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Despite its being a leading cause of nosocomal and community-acquired infections, surprisingly little is known about Staphylococcus aureus stress responses. In the current study, Affymetrix S. aureus GeneChips were used to define transcriptome changes in response to cold shock, heat shock, stringent, and SOS response-inducing conditions. Additionally, the RNA turnover properties of each response were measured. Each stress response induced distinct biological processes, subsets of virulence factors, and antibiotic determinants. The results were validated by real-time PCR and stress-mediated changes in antimicrobial agent susceptibility. Collectively, many S. aureus stress-responsive functions are conserved across bacteria, whereas others are unique to the organism. Sets of small stable RNA molecules with no open reading frames were also components of each response. Induction of the stringent, cold shock, and heat shock responses dramatically stabilized most mRNA species. Correlations between mRNA turnover properties and transcript titers suggest that S. aureus stress response-dependent alterations in transcript abundances can, in part, be attributed to alterations in RNA stability. This phenomenon was not observed within SOS-responsive cells.
Staphylococcus aureus is a leading cause of nosocomial and community-acquired infections. The organism owes its ability to cause disease, in part, to the production of a repertoire of virulence factors that modulate its ability to colonize host/inert surfaces, thwart host defenses, and disseminate to satellite sites (6, 39). Another facet of S. aureus pathogenesis is the organism's ability to maintain cellular homeostasis while enduring environmental challenges, such as changes in host cell core temperature or exposure to phagocyte-mediated reactive oxygen species (58). Although considerable progress has been made in defining S. aureus virulence factors and their regulatory networks, surprisingly little is known about the organism's ability to cope with environmental stresses.
Studies from Escherichia coli and, to a lesser extent, Bacillus subtilis indicate that bacteria have developed highly orchestrated responses to environmental stresses, which when elicited alter the organism's cellular physiology in a manner that enhances survival. For instance, DNA-damaging agents trigger induction (derepression) of the SOS response (reviewed in reference 16). Components of the SOS response increase the cell's capacity to inhibit cell division, repair DNA damage, and replicate noninstructive DNA lesions in an error-prone manner. Bacteria cope with conditions of nutrient limitation by eliciting the stringent response, which both reduces the cellular protein synthesis capacity and increases amino acid biosynthesis when substrates for protein synthesis are lacking (42, 52). Products of the cold shock response restore translation apparatus function, which is compromised at low temperatures, and resolve low temperature-mediated mRNA secondary structures that would otherwise impede the translation machinery (25, 26). At elevated temperatures, cells express heat shock factors that degrade/restructure heat-denatured proteins as well as factors that restore temperature-mediated alterations in chromosome topology (33, 61). It is currently unclear if S. aureus elicits similar responses and/or has developed novel strategies to cope with DNA damage, starvation, and temperature changes.
Recent studies indicate that bacterial stress responses are not merely controlled at the level of transcript synthesis. Rather, some responses modulate target mRNA stability to influence protein production (reviewed in reference 53). Perhaps the best example of this involves production of the major E. coli cold shock protein, CspA, an RNA structure-resolving protein that accounts for 13% of the total cellular protein at low temperatures (18). Under cold shock conditions, increased cspA mRNA stability, as opposed to changes in transcript synthesis, primarily accounts for the amount of CspA produced (14). Similarly, Klebsiella pneumoniae nitrogen fixation protein production corresponds to regulated mRNA turnover (10, 28, 29). Moreover, in Vibrio angustum, the cellular response to nutrient depravation is regulated by altering mRNA stability (53).
The focus of the current work is to define the members of S. aureus stress responses and their mechanisms of regulation. Doing so may provide a better understanding of the organism's ability to adapt to environmental challenges and provide novel strategies for the therapeutic intervention of bacterial infections. Accordingly, Affymetrix GeneChips were used to define the S. aureus SOS, stringent, cold shock, and heat shock responses and to measure the mRNA turnover properties of each response. The results indicate that each stress response influences the expression of distinct cellular processes, subsets of virulence factors, and antimicrobial resistance determinants. Many stress-responsive biological processes appear to be conserved across bacteria, whereas others are unique to S. aureus. Induction of the S. aureus cold shock, heat shock, and stringent responses caused dramatic global changes in mRNA turnover. This suggests that stress-mediated changes in mRNA abundances can, in part, be attributed to alterations in RNA stability as opposed to or in addition to changes in transcript synthesis. This phenomenon was not observed under SOS response-inducing conditions. Sets of S. aureus small stable RNA (SSR) molecules with no obvious open reading frames were also components of each stress response. Given the importance of SSR-like molecules in other organisms, it is likely that these stable RNA species influence stress-responsive functions (1, 51, 60).
Bacterial strains. S. aureus strain UAMS-1 is a well-characterized methicillin-susceptible clinical osteomyelitis isolate (2, 3, 5, 7, 46).
Growth conditions.Overnight cultures of UAMS-1 cells were diluted 1:100 in 200 ml fresh brain heart infusion (BHI) medium and were incubated at 37°C at 225 rpm with a flask-to-medium-volume ratio of 5:1. Once cultures reached mid-log phase (optical density at 600 nm, 0.25), they were challenged with either mupirocin (60 μg ml−1; AppliChem, Cheshire, CT) or mitomycin C (1 μg ml−1; Sigma-Aldrich, St. Louis, MO) and were subsequently incubated at 37°C for 30 min with aeration for induction of the stringent or SOS response, respectively. For induction of the cold shock and heat shock responses, cultures of UAMS-1 were grown to mid-log phase (as described above) and were subsequently incubated for an additional 30 min with aeration at 10°C and 42°C, respectively. Following induction of each response, rifampin (200 μg ml−1; Sigma-Aldrich) was added to arrest transcription, and 21 ml of cells was removed at 0, 2.5, 5, 15, and 30 min post-rifampin treatment. Twenty milliliters of each aliquot was added to 20 ml ice-cold acetone-ethanol (1:1) and stored at −80°C overnight; 10−1 and 10−5 dilutions of the remaining 1 ml were plated on BHI-rifampin (200 μg ml−1) agar and BHI agar, respectively. Plates were incubated overnight at 37°C, and viable CFU ml−1 were calculated to ensure that cell proliferation was halted by the addition of rifampin. If rifampin-resistant colonies were detected, the experimental samples were discarded, and the experiment was repeated.
Antibiotic susceptibility assays.Fifty milliliters of mid-log-phase UAMS-1 cells was either mock treated or challenged with mupirocin (60 μg ml−1) to induce the stringent response (as described above). Next, mock- and mupirocin-treated cultures at time zero (T0) were incubated in the absence or presence of either rifampin (200 μg ml−1) or ciprofloxacin (1.3 μg ml−1, which is the MIC) for an additional 4 h. Cell viability was monitored by determining the total CFU ml−1 at T0 and every hour thereafter. All susceptibility assays were repeated at least twice.
Microarray studies.Total bacterial RNA was isolated from each sample, labeled, and hybridized to Affymetrix S. aureus GeneChips (Santa Clara, CA) as previously described (46). The S. aureus GeneChips used in this study are the most comprehensive commercially available Affymetrix arrays, representing genomic sequences from S. aureus strains NCTC 8325, COL, N315, and Mu50 as well as intergenic regions. The experiment for each response was repeated twice (biological replicates), and posttranscriptional arrest samples were prepared from each biological replicate. GeneChip signal intensity values for each qualifier at each time point (both pre- and posttranscriptional arrest) were then averaged and normalized to Affymetrix spike-in signals, using GeneSpring 6.2 software (Silicon Genetics, Redwood City, CA). The half-life of each transcript was calculated as the time point at which the T0 signal decreased by a factor of 2, as previously described (46, 49).
Real-time PCR.Quantitative real-time PCR primers are shown in Table 1. Real time-PCRs were performed as previously described (46). Briefly, 25 ng of total bacterial RNA was reverse transcribed, amplified, and measured using a LightCycler RNA Master SYBR green I kit (Roche Applied Science, Indianapolis, IN) following the manufacturer's recommendations. As an internal control, 25 pg of RNA was used to quantitate rRNA. Transcript concentrations were calculated using LightCycler software, with a LightCycler control cytokine RNA (Roche Applied Science) titration kit as a standard, and were then normalized to the 16S rRNA abundance.
Cold shock response.To characterize the ability of S. aureus to adapt to changes in temperature, we first identified members of the organism's cold shock response. Accordingly, S. aureus strain UAMS-1 was grown at 37°C to the mid-log phase of growth, at which point cell cultures were incubated at 10°C for an additional 30 min to induce the cold shock response. Affymetrix S. aureus GeneChips were used to compare the transcript titers of cold-shocked and unshocked cells (grown at 37°C).
The cold shock condition studied did not appreciably affect cell viability (data not shown) but did increase the mRNA titers of 46 genes (Table 2). Transcription of the cold shock gene cspB was induced 9.3-fold at the lower temperature, confirming that the conditions used were appropriate for studying the S. aureus cold shock response. Transcription of the cold shock gene cspA was also upregulated 2.0-fold, but this was not considered significant by the t test (P ≤ 0.05). This correlates with E. coli cspA expression; cspA is strongly induced at 25°C but is marginally upregulated (at the transcriptional level) at a lower temperature (15°C) (57). The majority of cold shock-induced transcripts (36%) included hypothetical or conserved hypothetical genes; the latter are conserved within all publicly available sequenced S. aureus genomes. Two members of the cid regulon, lrgA and lrgB, which are believed to counteract the cell's programmed cell death machinery, were induced (23), as were four competence orthologs (SACOL0813, SACOL0814, SACOL1601, and SACOL1644). Several virulence determinants were induced during cold shock conditions, including two pathogenicity island genes (SACOL0901 and SACOL0902), an enterotoxin gene (seo), a lipase gene (lip), and a sortase gene (srtA). Two putative antimicrobial resistance determinants, mepA (27) and a beta-lactamase-like gene (yycJ), were induced by cold shock. The SOS repressor LexA (59) and a general stress-inducible protein (SACOL0958) that is predicted to bind mRNA were also upregulated at the low temperature. Real-time PCR confirmed that srtA and cspB transcripts were induced 2.2- and >1,000-fold, respectively, by cold shock conditions (data not shown). A total of 416 transcript titers decreased in response to low temperature (see Table S6 in the supplemental material).
Heat shock response.Next, we identified transcripts that are induced in heat-shocked UAMS-1 cells. To do so, mid-log-phase cells (grown at 37°C) were incubated for 30 min at 42°C to induce the S. aureus heat shock response. The transcript profile of heat-shocked cells was then compared to that of unstressed cells. Induction of the heat shock response did not affect cell viability (data not shown) but did induce the transcription of 98 genes.
As shown in Table 3, three well-characterized heat shock response genes, ctsR, clpB, and clpC, were upregulated 3.1-, 5.0-, and 2.3-fold, respectively, during growth at the elevated temperature, suggesting that the conditions tested are appropriate for studying aspects of the S. aureus heat shock response (12). Among the genes induced by heat shock were a number of putative S. aureus virulence factors, including (i) the alpha-hemolysin gene (hla), (ii) pathogenicity island genes (SACOL0900 and SACOL0901), (iii) an LPXTG motif-containing gene (SACOL2668), and (iv) members of the urease system (ureA-ureG), which are strongly upregulated in S. aureus biofilms (3). Thirty-six hypothetical or conserved hypothetical proteins were induced by heat shock conditions. Eleven cold shock genes were also induced within heat-shocked cells, indicating that they may constitute members of a general temperature-mediated stress response. Included among these were six conserved hypothetical genes, a pathogenicity island gene (SACOL0901), the MarR family regulator gene, and two competence orthologs (SACOL0813 and SACOL0814). Real-time PCR confirmed that clpC and ctsR were upregulated 65- and 95-fold, respectively, under heat shock conditions (data not shown). Forty-two transcripts decreased in response to heat shock conditions (see Table S6 in the supplemental material).
Stringent response.Mupirocin is an antimicrobial agent that induces the staphylococcal stringent response by inhibiting isoleucyl tRNA synthetase, thereby increasing the cellular concentration of uncharged tRNAIle molecules (8, 11). Using real-time PCR, we determined that 30 min of mupirocin treatment (60 μg ml−1) increased the transcript titers of the stringent response control factor relA and the ilv operon (3- and >1,000-fold, respectively), whereas rpsL transcript levels were decreased (5-fold); cell viability was marginally affected (data not shown). These results mimic the transcription profile for mupirocin-mediated E. coli stringent response induction and were the same conditions used by Crosse and colleagues to induce the stringent response in S. aureus strain 8325-4 (11, 47). Lower mupirocin concentrations did not appreciably alter the transcript titers of relA, ilvA, and rpsL, whereas higher concentrations increased toxicity.
Mupirocin treatment induced 248 open reading frame transcripts (Table 4). As expected, the stringent response element relA was upregulated (2.4-fold). Likewise, members of the ilv (ilvA-ilvD and ilvN), leu (leuA-leuD), and thr (thrB and thrC) operons were strongly upregulated by mupirocin challenge (30- to 157.1-fold); all have been shown to be responsive to isoleucyl tRNA limitation (47). Collectively, these results suggest that the conditions used were appropriate for studying components of the S. aureus stringent response. Eliciting the stringent response induced several classes of gene products, including (i) 37 transport proteins, (ii) 10 previously characterized virulence factors, (iii) 18 regulatory molecules, and (iv) 10 peptidases. The last class is a hallmark of the stringent response (22). Among the elevated transport proteins were three putative drug efflux pumps, i.e., NorA (10.1-fold), MepA (5-fold), and an EmrB/QacA-like protein encoded by the SACOL2413 gene (36, 64). Two loci, encoding a set of putative ABC transporter proteins (SACOL0504 to SACOL0506; average induction, 124-fold) and a set of oligopeptide transporter proteins (SACOL0991 to SACOL0995; average induction, 73-fold), were among the most dramatically upregulated transcripts in mupirocin-challenged cells. The virulence determinants of the stringent response included autolysin (alt), fibrinogen binding protein (fbp), sortase A (srtA), components of the intracellular adhesion locus (icaA and icaB), and extracellular proteases (sspA-sspC). Among the transcription factors that were upregulated were three well-characterized virulence factor regulators, i.e., sarR (3.2-fold), sarZ (8.7-fold), and a component of the agr locus (agrA; 3.1-fold). Real-time PCR demonstrated that the norA mRNA titer was induced 36.2-fold following induction of the stringent response (data not shown). A total of 814 transcripts decreased in abundance under stringent conditions (see Table S6 in the supplemental material).
SOS response.Mitomycin C is an antimicrobial and anticancer agent that causes DNA intra- and interstrand cross-linking as well as monofunctional alkyl lesions and is a potent inducer of the bacterial SOS regulon (41). To define the optimal conditions for induction of the SOS regulon, log-phase UAMS-1 cells were treated with 0.5, 1.0, 2.5, or 5 μg ml−1 mitomycin C for 30 min. Real-time PCR was used to compare recA and uvrB transcript titers at each drug concentration to those in mock-treated cells; recA and uvrB are well-characterized components of the SOS response (24, 32, 48). The cell viability of each mitomycin C-challenged sample was also compared to that of mock-treated cells. It was determined that 1 μg ml−1 mitomycin C induced both recA (2.0-fold) and uvrB (15-fold) transcription and simultaneously produced the least amount of toxicity (data not shown), suggesting that these conditions were appropriate for studying mitomycin C-mediated induction of the SOS response. Accordingly, log-phase UAMS-1 cells were treated with 1 μg ml−1 mitomycin C for 30 min, and transcript titers were compared to those for untreated cells.
A total of 73 genes were induced by mitomycin C challenge (Table 5). Among these were the genes for the SOS repressor protein LexA (4.6-fold), components of the nucleotide excision repair machinery, namely, UvrA (2.4-fold) and UvrB (4.1-fold), the single-stranded binding protein (ssb; 44.5-fold), and the recombination repair proteins SbcC (4.9-fold) and SbcD (4.3-fold), all of which are known members of the bacterial SOS response (13). Additionally, a umuC-like gene (SACOL1400) was dramatically upregulated (36.2-fold) by mitomycin C challenge; UmuC is a component of the E. coli SOS response that promotes replicative lesion bypass of noninstructive DNA lesions (44). Collectively, these results suggest that the conditions used were appropriate for studying the SOS system.
Among the SOS-induced transcripts were several bacteriophage replication/packaging genes, including those for N315 SA1791 (42.1-fold), which has homology to the replication initiation protein DnaB, a dUTPase (COLSA0357; 34.3-fold), putative phage tail components (SAV1954 [4.3-fold] and SAV1955 [5.8-fold]), and a small terminase protein (SACOL0906; 8.6-fold). Induction of these phage transcripts correlated with the dramatic SOS-mediated upregulation of a putative phage antirepressor gene, SAV1994 (42.5-fold). Two loci, each harboring a set of genes, were among the highest SOS-induced transcripts. They included members of a bovine pathogenicity island (SACOL0901 to SACOL0904; average induction, 12.5-fold) and a set of genes encoding conserved hypothetical proteins (N315 SA1767 to SA1780; average induction, 12.9-fold). A putative Holliday junction resolvase gene (rusA) was also dramatically upregulated (67.8-fold). These results were validated, in part, by real-time PCR, which demonstrated that uvrB was upregulated in SOS-induced cells (41.5-fold) (data not shown). RecA transcript titers saturated the microarray, and thus we could not accurately determine what, if any, effect mitomycin C challenge had on recA expression. Nonetheless, real-time PCR did indeed indicate that recA was upregulated (2.4-fold) in mitomycin C-challenged cells, as opposed to the case in untreated cells. As shown in Table S6 in the supplemental material, 453 transcripts decreased in response to SOS-inducing conditions.
Global effects of stress responses on RNA half-lives.As stated above, studies have linked stress response-mediated changes in protein production to alterations in target transcript mRNA stability, suggesting that modulating mRNA turnover plays a role in bacterial adaptability to environmental challenges. Admittedly, most of those studies have been limited to a few transcripts. Nonetheless, we set out to determine whether induction of the S. aureus cold shock, heat shock, stringent, or SOS response globally influences mRNA turnover. To do so, either log-phase UAMS-1 cells were mock treated or the corresponding stress response was induced (conditions described above). Rifampin was then added to inhibit de novo transcript synthesis, as previously described (46). Aliquots were removed at 0, 2.5, 5.0, 15, and 30 min post-transcriptional arrest, and cell viability and rifampin resistance were measured (see Materials and Methods). Total bacterial RNA was isolated, and the mRNA half-lives of transcripts produced in mock-treated, cold-shocked, heat-shocked, stringent response-induced, and SOS response-induced cells were determined using Affymetrix S. aureus GeneChips as previously described (46, 49).
The results (Fig. 1) indicate that log-phase transcripts are degraded rapidly within mock-treated cells; 89.7% of all transcripts had half-lives of ≤5 min, 206 transcripts (9.2%) had intermediate half-lives (>5 min but ≤30 min), and 25 (1.1%) RNA species were stable (half-lives of >30 min). These results are in agreement with previous studies using custom-made S. aureus GeneChips (Saur2a), which found that the half-lives of 89.6% of all UAMS-1 log-phase transcripts were <5 min, those of 9.5% of transcripts were intermediate, and those of 0.7% of transcripts were stable (>60 min) (46). Induction of the SOS response did not appreciably affect global RNA turnover properties, whereas induction of the heat shock, cold shock, and stringent responses appeared to dramatically stabilize RNA species (Fig. 1). Within heat-shocked cells, 38.5% of log-phase transcripts had half-lives of ≤5 min, 54.5% demonstrated intermediate rates of mRNA turnover, and 7.1% were stable. Similarly, the half-lives of transcripts in stringent response-induced cells were as follows: 42.9% were ≤5 min, 45.7% were intermediate, and 11.4% were stable. Cold-shocked cells had a unique RNA turnover profile, as only 0.7% of transcripts had half-lives of ≤5 min, while the majority (64.1%) had half-lives of between 5 and 30 min or were stable (35.1%).
One possible explanation for the observed stress-mediated increases in mRNA stability could be that rifampin is not active within heat-shocked, cold-shocked, and stringent response-induced cells. Indeed, most of these stress conditions induce transport functions and/or drug efflux pumps (stringent response; NorA and MepA). However, several lines of evidence suggest that the efflux of rifampin does not contribute to this phenomenon and that the antibiotic arrests de novo transcript synthesis under each stress condition. First, Williams and Piddock have shown that efflux inhibitors do not influence rifampin accumulation within S. aureus cells (63). Second, a large portion of the transcriptome is rapidly degraded (half-lives of ≤5 min) under both heat shock and stringent response conditions, suggesting that de novo transcript synthesis is arrested. Third, rifampin challenge has similar effects on unstressed and stressed cell proliferation at 2.5, 5.0, 15, and 30 min post-rifampin treatment (data not shown). Moreover, the results in Fig. 2 demonstrate that induction of the stringent response confers resistance to the fluoroquinolone ciprofloxacin, presumably via norA and/or mepA upregulation, but does not reduce rifampin susceptibility. More specifically, UAMS-1 viability was decreased 2,600-fold during prolonged exposure (3 h) to ciprofloxacin (Fig. 2A). In contrast, induction of the stringent response decreased UAMS-1 susceptibility to ciprofloxacin, resulting in a threefold reduction in cell viability (Fig. 2B). In contrast, induction of the stringent response had no measurable effect on S. aureus rifampin susceptibility; cells challenged with rifampin demonstrated dramatic reductions in cell viability, in both stringent response-induced and noninduced cells (compare Fig. 2A and B). Finally, these results fit directly with studies of other organisms (53). Collectively, these results suggest that the stringent, cold shock, and heat shock responses influence molecular components that influence mRNA turnover in S. aureus cells.
The global increases in mRNA stability in stringent response-induced, cold-shocked, and heat-shocked cells could be explained by two other scenarios, as follows: (i) these stress conditions induce/activate cellular RNA-stabilizing capacities or (ii) the conditions repress RNase production/function. Interestingly, we found that homologues of putative B. subtilis RNase genes (15 genes) are positively regulated by the alternative sigma factor σB (P. M. Dunman, P. D. Olson, and K. L. Anderson, unpublished data). However, as shown in Fig. 1, no global differences in mRNA turnover were observed between UAMS-1 sigB+ and UAMS-1 sigB mutant cells. Thus, it is likely that stress-induced σB-dependent alterations in RNase expression do not account for differences in mRNA stability.
Correlation between stress responses and target transcript stability.Table S6 in the supplemental material lists all loci represented on the S. aureus GeneChip that were up- or downregulated by each stress response and the RNA half-lives of these transcripts under each stress condition as well as in mock-treated cells. A comparison of stress-mediated changes in transcript titers and their corresponding RNA turnover properties indicated that stress response-dependent alterations in transcript abundances can be attributed, in part, to alterations in RNA stability. In other words, induction of a stress response appears to alter both transcript synthesis and stability, suggesting that modulating RNA turnover may be an important component of the ability of S. aureus to cope with environmental challenges.
More specifically, as shown in Table S6 in the supplemental material, 164 of the 277 stringent response-induced transcripts are also expressed in mock-treated cells. A comparison of their RNA half-lives indicated that 147 (89.6%) of these transcripts are more stable when the stringent response is elicited than in mock-treated cells. Similarly, 65 heat shock-induced transcripts were also detected within mock-treated cells. Sixty-three (96.9%) of these transcripts were more stable in heat-shocked cells than in mock-treated cells. Most (56%) cold shock-induced transcripts were not detected in mock-treated cells, and thus their RNA half-lives could not be compared. The remaining 28 transcripts were more stable under cold shock conditions than in mock-treated cells. Sixty-two SOS-induced transcripts were detected within mock-treated cells. A comparison of their half-lives found that five (8%) SOS-induced transcripts were more stable in mitomycin C-stressed cells than in mock-treated cells. Real-time PCR was used to validate these results in part. As shown in Table 6, GeneChip-based RNA turnover measurements correlated with real-time PCR-determined RNA half-lives for each mRNA species analyzed.
We also investigated whether stress response-mediated decreases in transcript titers correlated with increased rates of mRNA turnover. Although induction of the stringent response globally increased RNA stability (Fig. 1), 20% of the stringent response-downregulated transcripts were degraded by the first posttranscriptional arrest sampling time (half-lives of ≤2.5 min) or were more rapidly degraded under stringent response conditions than under mock treatment conditions (see Table S6 in the supplemental material). Forty-three genes were downregulated within heat-shocked cells. Of these, 8 transcripts were not detected under heat shock conditions, and a comparison of mRNA half-lives of the remaining 35 transcripts indicated that 3 (8%) were less stable within heat-shocked cells or had half-lives of ≤2.5 min. No cold shock-repressed transcript demonstrated more rapid turnover under cold shock conditions. RNA turnover was more rapid under SOS-induced conditions than in mock-treated cells for 15% of the SOS-repressed transcripts.
Stable RNA species.We have previously shown that log-phase UAMS-1 cells produce a set of SSR (half-lives of >60 min) molecules that are not expected to code for protein products (46). Given the importance of the S. aureus agr-encoded RNAIII molecule and small noncoding RNAs within other pathogens, it is likely that many of these molecules play an important role(s) in S. aureus biological processes. As shown in Table S8 in the supplemental material, 126 stable transcripts (half-lives of >30 min) that map to short S. aureus intergenic regions but have no defined function were identified to be produced under mock and/or stressed conditions.
Most SSRs were found to be stress-responsive; 12 small stable RNAs were produced in untreated cells, whereas 90% of SSRs were produced in response to stress. More specifically, two RNA species were stable under all conditions examined. Six transcripts were stable under four of the five conditions. Five transcripts were stable under three conditions, with the majority of these (4) being stable under stringent response, cold shock, and heat shock conditions but having half-lives of between 2.5 and 15 min under mock treatment and SOS-induced conditions. Eleven RNA species were stable under two conditions studied, and 102 transcripts were stable under one condition. Based on the surrounding genomic content and directionality of each SSR, it is likely that some stable RNA molecules are cotranscribed as part of an operon, whereas others are more likely to behave as antisense RNA molecules.
Infection is a dynamic process, during which the invading organism is subjected to an array of environmental challenges. Bacteria have developed highly orchestrated processes to respond to environmental stresses, which when elicited alter the cellular physiology in a manner that enhances the organism's survival and its ability to cause disease. Much effort has been devoted toward defining the members of bacterial stress responses by identifying genes whose transcript synthesis is controlled in a stress-specific manner. Only recently has it become recognized that modulation of mRNA degradation is a highly regulated process that also plays an essential role in many stress responses.
Despite S. aureus being a leading cause of nosocomial and community-acquired infections, surprisingly little is known about S. aureus stress responses. Here we have used Affymetrix S. aureus GeneChips to define transcript species that are altered under cold shock, heat shock, stringent, and SOS response-inducing conditions. In addition, we have defined the mRNA turnover characteristics of each response and identified a set of small stable RNA molecules with no obvious open reading frames that are produced as a component of each stress response.
Collectively, our results suggest that S. aureus stress response-dependent alterations in transcript abundances can be attributed, in part, to alterations in RNA stability. This was especially true for conditions of heat shock, cold shock, and stringent response induction, where most (89 to 100%) stress-induced transcripts had increased stability compared to those in untreated cells. Admittedly, it is not yet clear what, if any, effect the observed alterations in transcript stability have on protein production. However, studies of other organisms suggested that modulation of RNA turnover directly influences protein abundance. Thus, it seems likely that the ability of S. aureus to modulate mRNA turnover in a stress-responsive manner correlates with changes at the protein level. Nonetheless, currently it would be premature to interpret the effects of stress-mediated mRNA stabilization on the cellular physiology of S. aureus cells, simply because we do not know whether increases in transcript stability increase or decrease protein production (of all or subsets of mRNA species). However, our expression data do provide many insights about how S. aureus copes with various types of stress.
In general, our results suggest that, like the case for other bacteria, S. aureus stress responses are distinct, but response members do overlap. Moreover, there is a high degree of similarity between the ways that different bacteria cope with environmental stresses. For instance, induction of the S. aureus cold shock response profoundly stabilized most RNA species, increased the transcript titer of the SOS repressor LexA, and decreased expression of the stringent response control factor relA. This suggests that cold shock conditions repress both SOS and stringent responses. Studies have demonstrated that cold-shocked E. coli cells behave similarly, as low temperatures increase RNA stability and reduce RelA activity (61). Although low temperature appears to repress the stringent response, 27 S. aureus cold shock response genes were also components of the stringent response (see Table S6 in the supplemental material), indicating that they may represent members of a generalized stress response. Included among these 27 genes was the cold shock factor cspB. In E. coli, CspB and the major cold shock protein, CspA, are believed to act as RNA chaperones, although their RNA binding specificities differ (25, 43). Transcription of CspA was only marginally induced in cold-shocked cells, yet cspA mRNA was profoundly stabilized during low-temperature growth (half-life of <2.5 min at 37°C versus >30 min at 10°C), which based on E. coli cspA studies, suggests that CspA production was increased. Indeed, preliminary proteomics studies indicated that CspA levels are dramatically increased within cold-shocked UAMS-1 cells (S. Slater and P. M. Dunman, unpublished). Interestingly, cspA mRNA was also significantly stabilized in stringent response-induced cells (see Table S6 in the supplemental material), with an intermediate half-life. Given that cspB expression and cspA mRNA stability correlate with decreased mRNA turnover within both cold-shocked and stringent response-induced cells, it is conceivable that CspB and/or CspA may directly modulate transcript stability.
Induction of the cold shock response primarily increased the transcription of genes with no previously determined function. These gene products may play a role in rescuing stalled ribosomes, which is a requirement for cellular survival at low temperatures. Cold shock conditions also induced the expression of a protein with an S1 RNA binding domain, which is thought to mediate single-stranded RNA and RNA-pseudoknot binding (45) and may contribute to RNA stability at low temperatures. As shown in Fig. 3A, the predominant effect of cold shock conditions was the general decrease in mRNA titers involved in most cellular functions, despite globally increasing mRNA stability. This suggests that low temperatures promote basal S. aureus gene expression, but because RNA species are stable, templates for translation are available for the cell to efficiently respond to changes in growth conditions without having to expend energy for de novo gene expression.
Heat shock conditions induced clpB and clpC transcription. ClpC is proposed to play a role in targeting heat-denatured proteins for degradation by ClpP (15). ClpB is believed to interact with DnaK and other heat shock proteins to mediate solubilization of protein aggregates (19, 37, 38). Although neither clpP nor dnaK transcripts were induced by the heat shock conditions studied, their transcripts were stabilized at the higher temperature (data not shown). The upregulation of clpB and clpC suggests that, like the case for other bacteria, coping with aberrant proteins is a vital component of S. aureus's ability to endure elevated temperatures. The urease operon (ureA-ureG), whose gene products convert urea to ammonia and CO2, was also upregulated at the high temperature. The thermodependent protein denaturation properties of urea have been well documented. Thus, it is conceivable that heat-shocked cells may guard against urea-based protein denaturation by metabolizing endogenous urea. As shown in Fig. 3B, amino acid biosynthetic genes are in the largest class of genes that are upregulated at elevated temperatures, suggesting that replenishing substrates for protein synthesis is also a priority under heat shock conditions. Paradoxically, the heat shock repressor protein CtsR was also upregulated under heat shock conditions. Frees and colleagues observed this same phenomenon and suggested that CtsR may need a cofactor for repressor function (15). No obvious contributor to the observed increase in heat shock-mediated mRNA stability was found. It is possible that elevated temperatures cause the denaturation of mRNA secondary structures that are recognized by the S. aureus RNA degradation machinery.
Induction of the SOS response increased the expression of nucleotide excision repair pathway and recombinational repair components, suggesting that, like the case for other organisms, repairing DNA damage is an important aspect of S. aureus SOS-induced cells. In a related study, the S. aureus transcriptional response to the oxidizing agent hydrogen peroxide, which also induces the bacterial SOS response, was determined for strain NCTC 8325 (9). The current work differs significantly from that study in several respects. First, H2O2 damages DNA, lipids, and proteins (50). Thus, in addition to inducing the SOS response, H2O2 also induces other bacterial oxidative stress responses (50), whereas the SOS-inducing agent used here, mitomycin C, primarily causes DNA damage (54, 56). Second, the former study was based on S. aureus strain NCTC 8325, which is functionally deficient for production of the primary stress response sigma factor, SigB (17). SigB has been shown to modulate the expression of a number of genes, including many that contribute to S. aureus pathogenesis (4). Moreover, Giachino and colleagues have shown that by virtue of their SigB deficiency, NCTC 8325 cells are less tolerant to DNA-damaging agents, suggesting that the strain may also be deficient in SOS functions (17). Comparison of our results to the transcriptional changes within H2O2-challenged NCTC 8325 cells suggested that SigB does not play a major role in DNA damage-mediated repair functions or the replication of DNA lesions; both studies found that repair components and a homologue to the replication protein UmuC (incorrectly annotated as UvrA in the preceding study) are induced (30). The current work also suggests that SOS induction activates the expression of numerous bacteriophage (Fig. 3D) and pathogenicity island genes. The concerted functions of these factors may account, in part, for the observation that SOS induction results in phage-mediated pathogenicity island dissemination among staphylococci (34, 55).
Striking features were observed within S. aureus stringent response-induced cells. As shown in Fig. 3C, amino acid biosynthetic processes were dramatically upregulated, and members of the translation machinery were downregulated (25 genes), mimicking the effects in mupirocin-treated E. coli cells (47). This implies that S. aureus copes with nutrient-limiting conditions by slowing bulk protein synthesis while increasing the cellular concentration of free amino acids. It was also observed that secreted proteases (i.e., SspA, SspB, and SspC) and transport processes are upregulated (Table 4). These functions may constitute another mechanism by which S. aureus increases its cellular amino acid concentration; the organism may increase digestion of extracellular proteins within its milieu and transport degradation products into the cell to bolster the abundance of amino acids. Another aspect of induction of the stringent response was the profound increase in mRNA stability of most transcripts, but as stated above, we do not yet know the consequences of this phenotype. Interestingly, we found that 60 μg ml−1 mupirocin is optimal for stringent response induction within UAMS-1 cells, which produce high levels of SigB. Crosse and colleagues independently found that the same experimental conditions effectively induce the stringent response within 8325-4 cells, which are SigB deficient (11). This suggests that SigB is not likely to play a major role in stringent response induction.
Each stress condition studied caused alterations in virulence factor expression. In general, S. aureus virulence determinants are expressed in a cell density-dependent manner. Cell surface components that are involved in attachment to host tissue are expressed within exponential-phase cultures, whereas extracellular virulence enzymes/toxins are preferentially produced during the postexponential growth phase (40). Within cold-shocked log-phase cells, extracellular virulence factor regulators (mgrA, sarA, and saeRS) were significantly downregulated in comparison to those in cells grown at 37°C, presumably ensuring low-level production of extracellular virulence determinants. The expression of cell surface virulence factors, including clfA, clfB, and fnbA, did not change at the low temperature. The best-characterized upregulated virulence determinant was sortase (srtA), which is an enzyme that tethers cell surface components (such as host attachment components) to the cell wall (35). Taken together, these results suggest that cold-shocked cells are poised to express cell surface factors on their exterior. Within heat-shocked cells, clpC and the urease operon were upregulated, and as stated above, it is possible that these factors influence the protein turnover properties of the cell. A number of uncharacterized pathogenicity island genes and the alpha-hemolysin gene (hla) were also upregulated at elevated temperature. No known virulence determinants were downregulated under heat shock conditions. Further characterization of the heat-dependent increase in overall mRNA stability may provide a better understanding of pathogenic features of heat-shocked cells. Induction of the S. aureus stringent response upregulated the production of various extracellular virulence determinants, including several proteases. This suggests that stringent response cells are poised to degrade host tissues.
The importance of short noncoding RNAs (microRNAs) in regulating eukaryotic cell development, cell death, and chromosome silencing is well documented. Recent studies have demonstrated that microRNAs also play essential roles in prokaryotic processes, including the regulation of bacterial stress responses and pathogenesis (reviewed in reference 20). For instance, noncoding RNAs are required for Vibrio sp. quorum sensing and Pseudomonas aeruginosa iron homeostasis (31, 62). Within E. coli, over 60 microRNAs have been identified, most of which are thought to act by binding to a protein and modifying its activity or by base pairing with mRNAs and affecting target transcript stability and translation (reviewed in reference 21). We recently identified eight putative S. aureus noncoding small stable RNA molecules (46), which taken together with the results of the current study, suggests that S. aureus produces an array of SSRs both during log-phase growth and in a stress-responsive manner. Based on the genomic context of SSRs, it is likely that many behave as antisense molecules, whereas others are components of operons (see Table S8 in the supplemental material). Studies are currently under way to better characterize these molecules and determine what influence, if any, they have on S. aureus biological processes and stress responses.
We thank Gail Crowell for technical assistance.
This work was partially supported by a University of Nebraska Medical Center Assistantship to K.L.A.
- Received 28 April 2006.
- Accepted 3 July 2006.
- Copyright © 2006 American Society for Microbiology


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Winner of December challenge
6 comments:
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subset: commoncrawl


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All British made and designed to last
The 2-in-1 garden tool designed to make your life much easier when weeding and planting bulbs in lawns and grassy areas.
£57.60 each plus post and packing
Mainland UK only. View delivery costs
This device was designed and developed for the easy planting of bulbs into lawns and grassy areas. It can be used for other types of planting at the gardeners' discretion. It is recommended in dry weather, that the area to be planted should be thoroughly watered the day before, thus making it easier to push the Little Plugger into the ground.
For planting bulbs up to 2 inch or 51mm diameter.
This device is not suitable for use in very stony ground.
British made. This product is made from UK sourced materials and is entirely manufactured in the UK.
Length overall:
925mm approx
Width overall:
266mm approx
Materials used:
All metal construction
Powder coated for durability
2kg approx
See how Little Plugger makes light work of bulb planting and weeding in your lawn or grassy areas with hardly any bending, saving backache you might normally experience.
Such a time saving device, so simple to operate leaving the grass quite undisturbed after planting. The lawn weeding function is such a bonus, with no weed to dispose of and no hole to fill. I wish I'd had this tool years ago.Mr. Jamie Sturdevant, Head Gardener and Landscaper, Hampshire
Excellent design, so easy to use. Our lawn will be very colourful in the spring and weed free.Mr. and Mrs. Weatherhead of West Berks


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Bowl barrow 20m south of Flanchford Road: part of Reigate Heath round barrow cemetery
- Heritage Category:
- Scheduled Monument
- List Entry Number:
- 1008871
- Date first listed:
- 16-Nov-1934
- Date of most recent amendment:
- 23-Feb-1993
The above map is for quick reference purposes only and may not be to scale. For a copy of the full scale map, please see the attached PDF - 1008871.pdf
The PDF will be generated from our live systems and may take a few minutes to download depending on how busy our servers are. We apologise for this delay.
This copy shows the entry on 10-Aug-2020 at 12:16:15.
The building or site itself may lie within the boundary of more than one authority.
- County:
- Surrey
- District:
- Reigate and Banstead (District Authority)
- National Grid Reference:
Reasons for Designation
Round barrow cemeteries date to the Bronze Age (c.2000-700 BC). They comprise
closely-spaced groups of up to 30 round barrows - rubble or earthen mounds
covering single or multiple burials. Most cemeteries developed over a
considerable period of time, often many centuries, and in some cases acted as
a focus for burials as late as the early medieval period. They exhibit
considerable diversity of burial rite, plan and form, frequently including
several different types of round barrow, occasionally associated with earlier
long barrows. Where large scale investigation has been undertaken around them,
contemporary or later "flat" burials between the barrow mounds have often been
revealed. Round barrow cemeteries occur across most of lowland Britain, with a
marked concentration in Wessex. In some cases, they are clustered around other
important contemporary monuments such as henges. Often occupying prominent
locations, they are a major historic element in the modern landscape, whilst
their diversity and their longevity as a monument type provide important
information on the variety of beliefs and social organisation amongst early
prehistoric communities. They are particularly representative of their period
and a substantial proportion of surviving or partly-surviving examples are
considered worthy of protection.
Despite the possibility of partial excavation, the bowl barrow 20m south of Flanchford Road is an integral part of the Reigate Heath round barrow cemetery and contains archaeological remains and environmental evidence relating to both the monument and the landscape in which it was constructed.
The monument includes one of seven bowl barrows situated on the crest of a
slight ridge of Greensand and forming part of a dispersed linear round barrow
cemetery aligned north-west to south-east on Reigate Heath. The barrow has a
small flat topped mound 15m in diameter and 0.4m high with a slight central
hollow suggesting that the barrow was once partially excavated. There has
been some disturbance to the northern edge of the mound resulting in its
uneven shape. Surrounding the mound is a ditch from which material was
quarried during the construction of the monument. No longer visible at ground
level the ditch has become infilled over the years but survives as a buried
feature 2m wide.
This could be one of the four barrows on Reigate Heath partially excavated in
1809 before the planting of pine trees. In two of the mounds burnt bones were
found and in the largest barrow a circular hole 0.5m in diameter and 0.4m deep
containing ashes and charred wood was discovered cut into the natural rock
beneath the mound. Three of the pine trees planted on the mound survive.
MAP EXTRACT The site of the monument is shown on the attached map extract. It includes a 2 metre boundary around the archaeological features, considered to be essential for the monument's support and preservation.
The contents of this record have been generated from a legacy data system.
- Legacy System number:
- 20165
- Legacy System:
Books and journals
Grinsell, L V, 'Surrey Archaeological Collections' in Surrey Barrows 1934-1987: A Reappraisal, , Vol. 79, (1987)
This monument is scheduled under the Ancient Monuments and Archaeological Areas Act 1979 as amended as it appears to the Secretary of State to be of national importance. This entry is a copy, the original is held by the Department for Digital, Culture, Media and Sport.
End of official listing


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- Arrival2:00 p.m.
- Check Out10:00 a.m.
- Total26 Rooms
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We are looking for the best deals....
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¡Coupon Available!It applies when your payment.
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El Pueblito Iguazu Hotel Tematico is surrounded by exuberant flora and fauna and is an excellent option for nature lovers visiting the area. This ecologically sustainable hotel has a restaurant that serves gourmet and local cuisine, a swimming pool, spa, event facilities and onsite parking. Guests of this Puerto Iguazu hotel can also enjoy the convenience of an onsite laundry and WiFi throughout. Each rustic guestroom comes with heating, a TV, a mini-fridge and a private bathroom with a tub and shower.
Extra Charge.
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Sauna and steam room


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Rajasthan is located in the western region of India touching the Thar Desert where the climate is dry and the area is covered with thorny bushes and trees. The shade of Rajasthan is not possible to overlook and the effect of emerald green, yellowness and saris is simply attractive. Make your holidays in the most royal state by booking this Rajasthan Students Tour Package which is especially planned keeping the students in mind. Rajasthan is a rich in its culture and heritage and for its historical forts, palaces and art and dance forms. Rajasthan is all about royal Rajput traditions and their culture, vivid colours, camels and festivities. Rajasthan is also famous for historical monuments and forts. Festivals of Rajasthan include appealing rituals, ethnicity and vigour. Rajasthan is a fantastic destination for the students travelling educational tours and excursions. Tatla Trip offers wide range of Rajasthan Student Tour Packages for students to understand its rich historical, cultural, wildlife discoveries.
Rajasthan Students Tour Packages in particular covers all those things that will prove to be utterly pleasurable for the students planning a fun filled trip with their friends or batch mates. The places to visit and activities are all arranged in a way that will make your trip a memorable one. So, here we will be discussing with you all the services that will fit your requirements and what all places you need to visit when in Rajasthan. Tatla Trip Rajasthan Tourism gives an opportunity to the travellers to experience the beauty of the most exotic destination of Rajasthan in refined lodging and delicious cuisine at budgeted price. It manages numerous restaurants, cafeterias and also organizes package tours, fairs, festivals, and entertainment, shopping and transport services as per the requirement.
For more details, Visit our Page https://www.tatlatrip.com/student-tours/


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One preferred tactic for addressing laws you don't like is to cut off funding to the point where those laws are no longer enforceable. Budgets are statements of priorities. The past ten years have seen sharp budget cuts at the NYSDEC — The New York State Department of Environmental Conservation. As a result, according to a report recently issued by The Environmental Advocates for New York, "The DEC is looking less, and finding less."
Governor Cuomo has sought to compensate for the cuts by having businesses self-report. But it is a questionable strategy to expect polluters to report on themselves. It is also questionable to so slash staff that poaching can proceed practically unchecked. Easing up on environmental enforcement is seen narrowly as being 'business friendly.' But a ravaged environment, where the water is undrinkable, the water not fit to swim or fish in, is really bad business. If the appeal of living on Long Island vanishes, all businesses will suffer greatly. It is precisely by vigilantly protecting the environment that we can assure Long Island, New York, and this nation's economic future.
With such a tremendous need for jobs, and with many many billions of dollars of environmental remediation to implement given all that we have done and are doing to despoil the environment, it would seem that Albany (and our nation ) has its budget priorities precisely backwards.


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Hello Games, creator of the somewhat notorious space exploration game No Man's Sky, has added a new update that brings several new elements to the gameplay experience. These include ground vehicles, improved graphics, support for PlayStation 4 Pro, and various new survival-oriented modes.
The trailer for all this new content shows off the Roamer, a rover-like vehicle capable of traversing rough terrain extremely quickly and allowing players to explore planets much more quickly; the Nomad, a hovering scout vehicle that is even faster than the Roamer; and the Colossus harvester, which can carry multiple resources to facilitate long mining missions. Existing spaceships can now be specialized to fill specific roles as well.
Additionally, there are now multiple modes to choose from, including the normal baseline mode, a survival mode, and a permadeath mode where players only have one life. The game also now has support for the PS4 Pro, with improved graphics for lighting, ambient occlusion, and 4K resolution. There is a photography mode as well which allows players to change the time of day and add filters to the images they take.
If you were unimpressed with No Man's Sky at launch, will this update convince you to give it another try? Let us know what you think of it below!
Source: PlayStation Blog
Our Verdict


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BayToday, in partnership with the North Bay Humane Society, brings you a regular feature highlighting a special pet that is in need of adoption.
Sheila is domestic short hair mix cat.
This female with the unusual markings is grey and white in colour and medium sized.
She is not neutered or declawed.
Sheila is friendly, lovable and calm.
Unable to adopt? Share this feature on social media so each animal can get the exposure it needs and find a loving home.
The Adopt Me segment in North Bay is proudly sponsored by McDougall Energy, your local Esso branded reseller for heating oil, propane and fuel.
McDougall Energy understands that while they can warm your home, adopting a pet can warm your heart. Visit www.mcdougallcorp.com to learn more about McDougall Energy and their community involvement.


text:
Source: Public Health England
Follow this link for fulltext
Date of publication: October 2014
Publication type: Website
In a nutshell: Healthier Lives is a new interactive tool which shows the large variation in prevalence and treatment of hypertension and diabetes around England as well as the provision of the NHS Health Check to prevent heart disease and other conditions. The data allows people to see how their GP surgery, CCG and Local Authority are performing compared to the national average.
Length of publication: 1 webpage
Some important notes: Please contact your local NHS library if you cannot access the full text. Follow this link to find your local NHS library.
Acknowledgement: Press release


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The recently renovated Bank Of Ireland Branch has now been transformed into the Left Bank Bar and Nightclub. A world-famous Bar & Grill in Kilkenny, Ireland.
After Opening its doors on the 8th of May 2008 Left bank has established itself as the most popular events and nightlife centre in Kilkenny with the most unique bar and central location in the centre of Kilkenny city. It is the ideal place for a party, hen party or a family celebration in Kilkenny.
The impressive main bar with a lively atmosphere is ideal for either a mid-week drink or a group night out at the weekend. Our Bar food menu is available until 10 pm Monday - Saturday & 9 pm Sunday, with our sister restaurant Rive Gauche serving until 10 pm Monday to Saturday and 9 pmSunday.
Louie's Backyard (outdoor area) is a fully heated and covered smoking area with its own outdoor fireplace and both an indoor and outdoor bar.
LB2 Social Club located on the first floor has a beautifully designed Nightclub opened every Friday & Saturday night. Its a popular destination for locals and visitor's alike with Kilkenny's top DJ's playing a mix to suit all ages.
LB2 Left Banks Late Night Bar hosts our Cocktail Lounge is open every Friday & Saturday night.
This function room can be transformed to suit any style of event. This room, with no hire charge, can easily accommodate up to 350 people.


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Prime Minister Narendra Modi's visit to Chhattisgarh to marks the state's 16th foundation day on November 1, 2016. He launched the Saur Sujala Yojana in Chhattisgarh in order to provide solar powered irrigation pumps to farmers at a subsidized price. He also inaugurates jungle safari, statue of Pandit Deendayal Upadhyaya and Naya Raipur BRTS project in Chhattisgarh. Chhattisgarh was the first state to implement the Saur Sujala Yojana .Under the scheme, solar powered irrigation pumps of 3HP and 5HP capacity worth Rs 3.5 lakh and Rs 4.5 lakh respectively would be distributed to the farmers by March 31, 2019.
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The UK's Leading Australian Wine Specialist
Red Wine
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Josh Cooper SALE
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Barossa Valley
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Customer Testimonials
Medal Winning Wines
Last Bottles
Buy Australian Wine Online
Château Grand-Puy-Lacoste sits in the Pauillac appellation of Bordeaux and being one of the oldest estates in the Médoc, it is steeped in a rich history. The vineyards are planted with 75% Cabernet Sauvignon and 25% Merlot with many of the vines having an average age of almost 40 years. In 1978, the château owner Raymon Dupin, sold the property to the Borie Family, who were renowned for their fantastic wines at Ducru-Beaucaillou and Haut-Batailley. Today the estate is owned and run by François-Xavier Borie. "I do what I love, what I believe in. We want to build Grand-Puy-Lacoste's renown on consistent quality rather than showy effects. I'm suspicious of wines made for competitions; I prefer that Grand-Puy-Lacoste create a reputation based on the long term, vintage after vintage. Pretentiousness, fleeting fashion and short-term media attention is not what we do."
Let us know, chances are we can source it for you...


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Sue Bryce Education – Lighting – Natural, Constant, and Strobe (Full) | File Size 484.2 MB| Free download
Natural, Constant, and Strobe
- Do you ever feel that there just isn't enough light in your studio to work with? Even if you've never thought seriously about using artificial light, Sue walks you through a few different lighting setups to consider when the daylight just isn't measuring up. Then, compare and contrast each shoot to find a scenario that will work best for you.
Shoot with Kino Light (14:20)
Sue shows us the surprising results of using a constant light source while shooting a portrait of Dani. This is a great solution if you're worried about the amount of light in your studio, or if you just need a boost of natural light.
Items Used:
– Kino Flo
– C stand & sand bag
– white Vflats (available at local camera supply)
– backdrop
Key Things to Take Away:
Place at an angle, nice wash of light similar to natural
Can use for video light as well
Great if you're worried about the amount of light, or need a boost of natural light
Shoot with Kino + Scrim (5:47)
When we add the scrim in front of the constant light source, it forces the light to become more spread out and the result is a bit softer. Even though adding the scrim adds additional cost, Sue explains that it could very well be worth it to shoot at any time of day.
Items Used:
– Kino Flo, three blubs
– Scrim
– C stand & sand bag
– white & black Vflats (available at local camera supply)
– backdrop
Key Things to Take Away:
Incredible light to shoot any time of day
Light source is being spread out, and it's soft
Shoot with Strobe (7:24)
Using the same setup as the constant light, Sue uses her strobe to bounce light onto the client. Watch as she makes small adjustments to get the light just right, and gives you insight on how the strobe can enhance your studio.
Items Used:
- Strobe, through scrim, without the modifier
- 59" elinchrom deep dish octobox
- white and black V flats (available at local camera supply)
- ND Filter
Key Things to Take Away:
-Using these lights can save you a lot of time in post-processing
-They also can really enhance your studio – big plus is that you can shoot any time of day
-Don't spend money on things that won't help you make more money
Retouching (14:03)
Go through the photos taken with natural light, constant light, and the strobe. See the difference in detail and compare and contrast which lighting scenario you like the best.
Items Used:
- Adobe Photoshop
- Preview
Key Things to Take Away:
-The goal is not to get away from your favorite style of lighting. The goal is to find a solution to shooting outside of daylight hours.
|Constant_and_Strobe_FULL.torrent (download)
Course content: https://suebryceeducation.com/course/natural-constant-and-strobe/
Direct Download
|Constant_and_Strobe_FULL.zip (download)


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Supported by
Félicien Kabuga, 84, had been on the run for 23 years, since he was indicted by the International Criminal Tribunal for Rwanda on multiple charges of genocide.
By Marlise Simons and Norimitsu Onishi
Twenty-five years after they participated in one of the world's most atrocious instances of mass violence, their experiences in Rwanda show that peace and reconciliation are possible.
By Hollie Nyseth Brehm and Laura C. Frizzell
The French president said he wanted April 7 to become an annual day of commemoration for the massacres, which remain a source of tension between France and Rwanda.
By Aurelien Breeden
As world leaders have expressed regret over failing to stop the massacre of as many as one million people in Rwanda, its president, Paul Kagame, has entrenched his power and punished dissent.
By Alan Cowell
Twenty-five years after the genocide, its effects are shaping a new generation.
By Jonathan Torgovnik
Scholastique Mukasonga's newly translated memoir is about the impact of the Rwandan genocide, during which 37 of her family members were killed.
By Parul Sehgal
President Paul Kagame's government ordered the release of Victoire Ingabire Umuhoza, leader of an opposition party, and Kizito Mihigo, a musician and activist.
By Richard Pérez-Peña
Clemantine Wamariya crisscrossed Africa with her teenage sister, enduring hunger, poverty, violence and trauma. Her memoir, "The Girl Who Smiled Beads," recounts her journey.
By Alexis Okeowo
The findings of an independent study could exacerbate a long diplomatic feud over accountability for one of Africa's worst atrocities.
By Jina Moore
President Paul Kagame's victory reflected what rivals called the stifling of dissenting views, and showed the glaring absence of a viable opposition.
By Zack Baddorf


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- Effective Cooling fans design to highly reduce the compression temperature.
- Durable cast iron crankshaft and pump – excellent wear-resistance.
- User-friendly – easy operation and maintenance.
- Various options for different application.
Control System
- Pressure Switch System "P" Type
The automatic pressure switch will stop and restart the compressor according to air demand.
It is normally recommended for intermittent operation and smaller compressor units.
|2.2kw / 3hp, 415V, 3 Phase
|Working Pressure (kg/cm²)
|12 Bar (16 Bar Maximum)
|Compressor Speed
|960 rpm
|270 L/min
|Tank Capacity
|237 Liter
|Compressor Block Physical Dimension
(L x W x H)
|500mm x 330mm x 400mm
|Weight (Bare Head only)
|Compressor Unit Physical Dimension
(L x W x H)
|1470mm x 550mm x 1100mm
|Weight (Whole Unit)


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Retiring on time is no accident. It's the work of long-term thinking and careful planning, whether you go about it on your own or hire an advisor to help out.
Even those who do hire advisors would be better served to understand the mechanics of saving and investing. Think you know a thing or two about the matter? Take this five-question financial savvy quiz and find out:
1. A retirement account of $200,000 means you can safely withdraw what amount on an annual basis and expect it to last for 30 years?
a. $10,000
b. $12,000
c. $8,000
d. $5,000
Answer: C. $8,000. Financial advisors counsel taking no more than 4% of your retirement savings per year ($200,000 x 0.04 =$8,000). Conservatively invested, that level of withdrawal means the money should last for three decades. Take more, and you are counting on a higher, and thus riskier, investment return. Or you run out of money sooner.
2. The longer I wait to take Social Security, the more I will get paid once I begin benefits.
a. True
b. False
Answer: A. True. Many people take their retirement benefits beginning at age 62, the earliest possible age. However, if you were born in 1943 or later you will be paid an estimated 8% more money for each year you wait until the ultimate retirement age of 70 (your "full" retirement age may be sooner). You are likely to collect for fewer years, it's true, but you might want to work for a few years past 62 in order to build up private retirement savings in a 401(k) or IRA.
3. A reasonably risk-adjusted portfolio that holds more stocks than bonds is likely to double your investment within what period of years?
a. Around 12 years
b. Around 10 years
c. Around 4 years
d. Around 7 years
Answer: B. Around 10 years. Many investors nearing retirement today have become used to stock market returns of 10 percent or more and doubling every seven years. However, the long-term return on stocks with dividends reinvested is about 6.6%. Held with bonds and rebalanced regularly, such a portfolio is likely to double your money in 10 years, not seven.
4. My 401(k) plan at work has fees that cost me what percent of my retirement savings balance every year?
a. Zero, it's free.
b. Less than a half of 1%.
c. About 1%
d. Easily more than 1%
Answer: D. Easily more than 1%. In fact, 401(k) plan fees average 1.5%. Large-company plans tend to be the least expensive, while small company offerings can cost as much as 3.86%. That money is deducted from your total portfolio balance, not from the potential earnings in a given year.
5. The single most important investment I can make for retirement is to…
a. Own good life insurance
b. Have a paid-up mortgage
c. Maximize tax-deferred and tax-free savings
d. Build a cash emergency fund
Answer: All of the above, but mostly C. Maximize tax-deferred and tax-free savings. Life insurance is useful if something happens to you, but that is unlikely. Paying off a mortgage is great, but your interest rate is probably very low and there are tax benefits to paying it down slowly. Everyone needs a cash emergency fund of at least three months (six months is better). However, all of these pale in comparison to a 401(k) or IRA which earns you a tax break now or is tax-free later and compounds at a market rate of return.
Feeling better about your financial savvy? Great! Didn't do so well? I recommend a trip to the public library. Try The Elements of Investing to start. It's a short read and easy to understand, yet the concepts are important and timeless. Then start making choices that will get your retirement game plan on track.


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Mercedes-Benz Trucks honoured by HRH The Princess Royal for commitment to training
2nd December 2016
Mercedes-Benz Trucks has been commended for its commitment to training and skills development by Her Royal Highness The Princess Royal, at a ceremony at St James's Palace in London.
The Milton Keynes-based business was one of 34 organisations honoured with a Princess Royal Training Award for creating lasting impact by successfully linking their skills development needs to business performance.
The winners, including prestige car manufacturers, healthcare organisations and charities, all demonstrated that training can make a significant contribution to productivity and business success.
Read the full story on Commercial Fleet.back


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Raymond Weil
Certified Official Agent
The RAYMOND WEIL watch range at Arnik Jewellers features a wide choice of men's watches and ladies' watches. The men's range features a variety of styles from classic chronograph designs with leather straps to stainless steel bracelet style watches featuring modern design elements. The ladies' collection features slightly smaller dials and bracelet style straps.
Mr Raymond Weil launched the watch brand back in 1976 in Geneva Switzerland. Raymond's son-in-law Oliver Bernheim joined the business in 1982, followed by RAYMOND WEIL's third generation in 2006, Elie and Pierre Bernheim ensuring family continuity. The luxurious watch brand is the result of three generations whose combined passion has maintained the company's heritage and international status.
Situated in Geneva, the home of Switzerland's finest watch makers RAYMOND WEIL combines the elements of Swiss excellence as well as beauty. With an award winning precision movements campaign RAYMOND WEIL'S attention to detail and quality is constant. Swiss watch making skills and knowledge is reflected in RAYMOND WEIL'S craftsmanship and technical material choices.
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Tweens + Teens
5 Friend Issues Your Tween Daughter Will Encounter
Navigating a tween daughter through her friendships can sometimes make me look back at toddler playdates as so much easier. We were right there and we could intercept the struggle for the same toy or help them climb and slide next to their playground buddies. Plus, most things could be solved with a good snack or a long nap.
The friend issues for our tween daughters are more complicated. We usually only hear our daughter's side of the story and it's often accompanied by a host of emotions. Sometimes we have to be a noticer – sense when something's off and then probe with gentle questions.
It's so important for us to teach our tween girls how to handle friendships. The tween years are full of change that create sticky friendship issues. Let's look at 5 friend issues your tween daughter will encounter and how to help navigate through them.
1. Your tween discovers her friend shared her secret.
Your daughter feels betrayed, hurt, and angry. She may be embarrassed that others now know something so personal about her. Don't dismiss these emotions, even if the shared secret seems childish. Talk about the importance of integrity and keeping confidences. Suggest that she talk to her friend after she's fully processed her emotion. And finally, caution her about sharing with this friend again, reminding her that she has control over what she shares and what she doesn't.
2. Your tween's friend wants to exclude another girl.
Tweens often enjoy the camaraderie of a club and don't realize how hurtful it is to be excluded. This is an opportunity to help your daughter develop compassion by seeing it from the excluded girl's point of view. How can she maintain a friendship with one while including the other? Suggest she lead by example by inviting the excluded girl to eat at their lunch table or join their circles of conversation.
3. Your tween is the new girl.
Walking into a new situation or new place is just plain hard. Giving your tween daughter tools now will help her all through life. I've heard it said that most people enter a room and think, "Here I am!" That kind of self-focus makes being the new girl even harder. Instead, teach your tween to enter the room thinking, "There you are!" Being other-oriented will help our girls to start conversation or even find someone else who's new and needs a friend.
4. Your tween's friend makes an uncomfortable (or unsafe!) dare.
The old truth or dare game often goes too far. Prepare your tween in advance for this one. It's okay and right to say no when a dare is inappropriate or unsafe. Assure your daughter the most courageous thing she can do is to stand alone in the face of peer pressure to do something she doesn't want to do. Give her words beforehand so she can say no to any dare with confidence.
Assure your daughter the most courageous thing she can do is to stand alone in the face of peer pressure to do something she doesn't want to do.
5. You see red flags with the friendship.
Sometimes our tweens may not come to us with a specific issue but we begin to see red flags in the friendship. Maybe you notice your daughter's personality changes after time with the friend or she bottles up and won't talk about time with this friend.
This happened with my own tween. She began asking sensitive questions one afternoon and as we talked openly, she burst into tears. She'd felt uncomfortable with peer pressure in a blossoming friendship, had felt trapped and now felt guilty for being in the situation. Surprisingly though, a few weeks later she began asking to see this friend. I knew the friendship was unhealthy, and I said a gentle but firm no. As moms, we need to create healthy boundaries in friendship for our tween daughters when we see red flags. Here's an easy hands-on illustration to teach your tween how to choose good friends.
What other ways are you encouraging your tween daughter in difficult friendships?


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Motorcycle magazines focus on the greatest, motorcycle websites focus on the latest. We focus on what we think you'll like. bikerMetric does too. We feature the men and women doing amazing stuff with motorcycles who wouldn't otherwise get the exposure ...Read More »
3D Printed Motorcycle
Designer and artist Jonathan Brand has created a 3D printed motorcycle that gearheads, motorcycle enthusiasts, 3D printing fans, and interior designers will all appreciate. From his studio in New Haven, CT, Jonathan Brand has printed out a full-scale replica 3D ...Read More »
Motorcyclist Rides to Hospital With Knife in Head
We don't typically post motorcycle news, but this is pretty amazing stuff. Warning: this is also the gorriest story we've had on here since someone tried to decapitate a motorcyclist in Toronto, Canada. A Brazilian man with an eleven-inch knife ...Read More »
A Marketing Move That Actually Helps Motorcyclists
Allstate's ONE program has installed 167 "Watch for Motorcycles" signs in 37 different cities across 19 states. Not bad at all!Read More »
Toronto Motorcycle Parking – Please Sign Here
Since 2005, motorcyclists in Toronto have enjoyed free parking. It was a move made by city hall almost ten years ago to encourage downtown commuters to take more traffic efficient means of transportation. In August 2014 alone the city approved ...Read More »
This Pug Does a Better Cruiser Impression Than You
We normally pride ourselves on posting top quality informative content for those who take motorcycling as a way of life …but… I bet this pug does a better V-Twin cruiser impression than you do! In other (much more important) news ...Read More »
YouMotorcycle Turns Five Years Old!
In 2009 two blossoming motorcycle websites made an impression on me. One was Jasper Bram's Helmet Hair Blog. The other was Trent Reker's bikerMetric. Bram always seemed to have his finger on the pulse of exactly what was cool, trending, ...Read More »
Win One of Three Free Harley-Davidson Street 750s
Deeley Harley-Davidson Canada has once again partnered with the Movember in support of men's health. That means you've got a chance to Win a Free Harley-Davidson Street 750, one of the first of these motorcycles to arrive in Canada.
Deeley's been taking the Movember partnership seriously. Over the past three years, the H.D. network (associates, retailers, and H.O.G. Members) have contributed over $500,000 in support of the campaign. Nice!
Weekend Roundup
Another sweet summer weekend has come and gone. If you've been offline for the past 72 hours while out riding (as you should have been), here's what you missed:Read More »
Motorcycles on Pinterest
Got motorcycles? Got Pinterest? We're all over it. See anything you like? Join us!
Visit YouMotorcycle's profile on Pinterest.


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Odyssey, July 6, 10:00 am, Simpson House
"What's It like to Visit Transylvania?"
We'll have a selection of people who first went to visit our partner village in '91, '94, '04, '12, and '13–and they'll share what that experience was like. Maybe after this session with your friends, you'll decide to put out $3000 for a new experience! If you are interested, email email@example.com or firstname.lastname@example.com


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Product Description
Street Flags
Street Flags are a great way to advertise whilst on the move. Ideal for promoting events, charity awareness, your stand location at an exhibition or keeping a tour group together.
The sturdy and lightweight back pack comes with poles for all four different flag shapes which are easily attached using the poles supplied. As a result you can easily swap between a variety of graphics and flag shapes using the same hardware. The Street Flag back pack is supplied with reinforced straps and flag mounting system. An adjustable belt and comfortable padding makes these systems comfortable to wear.
The Edge, Feather and Quill flags are all dye sublimation printed onto fabric. Therefore this means that the image is show through on the reverse of the flag. The X-Banner is printed on banner vinyl so is white on the reverse of the flag.
Lead time on this product is 10 working days, if your order is urgent please call prior to placing your order.
Edge: 1185 x 480mm
Feather: 1268 x 310mm
Quill: 1268 x 310mm
X-Banner: 1280 x 650mm
Artwork Specifications: Download Street Flag Artwork Guide
Outdoor Banner Stand:
Adventurer Outdoor Banner Stand.


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GPs tell patients with coughs and colds to 'stay away'
GPs have warned patients with coughs and colds to stay away from surgeries - with cases at a seasonal high.
They said coughs in particular have been leading to people contacting their local surgeries "in droves", when in reality they do not need to.
"Coughs and colds simply have to run their course," said Dr Steve Bassett, who is based in Swansea.
Only those whose symptoms become more serious, such as fever, should contact GPs.
In most circumstances, plenty of rest and fluids, keeping warm - and time - are the only cures, said Abertawe Bro Morgannwg University Health Board, which covers services from Swansea to Bridgend.
Antibiotics are also ineffective against common chest infections, so a trip to the GP surgery is "pointless, and takes up an appointment slot which a really sick patient might need".
Dr Bassett, who is also the lead GP for the new non-emergency 111 pilot service in the area, added: "If you have a cough - even if you are producing green phlegm - if you are eating and drinking normally and are otherwise pretty well, then we don't need to see you.
"It's only if you have other symptoms, such as a fever as well as a cough, or have problems breathing or are vomiting, that we may need to investigate further."
Andrew Evans, chief pharmaceutical officer for Wales, encouraged people to ask their local pharmacist for advice.
"They are experts in medicines and their use and are able to give advice on a range of conditions without the need for an appointment," he said.
And chief medical officer Dr Frank Atherton said people can use an NHS app to understand which service they need.
"In many cases, people go to emergency units when they could have received the same advice from a local pharmacist or been seen and assessed in a minor injuries unit, GP surgery out of hours doctor.
"The message is clear. Choose well to help save lives."
Mark Walker, deputy medical director and GP for Betsi Cadwaladr University Health Board, said there is little GPs can do to help patients with the common symptoms of a cold.
"Visiting a GP seeking treatment for a cough makes it harder for others who need an appointment to get the treatment they need. We're also aware of people who have failed to make a GP appointment turning up at Emergency Departments, which again has a knock-on effect for waiting times."
Cardiff and Vale health board also said many people were going to their GPs with a persistent cough, and stressed there was very little a GP can do.
Cwm Taf University Heath Board said it was managing a high level of demand, as expected in winter.
The advice also applies to children under five with a cough or cold who are otherwise active and eating normally. They do not need a visit to the doctor.
Earlier this week Cardiff and Vale health board warned University Hospital of Wales' emergency unit was "extremely busy" after the new year's extended holiday and asked the public to "play their part" in making use of local chemists.
A spokesman said the hospital was still "pretty busy" and urged patients to choose well.
In the Hywel Dda University Health Board area, which covers Carmarthenshire, Ceredigion and Pembrokeshire, patients are being urged to use a new community pharmacy service if they have a low-level injury or illness, in a bid to free up pressure on A&E departments.


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Elvin Bishop
9 articles
Led Zeppelin, Brian Auger's Trinity, Elvin Bishop: Rose Palace, Pasadena CA
Live Review by John Mendelssohn, Los Angeles Times, 6 May 1969
Rock Show Staged at Pasadena Rose Palace ...
Chuck Berry, Elvin Bishop, John Mayall: Fillmore East, New York NY
Live Review by Ian Dove, Billboard, 18 October 1969
Berry, Bishop — Musical Brothers ...
Santana, Elvin Bishop: Civic Auditorium, Santa Monica CA
Live Review by John Mendelssohn, Los Angeles Times, 3 March 1970
Santana Plays Two Rock 'n' Roll Shows ...
ZZ Top, Elvin Bishop: Long Beach Arena, Long Beach CA
Live Review by Richard Cromelin, Los Angeles Times, 27 August 1974
ZZ Top a Sell-Out Sleeper ...
Elvin Bishop: The Bourbon Street Irregulars
Report and Interview by Jim Esposito, unpublished, April 1975
2009 note: CREEM flew me up to New Orleans to write a story on Elvin Bishop. Rejected this. Editor told me: "You didn't say why ...
Review by Bob Fisher, New Musical Express, July 1975
ELVIN BISHOP'S place in the scheme of post-Beatles US Rock has been pretty much undervalued over the years. This is probably owing to his uncanny ...
Elvin Bishop: Hometown Boy Makes Good
Review by Max Bell, New Musical Express, 15 January 1977
YES INDEED I do believe we've got ourselves a good one here. Elvin Bishop has resisted the slightly formulaic limpness of Struttin' My Stuff ...
The Elvin Bishop Band: Hometown Boy Makes Good! (Capricorn)
Review by John Morthland, Creem, March 1977
IT TOOK ELVIN Bishop years to hit his groove, years in which he played every role from overbearing ham to dependable sideman to journeyman pro ...
An Interview with Elvin Bishop
Interview by Carl Wiser, Songfacts, 13 September 2007
HOW DO YOU write a blues song? Elvin Bishop, a founding member of the Paul Butterfield Blues Band before launching a successful solo career, is ...
back to LIBRARY


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President Trump is announcing his presidential reelection bid in Orlando, Florida, at 8:00 pm ET before a massive crowd of over 20,000 supporters ahead of the Democrat primary debates.
Join me LIVE tonight in Orlando, Florida at 8:00 P.M. Eastern as we kickoff #Trump2020. Enjoy!
— Donald J. Trump (@realDonaldTrump) June 18, 2019
Owen Shroyer provides live commentary of the rally:
Trump Gives Shoutout To CNN At Latest Rally – The Fakest Name In News! https://t.co/zXPN9MS1E4
— J Owen Shroyer (@allidoisowen) June 19, 2019
CNN Fully Triggered As Trump Rally Steals Their 5 Viewers https://t.co/mtExZjvrwu
— J Owen Shroyer (@allidoisowen) June 19, 2019
Watch Live: President Trump Defeats The Fake Polls With Record Florida Rally https://t.co/3tGcUthCAy
— J Owen Shroyer (@allidoisowen) June 19, 2019
Watch Live: Record Setting Trump Rally In Orlando Destroys Fake Polls #TrumpOrlandoRally https://t.co/YHYYrytOOv
— J Owen Shroyer (@allidoisowen) June 18, 2019
Reporter Millie Weaver is also on the ground at Orlando rally:
Orlando Trump Rally Interviews with supporters https://t.co/hxtUvurrek
— Millie Weaver (@Millie__Weaver) June 18, 2019
The Reopen America Back to School Special is now live! Earn double Patriot Points on our hottest items!


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0.1 kg
Warning: Last items in stock!
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Reinforced gears set.
Model 100:200 SUPER HIGH TORQUE.
TENACITY version.
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100% GUN FIVE impruved brand NEW LINE.
We only use the BEST materials for creating our products.
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mar 31, 2020CLOSING OF THE HOLIDAY 1st OCTOBER 2019
giu 15, 2019public saturday closing
mag 8, 2019PCC9 COMBO
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Back up limitless photographs and videos for absolutely free, up to 16MP and 1080p HD. Access them from any phone, tablet, or personal computer on – your photos will be protected, secure, and normally with you. This is a war, she says, "with so a lot of casualties that we really should contact it by its true name, this war with so many dead by police, by violent ex-husbands and partners and lovers, by people today pursuing power and profit at the point of a gun or just shooting initial and figuring out who they hit later." To get to the root of these American crises, she contends that "to acknowledge this state of war is to admit the need for peace," countering the despair of our age with a dose of solidarity, creativity, and hope.
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As brought to life in the bestselling Summoner series, the magic of summoning is also an art, with a story of its own. In this new chapter of the Saxon Tales series—a rousing adventure of courage, treachery, duty, devotion, majesty, love and battle, as observed by way of the eyes of a warrior straddling two worlds—Uhtred returns to fight as soon as once more for the destiny of England. An unprecedented cinematic journey ten years in the making and spanning the whole Marvel Cinematic Universe, Marvel Studios' Avengers: Infinity War brings to the screen the ultimate, deadliest showdown of all time.
Board the Millennium Falcon and journey to a galaxy far, far away in Solo: A Star Wars Story, an epic action adventure with the most beloved scoundrel in the galaxy. Shop a range of good quality interior closet doors at Lowe's for ProsFind barn doors, sliding and bifold doors, French doors and a lot more from leading brands. 16 x 80 interior prehung door – Welcome and thank you for these of you who have visited this web page, discussing photographs of homes, home designs, and wallpapers for your desktop as effectively as your mobile.
Back up unlimited pictures and videos for cost-free, up to 16MP and 1080p HD. Access them from any phone, tablet, or laptop on – your photos will be safe, safe, and generally with you. Via a series of daring escapades deep inside a dark and hazardous criminal underworld, Han Solo befriends his mighty future copilot Chewbacca and meets the notorious gambler Lando Calrissian, in a journey that will set the course of one of the Star Wars saga's most unlikely heroes. 16 X 80 Mobile Residence 1998 SUNSHINE 3BR 2BA 16X80 MOBILE Household PERRY KANSAS Used. 16 x 80 inch interior door – We hope that, by posting this 16 x 80 inch interior door , we can complete fill your requires of inspiration for designing your dwelling.
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Google automatically detects your computer's location using its IP address , Place History (if it's turned on), and current areas you have searched for. Wallpaper 16 x 80 interior prehung door is a kind of material employed to cover and decorate the inner walls of properties, offices and other buildings, which is a single aspect of interior decoration. Right here an enigmatic band of warriors bear swords of no human metal a tribe of fierce wildlings carry males off into madness a cruel young dragon prince barters his sister to win back his throne a kid is lost in the twilight involving life and death and a determined lady undertakes a treacherous journey to defend all she holds dear.
16 X 80 Interior Door – This gripping, stranger-than-fiction espionage thriller brings to life the incredible correct story of Moe Berg, the experienced baseball player who became a Globe War II spy.


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When's the last time you had a conversation about sexually transmitted diseases? When's the last time you were tested?
The Sugar Bowl is a place to find sophisticated relationships. It is also a place to have a mature, evolved discourse on sexual health. Given the prevalence of this epidemic and the need for open, honest conversations about this subject, let's drop the stigma. You can have an STD and still have a healthy, wonderful life. This is both in and out of the bedroom. Prevention is the key to addressing STDs. This blog will detail why you should be aware of STDs. How to stay safe, and how to initiate a productive conversation with your partner.
This is a Big Issue
One reason STI's are so common is that the public doesn't know that much about them. In addition to that, we REALLY don't know how to talk about them. Many schools don't teach any kind of sexual education. Those that do can be more confusing than constructive. For example, my co-ed health class was taught by a flustered, retired football coach. We skipped over a lot of important information and were left with plenty of questions.
Ready for some shocking statistics?
Over 30% of Americans have an STD. Some sexually transmitted diseases, like syphilis, gonorrhea, and chlamydia, are spreading at epidemic levels. Over 2 million new cases were reported in 2016. Other sexually transmitted infections are even more common, as researchers estimate over 20 million new cases occur each year. However, many of these infections – think millions of cases – produce no symptoms, thereby going untreated. This underscores the importance of regular screenings.
To reduce your risk, you should use a contraception correctly, every time. Also, please be aware that STIs can spread through ANY exchange of ANY fluids, not just 'traditional sex.' Finally, contracting an STI doesn't make you a bad person. It certainly doesn't exclude you from love and intimacy.
Let's Talk about Sex
Now that you are up to speed on STDs, it is time to initiate that conversation with your Sugar Baby/Sugar Daddy. Whether you are just getting to know each other or have been exclusive for years. It shouldn't be taboo to talk about sexual health with a future or current partner. It is uncomfortable. Have this talk in a quiet, private place, where you are unlikely to be overheard or disturbed. Set the mood with some calming ambient music. Calm your nerves with a glass of wine. Then, tell your Sugar Daddy that you read an awesome article on sexual health and wanted to discuss safe sex. By creating a comfortable space, being well-informed on the issues, and going in with a plan, you're setting a good tone for the conversation.
End the talk by making a plan to both get tested. We ALL deserve safe sex. Regular STD screenings are crucial in reducing the spread of these diseases.
How to tell your SB/SD about an STD
First things first: yes, you can Sugar with an STD. (Remember, we checked our stigma at the top of this article!) Sugar Daddies and Sugar Babies are drawn from a large swath of life. So it is just a fact that a significant portion of this population has an STD. Check out the forums for more advice and experiences!
In any relationship, you should be very honest about this topic. One option is to put this information on your profile. While you will likely receive fewer messages, the messages you do get will be from people who won't see the condition as a dealbreaker. Additionally, this technique will make you more attractive to a partner with a similar affliction.
When having an in-person conversation, begin by briefly explaining the condition and how it affects you. Much of the fear and stigma associated with STDs is the result of misinformation. So this really is an important step.
Next, statistics are on your side, so make them work for you! For example, at least 3.7 billion people, almost half of the world population, has the HSV-1 virus, more commonly known as a strain of herpes. If a partner breaks things off because of HSV-1, odds are their next partner would carry the virus as well! Essentially, you are a 'safer' choice because you are aware of the condition, can mitigate the risks, and are on top of your sexual health.
Having this conversation demonstrates you care about your partner and respect their health, and those are very attractive qualities. Make sure to ask if your partner has any lingering questions or concerns before the topic changes.
STDs are 100% preventable, so let's prevent them. Get tested, be vocal, and use protection!


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Alex Zhirui Ji joined CRU in 2017 as steel analyst in CRU's Singapore office. She covers the South East Asian markets and interaction between SE Asia and other international key players.
Before joining CRU, she worked as precious metals analyst at Thomson Reuters. She speaks Mandarin and Cantonese, and has lived in various cities in China and Singapore. Alex holds a Masters in Business Journalism and is currently working towards the CFA qualification.


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. Filmed in Atlanta, Black Panther generated more than $83.9 million to the local economy and contributed more than $26.5 million in wages to more than 3,100 workers across the state. Production not only filmed in downtown Atlanta, but also on location at Piedmont Park, Castleberry Hill, Mechanicsville, West End, Vine City, Decatur, Marietta, Cartersville, and other locations across north Georgia and metro Atlanta.
As the first day of school rapidly approaches, an organization will work to ensure that students have the supplies they need.