text:
ENZYMES AND PROTEINS
Peptidoglycan O Acetylation and Autolysin Profile of Enterococcus faecalis in the Viable but Nonculturable State
John M. Pfeffer, Hendrik Strating, Joel T. Weadge, Anthony J. Clarke
John M. Pfeffer
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
Hendrik Strating
Joel T. Weadge
Anthony J. Clarke
For correspondence: aclarke@uoguelph.ca
The O acetylation of peptidoglycan occurs specifically at the C-6 hydroxyl group of muramoyl residues. Using a combination of high-performance liquid chromatography-based organic acid analysis and carbohydrate analysis by high-pH anion-exchange chromatography, we determined that strains of Entercoccus durans, E. faecalis, E. faecium, and E. hirae produce O-acetylated peptidoglycan. The levels of O acetylation ranged from 19% to 72% relative to the muramic acid content, and they were found to vary with the growth phase of the culture. Increases of 10 to 40% in O acetylation were observed with cultures entering the stationary phase. Cells of E. faecalis in the viable but nonculturable (VBNC) state had the highest levels of peptidoglycan O acetylation. The presence of this modification to peptidoglycan was shown to inhibit the action of hen egg white lysozyme in a concentration-dependent manner. Zymography using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the production of specific autolysins in E. faecalis. Differences in the expression of specific autolysins were observed with the age of the culture, and VBNC E. faecalis produced the highest levels of these enzymes. This technique also permitted classification of the enterococcal autolysins into enzymes that preferentially hydrolyze either O-acetylated or non-O-acetylated peptidoglycan and enzymes that show no apparent preference for either substrate type.
The gram-positive, gut commensal bacterium Enterococcus faecalis is the causative agent of 90% of all enterococcal infections, which include endocarditis, bacteremia, and urinary tract infections. Of these, urinary tract infections are the most common, and the majority of them are acquired nosocomially. E. faecalis infections pose problems for the clinician because of their resistance to multiple antibiotics, sometimes including vancomycin, the drug of last resort for many gram-positive infections (recently reviewed in references 14, 19, and 32). To exacerbate this situation, this bacterium has been shown to persist in a mouse model for extended periods of time in the kidney (26). This persistence is thought to result from the cell's ability to enter the viable but nonculturable (VBNC) state, a feature first described for E. faecalis by Lleo et al. (30). VBNC E. faecalis, like other VBNC pathogenic bacteria, appears to retain its pathogenicity genes and is able to resume active growth upon restoration of the optimal environmental conditions (reference 30 and references therein). While in this state, E. faecalis cells have an irregular morphology, and there are concomitant alterations in the molecular architecture of the peptidoglycan sacculus (39). However, the changes observed in the peptidoglycan composition do not readily account for the persistence associated with the VBNC enterococci. One possible explanation for the apparent resilience of VBNC E. faecalis could involve a modification to the peptidoglycan, such as its O acetylation, that has not been characterized previously.
The O acetylation of peptidoglycan occurs on the C-6 hydroxyl moiety of component N-acetylmuramoyl residues, producing the corresponding N,O-diacetylmuramoyl derivatives in the bacterial cell wall heteropolymer (Fig. 1). To date, a total of 28 species of both gram-positive and gram-negative bacteria are known to modify their peptidoglycan in this manner. These species include a number of important human pathogens, such as Staphylococcus aureus, Bacillus anthracis, Campylobacter jejuni, Helicobacter pylori, Neisseria gonorrhoeae, and all species of Proteus, Providencia, and Morganella (11, 12, 44). For each species and strain examined, the extent of peptidoglycan O acetylation is not stoichiometric but instead ranges typically between 20% and 70% relative to the muramic acid content. Nonetheless, this modification has been shown to inhibit the lytic action of the muramidases (lysozymes) associated with the innate immune system in a concentration-dependent manner (6, 16, 35, 36, 41). The O acetylation of peptidoglycan has also been demonstrated to inhibit the action of the lytic transglycosylases (5), which are ubiquitous bacterial enzymes associated with peptidoglycan metabolism (both biosynthesis and turnover) (4). The latter enzymes act like muramidase to cleave the β-1,4 linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues, but instead of catalyzing a hydrolytic reaction, they produce a 1,6-anhydromuramoyl product (21). Thus, the catalytic mechanism of the lytic transglycosylases requires a free C-6 hydroxyl group on the N-acetylmuramoyl substrate (Fig. 1). For this reason, O acetylation has been proposed to serve as one level of control for peptidoglycan metabolism (33).
Structure of peptidoglycans and their lysis by lysozyme and lytic transglycosylases. (A) Lysis of peptidoglycan by lysozyme and lytic transglycosylases (LT), resulting in the release of GlcNAc and either MurNAc or 1,6-anhydroMurNAc residues. (B) Structure of O-acetylated peptidoglycan. The presence of the C-6 acetyl moiety on muramoyl residues precludes the formation of 1,6-anhydromuramoyl products by lytic transglycosylases, while it also sterically hinders the binding of lysozyme. R, stem peptides associated with MurNAc residues.
In the current study, the presence and extent of peptidoglycan O acetylation were determined for four clinically important species of enterococci, E. faecalis, E. faecium, E. hirae, and E. durans. In addition, the role of O acetylation in the VBNC state of E. faecalis was investigated.
Materials.Ferric ammonium citrate was purchased from Fisher Scientific (Nepean, ON, Canada), and sodium azide was purchased from Acros Organics (Geel, Belgium). Tryptic soy growth medium, oxgall, esculin, and all other medium supplements were provided by Difco Laboratories (Detroit, MI), while all other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
Bacteria and growth conditions.Most of the bacterial strains used in this study (Table 1) were obtained from the American Type Culture Collection, Rockville, Md.; the only exception was the E. durans strain, which was obtained from the culture collection of the Department of Molecular and Cellular Biology (formerly Department of Microbiology), University of Guelph. To obtain pure viable cultures, lyophilized stocks of each species and strain were resuspended in 5 ml of tryptic soy broth (TSB), diluted 1/20 into fresh TSB, and incubated overnight at 37°C with shaking (200 rpm). Samples of each culture were then serially diluted to a dilution of 10−7, and 100-μl aliquots were spread onto selective Bacto bile esculin azide (BBEA) agar (15). Cell growth was monitored turbidometrically by determining the optical density at 600 nm (OD600) using a DU530 LifeScience UV/visible spectrophotometer (Beckman Coulter, Mississauga, ON, canada).
Extents of peptidoglcyan O acetylation in the entercocci
To obtain mid-exponential-phase E. durans, E. faecalis, and E. hirae cultures for cell wall analysis, overnight cultures (OD600, 1.4) were diluted 100-fold into 3 liters of fresh TSB and incubated at 37°C until the OD600 was 0.7. Stationary-phase cultures were cultured to an OD600 of 1.4, at which point they were incubated for a further 12 h to ensure uniformity. For E. faecium, which grew more slowly and to a lower density than the other species under the same conditions, mid-exponential and stationary cultures were grown to OD600 of 0.4 and 0.9, respectively.
The method of Signoretto et al. (39) was used to prepare VBNC cells of E. faecalis. Briefly, an early-exponential-phase culture (OD600, 0.3) was resuspended in filter-sterilized raw (well) water (Guelph aquifer) that had been autoclaved twice at 121°C for 20 min to obtain a final concentration of 4 × 107 to 5 × 107 CFU/ml and incubated at 4°C for 16 to 21 days. To confirm the VBNC state and to check for possible contamination, two 50-ml samples of each strain were passed through a 0.22-μm filter, and the cells recovered on the filter were placed on either BBEA or tryptic soy agar plates and incubated at 37°C for 5 days. The remaining cells were harvested by centrifugation (15,000 × g, 4°C) and monitored by phase microscopy for the expected change to an irregular morphology.
Isolation and purification of peptidoglycan.Cells were harvested by centrifugation (9,000 × g, 4°C) for 12 min and frozen overnight at −20°C. Whole-cell pellets were then resuspended in 100 ml 25 mM sodium phosphate buffer (pH 6.0). Insoluble peptidoglycan and associated teichoic acids were extracted using the boiling 4% sodium dodecyl sulfate (SDS) procedure described by Holye and Beveridge (22), taking care to preserve natural levels of O acetylation (9). The SDS-insoluble cell wall material (for convenience referred to below as peptidoglycan) was recovered by ultracentrifugation at 160,000 × g for 55 min at 25°C, washed at least four times with 25 mM sodium phosphate buffer (pH 6.0), and lyophilized.
Determination of the extent of peptidoglycan O acetylation.Isolated insoluble peptidoglycan was resuspended in 25 mM sodium phosphate buffer (pH 6.0) and fragmented by continuous sonication for 4 min using a Heat Systems XL2020 sonicator. Samples of insoluble peptidoglycan were treated with 500 mM (final concentration) NaOH for 30 min at the ambient temperature to release any ester-linked acetate and then subjected to ultracentrifugation (100,000 × g, room temperature) using a Beckman Airfuge (Beckman Coulter, Mississauga, ON, Canada). Released acetate was quantified both by high-performance liquid chromatography-based organic acid analysis using an Aminex HPX-87H Bio-Rad column as previously described (9) and using a Megazyme acetic acid assay kit (Megazyme International Ireland Ltd., Wicklow, Ireland). The extent of O acetylation was expressed as a percentage of the muramic acid content, which was determined by quantitative amino sugar analysis (10) of associated insoluble peptidoglycan pellets after hydrolysis in 4 M HCl at 96°C in vacuo for 18 h.
Susceptibility of peptidoglycans to lysozyme hydrolysis.Peptidoglycan was isolated and purified as a substrate from cultures of E. faecalis grown to the mid-exponential, stationary, and VBNC phases. Suspensions of the peptidoglycan samples were prepared by brief ultrasonication as described above. The turbidometric assay of Hash (20) was used to assess the susceptibilities of the purified peptidoglycan preparations in 50 mM sodium phosphate buffer (pH 6.0) to 200 μg/ml (final concentration) of hen egg white lysozyme (HEWL), as previously described (16). De-O-acetylated peptidoglycan was prepared by treating peptidoglycan with 40 mM NaOH for 2 h at room temperature, followed by adjustment of the pH to 6.0 and reisolation by ultracentrifugation (16).
Zymography of SDS-stable autolysins produced by E. faecalis and E. hirae.Peptidoglycans isolated from both E. faecalis and E. hirae, together with the corresponding de-O-acetylated forms, were used to assay separately the autolysin content associated with whole-cell lysates of the bacteria by zymography. In some experiments involving suspension of the peptidoglycan substrate in SDS-polyacrylamide gel electrophoresis separating gels at the typical alkaline pH (pH 8.6), the method of Bernadsky et al. (3) and Watt and Clarke (43) was used. In other experiments, in which it was necessary to preserve the natural levels of O acetylation in peptidoglycan, zymograms in which peptidoglycan was incorporated into the resolving gel of a discontinuous SDS-polyacrylamide gel electrophoresis system that was operated at a slightly acidic pH were prepared as previously described (40). Thus, [bis(2-hydroxyethyl)imino]tris(hydroxymethyl)methane (Bis-Tris) buffers at pH 6.8 and 6.3 were used for the separating and stacking gels, respectively, while the pH of both the N-2-acetamido-2-hydroxyethanesulfonic acid (ACES)-HCl anode buffer and the Bis-Tris cathode buffer was 6.8.
Extent of peptidoglycan O acetylation in enterococcal species.The peptidoglycans from different strains of four common species of pathogenic enterococci grown to the mid-exponential phase were isolated by the boiling SDS protocol, taking care to maintain the pH below 7 and thereby preserve natural levels of O acetylation. Following lyophilization, the preparations were subjected to analysis for ester-linked acetate. As shown in Table 1, bacteria representing each of these four species of Enterococcus were found to O acetylate their peptidoglycans. To our knowledge, this is the first report of O acetylation of peptidoglycan in any species of enterococci. The extent of this modification, relative to the muramic acid content, was found to range broadly from 19 to 72%. As observed with other species for which a number of strains have been analyzed, such as N. gonorrhoeae (35, 41) and Proteus mirabilis (9, 11), the extent of O acetylation in E. faecalis and E. hirae was strain dependent. Also similar to the other O-acetylated bacteria that have been studied, the levels increased as the cells entered the stationary phase. The increases ranged from at least 10% for E. faecium NRC 8012 (an increase from 46% to 57%) to almost 40% for E. hirae ATCC 10541 (an increase from 26% to 68%).
Susceptibility to muramidase.Consistent with the presence of ester-linked acetate, the enterococcal peptidoglycans were relatively resistant to hydrolysis by HEWL. As shown in Fig. 2, addition of HEWL to suspensions of insoluble peptidoglycan sacculi isolated from E. faecalis ATCC 19433 grown to the mid-exponential phase resulted in less than 20% solubilization within the first 15 min of the reaction, and the level reached approximately 50% after prolonged incubation. In contrast, prior removal of the O-acetyl groups from the peptidoglycan by mild-base hydrolysis resulted in complete solubilization within 15 min of enzyme addition under the same reaction conditions. It should be noted that the base hydrolysis treatment also removed any teichoic acid associated with the peptidoglycan, which, like acetate, is covalently attached at C-6 hydroxyl groups of muramoyl residues. Thus, both types of modification to the muramoyl residues likely contributed to the observed HEWL resistance. Nonetheless, peptidoglycans isolated from cells grown to the stationary phase and therefore having increased levels of O acetylation were even more resistant to HEWL-catalyzed hydrolysis, suggesting that this modification has an important role in conferring resistance. Similar observations were made with the other E. faecalis strains (data not shown).
Susceptibility of E. faecalis peptidoglycan to hen egg white lysozyme. Representative samples of peptidoglycan (approximately 0.5 mg/ml) in 50 mM sodium phosphate buffer (pH 6.0) were incubated at 25°C with 200 μg/ml (final concentration) of lysozyme, and the decrease in OD660 was monitored with time. The percentage of peptidoglycan solubilized was determined by comparison with the amount solubilized in an appropriate control to which lysozyme was not added. Peptidoglycans were isolated from E. faecalis ATCC 19433 grown to the mid-exponential phase (▴), the stationary phase (•), and the VBNC state (▪). ⧫, control (de-O-acetylated E. faecalis peptidoglycan prepared by incubation in 40 mM NaOH for 2 h).
These data are thus analogous to those obtained with both N. gonorrhoeae (6, 35, 36, 41) and P. mirabilis (9, 11, 16), in which resistance to HEWL was shown to be directly proportional to the levels of O acetylation. More importantly, however, the data help explain very early observations regarding large variations in the lytic response of enterococcal cells to HEWL (7, 42), which was later found to be dependent on the strain used and the growth phase from which the cells were obtained (17, 31). The fact that the previous studies were conducted using buffers with a basic pH further compromised the data due to the lability of the ester-linked acid in this environment. The observation that all enterococci are eventually lysed by mutanolysin, in contrast to the differential effects obtained with lysozyme, can also be accounted for by O acetylation because the action of mutanolysin (a muramidase produced by Streptomyces globisporus) is not affected by this modification to peptidoglycan. Finally, the fact that a single intravenous injection of mutanolysin resulted in complete resolution of acute arthritis and prevention of chronic joint disease induced by an athropathic dose of enterococcal peptidoglycan in a rat model (23) can also be accounted for by the presence of O acetylation. Although more active than HEWL, rat (and human) lysozyme does not efficiently hydrolyze O-acetylated peptidoglycan, thus permitting large fragments to persist in the mammalian host and thereby induce a variety of pathobiological effects (12, 16). The administration of mutanolysin, however, should lead to the immediate degradation of the circulating O-acetylated peptidoglycan sacculi into small fragments that may persist but are not arthropathic.
Autolysin profile of E. faecalis.As we demonstrated that the most clinically relevant enterococcal species, E. faecalis, O acetylates its peptidoglycan, we were curious to know if this bacterium produces a complement of autolysins with various specificities for the modified cell wall polymer. To investigate this, we utilized the recently developed zymogram procedure involving acidic separating gels and running buffers (40). This system retains the natural levels of base-labile O acetylation on the peptidoglycan substrate throughout electrophoretic separation of protein samples, thereby permitting differentiation of any autolysins with specificity for the modified substrate.
Identical samples of the total proteins present in E. faecalis ATCC 19433 grown to the late exponential phase were subjected to this electrophoretic system involving two separating gels, one containing O-acetylated E. faecalis peptidoglycan and the other containing de-O-acetylated material (prepared by mild-base hydrolysis). In both systems, at least five autolysin bands at apparent molecular masses of 105, 90.2, 88.0, 79.7, and 66.8 kDa were clearly visible in the separating gels counterstained with methylene blue (Fig. 3). Although this is the first report of E. faecalis autolysins, the presence of five lytic bands was somewhat surprising since only one major autolysin, a muramidase with an apparent molecular mass of 93.6 kDa, was previously known to be produced by E. hirae (formerly E. faecalis) (37). This E. hirae autolysin is processed into two smaller forms with molecular masses of 91.5 and 66.8 kDa (1, 8, 27). It is possible that the autolytic bands in Fig. 3 at apparent molecular masses of 90.2, 88.0, and 66.8 kDa correspond to the three forms of the E. hirae muramidase. Whereas the 105-kDa autolysin is probably a distinct enzyme that has not been characterized yet, it is possible that the 79.7-kDa autolysin represents another proteolytic form of the 90.2-kDa enzyme. In addition to the use of a different enterococcal species, the reasons for the discovery of two new putative autolysins could include the use of both an acidic electrophoretic system and different peptidoglycans for the zymography. In the current study, purified E. faecalis peptidoglycan sacculi were incorporated into the running gels of the zymograms, whereas Leclerc and Asselin (27) employed Micrococcus luteus cells in their zymogram analysis of E. hirae cell extracts. Indeed, these workers detected only one autolytic band at 67 kDa in zymograms of an identical cell extract when the cell walls of Corynebacterium sepedonicum were used as the substrate.
Zymogram analysis of E. faecalis ATCC 19433 autolysins under acidic conditions. Identical samples of the total proteins present in E. faecalis grown to the late exponential phase were subjected to the electrophoretic system involving two separating gels, one containing O-acetylated E. faecalis peptidoglycan (+OAc) and the other containing de-O-acetylated material (prepared by mild-base hydrolysis) (−OAc). The arrows and asterisks indicate the positions of autolysins with less and more activity with O-acetylated peptidoglycan, respectively.
While the acidic electrophoretic system used precluded the formation of tightly focused protein bands, as expected in basic systems, it was apparent that some of the intensities of the clearing zones were different, suggesting that there were variations in the specificities of the autolysins detected. Thus, the 90.2-kDa muramidase and its potential proteolytic forms, together with the 79.7-kDa autolysin, appeared to be more active with the de-O-acetylated peptidoglycan, whereas the newly discovered 105-kDa enzyme had greater specificity for the naturally O-acetylated material. In addition to these autolysins, a number of minor enzymes with lower molecular masses were barely detectable in the acidic zymograms. It is possible that some of these enzymes may be more active with the O-acetylated material, but further careful analysis is required for confirmation of this.
The finding that E. faecalis produces autolysins with different specificities for O-acetylated peptidoglycan is consistent with the hypothesis that this modification aids in the control of autolytic activity. With E. faecalis, it seems that the activity of the various forms of the so-called major muramidase (molecular masses, 90.2, 88.0, and 66.8 kDa) is controlled at the enzymatic level through O acetylation of the substrate, which, as shown in Table 1, increases with the age of the culture, thereby preventing autolysis as the cells remain in the stationary phase. To compensate for the lack of continued activity of this muramidase, which may still be required for routine maintenance of the peptidoglycan sacculus and/or insertion of various cell wall components, it is possible that the gene encoding the 105-kDa enzyme is then induced and expressed. Evidence for such expression of this enzyme in response to the state of the culture and, more specifically, the extent of peptidoglycan O acetylation is presented below.
O acetylation of VBNC E. faecalis.The cell wall of E. faecalis grown to the VBNC state has recently been characterized by other workers in terms of the chemical composition, and comparisons were made with exponentially growing and stationary cells (39). This analysis revealed that (i) the extent of peptidoglycan cross-linking was greater in VBNC cells, (ii) the lipoteichoic acid levels doubled, and (iii) the amount of teichoic acid remained unchanged. However, the conditions employed for these studies (i.e., the method employed for isolation of the peptidoglycan and the chemical treatments used for characterization of the peptidoglycan) precluded detection of O acetylation. Since it is now known that the peptidoglycan of E. faecalis is O acetylated, there is still the question of whether the cell wall layer becomes more recalcitrant to hydrolysis by increasing its level of O acetylation when it enters the VBNC state. This issue is all the more intriguing in light of the fact that teichoic acids are covalently linked to the peptidoglycan of gram-positive cells through phosphoester linkages to the C-6 hydroxyl group of muramoyl residues (18) (viz., the same site as the site for O acetylation) (11). With no observed increase in teichoic acid levels in VBNC cells, the potential for increased O acetylation would exist.
In the current study, cells of three strains of E. faecalis were grown to the VBNC state for further analysis of both their peptidoglycans and autolysin profiles. Thus, following a minimum of 16 days of incubation at 4°C in raw well water, the morphology of the cells became more irregular, as expected, and the cells were slightly elongated (30). Confirmation that the cells had entered the VBNC state was obtained by unsuccessful attempts to culture them back on either BBEA or tryptic soy agar.
The cells were harvested, and their peptidoglycan sacculi were recovered, taking care to preserve the natural levels of any O acetylation. Analysis of this material revealed that the extent of O acetylation had increased by 10 to 16% from the stationary-phase levels. Thus, the VBNC cells had the highest levels of modification, which ranged from 44 to 72% depending on the strain (Table 1). These levels of O acetylation are among the highest known so far for bacteria characterized for this modification under natural conditions (9, 11, 12). In this regard, the level of O acetylation of Staphylococcus aureus peptidoglycan has been observed to increase to approximately 70% upon administration of the bacteriostatic antibiotic chloramphenicol (24). Presumably, in both conditions of bacteriostasis in which cell growth and division have been halted, the maturation of peptidoglycan O acetylation is allowed to continue.
Hydrolysis and autolysis of VBNC E. faecalis.Not surprisingly given the high levels of O acetylation, the peptidoglycan associated with the VBNC E. faecalis was found to be recalcitrant to HEWL-catalyzed hydrolysis. As shown in Fig. 2, incubation of the O-acetylated peptidoglycan from strain ATCC 19433 (44%) with HEWL resulted in less than 12% solubilization over a 60-min period. With strain NRC 8023, which had 72% O acetylation in the VBNC state, the level of solubilization was less than 8% (data not shown). Hence, the extensive levels of modification protect the cell walls of the VBNC cells from exogenous hydrolytic agents and thereby permit the cells to survive in this otherwise vulnerable state. Such high levels of O acetylation also preclude any detrimental activity of the major endogenous muramidase produced by E. faecalis, which, interestingly, has been observed to be expressed at elevated levels in VBNC cells (39). As noted above, this enzyme appears to be inhibited by O acetylation of its substrate. Indeed, despite a reported 25 to 50% increase in the level of this muramidase, the rate of autolysis for VBNC cells was found to be only slightly higher than the rates of autolysis for either exponentially growing or stationary cells (39).
The increase in autolysin concentration associated with VBNC E. faecalis observed by Signoretto et al. (39) was confirmed in the current study. Figure 4 shows a zymogram for an experiment in which samples containing equal concentrations of E. faecalis cell sonicates obtained at different stages of growth were loaded into a separating gel containing E. faecalis peptidoglycan under basic conditions. Identical samples were loaded into a second zymogram gel, but this gel was stained with Coomassie brilliant blue after electrophoresis to confirm protein concentrations (data not shown). Under these conditions, in which the peptidoglycan substrate would have existed in its de-O-acetylated state, the three forms of the major muramidase were readily detected in the cells growing exponentially. When cells entered the stationary phase, the expression of this enzyme in all three states persisted, but at significantly reduced levels, with the smallest form predominating. In addition, four other lytic bands became evident. That this shift in autolysin production was not an artifact of unequal gel loading is clear from the obvious change in the proportions of the autolysins detected in the separate samples. One of the newly produced enzymes was the 105-kDa autolysin discussed above, which has specificity for both O-acetylated and unacetylated peptidoglycan. The three other enzymes, with apparent molecular masses of 62, 54, and 47.5 kDa, have not been detected previously. It is possible that one or more of these enzymes may represent latent forms of either the muramidase or the 105-kDa autolysin. Alternatively, they could represent unique autolysins that function specifically in stationary-phase cells.
Zymogram analysis of autolysins from E. faecalis ATCC 19433 grown to different culture states. Sonicates of equal amounts of cells from mid-exponential-phase (lane 1), stationary-phase (lane 2), and VBNC (lane 3) cultures were loaded into a zymogram gel containing peptidoglycan under basic conditions. The black and white arrows indicate autolytic bands with relatively low and high intensities, respectively.
Following conversion to the VBNC state, E. faecalis resumed expression of the major muramidase in all three of its forms while both maintaining production of the lower-molecular-mass autolysins and increasing production of the 105-kDa autolysin. In fact, production of the muramidase appeared to be at the highest level detected for all three cell states, which is consistent with the observations made by Signoretto et al. (39). Why E. faecalis resumes production of this muramidase at such high levels is not clear given our finding that the enzyme does not efficiently act on the highly O-acetylated peptidoglycan that exists in this VBNC cell state. This finding is especially puzzling in view of the cell's increased production of the 105-kDa autolysin, which hydrolyzes the O-acetylated wall polymer and thus has a housekeeping function. Moreover, although considered to be the major autolysin produced by E. faecalis, the muramidase is not essential for cell growth and plays no significant role in virulence (34). It has been proposed that this muramidase may aid in the persistence of VBNC E. faecalis in aquatic environments by participating at the cell surface in adhesion of the cells to surfaces, such as the chitin walls of copepods (38). No impairment of such adhesion was found with a Lyt mutant of E. faecalis, which produces the muramidase with apparently reduced hydrolytic activity (13); this finding resulted in dismissal of this proposal (38). However, the muramidase extracted from the Lyt mutant did not differ from the parent enzyme in terms of the ability to bind to cell wall substrates (13), leaving the possibility that the "dysfunctional" derivative of the muramidase may nonetheless be expressed and perform this binding function. Clearly, more studies are required to address this issue.
An alternative function for the increased production of the muramidase in VBNC E. faecalis could involve the role of the enzyme in predation. A variety of other bacteria are now known to release a complement of enzymes, including autolysins, when they are stressed, and these enzymes may both kill and degrade neighboring cells, thereby providing necessary nutrients. For example, Pseudomonas aeruginosa has been observed to release proteases, lipases, nucleases, and autolysins, including its major autolysin, in membrane vesicles when it is confronted with environmental stresses or administration of antibiotics (25, 28, 29). These enzyme packages have been demonstrated to be capable of attacking both other gram-negative bacteria and gram-positive bacteria. Hence, it is conceivable that VBNC E. faecalis in a nutrient-depleted environment releases its major autolysin, the 105-kDa muramidase, and its derivatives for the same purpose, thus preying on other bacteria while protecting itself from autolysis by increasing the level of peptidoglycan O acetylation.
Concluding remarks.The presence of peptidoglycan O acetylation in the enterococci has escaped previous detection and study. This is true despite the significant attention that these bacteria, especially the vancomycin-resistant enterococci, have attracted. The fact that this modification of the peptidoglycan sacculus appears to play a role in the control of autolysins and thus the growth and division of the bacteria (5, 33) underscores the need for further investigation. Little is known about the pathway for peptidoglycan O acetylation, but O acetylation has been shown to be a maturation event, occurring outside the cell within the sacculus (reviewed in references 11 and 12). This suggests that the enzyme(s) involved in the process is relatively exposed to exogenous agents, such as administered antibiotics. Hence, this enzymatic system might prove to be an attractive new target for the development of a new class of antibiotics effective against the enterococci, as well as possibly many other bacteria.
Recently, an integral membrane protein in S. aureus N315 was found to function as a peptidoglycan O-acetyltransferase (OatA) (2). Using OatA (accession number BAB43658) as a probe, we identified a gene encoding a potential homolog in the E. faecalis genome (accession number AE016949) with 34.4% identity and 69.5% similarity. Thus, our future studies will involve cloning of this hypothetical gene and determination whether its translated product does in fact also function as a peptidoglycan O-acetyltransferase.
This work was supported by an operating grant to A.J.C. from the Canadian Institutes for Health Research (grant 62772) and by a postgraduate scholarship (PGSB) to J.T.W. from the Natural Sciences and Engineering Research Council of Canada.
Received 15 September 2005.
Accepted 27 October 2005.
Béliveau, C., C. Potvin, J. Trudel, A. Asseli, and G. Bellemare. 1991. Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin. J. Bacteriol. 173:5619-5623.
Bera, A., S. Herbert, A. Jakob, W. Vollmer, and F. Gotz. 2005. Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus. Mol. Microbiol. 55:778-787.
Bernadsky, G., T. J. Beveridge, and. A. J. Clarke. 1994. Analysis of the sodium dodecyl sulfate-stable autolysins of select gram-negative pathogens by using renaturing polyacrylamide gel electrophoresis. J. Bacteriol. 176:5225-5232.
Blackburn, N. T., and A. J. Clarke. 2002. Families of lytic tranglycosylases. J. Mol. Evol. 52:78-84.
Blackburn, N. T., and A. J. Clarke. 2002. Characterization of soluble and membrane-bound family 3 lytic transglycosylases from Pseudomonas aeruginosa. Biochemistry 41:1001-1013.
Blundell, J. K., G. J. Smith, and H. R. Perkins. 1980. The peptidoglycan of Neisseria gonorrhoeae: O-acetyl groups and lysozyme sensitivity. FEMS Microbiol. Lett. 9:259-261.
Chesbro, W. R. 1961. Lysozymes and the production of osmotic fragility in enterococci. Can. J. Microbiol. 7:952-955.
Chu, C. P., R. Kariyama, L. Daneo-Moore, and G. D. Shockman. 1992. Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae. J. Bacteriol. 174:1619-1625.
Clarke, A. J. 1993. Extent of peptidoglycan O acetylation in the tribe Proteeae. J. Bacteriol. 175:4550-4553.
Clarke, A. J. 1993. Compositional analysis of peptidoglycan by high-performance anion-exchange chromatography. Anal. Biochem. 212:344-350.
Clarke, A. J., and C. Dupont. 1992. O-Acetylated peptidoglycan: its occurrence, pathobiological significance and biosynthesis. Can. J. Microbiol. 38:85-91.
Clarke, A. J., H. Strating, and N. T. Blackburn. 2000. Pathways for the O-acetylation of bacterial cell wall polymers, p. 187-223. In R. J. Doyle (ed.), Glycomicrobiology. Plenum Publishing Co. Ltd., New York, N.Y.
Cornett, J. B., B. E. Redman, and G. D. Shockman. 1978. Autolytic defective mutant of Streptococcus faecalis. J. Bacteriol. 133:631-640.
Courvalin, P. 2005. Genetics of glycopeptide resistance in gram-positive pathogens. Int. J. Med. Microbiol. 294:479-486.
Difco Laboratories. 1984. Difco manual, 10th ed. Difco Laboratories, Detroit, Mich.
Dupont, C., and A. J. Clarke. 1991. Dependence of lysozyme catalyzed solubilization of Proteus mirabilis peptidoglycan on the extent of O-acetylation. Eur. J. Biochem. 195:763-769.
Glick, A. D., J. M. Ranhand, and R. M. Cole. 1972. Degradation of group A streptococcal cell walls by egg white lysozyme and human lysosomal enzymes. Infect. Immun. 6:403-413.
Grant, W. D., and A. J. Wicken. 1968. Muramic acid phosphate and the linkage of teichoic acid to peptidoglycan in Bacillus stearothermophilus cell walls. Biochem. Biophys. Res. Commun. 32:122-128.
Hancock, R. E. W. 2005. Mechanisms of action of newer antibiotics for gram-positive pathogens. Lancet Infect. Dis. 5:209-218.
Hash, J. H. 1967. Measurement of bacteriolytic enzymes. J. Bacteriol. 93:1201-1202.
Höltje, J.-V., D. Mirelmen, N. Sharon, and U. Schwarz. 1975. Novel type of murein transglycosylase in Escherichia coli. J. Bacteriol. 124:1067-1076.
Hoyle, B. D., and T. J. Beveridge. 1983. Metal binding by the peptidoglycan sacculus of Escherichia coli K-12. Can. J. Microbiol. 30:204-211.
Janusz, M. J., C. Chetty, R. A. Eisenberg, W. J. Cromartie, and J. H. Schwab. 1984. Treatment of experimental erosive arthritis in rats by injection of the muralytic enzyme mutanolysin. J. Exp. Med. 160:1360-1374.
Johannsen, L., H. Labischinski, B. Reinicke, and P. Greisbrecht. 1983. Changes in the chemical structure of walls of Staphylococcus aureus grown in the presence of chloramphenicol. FEMS Microbiol. Lett. 16:313-316.
Kadurugamuwa, J. L., and T. J. Beveridge. 1996. Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria, including pathogens: conceptually new antibiotics. J. Bacteriol. 178:2767-2774.
Kau, A. L., S. M. Martin, W. Lyon, E. Hayes, M. G. Caparon, and S. J. Hultgren. 2005. Enterococcus faecalis tropism for the kidneys in the urinary tract of C57BL/6J mice. Infect. Immun. 73:2461-2468.
Leclerc, D., and A. Asselin. 1989. Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis. Can. J. Microbiol. 35:749-753.
Li, Z., A. J. Clarke, and T. J. Beveridge. 1996. A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division, and secretion in surface membrane vesicles. J. Bacteriol. 178:2479-2488.
Li, Z., A. J. Clarke, and T. J. Beveridge. 1998. Gram-negative bacteria produce membrane vesicles which are capable of killing other bacteria. J. Bacteriol. 180:5478-5483.
Lleo, M. M., M. C. Tafi, and P. Canepari. 1998. Nonculturable Enterococcus faecalis cells are metabolically active and capable of resuming active growth. Syst. Appl. Microbiol. 21:333-339.
Metcalf, R. H., and R. H. Deibel. 1972. Growth of Streptococcus faecium in the presence of lysozyme. Infect. Immun. 6:178-183.
Murray, B. E. 1990. The life and times of Enterococcus. Clin. Microbiol. Rev. 3:46-65.
Payie, K. G., H. Strating, and A. J. Clarke. 1996. The role of O-acetylation in the metabolism of peptidoglycan in Providencia stuartii. Microb. Drug Resist. 2:135-140.
Qin, X., K. V. Singh, Y. Xu, G. M. Weinstock, and B. E. Murray. 1998. Effect of disruption of a gene encoding an autolysin of Enterococcus faecalis OG1RF. Antimicrob. Agents Chemother. 42:2883-2888.
Rosenthal, R. S., J. K. Blundell, and H. R. Perkins. 1982. Strain-related differences in lysozyme sensitivity and extent of O-acetylation of gonococcal peptidoglycan. Infect. Immun. 37:826-829.
Rosenthal, R. S., W. J. Folkening, D. R. Miller, and S. C. Swim. 1983. Resistance of O-acetylated gonococcal peptidoglycan to human peptidoglycan-degrading enzymes. Infect. Immun. 40:826-829.
Shockman, G. D., J. S. Thompson, and M. J. Conover. 1967. The autolytic enzyme system of Streptococcus faecalis. II. Partial characterization of the autolysin and its substrate. Biochemistry 6:1054-1065.
Signoretto, C., G. Burlacchini, C. Pruzzo, and P. Canepari. 2005. Persistence of Enterococcus faecalis in aquatic environments via surface interactions with copepods. Appl. Environ. Microbiol. 71:2756-2761.
Signoretto, C., M. M. Lleo, M. C. Tafi, and P. Canepari. 2000. Cell wall chemical composition of Enterococcus faecalis in the viable but noncultruable state. Appl. Environ. Microbiol. 66:1953-1959.
Strating, H., and A. J. Clarke. 2001. Differentiation of bacterial autolysins by zymogram analysis. Anal. Biochem. 291:149-154.
Swim, S. G., M. A. Gfell, C. E. Wilde III, and R. S. Rosenthal. 1993. Strain distribution in extents of lysozyme resistance and O-acetylation of gonocccal peptidoglycan determined by high-performance liquid chromatography. Infect. Immun. 42:446-452.
Toennies, G., L. Iszard, N. B. Rogers, and G. D. Shockman. 1961. Cell multiplication studied with an electronic particle counter. J. Bacteriol. 82:857-866.
Watt, S., and A. J. Clarke. 1994. Initial characterization of two extracellular autolysins from Pseudomonas aeruginosa PAO1. J. Bacteriol. 176:4784-4789.
Weadge, T. J., J. M. Pfeffer, and A. J. Clarke. 2005. Identification of a new family of enzymes with potential O-acetylpeptidoglycan esterase activity in both gram-positive and gram-negative bacteria. BMC Microbiol. 5:49.
Journal of Bacteriology Jan 2006, 188 (3) 902-908; DOI: 10.1128/JB.188.3.902-908.2006
You are going to email the following Peptidoglycan O Acetylation and Autolysin Profile of Enterococcus faecalis in the Viable but Nonculturable State


text:
Gordon Parks-Inspired Arts Workshop
Monday, February 9th
(6:00-7:00, 7:15-8:30 p.m.)
Mozart Park Fieldhouse
2036 N. Avers Ave.
all ages, free & open to the public
You are invited to come to our next arts workshop, when we focus on the life and legacy of Gordon Parks, who was a photographer, filmmaker, poet and composer. We will:
Talk about Parks' biography, including his connection to Chicago;
Look at and discuss a some of Parks' photographs;
Watch a clip of one of his films; and
Write poetry and create visual art inspired by Gordon Parks.
Bring your musical instruments, your poetry, your art supplies! These workshops are interactive and multidisciplinary. We have supplies and equipment on hand that workshop participants can use -- including pencils, pens, paint, a piano, other musical instruments.
Location: Mozart Park is in Chicago's Logan Square neighborhood. It's on the north side of Armitage Ave. -- several blocks east of Pulaski Rd., just south of Dickens and Shakespeare Streets. Our workshop series happens in the room to the left of fieldhouse lobby; some workshop sessions may happen outside in the park, weather permitting.
Transportation & parking: Mozart Park can be reached by public transportation (such as the #73 Armitage Ave. bus, and not far from the Logan Square and Western Ave. stations on the CTA's blue line. Mozart Park has a parking lot on Armitage, east of Avers.
Registering for Chicago Portraits & Arts Journeys: You can register for this workshop series at the Chicago Park District website.
Additional info: You can contact us by clicking here (if you have questions about this workshop or to RSVP). Click here to find out more about the Chicago Portraits & Arts Journeys workshop series.
Above: Photographs by Gordon Parks. Courtesy of and copyright The Gordon Parks Foundation.
"Gordon Parks: Arts in the service of a social vision" by Steve Kloehn (UChicagoNews, 2/25/2014)
The Gordon Parks Foundation
Gordon Parks and Isaac Hayes discuss the Shaft theme
The Gordon Parks Museum
Gordon Parks Papers, Collection Summary (Wichita State University)
"A Lost Story of Segregated America From LIFE's First Black Photographer" by Eliza Berman (LIFE Magazine, 1/23/2015)
Oral history interview with Gordon Parks, 1964 Dec. 30 (Archives of American Art at the Smithsonian Institution)
Nelson Algren-Inspired Arts Workshop at Mozart Park
You are invited to come to our next Chicago Heroes & Arts Adventures workshop --
Monday, March 31 (6:00-7:15, 7:30-8:30 p.m.)
During this workshop, inspired by the great Chicago writer Nelson Algren, participants will:
Learn more about Algren's work and legacy;
Read passages from Algren's poetry and prose, and several letters from Simone de Beauvoir to Algren;
Watch and discuss several video clips: part of Studs Terkel's 1975 interview with Algren; a clip from The Road Is Nothing, the End Is All; and Willem DeFoe reading "The Lightless Room," a short story by Algren; and
Creatively explore Algren's work through creative writing and visual art.
Location: Mozart Park is on Armitage Avenue in Logan Square -- several blocks east of Pulaski Road, just south of Dickens and Shakespeare Streets. There is street parking by Mozart Park, and a parking lot is on the east side of Avers.
Please contact us by clicking here -- if you have questions about this workshop and/or if you would like to RSVP. Thanks!
"A Breakup Letter from Simone de Beauvoir" by Maria Popova
"But Never a Lovely So Real" by Colin Asher (The Believer)
Interview: Filmmaker Michael Caplan (chicagist.com)
"Life, Love, and Nothing in Between" by Kelly Kleinman (Chicago Reader)
"Love and Letters in Paris and Chicago" by Joel Henning (The Wall Street Journal)
The Man with the Golden Arm (dir. Otto Preminger, 1955)
Nelson Algren Committee
Nelson Algren Live! event clip featuring William Dafoe
Nelson Algren: The End Is Nothing, the Road Is All
"The Ninth Man Out: As baseball begins again, Nelson Algren turns 100" by Jeff McMahon (New City)
"The Secret Faces of Poets in Nelson Algren's Chicago: City on the Make" by Jeff McMahon
Interview with Meg Duguid
Meg Duguid is an artist, performer and curator whose work has been presented at Defibrillator (including Bubbles Plus Bubbles Divided by Traffic), and collaborations with Out of Site.
Duguid received her BFA from the School of the Art Institute of Chicago and her MFA from Bard College. She has performed and exhibited at the Museum of Contemporary Art in Chicago, the Hyde Park Art Center in Chicago, Macy's on State Street in Chicago, the DUMBO Arts Festival in Brooklyn, and 667 Shotwell in San Francisco. She has screened work at Synthetic Zero in New York, Spiderbug in Chicago, and at the Last Supper Festival in Brooklyn. From 2009 to 2011 she ran Clutch Gallery, a 25 square-inch white cube located in the heart of her purse; since then she has lent my purse to others to curate and carry. She lives and works in Chicago, IL with her husband and three cats.
Recently Borderbend intern Ellyn Leahy interviewed Meg Duguid about her background, curating Clutch Gallery, and other projects.
Leahy: What artists most inspire you?
Duguid: Mierle Laderman Ukeles, Marcel Duchamp, Aleksandra Mir, Pina Bausch.
Leahy: You've been living here in Chicago for a few years now. What is your favorite thing about living and working in Chicago?
Duguid: My favorite thing about living in Chicago is the fact that my husband lives here and all of my stuff is here. My favorite thing about working in Chicago is the community's ability to entertain anything as art. It makes the discussion about what art is and how it functions really great.
Leahy: "Recipe Roulette," which you and Catie Olson performed during the 2010 Chicago Calling Arts Festival, was wonderful. What role does humor play in your art?
Duguid: Recipe Roulette was a collaborative piece that Catie Olson and I created where I made chocolate bon bons while Catie hula-hooped. Every time the hoop would drop, I would drop a bon bon on the floor in solidarity with Catie's motions and also locking her movement into a certain part of the floor as the bon bons built up. This was a sister work of Jump Jump Pie Pie, that we performed in Brooklyn where we each made an edible mud pie while the other kept pace by jumping rope in high heels.
above: photos of Meg Duguid and Catie Olson performing Recipe Roulette
Catie and I investigate the create work using an iterative structure; we let our play-on-word conversations lead our projects. Our collaborative rapport is as important as the final events themselves. During our process the iterative word piles that we create allow our personal and political lives to float to the top as well as let us frame them through our own diverse experiences with different media and mediums. At first glance our work seems fun, but there is more to it than that; on closer inspection you will find that not only are we two women creating humorous work but we tend to reference word play, the body, and food as well.
Leahy: How did you first get involved with curating, and how does it affect your other work?
Duguid: I have personally curated and organized a few projects over the years. Usually I put things together because there were a number of folks I wanted to work with or put a show together with.
Beach by Kim Guare, presented by Clutch Gallery
photo credit: Emma Robins
In 2009, I moved back to Chicago and conceived Clutch, which is the longest-running curatorial project I have done. Clutch Gallery is a 25-square-inch space located in the heart of my purse. This curatorial project was dedicated to exhibiting contemporary art of all media. Clutch opened in December 2009 and was initially intended to maintain regular programming through December 2010, but I continued to carry and program it until the end of 2011 in the belief that it would die a fitting and natural death by wearing out from daily use.
During my time with Clutch, I showed 23 artists in 22 shows. I was responsible for each job in my gallery—I was preparator, gallery assistant, director, and marketing director, but most importantly I was a performer.
With Clutch, my intent was to play on the grand history of artist-run spaces in this city while extending my own practice as a performance artist. Clutch allowed me to create a scenario in which my daily interactions with people could become a performance at any time.
Clutch had an element of constant duration. The performance was larger than just opening up the piece to someone at any moment or explaining the nature of the work. Essentially as a performer, the duration of the piece was both constant and not at all. The potential energy of just holding the space, maintaining the space, and sitting near the space allowed its performative potentiality to radiate into all things Clutch, and the moment someone approached Clutch's conceptual sphere, the person became a part of it whether the purse was in a state of rest or motion. During my time with Clutch I performed alone, with shopkeepers, baristas, TSA agents, thieves, the Secret Service, artists, family, and many others.
Last fall I decided to stop carrying Clutch. So I put it up for others to carry. Emma Robbins is currently carrying and curating Clutch Gallery.
above: photos from Meg Duguid's
Sounds like Mustache
Leahy: Why do you often choose to use multiple media in your work -- for example, combining performance art, photography, and drawing in the Episode Series?
Duguid: I believe that documentation is paramount to my art making, and I'm invested in the ways that documentation and art can be integrated in a single practice. At one point I felt comfortable talking about my work as performance art. But now I have found that the term performance art no longer encompasses what it is I do. I am actively seeking to articulate the relationships I am making between the performative and photography, video, sculpture, and drawing.
Drawing has proved to be a really effective tool for me in a few ways. First, for creating objects that stand in for the real deal. A mustache can suddenly just be a drawing of one held up in front of one's face, and that's enough for the viewer to say "Hey, look. A mustache."
Second, drawing is a really nice tool to use in conjunction with photographs of an action. In 2003, I started a series of street performances based in early filmic comedy. These performances derived from the simple question of what happens when you take a comedic moment out of its media context and re-present it in real life. While performing these acts in public, I have found that video cameras become too intrusive and upset the performance, usually contextualizing it as reality television, rather than being a calculated moment. As a result I switched my format to still photography, which allowed my documenters to remain as unobtrusive as tourists or other passersby.
When I got the photos back, I began to ask myself, how do I make this more than just documentation? It's this question that led me to start erasing the performers out of the images and drawing them back in. The result is a photograph of an actual place and time with a comic book performer doing the action. It plays with the idea of what is real and what isn't, as well as starts to refer to the idea of comic in all its meanings.
In the autumn of 2007, I staged a 15-person slapstick performance in Battery Park in New York City. The performance consisted of an all-female cast, five photographers, and the backdrop of the Statue of Liberty. I am currently in the process of creating a comic book from it. There are about 200 images in the book, so this piece has taken up various portions of the last five years, and I am finally going to be ready to unveil it by the end of this year.
Leahy: Last October you had a performance art piece called "Bubbles Plus Bubbles Divided by Traffic," which happened at Defibrillator during Chicago Calling. Would you mind giving us a glimpse into the creative process behind that piece?
above: photos from Bubbles Plus Bubbles
Divided by Traffic
Duguid: This work was twofold for me. I have been thinking about the performative quality of action painting lately, and I am really interested in how a piece of documentation from a previous work can carry over to the next work. The work in Defibrillator was called Bubbles Plus Bubbles Divided by Traffic, and it took place in the two windows at Defibrillator. This performance consisted of two men wearing Speedos, one in each window, making action paintings by blowing colored bubbles onto paper to a sound piece of traffic over bubble wrap.
Bubbles Plus Bubbles Divided by Traffic utilized a sound piece that was created as a part of Sounds Like Mustache that was performed at the Polish Triangle in August of 2011 as a part of the Out of Site Performance Series. In that performance I poured soap into the fountain in the triangle while bubble machines spewed bubbles into the triangle while a three-person mustache, a mustache that is so big two people must carry it so a third can wear it, walked on bubble wrap that was laid down throughout the triangle. The documentation of this work was the sound work Bubble Wrap Over Traffic.
Leahy: What are you working on now?
I have some work, including some of the newer parts of the episode series, going to the ZonaMaco Art Fair in Mexico City. Catie Olson and I are collaborating on a 10-course performance that is going to be performed at Defibrillator in September of 2012. And I am at the tail end of negotiating the rights to produce a screenplay that I have fallen in love with. I don't want to say what it is until the ink is dry on the contract, because you just don't know until the signature hits the dotted line, but if it all works out well, this will be the largest project I have ever taken on.
Bicycles & the Arts
The Borderbend Arts Collective has presented two "Bicycles & the Arts" events -- during the Chicago Calling Arts Festival in 2010 and last year. Borderbend is co-presenting a third "Bicycles & the Arts" event, in partnership with Working Bikes Cooperative. That event happens on September 28, during the Seventh Annual Chicago Calling Arts Festival.
Bicycles and the arts have commingled a lot for more than a century -- such as in film, literature, sculpture, and music. Here are some highlights which span more than a century. Alfred Jarry was a French writer who wrote the play Ubu Roi and came up with the concept of pataphysics; he was an avid bicyclist, and bikes appear in many of his writings. Another French artist named Marcel Duchamp created a sculpture entitled Bicycle Wheel in 1913; that sculpture was among Duchamp's many readymades which have changed the way people think about art. Several decades later, Italian filmmaker Vittorio De Sica directed The Bicycle Thief (1948), an iconic Italian film which "works as a sentimental study of a father and son, a historical document, a social statement, and a record of one of the century's most influential film movements." In 1960, Swiss artist Jean Tinguely unveiled a kinetic sculptureentitled Homage to New York, which included dozens of bicycle wheels and other parts, and whichfell apart during a 27-minute performance. Have you seen the video of a 22-year-old Frank Zappa teaching Steve Allen how to play a bicycle, followed by a performance with musical bicycles and the accompaniment of the Steve Allen Show orchestra?
Today you can find examples of intersections between bicycles and art, in Chicago and around the world. Recently a collective in Rogers Park proposed that 15 bike racks be constructed, and that proposal won funding during the 49th Ward's participatory funding initiative; those bike racks are currently under construction. If you've ever seen or participated in a Critical Mass event, you've probably seen some interesting art bikes, including tall bikes. As you travel along North Avenue in the Wicker Park / Bucktown neighborhood, you will pass a colorful bicycle mounted on the side of a building, at the corner of North and Wolcott Avenues. That bicycle, which hangs above the entrance of Rapid Transit Cyclechop, has fans in the bike wheel spokes, so the wheels are often spinning in the air. Local artist Ronnie LoBello built that bicycle 15 years ago.
Working Bikes Cooperative has several amazing bike artworks created by Matt Weber and others. Steven Lane curates an annual winter bike art show; that's always an exciting show to look forward to.
Examples of art bikes and bicycle art abound elsewhere here in the U.S. Artistically designed bike racks are being constructed in Rogers Park, and David Byrne has designed bike racks which have been installed in New York City. A group in Minneapolis has organized several annual Bike Art shows, and Tall Bike Posse in California organizes Art Bike Build events and bike art shows.Bicycle Inter-Community Action & Salvage in Tucson offers workshops where you can make inner tube wallets, tire belts, mobiles, sculptures, and jewelry out of recycled bike parts. In 2010 I saw Gabriel Orozco's Four Bicycles (There Is Always One Direction) at the MoMA, amazing.
photo of artworks on display during the "Bicycles & the Arts" event at Happy Dog Gallery (10.1.2010)
above: photos from the "Bicycles & the Arts" event during the 2010 Chicago Calling Arts Festival
Globetrotters can find plenty of examples of intersections between the arts and bicycles around the world. The Dekochari (Japanese art bike) is a unique phenomenon; here're some interesting pictures. Kunstrad (Art Cycling) competitions are happening in Europe.
Stay tuned for updates regarding the next "Bicycles & the Arts" event.
-- Dan Godston

redpajama_set_name: RedPajamaCommonCrawl


WELLINGTON, Fla., May 21, 2018 (GLOBE NEWSWIRE) -- KLX Inc. (the "Company") (NASDAQ:KLXI), a leading distributor and value added service provider of aerospace fasteners and consumables, and a provider of services and products to the oil and gas exploration and production industry, today reported its first fiscal quarter ended April 30, 2018 financial results.
On a consolidated basis, revenues increased 21.3 percent to $499.1 million, driven by an approximate 11.7 percent increase in ASG revenues and an approximate 74.0 percent increase in ESG revenues. Adjusted operating earnings and Adjusted operating margin were $75.5 million and 15.1 percent, respectively. Adjusted Net Earnings and Adjusted Net Earnings per diluted share increased 63.8 percent and 67.2 percent, to $57.0 million and $1.12 per diluted share, respectively.
"We are pleased to report that 2018 is off to a strong start as both consolidated revenues and operating earnings increased strongly in the first quarter as compared to the same period in the prior year. Our ASG segment delivered solid year-over-year revenue growth, driven by an approximate double-digit percentage increase in both commercial aerospace manufacturing and aftermarket revenues, while our ESG segment continued to generate strong revenue growth and margin expansion on both a year-over-year and sequential quarterly basis," said Amin J. Khoury, Chairman and Chief Executive Officer of KLX.
For the three months ended April 30, 2018, our ASG segment generated revenues of $388.1 million, an increase of approximately 11.7 percent compared to the same period in the prior year, driven by approximately 5.9 percent organic revenue growth. On a channel basis, aftermarket organic revenue growth of 6.6 percent was particularly strong, driven by a substantial increase in aircraft maintenance activity among MROs, repair shops and airlines in the Americas, Asia and Europe. Revenues from our OEM channel increased 5.4 percent on an organic basis, driven by an increase in activity from commercial aerospace manufacturing customers and initial revenues from the ramp-up of new programs. On a GAAP basis, ASG operating earnings were $54.7 million and operating margin was 14.1 percent. ASG's Adjusted operating earnings, adjusted to exclude one-time costs related to the strategic alternatives review and the transition to ASG's new global distribution and operations center, were $65.2 million, or 16.8 percent of revenues. ASG Adjusted EBITDA of $76.1 million was 19.6 percent of revenues.
For the three months ended April 30, 2018, as compared to the same period of the prior year, ESG revenues of $111.0 million reflected organic revenue growth of approximately 74.0 percent. GAAP operating earnings increased $16.6 million, from a loss of $(10.1) million during the first quarter of last year, to a positive $6.5 million in the current quarter, which includes the aforementioned one-time costs. Exclusive of such costs, ESG's Adjusted operating earnings were $10.3 million in the first quarter, a $20.4 million improvement over the same period last year. Adjusted EBITDA of $22.0 million improved by $20.7 million. As compared to the fourth quarter of 2017, revenues increased by 17.8 percent, Adjusted operating earnings improved by $9.1 million from $1.2 million to $10.3 million and Adjusted EBITDA improved by $9.3 million, or 73.2 percent, to $22.0 million, or approximately 20 percent of revenues.
As a result of the pending sale of the Aerospace Solutions Group (ASG) to Boeing, the Company will no longer be providing ASG segment level guidance.
The Company increased its 2018 ESG guidance for revenues, Adjusted operating earnings and Adjusted EBITDA. The Company's Fiscal 2018 ESG outlook excludes costs related to the strategic alternatives review, including spin-off related expenses.
This news release contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Exchange Act. Such forward-looking statements involve risks and uncertainties. The Company's actual experience and results may differ materially from the experience and results anticipated in such statements. Factors that might cause such a difference include those discussed in the Company's filings with the Securities and Exchange Commission ("SEC"), which include its Annual Report on Form 10-K and Current Reports on Form 8-K. For more information, see the section entitled "Forward-Looking Statements" contained in the Company's Annual Report on Form 10-K and in other filings. The forward-looking statements included in this news release are made only as of the date of this news release and, except as required by federal securities laws and rules and regulations of the SEC, the Company undertakes no obligation to publicly update or revise any forward-looking statements, whether as a result of new information, future events or otherwise.
This release includes "Adjusted Net Earnings" and "Adjusted Net Earnings per diluted share" to reflect net earnings before the non-cash charge associated with the revaluation of the Company's deferred tax assets, Costs as Defined, and the amortization and non-cash compensation expense, and to include the tax benefit from the amortization of tax-deductible goodwill. This release includes "Adjusted operating earnings," "ASG Adjusted operating earnings" and "ESG Adjusted operating earnings," which excludes Costs as Defined. This release also includes "Adjusted EBITDA," "ASG Adjusted EBITDA" and "ESG Adjusted EBITDA," which excludes non-cash compensation expense and Costs as Defined, which are "non-GAAP financial measures" as defined in Regulation G of the Securities Exchange Act of 1934, as amended (the "Exchange Act").
1 Costs and expenses related to review of strategic alternatives and transitioning to ASG's new global distribution and operations center.
2 For purposes of this calculation, tax benefit of goodwill deduction is limited to income tax at current effective rate for periods ended April 30, 2018 and April 30, 2017 of ~25.1% and ~37.7%, respectively.


text:
Serving in the spirit of Catherine McAuley
With studies, sports, and social activities, high school students' schedules are often packed full. Joan Fasulo, who recently graduated second in her class from Catherine McAuley High School in Portland, certainly knows what that is like.
"I've been sleeping a lot since graduation," she jokes.
But no matter how busy this 18-year-old's schedule becomes, Joan always makes sure there is time in it for service. Her extensive volunteer work recently earned her a Rotary Youth Leadership Award.
"When you see the people who need help, it's kind of hard not to want to help," she says.
Joan says she was raised in a household where service to others was a way of life.
"I think my mother and father taught us to give back. My mother has always volunteered, and they did a lot of work in churches when we were younger."
She recalls, for instance, at age four, accompanying her parents to the Ronald McDonald House where they were weekend managers. Joan began volunteering there herself when she was a high school freshman, working a few shifts each week.
"I cleaned and cooked, because it was usually around dinnertime after school, and I manned the desk, did paperwork and projects, and stuff like that," she says.
Joan says her family experienced firsthand the value of the Ronald McDonald House when she required heart surgery as an infant. The house, located in Portland, provides a place for families to stay while loved ones are in the hospital.
"It gives a lot of support for all the people going through similar things. You know, they'll see each other, and they'll talk," she says.
She says chatting and sharing with the families was her favorite part of serving there. "You hear a lot of stories. There were people there from all different walks of life," she says.
During summer vacation, Joan also volunteered at Camp Sunshine, a camp in Casco for children with life-threatening illnesses.
"I just signed up to be a volunteer, and I said, 'Just put me wherever you need me,'" she says. "I went there for a week. I worked the nursery and the 'tot lot,' so it was children from zero to age six."
A few times each month, Joan also lent a hand at the Good Cause Thrift Shop, which sells clothing and other goods. Proceeds go to Catherine McAuley High School, and the shop also provides direct assistance to a number of area agencies including Preble Street and the Ronald McDonald House.
"My mom used to volunteer there, so I went there. I went sometimes when she was there when I was younger, and then I did it more," she says.
In addition to the influence of her parents, Joan says Catherine McAuley High School creates an environment where service is encouraged.
"Catherine McAuley, herself, was a service lady, and they try to emulate that. We have our service weeks, and everybody had to do a mandated, certain number of hours. There are ample opportunities for you to volunteer and give back."
She says there is a great sense of community at the high school, which makes it less intimidating to join clubs and take part in activities.
"They always make everything fun. Everyone is, at least my class was, really close, so I like that a lot. And the teachers are really accessible," she says. "I think they encourage you to get a lot more involved than you would be."
During her years at McAuley, Joan tutored a student from St. Brigid School, was a member of the National Honor Society, participated in Mock Trial, was co-captain of the Math Team, and was vice president of the Social Action Club.
Her interest in social justice issues led Joan to spend the high school's service days at Preble Street, which serves those battling hunger and homelessness. It also prompted her, during the summer of her sophomore year, to participate in John Hopkins University's Center for Talented Youth (CTY) Civic Leadership Institute. The service learning program held in Berkeley, California, combined academics with community action.
"There were a lot of courses in social justice, change, and poverty and everything. So we would spend a day in the classroom and then do a day of service, then do a day in the classroom, and then a day of service."
While at the institute, Joan volunteered at the Boys and Girls Club in Oakland, did door-to-door canvassing for the Sierra Club, served breakfast through The Catholic Worker Movement, served at a shelter for displaced women in San Francisco, and helped sell Street Sheet, a small newspaper published by the Coalition on Homelessness.
"You get 30 copies, and you sell them for one-dollar a piece, and that's your income," she explains.
Joan describes selling the newspaper as an eye-opening experience.
"When we sold Street Sheet, people were so mean because they thought we were homeless. They wouldn't look you in the eye," she says. "I've never really experienced anything like that, so it was different to walk in somebody else's shoes. It was only for a day, but everybody in the group was pretty shaken up."
She calls the institute a turning point.
"It just clicked. We had speakers come in and talk to us and stuff," she says. "You weren't just doing work, you knew people; you were learning all these things."
Joan plans to study public policy when she attends the University of Virginia this fall. She says she would either like to become an environmental lawyer or do non-profit advocacy work of some kind.
She says she feels Catherine McAuley High School has prepared her well for the future, and says there is a lot she will miss.
"I've had good friends and just been close and had fun," she says. "It's so hard to explain what the special qualities of McAuley are. Unless you experience it, you don't get it."


SIAM Journal on Optimization 6 (1996),704-713.
Journal of the Operations Research Society of Japan,vol. 39, pp.188-212(1996).
Journal of Applied Mathematics and Stochastic Analysis,vol. 9, pp.143-157(1996).
Journal of Operations Research Society of Japan,,vol.39, pp.459-474(1996).
生物科学,47 巻,4号, 1996, pp. 181-190.
Computational Optimization and Applications 5(1996), pp. 5-37.
Mathematical Programming 72 (1996),pp. 1-15.
Journal of the Operations Research Society of Japan 39 (1996),pp. 213-229.
SIAM Journal on Optimization 6 (1996),pp. 446-460.
Nonlinear Optimization and Applications , G. Di Pillo and F. Giannessi (eds.), Plenum Press,,1996, pp. 155-170.
Journal of the Operations Research Society of Japan 39 (1996),pp. 407-427.
Journal of Optimization Theory and Applications 90 (1996),pp. 581-603.
SIAM Journal on Optimization 6 (1996),pp. 1106-1120.
Journal of the Operations Research Society of Japan 39 (1996),pp. 566-591.
International Journal of Systems Science 27 (1996),pp. 917-923.
Complex Systems, ,Vol. 10, pp. 73-90, 1996.
Proc. International Conference on Evolutionary Computation (ICEC'96), Nagoya, May 20-22, 1996, pp. 150-153.
The Tenth International Workshop on Qualitative Reasoning about Physical Systems, Stanford, CA, May 21-24, 1996, pp. 93-102.
in Proc. of World Congress of Neural Networks (WCNN'96), San Diego, CA, September 15-18, 1996, pp. 508-511.
Proc. International Workshop on Principle of Diagnosis (DX 96), Val-Morin, Canada, October 13-16, 1996, pp. 106-114.
in H. Asama Eds., Proc. of International Symposium on Distributed Auto nomous Robotic Systems (DARS'96), Wako, October 29-31, 1996, pp. 129-140.
in Proc. IEEE/RSJ International Workshop on Intelligent Robots and Systems (IROS'96), Osaka, November 4-8, 1996, pp. 1739-1746.
in Proc. of the 35th IEEE Conference on Decision and Control (CDC'96), Kobe, December 11-13, 1996, CD-ROM(ISBN 0-7803-3593-7).
in Y. Ishida Ed. ICMAS International Workshop on Immunity-Based Systems (IMBS96), Kyoto, December 10-13, 1996, pp. 2-10.
in Y. Ishida Ed. ICMAS International Workshop on Immunity-Based Systems (IMBS96), Kyoto, December 10-13, 1996, pp. 93-105.
オペレーションズ・リサーチ, 41 (1996), pp. 331-336.
Technical Report 95025, Graduate School of Information Science, Nara Institute of Science and Technology (September 1995, revised January 1997).
Technical Report 96010, Department of Applied Mathematics and Physics, Kyoto University (September 1996, revised February 1997).
Technical Report 97005, Department of Applied Mathematics and Physics, Kyoto University (March 1997).
システム制御情報学会特別講演, May, 1996, pp. 1-6.


text:
Sparta: War of Empires wallpapers
Follow Us on Instagram 0
F2PMeter: Is Sparta: War of Empires at War Truly Free to Play?
F2PMeter: Is Sparta: War of Empires at War Truly Free to Play? We're putting Sparta: War of Empires through our F2PMeter this time round. Apparently free to play (we'll soon see…), this browser-based strategy title allows players to gather resources, build cities and raise armies....
Sparta War of Empires: The Good & The Bad
Sparta War of Empires: The Good & The Bad Sparta: War of Empires is a free to play browser-based strategy game that challenges players to build up their own army of Spartan warriors in a feudal Greece. We take a look at the best and worst elements of this title in our latest Good & Bad...
Exclusive Sparta War of Empires Giveaway
Exclusive Sparta War of Empires Giveaway Do you want to lead your very own army and conquer your enemies' land? In collaboration with Plarium today we have for you this exclusive giveaway for new users accounts of Sparta: War of Empires. From now on until the end of August, by...
One Comment - "Exclusive Sparta War of Empires Giveaway"
Nikola July 17, 2014 at 6:54 PM -
This is not really giveaway, they are giving 200 gold from beggining
Play Sparta: War of Empires
Sparta: War of Empires profile
Sparta: War of Empires screenshots
Sparta: War of Empires videos
Sparta: War of Empires review
F2PMeter Sparta: War of Empires


In this eleventh poll of the Daily Dozen Disciplines, we will discuss how making time for spirituality can improve one's quality of life.
Spirituality is a very personal part of one's being. However, as individual as it may be to each person, we tend to believe in fundamentals of a spiritual nature as a group or religion. The dictionary defines spirituality as a "sensitivity or attachment to religious values." The word "values" is probably the most important part of the definition. Our values have a great influence on how we think, act, and make choices each day. Our values influence what we consider good or evil.
There are so many organized religions in this world. Most religions serve the purpose of laying out the ground rules for acceptable behavior for the good of mankind. There are also some so called religions that don't serve mankind very well. For the purposes of this poll, we will try to focus on those that promote good values.
Can making time for some moments of spiritual thought daily, strengthen one's character and resolve to face the challenges of life head on? Can one's quality of life get better from the strength gained from their spiritual life? Let us know how you feel about spirituality in this week's poll. This poll will end on Sunday 12/29/02. Watch for the results and summary by Monday morning 12/30/02.
Our poll produced an overwhelming combined 97% vote from our respondents, who believed that spiritual thoughts can have a positive impact and improve one's quality of life.
Now I do realize that the people who come to eHappyLife.com and take our polls may not represent the typical individual that resides on planet earth. Our audience is skewed toward middle age viewers who have a better appreciation toward good values. . Our viewers are more likely to practice spirituality in their lives than younger adults still searching for tangible evidence that a spiritual world even exists.
Regardless, I am happy to produce the results of this poll. Especially since spirituality has played such a major role in my life. From a small child I have always been able to have a happy connection and draw a great deal of strength through my spiritual thoughts. No one ever had to force me to think as I did. My actions were always voluntary. Even as a college student living away from home, I always attended Sunday Church Services alone each week. I did so because it made me feel good to have the discipline to do so. I always felt great after investing that one hour of prayer weekly as I connected with my spiritual world. To this day, I continue the same practice. I also connect spiritually on a daily basis, even if it's for a few moments a few times a day. I sincerely believe this connection greatly improves my quality of life.
This world has so many negative vibes going through the air. Spirituality can serve as a huge positive force to help you face the negative surrounding us. We should not let what goes on in the institutions of religion ever deter us from being spiritual. As stated above in the Commentary, spirituality is a very personal part of one's being. It's a relationship between you and your creator. Don't let any of the interference with the outside world ever disturb that relationship. Hope you enjoy a life full of spirituality and love.


text:
Capital Cities in Oceania
Oceania is the smallest of the continents in both area and population (if we do not count with Antarctica).
List of Oceanic capitals (Alphabetically sorted by country)
Fiji – Suva
Kiribati – South Tarawa
Marshall Islands – Majuro
Micronesia Federation – Palikir
Nauru – Yaren
New Zealand – Wellington
Did you know? Wellington is the world's southernmost capital. By the way, there are no other capitals at a distance from the nearest capital (Canberra in Australia as the closest "capital city neighbor" is 2326 km away). As a fun fact, it can also be mentioned that Wellington is also the capital of the world where it blows most.
Palau – Melekeok
Papua New Guinea – Port Moresby
Solomon Islands – Honiara
Samoa – Apia
Tonga – Nuku'alofa
Tuvalu – Funafuti
Vanuatu – Port Vila
4 Facts about Oceania (which you may not know)
Australia is by far Oceania's largest country, both in terms of population and area.
In addition to Australia, New Zealand and Papua New Guinea, the continent includes of more than 25,000 volcanic and coral islands scattered throughout the Pacific Ocean.
Mount Wilhelm (4509 masl) in Papua New Guinea is the highest mountain.
Australia is the driest continent on the globe (Africa is the second driest). By the way, do you know the difference between a continent and a continent? How can e.g. Australia being both continent and one of the countries of a continent (Oceania)?
Capital Cities in Europe
Capital Cities in Asia
Capital Cities in Africa
Capital Cities in South America
Capital Cities in North America
Oceania Population
List of Countries in Oceania and Australia
List of Cities and Towns in Alaska
List of Cities and Towns in Florida
List of Cities and Towns in Delaware
List of Cities and Towns in Virginia
List of Cities and Towns in Washington
List of Cities and Towns in Wisconsin


text:
Brown responds best to NHRA Pomona pressure
Rob Geiger
NHRA/AUTOIMAGERY
Antron Brown
Reduced to just one qualifying session by rain, NHRA Top Fuel pro Antron Brown made the most of his lone pass down Auto Club Raceway, posting the quickest 1,000-foot pass ever, a stunning 3.708 seconds, that puts him atop the elimination ladder.
Brown joins Funny Car racer Robert Hight (4.037 seconds) and Pro Stock ace Greg Anderson (6.588 seconds) on the low qualifier's podium at the 49th annual Kragen O'Reilly NHRA Winternationals, the first of 24 races on the NHRA's Full Throttle circuit.
"I'm so glad I was able to give that to these fans that stuck it out the last few days through all this rain," Brown said. "They loved seeing that 3.70 up there on the scoreboard and so did we.
"My team owner Mike Ashley said we're going to just let all the attention fall on Tony Schumacher and Larry Dixon and we'll just go out there and race our Matco Tools dragster, do our thing, and see what happens."
Under the guidance of new crew chief Mike Green, defending Top Fuel champion Tony Schumacher is fifth with a conservative 3.897 seconds at 311.99 mph. Shockingly, Larry Dixon and his Alan Johnson Al Anabi Racing team failed to qualify after suffering mechanical problems early in their run.
"To say we're disappointed might be the biggest understatement ever," said Dixon, the clear-cut preseason favorite. "It's unfortunate, but NHRA awards the same points at Phoenix (the next event) and we'll be ready to go."
According to Hight, crew chief Jimmy Prock swung for the fences even though it seemed certain there would be just one opportunity to make the field.
"It's so much pressure on the whole team and the driver when you know you're going to have rain-shortened sessions," Hight said, "but you know my crew chief Jimmy Prock shoots for the moon every time, and that worries me some times. That was really the case this time because we have a new setup and there's always a huge learning curve. To be No. 1 right now is just incredible to me."
Al Anabi Racing's Funny Car driver, Del Worsham, did much better than his stablemate Dixon, running the best elapsed time of his career, a 4.038 seconds, that placed his Toyota Solara second overall.
Ashley Force Hood (4.061 seconds) and her famous father John Force (4.092 seconds) rounded out the top four in their Castrol Ford Mustangs.
Anderson and his KB Racing teammate, Jason Line, obviously did their homework over the offseason, taking the first two spots on the ladder. Anderson's Summit GXP was considerably quicker than Line's identical mount, running a 6.588 to Line's 6.616.


text:
CNBC HD joins Sky
Business news channel CNBC HD simulcast has now joined Sky. As of today, Sky HD and Q users will see CNBC HD on channel 505, while the standard definition version has moved to channel 890. CNBC carries the latest from the financial markets, with live programming Monday-Friday from Asia, Europe and the USA, plus executive […]
By Expat • UK Media News • • Tags: CNBC HD, Comcast, Sky
Discovery to launch DMAX in UK
Discovery is launching DMAX as a free to air channel in the UK from January 16th. It launches with an adventurous tone of voice centred around 'more is more' for men who want to indulge their passions for survival, extreme lives, motoring and comedy. DMAX replaces Travel Channel, though the lTravel Channel will remain as […]
By Expat • Discovery, UK Media News • • Tags: DMAX UK, Freesat, Freeview, Sky
STV and Sky agree five-year strategic partnership
STV and Sky have today announced a five-year strategic partnership, which will give Sky customers in Scotland access to STV's regional variants in full HD, as well as STV's leading VOD service – STV Player – on their set top boxes for the first time. Sky customers in STV's broadcast licence areas will be able […]
By Expat • STV • • Tags: Scotland, Sky, STV HD
Sky prepares for no-deal Brexit
Sky Ireland has sent letters to broadcasters about their post-Brexit plans in case of a no-deal scenario – to ensure they retain licences to broadcast in both the UK and EU markets. According to The Guardian, Sky UK contacted US broadcasters like Disney and Discovery amid concerns that they would not have a licence to […]
By Expat • Ireland • • Tags: Brexit, Ireland, Sky
Now TV to offer Full HD
Now TV has confirmed it will be upgrading its streams to full HD later this year. As part of wider infrastructure changes within Sky that will enabling viewers to access the full Sky TV service without a satellite dish, Now TV will offer users the option to access Full HD streams from later this year. […]
By Expat • Now TV, UK Media News • • Tags: Full HD, Skt, Sky
CMA sets out Fox/Sky investigation issues
The UK's Competition and Markets Authority (CMA) has set out more detail about what it intends to examine in its investigation into the proposed takeover of Sky Plc by 21st Century Fox. On 20 September, Karen Bradley, the Secretary of State for Digital, Culture, Media and Sport referred Fox's proposed takeover of Sky to the […]
By Expat • UK Media News • • Tags: Competition and Markets Authority, Sky
FreeSports delayed on Freesat
Freesat viewers will need to wait until the second week in September to gain access to Freesports. On Twitter a spokesperson said "We're launching on Freesat on the 12th September. Not the 11th September as we originally thought." The channel launched on Thursday morning on Freeview channel 95, 75% of the UK can receive Freesports […]
By Expat • Sport, UK Media News • • Tags: Freesat, FreeSports, Freeview, Sky, Virgin Media
Sony Pictures to launch Pop Max
Sony Pictures Television Networks (SPTN) has launched Pop Max – a new dedicated kids channel, on 30th August. Pop Max, which will be available across Freeview, Sky and Freesat, is a rebrand of current channel Kix, designed to "evolve" SPTN's kids channel portfolio. According to SPTN, Pop Max will be a "cheeky comedy channel" targeting […]
By Expat • Sony Pictures TV, UK Media News • • Tags: Freesat, Freeview, Pop Max, Sky
ITV changes on Sky
ITV will reshuffle some of its channels on the Sky platform on 1st August 2017, the changes will see ITVBe move to CBS Drama's current EPG slot in many areas of the UK. The move on August 1st by ITVBe to channel 149, will also see many of ITV's +1 channels move up the Sky […]
By Expat • ITV, SKY • • Tags: ITVBe, Sky
BBC starts Red Button Tests
Test transmissions containing extra Red Button video streams that will become available to Sky and Freesat viewers during Wimbledon and Glastonbury have now commenced on satellite. The BBC has not confirmed the extent of its Red Button service for this summer, though the tests show five additional standard definition streams and one HD stream on […]
By Expat • BBC • • Tags: BBC Red Button, Freesat, Sky
ShowBiz TV closes
Entertainment channel ShowBiz TV has closed after selling its EPG slot slot on Sky. The channel, which was born out of the classic TV channel 'Bonanza Bonanza' and broadcast on Sky channel 266 and available free-to-air on digital satellite was also distributed in the Greater Manchester area on Freeview channel 80.
By Expat • UK Media News • • Tags: EPG Slot, ShowBiz TV, Sky
21st Century Fox's bid for Sky referred to European Commission
Karen Bradley, the UK Secretary of State for Culture, Media and Sport, is to refer 21st Century Fox's bid to acquire the 61 per cent of pay-TV operator Sky it does not already own to the European Commission on grounds of media plurality and adherence to broadcasting standards. In a formal Intervention Notice, Bradley said […]
By Expat • SKY • • Tags: 21st Century Fox, European Commission, Karen Bradley, Ofcom, Sky


text:
S. 744:
Border Security, Economic Opportunity, and Immigration Modernization Act
Published: Thu, Jan 23rd 2014 @ 12:54 pm EST
Sponsored by: Sen. Charles Schumer [D-NY] in the 113th congress
S. 744, the Border Security, Economic Opportunity, and Immigration Modernization Act, was a massive immigration reform bill, introduced by the 'Gang of 8' that would result in approximately 30 million new permanent work permits issued in the first 10 years if passed. The bill would grant legal status and worker permits to an estimated 11 million illegal aliens with an opportunity for green cards after 10 years and replace some family-based immigration categories with a merit-based points system.
Read more about Border Security, Economic Opportunity, and Immigration Modernization Act
H.R. 477:
Nuclear Family Priority Act
Sponsored by: Rep. Phil Gingrey [R-GA11] in the 113th congress
H.R. 477, the Nuclear Family Priority Act, would eliminate the family-based green card categories that are directly responsible for endless chains of family migration. The current annual limits on green cards are 78,000 for parents, 65,000 for adult brothers and sisters, 23,400 for married sons and daughters and 23,400 for unmarried adult sons and daughters. H.R.
Read more about Nuclear Family Priority Act
H.R. 425
Sponsored by: Rep. Sam Graves [R-MO6] in the 113th congress
H.R. 425, would restrict any federal funds from being used for the "Provisional Unlawful Presence Waivers of Inadmissibility for Certain Immediate Relatives" rule published by the Department of Homeland Security (DHS), which allows illegal aliens to remain in the U.S. prior to leaving to process immigrant visa applications.
Read more about H.R. 425
H.R. 692, Nuclear Family Priority Act, would eliminate current green card categories for adult brothers and sisters, married and unmarried adult sons and daughters, and create a special non-working visa for parents. By not providing an increase in any other category, overall immigration would decrease by more than 111,800 per year (1.118 million a decade). The numbers would also be indirectly reduced by this bill because there would be fewer spouses or parents of U.S. citizens that would be brought into the country by immigrants.
10 Prescriptions for a Healthy America Act
Published: Mon, Jan 27th 2014 @ 11:47 am EST
To establish requirements for any health reform legislation enacted by the Congress or the President during the 111th Congress.
Read more about 10 Prescriptions for a Healthy America Act
The bill would eliminate the extended family visa categories (e.g., married sons and daughters of citizens, etc.), thus ending "chain migration" as recommended by the bi-partisan Barbara Jordan Commission in 1997. Chain migration is the process where seemingly endless "chains" of foreign nationals are allowed to immigrate to the United States, since our laws allow citizens and lawful permanent residents to bring in their non-nuclear, adult family members. It is the primary mechanism that has caused legal immigration in this country to quadruple since the 1960s.
Filipino Veterans Family Reunification Act
Published: Mon, Jan 27th 2014 @ 2:44 pm EST
Sponsored by: Sen. Mazie Hirono [D-HI] in the 110th congress
The bill would exempt children of Filipino World War II veterans naturalized pursuant to the Immigration Act of 1990 from numerical limits on worldwide immigration.
To exempt children of certain Filipino World War II veterans from the numerical limitations on immigrant visas.
Read more about Filipino Veterans Family Reunification Act
Sponsored by: Sen. Herbert "Herb" Kohl [D-WI, 1989-2013] in the 110th congress
S. 703 would expand chain migration by expanding the definition of "immediate relative" for purposes of exemptions from the numerical cap. It would include children of U.S. citizens' parents accompanying or following them to join the parent.
Read more about S. 703
Sponsored by: Sen. Daniel Akaka [D-HI, 1990-2013] in the 110th congress
S. 671, Filipino Veterans Family Reunification Act, would exempt children of Filipino World War II veterans naturalized by the Immigration Act of 1990 from numerical limits on worldwide immigration.
H.R. 938, the Nuclear Family Priority Act, would end chain migration by restricting allocation of family-sponsored immigrant visas so that only spouses or children of a lawful permanent resident (LPR) alien can obtain such visas. Furthermore, it would reduce the number of family-sponsored immigrant visas available per fiscal year to 88,000 minus the number of aliens who were paroled into the United States for humanitarian reasons in the second preceding fiscal year.
(-) Remove Reduce Chain Migration filter Reduce Chain Migration
Challenge Status Quo
Apply Challenge Status Quo filter
Apply House filter


Gurugram-based online lifestyle media platform What's Up Life has raised fresh funding from actor and television personality Rannvijay Singh, who also joins the company as its brand ambassador.
Founded in 2014 by Gaurav H Luthra as What's Up Gurugram, What's Up Life (WUL) curates engaging content on their social media pages, editorials, and calendar to keep users updated with all the activities and events across categories.
At present, What's Up Life is among the top names in three out of five cities it is present in via Gurugram, Kolkata, and Hyderabad. Further, the startup plans to expand to Mumbai, Bengaluru, Pune, Chandigarh, and Jaipur in the next six months.
Venture Garage was the advisor to WUL for the fundraise and also helped in connecting with Ranvijay Singh via their network.
"Strategic investments can provide tremendous firepower especially in the early stages of a venture when the value-addition from the investor's end matters more than the capital. With Rannvijay onboard the WUL wagon, the business will get a significant boost," said Vineet Sagar, managing partner, Venture Garage.
Recently, What's Up Life raised $100K from Karan Puri, author, and Praveen Puri of Skyline Knowledge Centre, a Skyline Education Group Company.
The company plans to use the latest funding for company expansion as well as to expand its team to newer cities and create new business lines.
With the glitz and glam of television and Bollywood stars, several startups have received investments as well as brand endorsements from popular actors.
Aamir Khan has invested in furniture startup Furlenco, while leading actresses Alia Bhatt and Jacqueline Fernandez invested in fashion portal Stylecracker and juice maker and online portal Rakyan Beverages respectively.
Among early investors in the lifestyle sector from Bollywood are Karishma Kapoor, Ajay Devgn, and Shilpa Shetty. Karishma Kapoor had invested in a kids clothing and accessories portal Babyoye in 2011.
After cricket stars like Sachin Tendulkar, Virat Kohli, and MS Dhoni, Bollywood stars' endorsements have been considered a big catalysing power for companies for a long time.
Amitabh Bachchan, Shahrukh Khan, Akshay Kumar, Salman Khan, and Hrithik Roshan are some of the names that are in demand for brand endorsements in the startup ecosystem.
A report entitled Mobile Internet in India 2017 estimates the number of mobile Internet users in India to reach 478 Mn by June 2018. With such deep Internet penetration, digital media is growing by leaps and bounds in India.
The country's media and entertainment (M&E) sector is poised to touch $45.1 Bn by 2021, expanding at a CAGR of over 10.5%. A PwC report revealed that the segment has undergone substantial progress since 2016 when its valuation stood at $27.3 Bn.
With expansion on the cards, What's Up Life was looking for the right push and the recent investment and endorsement by Rannvijay Singh might just be what it needed.


The safety and well-being of the students and faculty at Shanghai Livingston American School are our highest priority. The school has employed specialists to identify health and safety issues at our new campus and their recommendations have been and will be implemented and monitored.
1. The campus location has been carefully selected so as to be away from heavy traffic, factories, power generators and other? environmental conditions.
2. The campus has been outfitted with specially-designed furniture with rounded edges, protected electrical outlets, anti-skid walkways and stairs, shatterproof windows, etc. The school is also free of asbestos and lead-based paints.
3. Students are supervised at all times. All visitors are required to check-in at the office and are given special passes to wear while on campus. Students are released only to individuals who have been approved by their parents/guardians.
4. Playground equipment and activities are chosen with safety considerations in mind.
5. There is a school emergency plan in place. Students, staff and parents are made aware of appropriate procedures in special circumstances. Practice emergency drills (fire, earthquake, and other threats to safety) are held regularly.
1. The school's heating/air-conditioning equipment circulates air which has been filtered of impurities.
2.?Campus food services are manned by specially trained personnel whose job it is to maintain the highest level of sanitation. Food offerings are tasty, wholesome and nutritious. Food preparation and service adhere to stringent rules, such as the segregation of raw and cooked foods, maintenance of proper temperatures and antiseptic cleaning of utensils and dishware.


text:
Shandria Brown
Birth Date: July 7, 1983
College: Lincoln University
Events: Track – 4 x 100m relay
Personal Best:
55m – 6.97 (indoor) at Fairfax, Virginia in 2002
60m – 7.43 (indoor) at the NCAA Division II Championships in 2003
100m – 11.33 (outdoor) at the NCAA Division II Championships in 2004
200m – 23.12 (outdoor) at Carbondale, Illinois
200m – 23.89 (indoor) in 2002 in Manhattan
2004 Season Best:
55m – 6.99 (indoor) at Carbondale, Illinois
60m – 7.45 (indoor) at Warrensburg, Missouri
100m – 11.52 (outdoor) at the National Championships
Career Highlights:
2003 – Champion at the NCAA Division II Championships in the 60m
2004 – Champion at the NCAA Division II Championships in the 100m
Champion at the NCAA Division II Championships in the 200m
Her performance at the NCAA Division II Championships this year went a long way in enabling her team from Lincoln University to win the overall title.
Tweet Print Send Email Share


Young and motivated professional ready to learn and grow within any roll. Maintains strong and consistent communication with clients to ensure satisfaction and efficiency. Experienced in educating both clients and prospects alike about existing product and new services. Engaged in thinking of ways to improve and enhance any aspect of my job, client experience or my performance.
Where can I find a Merchant Factors Account Executive resume example in New York, New York ?


The shooting of NTR, Nandamuri Balakrishna's much-awaited biopic on his father and legendary actor-politician NT Ramarao, took place for a couple of days in December last week. A special teaser, which was supposed to be out on NTR's death anniversary on January 18, was postponed indefinitely.
As per the latest updates, the Nandamuri family members had requested Balakrishna to rethink his decision of releasing the teaser on their father's death anniversary, which is not a happy occasion. Balakrishna immediately obliged his brothers and sisters' request and postponed the teaser's release.
This highly-anticipated biopic is being bankrolled by Balakrishna, Sai Korrapati and Vishnu Induri. The regular shooting will kick-start from August. Stay tuned for further updates.