcblk0: detected capacity change from 0 to 8018460672[ 1.166351] mmcblk0: p1 p2 p3 [ 1.259674] EXT3-fs: barriers not enabled[ 1.265411] kjournald starting. Commit interval 5 seconds[ 1.271331] EXT3-fs (mmcblk0p2): mounted filesystem with ordered data mode [ 1.278686] VFS: Mounted root (ext3 filesystem) readonly on device 179:2.


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Nothing says that the executive power and excessive luxury like black and gold in an office design. The design prevent too carried away, however, artistic accents soft and add creative interest to this conference, meeting and lounge area i29. The thematic focus on the idea of a spotlight is immediately obvious to a resident of the large circular ceiling lights of the faux shadow them on the ground and throw the spaces on the wall meant by their presence. This repeated theme also offsets what might otherwise be a dusty, overly elegant space.
Ultimately, this design is the right balance between conceptual and practical, artistic and functional. It serves its purpose well enough light, chairs, work and storage, but still provides a radically unique office interior.
The most Beautiful Christmas Tree Decorating Collection
Decorate our Christmas tree every year is usually an all day affair, but as much as love Christmas. . . Read more »
Guest Decorating Articles – Home Decorating
Here are some great guest decorating articles submitted by our home decorating friends. . . Read more »
Interior Design: Holiday Decorating Tips: Christmas Candles
Do you love the winter holiday season or does this time of year cause you stress? One way to lower your holiday stress, using fewer decorations, decreases your "just have to much to do" list. . . Read more »
Best Interior Design – Bathroom TV collection, modern gadget for your Luxury Bathroom
Best Interior Design – Bathroom TV collection, modern gadget for your Luxury Bathroom. . . Read more »
Decorating with coat racks and vintage clothing for a unique accent to your home
When most people think of coat racks, they think strictly of hanging coats, hats, and scarves. . . Read more »
Home Interior Designs Inspiration Ideas
Tags: #Glamourous Black Gold Office #Interior Design #Luxury Bathroom


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Flawless Automotive Repair Inc
ReScore Reviews™ (3)
99% Would Recommend
Latest Review 2 days ago
Review for Flawless Automotive Repair Inc
Marc N. on 10/20/2014
Marc said this business is great and professional. The staff is some of the best he has ever encountered and their facility is neat as a pin. They solve problems, are reasonable and affordable. They stand behind their work one hundred percent, are honest and he has taken his vehicle to them for 8 years.
Vehicle: BMW M5
Category: Service
Service Date: 09/30/2014
Review Created: 10/20/2014
This review was collected via phone interview by Customer Research Inc.


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Laura Quist is a research coordinator for the Community Research Institute at the Dorothy A. Johnson Center for Philanthropy at Grand Valley State University in Grand Rapids, Mich. Quist joined the Johnson Center in 2015. Previously, she was the communications coordinator and research assistant for the Transition to Independence program at Wayne State University in Detroit, MI.
She earned her bachelor's at Grand Valley State University in Sociology and holds a Master's in Social Work from Wayne State University. She continues to write about the challenges faced by current and former foster youth in Michigan.
Quist is from Grand Rapids and grew up in Alger Heights. She loves spending time with friends and family, especially her two young nieces, Violet and Ivy. When she's not working, she enjoys walking her dog, taking trips up north, and enjoying all the great food and drink Grand Rapids has to offer.


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Hackvalue - Tales from the trenches of specialized web hosting and development
Hackvalue is the corporate blog of Loco, a company that does lots of
interesting stuff gravitating around Free and Open Source software and
licensing, web development, and anything else that we find
In this particular part of the blog all content is English, but if you have no problems reading Dutch, then the Dutch part of this blog might also be interesting for you. It caters to a slightly different crowd.
If you're wondering why we use the name Hackvalue, please read the FAQ.
Book review: Everything you know about CSS is wrong
Tags: book review, css, web, webdesign, webdevelopment
Joor Loohuis, January 3, 2010, 6148 views.
While 'Everything you know about CSS is wrong' may be a presumptuous title for a book, it discusses an important development in the layout of websites and applications. The book provides an in-depth discussion of the advantages of CSS table display styles.
Automated certificate expiration checks on CentOS
Tags: administration, certificates, linux, ssl
Joor Loohuis, December 20, 2009, 11746 views.
It is essential to keep track of the expiration dates of SSL certificates, and so it's useful to have a periodic check on all certificates in use on a system, that will alert systems administrators when a certificate is about to expire. This article shows one way of setting up such a procedure for CentOS and other RHEL derivatives.
Bad requests from the Reed Elsevier search engine
Tags: crawler, HTTP, search engine, SEO
Joor Loohuis, December 3, 2009, 4872 views.
Over the last weeks we're seeing ever increasing amounts of strange requests from a crawler that runs on IP addresses used by Reed-Elsevier in Australia. Time for some online forensics.
One off system backup methods
Tags: administration, fedora, rsync, ssh
Armijn Hemel, December 1, 2009, 6445 views.
Before a new system install it is smart to do a backup of important personal data. There are various methods how you can do this.
Off-by-one errors in background placement in web browsers
Tags: browser, bug, Chrome, css, Firefox, Internet Explorer, Safari, web
Joor Loohuis, November 26, 2009, 8478 views.
The rendering engines in some popular web browsers contain a bug, where a background positioned in the center of a block element is sometimes offset by one pixel relative to an element that is positioned with automatic margins.
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Hawk Jones Trailer (1986)
01 January 1986 Action, Comedy 88 mins
'Hawk Jones' is one of the only movies ever made that has an all child cast! With an average age of 8 you won't believe what happens in this movie! The local gangster, Antonio Coppola, is threatening to take over the entire town of Minitropolis. Antonio and his gang of henchmen are causing destruction everywhere; turning the once quiet town upside down.
Watch the official Hawk Jones 1986 trailer in HD below or find more Hawk Jones videos on Vidimovie.