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The CsrR/CsrS two-component system of group A Streptococcus responds to environmental Mg2+
Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved February 3, 2003 (received for review October 14, 2002)
Group A streptococci control expression of key virulence determinants via the two-component sensor/regulator system CsrR/CsrS. The membrane-bound sensor CsrS is thought to respond to previously unknown environmental signal(s) by controlling phosphorylation of its cognate regulator component CsrR. Phosphorylation of CsrR increases its affinity for binding to the promoter regions of Csr-regulated genes to repress transcription. Here we show that environmental Mg2+ concentration is a potent and specific stimulus for CsrR/CsrS-mediated regulation. We studied the effect of divalent cations on expression of the Csr-regulated hyaluronic acid capsule genes (hasABC) by measuring chloramphenicol acetyltransferase (CAT) activity in a reporter strain of group A Streptococcus carrying a has operon promoter-cat fusion. Addition of Mg2+, but not of Ca2+, Mn2+, or Zn2+, repressed capsule gene expression by up to 80% in a dose-dependent fashion. The decrease in capsule gene transcription was associated with a marked reduction in cell-associated capsular polysaccharide. RNA hybridization analysis demonstrated reduced expression of the Csr-regulated hasABC operon, streptokinase (ska), and streptolysin S (sagA) during growth in the presence of 15 mM Mg2+ for the wild-type strain 003CAT but not for an isogenic csrS mutant. We propose that Mg2+ binds to CsrS to induce phosphorylation of CsrR and subsequent repression of virulence gene expression. The low concentration of Mg2+ in extracellular body fluids predicts that the CsrR/CsrS system is maintained in the inactive state during infection, thereby allowing maximal expression of critical virulence determinants in the human host.
Pathogenic bacteria are able to adapt rapidly in the host by up-regulating expression of gene products necessary for survival in the specific environmental niche defined by the site of infection. In the human host, environments encountered by bacteria can vary widely, for example, from the external skin to mucosal surfaces of the airway or the intestine, or deeper tissues and the bloodstream. Therefore, adaptation and survival of the bacteria hinge on their ability to probe the environment and respond appropriately. Group A Streptococcus (GAS or Streptococcus pyogenes) can produce a spectrum of clinical syndromes in humans that range from superficial infection of the pharyngeal mucosa to invasive infection of deep tissues or the bloodstream. GAS elaborate a repertoire of cell-associated (capsular polysaccharide, M protein, extracellular matrix binding proteins) and secreted products (pyrogenic exotoxins, enzymes, cytotoxins) that contribute to the pathogenesis of infection (reviewed in ref. 1). Regulated expression of these products may augment the organism's survival and its virulence in specific niches within the infected host.
Expression of several GAS virulence determinants is controlled by the CsrR/CsrS two-component regulatory system (also called CovR/CovS) (2–5). First reported as a regulator of the has operon that directs synthesis of the hyaluronic acid capsule, the Csr system also controls expression of secreted and cell-associated GAS proteins including streptokinase (ska), the integrin-like protein/IgG protease, Mac or IdeS (mac or ideS), streptolysin S (sag operon), and streptodornase (speMF) (4–7). Csr-mediated regulation of cysteine protease (speB) has been observed in some strains, but not in others (2, 4, 5). DNA microarray analysis suggests the system influences, directly or indirectly, expression of 15% of chromosomal genes (8).
Of 13 putative two-component systems identified in the GAS genome, the CsrR/CsrS system is the best characterized to date and, together with the FasBCA system, one of only two such systems linked to virulence (9, 10). By analogy to well characterized two-component sensor/regulator bacterial systems, the predicted extracellular domain of the membrane-bound sensor component CsrS is expected to bind an extracellular ligand or to sense an environmental condition. This interaction is predicted to trigger autophosphorylation of the cytoplasmic domain of the CsrS protein. The phosphate group is then transferred on to the receiver domain of the cognate regulator CsrR. The regulator component CsrR, when phosphorylated, shows increased binding affinity for the promoters of several genes proposed to be regulated by the system, including the has operon promoter (3, 6, 11). Both CsrR and CsrS mutants exhibit increased transcription of Csr-regulated genes, a phenotype that indicates the system acts to repress target gene expression through its regulator component, CsrR.
A critical issue in understanding the potential function of the Csr system in virulence gene regulation in vivo is the identification of the environmental signal(s) sensed by CsrS. As is the case with many bacterial two-component systems, the identity of such signal(s) for the Csr system has, until now, been completely unknown. In this investigation we used a GAS reporter strain that carries a fusion of the has operon promoter to the gene encoding chloramphenicol acetyltransferase (cat) to identify the environmental signal(s) that interact with the sensor component CsrS. The results of these studies, together with experiments measuring transcription of other Csr-regulated genes, support a model in which environmental Mg2+ binds to CsrS to trigger the phosphorelay that results in repression of Csr-regulated genes.
Materials and Methods
Bacterial Strains and Growth Conditions.
GAS strains used in this study are listed in Table 1. DLS003 is an encapsulated M-type 3 strain isolated from a patient with necrotizing fasciitis (12). Derivative strain 003CAT carries on its chromosome a fusion of a 688-bp fragment, encompassing its autologous has operon promoter sequences with the 5′ portion of hasA, to the cat sequence (13). GAS was grown at 37°C in Todd-Hewitt broth (Difco) supplemented with 0.5% yeast extract (THY), or on THY agar supplemented with 5% defibrinated sheep blood. For cloning experiments, Escherichia coli DH5α was grown in Luria–Bertani (LB) broth (Difco) or on LB agar. When appropriate, antibiotics were added at the following concentrations: ampicillin, 50 μg/ml; kanamycin, 50 μg/ml for E. coli, 300 μg/ml for GAS; erythromycin, 200 μg/ml for E. coli, 1 μg/ml for GAS; chloramphenicol, 20 μg/ml for E. coli, 5 μg/ml for GAS.
CAT Assays.
GAS cytoplasmic extracts were prepared as described (13) with slight modifications. Briefly, bacteria grown overnight on THY-blood agar were inoculated in THY broth and collected by centrifugation (1,250 × g; 8 min) at mid-exponential phase growth (A600 = 0.3–0.4) unless otherwise stated. Pellets were washed in 1 ml Tris (10 mM, pH 7.8) and resuspended in 180 μl of the same buffer, to which 20 μl (≈15 units) of mutanolysin was added. Bacteria were treated for 1 h at 37°C and then centrifuged at 18,000 × g for 5 min, and the supernatant carrying cytoplasmic extracts was collected. Determination of CAT activities in 3–5 μg of total protein extract was performed by using the spectrophotometric assay described by Shaw (14). To control for the effects of the different chemicals used on both mutanolysin and CAT activity, control bacterial cultures received the chemical studied immediately before collection of samples. Cytoplasmic extracts from the non-CAT expressing wild-type GAS strains DLS003 and 87-282 did not produce any background CAT activity, nor did the CAT-expressing strains 003CAT and 282CAT when chloramphenicol, the acetyl acceptor in the enzymatic reaction, was omitted from the reaction mix. Results were recorded as milliunits of CAT per mg total cell protein per min (mu/mg/min).
Measurement of GAS Cell-Associated Capsular Polysaccharide.
Cell-associated hyaluronic acid was measured as described (15) with slight modifications. Briefly, 6 ml of GAS culture grown in THY to mid-exponential phase (A600 = 0.35–0.4) were harvested by centrifugation, washed in 10 mM Tris buffer (pH 7.5), resuspended in 300 μl of the same buffer, and mixed on a vortex mixer in an equal volume of chloroform. The hyaluronic acid content of the aqueous phase was then measured spectrophotometrically at 640 nm by using 1-ethyl-2-[3-(1-ethylnaptho-[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]-naptho-[1,2-d]- thiazolium bromide (Stains-All, Sigma). The values shown represent the values obtained for each strain tested minus the background values obtained by using the acapsular mutant strain DLS003hasA∷ΩKm−2 (12).
Measurement of Total Magnesium and Calcium Concentrations in Growth Media.
Magnesium and calcium concentration measurements in THY broth were performed at the Brigham and Women's Hospital Chemistry Laboratory (Boston) by using an Olympus AU640 analyzer according to the manufacturer's instructions. Mg2+ bound to xylidyl blue at pH 11. 4 was measured bichromatically at 520/800 nm, whereas Ca2+ bound to Arsenazo III (2,2-[1,8-dihydroxy-3,6-disulphonaphthylene-2,7-bisazo]-bisbenzenearsonic acid) at pH 6.5 was measured bichromatically at 660/800 nm. To control for the accuracy of the measurements, magnesium and calcium concentration measurements in chemically defined medium were determined in parallel.
DNA Techniques.
Plasmid pCR2.1 is a linearized E. coli vector used for direct cloning of PCR products (Invitrogen); pUC19 is a high copy number cloning vector (16); pBRΩKm−2 is a pBR322 derivative carrying the ΩKm−2 cassette (17); and pJRS233 is a temperature sensitive E. coli-Gram-positive shuttle vector provided by June Scott (18). Plasmid DNA was obtained from E. coli by using the Qiagen miniprep or maxiprep kit according to manufacturer's instructions. GAS chromosomal DNA was prepared as described (19). Restriction endonuclease digestions, DNA ligations, transformations of chemically competent E. coli, and Southern hybridizations (ECL, Pharmacia) were performed by using standard protocols (20). GAS electrocompetent cells were prepared and transformed by using Gene Pulsar II (Bio-Rad) as described (13).
PCR was performed with Taq DNA polymerase (Life Technologies) on a GeneAmp PCR 2400 System (Perkin–Elmer) for 32 cycles at the following conditions: 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min. Primers used in PCR are shown in Table 3, which is published as supporting information on the PNAS web site, www.pnas.org.
Construction of a csrS Mutant.
The sensor component gene csrS of GAS strain DLS003 was inactivated by inserting in its 5′ end the kanamycin resistance cassette ΩKm−2. In brief, a 1.1-kb DNA fragment encompassing the 3′ end of csrR and the 5′ end of csrS was amplified from DLS003 chromosomal DNA by using primers csrR-F1 and csrS-R1 (Table 3). The amplicon was cloned into vector pCR2.1 (Invitrogen), released by EcoRI digestion and ligated into pUC19 to give plasmid pUCcsrS. The ΩKm−2 cassette obtained from vector pBRΩKm−2 (17) by SmaI digestion was then inserted into the EcoRV site 310 bp downstream of the csrS start codon. The resulting csrSΩKm−2 construct was released by PvuII digestion and cloned into SmaI-digested pJRS233 (18) to give plasmid pJRcsrSΩKm−2. The fidelity of the final construct containing the 5′ end of csrS disrupted with ΩKm−2 was verified by DNA sequencing. pJRcsrSΩKm−2 was introduced into DLS003 by electroporation and the csrS mutant obtained as described (2). Replacement of the wild-type csrS copy with csrSΩKm−2 and loss of pJRS233 sequences was verified by Southern hybridization.
DNA Sequencing.
Sequencing of plasmid DNA was performed at the Brigham and Women's Hospital Automated Sequencing and Genotyping Facility. Primers used for sequencing are listed in Table 3.
RNA Dot Hybridizations.
Total bacterial RNA was isolated by using the Rneasy mini kit (Qiagen) according to manufacturer's instructions, except that bacteria were lysed by shaking with glass beads on a dental amalgamator (Patterson Brand). RNA samples were treated with 5 units of DNase I (Invitrogen) for 30 min at room temperature to remove any contaminating DNA. The final concentrations of the RNA obtained were adjusted to 100 ng/μl, and the samples were divided into aliquots and frozen at −80°C until needed. RNA was blotted onto Hybond N+ membranes (Pharmacia) by using the Bio-Dot Microfiltration apparatus (Bio-Rad). DNA probes were generated by PCR using 003CAT chromosomal DNA as template, and were radiolabeled with [α-32P]dATP by random priming using the RadPrime DNA Labeling System (Invitrogen). The primer pairs used to generate probes for rpsL, recA, emm3, csrR, csrS, hasB, ska, sagBCD, and speB are listed in Table 3. Hybridizations were carried out in Church buffer at 60°C for 16 h followed by one wash in 1× SSC − 0.1% SDS at room temperature and three washes in 0.5× SSC − 0.1% SDS at 60°C (20). Negative controls consisted of 2 μg total E. coli RNA; positive controls carried 0.3 ng of unlabeled denatured probe DNA in addition to 2 μg total E. coli RNA.
GAS Capsule Gene Expression Is Repressed at High Mg2+ Concentrations.
In an effort to identify the signal(s) that regulate virulence gene expression in GAS through the two-component system CsrR/CsrS, we studied the effect of divalent cations on expression of the csr-regulated capsule gene operon (hasABC). Divalent cations such as Mg2+ and Ca2+ are required for fundamental biochemical reactions in bacteria, but also function as environmental stimuli to which two-component systems of certain pathogenic species respond by altering expression of genes involved in virulence (21–23). Capsule gene expression levels in GAS were quantified by measuring CAT activity in cell extracts of strain 003CAT that carries on its chromosome a fusion of its autologous has operon promoter to the gene encoding chloramphenicol acetyltransferase (13). To determine whether the divalent cation concentration influenced capsule gene expression, we compared the CAT activity in extracts of 003CAT grown in unsupplemented broth with that from the same strain grown in broth supplemented with additional amounts of one of four divalent cations: Mg2+, Ca2+, Mn2+, or Zn2+. During growth in THY broth, GAS capsule gene expression is maximal at mid-exponential phase after which it declines to a very low level in stationary phase. Maximum CAT activity consistent with high capsule gene expression was observed in cell extracts of 003CAT obtained at mid-exponential phase growth (see below). However, when 003CAT was grown in broth supplemented with MgCl2 (or MgSO4), capsule gene expression was reduced in a dose-dependent fashion. CAT activities for bacteria grown in 5 and 20 mM additional MgCl2 were reduced by 50% and 78%, respectively, compared with those for bacteria grown in THY broth without additional MgCl2 (Fig. 1). Growth rate, viability, and final cell density reached at stationary phase remained unaffected. Capsule gene expression did not increase when the Mg2+ concentration in THY broth was lowered by the addition of EDTA. Instead, capsule gene expression decreased, as reflected by a 3-fold reduction in CAT activity in extracts of bacteria grown in THY-0.5 mM EDTA. This reduction in capsule gene expression was not attributable to Mg2+ depletion, however, because wild-type CAT activity was restored during growth in THY-0.5 mM EDTA supplemented with 0.5 mM CaCl2 (data not shown). Therefore, capsule gene expression levels were maximal in THY broth without additional Mg2+. The total Mg2+ concentration in THY broth was measured at 0.97 ± 0.02 mM, a level that is similar to the physiological Mg2+ concentrations encountered by GAS in human serum and extracellular body fluids (0.7–1 mM) (24).
The reduced CAT activity of 003CAT at high MgCl2 concentrations correlated with reduced capsule production: the amount of cell-associated capsular polysaccharide measured on cells grown in 5 and 10 mM additional MgCl2 was 22% and 15%, respectively, of the amount on cells grown in THY broth without additional MgCl2 (Fig. 1). Repression of capsule expression was also evident during growth of 003CAT on THY-blood agar supplemented with MgCl2. Colonies on medium supplemented with 10 mM MgCl2 were small and dry, an appearance indistinguishable from that of colonies of an isogenic acapsular mutant strain DLS003hasA∷ΩKm−2 (12).
Down-regulation of capsule gene expression was specific to Mg2+ as no repression was evident when bacteria were grown in THY broth supplemented with 5 mM or 10 mM additional CaCl2. On the contrary, capsule gene expression increased by ≈40% when an additional 1 or 2 mM CaCl2 was provided (Fig. 1). Because the total concentration of calcium in unsupplemented THY broth was measured at 0.36 ± 0.02 mM, addition of 1 mM CaCl2 produced a total Ca2+ concentration near the physiological range found in human serum and extracellular body fluids (1–1.3 mM) (25). Addition of Mn2+ up to 0.5 mM or Zn2+ up to 0.1 mM did not alter capsule gene expression levels or the amount of cell-associated hyaluronic acid. Addition of either of the two cations at higher concentrations reduced bacterial viability as determined by counts of colony-forming units, growth rates, and final cell densities reached in culture at stationary phase.
Csr-Regulated Genes Are Repressed by High Mg2+ Concentrations in Strain 003CAT.
To investigate whether expression of other Csr-regulated genes was repressed at high Mg2+ concentrations, we studied expression not only of the hyaluronic acid synthesis genes (hasABC), but also of streptokinase (ska) during exponential phase and streptolysin S (sagA) during stationary phase. Expression of both Csr components, CsrS and CsrR, was also monitored at both growth phases. In addition, the effect of Mg2+ on expression of M-type 3 protein (emm3) that is regulated by a Csr-independent mechanism was studied (4, 5, 26). GAS strain 003CAT was grown in THY broth with or without supplemental MgCl2 (15 mM), and total RNA was collected at mid-exponential (A600 = 0.3) and stationary (A600 = 0.8) phase. Four-fold serial dilutions of total RNA were blotted onto nylon membranes and hybridized to a radiolabeled DNA probe specific for each gene or operon of interest. The probe for the capsule gene cluster was to the middle gene (hasB) of the has operon (27). The probe for streptolysin S was to the gene cluster sagBCD located downstream of the streptolysin S structural gene (sagA) in the sagABCDEFGHI operon. Streptolysin S activity requires expression of the genes downstream of sagA; therefore, transcript levels for sagBCD reflect streptolysin activity more accurately than sagA transcript itself (28). As a control, the RNA was also probed for two housekeeping genes, rpsL and recA, neither of which is known to be regulated by Csr. As shown in Fig. 2, expression of rpsL and recA was not altered during either exponential or stationary phase in the presence of 15 mM additional MgCl2. By contrast, growth in supplemental Mg2+ was associated with dramatic repression of the Csr-regulated capsule and streptokinase genes at exponential phase and of streptolysin S at stationary phase. Based on the relative intensity of the hybridization signals, the amount of mRNA produced under high Mg2+ conditions was estimated to be reduced 4- to 16-fold for each of the three genes compared with that from cells grown in unsupplemented broth. As expected, expression of streptokinase and capsular polysaccharide was maximal at exponential phase and very low or absent in stationary phase, whereas the converse was true for streptolysin S. The finding that Mg2+ repressed not only capsule gene expression, but also expression of streptokinase and streptolysin S suggests strongly that the effect was mediated by the Csr system.
Conflicting data have been reported regarding Csr regulation of speB and of the csrRS locus itself. Whereas speB expression is repressed by Csr in some strains, it appears not to be affected by Csr in others, including DLS003 (2, 4). CsrR was previously reported to repress its own expression, as increased csrRS transcript levels were observed in a csrR mutant (4). However, a subsequent study indicated that phosphorylated CsrR did not bind to its own promoter and therefore did not repress its own expression directly (6). In the present investigation, we found the transcription levels of speB and of the csrRS operon, as well as expression of the Csr-independent emm3, remained unaffected at high Mg2+ concentrations (data not shown).
Expression of Csr-Regulated Genes Is Not Repressed by High Mg2+ Concentrations in a csrS Mutant.
If the hypothesis is correct that Mg2+ down-regulates gene expression by signaling through the CsrR/CsrS system, Csr-regulated genes that are repressed by Mg2+ in the wild-type strain 003CAT should be unaffected by high Mg2+ in a mutant strain that lacks the CsrS sensor. To test this prediction, we constructed an isogenic sensor component mutant (003csrSΩ) by inserting the ΩKm−2 cassette (17) within the coding sequence of csrS near the 5′ end of the gene. In contrast to wild-type strain 003CAT, the sensor component mutant strain showed no decrease in production of capsular polysaccharide at Mg2+ concentrations up to 10 mM (Fig. 3). In further experiments, we examined the expression of Csr-regulated genes in 003csrSΩ grown in the presence or absence of 15 mM MgCl2. As shown previously for csr mutants, expression of hasABC, ska, and sagABCDEFGHI during growth in unsupplemented medium was increased relative to that for wild-type strain 003CAT. However, in contrast to the results obtained with 003CAT, growth of the csrS mutant in 15 mM Mg2+ had no effect on expression of the Csr-regulated genes, hasABC, ska, or sagABCDEFGHI (Fig. 2). The fact that inactivation of csrS abrogated Mg2+ regulation provides strong evidence that Mg2+ signals repression of Csr-regulated gene expression through its interaction with the membrane-bound sensor protein, CsrS.
Kinetics of Mg2+-Mediated Capsule Gene Repression.
To study the kinetics of transcriptional regulation in response to a change in Mg2+ concentration, we grew duplicate cultures of 003CAT to early exponential phase (A600 = 0.15) at which point one culture was exposed to 15 mM MgCl2. Sequential bacterial samples were collected from both Mg2+-treated and untreated cultures and CAT activity was determined at each time point. As shown in Fig. 4, the rate of growth was similar with or without supplemental MgCl2. CAT activity for bacteria grown in THY with no additional Mg2+ increased from early exponential phase to reach a maximum 90 min later and then dropped sharply at early stationary phase, a pattern that is also reflected in the amount of cell-associated hyaluronic acid produced (Fig. 4). In contrast, CAT activity in bacterial samples collected after addition of MgCl2 remained relatively unchanged for the first 15 min and then declined over time. At 60 min and 90 min after addition of MgCl2, CAT activities were reduced by 71% and 75%, respectively, compared with those recorded at the same time points for bacteria grown without additional MgCl2. These results indicate that repression of Csr-regulated gene transcription occurs within minutes of a change in environmental Mg2+.
High Mg2+ Concentrations Repress Capsular Polysaccharide Expression in Diverse GAS Isolates.
Because the csr system is highly conserved among GAS strains, we reasoned that Mg2+-dependent repression of capsule synthesis should be demonstrable in other GAS strains in addition to 003CAT. To test this hypothesis, we examined capsule production during growth in THY supplemented with 10–20 mM MgCl2 in a series of GAS isolates of various M protein types. We tested capsule gene expression in strain 282CAT, derived from the heavily encapsulated strain 87-282 (M type 18), that also carries a fusion of its autologous has operon promoter to cat (13). CAT activity in 282CAT extracts from exponential phase cells grown in THY were 2- to 3-fold higher than those for 003CAT extracts, consistent with the higher amounts of cell-associated hyaluronic acid produced by the former strain (13) (see below). Similar to the results obtained for M type 3 strain 003CAT, capsule gene expression in 282CAT decreased dramatically during growth in broth supplemented with MgCl2. Somewhat higher concentrations of MgCl2 were required to achieve maximum repression of capsule gene expression in 282CAT compared with 003CAT: 20 mM MgCl2 was required for a 3-fold decrease in CAT activity for 282CAT, whereas 10 mM MgCl2 produced a similar decrease in the less encapsulated strain 003CAT. Because the has operon is transcribed more actively in 282CAT than in 003CAT, it is possible that higher MgCl2 concentrations are necessary to produce sufficient amounts of phosphorylated CsrR for maximal repression of gene expression in 282CAT. Reduced capsule gene expression by 282CAT during growth in high Mg2+ correlated with markedly reduced production of cell-associated hyaluronic acid (Table 2). Similar Mg2+-dependent repression of capsule synthesis was also observed for three additional GAS strains of M types 1 and 3 (Table 2).
The evidence presented here suggests that environmental magnesium regulates virulence gene expression by GAS through the CsrR/CsrS two-component system. The fact that high Mg2+ concentrations stimulated repression of Csr-dependent virulence genes in the wild-type GAS strain but not in an isogenic CsrS mutant points to a direct interaction of Mg2+ with the putative sensor component protein. The interaction is likely to involve the extracellular domain of CsrS in a fashion analogous to that described for the periplasmic domain of the PhoQ Mg2+ sensor of Salmonella enterica serovar Typhimurium (21, 29). Both CsrS and PhoQ belong to the EnvZ family of sensor kinases, and CsrS is similar to PhoQ in several ways: the two sensor proteins are of similar size; each has two predicted membrane spanning domains near the N terminus; both have a predicted extracellular or periplasmic domain of similar size (146 and 151 aa for PhoQ and CsrS, respectively). The two proteins share 43% similarity over 300 aa by blastp analysis (30). Within the predicted extracellular domains of the two proteins, there is particularly striking similarity in acidic residues, which have been implicated in cation binding. Ten of 20 acidic amino acids within the predicted extracellular domain of CsrS are similar or identical in PhoQ (Fig. 5, which is published as supporting information on the PNAS web site). PhoQ of E. coli has been proposed to bind Mg2+ on its periplasmic domain via a cluster of seven amino acids, six of which are acidic (31). Such a cluster is also found in the PhoQ periplasmic domain of S. enterica serovar Typhimurium, but not in some other PhoQ homologues that are also able to respond to Mg2+ (23, 32). Thus, a specific motif of acidic residues required for Mg2+ binding has not been defined.
Although there are striking parallels between CsrR/CsrS and the PhoP/PhoQ system, there are also important differences that may reflect the specific adaptive functions of these regulatory systems for pathogens that employ different strategies for survival within their animal or human hosts. In S. enterica serovar Typhimurium, binding of Mg2+ to PhoQ promotes dephosphorylation of PhoP, which renders it inactive as a transcriptional activator of PhoP-induced genes (23). By contrast, binding of Mg2+ to CsrS results in repression of CsrR-regulated genes, presumably by increasing phosphorylation of CsrR. So, although the net result in both cases is repression of virulence gene expression by high Mg2+, repression is achieved through opposite effects on phosphorylation of the respective regulator proteins. Another important difference between the two systems is the range of magnesium concentrations to which they respond: maximal repression of PhoP-induced genes in Salmonella occurs during growth in Mg2+ concentrations of ≈1 mM or greater, whereas the Csr system is fully active in repressing gene expression only at Mg2+ concentrations ≥10 mM. It has been suggested that responsiveness of the PhoP/PhoQ system is optimized for survival of Salmonella within the phagosome where Mg2+ concentrations are likely to be in the micromolar range (33). Although GAS may enter a variety of eukaryotic cells, they fail to proliferate intracellularly and their intracellular survival is limited (15, 34). The Mg2+ concentration at which the Csr-regulated genes are maximally expressed (≈1 mM) is approximately that of human extracellular fluids, as might be expected for an extracellular pathogen. Furthermore, whereas one of the functions of the PhoP/PhoQ system is induction of expression of two magnesium importer systems (MgtA, MgtCB) to permit Salmonella survival in very low Mg2+ concentrations, no such system for scavenging magnesium has been identified in GAS. Rather, the CsrR/CsrS system appears to be optimized to ensure maximum expression of virulence factors under conditions encountered by GAS in mucosal secretions and extracellular fluids and not under conditions of higher Mg2+ concentrations such as the natural environment outside the body.
Expression of the GAS genes that direct synthesis of capsular polysaccharide, streptokinase, and streptolysin S was strikingly repressed in the presence of high Mg2+ concentrations. However, expression of the two Csr components in the same conditions was not affected. Although some studies have suggested autoregulation of the csr system, the CsrR protein appears not to interact directly with the csr operon promoter (6). The apparent lack of Mg2+ regulation of the csr locus itself suggests that the repressive activity of the system depends primarily on the phosphorylation state of CsrS which governs phosphorylation of CsrR, rather than on the relative abundance of the CsrS and CsrR proteins. Repression of capsule gene expression in GAS subjected to high Mg2+ concentrations was evident within minutes. Such rapid repression is consistent with the proposed model of Mg2+ interacting with CsrS already present on the cell membrane and initiation of the phosphorelay cascade. Magnesium is abundant in the natural environment and may signal GAS to adapt for survival in the external environment. Signaling through the Csr system may permit GAS to rapidly down-regulate production of host-specific virulence factors and divert energy to alternative metabolic pathways important for survival outside the human host.
The csr system response appears to be specific to Mg2+, because other divalent cations tested did not affect capsule gene expression at nontoxic concentrations. Whereas high Mg2+ resulted in down-regulation of Csr-regulated genes, we observed a modest increase in capsule gene expression as the Ca2+ concentration was increased to a level similar to those found in human extracellular fluids. It remains to be determined whether the GAS response to Ca2+ is Csr-dependent. If so, both Mg2+ and Ca2+ concentrations encountered by GAS in the human host are ideal for maximum Csr-regulated gene expression.
Although the experiments presented here were performed in vitro, Mg2+-dependent regulation of virulence gene expression by GAS is expected to operate also in vivo during infection. Expression of Csr-regulated virulence genes was maximal, hence, the csr system was minimally induced, at ≈1 mM Mg2+, a concentration that is similar to the physiologic Mg2+ levels in body fluids such as mucosal secretions and the blood (24). A previous study demonstrated that expression of the Csr-regulated has operon is rapidly induced after introduction of GAS into the mouse peritoneum or the baboon pharynx (35). Although the precise stimulus for up-regulation of capsule gene expression was not defined, the results are consistent with those of the current investigation. In recent experiments, we also observed Mg2+-dependent repression of capsule gene expression in GAS grown in 10% or 50% human plasma (data not shown). Thus, it appears unlikely that other factor(s) in plasma inhibit the down-regulatory effects of high magnesium on Csr-regulated genes. Rather, the low concentrations of Mg2+ at both deep tissue and mucosal sites ensure that CsrR will remain in the unphosphorylated state that has low affinity for Csr-regulated promoters, thereby permitting high-level expression of the has operon and other Csr-regulated virulence genes.
Identification of magnesium as a specific environmental stimulus that controls Csr-mediated regulation of GAS virulence determinants expands our understanding of the complex interactions between GAS and the human host. Successful adaptation of a pathogen requires dynamic modulation of metabolic activities and expression of critical virulence determinants in a manner that optimizes bacterial survival in the various environmental conditions the organism confronts. Our data implicate environmental Mg2+ as a stimulus that signals GAS to up-regulate expression of Csr-regulated virulence factors under conditions encountered during human infection. To our knowledge, this is the first specific signal demonstrated to interact with any two-component regulatory system in this important human pathogen.
We thank D. Wessels for technical assistance and R. Ross and J. Cocchiaro for helpful discussions. This work was supported by National Institutes of Health Grant AI29952 and by a grant-in-aid from the American Heart Association.
- group A Streptococcus;
- chloramphenicol acetyltransferase
- Received October 14, 2002.
- Copyright © 2003, The National Academy of Sciences
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text:
Trumpet 3 missing Notes
edited November 2018 in General Questions
I seem to have been hit by a bug that supposedly only is relevant for Breath controller users. But for those it results in dropping notes while playing (especially when doing trills) which is really displeasing.
I suppose the problem can be reproduced with a Breath controller or with any DAW by Midi programming, so I just provide a description how to create the behavior.
- no blowing (BC = 0), no key pressed
- press a key on the keyboard
- increase BC to produce a sound
- press another key .e.g. higher (legato upwards)
- release th second key (legato downwards = trill)
- stop blowing (BC -> 0, still holding the first key
- increase BC intending to trigger the sound creation. IMHO now the key still pressed should (again) be used to determine the note.
But no sound is heard.
I suppose the problem can be reproduced with a Breath controller or with any DAW by Midi programming, so I just provide a description how to create the behavior.
- no blowing (BC = 0), no key pressed
- press a key on the keyboard
- increase BC to produce a sound
- press another key .e.g. higher (legato upwards)
- release th second key (legato downwards = trill)
- stop blowing (BC -> 0, still holding the first key
- increase BC intending to trigger the sound creation. IMHO now the key still pressed should (again) be used to determine the note.
But no sound is heard.
Any help on this issue ? (Do other libraries also feature this bug ? Maybe it's a Kontakt problem ???)
Bump again......
No idea how to overcome this problem ?
Hi, Michael. I use wind controller and can confirm this doesn't happen there, likely because it sends unique key up/down on each note of the trill. I did try on BC mode though and can reproduce what you describe. It only seems to happen after a trill though where you never release note#1. The only workaround I see is to release and re-press the original note. Does appear to be inconvenient as the BC->0 normally appears to work as expected until you happen to play a trill as you describe which would be frustrating with live playing.
I doubt my answer helps you much but wanted to let you know that I can reproduce what you described.
Many thanks for testing and confirming the problem.
When playing with keyboard and breath controller, you can hardly avoid the situation, as this results from "naturally" performing with this equipment. After some decent training with the BC you will always trigger the notes with breath, pressing the key in advance or holding it pressed whenever appropriate. With this technique (and due to the multiple real time controls the TEC BBC v2 offers) I feel using the BC you can play even more versatile and "natural" then with a wind controller. (I do have a wind controller, but retired it. )
I also use multiple SWAM instruments, and those do not feature this issue.
So I suppose this needs to be called a bug of the Kontakt bases Sample Modeling Trumpet, and only can be mended by the Sample Modeling engineers.
I desperately hope for help from SM !
I can also confirm that Horns and Trombones behave this way as well when in BC mode, so it's not specific to Trumpet.
I just noticed this as well. I am using Kontakt 5.7.1 Perhaps that is the problem?
It seems to be due to the last Kontakt 5.7.1 update. Retriggering is no longer working in BC mode. Could you please try WC mode as a provisional workaround.
Of course in WC mode, triggering a note will occur by pressing a key, rather than when CC2 overcomes a threshold value. We will immediately contact Native Instruments for a fix.
Giorgio Tommasini wrote: We will immediately contact Native Instruments for a fix.
So this in fact is a Kontakt issue !
Hi all.
Can you first verify that retriggering, i.e. playing a note, overlapping another note (legato), releasing this second note and therefore coming back to the sound of the first note, is indeed working in BC mode with the Trumpet3, or Trombone3, in the latest build of Kontakt 5.7.1 (R35)?
Please let us know.
I'm on 5.7.1 (R35). What I see is this when in breath control mode:
1) Press key down D3, but CC2=0.... no note sounds, as expected.
2) While D3 is held, increase CC2, note starts, as expected.
3) While D3 is held and CC2 constant and non-zero, press key down E3. E3 sounds, as expected.
4) While D3 and E3 are still held down and CC2 still constant and non-zero, lift key E3. D3 sounds again, as expected.
5) While D3 is held and CC2 constant and non-zero, press key down E3. E3 sounds, trilling as expected
6) While D3 and E3 are still held down, bring CC2 to zero, then back up. E3 sounds, as expected.
7) While D3 and E3 are still held down and CC2 still constant and non-zero, lift key E3. D3 does NOT sound, unexpectedly.
Alternately as step 5) While D3 is still held down, bring CC2 to zero, then back up. D3 does NOT sound, unexpectedly.
So trill retriggering works until you get a CC2=0 in the mix.
I could do a Reaper JSFX script that prevents CC 2 from going to zero but use 1 as a minimum.
I also could so a script that with CC 2 = zero sends a note-off for the last note detected and with CC going non zero sends a note on for that note (unless another note on had been detected in between).
But this of course is a clumsy work around; a decently working instrument would be a lot nicer.
If this really is a Kontakt issue and can't be mended by SM, that would be really bad news, as I doubt NI will react in an acceptable time frame, while I do trust in SM.
Thanks John, you perfectly nailed down the issue.
As you know, in BC mode, a note is triggered when CC2 overcomes a certain threshold (about 9). When it falls below the threshold a note-off is issued.
If the key is still pressed and the time lapse is less than the sample length, about ten seconds, a new note-on is issued.
This is correct, and corresponds to what has been requested by many. If the threshold had been set to zero, one could not control note on/off with the BC.
Retriggering in Kontakt 7.5.1 (r35), in BC mode, effectively works until CC2 does not drop below the note-off threshold. If this occurs after any legato, and therefore after trills, the instrument resets to the initial state. In this case raising again CC2 above the threshold, even if a key is still pressed, does no longer yield a note-on. One needs to press again a key to restore the normal behavior.
The same issue appears in previous versions of Kontakt, as for example 5.5.2.
We will try to fix it in a next release of all our Brass.
Thanks all for reporting this.
Giorgio Tommasini wrote: We will try to fix it in a next release of all our Brass.
Great !
So we don't need to wait for NI to fix anything.
I really hope that such a release will be available really soon, as playing with the BC is decently hampered by that issue.
Interestingly enough, this issue is present since the very first release of version 3 of all our Brass. They behave exactly in the same manner in Kontakt 4. Nobody reported it so far ;-)
It's definitely not related to Kontakt, my mistake. Anyway, we will try to fix it, as said, in a next release. Is there anything else that you would like to see in the future update?
Giorgio Tommasini wrote: Nobody reported it so far ;-)
Seemingly there are not many Breath Controller users who do enough live playing to develop the "advanced" technique of leaving the key pressed and re-trigger the note by breath, and be hit by the problem.
In fact I did an extensive BC playing training with SWAM instruments, that do not feature this problem, and hence I was able to get used to this playing style, and when staring to use the Trumpet, I found that certain notes in the stream did not play. After that, it took me a while to find out the exact way to recreate the problem.
Thanks for listening to your users !
As it seems to last some time until the SM devs can create an update, I did a Reaper plugin.
Maybe it's not perfect, but it helps a lot with that issue,
</s><i> </i>desc:MIDI Trumpet Bugfix //tags: MIDI processing filter //author: MSchnell @init CC_MSG = 0xB0; // 11; EXPRESSION = 2; NOTE_ON = 0x90; NOTE_OFF = 0x80; minnote = 52; maxnote = 84; notes = 0; notescount = maxnote - minnote + 1; killed = 0; e0 = 0; @block while (midirecv(mpos, msg1, msg2, msg3) ) ( statusHi = msg1 & 0xF0; channel = msg1 & 0xF; (statusHi!=0xF0) && statusHi ? ( // Time code etc // x = StatusHi; mute = 0; statusHi == CC_MSG ? ( // CC Message msg2 == EXPRESSION ? ( msg3 ? ( e0 = 0; // expression > 0 killed ? ( killed = 0; i = minnote; loop (notescount, vel = notes[i]; vel ? ( // notes[i] = 0; midisend(mpos, NOTE_OFF | channel, i, 0); midisend(mpos, NOTE_ON | channel, i, vel); ); i += 1; ); ); ) : ( e0 = 1; killed = 1; ); ); ) : statusHi == NOTE_OFF ? ( notes[msg2] = 0; !e0 ? killed = 0; ) : statusHi == NOTE_ON ? ( notes[msg2] = msg3; !msg3 ? ( !e0 ? killed = 0; ) : ( killed = 0; ); ); !mute ? midisend(mpos, msg1, msg2, msg3); ); ); <e>
Giorgio Tommasini wrote: Anyway, we will try to fix it, as said, in a next release.
I am more and more using the Trumpet with good results, as I get some degree of training with it.
My workaround Reaper script does help a lot (Otherwise I could not use the trumpet), but it's not
perfect. From time to time I still get missing (unplayable) notes.
Any news regarding an update ?
Giorgio Tommasini wrote: We will try to fix it in a next release of all our Brass.
Any hope ... ?
Monthly reminder:
Still desperately waiting for this updated ...
Yet another monthly reminder:
Still desperately waiting for this updated ...
Anybody still alive round here ?!?!?
Yet another monthly reminder:
Any hope for a fix for this confirmed bug ?
Anybody still alive round here ?!?!?
Yes, we're alive, and working hard, Michael. But we have a scale of priorities. And we're going to respect them.
It will take time, but these minor bugs, or limitations, will be fixed, rest assured,
Thanks for the message. I suppose you do understand that I am (we all are) slightly alienated by the silence....
Staying confident...
Yet another monthly reminder...
Sad Face, still no solution?
This is quite unusual for this firm. They're usually rather fast on bug fixes :shock:
Yet another monthly reminder...
Monthly reminder for October 2018...

subset: commoncrawl


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William Raveis Advances in Massachusetts with 2nd Key Appointment
Posted by Joanne Paone — July 10, 2008
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Andrew Puhanic — The Globalist Report — December 2012
A new study published by the Center for the Study of Carbon Dioxide and Global Change has concluded that the impact of Global Warming on Earths natural resources is much less than predicted and is in fact responsible for improving plant growth and development.
The study argues that the United Nations has lied to the public about claims that current temperature levels and changing precipitation patterns are beginning to stress Earth's natural and agro-ecosystems by reducing plant growth and development.
The study also argues that the United Nations has misled the public with its claims that the world needs to drastically reduce the ongoing rise in the air's CO2 content because crops will fail, food shortages will become commonplace, and many species of plants (and the animals that depend on them for food) will be driven to extinction.
(To continue reading about the studies findings, click here)


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Have you ever seen Animal Planet's Cats 101 show about Savannahs? Have you received a phone call about a lost Savannah cat today? There is a lost Savannah cat, somewhat famous too, as she is the mother of Mondo, the lost cat from the Cats 101 show on Savannahs. Katie went missing from Lemon Avenue. If you have seen her, please call 619-469-9547. She needs medicine )-:
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A NARROW mountain lane has been damaged and turned into an "international playground" for 4x4s and satnav-guided lorries, angry villagers claimed yesterday.
Residents living at Allt-y-Badi near Llangollen say their quality of life has been "destroyed" after a route once used only by walkers became impassable through its popularity with heavy vehicles and off-roaders.
But, although the single-track has now been reduced to potholes and is passable only by 4x4s equipped with winches, residents hope a new website has finally helped resolve the situation.
Villager Sally Clark and her company director husband Keith fear Grade-II listed walls surrounding her 400-year-old property and lining the lane itself are threatened by the HGVs.
She said, "Until the last few years the lane wasn't used as a road. Before the 4x4s used it the road was a green lane.
"Most of it was covered by grass and it was used by horses and walkers and not by traffic.
"When the traffic comes it breaks up more of the road surface and it's churning up the banks at the side and, when there is heavy rain, everything gets washed up and blocks up the drains near our properties. We get silt, mud and debris filling up the street."
She often spends hours helping lorry drivers negotiate their way out after becoming wedged in the lane.
Describing the thoroughfare as an "international playground", she claimed excess water washes eroded materials down the length of the lane onto neighbouring land.
Mrs Clark, 56, finally lost patience on February 14, and turned to fixmystreet.com after a 20-tonne lorry was misdirected down the route by satnav.
The website encourages residents to publicise gripes about local problems online.
It automatically forwards the complaint to the relevant local authority – Denbighshire County Council in Mrs Clark's case.
Writing about the incident on the site, Mrs Clark said, "The police were called and expressed their 'concern' that after two years the problem remained unsolved. I am appalled by the attitude of Denbighshire County Council."
Around four residents live along the lane and one, who did not wish to be named, claimed the local authority had now been "embarrassed" into action by the complaints on fixmystreet.com
They said, "The complaints have been on-going and we have written letters before to the council and they had always been quite dismissive.
"In the past we've been fobbed off by being told things like, 'There's nothing we can do' or 'Maybe we can do something'."
A spokesperson for Denbighshire County Council said, "We are aware of the concerns of some local residents. Allt y Badi is a rural country lane that is unsuitable for large and heavy vehicles. Unfortunately, a number of lorries that are fitted with GPS systems have been using the road to travel from Llangollen to Glyn Ceiriog.
"The council has already erected a sign which warns motorists that the road is unsuitable for heavy vehicles. It has also contacted a number of GPS companies who have now amended their route records and should result in less motorists using the road. The council also intends to upgrade advisory signage in the area."


text: Avenger Assembled Posted January 4 "Hmph." Watchdog made a noise slightly muffled by her helmet, Ashley doing her best to hold back the surreal feeling of hysteria she'd felt ever since she'd realized Danica had just walked straight into the agency command post. "Caught them off-guard," she finally growled, after pushing through what sounded like a faint choking sound. "...did the job, didn't it?" Judy frowned, then murmured with a little more edge than she usually used with her sister, "Ah don't think talkin' like you were gonna shoot people is helping any!" "We'll talk about this later," said Ashley, coughing slightly as her tone grew more reassuring. "You know I wasn't going to shoot those people." Well maybe Graves and his stupid idea of using the damn Claire's as the command center! Share this post Link to post


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Fitness can be the other "f" word. It's difficult, hard and requires personal discipline which is something not a lot of people have these days. Fitness doesn't mean that you have to live in a gym or eat nothing but salad. This article will show you some great easy ways to incorporate fitness into your life.
If you want to get in shape but don't have money for a gym membership or fancy exercise equipment, don't fret. There are plenty of exercises you can do without needing any of this. You can run, walk, do sits or push-ups. Don't let a lack of money get in your way of looking and feeling great.
A great way to stay fit is to take periodic pictures of yourself. By looking at pictures of yourself, you'll be able to track your progress and they'll also keep you motivated. You can also share these pictures with others to help motivate them in their fitness goals.
You can increase the likelihood that you achieve your fitness goals if you plan out and stick to a daily routine. By doing the same things at the same times, you will fall into a pattern that is more difficult to break than it is to keep. By doing this your fitness routine will become second nature.
If you are looking to get physically fit, try a Zumba class. Zumba is a very popular new class offered in most gyms across the country. It incorporates Latin dance with aerobic moves. Even non-dancers enjoy the movements and everyone who tries it, comes out of the class burning hundreds, if not thousands, of calories.
Investing into a set of free weights for your household can help you a lot. If you don't have time to go to the gym then you can work at the start or end of your day. Free weights around the household can also give you more motivation to workout to your true potential because you don't have a bunch of strangers staring at mk677 you.
If you are a regular runner make sure that you replace your running trainers at least every 500 miles. If you don't replace your trainers when the soles have worn out, you will be running in an unstable trainer which could lead to knee injuries and ankle pain. A worn out shoe sole can also cause the bottoms of your feet to be covered in blisters and hard crusty callouses.
Make sure you're using the proper forms when you're exercising. If you try to cut corners, you're only cheating yourself. Cutting corners means you're not going to get the full benefit of your workout. Not only that, but you risk injuring yourself and putting your fitness goals in the toilet.
Make sure to replace your workout shoes after a while to avoid having major knee injuries. It is generally suggested that you determine an expiration date of sorts on your workout shoes. To calculate this, figure that shoes generally last for about 500 miles. Take the number 500 and divide it by your weekly mileage to see how long your shoes should last.
When you need shoes for working out, be sure that they fit properly. Go shoe shopping in the evening time. When it is late in the day, your feet are the largest. When trying on the shoes, be sure that you can wiggle your toes and that you have about a half inch of space between your longest toe and the shoe.
Most people will feel extremely tired or exhausted after a workout. You will most likely want to take some sort of pain killers like Advil or aspirin. Scientists have shown that this actually has little to no effect on relieving pain is no more helpful than a placebo.
Whether you are maintaining your health or trying to shed some weight, it is important to keep your mind focused only on the positive aspects of your fitness program. Do not view your daily job or rigorous training session as a punishment; you will start to resent it early on. Instead, try to visualize your fitness routine as a privilege and a support system in your journey.
Running is one of the best ways to get fit. While running, there is a simple formula you should follow regarding the speed and pace of your run. You should start out slowly, then in the middle, you should increase speed, then at the end run as fast as you can.
As you get older, you need to devote more time to stretch during warm-ups and cool-downs. Your body's muscles become more stiff as you age. This is irritating but unavoidable. Fortunately, you can take steps to counteract the effect by holding each of your stretch positions a little longer, as you get older.
Stay limber by stretching often, and if you are getting older, hold your stretches for longer periods of time. Your muscles will remain warm, strong and loose, and you will be able to work out more vigorously. Stretching can also help reduce or prevent soreness of the muscles and increases flexibility.
Being physically fit is a personal choice. It doesn't mean that you have to look like a body builder or guzzle down protein shakes by the liter. As this article has pointed out, there are some great, easy and simple ways that you can be much more fit and healthy on a daily basis.


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LOBSTER Workshop on Monitoring and Measurement
This workshop will present the pilot Internet monitoring infrastructure being developed in Europe by the LOBSTER project. It will show how passive monitoring can be utilised on multi-gigabit links, outline anonymisation techniques, and demonstrate the new applications developed for characterising traffic. It will also look more widely at the challenges of high-speed passive monitoring, and the research possibilities in this area.
|Tuesday, May 16 from 14:00 to 15:30
|Archived streams are available per presentation.
|Kevin Meynell TERENA (NL)
Presentations in this session:
|LOBSTER: Large Scale Monitoring of Broadband Internet Infrastructure
|Evangelos Markatos
|(FORTH-Foundation for Research & Technology Hellas )
|Streamline - an Efficient Stream-processing Framework
|Herbert Bos
|(Vrije Universiteit Amsterdam)
|Ruler: Language-based Anonymisation
|Herbert Bos
|(Vrije Universiteit Amsterdam)


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PKF Cooper Parry
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Whether it's about who you're getting married to, what is his education, why is your skin looking like that. who have regular problems because all of us have them. I think it's important for me.
People fled the countryside because of the association of the area with the absence of prospects of receiving an education, unemployment. However, in the past few years, Italy has witnessed an.
May 13, 2019 · There are numerous reasons why post-secondary education is important, though this often comes down to requirements for employment in various industries and professions. In some professions, this level of education may be required to gain certification or licensure, which is often required to work in these professions.
Educational Methods and Theories / History of Italy / Education / Renaissance. 3 main reasons why education is important? many people wants education to enhance their knowledge in their career.
While acknowledging this conversation is important, many science. inclusive practices enhance student learning and decrease social disparities in STEM education, ultimately supporting STEM.
IEPs Are Important Because Special Education Students Are Important. The importance of an IEP cannot be understated. IEPs are education plans developed specifically for a special needs child by a team of professionals and family members. IEPs are unique in that they focus on a child's strengths, include measurable goals— including non-academic goals—.
They will cover why simple self-care, such as getting enough sleep. alongside relationships and sex education. "It will help children learn how to look after themselves, physically and mentally,
Finding a Job. Although a bachelor's degree is not necessary for getting some positions, it can still be an important tool for gaining employment. When two job candidates have equal qualifications, an employer is often more likely to choose the candidate with the college degree. In addition, even in job where a college degree is not relevant,
HP's education Chromebooks aren't the flashiest computers, but they're hugely important to the schools and thousands of students who use them. That's why it's good to see. Image: HP Also getting an.
In college, it can be hard to remember due dates, assignments and tasks for each class when you don't have the same classes every day or professors continually reminding you of important dates.
From where I sit, I've seen careers blossom by getting involved with. to my professional journey? Why or why not?" Pay attention to what you can start to bring forth that you may have been.
Apr 30, 2013 · Community Centered: 23 Reasons Why Your Library Is the Most Important Place in Town. by Julie Biando Edwards, Melissa S. Rauseo, & Kelley Rae Unger on April 30, 2013
The standard deviation is a commonly used statistic, but it doesn't often get the attention it deserves. Although the mean and median are out there in common sight in the everyday media, you rarely see them accompanied by any measure of how diverse that data set was, and so you are getting.
Funds raised at the four-day exhibition will be handed over to Dubai Cares 'Bookings 2030' campaign which will ensure quality.
Feb 07, 2015 · Well I already had written this answer earlier somewhere but this answer makes more relevance here. Because of the following reasons one should read. 1)It improves Focus and Concentration. When you read , all of your attention is focused on.
A Video Community for Why Open Education Matters. Create a short video that explains the benefits and promise of Open Educational Resources for teachers, students and schools everywhere.
Bibb County Alabama Board Of Education A Bibb County. be for education and conservation," UWA President Ken Tucker said. The land will be tentatively named UWA's Center for Biology and Conservation Field Study. The executive committee. May 24, 2019 · Bibb County High School is proud to be able to offer the 2ND Annual Ben Jones football camp. Ben is a former
Places like Harvard, she says, are "already so difficult to get into, and legacy admissions just make it. The reality is.
Finn could get hurt. That's why we put the option in episode two to call her again and follow her on Facebook, in case you.
Second Grade Social Studies Standards Teachers Having Affairs With Students "We can do universal child care for every baby zero to 5, universal pre-K, universal college and knock back the student loan. Have a tip, story, suggestion. "When Boston Public Schools moved to close the West Roxbury Education Complex last year, students and teachers from the Urban Science Academy and
There are however several important steps. budget There's no getting around it: college is expensive! Today's parents know this, which is why many will start saving when their kids are young to.
Why is college important? Education holds the key to your child's future. Education can help your child reach his/her life goals and dreams. Education will help your child choose what he/she wants in life. Here are 10 key reasons why going to college is. job, maintain a job, and get.
What is media literacy, and why is it important? Get expert advice and tips from Common Sense Media editors.
Now, the research arm of the federal Education Department says that getting a students' name right is essential to ensure they're getting the services they need. A recently released guide from the.
Education is very important for our lives. Without education people wouldn't have their high-developed machine and would believe that the Earth is a planet around that all another planets are moving. Why people want to have their high education? As for me I have my reasons why I want to get high education. First reason I want to get high education is for career purpose.
This is about getting ready for the next level faster and more efficiently. Both of my parents went to college. My mom got.
And do not get me wrong, I am very proud of my achievements. Where is the Harvard Business School equivalent for emotional education? Why shouldn't you prepare for emotional conflicts and.
Best University For Mass Communication They have no memory of alleged weapons of mass destruction. faculty members and staffers at UT Austin who work with. Chief Leo Ekpenyong, a Post-Graduate student of the Mass Communication Department of Baze University. in line with global. May 03, 2019 · To train and groom the next generation of media leaders, the Bennett University by
International education is probably the most important thing. It's about foreign languages, it's being able to work and travel in other countries, it's being able to understand what other people are saying, it's about listening, which is very important as we move forward in a much more complex world.
Why Getting a College Degree Is So Important? With tuition fees getting higher and student loans putting a heavier toll on future Masters and Doctors, more and more students choose to seek full-time job opportunities after high school, without enrolling into college.
Importance Of Adult Education In India Importance of Education Speech 2. Good morning to the Excellencies, my respected teachers and my dear friends. I would like to speech over importance of education at this great occasion in front of you. Education is very important to all of us. Our parents make us learn many things at home and then send us
Jan 24, 2014 · 10 Reasons Why Arts in Education Is so Important for Kids. 3. Motor Skills. This applies mostly to younger kids who do art or play an instrument. Simple things like holding a paintbrush and scribbling with a crayon are an important element to developing a child's fine motor skills. According to the National Institutes of Health,
Corey Alexander (Apr 14, 2014 7:51 PM) – Read by: 13 Why was education so important to African-Americans? Prior to the end of slavery many slaves were still illiterate. After President Abraham Lincoln issued the Emancipation Proclamation African Americans in slave states were considered free.
Kingwood College Continuing Education The Dallas County Community College District (DCCCD) is a network of seven community colleges in Dallas County, Texas (). It is headquartered at 1601 S. Lamar St. in Dallas.The Colleges of the DCCCD serve more than 70,000 students annually in academic, continuing education and adult education programs. The Colleges of Dallas County Community College District
But, have we stopped to think about why it's so important to get into the "right" schools? Yes, some schools have better academic rigor than others. That's clear. You will get a better education at.
I previously wrote about tips and tricks you can use to build a daily routine that works for you. But that article doesn't wholly address the question of why having a daily routine matters in the first place. Successful people are sticklers for routines, but why is that?
as well as the importance of health, education and bullying prevention. The Dayton Valley Dog Park will be hosting their annual Barking Lot Sale tomorrow, Saturday, May 18, complete with a slew of.
Why are they willing. Japan.well, you get it, will have to chance to see one another again or meet fellow exchange.
Maybe it's just that naturally smart people tend both to get a lot of education and to earn a. even when you control for income. That's why keeping its price low and enrollment accessible is so.
Jan 02, 2018 · Why It's So Important to Negotiate Your Starting Salary Many job seekers make this common mistake.
I'll explain why SSL certificates. Despite this important component of SSL, many end-users and organizations have delayed their adoption due to the price of the certificates and the complexity of.
A Look Under the Microscope Nutritional magnesium is deeply involved in energy production, oxygen uptake, central nervous system function, electrolyte balance, glucose metabolism and muscle activity, including that all important muscle—the heart.
Supporting HIV & AIDS education is one of FACT's most important missions. This area will shortly include detailed information about prevention of infection, how and where to get confidentially tested in the Greater Lehigh Valley area, and links to information on how to live and cope with HIV.
Even though parents still are shy to discuss sex with their children, here is why it is important. Sex is definitely one of the most thought about subject but the myths related to it make us feel bad.


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Summer is music festival season, which means fun in the sun, good music, and festive summer drinks in hand, and nobody knows how to make a fun summer cocktail like BACARDÍ Rum.
BACARDÍ BAY is popping up at music festivals all over the country, giving concert-goers an experience like no other with its Latin-Caribbean oasis right in the middle of the action.
BACARDÍ BAY is the place to be. Decked out with tropical prints, games, Rum bars, and photo ops galore, BACARDÍ BAY is the perfect place to gather with friends, take in the festival and sip on an amazing rum cocktail. Whether guests want to hang out, dance or experience the #SoundofRum, the BAY is bringing music lovers together and encourages them to "Do What Moves You."
The festival pop ups incorporate the brand's Do What Moves You elements into the programming. These campaigns integrate music and dance (this summer's Pass The Beat campaign features a new track, Make it Hot, from Major Lazer and Brazilian singer Anitta) and drive a strong connection between all of the festivals and the programming happening throughout the summer.
BACARDÍ El Coco
• 1 part BACARDÍ Límon Rum
• 1 part orange juice
• 1 part cranberry juice
• 1 part ginger-ale
• 1 part coconut water
• Garnish with lemon wedge
• Directions: Mix ingredients over ice
BACARDÍ Summer Sounds Cocktail
• 1 part BACARDÍ Superior Rum
• 3 parts pink lemonade
• 3 parts soda water
• Garnish with 6-8 mint leaves
• Top with 1-2 raspberries
• Directions: Mix ingredients over ice
BACARDÍ BAY was on site at the Cavendish Beach Music Festival on Prince Edward Island from July 5-7. The multi-day concert featured 51 musicians, including headliners Carrie Underwood, Eric Church and Hunter Hayes, and brought in more than 60,000 people.
While they danced and sang along to the country music, P.E.I. concert-goers sipped on BACARDÍ BAY cocktails like BACARDÍ Summer Sounds Cocktail (made with pink lemonade) and BACARDÍ El Coco (made with coconut water). Despite some rainy weather, many attendees have happily attended the outdoor concert for the last 11 years.
BACARDÍ BAY was also a hit at FVDED in the Park in Surrey, B.C. from July 6-7, when more than 40,000 hip-hop and electronic music fans gathered at Holland Park.
Next up, you can find the BACARDÍ BAY at Osheaga Music Festival in Montreal, Que., from August 2-4, and at ÎleSoniq in Montreal on August 9 & 10.
Can't make it to these music festivals this year? Why not pick up your favourite BACARDÍ Rum and try out on of the BACARDÍ BAY cocktails yourself.
BACARDÍ Coconut & Pineapple
• 1 part BACARDÍ Superior Rum
• 4 parts pineapple juice
• Garnish with lemon wedge
• Directions: Mix ingredients over ice
BACARDÍ Cuatro & Soda
• 1 part BACARDÍ Anejo Cuatro
• Soda water
• Garnish with lime wedge
• Directions: Mix ingredients over ice
BACARDÍ Oakheart & Ginger
• 1 part BACARDÍ Oakheart Spiced
• 4 parts ginger-ale
• Garnish with lemon wedge
• Directions: Mix ingredients over ice


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Hello, I recently bought the box, with a key for F-Secure SAFE. I got home, registered and followed the box's instructions correctly. But I still get the message saying that my key is invalid, after writing the campaign name and the code.
Product: F-Secure SAFE, 1 year for 5 devices.
Campaign name: ccity
Key: *****-*****-*****-*WAHB
EDITED: Misunderstanding
According to the Chat Contact Page Global English support hours are Mon-Fri, 6-16 (GMT), which I assume means 6am - 4pm.
Sorry, but I don't think you'll get a response from F-Secure here before the morning, as they're not usually around at weekends.
Hi Nis,
Sorry to hear about your trouble!
If you have tried to register using the correct and complete campaign code and the key using the box instructions, and if the issue persists, I would recommend to contact our support through a support request or chat with our Experts.
They might have to check your key and the campaign code and assist you further on the installation.
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Search Results for 'Jen Randall'
2 results found.
Ireland's only adventure film festival comes to Galway
LEO HOULDING, whose audacious ascents on epic mountain faces, documented in award winning films like The Asgard Project, The Wildest Dream, and The Last Great Climb, is coming to Galway for the Why Not? Adventure Film Festival.
Banff Mountain Film Festival
THE BANFF Mountain Film Festival, a feast of documentaries from the global mountain and sports community, celebrating a love of the outdoors and the spirit of adventure, returns to Galway.


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Roy Batty
Daily Stormer
March 22, 2019
These days, no one ever knows if "hate crimes" are real or Jussie Smollett type hoaxes. Minorities are trying to get sympathy by claiming people hate them, while actually, people do hate them.
This one is a bit suspicious, you have to admit.
Mosques have been targeted in overnight attacks in Birmingham, police have confirmed. Counter-terrorism officers are investigating the incidents, but no motive has been attributed.
An initial attack was reported at a mosque on Birchfield Road 2.32am local time and, as officers were responding, additional reports were received of a man smashing windows at another mosque on Slade Road at approximately 3.14am local time.
Police were dispatched to patrol the streets citywide in an effort to prevent further destruction, and patrolling officers discovered damage at other places of worship in the Witton Road, Aston Broadway areas of the city.
First off, I've never heard of a Christian sledgehammer attack before.
If this really happened, then it's a first.
The location of the crime is also suspicious: Birmingham.
There are no White people left in Birmingham, to my knowledge. None with any fight left in them, at least.
This attack will almost certainly work towards the benefit of Moslems.
For the low, low cost of replacing a few broken windows, it will justify the arrests of another dozen Boomers who wrote naughty things on Facebook. And it will mean more cops being sent to sensitivity training and more money spent on neighborhood outreach (bribing tribal chieftains and imams to prevent them from letting their youths riot in the streets) and more White people bashing in the media.
Worse isn't better in this instance.
Every single time a prank happens IRL we need to soberly assess whether it helps the situation or hurts it. Whether it was a false flag or not. Blanket statements like, "the worse, the better" do not help us get to the bottom of anything and should be avoided.
I tell you what though, if they fail to find a culprit for this crime, you'll know that the Moslems did it themselves.