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Matrix metalloproteinases (MMPs) 2 and 9 are two gelatinase members which have been found elevated in exudative pleural effusions. In endothelial cells these MMPs increase paracellular permeability via the disruption of tight junction (TJ) proteins occludin and claudin. In the present study it was investigated if MMP2 and MMP9 alter permeability properties of the pleura tissue by degradation of TJ proteins in pleural mesothelium.
In the present study the transmesothelial resistance (RTM) of sheep pleura tissue was recorded in Ussing chambers after the addition of MMP2 or MMP9. Both enzymes reduced RTM of the pleura, implying an increase in pleural permeability. The localization and expression of TJ proteins, occludin and claudin-1, were assessed after incubation with MMPs by indirect immunofluorescence and western blot analysis. Our results revealed that incubation with MMPs did not alter neither proteins localization at cell periphery nor their expression.
MMP2 and MMP9 increase the permeability of sheep pleura and this finding suggests a role for MMPs in pleural fluid formation. Tight junction proteins remain intact after incubation with MMPs, contrary to previous studies which have shown TJ degradation by MMPs. Probably MMP2 and MMP9 augment pleural permeability via other mechanisms.
Matrix metalloproteinases (MMPs) 2 and 9 are two gelatinase members which have been measured and found to be elevated in pleural exudates of different origin (parapneumonic, malignant, tuberculous). MMPs consist a family of proteolytic enzymes that break down virtually all the protein components of the extracellular matrix. The balance between matrix deposition and degradation is tightly regulated in human tissues and a disruption of this balance has been implicated in several pathological conditions such as cancer, cardiovascular diseases and arthritis [1,2]. MMP2 and MMP9 are thought to be involved in pleural fluid accumulation in the pleural cavity. The disruption of the integrity of the mesothelial layer or the underlying basement membrane, and therefore the facilitation of fluid influx into the pleural space has been proposed as a possible mechanism [3], although no study has been so far conducted.
MMPs have been correlated with the induction of increased capillary permeability in several inflammatory conditions, such as brain and myocardium ischemia injury and diabetic retinopathy [4-6]. In in vitro studies of endothelial cells, MMPs increase paracellular permeability by disrupting tight junction barrier [5,7,8]. Tight junctions (TJs) are a specific type of cell-cell contacts that obstruct paracellular pathway for solute diffusion and they regulate the paracellular passage of small molecules such as water and ions [9]. The two major constituent proteins of TJs are occludin and claudin and the disruption of these proteins in several culture systems has been correlated with increased water and solute flux [9].
This study was designed in order to investigate if MMP2 and MMP9 increase the permeability of sheep pleura and thus contribute to the pathogenesis of pleural effusion formation. The effect of MMP2 and MMP9 on TJ proteins occludin and claudin-1 was examined in order to investigate if MMPs alter paracellular permeability of the mesothelial layer.
MMP2 and MMP9 decrease the transmesothelial resistance of parietal and visceral sheep pleura
We treated parietal and visceral sheep pleura specimens with increasing doses of MMP2 and MMP9 (0.1, 1, 10 and 20 ng/ml) and the RTM was measured over a 40-min period. The RTM decreased in all concentrations studied and this decrease occurred at both parietal and visceral pleura and on both the apical and basolateral side. The decline in RTM suggests for an increase in the permeability of the tissue. As control RTM , we regarded the RTM of the tissue before MMP was added. The mean RTM of parietal pleura was calculated to be 15 ± 3 Ωcm2 and that of visceral pleura 17 ± 4 Ωcm2. Sheets of pleura specimens, at which no MMPs where applied, showed a stable value of RTM at least for a period of 40 min (Figures 1 and 2, control experiments).
Figure 1. Effect of 0.1 ng/ml MMP2 and MMP9 on the transmesothelial resistance (RTM) of sheep pleura. A time-dependent decrease in transmesothelial resistance (RTM) occurred at both parietal and visceral pleura and on both the apical and basolateral side. For MMP2 the decrease on the apical side of parietal pleura is not significant. Data are given as mean ± SD (n = 6). ns: non-significant, * p < 0.05, ** p < 0.001 and *** p < 0.0001 compared to control RTM (ANOVA test with Dunett post-test).
Figure 2. Effect of 20 ng/ml MMP2 and MMP9 on the transmesothelial resistance (RTM) of sheep pleura. A time-dependent decrease in transmesothelial resistance (RTM) of sheep pleura occurred after the addition of MMP2 or MMP9 at concentration 20 ng/ml. The time-dependent response occurred at both parietal and visceral pleura and on both the apical and basolateral side. Data are given as mean ± SD (n = 6). ns: non-significant, * p < 0.05, ** p < 0.001 and *** p < 0.0001 compared to control RTM (ANOVA test with Dunett post-test).
A time-dependent response occurred both apically and basolaterally for all four concentrations studied. After the addition of MMPs, the RTM decreased significantly and declined progressively thereafter up to the 40 min of incubation when the experiment was terminated (Figures 1 and 2). A significant drop in resistance occurred within 5-15 min for both concentrations 0.1 (Figure 1) and 20 ng/ml (Figure 2). However a delayed response was observed when 0.1 ng/ml MMP2 was incubated with the apical side of the visceral membrane (Figure 2b). The effect of 0.1 ng/ml MMP2 on the apical side of the parietal pleura was not significant (Figure 1a). The changes in RTM for concentrations 1 and 10 ng/ml were comparable to these for 0.1 and 20 ng/ml (data not shown).
A dose-dependent response was observed for both MMPs studied (Figure 3): for MMP2 the decrease in RTM was greatest on the apical side of parietal pleura at concentration 20 ng/ml (45 ± 12% of the control value). For MMP9 the greatest effect was observed on the basolateral side of the parietal pleura at the low concentration of 0.1 ng/ml (59 ± 17% of the control value).
Figure 3. Dose-dependent decrease in transmesothelial resistance (RTM) of sheep pleura after the addition of MMP2 or MMP9. Different concentrations (0.1, 1, 10 and 20 ng/ml) of MMP2 and MMP9 were added at parietal and visceral pleura and on the apical and basolateral side and the RTM was measured on the 40 min. The changes in RTM are expressed as percentage difference of the control value (100%), which is the RTM value just before the addition of MMP. Data are given as mean ± SD (n = 6). ns: non-significant, * p < 0.05, ** p < 0.001 and *** p < 0.0001 compared to control RTM (paired t-test).
TIMP2 partially prevented the decrease in RTM induced by MMPs
Next, the MMP inhibitor TIMP2 (200 ng/ml) was applied to the pleura either alone or simultaneously with MMP2 at concentration 20 ng/ml or MMP9 at concentration 0.1 ng/ml. These concentrations of MMPs were chosen because of their maximum effect on RTM. TIMP2 was shown to partially prevent the MMP2 (Figure 4) and MMP9-induced (Figure 5) fall in RTM. This effect was obvious on both the parietal and visceral pleura. Moreover, the application of TIMP2 by itself tended to increase pleural permeability, particularly at visceral pleura and at its basolateral side, as the RTM was 50 ± 5% of control value on the 40th min (Figure 5d).
Figure 4. TIMP2 reverses partially the effect of MMP2 on pleural permeability. TIMP2 was added at concentration 200 ng/ml at on the apical and basolateral side of parietal(a) and visceral(b) pleura. MMP2 was added at the same time at concentration 20 ng/ml. TIMP2 seems to prevent partially the decline in RTM induced by MMP2 on the 40th minute of the experimental procedure. Data are given as mean ± SD. n = 6 for MMP2, n = 3 for combined MMP2 and TIMP2, n = 3 for TIMP2.
Figure 5. TIMP2 reverses partially the effect of MMP9 on pleural permeability. TIMP2 was added at concentration 200 ng/ml on the apical and basolateral side of parietal(a) and visceral pleura(b). MMP9 was added at the same time at concentration 0.1 ng/ml. TIMP2 seems to prevent partially the decline in RTM induced by MMP9 on the 40th minute of the experimental procedure. Data are given as mean ± SD. n = 6 for MMP9, n = 3 for combined MMP9 and TIMP2, n = 3 for TIMP2.
MMP2 or MMP9 do not alter occludin and claudin-1 immunostaining at cell cultures
We next investigated if the increase in transmesothelial permeability, as occurred by Ussing chamber experiments, is accompanied by loss of tight junction proteins by indirect immunofluorescence. The mesothelial cells in control experiments showed a clear membrane pattern for occludin (Figure 6) and claudin-1 staining (Figure 7). The staining was continuous and decorated the cell periphery. Because cells were well grown to confluency and occludin and claudin-1 are located at sites of lateral membrane with cell-cell contact, a continuous line resembling to "honeycomb" pattern was obvious. Non-specific staining of the nucleus occurred. The incubation of mesothelial cells with MMPs had no effect on occludin and claudin-1 immunostaining (Figures 6 and 7, respectively), implying that MMPs do not alter occludin and claudin-1 localization at tight junctions.
Figure 6. Immunofluorescent microcopy of pleural mesothelial cells treated or not with MMP2 and MMP9 using an anti-occludin antibody. Both control cells and cells treated with MMP2 or MMP9 displayed a continuous staining at cell periphery. Nuclei were stained with Dapi.
Figure 7. Immunofluorescent microcopy of pleural mesothelial cells treated or not with MMP2 and MMP9 using an anti-claudin-1 antibody. Control cells displayed a continuous staining at cell periphery. The same pattern occurred also for cells treated with MMP2 or MMP9. Nuclei were stained with Dapi.
MMP2 or MMP9 do not alter occludin and claudin-1 expression at mesothelial cells
Western blotting of mesothelial cell extracts showed no alteration for occludin and claudin-1 staining (Figure 8). Occludin migrated as two main bands with apparent molecular weights from 60 to 65 KDa. An additional band at about 85 KDa was also detected (Figure 8a). Claudin-1 was detected as a 28 kDa band (Figure 6b). Neither loss of band intensity nor the appearance of new protein fragments was observed. These findings imply that MMP2 and MMP9 do not degrade TJ proteins.
Figure 8. Immunoblot analysis of TJ proteins after treatment with MMPs. Occludin appears as a 65 KDa protein (a) and claudin-1 migrates as a 28 KDa band (b). lane 1 = control, no incubation, lane 2 = control, incubation for 1 h at 37°C, lane 3 = incubation with MMP2 for 1 h at 37°C, lane 4 = incubation with MMP9 for 1 h at 37°C. Equal amounts of protein were loaded to each lane (15 μg protein/lane). Antibody against β-actin was used to control the equal amounts of proteins in the lysates (c). No alteration to the tight junction proteins occludin and claudin-1 was observed after incubation with MMPs.
Discussion and Conclusions
In the present study MMP2 and MMP9 decreased the transmesothelial resistance of parietal and visceral sheep pleura in in vitro experiments. This decrease supports that MMPs augment pleural mesothelial permeability. The paracellular pathway, through which mesothelial permeability may increase, was studied. After incubation of primary mesothelial cells with MMP2 or MMP9, mesothelial monolayer integrity was not disrupted and the expression of TJ proteins occludin and claudin-1 in the cytoplasmic membrane remained intact.
MMP2 and MMP9 were selected for our study because they have been found elevated in exudative pleural effusions of different origin [3,10,11]. MMP2 is expressed and secreted constitutively in the pleural cavity by mesothelial cells [12]. In exudates high levels of MMP2 may be due to increased expression as a result of stimulation of mesothelial cells by cytokines or other cells, such as mononuclear cells, might collaborate in MMP2 release [10]. MMPs can degrade almost all components of the extracellular matrix (ECM) and to date it is widely known that MMPs can cleave not only ECM components, such as collagen and elastin, but also non-ECM protein substrates, such as cell surface molecules, ECM-bound growth factors and cytokines released on the ECM [13,14].
In the present study the decrease in transmesothelial electrical resistance of sheep pleura that occurs after incubation with MMP2 or MMP9 suggests for an increase in the permeability of the pleura. As expected for an enzyme, the decrease in resistance of the pleura was time-dependent with the maximum decline in resistance occurring on the 40 min. Regarding the dose-dependent effect, a remarkable difference between MMP2 and MMP9 occurred: for MMP2 the greatest increase in permeability occurred at the highest concentration studied, which is 20 ng/ml. On the contrary, for MMP9 the greatest increase in permeability occurred at the lowest concentration studied, which is 0.1 ng/ml. The reason for this discrepancy is not elucidated but we should take into account differences between enzyme kinetics and substrate specificity.
TIMP2 is a tissue inhibitor of metalloproteinases and inhibits MMPs activity by binding noncovalently to their active site [15]. TIMP2 is found in exudates whereas in transudates its levels are usually no detectable [3]. The application of TIMP2 on the pleura inhibited the decrease in RTM which was induced by MMPs on both the parietal and visceral pleura. This finding enhances our previous finding and suggests that the decline in RTM is an effect caused by MMPs and not a non-pecific result. TIMP2 was selected at the present study because it is 2-9 times more effective than TIMP1 for the inhibition of MMP2 and MMP9 activity [16]. Moreover, TIMP2 tended to increase pleural permeability particularly at visceral pleura and at its basolateral side, when it was applied alone at the pleura. This finding can be interpreted considering the growth-promoting-activity that has been attributed to TIMP2 [17]. A wide range of human, bovine and mouse cells proliferate when incubated with TIMP2. Indeed, receptors of TIMP2 have been identified at the surface of the above cells [18]. One possible explanation is that TIMP2 acts on mesothelial cells via receptor-binding and influences mesothelial permeability, as is the case for numerous growth factors.
We next looked for a possible mechanism explaining the impact of MMPs on the transmesothelial resistance and asked whether TJ proteins, occludin and claudin-1, may be hydrolyzed by MMPs. TJs are a specific type of cell-cell contact which are located in the most apical region of the lateral plasma membrane. The paracellular passage of small molecules, such as water and solutes, is highly regulated by the TJ proteins, including occludin and claudin-1 [9]. Indirect immunofluorescence experiments for occludin and claudin-1 showed a continuous staining at cell periphery which was not weakened and remained at the cell borders after incubation with MMPs. Similarly, western blot revealed that these proteins are expressed at pleural mesothelium under normal conditions and are not proteolytically disrupted by MMPs. More specifically for occludin, western blot analysis revealed three distinct bands: at 60 KDa, 85 KDa and a broad band at about > 60 KDa. These bands may be due to occurrence of splice variants or post-transational modification, i.e phosphorylation. Previous studies have shown that occludin is widely phosphorylated on serine and threonine resides and modulation of occludin phosphorylation regulates cellular localization and paracellular permeability [19,20]. Some degree of phosphorylation may also be the case in the present study. As far as the 80 KDa band is concerned, we cannot rule out the possibility that this band corresponds to a protein complex between occludin and claudin (MWs 60 and 28 KDa respectively). Occludin and claudin do not interact directly to each other, but they crosslink by integral TJ proteins, such as ZO-1, ZO-2 and ZO-3 [9]. The intensity of the above band increased after MMPs incubation, implying that MMPs interfere with occludin and claudin interactions. Because the 80 KDa band appeared enhanced to all three western blot experiments that were performed, it is less likely to represent an artifact. The mesothelial cells used for immunofluorescence and western blotting derived from upper-, middle- and lower-heighted visceral pleura and no difference between them occurred.
Our data is contradictory to previous results which have revealed MMPs as major contributors to the control of paracellular permeability by proteolytic degradation of TJ proteins. More specifically, MMPs have been correlated with an increase in capillary permeability that follows ischemia-reperfusion injury in brain [8], in myocardium [4], in lung [21] and in kidney [22]. These studies attributed to MMPs an elementary role in the inflammatory process and the breakdown of the paracellular capillary permeability during inflammation. In some studies a selective cleavage of TJ proteins occurred [7,23] but the results of our study clearly showed that TJ integrity is not disrupted at mesothelial cells by MMPs. It is possible that other substrates, different from occludin and claudin-1, are a molecular target for MMPs at pleural mesothelium. For example, MMPs have been found to disrupt or reorganize the basement membrane of endothelial cells and thus result to increased permeability [24,25]. Moreover, adherent junctions are also degraded by MMPs and their hydrolysis leads to TJ disassembly and to increased permeability [22,26].
The limitations of the present study are the followings: Firstly, the Ussing chamber technique investigates permeability alterations provoked on mesothelial membrane, which consists a confluent membrane between the apical and basolateral compartment of a chamber. However, under in vivo conditions it is possible that MMPs act not only on mesothelial cells but also on vascular capillaries lying beneath the basement membrane. Secondly, the precise role of the mesothelial layer at pleural fluid turnover is not fully established. Although we used to believe that mesothelial cells are leaky and display no resistance at pleural fluid passage, more recent investigations indicate that the permeability to solutes of mesothelium is of the same order of magnitude as that of the capillary endothelium [27,28]. This means that pleural fluid is a filtrate of pleural capillaries and mesothelium too. Moreover, damage of pleural mesothelial monolayer by lipopolysaccharide (LPS), thrombin and bacteria increase pleural permeability to proteins and demonstrate to play a central role in the formation of effusions [29,30]. Finally, sheep pleura resembles to human pleura as far as morphology and function is concerned. On both pleurae the blood supply comes from the systemic circulation and microscopically two different types of mesothelial cells are found: the cyboidal cells with less developed TJs and flattened cells with more TJs [31]. However, further studies should be performed at human pleura in order to confirm the results of the present study.
Our findings imply an important role for MMPs in the pathogenesis of pleural effusions. Under normal conditions, MMPs and especially MMP2 are found only in small amounts in the pleural fluid. The balance of MMPs in pleural fluid may serve the degradation and turnover of ECM that underlies mesothelial cells and which normally occurs in low rates. However in pleural exudates MMP2 and MMP9 levels increase, as shown from previous studies, and a role for MMP2 and MMP9 in pleural fluid formation is proposed by our study. Tight junctions do not apparently loosen in mesothelial cells after the addition of MMPs. It is possible that other mechanisms exist through which MMPs increase mesothelial permeability. The revelation of the mechanims by which MMPs compromise the mesothelial barrier will provide a better understanding of the pathogenesis of pleural fluid formation.
Specimen collection and preparation of sheep pleura
Intact sheets of visceral and parietal sheep pleura were obtained from adult female sheep. The samples were collected from the slaughterhouse immediately after the death of the animals (time of warm ischemia close to 0 minutes). Pieces of parietal pleura were carefully stripped from the chest wall whereas those of visceral pleura were carefully stripped from the underlying lung. Parietal and visceral pleura were examined for evidence of holes or adherent tissue and were discarded if they were not intact. Immediately after removal, the pleural tissue from the animals was placed in oxygenated Krebs-Ringer bicarbonate (KRB) solution at 4°C and transferred to the laboratory within 30 minutes. The KRB solution was balanced at pH 7.4 and bubbled with 95%O2-5%CO2. The solution contained (in mM) 117.5 NaCl, 1.15 NaH2PO4, 24.99 NaHCO3, 5.65 KCl, 1.18MgSO4, 2.52 CaCl2, and 5.55 glucose.
Electrophysiological transmesothelial measurements
The effect of MMPs on pleural permeability was studied by conducting Ussing experiments under open circuit conditions. The pleura was mounted carefully in Ussing chambers (K Mussler Scientific Instruments, Aachen, Germany) with an opening surface area of 1 cm2. Tissues were bathed with 4 ml KRB solution on each side of the membrane and were continuously oxygenated with 95%O2-5%CO2. Two pairs of Ag/AgCl electrodes monitored the transmesothelial potential difference (Pd, in mV) and the transmesothelial resistance (RTM, in Ωullet cm2) under open circuit conditions.
Transmesothelial electrical parameters were measured before and after the addition of active MMP2 or MMP9 (Calbiochem, San Diego, California, USA) at four different concentrations 0.1, 1, 10 and 20 ng/ml. In some experiments TIMP2, a tissue inhibitor of metalloproteinases, was added at concentration 200 ng/ml. MMPs and TIMP2 were added both apically and basolaterally and the electrical parameters were monitored over a period of 40 minutes (at minutes 5, 10, 15, 20, 25, 30, 35, 40). After the addition of the above substances, alterations in the RTM were expressed as the change from the starting value. Activity of matrix metalloproteinases is temperature-dependent, therefore measurements of transmesothelial electrical parameters were conducted at 37°C. The mesothelial cell membrane facing the fluid side is here called the apical membrane, and that facing the blood side is called the basolateral membrane. The voltage response to applied current pulses of 50 μA amplitude and 200 msec duration was measured. The transmesothelial resistance was calculated deducting automatically the resistance of the solution.
Cell cultures
Primary cultures of sheep pleural mesothelial cells were prepared (modified from Stylianou et al [32]). Briefly, specimens of intact visceral sheep pleura were obtained from the slaughterhouse immediately after the death of the animal. The procedure was performed under sterile conditions and pieces of approximately 6 cm2 were placed in M199 media (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The specimen was transferred on ice to the laboratory, washed with PBS and subjected to enzymatic disaggregation. The specimen was in this incubated for twenty minutes at 37°C in disaggregation solution that contained 0.125% trypsin, 0.01% EDTA and 0.1% glucose in PBS solution. After incubation the pleura membrane was discarded and the suspension was centrifuged at 100 × g for five minutes at 4°C. The resultant supernatant was discarded and the cell pellet was suspended in 10 ml prewarmed M199 media supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.4 μg/ml hydrocortisone, 10 mg/ml insulin, 50 mg/ml transferin and 200 mM L-glutamine and subjected to recentrifugation at 100 × g for five minutes at 4°C. Finally the supernatant was discarded, the cell pellet was resuspended in 5 ml of the same medium and seeded in 25 cm2 tissue culture flasks. The medium was changed every third or fourth day. Cultures contaminated with spindle-like cells, representing probably fibroblasts, were discarded. Confluency of the cells was regarded as a prerequisite for their experimental use. Passages 1 and 2 were used for immunofluorescence experiments and western blot.
Indirect immunofluorescence
For immunofluorescence experiments, mesothelial cells were grown on fibronectin-coated glass coveslips and confluency was reached about 7 days later. After two subsequent washes with PBS, cells were incubated in serum-free media with MMP2 or MMP9 at concentration 10 μg/ml for one hour at 37°C and then were fixed with 3% formaldehyde for 5 minutes at room temperature. Control cells received no MMP treatment and were incubated with serum-free media. After washing, the cells were permeabilized with 1% Triton X-100 in PBS for 15 minutes at 4°C. Samples were washed with PBS and soaked in blocking solution, which consisted of 3% BSA and 0.1% Tween-20 in PBS for 16 hours. Coverslips were incubated with polyclonal anti-occludin (dilution 1:50; Zymed Laboratory, San Fransisco, USA) or polyclinal anti-claudin-1 antibody (dilution 1:50; Zymed Laboratory, San Fransisco, USA) in PBS containing 1% BSA/0.1% Tween-20 for 1 hour at room temperature. Cells were rinsed three times in 1% BSA/0.1% Tween-20/PBS and incubated with a FITC- or CY3-conjugated anti-rabbit IgG secondary antibody (dilution 1:50), for occludin and claudin repectively, for 30 minutes at room temperature. Finally coverslips were washed and mounted with Vectashield containing Dapi. Images were collected with a Leica DFC480 camera (LAS software version V2.3.1R1) on an Axioscope 40 Zeiss microscope.
Western blot
Mesothelial cells were grown in culture dishes and cellular extract was obtained as described previously by Giebel et al [5]. Briefly, mesothelial cells were washed with cold PBS and cellular extract was obtained after scraping the culture dish with 200 μl lysis buffer (0.1% Triton X-100 in 100 Mm PO4 buffer). The cellular extract was incubated for 30 min on ice and then was subjected to centrifugation at 100 × g for 30 min at 4°C. The pellet was discarded and the supernatant was divided into four equal aliquots each of which had a volume 20 μl. The protein concentration was measured by Bradford method (Bio rad) and each aliquot contained 15 μg proteins. Two aliquots were used as controls and one of them was incubated at 37°C for one hour. Additional aliquots received either MMP2 or MMP9 at concentration 5 μg/ml and were incubated at 37°C for one hour. At the end of incubation period, SDS sample buffer was added to all aliquots and incubation at 95°C for 3 min followed. Samples were loaded on 10% polyacrylamide gel for electrophoresis and then proteins were transferred to a nitrocellulose membrane. The quality of the transfer was controlled by Ponceau staining of the membrane. The membrane was blocked in 5% milk in PBS-Tween-20 for 30 min at room temperature and then incubated with primary antibodies (polyclonal anti-occludin and anti-claudin-1 at dilution 1:250) for 16 hours at 4°C. After 3 washes with PBS-Tween-20, the membrane was incubated for 1 hour at room temperature with HRP-secondary antibody (anti-rabbit IgG at dilution 1:3000) and the immunoreactive bands were detected with enhanced chemiluminescence (Roche).
Statistical analysis
Statistical analysis for quantative experiments was performed using GraphPad Prism 5. For permeability experiments, comparisons between control and MMP-treated tissues were made using one way ANOVA test with Dunnett post-test. For short-circuit experiments, the comparison between control experiments and experiments with application of MMPs was performed with unpaired t-test. Values of p < 0.05 were regarded as significant.
MMPs: Matrix metalloproteinases; TJs: Tight junctions; RTM: Transmesothelial resistance; SD: Standard deviation; TIMP2: Tissue inhibitor of metalloproteinases 2; ECM: Extracellular matrix; FBS: Fetal bovine serum.
Authors' contributions
EA wrote this manuscript. EP contributed to the performance of indirect immunofluorescence and western blotting. CH, KG and PAM conceived the study and participated in the design of the experiments. All authors read and approved the final manuscript.
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Eur Respir J 1997, 10:2411-2418. PubMed Abstract | Publisher Full Text
Antony VB: Immunological mechanisms in pleural disease.
Eur Respir J 2003, 21:539-544. PubMed Abstract | Publisher Full Text
Wheeldon EB, Mariassy AT, McSporran KD: The pleura: a combined light microscopic and scanning and transmission electron microscopic study in the sheep. II. Response to injury.
Exp Lung Res 1983, 5:125-140. PubMed Abstract | Publisher Full Text
Stylianou E, Jenner LA, Davies M, Coles GA, Williams JD: Isolation, culture and characterization of human peritoneal mesothelial cells.
Kidney Int 1990, 37:1563-1570. PubMed Abstract | Publisher Full Text


text:
Facebook Calls Ownership Lawsuit 'Frivolous'
Facebook is facing a lawsuit from a New York man who claims he owns an 84-percent stake in the popular social networking website–a lawsuit that they dismiss as "frivolous" but one that has nonetheless led to a judicial order blocking transfer of the company's assets.
The lawsuit was filed on behalf of Wellsville, New York native Paul Ceglia on June 30. Ceglia claims that, in April 2003, he signed a contract with Facebook founder Mark Zuckerberg in order to design a website to be called either "The Face Book" or "The Page Book." Said website was to be completed by January 1, 2004, and in exchange for his services, Ceglia claims he was promised $1,000, 50-percent of the business up front, and an additional one-percent each day until the work was complete.
According to the lawsuit, which was filed in Allegany County Supreme Court, the website was eventually launched as thefacebook.com on February 4, thus granting Ceglia an 84-percent stake in the company.
Representatives from Facebook released a statement calling the lawsuit "completely frivolous" and stating that the company would "fight it vigorously," according to an AFP article published Monday. Furthermore, company official Barry Schnitt told Reuters, "The order will not affect our ability to do business but we do not believe it is legally supported and we have moved to have it vacated."
According to Reuters reporter Alexei Oreskovic, "In December 2009, New York Attorney General Andrew Cuomo obtained a temporary restraining order against a wood-pellet fuel company owned by Ceglia and his wife Iasia, of Wellsville, New York, according to a press release"¦ Cuomo's suit alleged that Allegany Pellets took $200,000 from customers and failed to deliver any products or refunds."
"This company and its owners repeatedly lied to consumers and continued to solicit new orders despite an inability to deliver wood pellets that were bought and paid for months before the winter heating season began," Cuomo told the AFP recently.
On Monday, Facebook unveiled a new "panic button" feature in partnership with the United Kingdom's Child Exploitation and Online Protection Centre (CEOP). The ClickCEOP feature, as it is known, appears on a user's profile page when it is added or bookmarked and is designed to allow users between the ages of 13 and 18 to notify Facebook officials if they encounter "suspicious or inappropriate behavior," according to a report by Reuters' Kylie MacLellan.
"There is no single silver bullet to making the internet safer but by joining forces with CEOP we have developed a comprehensive solution which marries our expertise in technology with CEOP's expertise in online safety," Facebook vice-president Joanna Shields told members of the media on Monday.
"Together we have developed a new way of helping young people stay safe online and backed this with an awareness campaign to publicize it to young users," she added. "It is only through the constant and concerted efforts of the industry, police, parents and young people themselves that we can all keep safe online."
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Half-Life 2 Purchase Options Revealed, CS:Source Full to come out Next Week
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Location: everywhere
Posts: 2,216
Re: Half-Life 2 Purchase Options Revealed, CS:Source Full to come out Next Week
Originally Posted by
I wish all publishers used software similiar to steam. I love the fact that I can download the game to any computer I own then just log on and play with no cd's needed. I think its a great idea
mmm.. i really dont like the idea of steam at all.. it can be used to charge you (pay to play) per day/per month/per computer or Cdkey. per update any bizarre way they choose.. similar to everquest when you need to pay every month to play the game. or pay more for accounts with more privileges. pay to play the single player... or pay for any MOD. what about LAN parties ? would everyone need to be looged to steam to play? most likely.. there is an interesting discussion around here about this ..
never thought the legal Fight between Valve and Vivendi was that serious.. and there is a good chance that Vivendi will lose.. since Counterstrike source will be released pretty soon by steam and Vivendi can't do anything about it ,the information everywhere is that they only control the Singleplayer part which is ->HL2. if Vivendi delay the game to pressure Valve ,they will lose more money ,since people will not wait for Hl2 and will buy the MP first. and if Vivendi announce Gold now for Hl2 ,Valve will have 3-4 weeks of advantage selling the full game first by steam accounts. all in all it doesnt look good for the gamers ,because Vivendi is a Big publisher and they have the resources to make the life of the entire project (its evolution and future) very dificult if they lose Big money with their deals.
so wait and see.. probably the multiplayer part CS source might be released as soon as next week ,or the other..and the single player will come a few weeks later online if Vivendi can't manage to stop Steam. but its doesnt look good inmho positive for gamers this legal battle ,because Valve and Vivendi fight to get more money from the gamers migh end screwing people ,migh force some of them to buy the game 2 times to have it completely.. both halfs
a side note, its new to me that Hl2 game doesnt have a multiplayer
learned today that CS source is not only the multiplayer part ,(which i knew) ,but that it is the only one.
which is dissapointing ..since i enjoyed Hl1 singleplayer and never liked CS mod at all.. figure that.
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The Public Employees Federation, New York's second-largest state workers union, is weighing in on this morning's reports that Gov. Andrew Cuomo is thinking of following in his father's footsteps and seeking a massive reduction in the state workforce in his first executive budget.
In a prepared statement, PEF President Kenneth Brynien said he has received "no formal notification" regarding any proposed layoffs.
He warned that slashing the public payroll by 10,000 to 15,000 positions would not only "cripple the delivery of essential services," but would also likely have the opposite effect of a key Cuomo initiative: Growing the state's economy.
Read more...


text:
How to Make Gluehwein
Gluehwein is a common German drink, also known as Hot spiced wine. This makes one serving, and is an easy to make treat.
- 1 cup (236 ml) wine. (White or red)
- 1 whole clove (Make it 3/4 if you do not want it to be very strong)
- 1 small piece of a lemon peel
- 5 sugar cubes (More or less depending on your taste)


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Let your system tray remind you to take a break.Windows Version 3.1 ... Added: 08/08/12Total Downloads44,484Last Week41
SketchBook Pro is a powerful and easy to use drawing, painting, and sketching application.Mac Version 7.0.1 ... Added: 08/11/14Total Downloads11,573Last Week23
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text:
Stow location for batteries on PSC 34
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Re: Stow location for batteries on PSC 34
We have two 125Ahr group 29 batteries in the port locker and another to starboard of the rudderpost on a shelf in the engine compartment. The battery to starboard of the rudderpost is accessed through a closed cutout at the foot of the quarterberth. They are all from Sams, and we replace them every three years as a matter of course. At that point they have been cycled perhaps 3x175 times. They are the house battery and are charged by the engine alternator (or the 75W solar panel). To starboard of the ruddrerpost is an Optima group 34M battery. It is the engine starting battery and is charged through a Xantrex Echo-Charge. The shelf that the batteries sit on is in the spot where wheel steered PSC 34s have their quadrant. We have engine drive refrigeration and LED lighting, so we have more than enough battery capacity.
Bill Murdoch
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The "Coins for Kenzie" and "Hats On For Kenzie" campaigns, spearheaded by local teen Megan Moore to raise money for 8-year-old McKenzie Barbre have netted a nice chunk of change. Barbre has had two bouts with eye cancer.
Moore presented a check for $1,531 to Barbre's family Tuesday after just a little over a month of fundraising efforts. McKenzie's family will use the money to pay for medical bills.


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More Products By HARTZ:
Average rating from 3 reviews.
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Hartz Oinkies
Average rating from 11 reviews.
(4.6 out of 5, based on 11 reviews)
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Reader's Reviews (11)
Our dogs love these treats
posted 6 years ago
We give both of our dogs these treats and they like them. They usually eat the chicken part off right away and then come back a little while later to eat the rest of the treat. These treats have no dyes and do not stain the carpet. It keeps the dogs busy for a while and helps to reduce the plaque and tartar on their teeth. You can also find off brand varieties as well that are cheaper than the Hartz brand.
Pros: Great taste, non-staining, no dyes
Cons: None
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We love them
posted 6 years ago
We alternate between chews and products to keep things fresh for our dogs. These are one of the favorite chews in our home. They last a lot longer than many busy bones. Cost is reasonable as well.
Pros: They last longer than some chews on the market
Cons: Make larger sizes?
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Pink is my favorite color these are wonderful.I will recommend a good website fou you: www.9footballshirt.org
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Reader Survey Results
Last March we asked readers for feedback about what they wanted to see happen with Linux. We got responses from all over the world—Israel, Italy, Belgium, UK, Netherlands, Japan, Thailand, Austria, Germany, and the US. Although our survey wasn't entirely scientific, this response hints at the wide-spread implementation of Linux.
Responses showed that the first priority for Linux users is the porting of a WYSIWYG word processor. Microsoft Word was the number one choice, followed by WordPerfect, AMIPro, and FrameMaker. The second category on the wish list was a good database program, with the number one choice Microsoft Excel. Requests for other ports included CAD programs, Motif, fax software, and Lotus 1[hy]2[hy]3. See "Stop the Presses", this issue, for an update on WordPerfect for Linux, and "Moo-tiff Development Environment", this issue, and "Motif for Linux", July, 1995, for reviews of Motif ports for Linux.
Emacs was the most frequently-listed application used by readers. Maybe it has a built-in command for filling in "emacs" on any questionnaire. Netscape was the next most frequently-listed application. Many other Net and Web tools were included in the 63 different applications people listed. Text processing tools, e-mail tools, graphics tools, and databases were also popular. Only four readers listed vi variants, maybe because vi users think of it as a utility rather than an application. This will probably provide plenty of fuel for Emacs versus vi discussions.
Readers used a montage of operating systems—Linux was the most popular, of course, with other flavors of Unix (SunOs, Solaris, Ultrix, Dynix, etc.) and DOS also showing up frequently. OS/2, VMS, Mac/OS, Windows NT, and NeXT were also listed. Twenty-six different operating systems, including all the Unix variants, were listed.
Linux Journal readers program in a variety of languages. C was most popular, followed by C++, Tk/Tcl, perl, and FORTRAN. FORTRAN? Twenty-five different languages were listed.
If nothing else, these results show that Linux Journal readers, and by extension Linux users in general, are a diverse bunch.
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- Goodbye, Pi. Hello, C.H.I.P.


text:
Apple deals: iMac, HD display, iPod shuffle
updated 11:55 pm EDT, Tue July 1, 2008
Apple deals on iMacs
Reconditioned iMacs in a range of sizes and prices are now available at the Apple Store. Aluminum-cased iMacs start at $949 for the 20-inch 2GHz Intel Core 2 Duo model with 1GB of memory and 250GB hard drive and go up to $1,899 for the 24-in. 3.06GHz model that has 2GB of memory and a 500GB hard drive. One configuration in white is available for $1,119 that has a 24-inch display 2.16GHz Intel Core 2 Duo processor, 1GB of memory and a 250GB hard drive. All models have a built in iSight webcam, 8x SuperDrive and include an Apple remote, Apple keyboard and a Mighty Mouse.
Apple certified refurbished HD displays are available in 20-in. (1680 x 1050 resolution) for $499, 23-in. (1920 x 1200 resolution) for $749, and 30-in. (2560 x 1600 resolution) for $1,1499. Each display is set in an aluminum enclosure and includes two FireWire 400 ports and two USB 2.0 ports. Previous generation 1GB iPod shuffles in blue, green, and silver are only $39. They hold up to 240 songs with up to 12 hours of music playback and include earphones as well as a USB cable. Other items include the wireless Mighty Mouse for $59 and AirPort Express base station for $79.


text:
Informal, unscientific survey
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Re: Informal, unscientific survey
Oh, this is a thread which has the potential for a lot of anger and resentment on both sides. I bet some have read it and decided not to "go there" by not responding at all. As I write I am wondering if I should, too.
I guess I'll continue by reminding myself of the context: this is a small New England seaport, not NYC Harbor where harbor security is generally on high alert; all harbormasters have a challenging job that can raise their blood pressure quite easily. I once interviewed for an Assistant Harbormaster position and immediately gained much respect for the fellow who held the top position. He was a young ex-cop with a very even demeanor. His description of dealing with several rowdy and disrepectful boaters who've had at least one too many on July 4th or Labor Day showed his judgement and diplomatic skill (it was around 25 years ago, before DUI enforcement on those waters); his description of potentially having to rescue a senior member of the community suffering a heart attack aboard a boat in the confused seas of a harbor's entrance at twilight showed how demanding things could get. That said ...
1. Never in 38 years.
2. I know of no one, but this isn't something many people would like to admit unless they were clearly mistreated or the harbor police have earned a reputation of being overzealous.
3. Harbors with known "strict enforcement" would likely be less attractive to me. I've met such officers and found their style abusive and offensive. The harbormaster you describe, above, seems to have a good understanding of the "serve and protect" credo and I think that community is lucky to have him. Like Tempest, "FWIW...."
Last edited by Michael K; 04-22-2013 at
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la times
Each week, we bring you a selection of the best images from around the world in Framework's The Week in Pictures.
Enjoy the journey with us.
wsj photojournal
SEALING A DEAL: Sheikh Hamad Bin Khalifa al-Thani, the emir of Qatar, used his phone to photograph Qatar's minister ...Photos of the Day: April 9
la times
Monday's Pictures in the News begins in Tunisia, where protesters hold Tunisian flags as they sit in front of police in Tunis, the capital. Police fired tear gas Monday to disperse a rally on a central Tunis avenue where demonstrations are banned. Hundreds of demonstrators, marking Martyrs' Day and protesting against the ban imposed on March 28, sought shelter in neighboring streets and shops.
Jewish men wrapped in prayer shawls attend the annual Cohanim prayer, or Priest's blessing, for the Pesach ...Pictures in the News | April 9, 2012
flak photo
Kim Kyung-Hoon / Reuters
Saudi Arabia's Wojdan Shaherkani arrives ahead of her women's +78kg elimination round of 32 judo match against Puerto Rico's Melissa Mojica at the London 2012 Olympic Games on August 3, 2012.
Mitch Goldich, NBC Olympics reports — After a week of controversy and hand-wringing — first declaring she would fight without a hijab, then saying she would withdraw if not allowed to wear one — Wojdan Shaherkani finally made history this morning, as the first Saudi Arabian woman ever ...Wojdan Shaherkani makes history as first Saudi Arabian woman in Olympics
Amer Hilabi / AFP - Getty Images
A Saudi youth sits next to his sport utility vehicle lifted with stones and bricks in the tourist area of al-Habla, Saudi Arabia on July 12, 2012. Young Saudi males gather in al-Habla for a summer camp to escape the Gulf heat and spend time mounting their cars with rocks for display.
.Saudi youth escape heat and mount vehicles
F STOPPERSGrowing up in Bosnia during 1992-1995, photographer Ziyah Gafić could not participate in the war – only stand by on the sidelines. Having no academic background in photography, he became very interested in Muslim culture and while working on his project, "Troubled Islam", he got a very unique opportunity to shoot and interview the women [...] [BTS Video] Life For Saudi Arabian Women
feature shoot
Photographer Ziyah Gafic was born in Sarajevo, where he graduated in Comparative literature. Since 1999 he has been traveling extensively and has covered major events in more than forty countries. This video profile is from VICE's Picture Perfect series and documents Gafic's journey to photograph successful women in Saudi Arabia.Saudi Arabian Women Photographed by Ziyah Gafic
360 cities360° panorama by Cansız Hayal (360TR.COM).
RANGEFINDER FORUMOk, so i got this job, working as an architect (that's what i do), for 6 months in Saudi Arabia, al Khobar.
Fayez Nureldine / AFP - Getty Images
Saudi police officers monitor screens connected to cameras, at the Command and Control Center in Mina, outisde the holy city of Mecca, on November 7, 2011, as millions of Muslim pilgrims take part in the annual hajj.
Fayez Nureldine / AFP - Getty Images
An aerial view shows Muslim pilgrims walking around the Kaaba in the Grand Mosque in the holy city of Mecca during the annual Hajj pilgrimage rituals on November 7, 2011.
Read more about the CCTV camera system used ...Saudi police use security cameras during Hajj in Mecca
msnbcRich Shulman writes
I guess that's what you would expect him to say. Full story.
Prince Nayef bin Abdulaziz al-Saud, newly appointed Crown Prince speaks at an intrerview at the Riyadh Governor's Palace, October 29.
Fahad Shadeed / Reuters
Saudi men turn out in large numbers to show support and allegiance to newly appointed Crown Prince of Saudi Arabia, Prince Nayef bin Abdulaziz al-Saud at the Riyadh Governor's Palace, October 29.New Saudi crown prince says Kingdom to remain stable
FLICKR1935 Auburn 851 Boattail Speedster (IBN MANSI Showroom - Jeddah, Saudi Arabia) by Hosam Al-Ghamdi .:|:. حسام الغامدي
photoradar forumGood morning
msnbcRich Shulman writes
This challenge to the Saudi driving ban reminds of the civil rights movement in the United States in the 1960s. Each day the women become bolder in their effort to defy the driving ban. Yesterday, Secretary of State Hillary Clinton praised the women for their bravery.
Fahad Shadeed / Reuters
Female driver Azza Al Shmasani alights from her car after driving in defiance of the ban in Riyadh June 22. Saudi Arabia has no formal ban on women driving. But as citizens must use only Saudi-issued ...Activists: 2 Saudi women take drive in capital
Athar Hussain / Reuters
A policeman is reflected in a window of a car shattered by bullets, currently parked in a police station, in which a Saudi diplomat was travelling in when he came under attack by gunmen, in Karachi, Pakistan on May 16. Gunmen on motorcycles attacked the car belonging to the Saudi Arabian consulate in the Pakistani city of Karachi on Monday killing a Saudi diplomat, police and the Saudi ambassador said.
KARACHI — Gunmen on a motorbike shot and killed a Saudi diplomat as he was ...Gunmen kill Saudi diplomat in Pakistan
RAHEB HOMAVANDI / Reuters
EDITORS' NOTE: Reuters and other foreign media are subject to Iranian restrictions on leaving the office to report, film or take pictures in Tehran.
An Iranian spectator (C) wearing a shirt with picture of founder of the Islamic Republic Ayatollah Ruhollah Khomeini shouts to oppose the presence of Saudi Arabian troops in Bahrain during the AFC Champions League soccer match between Iran's Persepolis and Saudi Arabia's Al-Ittihad at Tehran's Azadi stadium May 3, 2011.
BEHROUZ ...Protest at Iran soccer game about Saudi involvement in Bahrain quashed by police


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We take a look back at the occasions in the Premier League that West Bromwich Albion took at least a point from the Gunners
West Bromwich Albion's collective performance at Manchester United was outstanding - but their next opponents could allow a different style of play
The Old Trafford hero's current deal is ticking down rapidly
West Bromwich Albion's big Swede saw the solitary goal at Old Trafford deflect in off his leg from Chris Brunt's free-kick
West Bromwich Albion reached the desirable 40-point mark with an excellent 1-0 win over Louis Van Gaal's Red Devils
West Bromwich Albion boss Tony Pulis has revealed he'll be forced into making alterations for the trip to Manchester United
The latest Premier League transfer talk
Former Villa star calls on Paul Lambert's team to go on the attack against in-form Manchester United on Saturday
The latest top flight tittle-tattle
The stats don't lie! WBA have a diamond in Saido Berahino
Baggies midfielder Craig Gardner says striker was better than Louis Van Gaal's galacticos
Brian Dick's full-time report with pictures from The Hawthorns as a late blow forces Albion to settle for a point against Man Utd
The return of the Premier League means one thing - the return of Fantasy Football!
Here's how you can create a winning Fantasy Football team for the 2014/15 Premier League season
Chris Lepkowski gives his view on the comprehensive defeat at home to Manchester United
West Brom: Chris Lepkowski looks back at the history of games against Manchester United
The Dutch forward is currently suffering with an illness on international duty
Aston Villa not underestimating the Manchester United challenge, despite van Persie injury
Stats, head-to-head, flashback, team news and more
Manchester United boss defends Ashley Young over dive accusations
Everything you need to know ahead of Manchester United v West Bromwich Albion
Tom Ross gives his thoughts on the departure of Wolverhampton Wanderers manager Dean Saunders
ANALYSIS: Gregg Evans takes a look at the impact of Aston Villa striker Gabby Agbonlahor
Paul Lambert praises his Aston Villa players for not giving up against Manchester United
Paul Lambert's claret and blues can't stop United's Premier League title celebrations
Mel Eves looks at the impact of Aston Villa striker Christian Benteke and why he deserves PFA award
Latest West Bromwich Albion team news and a quick look at the opposition
West Bromwich Albion keeper Ben Foster warns Baggies pals about the 'late' Manchester United goal threat
Goal hero Demba Ba insists he will be happy even if he is not finding the back of the net as long as Newcastle are winning.
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Enviro-Tote, Inc.
We love this stylish poodle, designed by Kristina Fiore, ready to greet the sun in shades.
From beach bag to impromptu picnic basket, the tote is a summer essential. To celebrate the style of the season, we asked our Pinterest community to design a chic, casual tote. We're thrilled to share the best and brightest winning creations here -- voted on by you!


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Join free webinar on 28 May on passenger experience and future air travel.
Why closing gaps in the Tokyo Convention will be a deterrent to unruly passenger behavior.
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Improve baggage performance. From quick wins to strategical options, custom solutions for airlines, airports and ground handlers.
We're adding new IATA training centers, partner locations and distance learning options to bring our training closer to you.
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Tony Tyler's remarks at AVSEC World, which took place in Washington, on 27 October
Press Release No.:
Date: 8 November 2007
CANCUN - The International Air Transport Association (IATA) called for greater transparency in the Latin American airline industry to form a sounder basis for key policy decisions.
"We must be open and honest about the economic and social benefits that we bring; and about the policy issues that we face. Facts and figures demonstrate policy failures of the past - including failed airport privatisations," said Giovanni Bisignani, CEO and Director General of IATA.
IATA partnered with the Latin American Civil Aviation Commission (CLAC) to release a series of new economic studies during the annual general meeting of the Latin American Air Transport Association (ALTA) focused on five countries: Bolivia, Chile, Colombia, Mexico and Panama.
In Latin America, airlines support 2.7 million jobs and US$157 billion in related business. "By explaining ourselves better we can help governments avoid policy myopia so that together we can build an even stronger base for future growth," said Bisignani.
Bisignani urged attendees to work hard in four important areas:
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Cray Eugene Williams is wanted on a federal arrest warrant out of Kansas City for conspiracy to distribute marijuana.
According to authorities, Williams is considered armed and dangerous. He has ties to the Kansas City area, but is known to travel to Houston, Texas, Las Vegas, Nevada and Los Angeles, California.
Williams, 5'9" and 220 pounds, has tattoos on both hands and wrists. He also has the letters "MGB" tattooed on his neck.
The FBI says Williams also goes by the names McKinley Kingsley, Jerome Parris and Jason Williams.
Call 816-474-TIPS with any information that could lead to an arrest.


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Too drastic, Egyptian envoy says of Filipinos' evacuation
More News from Tarra Quismundo, Tina G. Santos
MANILA, Philippines—While saying an evacuation order was too drastic for the situation, Egypt has expressed its deference to the Philippines' decision to initiate mandatory repatriation amid continuing violence across the Arab country.
In an interview, Egyptian Ambassador to Manila Mahmoud Mostafa Ahmed assured the Philippines that his country would protect Filipinos caught in the violence, emphasizing that the interim government was exerting all efforts to contain what he described to be pockets of violence in his homeland.
"We respect what the Philippines issued. Despite that, we think that most Filipinos will not be willing to come back," Ahmed told reporters at a briefing at the Egyptian Embassy in Makati City yesterday.
Emergency visits
"The Secretary (Foreign Secretary Albert Del Rosario) has made two visits in 12 days to Cairo, and he raised the fourth degree [of emergency alert] based on his assessment. Despite that, my point of view is it (the situation) doesn't deserve it," he said.
Other than the Philippines, only Thailand has ordered a mandatory evacuation of its citizens following clashes between supporters of deposed Egyptian President Mohammed Morsi and security forces, which has led to the hundreds of deaths and destruction of churches, courts, schools, hospitals and other critical infrastructure across Egypt.
On Aug. 19, Del Rosario placed Filipinos in Egypt under Crisis Alert Level 4, the highest emergency alert for Filipinos overseas, after seeing for himself the "marked deterioration in peace and order, exacerbated by complex political challenges" in Egypt.
Ahmed said he understood the government's duty to protect Philippine citizens abroad, but he believed many will decide to stay.
"They are enjoying the full support of the Egyptian government and they enjoy complete freedom to go wherever they want," said the envoy.
The official government count of Filipinos in Egypt is at 6,000, but Ahmed said the actual figure might be more than this.
He also clarified that most of Egypt was generally safe and that violence, much of which he attributed to "terrorists" from Morsi's supporters in the Muslim Brotherhood, were limited to only a few areas.
Still under control
"The airports are working normally. The violence in Cairo is limited to certain focused areas, not the whole Egypt. We are not saying that we are now living in paradise. The level of violence is still under control," said Ahmed.
Decrying criticism about the interim government's crackdown, he said there was no other choice but to disperse the thousands of Morsi supporters who had been blocking major thoroughfares around Egypt for nearly two months.
He said the protesters were caught carrying arms and had started gunfire at the start of the crackdown earlier this month.
"We are still considering ourselves to be in a war against terrorism. We hope it'll improve in the next days and I hope they will come back to reason. Our future should be inclusive and this is what we are calling for and working at," said Ahmed.
New future
"The government is trying to contain the situation by any means. They are all Egyptians. We are going to deal with them according to the law… We are going to deal with them with tolerance, good spirit to convince them that what has passed has passed and let us look to the new future a new horizon," he said.
So far, five Filipinos have returned from Egypt since the evacuation order. At least 15 are expected to arrive today while nine are leaving Cairo for Manila on Sept. 1.
One Filipino-Egyptian was injured by a stray bullet amid the violence earlier this month, but she has been discharged from the hospital.
According to the Overseas Workers Welfare Administration (Owwa), the 15 Filipinos, mostly carrying student visas, will arrive at the Ninoy Aquino International Airport via a Gulf Air flight.
Coming in trickles
The repatriates will be met at the airport by the Owwa repatriation team that will assist them in their arrival as they go through the customs and immigration processes.
The Department of Foreign Affairs (DFA) earlier said Filipinos in Egypt were coming to the Philippine Embassy in Cairo in trickles to sign up for repatriation.
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Swaddling a baby
Filed under: Newborns, Babies, Your Pregnancy, Resources, Expert Advice: Toddlers & Preschoolers, Nutrition: Toddlers & Preschoolers, Development/Milestones: Babies, Research Reveals: Toddlers & Preschoolers, Gear Guides: Toddlers & Preschoolers, Gear Guides: Babies, Activities: Toddlers & Preschoolers, Behavior: Toddlers & Preschoolers, Development: Toddlers & Preschoolers, Health & Safety: Toddlers & Preschoolers, Expert Advice: Babies, Toddlers Preschoolers, Baby-sitting, Health & Safety: Babies, Feeding & Sleeping, Day Care & Education, Research Reveals: Babies
This is called swaddling a baby. We did this for our first two and I assumed that I would remember how to do it for our third who was born earlier this week. Alas, I am old and forgetful, so the process didn't come back to me. Now, I could have just asked a nurse to show me how to do it, but being the complete nerd that I am, I turned instead to the internet. There are quite a few videos on YouTube that demonstrate various ways to swaddle an infant.
The one that I like best is the ultra swaddle, although the blankets at the hospital weren't long enough to do it right. If you're using a really big blanket, the Baby Burrito video makes it look pretty easy. Taking the simple swaddle one step further, this video uses a second blanket to create a burka-like wrap that seems pretty secure. Lastly, here's a fun pastime for new parents and their friends -- a swaddle battle. See who can swaddle the kid the best, the fastest, and the most successfully.
After watching all these videos, you'd think I'd be a swaddle master, but unfortunately, I'm not. It's not all my fault, however; I have a very uncooperative partner -- one who likes to wave his arms about and kick his legs like mad. Still, I'll keep practicing and, someday, I'll get it right. Probably right about the time he's ready to go off to college.
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5-10-2008 @ 9:53PMKathy Abbott said...
As a Lactation Consultant I don't recommend swaddling a new born. Babies like to bring their hands up towards their mouth when they are getting hungry. This is a feeding cue. Crying is the very last sign of hunger. It can be very difficult to get a crying baby to latch onto the breast. By keeping a sleeping baby tightly swaddled you run the risk of missing early feeding cues and having to feed a distraught infant (crying is more a sign of stress than of hunger). Better to keep the baby skin to skin as much as possible, and at the very least leave the baby's arms exposed.
Kathy Abbott, IBCLC
5-11-2008 @ 2:09AMKat said...
I was never good at swaddling, horrible at it. with my second child I went out and got swaddle blankets. they have a little pocket for the feet, wrap the rest around the baby, it sticks with velcro and stays on...it was a lifesaver!
5-11-2008 @ 3:57AMSherry said...
My kids both hated swaddling and would scream and kick like they were being murdered if you even tucked a blanket around them. They liked their personal space and always slept with their arms thrown up over their heads. If the kid doesn't like it, which it seems he doesn't, why bother?
5-11-2008 @ 8:45PMkelly jeanie said...
Oh my goodness, I had no idea that even something as innocuous as swaddling could be considered controversial.
5-11-2008 @ 9:54PMHolly said...
Swaddling was wonderful...but the bigger my daughter got, the more challenging it was to keep her wrapped tight. We got the Miracle Blanket and loved it. I wrote about it on my blog. You can even find a discount for purchasing it there.
I bought three of them, so we would always have a clean one.


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Brush Up on History at Paul Revere House
Built in 1680, famed silversmith's house a Freedom Trail favorite
On the evening of April 18, 1775, Dr. Joseph Warren summoned Paul Revere, then employed by the Boston Committee of Correspondence and the Massachusetts Committee of Safety as an express rider carrying news, messages and documents. The task – to ride to Lexington, Massachusetts to alert Samuel Adams and John Hancock that British troops were marching to arrest them, immortalized the talented silversmith and his midnight ride. Those hazy on the historic particulars or just craving a taste of old Boston can find them at the Paul Revere House in Boston's North End.
Built in 1680, the Paul Revere House at North Square is believed to be among the oldest in Boston. One of the most popular attractions along Boston's Freedom Trail, the house was built on the site of the former parsonage of the Second Church of Boston. Increase Mather, the minister of the Second Church and later president of Harvard College, and his family (including his son, Congregational minister and author Cotton Mather) occupied the parsonage from 1670 until it was destroyed in the Great Fire of 1676. Paul Revere bought the house in 1770, moving in with his growing family, which at the time included his wife, Sarah, their five children, and his mother, Deborah. After Revere sold the home in 1800, the ground floor housed a series of shops, among them a candy store, a cigar factory, a bank, and a vegetable and fruit business. In 1902, Paul Revere's great-grandson, John P. Reynolds, Jr., purchased the building to ensure that it would not be demolished, and the Paul Revere Memorial Association was formed to preserve and renovate the building.
In April 1908, the Paul Revere House opened its doors to the public, and the association oversees its upkeep and day-to-day operations. The restored dwelling resembles its late 17th-century original—nearly 90 percent of the structure, including two doors, three window frames, and portions of the flooring, foundation, inner wall material, and raftering, are original. The period furnishings in the upstairs chambers belonged to the Revere family. In the courtyard are a 900-pound bell, a small mortar, and a bolt from the USS Constitution, all crafted by Paul Revere & Sons.
In addition to being a silversmith and a goldsmith—trades passed down from his father—Revere was a copper plate engraver, an illustrator, and an importer, and for nearly a decade he worked as a dentist, cleaning teeth and wiring in false ones. Through his lodge, active Freemason Revere grew close to members of the revolutionary movement and occasionally reported to them on the whereabouts of British soldiers. But it wasn't until a century later that Revere, who went on to become a successful industrialist, was regarded as a hero of the American Revolution. His ride was immortalized in Henry Wadsworth Longfellow's poem "Paul Revere's Ride," first published in 1860 in the pages of The Atlantic Monthly.
The Paul Revere House is at 19 North Square, Boston. The house is open daily from 9:30 a.m. to 5:15 p.m. from April 15 to October 31 and from 9:30 a.m. to 4:15 p.m. from November 1 until April 14. Admission is $3.50 for adults, $3 for seniors and students, and $1 for children. Take the Green Line to Government Center or Haymarket stations, the Blue Line to Government Center or Aquarium stations, or the Orange Line to State or Haymarket stations.
More information can be found here.
This article was originally published June 13, 2011.5 Comments


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Download Juventus' official apps for your chance to lead the team to glory on "Fantasy Manager 2014", talk with your Juve-supporting mates on "Juventus Live", recreate famous goals from last season with "Score!" and much, much more!
Juventus Live allows up to 4 friends to TALK while watching a Juve game on TV (voice, not text!) The experience is enriched with real time statistics and information about the game, and news during the entire week. Juventus Live is the perfect, complimentary companion to your TV.
Try Juventus Live free now!
Let's be honest, watching Juve playing on TV is great, but wouldn't it be much better in the company of other Bianconeri fans? Well, we have the perfect solution for you! Now you can watch Juve on TV, while TALKING to your friends; and see stats about the game.
No distracting text or tweets, just the voices of your buddies so you can finally yell GOOOOOOAL together again!
Hurrà Juventus, the club's official bilingual fanzine, is now available in an all-new digital format. Designed for smartphones and tablets, it allows you to enjoy videos and photo galleries as you read through the magazine dedicated to all things Bianconeri. You can access all this exclusive content through the app, which is available from the App Store and Google Play in Italian and English for just €4.49.
Are you up to managing Juventus?
From today you can find out thanks to Juventus Fantasy Manager 2015, the managerial game which allows you to fill the roles of manager, sporting director and chief executive simultaneously
With Juventus Fantasy Manager 2015 you are able to:
- Create your own squad, delve into the transfer market and purchase players from other teams
- Train your players and keep them in peak condition before selecting your best eleven
- Play fixtures and tournaments against your friends or other opponents from around the world
- Buy equipment for the team
- Construct and reshape club structures and services
And remember: if you have any doubts, Massimiliano Allegri is on hand to provide you with his expert opinion.
In this game the player manages his or her own football club and is responsible for finances, team, staff, stadium complex and everything that a football club in real life deals with. Matches take place against other players in real time and players compete in leagues, tournaments, friendlies and special challenge matches against real clubs. A substantial part of the game are card packs that can be purchased in the in game store.
These cards can form booster packs, skill upgrades or upgrades for premium items.
The player can use his fictional team against Juventus Football Club in a special challenge match. The Player accesses the challenge by purchasing a special coupon from the card store in the game and this allows them to make an attempt to beat the Juventus squad, which features player images and statistics from the team. If the player wins the match they earn a special branded trophy which also contains a counter showing how many times they completed the challenge.
Score! is a football simulation game designed for tablets and mobile devices. Instead of flicking or pressing a button, the player draws the balls path to the goal on the screen, in order to recreate goals and set pieces with precision and accuracy. Currently the game features over 300 goals from all over the world, including domestic leagues, European cups and International tournaments. There are Amateur, Pro and Bonus modes and a new multi-player mode that lets player challenge their friends via Game Center, and share their achievements via Facebook and Twitter. The game supports full Retina Display graphics and users over 500 motion-captured animations and true to life immersive commentary.
Player will be able to purchase a branded pack of Juventus goals, recreated from actual goals from the 2012/13 season. Player kits will be accurately represented based on the team strip for that season.
Play Zen Pinball, virtual simulation of pinball with 3D modeling and special effects.
Sounds, colors and effects will bring you at the Juventus Stadium thanks to the Juventus custom pack. Play with the Bianconeri champions to gain more points, smash the other teams and become champion of the Super League of Pinball.
Football Kicks Title Race allows you to take charge of a full team of players, improve their performances with intensive training and play exciting matches, where YOU can make the difference over 90 minutes, providing inch-perfect passes to score net-bursting goals.
As well as player management, Football Kicks Title Race enables you to expand your team's stadium to attract more fans and make additional revenue for you to spend on star players.