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MOLECULAR AND CELLULAR PATHOGENESIS
Hyperproduction of Alpha-Hemolysin in asigB Mutant Is Associated with Elevated SarA Expression inStaphylococcus aureus
Ambrose L. Cheung, Yueh-tyng Chien, Arnold S. Bayer
Ambrose L. Cheung
Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, New York 10021 1 ;
Yueh-tyng Chien
Arnold S. Bayer
St. John's Cardiovascular Research Center 2 and Division of Infectious Diseases, 3 Harbor-UCLA Medical Center, Torrance, California 90509; and UCLA School of Medicine, Los Angeles, California 90024 4
To evaluate the role of SigB in modulating the expression of virulence determinants in Staphylococcus aureus, we constructed a sigB mutant of RN6390, a prototypic S. aureus strain. The mutation in the sigB gene was confirmed by the absence of the SigB protein in the mutant on an immunoblot as well as the failure of the mutant to activate ςB-dependent promoters (e.g., the sarC promoter) ofS. aureus. Phenotypic analysis indicated that both alpha-hemolysin level and fibrinogen-binding capacity were up-regulated in the mutant strain compared with the parental strain. The increase in fibrinogen-binding capacity correlated with enhanced expression of clumping factor and coagulase on immunoblots. The effect of thesigB mutation on the enhanced expression of the alpha-hemolysin gene (hla) was primarily transcriptional. Upon complementation with a plasmid containing the sigBgene, hla expression returned to near parental levels in the mutant. Detailed immunoblot analysis as well as a competitive enzyme-linked immunosorbent assay of the cell extract of thesigB mutant with anti-SarA monoclonal antibody 1D1 revealed that the expression of SarA was higher in the mutant than in the parental control. Despite an elevated SarA level, the transcription of RNAII and RNAIII of the agr locus remained unaltered in thesigB mutant. Because of a lack of perturbation inagr, we hypothesize that inactivation of sigBleads to increased expression of SarA which, in turn, modulates target genes via an agr-independent but SarA-dependent pathway.
Staphylococcus aureus is a major cause of human infections, such as superficial abscesses, pneumonia, endocarditis, and sepsis (6). The control of a multitude of extracellular and cell wall virulence determinants inS. aureus is growth phase dependent. In particular, cell wall proteins are normally synthesized in the logarithmic phase, while exoproteins are generally produced postexponentially. The growth phase dependence of these virulence factors is mediated in part by global regulatory loci, such as sar (12) andagr (22). These modulators may either interact with the target gene directly (e.g., RNAIII with hla[alpha-hemolysin gene] mRNA) or control another regulatory molecule (e.g., sar regulation of the agr gene product) which, in turn, alters the transcription of the target gene.
The sar locus is composed of three overlapping transcripts, designated sarA (0.56 kb), sarC (0.8 kb), andsarB (1.2 kb), initiated from the P1, P3, and P2 promoters, respectively. Because of this multiplicity of promoters, the activation of sar leading to the expression of SarA, the majorsar regulatory molecule, is complex and may be growth phase dependent. Whereas the sarB transcript and the more abundantsarA transcripts are maximally expressed during the exponential phase, the transcription of sarC from the P3 promoter is most active during the postexponential phase (3). Additional transcriptional analysis indicated that the P3 promoter is ςB dependent (17, 20, 25).
In contrast to the primary sigma factor (ςA), which is required for the expression of housekeeping genes, SigB (ςB) is an alternate transcription factor that has been shown to respond to environmental stresses (e.g., stationary phase of growth) in gram-positive bacteria (20). The core RNA polymerase associated with a particular sigma factor recognizes a specific set of promoters with conserved sequence motifs to initiate the transcription of genes programmed to respond to certain environments (20, 22). For Bacillus subtilis, ςB activity is regulated posttranslationally by complex networks of protein-protein interactions governed by a variety of environmental stresses (1, 20). Because one of the promoters (P3) within the sar locus is ςB dependent, it is conceivable that the SigB protein influences sar expression. As the sar locus activates the synthesis of alpha-hemolysin at the transcriptional level, presumably in part through the interaction of SarA with the agr locus (15), we speculate that sigB may modulate sarA expression and the ensuing hla transcription.
In this study, we report the construction and characterization of asigB mutant of RN6390, a prototypic S. aureusstrain. The specificity of the mutation was confirmed by the absence of the SigB protein on an immunoblot, but the protein was restored in the mutant by a shuttle plasmid carrying the sigB gene. Phenotypic analysis revealed that the sigB mutant strain secreted more alpha-hemolysin than the parental strain, as determined by immunoblotting and Northern analysis. Complementation of the mutant with the sigB gene in trans reestablished alpha-hemolysin expression to near parental levels. Interestingly, the hyperproduction of alpha-hemolysin coincided with elevated SarA expression in the sigB mutant. Using the rabbit endocarditis model, we found that the sigB mutation was stable in vivo. We hypothesize that the hyperproduction of alpha-hemolysin in S. aureus as a result of the sigB mutation is mediated by an increase in the SarA level which, in turn, enhances the transcription of hla via a direct pathway (i.e.,agr independent).
Bacterial strains, plasmids, and growth media.The bacterial strains and plasmids used in this study are listed in Table1. Phage φ11 was used as the transducing phage for S. aureus strains. CYGP, 0.3GL medium (26), and tryptic soy broth (TSB) were used for the growth of S. aureus strains, while Luria-Bertani medium was used for growing Escherichia coli. Antibiotics were used at the following concentrations: erythromycin at 5 μg/ml and ampicillin at 50 μg/ml.
Strains and plasmids used in this study
Genetic manipulations in S. aureus. A sigBmutant of RN6390 was constructed as described previously (12) by transducing the parental strain with a phage lysate of strain RUSA168 carrying the sigB mutation (30). Transductants were selected on agar containing erythromycin. Correct insertion of Tn551 into thesigB locus of RN6390 was confirmed by Southern blotting with Tn917 and sigB probes as described previously (12). One transductant, ALC1001, was chosen for further studies.
To complement the sigB mutation in ALC1001, we introduced into the shuttle plasmid pSK236 a 1.6-kb fragment (derived from pDG148) containing the pSpac promoter followed by the polylinker site and thelacI repressor of E. coli, yielding pALC1456. Plasmid pALC1496 was constructed by cloning the sigB open reading frame into the multiple cloning site of pALC1456. The shuttle plasmid was electroporated into RN4220 and then transduced intosigB mutant ALC1001 with phage φ11 as described previously (12). The presence of the recombinant plasmid was confirmed by restriction mapping.
Production of anti-SigB monoclonal antibodies.Based on the published sequence (30), we amplified by PCR the 768-bpsigB gene with the following primers: 5′-GCCAT2687ATGGCGAAAGAGTCGAAATCAGCT2710-3′ (NdeI site underlined) and 5′-GCGGATCCCTA3454TTGATGTGCTGCTTCTTG3437-3′ (BamHI site underlined). The correct PCR product, verified by automated sequencing, was cloned into theNdeI-BamHI sites of expression vector pET14b (Novagen, Madison, Wis.) and transformed into E. coliBL21(DE3).pLys.S. The resulting plasmid, pALC1270, contained an N-terminal His tag and the entire sigB coding region.
Recombinant protein expression was induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG) (1 mM) to a growing culture (30°C) at an optical density at 600 nm (OD600) of 0.5. Three hours after induction, the cells were harvested, resuspended in binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl [pH 7.9]), and sonicated on ice. After removal of the cellular debris by centrifugation (15,000 × g for 15 min), the clarified supernatant was purified on a nickel affinity column in accordance with the manufacturer's instructions. The protein (≈30,000 kDa) eluted from the column with elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl [pH 7.9]) appeared homogeneous on a sodium dodecyl sulfate gel (data not shown) and was authenticated by sequencing of the N-terminal 15 residues after the His tag had been removed by digestion with thrombin.
Purified SigB was used to immunize two (BALB/c × SJL/J) F1 mice (100 μg each) to obtain monoclonal antibodies as described by Jones et al. (21). The titers of the immune sera were determined by an enzyme-linked immunosorbent assay (ELISA) in which diluted sera were added to microtiter wells precoated with purified SigB (5 μg/ml). After hybridoma fusion, antibodies from limiting dilutions were screened by an ELISA with immobilized SigB protein. Monoclonal antibody 2D7 was purified from culture supernatants with a protein A affinity column (21) and tested for reactivity with purified SigB on immunoblots.
Phenotypic analysis of the sigB mutant and its isogenic parent.Several virulence traits of the sigBmutant were evaluated. First, the production of alpha-, beta-, and delta-hemolysins in the sigB mutant and its parental counterpart was assessed by cross-streaking the tested strain with indicator strains as described previously (9). To verify the production of alpha-hemolysin on immunoblots, equivalent volumes of extracellular proteins that had been harvested at the stationary phase and concentrated 50-fold by use of a Centriprep concentrator (Amicon Inc., Beverly, Mass.) were blotted onto nitrocellulose and probed with rabbit anti-alpha-hemolysin antibody (a gift from B. Menzies, Nashville, Tenn.) diluted 1:2,000 and then with the F(ab)2fragment of the goat anti-rabbit antibody–alkaline phosphatase conjugate (Jackson ImmunoResearch, West Grove, Pa.) as described previously (7). Reactive bands were visualized as described by Blake et al. (5).
Taking advantage of the cytolytic effects of alpha-hemolysin upon rabbit erythrocytes and platelets (2, 4), two functional assays for alpha-hemolysin production were performed. First, alpha-hemolysin levels were quantitated by assaying the hemolytic titers of serially diluted stationary-phase culture supernatants for 1% washed rabbit erythrocytes as described previously (2,4). Purified alpha-hemolysin was used as the positive control. The data were expressed as mean units of hemolytic activity per milliliter of culture supernatant from two separate runs. The hemolytic units were defined as the reciprocal of the highest dilution of the culture supernatant causing 50% erythrocyte lysis. Second, the extent of alpha-hemolysin production was ascertained by measuring platelet lysis spectrophotometrically (at OD600) upon exposure to bacterial supernatants as described previously (2).
In addition to hemolysins, we also measured other putative virulence traits, such as fibronectin- and fibrinogen-binding capacities. The fibronectin-binding capacity of the isogenic pair was compared with an125I fibronectin-binding assay as described previously (9). The fibrinogen-binding capacity was determined semiquantitatively by immunoblotting. Cell wall proteins from equivalent numbers of bacterial cells grown overnight were extracted as described previously (10). Equivalent volumes of cell wall extracts (10 μl) were resolved on sodium dodecyl sulfate gels and transferred to nitrocellulose. The nitrocellulose membranes were then blocked with blocking buffer (0.01 M Tris, 0.5 M NaCl, 0.5% Tween 20 [pH 8.2]) containing 1% bovine serum albumin for 1 h at 37°C, incubated at 37°C with fibrinogen (1 mg/ml) in the same buffer, and washed three times with blocking buffer and finally with goat antifibrinogen antibody conjugated to alkaline phosphatase (1:1,000) as described previously (13). As both coagulase and clumping factor bind fibrinogen (14, 29), we assayed for the presence of these proteins in cell wall extracts by immunoblotting. Nitrocellulose membranes containing cell wall extracts were incubated with blocking buffer containing 0.1% human serum (for the blocking protein A-Fc interaction) for 1 h at room temperature. Rabbit anticoagulase (1:1,000) and anti-ClfA (1:1,000) antibodies were then added to separate blots, followed by goat anti-rabbit antibody–alkaline phosphatase conjugate (1:10,000). All reactive bands were visualized as described by Blake et al. (5).
Isolation of RNA and Northern blot hybridization.Overnight cultures of S. aureus were diluted 1:50 in CYGP and grown to the mid-log (OD650, 0.7), late log (OD650, 1.1), and postexponential (OD650, 1.7) phases. The cells were pelleted and processed with a FastRNA isolation kit (Bio 101, Inc., Vista, Calif.) in combination with 0.1-mm-diameter sirconia-silica beads in a FastPrep reciprocating shaker (Bio 101) as described previously (8). Ten micrograms of each sample was electrophoresed through a 1.5% agarose–0.66 M formaldehyde gel in morpholinepropanesulfonic acid (MOPS) running buffer (20 mM MOPS, 10 mM sodium acetate, 2 mM EDTA [pH 7.0]). Blotting of RNA onto Hybond N+ membranes (Amersham, Arlington Heights, Ill.) was performed with a Turboblotter alkaline transfer system (Schleicher & Schuell, Inc., Keene, N.H.). For detection of specific transcripts (agr, sar, and hla), gel-purified DNA probes were radiolabeled with [α-32P]dCTP by the random-primer method (Ready-To-Go labeling kit; Pharmacia) and hybridized under high-stringency conditions (7). The blots were subsequently washed and autoradiographed.
Preparation of cell extracts and immunoblot analysis of SigB and SarA in the sigB mutant.Cell extracts were prepared for strain RN6390 and the corresponding sigB mutant. After being pelleted, the cells were resuspended in 1 ml of TEG buffer (25 mM Tris, 5 mM EGTA [pH 8]), and cell extracts were prepared from lysostaphin-treated cells as described previously (15).
Cell extracts were immunoblotted onto nitrocellulose membranes as described above. For the detection of SigB and SarA, monoclonal antibodies 2D7 (1:1,000 dilution) and 1D1 (1:2,500 dilution), respectively, were added to an immunoblot and allowed to incubate with the membrane for 3 h, followed by another h of incubation with a 1:10,000 dilution of goat anti-mouse antibody–alkaline phosphatase conjugate. The reactive bands were visualized as described previously (5).
Determination of SarA levels by a competitive ELISA.Microtiter wells were coated with purified SarA protein (ca. 3 μg/ml) in 0.1 M Tris–0.3 mM MgCl2 (pH 10) for 3 h at 37°C. After being washed with phosphate-buffered saline (PBS)–Brij (pH 7.4), the wells were blocked with PBS-Brij containing 1% bovine serum albumin overnight at 4°C. The optimal dilution of antibody, defined as the dilution at which 50% of the antibody (i.e., 50% of the maximum amount of bound antibody) bound to the protein-coated wells, was determined to be 1:20,000. Using anti-SarA antibody 1D1 at the optimal dilution (1:20,000), we constructed a standardized curve by adding known quantities of purified SarA (0 to 25 μg/ml) to compete for binding to the anti-SarA antibody, resulting in inhibition of binding to the immobilized SarA protein. For the competitive assay, triplicate cell extracts prepared from individual sar mutant clones (see above for preparation methods) were substituted for purified SarA. The protein concentrations in the cell extracts were determined with a protein assay kit from Bio-Rad Laboratories, Hercules, Calif. By comparing the levels of inhibition to the standardized curve, the relative concentration of the SarA protein in each extract was derived.
Rabbit model of endocarditis.To ascertain the stability of the sigB mutation in vivo, we chose the rabbit model of endocarditis. Briefly, a bacterial suspension harvested from an overnight culture was diluted in PBS, and bacterial numbers were confirmed by plate counting. Endocarditis on the aortic valves of New Zealand White rabbits (2 kg) was induced by catheterization as previously described (9). At 48 h postcatheterization, groups of animals (three each) were separately challenged intravenously with an inoculum of either 2 × 105 or 2 × 106 CFU. Catheters remained in place until animals were sacrificed by lethal intravenous injection of sodium pentobarbital (100 mg/kg of body weight). At the time of sacrifice (48 h postinfection), aortic valves and left ventricular vegetations from infected animals were removed, pooled, homogenized, and quantitatively cultured. Some colonies were then examined for the retention of the sigBmutation after in vivo passage by Southern (with a Tn551probe) and Western blotting.
Construction of a sigB mutant of RN6390.The mutation in a Tn551 insertion mutant, RUSA168, was previously mapped to the sigB gene of S. aureusCOL (30). To assess the phenotypic effect of thesigB mutation in a genetic background with well-defined virulence determinants (9), we elected to transduce the mutation from RUSA168 to RN6390 to yield mutant ALC1001. Southern blot hybridization with a Tn917 probe which shows significant homology with Tn551 revealed that the transposon insertion in ALC1001 was analogous to that found in RUSA168 (data not shown). To further confirm the mutation, we made use of the observation that the P3 promoter of the sar locus of S. aureus is ςB dependent (17, 25). Northern analysis revealed that P3-initiated sarC transcription was absent in strain ALC1001, whereas the ςA-dependent P1 promoter of sar was unaffected (Fig. 1). Complementation studies with plasmid pALC1496, carrying the sigB gene, revealed that complemented mutant ALC1497 showed sarCtranscription. Of note, the pSpac promoter was found to be active inS. aureus even in the absence of the inducing agent IPTG, suggesting that, contrary to observations made for B. subtilis (18), this promoter is leaky in the staphylococcal background.
Northern analysis of sar transcripts ofS. aureus RN6390, its isogenic sigB mutant ALC1001, and complemented mutant ALC1497. Northern analysis revealed that the sigB gene was transcribed in ALC1497 (data not shown). Ten micrograms of total cellular RNA obtained at the stationary phase (OD650, 1.7, as determined with an 18-mm borosilicate glass tube) was applied to each lane. The probe was a 730-bpsarA fragment (nucleotides 620 to 1349, based on the published sequence) (3). Because the transcription ofsarB, the largest transcript within the sarlocus, was minimal during the late log and stationary phases, only data for sarA and sarC transcripts are shown.
To ascertain if the mutation in ALC1001 was indeed due tosigB, we probed a cell lysate of the mutant with anti-SigB monoclonal antibody 2D7 in an immunoblot. As shown in Fig.2, SigB was not detectable in the mutant, while the parental strain (RN6390) as well as the complemented mutant strain (ALC1497) were found to contain SigB, as confirmed by the reactivity of the cell extract with the anti-SigB antibody. Additionally, SigB was not found in the mutant strain carrying the vector alone (data not shown). Consistent with the observation that SigB, being a regulatory molecule, is present in a low quantity in cells, we were able to discern the presence of SigB only upon loading at least 50 μg of cellular proteins from the parental strain.
Immunoblot of cell extracts of the parental strain,sigB mutant strain ALC1001, and complemented mutant strain ALC1497 probed with anti-sigB monoclonal antibody 2D7 (1:1,000 dilution). About 50 μg of cellular proteins was applied to each lane. The experiment was repeated two times, with essentially the same results. M. W., molecular weight. Numbers at left are in thousands.
To determine the stability of the sigB mutation in vivo, we infected rabbits that had been previously catheterized to produce thrombotic endocarditis with ALC1001. After harvesting the bacteria obtained from the cardiac vegetations of this animal model, we confirmed by Southern and Western analyses that the sigBmutation remained stable after in vivo passage (data not shown).
Phenotypic characterization of sigB mutant ALC1001.When ALC1001 and RUSA168 were streaked on blood agar plates, it was observed that the clear zone of hemolysis surrounding 24-h cultures was significantly enhanced in both sigBmutants compared with the respective parental strains. As the virulence determinants of RN6390 are well described (9), we chose to focus our analysis on sigB mutant ALC1001. In quantitative hemolysis assays for alpha-hemolysin, the culture supernatant of mutant ALC1001 yielded a mean hemolytic titer of 1,560 U/ml, while the corresponding parental strain titer was 490 U/ml. Purified alpha-hemolysin yielded a titer of 22,736 U/mg of protein. As an additional assay for the functional aspects of alpha-hemolysin, we also found that the culture supernatant of ALC1001 had a greater capacity to lyse platelets than the isogenic parental strain, as monitored by a decrease in the OD600 in a turbidimetric assay (Fig.3). An immunoblot of equivalent amounts of extracellular proteins of ALC1001, RN6390, and complemented strain ALC1497 disclosed that alpha-hemolysin was synthesized at a higher level in the sigB mutant than in the parental strain but was present at near the parental level in ALC1497 (Fig.4A). In measuring the transcriptional activity of hla by Northern blotting, we also found that thehla mRNA level of ALC1001 was higher than that of RN6390 (Fig. 4B). Complementation of sigB mutant ALC1001 with asigB-carrying plasmid (pALC1496) expressing thesigB transcript (data not shown) restored hlatranscription to the parental level (Fig. 4B). Similar results on the expression of alpha-hemolysin were observed with RUSA168 complemented with the sigB-carrying plasmid pALC1496 (data not shown). Collectively, these data imply that the sigB mutation is associated with enhanced hla expression initiated at the transcriptional level.
Lysis of platelets monitored on the basis of OD600. The supernatants of 18-h bacterial cultures in TSB were mixed with platelets (109 platelets/ml). Platelet lysis was monitored by measuring the decrease in the OD600over time (2). The medium and purified alpha-toxin served as the negative and positive controls, respectively. Symbols: diamonds, TSB control plus washed platelets; squares, RN6390 (supernatant) plus washed platelets; triangles, SigB (supernatant) plus washed platelets; circles, purified alpha-toxin (1 mg/ml) plus washed platelets.
Western and Northern analyses of the alpha-hemolysin gene product. (A) An immunoblot of extracellular proteins of ALC1001, its isogenic parent RN6390, and complemented strain ALC1497 from the late log phase was probed with rabbit anti–alpha-hemolysin antibody (1:2,500 dilution). (B) Ten micrograms of total cellular RNA obtained from the late log to early stationary phases was applied to each lane. The probe was a 3-kb EcoRI-HindIII fragment of the alpha-hemolysin gene (7). Similar complementation results were obtained with another sigB mutant (RUSA168) and plasmid pALC1496. These experiments were repeated three times, with similar results. The results of a representative experiment are shown.
The effect of the sigB mutation on beta-hemolysin secretion, as assayed on immunoblots, was more equivocal, with only a marginal increase in ALC1001 and no change in RUSA168 compared with the results for the respective parental strains. In comparison to that in RN6390, the expression of delta-hemolysin (hld) in ALC1001 on cross-streaked blood agar plates was not altered (see below for the RNAIII transcript encoding hld).
As cell wall-associated proteins such as fibronectin- and fibrinogen-binding proteins likely play a role in mediating the binding of S. aureus to catheters and host valvular tissues (9,11, 24, 28), we also assessed the ability of the isogenic pair to bind fibronectin and fibrinogen in vitro. The binding of mutant strain ALC1001 to radiolabeled fibronectin was similar to that of parental strain RN6390 (11,480 ± 856 [mean ± standard error of the mean] cpm versus 11,455 ± 816 cpm). In contrast, thesigB mutant bound more fibrinogen than its parental counterpart (Fig. 5A). Recognizing that multiple fibrinogen-binding proteins may be affected by thesigB mutation, we probed immunoblots containing cell wall extracts of the isogenic pair with anti-clumping factor (ClfA) and anticoagulase (Coa) antibodies (Fig. 5B and 3C). In comparison to that in RN6390, the expression of ClfA and Coa in sigB mutant ALC1001 was augmented, confirming the observation that thesigB mutant strain has a higher fibrinogen-binding capacity than the isogenic parental strain.
Immunoblots of cell wall extracts of RN6390 and ALC1001 probed with fibrinogen (A), anti-ClfA antibody (B), and anti-Coa antibody (C). Equivalent amounts of cell extracts were applied to the lanes. The positive controls were fibrinogen (A) and purified coagulase (C). M.W., molecular weight. Numbers at left are in thousands.
Expression of agr and sar insigB mutant ALC1001.The expression of alpha-hemolysin is influenced by global regulatory loci such as agr andsar. To assess if the sigB mutation in ALC1001 affected the ability of these loci to modulate alpha-hemolysin expression, we performed Northern blotting and showed that the expression of RNAII and RNAIII of the agr locus was not altered in the mutant strain relative to the parental strain (data not shown).
We have shown that the sarC transcript was absent insigB mutant ALC1001 but was restored upon complementation (Fig. 1). Concomitant with the decrease in sarCtranscription in sigB mutant ALC1001 was the repeated observation of an increase in sarA transcription in that strain (Fig. 1) as well as in RUSA168 (data not shown). Notably,sarA transcription was present at near the parental level in complemented mutant ALC1497. In contrast to RNA-mediated control of theagr locus, genetic analyses have indicated that the major regulatory molecule of sar is the SarA protein (7,15). To assess SarA expression, we probed an immunoblot of cell extracts of the isogenic pair with anti-SarA monoclonal antibody 1D1. The binding epitope of this antibody was recently mapped to residues 16 to 43 of the N terminus of the SarA molecule (16). Our data indicated that the expression of SarA, as determined by immunoblotting, was higher in ALC1001 than in its isogenic parent RN6390 (Fig.6). This pattern of SarA expression held true in three repetitions of the experiment. Using a recently described competitive ELISA (16) and the cell extracts of these two strains as competitors of 1D1 binding to immobilized purified SarA, we confirmed that the SarA level was elevated in mutant strain ALC1001 (280 ± 25 ng/mg of extract proteins) compared with the parental strain (170 ± 9 ng/mg). Collectively, these data support the notion that a sigB mutation results in enhanced SarA expression, thereby leading to an ensuing increase in alpha-hemolysin expression via an agr-independent mechanism.
Immunoblot of cell extracts of RN6390 and ALC1001 probed with anti-SarA monoclonal antibody 1D1 (1:2,500 dilution). About 30 μg of protein was applied to each lane. The positive control was purified SarA protein (14.5 kDa). M.W., molecular weight. Numbers at left are in thousands.
The sigB locus of S. aureus, contrary to the eight-gene sigB operon of B. subtilis(rsbR-rsbS-rsbT-rsbU-rsbV-rsbW-sigB-rsbX), comprises in sequential order four open reading frames which show sequence similarity to the rsbU-rsbV-rsbW-sigB gene cluster ofB. subtilis (Fig. 7) (30). Transcriptional analysis indicated that thesigB operon in B. subtilis is transcribed from two distinct but convergent promoters, the ςA and ςB promoters upstream of rsbR and rsbV, respectively. Although sequence analysis of the sigB locus of S. aureusdisclosed potential ςA- and ςB-like promoter sequences upstream ofrsbU and rsbV, respectively, the transcription start sites attributed to these putative promoters have not been experimentally confirmed. Irrespective of whether these two putative promoters or possibly another or others are active, it is likely thatsigB is the last gene encoded with the mRNA message.
Organization of the sigB operon in B. subtilis and S. aureus. In contrast to those inB. subtilis, the functions of the putative rsbU,rsbV, and rsbW genes in S. aureus have not been demonstrated. Similarly, the putative ςA and ςB promoters in S. aureus have not been experimentally confirmed.
Because the Tn551 insertion site in sigB mutant RUSA168 has been mapped to approximately nucleotide position 3254 (30) (or 189 residues from the initiation codon and 67 residues from the termination codon), we wanted to assess if the SigB protein was indeed absent in sigB mutant ALC1001. Immunoblot analysis with anti-SigB monoclonal antibody 2D7 revealed the absence of the SigB protein in the mutant strain compared with parental strain RN6390 (Fig. 2). This finding was also confirmed with an additional blot probed with mouse anti-SigB polyclonal antibody (data not shown), indicating that it is unlikely that SigB is synthesized as a truncated form. As the transposon insertion site in the sigB mutant is near the 3′ end of the sigB coding region andsigB may be the last gene transcribed in the polycistronic message, it is unlikely that the Tn551 insertion in the mutant significantly alters the transcription and translation of genes upstream (i.e., rsbU, rsbV, and rsbW). As an additional confirmation that sigB was altered in mutant ALC1001, we took advantage of the fact that the sarCpromoter within the triple-promoter system of sar is ςB dependent (17, 25). Predictably, the sarCtranscript was absent in mutant ALC1001 but was restored upon complementation with a plasmid carrying a functional copy of thesigB gene (Fig. 1). Collectively, these data clearly indicated that mutant ALC1001 is defective in the synthesis of SigB.
In analyzing some of the phenotypes related to virulence insigB mutant ALC1001, we found that both extracellular and cell wall proteins were altered. In particular, the capacity to bind fibrinogen as well as the production of alpha-hemolysin were enhanced in the sigB mutant compared with the parent. The augmented fibrinogen-binding capacity was probably mediated by upregulation in the expression of both clumping factor and coagulase in sigBmutant ALC1001 (Fig. 5). Clearly, the sigB mutation was linked to elevated expression of functional alpha-hemolysin at the transcriptional level in two S. aureus strains (ALC1001 and RUSA168) that we examined (Fig. 4B). This linkage was confirmed by complementation studies showing that the hyperproduction of alpha-hemolysin in the mutant could be restored to near the parental level when sigB was supplied in trans on a shuttle plasmid. In addition to these phenotypic alterations, Kullik et al. (23) recently reported that a cytoplasmic protein identified as alkaline shock protein 23 was missing in asigB deletion mutant, while staphylococcal thermonuclease, an extracellular protein, was secreted at a higher level than in the nonmutated controls. However, despite the pleiotropic nature of SigB in modulating a variety of genes, our results demonstrated that the effect of a sigB mutation on target gene expression can also be selective. For instance, a mutation in sigB in S. aureus can upregulate the expression of alpha-hemolysin but leave beta-hemolysin expression relatively unaltered. Similarly, such a mutation can enhance the expression of coagulase and clumping factor but not fibronectin-binding proteins.
It was recently shown that the SigB protein of S. aureus can activate ςB-dependent promoters in vitro (17). In addition, we have shown (25), as have others (17), that one of the promoters within the sarlocus (i.e., the sarC, or P3, promoter) is ςB dependent. Because of the observation that a sigB mutant has phenotypes consistent with the upregulation of sar (increased alpha-hemolysin production and increased fibrinogen-binding capacity), it seemed reasonable to explore if SigB may directly or indirectly modulate global regulatory loci, such as sar and/oragr, to effect these phenotypic changes. However, scrutiny of the agr promoter region did not reveal any ςB-dependent promoter sequences. Additionally, Northern analysis indicated that the transcription of RNAII and RNAIII in the agr locus was not altered in ALC1001, implying that SigB does not modulateagr-related transcription.
The sar locus has a triple-promoter system for which the major regulatory element is the 14.5-kDa SarA protein (124 residues) (3, 7, 15). The sarA gene is encoded by each of three transcripts initiated from three distinct promoters, designated P1, P2, and P3 (3). In particular, the ςA-dependent proximal P1 and distal P2 promoters of sar are maximally expressed during the exponential phase, while ςB-dependent P3 promoter expression peaks postexponentially (7, 25). We speculate that differential promoter activation within sarmay lead to disparate expression of the SarA protein (16,25), the major regulatory molecule in the control of hemolysin production in S. aureus (7). To assess if asigB mutation would lead to an increase in SarA expression and hence overproduction of alpha-hemolysin, we first established by immunoblotting and an ELISA that the SarA level was higher in thesigB mutant than in the parental control. Based on the phenotypes related to sar (9), a higher SarA protein level will likely enhance the expression of alpha-hemolysin (16). This notion was confirmed by Northern and Western analyses showing that the expression of hla was increased in a sigB mutant but was restored to normal levels upon complementation with a plasmid carrying sigB. Notably, the effect of a higher SarA level on target gene expression could not be mediated by agr, as evidenced by the fact thatagr-related transcription was no different in mutant ALC1001 than in RN6390. In previous studies (9, 14), we inferred from phenotypic analysis that the sar locus may have its own direct effect on target gene expression (e.g., beta-hemolysin and fibrinogen-binding proteins). In addition, we demonstrated that the SarA protein may bind to target gene promoters (e.g., the fibronectin-binding protein gene) in gel shift assays (14). Collectively, these data suggested that an increase in alpha-hemolysin expression as well as an upregulation of fibrinogen-binding proteins may occur by an agr-independent but SarA-dependent mechanism. As agr is not substantially altered in thesigB mutant, it is likely that a factor(s) other than SarA also is involved in the expression of genes perturbed by SigB. Because SigB participates in the general stress response of gram-positive bacteria (20), it seems plausible that ςB-dependent genes act in concert with SarA to modulate target gene expression.
The mechanism by which inactivation of sigB leads to elevated SarA protein levels is not fully understood. Usingsar promoter-XylE reporter fusions, we recently showed that a combined P3-P1 promoter is less active than a P1 promoter alone, suggesting that the P3 promoter may have a downregulatory effect on transcription from the P1 promoter of sar. A careful analysis of the sequence between the P3 and P1 promoters revealed a 16-bp inverted repeat. Gel shift studies revealed that a 12-kDa protein binds to this repeat (25). Additional genetic analysis indicated that the 12-kDa protein may be a repressor (unpublished data). Based on these data, we hypothesize that the P3 promoter (from which the sarC transcript is generated), upon activation by SigB, may undergo conformational changes to allow binding by the repressor protein. Presumably, a lack of SigB may interfere with the binding of this repressor protein, resulting in enhanced transcription from the P1 promoter and the ensuing elevated SarA expression. In concordance with this hypothesis is the observation that the level ofsarA transcription was found to be consistently higher insigB mutant strain ALC1001 than in the parental strain but returned to near the parental level upon complementation (Fig. 1). Additional experiments to address the interaction between SigB and this repressor protein are currently in progress.
We thank Alexander Tomasz for providing the sigBmutant strain RUSA168; Bill Haldenwang for supplying pDG148; Tim Foster for providing anti-ClfA antibody; H. Igarishi for providing anti-coagulase antibody; Michael Yeaman for providing guidance in the platelet lysis assays; and Kelly Eberhardt, Cong Li, and Iri Kupferwasser for excellent technical assistance.
This work was supported in part by grants-in-aid from the American Heart Association and the New York Heart Association and by NIH grants AI30061 and AI37142 to A.L.C. and by NIH grant AI39108 to A.S.B. Y.-T.C. was supported by a New York Heart Participatory Laboratory Award. A.L.C. is a recipient of the Irma T. Hirshl Career Scientist Award as well as the AHA-Genentech Established Investigator Award from the American Heart Association.
Editor: E. I. Tuomanen
Received 9 October 1998.
Returned for modification 4 December 1998.
Accepted 14 December 1998.
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Infection and Immunity Mar 1999, 67 (3) 1331-1337; DOI:
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"It's slowed right down," Merlin commented. "But you're right. Ice pack on the nose should shrink the capillaries. Direct pressure … " She shook her head.
Murray looked a sorry sight with a wad of toilet paper shoved up each nostril and an ice pack held to his face.
"Should we take him to the hospital?" Andrew suggested.
"No." Murray carefully shook his head.
"Are you taking aspirin, Murray? Warfarin? They all thin the blood. What about any medication like that?" Merlin asked.
"No," he repeated in a dull voice.
Merlin put one hand on his forehead and another over his nose. She felt the icy cold beneath one hand melt away into a slight warmth.
Please. If I have one gift left, let it be that of healing, she prayed.
After ten minutes, the wads were removed completely and the nosebleed had stopped.
A quarter of an hour after that, Murray was asleep, his head resting on a towel on the kitchen table. Andrew gestured with his head toward the door and Merlin went with him.
"I'd still like to be prepared. What thing?" he persisted.
"They're for real?" he wondered, his eyes widening.
Profelis was there inside an hour.
"You can't smell it?" he asked, his voice sympathetic.
"Just wait with him. I figured we'd need help. We can't both watch Murray every second, day an' night. With three, we can take it in shifts," she answered lightly, shrugging.
"What're your plans for this year?" Merlin asked.
Back in the kitchen, Profelis was circling the kitchen table. He could smell the reek of evil but, while it wasn't fresh, it wasn't stale either. Murray was smothered by it. It lay draped around him, under him, over him, even thru him. Profelis had no idea how to remove it. For now, he gently held his hands over Murray's head and upper back, then poured healing energy into him. When he was done, he checked him again and the reek was punctured in several places. He moved away and began to examine other aspects.
Murray's emotions were a strange mix. Resignation, frustration, a perverse and stubborn hope, and a keen urgency to be doing something. He was also tired and run down, but obstinately refusing to buckle. It was the hope and obstinacy which had prevented the stink from claiming every cell in his body. Right now, Murray Snowden had it balanced. Maybe Profelis had helped tip the balance a little more in his favor but he couldn't be sure. That much exposure to evil usually found other avenues thru which to strike back, and Profelis, and soon Alopex, would find themselves fighting a running battle.
"Would you and Professor Ellis like coffee in the lounge?" Andrew inquired.
"That'd be great. Thank you," Merlin accepted and went out again with Profelis following in her wake.
"Does he know about us?" Profelis whispered.
As Derek, Rachel, Nick and Anna settled back to wait for their flight to be called, and as Merlin, Andrew and Profelis guarded Murray from fresh attacks, it was just past seven that evening in Paris and Alex was about to call room service.
Nick's phone call had surprised her yet hadn't surprised her at all. The surprise had been initial – hearing his voice. She hadn't been surprised to hear what he'd had to say. Alarmed, yes. Intrigued, definitely. That intrigue had grown on her during Tuesday. She'd alerted the Paris house that she was being recalled within the next few days and she'd do what she could in the time she had left, and she'd reassured them that they really didn't need her to be there. They were more than capable of doing this themselves. She thought they'd believed her. She hoped they did, because her mind was being wrestled away from the hidden vault under Sacre Coeur to wander beneath the bleached sky of the Valley of the Kings. An Ancient Egyptian curse, a previous undiscovered tomb, and she was going there.
Alex had never been to Egypt. She knew a lot about it but she'd never been. She was looking forward to seeing the pyramids and gazing up at the Sphinx before flying south again. In the meantime, however, she could do some research. The French had explored extensively in Egypt with Napoleon. It had been a Frenchman who had first cracked the mystery of hieroglyphs when he'd translated the Rosetta Stone. Nick had said they'd be at least one day in London which meant they wouldn't be going on to Paris until Thursday at the very earliest; more likely, it would be Friday. That meant Alex had two days to dig up what she could from any records in the French archives.
Unfortunately, she couldn't make a start on that now. It was closed for the night. She had her laptop, however, so she called room service and ordered dinner, then booted up her computer and began work. Nick hadn't given her much information because, at the time he'd called, he didn't have it to give. Alex decided to start with looking at curses in relation to Ancient Egypt.
The most famous one, of course, was that linked to the boy pharaoh – Tutankhamun. It was fascinating reading, if a little macabre. Yes, most of it had been explained away by science. No such thing as a curse, they'd scoffed. However … Science couldn't deny it was a lot of coincidence. The tomb had contained a plaque bearing a curse. Lord Caernarvon, patron of the excavation, had died of an infected mosquito bite shortly after. At the same moment, the lights had gone out everywhere in Cairo and, thousands of miles away at his home at Highclere, his pet dog had howled and dropped down dead as well. Howard Carter, leader of the excavation, had survived but his pet canary was attacked and killed at his home by a cobra. Other members of the excavation team had suffered sickness and ill health had dogged them for years. The one guy who appeared to escape retribution was Carter but he was beset with personal failure for the rest of his life and, after his death, his reputation was at least smeared. The curse didn't end with the team who'd found the tomb and revealed such awesome treasures to the world. It continued to, allegedly, work into the Sixties and Seventies – people who had dealt with the treasure in the Cairo Museum died suddenly of heart attacks.
Alex read the scientific attempts to explain. Mercury poisoning. Lead poisoning. Radioactivity. There was even the very latest discovery of a white powder which was refined gold and reputed to have fantastic powers. There was a theory just published that a pot of this powder was in the Ark of the Covenant and it was this which gave the Ark its magical powers. According to the same source, pharaohs used to eat this powder, and that the powder was also the manna which sustained the Israelites during the exodus and years of wandering.
Alex knew that magic was supposed to have begun in Ancient Egypt and the pharaohs had magicians in their palaces. The priests interpreted the will of the gods and the magicians tried to find ways around it. Maybe they'd worked in conjunction with the priests to make a curse work over centuries, something intangible which would still protect the dead kings and queens as they dwelt in the afterlife.
She believed it was at least possible. Alex wasn't the kind of woman to instantly dismiss anything as impossible, unbelievable or completely unfeasible. Once, plaques bearing a curse had been put in tombs. A joke? A desperate, last ditch attempt to avert tomb robbers? Or was it that, at the time, people had believed in these curses? Science, time and distance had pulled their teeth, but that didn't mean they couldn't work. It just meant people dismissed curses as impossible, unbelievable and primitive. The Ancient Egyptians were definitely not primitive.
Her dinner arrived and she ate while she worked. She made notes of the various dynasties and pharaohs, and then looked up references to each one, noting down the major events – social, building, political and warfare – of each reign. She made notes of the main cities because they tended to come and go in favor, and, coupled with this, the main gods in favor too.
Before she knew it, the time was after midnight. Even so, it was with reluctance that Alex shut down the computer and prepared for bed. This was a puzzle with claws and teeth. Five had already died. More could die. The discovery was recent, the deaths even more so. This curse – if it was a curse – was real and powerful. Alex was going to Egypt .. and she may also be going into terrible danger.
Murray woke up in time for a late lunch and pronounced he was feeling his old self again. He looked pale but not resigned to his fate.
"You sure?" Merlin asked as they ate.
"Means there was, somewhere, a blue skinned man," Murray concluded.
Nick was sleeping. Anna was sleeping too. Long haul flights were boring. Rachel was reading a journal and concentrating on the article. Derek was frowning. Something was nudging him for attention but he couldn't focus on what exactly it was.
"Rachel," he murmured, his voice soft so not to disturb Anna who sat beside him.
"Yeah." She put down the journal and looked at him.
Rachel turned in the other direction and put a hand on Nick's arm. He started awake at once, sucking in a breath and blinking, every muscle tensed for action. Rachel snatched her hand away and Nick gave her a sheepish grin.
Nick reached under the seat in front to retrieve his carry on bag, opened it and removed the master file. It was getting thicker. Quickly, he sorted thru the printed sheets and handed a few to Rachel who passed them on to Derek. Frowning, Derek studied them, carefully looking at each. These weren't vacation pictures. There was no one standing, grinning broadly, in front of the painted wall. These were important records and they only showed the walls of the chamber.
"What is it?" Rachel asked and Nick eased a little closer as well.
Rachel saw a plain plastered wall with rough sketches on it, and she considered that Ancient Egypt was a structured, ordered civilization. It was Nick who answered.
"What's behind the eastern wall?" Rachel wondered.
Derek shuffled thru the printouts until he came to the shot of the finished painting. He had looked thru them briefly when he'd printed them from the email from the National Institute but he hadn't studied them this closely. There were quite a few of the eastern wall. Close ups and distance shots. He concentrated on the latter for now.
Profelis was, at that moment, laying on a bed in one of the guest rooms. Or Evan was. Profelis was over the river, paying a visit to what had become a thriving resource in the community.
"Hello," Joseph greeted as he opened the door.
"Peri sent me," Profelis said, almost like some kind of password.
"She's very well. I'll tell her you asked. You've built up quite a business," Profelis commented.
Profelis nodded as he'd seen this for himself. In fact, the halfway house Joseph had set up was looking a lot like a certain place of former employment.
"Do you know why they're still angry?" Profelis asked, following the former butler from the kitchen.
Joseph retreated to the other side of the room where he couldn't hear what was exchanged but could keep an eye on his guests. Profelis looked into a pair of mild blue eyes set in a wind and sun burned face. George Daly wore shorts and a short sleeved shirt, a bandana knotted around his throat.
"Why did you come then?" Profelis inquired, gesturing the group to one of the sofas while he took the armchair.
"Where is Murray?" Gayle inquired.
They glanced at each other, then they faced Profelis again.
Profelis listened to their side of events which closely marked Murray's version. Occasionally, Phil, Gayle and Rebekka chimed in with personal anecdotes.
He fell silent again, frowning, his lips moving but more in agitation.
"Have you seen him?" Profelis inquired.
"Except Murray," Gayle pointed out.
"Posthumously," Phil commented, folding his arms.
"Do you have problems with your eyes, Professor Daly?" Profelis inquired.
Like the others, Derek had put his watch to London time at the start of the flight. It was already after midnight in the UK so Derek closed his eyes and settled to sleep. He wanted to be alert when they landed. There was a lot to do in London, and not much time to do it. Rachel was dozing beside him and Nick had long ago developed the habit of being able to sleep at a moment's notice. Anna seemed to be another one with the ability to sleep anywhere, any position, any time.
Derek had given all the printouts back to Nick. But he still saw them. They floated before his eyes, almost teasing him. The answers are all here, they seemed to say. You just have to find them.
At some point in this reflection/dream, the color bled away to black, white and shades of gray, and the images he saw changed. They became sharper, faster. Flashes.
George Daly picking up the hammer and smiling in a kind of proud triumph. At his shoulders stood Murray and a young, sun bronzed man. They were nodding. Behind them, two women, one young, one older. They were all dirty and dusty, streaked with sweat. Around the top of a stairwell, a group of Egyptians in pale robes stood watching, craning their necks.
No, Derek shouted silently, don't break the seal!
George Daly swinging the hammer. The plaster cracking and crazing. Murray turning away to protect his face from flying splinters. And the earth groaning a warning, loud enough to drown out the raucous cheers of the native help. The hammer swinging again, and the earth answering.
George glancing back, saying something and smiling about it. Hefting the hammer and bringing it round, punching a hole, small, no bigger than a fist.
Derek felt his chest tighten as he stood there, an invisible witness to the swinging of the hammer, the hole growing larger. George Daly reaching back for a flashlight and shining it into the opening he'd made.
Dark, dark, doom and death … They couldn't see it but Derek could. A thick stream of black smoke or fog curled out of the hole, writhing around each, entering their bodies thru nostrils and mouths, even pores of the skin. The late afternoon sun seemed to grow dim, as if a haze of cloud had covered it.
Derek wasn't sure what happened next. His vision dream changed, flashing from the past to some other time. A world shrouded in darkness. People dying, plants, trees, animals .. all dying. The air was thick to breathe because they seemed to be choking. And it seemed hot, hotter than usual. Water was boiling away. No stars overhead. And it was silent, so silent. The earth was scared and kept its ancient voice hidden. Even its slow heartbeat seemed to falter.
Color crept back and his vision ended, returning to a dream. Yet it was more than that. Anubis with his jackal's head. Thoth, the ibis headed god. Horus with the head of a falcon. They stood around him, a triangle with Derek in the center. They didn't speak, merely looked at him.
What? What is it you want me to do?
The trust has been betrayed. The pact is broken.
He frowned, his eyes moving rapidly beneath the closed lids.
Thoth stepped nearer and Derek swallowed. Thoth, the god credited with inventing writing, reading, medicine and magic. The Great Scribe … Derek felt the weight of a hand on his shoulder and it crushed him because it was also the weight of the world.
But .. what am I do to? Recapture the evil .. yes, but how? How was it held in there while the door was built?
The three gods drifted away, leaving him on his knees, on his own in a small circle of light amid total blackness.
Doom, doom, dark and death … The earth does not speak. The sun hides, or is driven into captivity. And I must find black smoke and cage it before everything dies … The weight of the world is upon me.
He jerked, his eyes flying open.
"Were you dreaming?" Anna asked.
Derek went to answer but couldn't. For a split second there, he was sure he had been dreaming and maybe even more than that, but he'd woken so suddenly that, if he had, it had fled from his memory.
"Okay," she grinned and settled back again.
Derek checked the time and closed his eyes once more. Nothing important. Yes. If it was, he'd've remembered.


saBaI saOvaa ka mauFt mualyaaMkna pRRdana ikyaa jaayaogaa .
Careful review of spelling, capitalization, punctuation, antecedents, case, number, and subject-verb agreement.
Proofreading plus recommendations of word choice, sentence syntax and paragraph coherence to achieve diction, clarity, and logic.
Proofreading, copyediting, plus rewriting and restructuring text to achieve coherent and unified presentation.
ek saPaYT AaOr saMgaizt pRstuit ko ilae saMSaaoQana saMpadna tqaa punaRlaoKna evaM laoK ka punaR$paMkna krnaa .


text:
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Posts Tagged 'WSOP-TOC'
TOC Alternatives
Tao of Pokerati
by Dan Michalski, Jul 4, 2010 | 1:48 pm
Is somebody winning the TOC? Do we care? Not saying I won't be watching it once it gets edited down for TV, but I thought we were supposed to be at a final table by now, not down to 17 10. With more than a week of retrospective and a third of the field eliminated remaining, the preferable alternatives for the 2011 WSOP seem obvious:
a) Hold the TOC at the very beginning of the WSOP, under some premise like who's gonna kick off the Series with a little extra freeroll juice?
b) Hold the TOC during the November Nine. Add a little Harrah's-branded excitement to that week, ya know, and give top pros a reason to make sure they're part of it all? Also would give some eligible winners a chance to qualify while their stock is hot …
With this and three other final tables going on today … WTF happened to that "day off" we were promised?
Episode 44: TOC Rubbernecking
[audio:tao/TOP_W10_44_TOC_Rubbernecking.mp3]
Dan showed up to the Rio super early for a Sunday just in case the the DOJ decided to bust Full Tilt honchos Howard Lederer and Jesus Ferguson. Pauly compared his morbid fascination of the potential perp walk to watching a NASCAR race in order to see a big wreck.
Episode 45: Bad Beat Bar WTF!
[audio:tao/TOP_W10_45_TOC_EndDay1.mp3]
Dan and Pauly recap the brief events of Day 1 of the TOC, and the slight confusion surrounding the change in schedule.
Be sure to catch up with all the episodes you've missed in the TOP podcast archives.
Tags: 2011 WSOP Schedule, DOJ, poker-on-tv, Tao of Pokerati, tournament schedules, WSOP-TOC
WSOP Tournament of Champions top 20 vote-getters revealed
by Kevin Mathers, Jun 16, 2010 | 4:05 pm
This afternoon, the WSOP announced that the top 20 players receiving votes for their Tournament of Champions. The list is full of players (along with votes received) known by the casual poker player around the peak of the poker boom, circa 2005:
1. PHIL IVEY	16,267
2 DANIEL NEGREANU	16,239
3. DOYLE BRUNSON 13,796
4. PHIL HELLMUTH 12,673
5. CHRIS FERGUSON	11,585
6. ALLEN CUNNINGHAM	10,486
7. JOHNNY CHAN 10,434
8. SCOTTY NGUYEN 9,834
9. BARRY GREENSTEIN	9,806
10. JOHN JUANDA 8,835
11. ERIK SEIDEL 8,802
12. JENNIFER HARMAN 8,206
13. HUCK SEED 8,180
14. DAN HARRINGTON 7,342
15. T.J. CLOUTIER 6,281
16. SAMMY FARHA 6,085
17. HOWARD LEDERER	5,596
18. GREG RAYMER 5,404
19. JOE HACHEM 5,272
20. ANTONIO ESFANDIARI 5,129
These 20 will join Annie Duke, Mike Sexton, Mike Matusow, Joe Cada, Barry Shulman, WSOP Academy qualifier Bertrand "ElkY" Grospellier and WSOP.com online qualifier Andrew Barton on June 27th. In a change from the original schedule, the field will play four levels on the 27th, then play an additional four levels the following day. The remaining players will return on July 4th to play down to a winner with the final table of nine sharing in the $1,000,000 prize pool. The full press release is available here.
Tags: Harrahs, tournament operations, voting, WSOP-TOC
Everest Poker Fires Second Bullet with New Harrah's Lawsuit
Current WSOP felt dispute cause of streaming final table delay?
by Dan Michalski, Jun 13, 2010 | 10:48 am
Former and/or current WSOP table sponsor Everest Poker has filed another lawsuit against Harrah's … claiming the American gaming giant is infringing on trademark rights by playing the 2010 WSOP (and filming some of it) with Everest logos on and around all the tables.
This suit, filed in Las Vegas federal court, is the latest strike in a legal tit-for-tat between that adds to a list of matters on the table and off making 2010 arguably the most challenging WSOP year since Harrah's took over.
Everest first sued Harrah's on April 1, claiming breach of contract after the French ESPN affiliate electronically replaced Everest logos during 2008-09 WSOP broadcasts with "virtual signage" from Full Tilt, an Everest competitor. On those grounds, Everest refused to pay the final $8.4 million on a three-year contract, money the WSOP was expecting before this spring. That suit came, of course, shortly after the WSOP announced its $1 million "TOC" freeroll.
Buffering: So far the only live-streaming WSOP action has been from the WSOP-Academy sponsor's exemption TOC sit-n-go, which took place on a single table with their own felt, not one bearing the marks of Everest Poker.
Harrah's denied those claims, and followed with a suit of its own against Everest, alleging breach of contract and other infractions while seeking the promised Everest funds. And now, as mentioned, Everest has added further litigation to the Everest vs. Harrah's docket, all of which is pending.
Tags: everest poker, France, Harrahs, live streaming video, poker business, poker-media, poker-on-tv, wsop-academy, WSOP-TOC
(Way) Outside the WSOP – Day 14 Evening Edition
A short recap of Thursday's afternoon action, with congratulations going to Bertrand "ElKY" Grospellier, who won the one-table SNG for the final WSOP Tournament of Champions seat, defeating Michael Mizrachi heads-up.
DeWitt leads 5k NL final table
Jason Dewitt (2,600,000) holds a slight chip lead over Jeff Williams (2,550,000) with 8 players remaining in the $5,000 No-Limit Holdem final table. David Benefield (1,265,000) and Amit Makhija (720,000) are others looking to take down the title.
Brussard leading 2k limit holdem
The $2,000 Limit Holdem event is down to 48 players, with the money bubble burst when 45 remain. Andrew Brussard leads with 158,000 in chips, followed by Daniel Makowsky (140,000), Matt Matros (80,500), and Joe Serock (71,500) among the notables.
Danzer leads 10k 2-7 Lowball
George Danzer (200,000) leads the 34 remaining players in the $10,000 2-7 No-Limit Lowball World Championship. He's followed by David "Bakes" Baker (187,000), Eric Cloutier (145,000), Billy Baxter (101,000) and Daniel Negreanu (97,100) among the recognized names returning shortly from dinner break.
$1,500 PLO Day 1
The $1,500 Pot-Limit Omaha event drew a field of 889, with the top 81 making the money, with a first place prize of $256,919. Half of the field has already been eliminated before the dinner break, with the unofficial chip leader Wade Townsend with 55,000. Jordan Morgan (44,500), Fabrice Soulier (40,000) and "Miami John" Cernuto (32,000) among the names you've heard of in the top 10. Four more levels of play are left, with a chance that the money bubble will be burst later tonight.
$1,500 Stud Day 1
A field of 408 started the $1,500 Seven Card Stud event this afternoon, with the last 40 remaining earning more than they paid. In early action, Sorel Mizzi is the unofficial leader with 10,000, followed by Cyndy Violette (8,000), Allen Kessler (7,000) and Humberto Brenes (6,100). The field will play eight levels before calling it a night.
Tags: 2010 WSOP, Allen Kessler, amit makhija, cyndi-violette, Daniel Negreanu, david-benefield, elky, fabrice-soulier, final-table, limit-holdem, Miami John Cernuto, Michael Mizrachi, NL 2-7 Single Draw, no-limit hold'em, Pot Limit Omaha, WSOP-TOC
27th seat in WSOP TOC to be decided via SNG
by Kevin Mathers, Jun 8, 2010 | 9:52 am
Voting for the WSOP Tournament of Champions closes on June 15, and the final seat in the 27-player field will be determined in a nine-person tournament on June 10th. The nine players taking part in the tournament June 10, with the winner playing the $1,000,000 TOC June 27 are:
Tom Dwan
Michael Mizrachi
Bertrand "Elky" Grospellier
Paul Wasicka
The tournament will be filmed for the online version of WSOP Academy, with commentary provided by Ali Nejad and Phil Hellmuth. You can also follow the action as it happens on WSOP Academy's Twitter. More information can be found at WSOP.com.
Tags: andy-bloch, Bertrand Grospellier ("Elky"), don cheadle, Gus Hansen, Liv Boeree, Michael Mizrachi, paul-wasicka, sorel-mizzi, Tom Dwan, WSOP-TOC
Coming Soon: The Ivey Room at Aria
Opening May 22 with $1 million invitational freeroll
by Dan Michalski, Apr 19, 2010 | 6:11 pm
Remember those rumors about Phil Ivey plausibly bringing the Big Game at Bellagio to the new high-stakes room at Aria? Well … unless these pricey invitations mailed out to an undisclosed few are just some sort of elaborate belated April Fool's joke, they certainly seem to be true … with a poker christening in the form of a million-dollar freeroll that could-well rival the WSOP-TOC.
Take a look … The Ivey League $1,000,000 Poker Invitational … coming May 22:
More details in NVG at 2+2.
Tags: Aria, Big Game, Las Vegas poker rooms, The Ivey League Million-Dollar Invitational, The Ivey Room, WSOP-TOC
All-Star Players
The Poker Beat
by Dan Michalski, Mar 30, 2010 | 3:50 am
If you missed it last week, The Poker Beat crew gathered via Skype to talk some poker legal news … Carlos Mortenson's WPT domination (and where he stands overall in the poker) … and we invite folks via the Poker Road Forums to compete with us for a piece of our TPB prop bet picking the likely 20 to make the WSOP TOC.
NOTE: I predict 146 players in the WPT-Bucharest main event … turned out to be 161.
[audio:http://www.pokerroad.com/download/the-poker-beat:56]
Click below for my picks for the most-of-20 prop bet. And click here to submit your own and give yourself a chance at nabbing a piece of our prize pool.
Tags: Carlos Mortensen, Prop Bets, The Poker Beat, WPT, WSOP-TOC
Donkeybomber Looking to Buy His Way in to TOC
Schneider seeks freeroll backing deal with his fans
by Dan Michalski, Mar 24, 2010 | 4:00 pm
Supposedly some 140,000 ballots came in during the first week of WSOP TOC fan voting. Our eligible pal Tom Schneider's not in the Top 50 yet, let alone the all-important Top 20. As Harrah's continues its First Annual Worldwide WSOP.com Email Harvest Tournament of Champions All-Star game, many pros are realizing that they may not get in just on the merits of being tied for 39th among all-time bracelet winners (with dozens of others holding two) and having a twitter account.
So DonkeyBomber is making a desperate unique attempt at getting himself on this uber-elite invite list … check it out … this just showed up in my inbox from everyone's favorite 2007 Player of the Year:
This year, the World Series of Poker is having a Tournament of Champions. Only 27 players will play and 21 [sic.] of them must be voted in.
Here's the deal.
You vote for me, Tom Schneider, and ONLY ME, and you will be part of the pool that will split half of what I win. First place pays $500,000. 50% of what I win will be split by people who have voted for me. If I win $500,000, $250,000 will be split by my voters. It's that simple, and if I get voted in, I will be competing against only 27 players.
The site below is where you vote.
http://www.wsop.com/TOC/TOP50/
They will send you an email with a code in it. That code must be entered when voting.
After you have voted. Send this email to me and the verification email you receive from the WSOP site to tom@bigstackmedia.com and you will be part of the pool. I really would like to play in this tournament and your vote is very important to me. That's why I'm paying for it.
One more important request. Please email this to any friends or family you have that you think might find it fun to own a piece of someone playing in the WSOP Tournament of Champions.
Please vote and then wish me luck.
2007 WSOP Player of the Year
GL Tom! I will so laugh if you make it to #21 and think you're in when you're not. I kid, I kid — I haven't even voted yet, nor decided how I will. If you win, I'm concerned about the cost of stamps eating away profits.
Click below for the current Top 50 (in random order):
Tags: backing-deals, Tom-Schneider, Twitter, WSOP-TOC, WSOP.com
WSOP Announces New TOC Format
Fan ballot to determine spots in million-dollar freeroll
by Kevin Mathers, Mar 15, 2010 | 8:12 am
The big announcement that the WSOP announced on Twitter over the weekend has now turned up online. The 2010 WSOP Tournament of Champions will have 27 players will meet in June to play for a $1 million prize pool with the winner earning $500,000. The twist for this year is that the public will vote for the remaining field of 20 WSOP bracelet winners. A list of the 521 living bracelet winners is available here , along with the current top 50 in online voting. Voting starts at noon ET March 15 and runs through June 15th at midnight. The other spots have been filled by these players:
Joe Cada – 2009 WSOP ME winner
Barry Shulman – 2009 WSOPE ME winner
Mike Sexton – 2006 WSOP TOC winner
Mike Matusow – 2005 WSOP TOC Winner
Annie Duke – 2004 WSOP TOC Winner
Two sponsor exemptions (the WSOP will announce details on how these seats will be awarded shortly)
The TOC will begin on June 27th at noon, and play down to the final table. Play will then resume July 4th (the day before the WSOP Main Event begins) and play down to a winner. ESPN will film the action to air as a two-hour show on August 3rd. The entire press release is on page 2:
Tags: Annie Duke, Barry Shulman, espn, joe cada, mike sexton, press release, tournament-of-champions, voting, wsop-2010, WSOP-TOC
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redpajama_set_name: RedPajamaCommonCrawl


text:
Q: Time/hours syntax in JavaScript - how to do times that aren't on the hour I'm using jQuery to show a different message depending on whether the business I work for is open or not. Since the business only opens at 9.30, I need to be able to write this into the jQuery but so far I've only been able to google things that specify times on the hour.
var thehours = new Date().getHours();
var themessage;
var open = ('nu open');
var gesloten = ('nu gesloten');

if (thehours >= 9.30 && thehours < 18) {
    themessage = open;

} else if (thehours >= 18 && thehours < 24) {
    themessage = gesloten;

} else if (thehours >= 0 && thehours < 9.30) {
    themessage = gesloten;
}

$('.bericht').append(themessage);

var thehours1 = new Date().getHours();
var themessage1;
var open1 = ('09.30 - 18.00');
var gesloten1 = ('18.00 - 09.30');

if (thehours1 >= 9.30 && thehours1 < 18) {
    themessage1 = open1;

} else if (thehours1 >= 18 && thehours1 < 24) {
    themessage1 = gesloten1;

} else if (thehours1 >= 0 && thehours1 < 9.30) {
    themessage1 = gesloten1;
}

$('.bericht1').append(themessage1);

These are two different messages that show either 'now open/now closed' and either the opening or closing times.
It works but only seems to show that's we're open between 10.00 and 18.00, not 9.30 and 18.00, so I wonder if the syntax on 9.30 is wrong.

A: A quick look at the docs for getHours would tell you that it returns the

... integer number, between 0 and 23, representing the hour for the given date according to local time.

So using 9.30 will never work (and if it did, you're using decimal numbers to represent that time - wouldn't it be 9.5?!)
So you need hours and minutes:

var date = new Date();
var hrs = date.getHours();
var mins = date.getMinutes();

if((hrs==9 && mins>30) || hrs >=10){
   console.log("Its past 930am")
}

Putting this together with the rest of your logic gives:

function areWeOpen(){
  var date = new Date();
  var hrs = date.getHours();
  var mins = date.getMinutes();

  if(((hrs==9 && mins>30) || hrs >=10) && hrs<18){
     return true;
  }
  return false;
}

console.log( areWeOpen() ? "We are open" : "We are closed");

Simple!

A: 9.30 is a floating-point number (i.e. 9 and 3 tenths), not hours and minutes in your code.
Since getHours() returns integer number of full hours, your code will act similar to:
if (thehours1 >= 10 && thehours1 < 18) {
    themessage1 = open1;

} else if (thehours1 >= 18 && thehours1 < 24) {
    themessage1 = gesloten1;

} else if (thehours1 >= 0 && thehours1 < 9) {
    themessage1 = gesloten1;
}

which will return unexpected results for time 09:00 - 10:00.
You need to manually check hours and minutes.
Something like this:
var thehours = new Date().getHours();
var theminutes = new Date().getMinutes();
var themessage;
var open = ('nu open');
var gesloten = ('nu gesloten');

if (thehours === 9 && theminutes >= 30) { // 09:30 - 10:00 open
    themessage = open;
} else if (thehours >= 10 && thehours < 18) { // 10:00 - 18:00 open
    themessage = open;
} else { // when we are not open - we are closed :)
    themessage = gesloten;
}

$('.bericht').append(themessage);

You don't need to create conditions for non-working hours, you can just find working hours, and use else for non-working.
As alternative, you can use some sort of "minute of day" term since it is easier to compare them:
function getMinuteOfDay(hour, minute)
{
    return hour * 60 + minute;
}

var now = new Date();
var nowMinuteOfDay = getMinuteOfDay(now.getHours(), now.getMinutes());

var isOpen = nowMinuteOfDay >= getMinuteOfDay(9, 30) && nowMinuteOfDay <= getMinuteOfDay(18, 00);
themessage1 = isOpen ? open1 : gesloten1;

redpajama_set_name: RedPajamaStackExchange


text:
Erika Jayne, née le 10 juin 1971 à Atlanta en Georgie, est une chanteuse américaine.

Elle participe à l'émission de télé-réalité Les Real Housewives de Beverly Hills.

Elle est l'ex-femme de , un avocat américain.

Biographie

Filmographie
 Films
 1995 : Alchemy (TV) : Kitty
 1996 : Lowball : Kate
 2016 : Sharknado 4 (Sharknado 4: The 4th Awakens) (TV) : Frances

 Séries TV
 1990 : New York, police judiciaire (Law & Order) : Une jeune fille (saison 1, épisode 1)
 1996 : Haute Tension (High Incident) : Cindy Butterworth (saison 2, épisode 19)
 2016 - 2018 : Les Feux de l'amour (The Young and the Restless) : Farrah Dubose (3 épisodes)
 2018 : Hit the Floor : Beverly (saison 4, épisode 6)

 Émissions de télé-réalité
 2015 - présent : Les Real Housewives de Beverly Hills (The Real Housewives of Beverly Hills) (depuis la saison 6)
 2016 : Aquamen : les rois des aquariums (Tanked) (saison 11, épisode 3)
 2017 : Dancing with the Stars (saison 24 - semaines 1 à 5, éliminée)
 2018 : Hollywood Medium (saison 3, épisode 5)

Références

Liens externes

Chanteuse américaine du XXIe siècle
Participant à Les Real Housewives de Beverly Hills
Participant à Dancing with the Stars (États-Unis)
Naissance en juillet 1971
Naissance à Atlanta

redpajama_set_name: RedPajamaWikipedia


"Mrs. Sievers is the sole support of her mother and has a son-in-law in the Army." 4x5 Kodachrome transparency by Jack Delano.
"Viola Sievers, Chicago & North Western wiper, giving a giant 'H' class locomotive a bath of live steam at Clinton, Iowa."


text:
Millar, Stephen 2020. Popular music as a weapon: Irish rebel songs and the onset of the Northern Ireland troubles. In: Mangaoang, Áine, O'Flynn, John and Ó Briain, Lonán eds. Made in Ireland: Studies in Popular Music, New York: Routledge, pp. 130-141.
Calo, Francesca, Steiner, Artur, Millar, Stephen and Teasdale, Simon 2020. The impact of a community‐based music intervention on the health and well‐being of young people: A realist evaluation. Health and Social Care in the Community 28 (3) , pp. 988-997. 10.1111/hsc.12931
Millar, Stephen R. 2020. Sounding dissent: rebel songs, resistance, and Irish republicanism. Music and Social Justice, Ann Arbor: University of Michigan Press. 10.3998/mpub.11393212
Power, Martin J., Widdop, Paul, Parnell, Dan, Carr, James and Millar, Stephen R. 2020. Football and politics: the politics of football. Managing Sport and Leisure 25 (1-2) , pp. 1-5. 10.1080/23750472.2020.1723437
Millar, Stephen, Steiner, Artur, Caló, Francesca and Teasdale, Simon 2019. COOL Music: a 'bottom-up' music intervention for hard-to-reach young people in Scotland. British Journal of Music Education , pp. 1-12. 10.1017/S0265051719000226
Millar, Stephen R. 2018. 'Music is my AK-47': performing resistance in Belfast's rebel music scene. Journal of the Royal Anthropological Institute 24 (2) , pp. 348-365. 10.1111/1467-9655.12814
Carr, James, Power, Martin and Millar, Stephen 2017. More than a game: football, politics and popular culture. Brainstorm
Millar, Stephen R. 2016. Let the people sing? Irish rebel songs, sectarianism, and Scotland's Offensive Behaviour Act. Popular Music 35 (3) , pp. 297-319. 10.1017/S0261143016000519
Millar, Stephen R. 2016. Irish Republican music and (post)colonial schizophrenia. Popular Music and Society 40 (1) , pp. 75-88. 10.1080/03007766.2016.1229098
Millar, Stephen 2016. Kate Horgan.The Politics of Songs in Eighteen-Century Britain, 1723-1795. Poetry and Song in the Age of Revolution Number 4 [Book Review]. Blake: An Illustrated Quarterly 50 (1)
Millar, Stephen R. 2015. Musically consonant, socially dissonant: orange walks and Catholic interpretation in West-Central Scotland. Music and Politics 9 (1) 10.3998/mp.9460447.0009.102
Goodall, Kay, McKerrell, Simon, Markey, John, Millar, Stephen R. and Richardson, Michael J. 2015. Sectarianism in Scotland: A 'West of Scotland' problem, a patchwork or a cobweb? Scottish Affairs 24 (3) , pp. 288-307. 10.3366/scot.2015.0079
Millar, Stephen R. 2015. 'I Forbid You To Like It:' The Smiths, David Cameron, and the politics of (mis)appropriating popular culture. Echo: A Music-Centered Journal 13 (1)
Goodall, Kay, Hopkins, Peter, McKerrell, Simon, Markey, John, Millar, Stephen R., Richardson, John and Richardson, Michael J. 2015. Community experiences of sectarianism. [Project Report]. London: Scottish Government Social Research. Available at: https://www.gov.scot/Resource/0047/00470344.pdf


text:
The Glorified Death of the Fallen Warrior
Para ver este video, habilita JavaScript y considera la posibilidad de actualizar tu navegador a una versión que sea compatible con video HTML5
The Bible's Prehistory, Purpose, and Political Future
Universidad Emory
4.7 (171 calificaciones) | 16K estudiantes inscritos
Transcripción del video
With its walls razed to ground by Babylon's armies, Jerusalem joined a long line of ancient vanquished cities—from Ur and Nineveh and Persepolis to Babylon itself. While some recovered from the destruction, others did not. But none responded to political catastrophe by fashioning the kind of elaborate and enduring monument to their own downfall that we find in the Bible. Most conquered populations viewed their subjugation as a source of shame. They consigned it to oblivion, opting instead to extol the golden ages of the past. The biblical authors in contrast reacted to loss by composing extensive writings that acknowledge collective failure, reflect deeply upon its causes, and discover thereby a ground for collective hope. Working through colorful biblical and ancient Near Eastern texts, and drawing on an array of comparative examples, the course illustrates the thoroughgoing manner with which biblical authors responded to defeat by advancing a demotic agenda that places the community at the center. The aim of the biblical authors was to create a nation, and they sought to realize this goal via a shared text, which includes stories and songs, wisdom and laws. This corpus of writings belongs, without a doubt, to humanity's greatest achievements. Whereas the great civilizations of the Near East invested their energies and resources into monuments of stone that could be destroyed by invading armies, the biblical authors left a literary legacy that has been intensively studied until the present day. More important, these authors' visionary response to defeat brought to light a radical new wisdom: the notion that a people is greater than the state which governs it, and that a community can survive collapse when all of its members can claim a piece of the pie and therefore have a reason to take an active part in its collective life.
4.7 (171 calificaciones)
A deeply thought provoking and challenging course that exceeded my expectations, chiefly, by the way it helped me connect the ancient past with the present.
Thumbs Up¿Útil?
The first of many classes I intend to take on this subject. Please have others.\n\nA great class that brought many new thoughts and many revelations.
De la lección
Reinventing the Hero
In this module, we will begin by exploring a clue that adds further support to the general thesis of this course (i.e., that the Bible is a project of peoplehood in response to the defeat of the state). That clue is the absence of martyrdom and glorious death in the biblical narratives. We will see how the biblical authors reshape their history as they fashion narratives and law codes that promote "name-making" through procreation rather than heroic death. Through values that we take for granted today, the authors work to ensure the preservation of their people under conditions of foreign rule. Upon completion of this module, learners will be able to: 1) Identify narrative texts and law codes that relate to procreation, heroic death, and the expanded roles for both men and women, 2) Differentiate between the ideals of heroism found in the Bible and those found in non-biblical text, and 3) Understand that these values emerge out of pragmatic concerns related to corporate survival and the formation of a new kind of political community.
The Biblical Authors Reinvent the Hero7:11
Commemorating the Fallen Soldiers5:47
The Glorified Death of the Fallen Warrior8:51
The Bible's Treatment of Heroic Death6:09
Dr. Jacob L. Wright
Associate Professor of Hebrew Bible
Prueba el curso Gratis
Seleccionar un idiomaEspañol (Spanish)Inglés (English)Portugués (de Brasil)RumanoSerbio
Like the ancient myth of Achilles, much of the Gilgamesh tradition promotes this ethos of the warrior, with it's accompanying ideal of an individual, heroic, tragic death, similar to the death of Patroclus in the Iliad. Near the end of the 12th tablet version, Gilgamesh asks. The 12th tablet version is the full version of, the Gilgamesh Epic. He, Gilgamesh, ask, near the end of this version, have you seen the one who fell in battle? And Enkidu who, his friend, responds, I have seen him. His father and his mother hold him in high honor. They lift up, literally lift up his head. And his wife mourns his death. This is something that is, that a warrior would probably look forward to and like to hear. To this scene, one may compare the death of Enkidu, as a consequence of his battle with Humbaba. Gilgamesh commands, not only the artisans to construct the monument, but also the inhabitants of Uruk, the city, together with all nature, to mourn his death, and sing sweet songs in his honor. The various Sumerian versions of the death of Gilgamesh that are older, contain many such praises, and laments for the fallen hero, and were probably used to bewail the death of other warriors, and it, they may be the oldest part of the Gilgamesh tradition. Now such glorification, of individual heroic death, has to be sure a long legacy. Within Europe, it's found within the Greco-Roman, warrior tradition. Thus in addition to Homer, which I just mentioned, the Spartan poet Tyrtaeus, from about 625 B.C.E., he expressed, in his so called Code of the Citizens Soldier, who dies for the immortal glory awarded him by the polis, his parents, and his posterity. He expresses the ideals of this soldier, and it goes as thus. And he who so falls among the champions, and loses his sweet life, so blessing with honors, polis, his father, and all his people, why such a man is lamented, alike by the young and the elders, and all his polis, all the city, goes into mourning and grieves for his loss. His tomb is painted out with pride, and so are his children, and his children's children, afterward, all the race this is his. His shining glory is never forgotten, his name is remembered, and he becomes an immortal, though he lies under the ground. So, here, in this poem, from Tyrtaeus, we have a warrior who dies and leaves children, and has family, and his mother and father. But really what gives him his immortality is, is not his, his children, which are mentioned in the, piece. But rather, the fact that he died for his polis, he died an honorable death. And now honorable death, predestines him, to an eternal name, eternal life. In her book, the Invention of Athens. Nicole Loraux, shows how the death of the individual citizen's solider, became in Athens, an occasion to praise the city, and thereby to articulate a collective identity and values to which a citizen should aspire. And it's called the epitaphios logos, of the fifth and the fourth centuries. These are funeral orations, delivered by an elected orator. To celebrate the war dead of that year. And the best known example, perhaps many of you know it already, is attributed to Pericles, after the first year of the Peloponnesian war. And we find it in Thucydides, in his histories, Pericles' oration, is an extended glorification of Athens. As the city for which these citizens died. And it urges the living to keep up the good fight. Quote, so died these men as became Athenians. You, their survivors, must determine to have as unfaltering a resolution in the field, though you may pray that it may have an happier issue. For this offering of their lives, made in common by them all, they each of them individually received that renown, that name, which never grows old. And for a sepulchre, not so much that in which their bones have been deposited, but that noblest of shrines wherein their glory is laid up to be eternally remembered upon every occasion on which the deed, story shall fall for its commemoration. The translation is by R Crawley. Commemoration of the war dead occupies such a central place in public ritual and space, ceme, cemeteries and monuments, and what have you, that it may be said that glorification of sacrifice on the battlefield, of the willingness of men to die and now women to die, but in the past, men to die and women to send their men to die, on the behalf of the country, is one of the chief expressions of statehood throughout history. This willingness to die. In as much as states must demand of their subjects, a readiness to die, it is not surprising that bravery and courage are often elevated to the highest civic virtues. In periods when the fighting was performed by an elite few, or the aristocracy, courage and manliness were seen as the mark of nobility. And what nobility meant was that, they had access to more land and had more wealth because they're the ones who protected the country. And as participation, embattled broad into the modern period. And in some places as citizen armies emerged, courage and manliness were democratized, so that all, it was incumbent upon all male citizens to be manly, and to serve, and to fight, and it was not something that was just confined to the nobility. The glorification of marital valor, and the cult of the war dead, have pro, provoked the scent and antipathy. In modern society, as well as in ancient society, thus Pericles' funeral oration, which I just asided, appears to be satirized in Plato's menexenus. Various streams of the Gilgamesh tradition which I've been mentioning a lot in, in the previous segments various streams of this tradition, challenged the ethos of the hero, seeking to make an enduring name for himself through marshall valor. An old Babylonian text, depicting the encounter between Gilgamesh, who was still mourning for his fallen warrior companion Enkidu, and the divine inn tavern keeper, Siduri. She's a divine she's a deity, and she's a tavern keeper, and has a long story we won't get into. And so, this divine tavern keeper, Siduri, responds to the ethos, the warrior ethos that Gilgamesh represents, the ethos of going out, and seeking an opportunity to die in battle, so that one can have an enduring name, that one, an everlasting name immortality. And, as an alternative to the restlessness of this warrior. Whose desire for an enduring name is match by his non-reproductive sexual exploits. He's involved in all kinds of activities, that really do not produce children. Siduri sets forth the enjoyment of a non-heroic life. One that seeks pleasure in food, wife and child. And, let me just read, that episode, tho, those lines for, for you. Gilgamesh, whither do you rove? The life you pursue you shall not find. When the gods created mankind, mortality they appointed for mankind, im, immortality, in their own hands they kept. You, Gilgamesh, let your stomach be full. Day and night, enjoy your meal. Day and night, keep on being festive. Daily make a festival. Party everyday. Day and night dance and play. Let your clothes be clean, let your head be washed. Don't be mourning. In water you may be bathe. Look down at the little one who holds your hand, your son, your child. Let a wife be ever festive in your lap. Probably a reference to sexuality. This is the lot of humans. That's from tablet three. Tzvi Abusch, a famous interpreter of the Gilgamesh Epic, and great [INAUDIBLE] who taught for many years at Brandeis University, offers a compelling interpretation of this text. Quote, the sexual act, is now also a procreative act, which brings into being the posterity. And future signaled by the child, the child in one's hand. That Siduri mentions. Progeny implies death, but a child is also a form of immortality. And in our passage, this is the only kind of immortality that Gilgamesh can hope for, unquote. An enduring name is to be made not in the death of the warrior, but rather in the birth of a namesake, of a name to carry on the warrior who dies. This text may not be an explicit critique of the glorification of death on behalf of the state, as in the Egyptian parody of professions that I just discussed. Yet rulers who depended on the willingness of their subjects to fight to the death, and who promised them an immo immortal name, in return for such sacrifice, will cer, certainly not have been very appreciative of its sentiments.
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Westminster, Colo., December 2, 2011, Media Breakaway, LLC, a leader in online performance marketing, today announced that it is supporting two local charitable organizations this holiday season through its community involvement program - Media Breakaway Makes a Difference.
The goal of the Media Breakaway Makes a Difference program is to provide support for charitable groups and other worthy causes that make a positive impact on the local Denver community. Since its launch in 2007, the program has provided assistance to a number of organizations, including Habitat for Humanity, the Salvation Army, the Avon Walk for Breast Cancer and Denver's The Children's Hospital, just to name a few.
This year, Media Breakaway employees will be spending time with the children of the Mount St. Vincent Home. Mount St. Vincent is a government funded residential home for abused, neglected or challenged boys and girls. Most children range in the age group of 5-12, but some are as old as 18. While spending time with the children, employees will be helping them build gingerbread houses.
"Supporting the local community is one of the most important and rewarding activities that we can achieve as a company and as individuals," said Steven Richter, President of Media Breakaway, LLC. "We are very excited to be able to bring some joy to the children at the Mount St. Vincent Home this holiday season."
In addition to these volunteer activities, the company will also provide support to the Salvation Army by participating in their annual Giving Tree Program.
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