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Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.
Streptococcus pneumoniaeis the major cause of community-acquired bacterial pneumonia, and it is also responsible for otitis media and meningitis (2). Capsular polysaccharides were the first virulence factors to be identified. The capsule is thought to protect the bacteria from the host immune system by preventing phagocytosis (17). Purified capsular extracts do not have an inflammatory or toxic effect (31, 32). Among the proteins considered to be virulence factors (17, 45) are pneumolysin (3, 7, 12), autolysin (4, 12, 56), hyaluronidase (5), pneumococcal surface protein A (PspA) (8), PsaA (6), neuraminidase (10), immunoglobulin A1 (IgA1) protease (46, 59), and pyruvate oxidase (55), although for some of them a role in virulence has not been demonstrated.
Recent advances in the field of bacterial pathogenesis have allowed a large-scale identification of new virulence genes in different bacterial species. The methods developed are based on the concept that specific gene products are required for each stage of an infection process and that their expression is often regulated by the different environmental conditions encountered in the host. Mahan et al. (38) developed a system called IVET (in vivo expression technology) aimed at identifying bacterial genes that were preferentially expressed in the host during infection and were poorly transcribed under laboratory conditions. IVET was originally developed for use with Salmonella typhimurium (38) and then applied to Vibrio cholerae (11) andPseudomonas aeruginosa (58). Hensel et al. (28) expanded the concept of tagging originally developed by Walsh and Cepko (57) to monitor the fate of clonally related neocortical cells during brain development, developing a strategy to identify virulence genes by negative selection. This system, called STM (signature-tagged mutagenesis), exploits a pool of transposons in which each transposon is tagged with a unique DNA sequence so that the resulting insertion mutants are marked with a different DNA sequence. This permits the identification of bacteria recovered from hosts infected with a mixed population of mutants.
Tagged insertion mutants are combined into pools, which are used to infect the animals. At a defined time point, bacteria are recovered from the animals. Tag sequences are amplified from each pool by using a radioactive label before and after the infection. These two labeled tag probes are hybridized to filters containing spotted genomic DNA from all mutants of the corresponding pool. Mutants whose tags are positive for hybridization with the probe from the original pool and negative with the one from the recovered bacteria are considered to be virulence attenuated. This system was originally used to identify genes involved in virulence in S. typhimurium (28) and recently applied to Staphylococcus aureus and V. cholerae (15, 42).
S. pneumoniae has been studied for many years, yet its virulence mechanisms are not fully understood (17). Therefore, we have modified the original STM methodology to discover novel virulence factors in S. pneumoniae. Initially, we attempted to use for this purpose the encapsulated type 3 strain GP119, obtained by transformation from the nonencapsulated avirulent laboratory strain Rx1. Surprisingly, when GP119 was tested for virulence in a mouse septicemia model, it was found to be not virulent (unpublished observations); this result indicates that in factors in addition to the capsule are required for virulence. We then selected the encapsulated type 19F strain G54, a recent clinical isolate. This strain was chosen because of its high transformation rate (47) induced by using the 17-residue competence-stimulating peptide (26) and its virulence in a mouse pneumonia model, as assessed in our laboratory. The following two major changes were introduced in the original STM protocol (28).
(i) A library of tagged mutants was constructed by insertion-duplication mutagenesis using short random genomic DNA fragments inserted in a suicide plasmid vector bearing the molecular tag. The original transposon mutagenesis technique (28) was not applied to S. pneumoniae, since we did not expect transposons to insert randomly as shown forStreptococcus mutans (24) and Lactococcus lactis (49).
(ii) While in the original method (28) the filters corresponding to each pool had spots of genomic DNA from each mutant obtained by transferring bacterial colonies to the filters (colony hybridization), we used filters containing amplified tags from each mutant. This modification was necessary since in our hands colony hybridization generated a high background giving rise to false positives.
Using the modified STM technique, we identified 126 putative virulence genes from S. pneumoniae. Some of them corresponded to previously described pneumococcal virulence factors, while others showed homology to virulence genes found in other bacteria. In addition, we have identified genes not previously known to be involved in virulence.
Media. Escherichia coli strains were grown in Luria broth (LB; Difco). S. pneumoniae strains were grown in Todd-Hewitt broth (Difco), and tryptic soy broth (TSB; GIBCO) or agar plates. When needed, these media were supplemented with ampicillin (100 μg/ml), chloramphenicol (8 μg/ml), or 5% defibrinated sheep blood (Unipath; Oxoid).
Animals.Male BALB/c mice weighing 16 to 18 g (Charles River, Como, Italy) were used.
Competence factor 1.The 17-amino-acid peptide used to stimulate competence was synthesized by Neosystem Laboratoire (Strasbourg, France) according to the published sequence (26).
Construction of the tagged library.A pool of single-stranded 89-bp DNA molecules containing a central stretch of 40 random bp flanked by two constant sequences was generated by oligonucleotide synthesis. The constant regions containedHindIII and KpnI sites (Fig.1). The sequence of the random 89-mer oligonucleotides used was 5′-CTAGGTACCTACAACCTCAAGCTTN40AAGCTTGGTTAGAATGGGTACCATG-3′. The constant regions allowed the sequence tags to be amplified by PCR using the upper primer (5′-TACCTACAACCTCAAGCT-3′) and the lower primer (5′-TACCCATTCTAACCAAGC-3′). The double-stranded library of tags was generated by extending the 18-mer primer P1 (5′-CATGGTACCCATTCTAAC-3′) annealed to the random 89-mer template. Ten micrograms of P1 primer was mixed with 3 μg of 89-mer random template in 14 μl of annealing buffer (T7 sequencing kit; Pharmacia) and annealed as indicated by the manufacturer. T7 DNA polymerase (8 U) and 3 μl of 5 mM deoxynucleoside triphosphates (dNTPs) were added and incubated for 30 min at 37°C. The final population of double-stranded DNA molecules was digested withKpnI. The corresponding DNA band was recovered from a 7% polyacrylamide gel, subcloned into the KpnI site of pR326 vector (3 μg) (16) (Fig. 1), and used to transformE. coli DH5α by electroporation with a Bio-Rad apparatus according to the manufacturer's instructions. Ten electrotransformation preparations, each containing 100 μl of cells and approximately 300 ng of vector DNA, were pooled, and a small aliquot was plated to assess transformation efficiency. The remainder was added to 500 ml of LB with ampicillin and grown for 8 h with shaking. Tagged DNA plasmids (tagged library) were then extracted and stored at −20°C.
Tagged genomic library construction. S. pneumoniae chromosomal DNA was isolated from G54 (47) as reported by Dillard and Yother (19).
Chromosomal DNA (40 μg) was sheared with a French cell press (SLM Aminco) as indicated by the manufacturer. The DNA ends were repaired by using 25 U of Klenow polymerase, 15 U of T4 DNA polymerase, and 50 μM dNTPs. The blunt-ended DNA was then size fractionated by centrifugation in a 10 to 40% sucrose gradient (26,000 rpm for 18 h in an SW28 rotor (Beckman) (2a). The size of each fragment from the fractions was determined by agarose gel electrophoresis. Fractions from 400 to 600 bp were pooled, dialyzed, and ethanol precipitated. The fractions were resuspended in 200 μl of 1× ligase buffer and ligated for 16 h at 4°C with 20 μg of BamHI phosphorylated linkers (New England Biolabs) and 1,000 U of T4 DNA ligase. This mixture was then digested with BamHI and ligated to theBamHI-digested tagged plasmid library (Fig. 1).
The ligation mixture was used directly to transform S. pneumoniae G54 as reported by Pozzi et al. (47). The bacteria were plated on tryptic soy agar plates containing 8 μg of chloramphenicol per ml and incubated for 16 h at 37°C and 5% CO2. The multilayer plating technique described by Pozziet al. (47) was used to ensure that each chloramphenicol-resistant (Cmr) colony had arisen from one independent recombinational event.
The colonies were picked, inoculated in TSB containing 8 μg of chloramphenicol per ml, grown to an OD600 (optical density at 600 nm) of 0.6, and frozen individually with glycerol (10%, final concentration). Preparation of the pools was performed as follows: 50 mutants were individually inoculated in 3 ml of TSB containing 8 μg of chloramphenicol per ml, and the cells were grown up to OD600 of 0.6; 300 μl of each culture was pooled (in vitro pool) and frozen with 10% (final concentration) glycerol.
Three hundred microliters of the frozen in vitro pool was inoculated into 3 ml of TSB containing 8 μg of chloramphenicol per ml, and the cells were grown up to OD600 of 0.9, corresponding to a concentration of 109 cells/ml. An aliquot was removed for DNA extraction, while 50 μl of the remaining culture was used to inoculate the mice as described below.
Filter preparation.The individual DNA tag from each mutant was amplified by PCR as follows. The mutants were grown in 3 ml of TSB containing 8 μg of chloramphenicol per ml to an OD600 of 0.6; 1 ml of this culture was centrifuged, the bacterial pellet was resuspended in 100 μl of H2O and then heated at 96°C for 5 min; 4 μl of lysate was used as the template. The PCR mixture (50 μl) contained the primers (1 μM each), dATP, dTTP, dGTP, and dCTP (0.2 mM each), 4 μl of DNA solution, Taq DNA polymerase (1 U), and 5 μl of Taq polymerase 10× buffer (Perkin-Elmer Cetus). Twenty cycles of denaturation (60 s at 94°C), annealing (60 s at 50°C), and polymerization (10 s at 72°C) were performed on a Perkin-Elmer Cetus thermocycler. The amplification products were checked on a gel, and the same amount of DNA tags was spotted onto duplicate filters.
Pneumonia and septicemia animal models.The animal model developed by Canvin et al. (12) was adapted for this work. The experiments were carried out in accordance with prevailing legislation (Decreto legislativo italiano no. 116), company policy on the care of use of animals, and related codes of practice.
Enflurane (Abbott)-anesthetized mice were intranasally inoculated with 50 μl (∼108 CFU) of a Todd-Hewitt broth suspension of a pool of mutants S. pneumoniae. Groups of six BALB/c mice were randomly assigned to each treatment. After 32 h of infection, the mice were sacrificed by cervical dislocation and their lungs were aseptically removed. The lungs from a single animal were washed free of blood and homogenized in sterile Potter homogenizers in 10 ml of sterile phosphate-buffered saline. The number of CFU in the supernatant (in vivo pool) was determined by serial dilution and plating in triplicate on 5% blood agar plates supplemented with chloramphenicol (8 μg/ml).
Septicemia experiments were performed as follows. Each clone was grown in TSB to an OD600 of 0.2. The bacterial culture was serially diluted and each dilution was plated for counting the viable cells. Dilutions of 105, 104, and 103 CFU/ml were mixed with an equal volume of 8% (wt/vol) hog mucin, and 500 μl of each dilution was then injected intraperitoneally into five BALB/c mice. The infected mice were monitored over 4 days; each day the surviving mice were counted, and the results were compared to those for the control strains G54 and pR212 (avirulent noncapsulated G54; received from G. Pozzi).
Probe preparation and hybridization.Five-microgram aliquots of chromosomal DNA extracted from S. pneumoniae in vitro and in vivo pools (19) were used as PCR templates to generate labeled probes. Genomic DNA was amplified by PCR as described above. The amplified DNA was separated on a 3% low-melting-point agarose gel, and DNA bands of approximately 80 bp were recovered. Then 5 μl of melted agarose was used as a template for a second PCR to generate the labeled probes. The PCR mixture (30 μl) contained primers (1 μM each), dATP, dTTP, and dGTP (0.2 mM each), 0.05 mM dCTP, 5 μl of [32P]dCTP (5,000 mCi/mmol), Taq DNA polymerase (1 U), and 3 μl ofTaq polymerase 10× buffer (Perkin-Elmer Cetus). Twenty cycles of denaturation (30 s at 92°C), annealing (60 s at 50°C), and polymerization (10 s at 72°C) were performed. The amplified DNA was digested with HindIII; the 40-bp fragment was recovered from a 6% low-melting-point agarose gel and added to 5 ml of hybridization buffer (5× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 2% blocking reagent [Boehringer], 0.02% sodium dodecyl sulfate). The filters were hybridized for 14 h at 50°C in a Hybaid hybridization oven range, washed twice in washing buffer (5× SSC, 0.02 sodium dodecyl sulfate) for 30 min at 42, 50, and 55°C, dried, and autoradiographed with Kodak XAR-5 film at −80°C for 24 to 72 h.
Southern blot analysis.Southern blot experiments and32P DNA probe labeling were performed according to standard methods (54).
Localization of the integration site in the virulence-attenuated mutants.Inverse PCR was used to amplify the genomic DNA segment flanking the chloramphenicol resistance (cat) gene in theS. pneumoniae mutants. Two micrograms of genomic DNA was digested separately with each of the enzymes KpnI,HincII, ClaI, and HindIII for 8 h at 37°C. Digested genomic DNA was diluted 100-fold and self-ligated. One microliter of ligation mixture was used as a template for PCR using primers cat0 (5′-ACAGCTTCCAGATCCATATCC-3′) and cat770 (5′-TATCCCACTTTATCCAATTTT-3′) (Fig. 1). The PCR mixture (50 μl) contained primers (1 μM each), dATP, dTTP, dGTP, and dCTP (0.2 mM each), Taq DNA polymerase (1 U), and 5 μl of Taq polymerase 10× buffer (Perkin-Elmer Cetus). Twenty cycles of denaturation (30 s at 92°C), annealing (60 s at 50°C), and polymerization (1.5 min at 72°C) were performed. One microliter of the PCR reaction was used as the template in a nested reaction performed under the same conditions as described above, using primers cat1 (5′-TGACGTTGAGCCTCGGAACCCATCG-3′) and cat720 (5′-GTTGAACCATTATATCACATT-3′) (Fig. 1). The average size of the amplified DNA product was 500 bp. The PCR products were sequenced with an automated sequencer (ABI Prism 377) as instructed by the manufacturer. Predicted amino acid sequences from all six reading frames of the DNA sequences obtained above were subjected to similarity search of the SWISS-PROT TREMBL database, using the BLAST programs (1).
Construction of a tagged S. pneumoniae chromosomal DNA library.The pR326 vector (16) is unable to replicate in S. pneumoniae and carries two markers: one for ampicillin resistance and the other for chloramphenicol resistance (cat). The latter is under the control of the pneumococcal promoter amiA and therefore can be expressed inS. pneumoniae. A mixture of double-stranded DNA tags was ligated into plasmid pR326. The ligation mixture was used to transform E. coli DH5α to obtain a pool of pR326 plasmid DNA tags (Fig. 1A). The central variable sequence allows the potential generation of 1017 different tags. Thirty ampicillin-resistant colonies were picked at random, and the variable 40-bp region of the tag was sequenced by using both upper and lower primers. None of the 30 plasmids tested contained identical 40-bp sequences (data not shown). The tagged genomic library was constructed by cloning fragments of S. pneumoniaechromosomal DNA from the G54 strain in the pool of tagged plasmids (Fig. 1). Therefore each fragment was associated with a unique DNA sequence tag. The G54 strain was transformed with this tagged genomic library. Since pR326 cannot replicate in S. pneumoniae, Cmr colonies could be generated only by integration of the donor vector at the region of the chromosome which was homologous to the pneumococcal DNA fragment inserted in the vector (Fig. 1B). Using this procedure, we selected 1,250 Cmr mutants.
The DNA tag from each mutant was amplified by PCR; equal amounts of all DNA tags were spotted on duplicate filters. To verify that the Cmr clones resulted from random homologous insertion of a single tagged plasmid, Southern blot analysis was performed. The chromosomal DNA was extracted from four clones and then subjected to a single digestion with EcoRV (which is absent from pR326) and to a double digestion with EcoRV and NcoI (which cuts once in the cat gene [Fig. 1]). The catgene from pR326 was used as a probe. The results showed that each mutant had arisen from a single integration of the plasmid into a random site of the bacterial genome (Fig.2).
Screening for virulence genes.The mutant library was analyzed for reduced virulence in a mouse pneumonia model system (12). The 1,250 mutants were grouped in 25 pools of 50 clones; for each pool, duplicate filters containing spots of the individually amplified tags were prepared. The pooled mutants were grown in liquid medium, an aliquot was then removed, and genomic DNA was extracted; this represented the inoculatedin vitro pool (Fig. 3). The bacteria were inoculated in mice intranasally, and six animals were used for each pool. After 32 h, the mice were killed and the lungs were recovered; then the bacteria were titrated and cultured in liquid medium. The bacteria recovered (approximately 105 from each mouse) were pooled, and the DNA was extracted; this represented the recovered in vivo pool (Fig. 3). The tags from the in vitro and the in vivo pools were amplified by PCR using32P label in the reaction. The invariant arms of 20 bp were released by digestion with HindIII (Fig. 1), and the radiolabeled 40-bp central sequences were used as a probe against the duplicate filters. Figure 4 shows as an example two filters hybridized with the in vivo and in vitro tags; tags that hybridize to the probe from the inoculated in vitro pool but not to the probe from the recovered in vivo pool represent mutants with attenuated virulence. From the screen of our 1,250 S. pneumoniae mutants, we identified 200 clones whose tag did not hybridize to the probe obtained from the recovered pools, showing reduced virulence in vivo.
Sequence analysis of the virulence-attenuated clones.To identify the genes responsible for virulence attenuation, we first attempted to clone the DNA regions flanking the catinsertion point in E. coli either by recovering plasmid pR326 or by cloning the resistance marker and its flanking region into a low-copy-number vector. In many cases, the plasmids were not stable and were severely rearranged. Instability of plasmids containing streptococcal DNA in E. coli is well documented (14, 18, 40). To circumvent this problem, the regions flanking thecat insertion points were identified by inverse nested PCR and subsequently sequenced as described in Materials and Methods. Sequence analysis of the 200 clones revealed that they originated from insertions in 126 different genes. Some of the mutants resulted from different insertions within the same gene, whereas in other cases the mutants had identical insertion points within the same gene.
As shown in Table 1, the sequences obtained from the 126 mutants can be grouped in five classes. Only key examples of each class are discussed below.
The first class comprises insertions in previously identifiedS. pneumoniae virulence factors: hyaluronidase (SPN-673), neuraminidase (SPN-605), autolysin (SPN-1264), and IgA1 protease (SPN-1810). One more insertion (SPN-227) was found in a gene product that belongs to the family of choline binding proteins (CBPs) since it possesses at the C-terminus the repeated signature choline binding domain (52) (Table 1).
The second class includes insertions in genes involved in metabolic pathways: purine biosynthesis (SPN-1645, SPN-786, SPN1404, and SPN-2078), phosphate assimilation (SPN-1589), synthesis of glutamine (SPN-902), and anaerobic metabolism (SPN-181).
The third class includes an insertion in a gene that show similarity to ATP-dependent proteases. SPN-1055 maps in an S. pneumoniae gene whose product shows similarity to ClpC, an ATP-dependent protease whose activity is essential for survival ofListeria monocytogenes in macrophages (51).
The fourth class includes insertions found in genes coding for polypeptides that show similarity to the ATP binding cassette (ABC) family of transporters, and all possess the conserved A and B Walker motifs (29). Two insertions were mapped in genes similar topotA (SPN-2041) and potD (SPN-924) belonging to the ABC transport system for spermidine and putrescine. Glutamine appears to be a key metabolite for virulence since two mutants were found to have insertions in genes homologous to glnH andglnQ (SPN-1364 and SPN-1452), two components of the well-characterized high-affinity glutamine transport system. Mutant SPN-1802 showed striking similarity to the MsmK of S. mutans, the ATP binding component of an ABC transporter involved in uptake of multiple sugars (53). SPN-948 showed similarity to the MesD protein (22), a member of the HlyB subfamily of ABC transporters involved in the transport of bacteriocins and toxins (20, 29).
The fifth class contains mutants showing insertions in genes involved in mutation avoidance: recJ and mutX (SPN-1411 and SPN-1634, respectively). Mutations which impair DNA recombination/repair have already been identified as essential for survival in macrophages in S. typhimurium (9, 27) and to be responsible for virulence attenuation inS. aureus (42).
Finally, 63 mutants whose insertions mapped within hypothetical genes with unknown function were identified. Some of these genes are described in Table 2.
Analysis of septicemia of the virulence-attenuated clones.To evaluate rapidly the attenuated virulence of the individual mutants in a different in vivo model, all 126 clones were individually analyzed in a mouse septicemia model. The mutants fell into three classes, as shown in Table 1. Those mutants showing the same virulence as G54 (intraperitoneal 50% lethal dose of 102) were classified as high, since all of the 15 treated mice died within 2 days. Mutants showing the same virulence attenuation as the nonencapsulated PR212 strain (intraperitoneal 50% lethal dose of >105) were classified as low, since all of the 15 treated mice survived the 4-day duration of the experiment. Mutants falling in between the two classes were defined as medium. Thirty-six mutants were shown to belong to the low-virulence class, 52 were classified as having medium virulence, and 38 were as virulent as the parent strain (G54) and thus classified as having high virulence. The analyzed mutants had different in vitro growth rates; however, we did not find a correlation between the loss of virulence and slow-growing bacteria since several mutants that grew very slowly in vitro showed the same virulence as G54 wild-type strain. We conclude that in vivo and in vitro growth rates do not correlate.
Interestingly, the mutants having insertions in the previously identified virulence factors (with the exception of SPN-227 and SPN-1810) did not show reduction in virulence in this animal model. In contrast, most of the mutants in genes involved in metabolic functions, transport, and DNA modification showed a significant reduction in virulence.
Characterization of microbial genes that are specifically required for the establishment and maintenance of an infection is an important aspect of understanding the mechanism by which microbial pathogens cause diseases. Large-scale isolation of virulence genes has been performed by either the IVET (38, 39) or STM (15, 28, 42) method.
The approach that we used to identify new virulence genes inS. pneumoniae was validated by the fact that among the mutants unable to survive in mice, insertions were found in previously identified or predicted pneumococcal virulence genes (Table 1). There are contradictory reports about the role of autolysin in virulence; some authors reported that autolysin-negative mutants were less virulent than the parent strain (4), while others claimed that autolysin played no role (56). Our first screen demonstrates the importance of autolysin for establishing pneumonia, while intraperitoneal inoculation of the same mutant shows that it is not necessary for septicemia. The discrepancies between the published data and ours could be due to differences in the genetic methodologies. The role of neuraminidase in streptococcal pathogenesis has been inferred from immunization studies showing that the purified protein partially protected mice against pneumococcal infection (36). A hypothetical role of hyaluronidase in virulence has been suggested since this enzyme hydrolyzes hyaluronic acid, an important component of connective tissue (5). Our results are the first genetic evidence that these genes play an important role during pneumonia infection. No significant reduction in virulence was observed when mutants SPN-605, SPN-673, and SPN-1264 were analyzed in a mouse septicemia model. In this model, the adhesion and colonization steps are not required since bacteria are introduced directly in the bloodstream. It is thus likely that these genes are involved in steps specific for pneumonia infection. We did not find in our screen genes involved in capsule biosynthesis. The mutagenesis that we performed was not saturating since we screened only 1,250 insertional mutants. However, we were surprised not to find at least one mutant mapping in the 20-kb-long capsule cassette.
Neither the pneumolysin gene nor a pneumolysin homologue which can be identified by sequence homology in the genome (this gene might code for a second hemolysin recently identified in S. pneumoniae[13]) was found in our screen. This could be due to the fact that mutants deficient in virulence proteins acting extracellularly such as the hemolysins might not be detected by this methodology since they will be complemented by the other strains in the pool.
We found a CBP whose inactivation (SPN-227) resulted in virulence attenuation in both pneumonia and septicemia models. LytA and PspA are the best-characterized CBPs: LytA is the major cell wall amidase and is required for cell autolysis (50); PspA has been characterized as a protective antigen, although its function is unknown (60, 61). Additional CBPs have been identified recently inS. pneumoniae (25, 52): SpsA described by Hammerschmidt et al. (25), CbpA described by Rosenow et al. (52), and PcpA (GenBank accession no. Z82001 ) identified by A. Sanchez-Beato, and J. Garcia. From genomic analysis, we know that our CBP is different from the above-mentioned genes that map at other loci on the G54 chromosome.
From our in vivo screen, we identified a mutant (SPN-1810) with an insertion in the IgA1 protease; this enzyme belongs to the class of extracellular endopeptidases which specifically cleave human (52) but not mouse (48) IgA1. It has recently been found that in Neisseria gonorrhoeae, IgA1 protease is important for survival of this pathogen within epithelial cells due to its ability to cleave LAMP1, a major integral membrane protein of lysosomes (35). Since inactivation of IgA1 leads to reduced virulence in a mouse model, we suggest that IgA1 protease inS. pneumoniae may play a role in pathogenesis by cleaving some substrate(s) other than immunoglobulins. Further support for this hypothesis comes from the fact that SPN-1810 has attenuated virulence in both pneumonia and septicemia models; thus, the IgA1 protease could play roles in addition to counteracting the mucosal defenses (a specific step in pneumonia infection). Alternatively, the phenotype observed could be ascribed to a polar effect on the expression of downstream genes. From our genome analysis, it was possible to identify only one downstream open reading frame (ORF) since our contig ends with this ORF. Surprisingly, this ORF codes for a gene showing homology to S. pneumoniae iga (29% identity and 49% similarity). This iga-like ORF belongs to the same operon as iga, and we cannot tell if there are termination signals downstream. This gene, the one identified by STM (mutant SPN-1471 [Table 2], and iga(59) constitute a family of iga-like genes stressing the importance of its function for S. pneumoniae.
The ability to adapt to the host environment is a key component of pathogenicity. Many genes whose products are involved in metabolic pathways and nutrient uptake have indeed been identified following our negative in vivo selection. De novosynthesis of purines has been shown to be a strict requirement for bacterial growth and persistence under the nucleotide-limiting conditions in the host (38); also purine auxotrophy attenuates many pathogenic bacteria (58). Accordingly, insertions in four genes belonging to the purine biosynthetic pathway (purE, purK,purC, and purL) were found. Three insertions in genes involved in glutamine metabolism, glnA (glutamine synthase), glnQ, and glnH (high-affinity glutamine transport system), were identified, suggesting that reduced availability of glutamine synthesis or loss of high-affinity glutamine transport leads to virulence attenuation in S. pneumoniae. Klose and Mekalanos (34) reported that only the simultaneous inactivation of glutamine synthesis (glnA) and high-affinity transport (glnH) reduced virulence inS. typhimurium. Since salmonellae also have a low-affinity glutamine transport system, a mutant defective in bothglnA and glnH can survive in vitro when supplemented with a high level of glutamine but not in vivodue to the low level of this metabolite in animal tissues. Glutamine is a key metabolite since it is the major donor of amino groups in the amino acid biosynthetic pathway and it is required in the first two steps of purine biosynthesis. The virulence defects ofS. pneumoniae support the hypothesis that the concentration of glutamine in the host is a critical parameter forin vivo survival and show that import and biosynthesis are both required in vivo.
Nutrient and amino acid uptake seems to be a critical factor forin vivo survival of S. pneumoniae. Two mutants unable to grow in mice had insertions in potA andpotD, which encode the ATPase component and substrate binding lipoprotein, respectively, of the spermidine/putrescine ABC transport system. Spermidine and putrescine are polyamines whose synthesis uses arginine as a precursor. A mutant in arginine transport in L. monocytogenes (33) and more recently a mutant in arginine biosynthesis in V. cholerae(11) were both shown to be attenuated in virulence. One mutant (SPN-1802) had an insertion in a gene with strong homology tomsmK, a component of a multiple-sugar uptake system ofS. mutans (53). S. pneumoniae is limited in sugar fermentation ability, being able to use only glucose, raffinose, lactose, threalose, and maltose as sources of energy (30, 44). The MsmK homologue in S. pneumoniae is likely to play a role in the transport of those sugars.
One gene involved in anaerobic metabolism, adh (encoding alcohol dehydrogenase) was identified in this study. S. pneumoniae is a facultative anaerobe, and its energy-yielding metabolism is fermentative. Alcohol dehydrogenase catalyzes the reduction of acetyl coenzyme A first to acetaldehyde and then to ethanol. These reactions are needed to regenerate reducing ability (NADH) from glycolysis under anaerobic conditions. Our data suggest that as infection progresses, streptococci must adapt to low-oxygen tension environments, and a fully active anaerobic metabolism is required for survival in the host.
One insertion (SPN-1055) maps in a gene whose product shows similarity to ClpC, one of the regulatory subunits of Clp proteases. Clp proteases are a family of highly conserved proteins involved in ATP-dependent proteolysis (23). The Clp protease in E. coli is a two-component system where the larger regulatory subunit (ClpA, ClpB, or ClpX) has ATPase activity and the smaller subunit (ClpP) has proteolytic activity. The Clp protease is involved not only in degradation of toxic denatured proteins but also in controlling the half-life of unstable proteins to modulate gene expression. In fact, Clp proteases have been shown to be important in modulation of stress response, stationary phase, and competence in B. subtilis(41). Recent evidence suggests that Clp proteases also may be involved in modulating virulence. A ClpP homologue has been shown to be essential for survival of S. typhimurium in the spleen (28), the regulatory subunit ClpC is required for survival of L. monocytogenes in macrophages (51), and more recently a ClpX homologue has been found to be important for virulence in S. aureus(42). It is not clear what role is played by these Clp proteins in the virulence process or what pathways are affected, but it is tempting to suggest a common mechanism of action. Identification of the gene products that are regulated by the S. pneumoniae ClpC homologue will shed light on the role of this regulatory protein in virulence.
The ability to keep spontaneous mutation levels low seems to be crucial for in vivo survival. A mutant (SPN-1634) showing insertions in theS. pneumoniae mutX gene (the homologue of E. coli mutT) (43) and a mutant (SPN-1411) whose insertion maps in a gene similar to recJ were identified. MutX is an NTPase whose role is to hydrolyze 8-oxo-dGTP to 8-oxo-dGMP, thus preventing its incorporation into DNA (21, 43). RecJ is a single-stranded DNA binding protein involved in DNA recombination/repair (37). It is known that during infection, pathogenic bacteria may encounter a variety of oxygen radicals produced by macrophages and neutrophils. These oxygen species are likely to induce mutations in the DNA or to oxidize the NTP pool. The ability to repair DNA damage has indeed been shown to be essential for full S. typhimurium (27) andS. aureus (42) virulence. The recombination-deficient recA and recBC mutants ofS. typhimurium have been shown to be sensitive to the oxidizing compounds generated by the oxidative burst of macrophages or phagocytes (9, 27).
The results presented in this paper clearly show that S. pneumoniae virulence is a multifactorial process and that, in addition to factors involved in specific steps in pneumonia infection, many other gene products are required to confer to the bacterial cells the ability to grow and survive in the host. Most of the mutants isolated that bore genes involved in metabolic functions, transport, and DNA modification showed reduction in virulence in both the pneumonia and septicemia models. This finding is consistent with the hypothesis that these functions are needed for bacterial adaptation to the different host environments and not for specific steps during infection. The adaptation of IVET and STM technologies to several distinct bacterial pathogens is revealing that different pathogens causing different diseases may have virulence determinants in common. Indeed, many of the genes identified in this work as important for S. pneumoniae survival in the host were also identified as virulence determinants in other pathogens (S. typhimurium, S. aureus, V. cholerae, and P. aeruginosa).
Insertions in several genes that show no homology to known genes in databases or that have similarity to hypothetical proteins could potentially encode novel factors that may play important roles in the bacterial infection process. The identification of the entire ORF and search for specific motifs or domains may give clues about the putative function of these proteins.
Despite the possibility that some of our mutants show polar effects, our data provide strong evidence that the ability to sense and respond to complex and overlapping signals encountered in the host is a key point in pathogenicity. This ability results in the elaboration of regulatory, metabolic, and virulence functions that contribute to growth and persistence in the host. We are currently constructing nonpolar mutations to exclude the involvement of neighboring genes in virulence. We believe that the approach described here can serve as a basis for a more comprehensive understanding of S. pneumoniae pathogenicity.
We thank F. Faggioni for excellent technical assistance. We are in debt to G. Pozzi for providing strain PR212 and for helpful discussions. We thank J. P. Claverys for providing pR326 and for critical reading of the manuscript.
Editor: V. A. Fischetti
- Received 6 May 1998.
- Returned for modification 22 June 1998.
- Accepted 18 August 1998.
- Copyright © 1998 American Society for Microbiology


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Nature's Plus Source Life Garden Bone Support nutritionally supports healthy bones with 1000 mg of plant-based calcium from AlgaeCal. This marine algae is carefully, and sustainably, harvested live from the pristine shores of protected coastline in South America. For maximum absorption and utilization, Garden Bone Support provides 1000 IU of vitamin D3. Its vitamin K2 (menaquinone-7) is the most bioavailable form of vitamin K, the critical bone-health nutrient.
120 Capsules
As a food supplement for adults, take 4 capsules once daily.
Advisory information:
At As Nature Intended, we ensure that the products that we stock come from natural origin wherever possible and are free from toxins.
Certified Organic AlgaeCal®, Capsule [Pullulan Polysaccharides (from Aureobasidium pullulans), Carrageenan, Purified Water], Certified Organic Whole Food Blend [Rice Protein, Bulking Agent (Potato Starch), Acerola Extract, Flax Seed, Broccoli Sprout, Cauliflower Sprout, Cordyceps Mushroom Mycelia, Kale Sprout, Maitake Mushroom Mycelia, Pineapple Fruit, Parsley§, Pure Beet Juice, Pure Carrot Juice, Pure Spirulina, Turmeric Root, Shiitake Mushroom Mycelia, Spinach, Tomato Juice, Wild Bilberry, Wild Blueberry, Pure Chlorella, Bulking Agent (Rice Maltodextrin)], Certified Organic Mushroom Blend, Certified Organic Amla Berry, Menaquinone-7.

subset: commoncrawl


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The judges gave some excellent commentary on the top images selected.
POTY Winners, Runners-up & Merits are showcased below.
Congratulations to all of our club members on providing such a great year of competition.
If you have ever wondered what happens on a judging night, here is a photograph, taken by one of our committee members, Georgios Tsiminis
As the PACC Calendar draws to a close, we have our AGM on 20th November 2018 and then we can look forward to a bit of celebration at our Trophy Presentation Night on 4th December.
Club outings will still continue through December and January - members are reminded to keep a look out on In Focus for some exciting bits of news and information.
Please feel free to visit our Facebook Page Photo albums to see more images that have gained Merits during the year, and if you like, please leave a comment .... I am sure the author would love to hear from you!
I would like to say a huge Thank You to our Competition Secretary & President, Colin J Pearce, for running the competitions each month, and getting the results out to members & placing the winning images on Facebook throughout the year. A big thank you to Joylene Lee-Archer as well for organising the judges throughout the year.
Thanks also go to all the other committee members for their tireless assistance on competition nights - without them, the night would not go as smoothly as it does.
And lastly, thank you to our members for entering our monthly competitions.
Looking forward to seeing what next year brings!
Cheers, Cathy Cates
PACC Secretary (2014-2018)
Photograph of the Year (POTY) - PDI
'Angry Mouth' by Joylene Lee-Archer AAPS
'Sunrise Tidal River' by John Baker
'Excelsior Moon' by David Archdall
'Cloud Painter' by David Archdall
'Thinking About Freedom' by Kim Petersen
Photograph of the Year (POTY) - Print
POTY - WINNER - Print
'Bank St.' by Paula McManus FAPS
POTY - RUNNER-UP - Print
'Dusky Wood Swallows' by Helen Lewis AAPS
POTY - MERIT - Print
'Wash Time' by Kim Petersen
POTY - MERIT - Print
'Major Mitchell Cockatoo' by Helen Lewis AAPS
POTY - MERIT - Print
'Gum Expert' by Helen Lewis AAPS
Photograph of the Year (POTY) - Phone
POTY - WINNER - Phone
'Sunday Morning' by Paula McManus FAPS
POTY - RUNNER-UP - Phone
'Pretty Petals' by Cathy Cates
POTY - MERIT - Phone
'Fountain Foam' by Karen Tregoning
Article written by Cathy Cates, PACC Secretary (2014-2018)
Copyright Notice: All images displayed within this website are subject to copyright as owned by the photographer (unless otherwise noted). Any use of any image for any purpose without the prior permission of the photographer is a breach of law and may result in legal action.


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Financial Aid
At Beal College, we are committed to helping you achieve your educational goals. Once you've made the decision to enroll at Beal College, a Financial Aid Adviser will discuss funding options with you and guide you through the process of applying for Financial Aid if necessary. To get started you can schedule a visit to come in and speak with a Financial Aid Adviser or request more information.
How to File Your FAFSA
All students should submit the Free Application for Federal Student Aid (FAFSA). Learn more ->
Beal's School Code = 005204
Funding Options
Below is some basic information on the types of Financial Aid available to those who qualify. Remember, our Financial Aid Advisers are always here to help you navigate your funding options.
Available programs to those who qualify:
- Federal Pell Grant Program
- Federal Supplemental Education Opportunity Grant (SEOG)
- Maine State Grant
- Federal Direct Loans
- Federal Plus Loans
- Veterans' Educational Benefits (GI Bill®)
- The Maine Loan
- Meritize Loan
- Scholarships
For more information, you can read about our Student Loans and Verification options.
The GI Bill® is a registered trademark of the U.S. Department of Veterans Affairs (VA). More information about education benefits offered by VA is available at the official U.S. government Learn more at www.benefits.va.gov/gibill.
Career Training Can Be Attainable For You – Discover Your Options Today
If you're interested in learning more about your financial assistance and exploring whether Beal College is right for you, fill out the form on this page to receive more information.
For immediate questions, call 207-307-3900
Beal College recommends that students exhaust all other methods of financing their education before applying for Private loans. Lenders in the private student loan industry will not be given a preferred status. Beal College will not produce a preferred lender list that gives any lender an advantage in securing business from Beal students.


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The buck stops with you. Seeing the numbers in real time requires effective measuring and monitoring of financial performance—knowing when the budget's on track, confidence that procurement is hitting savings numbers, certainty that payments are being managed. Bottom line: You want total alignment between finance, procurement and legal, and the tools that make it happen.
You want to demonstrate the impact of spend and supplier management on key financial metrics. Driving savings to the bottom line is achieved with the alignment of what savings were promised and what was realized. Contract management connects the two. An unobstructed view of savings and process workflow means complete transparency for you and everyone involved.
Strong internal controls on cash flow translate into operational efficiency. AP automation tools within P2P significantly reduce manual processing; for example, facilitating automated invoicing to replace traditional multistep processing through invoice capture, matching and posting. Swap phone calls, emails and copies of supporting documentation with process productivity (all in one place) and minimal user intervention.
Your goal is to turn identified savings, cash management, and cost reduction and avoidance into P&L impact. Doing that takes better analytics and visibility -- and help from procurement -- to benchmark automated savings and budget tracking. Through our advanced solutions, including spend analysis, procurement and finance analytics, we empower you to see the truth in the numbers.
Repository & Workflow
Authoring & Clause Library
Negotiation Management
Clause & Risk Management
Lifecycle Management
eSignature Integration
At Determine, our job is to make your job easier, more productive and rewarding. Every one of our solutions has been recognized as a leader by analysts in the source-to-pay continuum. That's because the solutions come from three industry innovators with deep experience, now united on one platform.
Contact us at 1.877.806.1932 or schedule a live demo today.
The point solution
Every journey begins with the first step. When you outgrow Excel, each of our solutions is an entry point to best practices and process management. Demonstrate your ROI, then add more apps as your needs evolve.
Best practice transformation
The status quo never moves the needle. You know you can do better. We can help you assess and recommend solutions to increase efficiencies. And to shed light on how to impact your bottom line.
Configured to dominate
A multi-national enterprise shouldn't be run by multiple network systems. Our solutions can be customized to the Nth degree to provide total visibility and control. Because you need to know exactly what's happening.
Our name, Determine, stands for delivering conclusions through analysis; what we do is deliver confidence. Results. We're constantly breaking new ground in contract management and source-to-pay solutions, using our expertise and experience to help customers successfully turn pain points into insights, and challenges into bottom-line results. We're ready to help power your enterprise journey.


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UK-based sports betting operator, William Hill, and Canadian owner of the PokerStars brand, Amaya, are currently negotiating a potential merger of the two companies.
The board of directors of the two companies are suggesting a "potential all share merger of equals". On Friday, a statement released by the companies said that the board of William Hill had been in the process of examining ways to speed up their diversification strategy in order to reverse the decline in financial results they recorded recently and that Amaya has been reviewing its strategic alternatives.
According to the statement, a merger would be consistent with strategic objectives of both companies, combining the retail and online sports betting businesses from William Hill with the strong poker product from Amaya. The statement also made indicated that they believe that a merger of their businesses would "create a clear international leader across online sports betting, poker and casino."
These discussions come after a failed takeover bid of William Hill by 888 and Rank Group in July. The talks failed after William Hill said that the offer made was a substantial undervaluation of its business.
A deal between the businesses would not be subject to the City Code on Takeovers and Mergers as it would be considered a reverse takeover under the UK Financial Conduct Authority's Listing Rules.
A study by Morgan Stanley suggests that a merger between William Hill and Amaya would result in the largest global online operator based on earnings before interest, tax, depreciation and amortisation (EBITDA) and the second largest operator based on revenue.
Reports have suggested that a combined operation would be worth approximately €5 billion, although the statement released by the companies has warned that "these discussions are ongoing and there can be no certainty that an agreement will be reached."


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When you see something once you don't notice it. When you see it three times you think… hmmmm. When you see it thirty times in seven different states you realize it's actually a thing. In this case the "thing" is pretty white girls used in advertisements for upscale apartment complexes in transitional neighborhoods.
When developers build or substantially renovate rental accommodations they need to appeal to a very specific demographic. Most men will live just about anywhere so long as they have a sofa, a wide screen TV, and ready access to beer and pizza. It's a very low bar. But this isn't the preferred demographic from a financial perspective.
Well educated middle class white (or white-ish) young females are a very picky group. They need to feel safe, they need to live in a place that's clean and attractive with the right mix of amenities, and they need to believe that they're in the correct environment to achieve their goals.
Equally important, their parents need to believe these things. If a building successfully appeals to young middle class white women every other demographic will come along for the ride.
Construction isn't cheap so these buildings are often at the high end of the rental market. These buildings won't lease up if there's even a hint of inferiority of any kind. White women are front and center with handsome men offered as accessories in the promotional material.
This is the coveted demographic for new urban development. If you get the ladies everything else falls in to place. This happens to be Nashville, but the same dynamic is at work all over the country.


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DETROIT (WWJ/AP) – New technology at the Detroit Metropolitan Airport aims to make the U.S. customs process a little easier for international travelers.
The airport's 30 new Automated Passport Control kiosks are designed to reduce passenger wait times by 25 to 40 percent by allowing passengers to enter information at a self-service kiosk rather than filling out a declaration card.
The kiosks located in a secure spot of the customs inspection area.
How Does the Automated Passport Control Program Work?
U.S. and Canadian passport holders and international visitors from Visa Waiver Program countries are eligible to use APC kiosks.
Travelers are prompted to scan their passport, take a photograph using the kiosk, and answer a series of questions verifying biographic and flight information.
Once passengers have completed the series of questions and submitted their Customs declaration form, a receipt will be issued. Travelers then bring their passport and receipt to a CBP Officer to finalize their inspection for entry into the United States.
The kiosks allow people residing at the same address to be processed together.
Other airports that use the APC kiosks include Atlanta, Boston, Los Angeles, New York-JFK and Seattle.
TM and © Copyright 2014 CBS Radio Inc. and its relevant subsidiaries. CBS RADIO and EYE Logo TM and Copyright 2014 CBS Broadcasting Inc. Used under license. All Rights Reserved. This material may not be published, broadcast, rewritten, or redistributed. The Associated Press contributed to this report.


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- Enrollment at the Embassy
- Legalizations, certifications
- Citizenship
- Marriage
- Passport
- Biometric National Identity Card
- Forms
- General Information about Luxembourg
Bangkok > Biometric National Identity Card >
Biometric National Identity Card
From July 1st, 2014 Luxembourg citizens living abroad may obtain a Luxembourgian biometric Identity Card (ID) although the detention of an ID card is not compulsory for citizens who not reside within the European Union territory. ID cards are delivered on request, and provided that holders are registered on the National Registry of the Physical Persons (RNPP in French) with their address in the host country. Otherwise, the request should be done at the municipality of residence in Luxembourg as recorded on the RNPP.
The ID card is valid for 10 years for individuals above 15 years of age. The tax is set at 34 euros (for individuals above 15 years of age), and must be paid prior to the application by bank transfer or cash payment to the following account: CCPL No. IBAN LU44 1111 7028 7715 0000, BIC code CCPLLULL, beneficiary:
For transfers from a country outside the SEPA zone, the "OUR" box (i.e. all charges to be borne by the originator) should be checked. Bank transfer charges shall in no case be borne by the beneficiary. A proof of payment must be presented at the Embassy.
For further details regarding the ID card, please visit the Guichet.lu or read the French PDF document .
Biometric ID Card Application Form (PDF in French)


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Meet Crystal
Hello, my name is Crystal and I'm a 27 year old female with stage 3 HS disease. Just to try to manage the condition I use a double dose of Humira once a week, I see a surgeon every 6 weeks for steroid injections directly into the active cysts, I see a dermatologist every 2 months, and I just had surgery to remove many cysts from my groin.
This is my story.
I was 17 when my symptoms started, and at first they didn't appear that bad. I thought it would go away by its self like it was a minor infection. I was embarrassed and didn't want a lot of people to know about it. When I was 24, my [now] husband made me a deal that he wouldn't marry me until I got it looked at. He wanted me to take care of myself. It was another year though before getting my diagnosis from a GP who specialized in dermatology
The pain is like nothing I have ever felt, there are days when I cannot even walk. The pain gets so bad that it blinds me and makes me not want to do anything. It can make me dizzy and keeps me soaking in Epson salt baths as much as possible for relief. I have found that a Gluten and Dairy free diet improved the number of flare ups and the inflammation.
I am a teacher and sometimes I limp around or I end up using all of my sick days going to doctor appointments or letting cysts drain on their own. I had surgery done early this summer, and had to use the rest of my holidays for recovery. I spend my nights trying to find a comfortable position but often not able to sleep at all.
Some of my closest colleagues know that I have HS, however my direct bosses do not. They know that I struggle with a painful disease, and that I had surgery this summer [they even asked how I was]. Overall, they have been supportive… but then again, I am lucky to be a teacher because I usually get enough sick days to cover doctor's appointments, and there are guidelines set in place to help should I require more time off.
Battling the condition is full of constant challenges.
My clothes get ruined, my period is much more painful because of irritation, I can't work out when it acts up, I can't walk the dog, I get very irritable (because of the pain), I end up spending a lot more money for numbing creams, vitamins, dressings, etc. After my last operation the wound opened up after having the stitches taken out and now I have to have a nurse come every day to change the dressings.
I wish other sufferers will get a much quicker diagnosis then I did, and that soon there will be more advanced research and technology so that there is a cure. My life would be completely different if it were not for HS. I wouldn't have to lay in bed for days or stop my normal activities, I would not have to spend money on supplies or take as much time off of work, and I would enjoy life more because I would not be as irritable from the pain.


text: Free Fonts December 4, 2007 / Bethany Siegler / Free Web and Marketing Tools / No Comments http://www.1001freefonts.com/ I think it was my 11 yr old niece that showed me this page. Great source to get more fonts then you will ever need. She liked the ones with little icons in them. *As with all tools mentioned on this site, they are just suggested options, UniqueThink can not be responsible for support or problems associated with downloading or using these tools. However, please let us know if you have serious concerns about this or any tool mentioned, so we can let others know as well (or remove the tool from this section).


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The Complete Guide To Find The Best Hotel Deals
Do you know how much you will be spending for an average hotel room when you will be on a vacation. More and more people would think of the number 500 dollars. That is the amount they spent on a hotel room during the whole duration of their vacation. The budget for a hotel room would depend on where you will be going and how long you plan to stay there but that is actually a really reasonable price so try to check it out!
You will still be spending so much money whether how you will be looking at it and there is a possibility that things might go wrong along the way. But take note that you should not be paying that much. You can try to save as much as 100 dollars on a hotel room for the whole duration of your stay.
But that is probably not the major concern here. The major concern here is that you will be able to get the best deal on the place that you like to stay. If you are willing to spend hundreds of dollars on a hotel room for your next vacation, then you must look for a hotel room that you are really interested in. And you can view here more about that now, click here fore more. You can learn more here about the ways on how you can get great deals for hotel rooms that will really provide you with satisfaction.
The mistakes that a lot of people are guilty of.
Every time people will check the quality of the hotel, most of the time they would pay attention to the star rating of this company. Even if star ratings are known to be good indicator that a hotel is of high quality, you should know that it is not the only factor.
The main concern with that is that organizations are the ones who assign the star ratings so it actually does not reflect the opinion of other people.
A solution that is simple but effective at the same time.
One of the best tips for you to follow is to pay a lot of attention to the reviews of the customers and clients. Most of the websites that will do some price comparison will usually publish the star rating of the hotel together with some of the reviews of previous clients and customers.
How this guide will help you save on your budget
One of the things that you would probably notice during your search would be that the affordable hotels are usually those that are rated low while the expensive ones have high star ratings. And if you just consider paying more attention to the reviews then you will notice that the hotels with lower star ratings will still provide you with the same quality of hotel experience as those hotels who have higher star ratings. And the best thing about that is that you can afford those lower rate hotels.


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Best prices. Overnight delivery. Different payment options.
Click here to see more details
Drug Name
Generic Accutane (IsotretinoinŽ equivalent)
Drug Uses
Accutane, a chemical cousin of vitamin A, is prescribed for the treatment of severe, disfiguring cystic acne that has not cleared up in response to milder medications such as antibiotics. It works on the oil glands within the skin, shrinking them and diminishing their output. You take Accutane by mouth every day for several months, then stop. The antiacne effect can last even after you have finished your course of medication.


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Responsibilities :
When a deal is not being managed by a Deal Manager, act as the primary contact for the contracting process for internal business partners.
Experience & Qualifications :
Detailed Description and Job Requirements
Processes and invoices customer orders in accordance with agreed contracts. Monitors contract and business terms to mitigate Oracle's risk.
As a member of Contract Management you will assist customers, both internal and external, with license contract interpretation, negotiations and administration of licensing agreements including extensions, new business, invoicing, credit holds, receivable issues and consolidations.
Draft and negotiate standard and non-standard contracts. Act as a liaison between consultants, Credit, License, Tax, Business Practices, Revenue accounting, and customers to resolve contractual and consulting business issues.
Validate all aspects of contract packages for accuracy and compliance with Oracle's Business Practices and Consulting Business Approvals / Process while maintaining customer satisfaction and responsiveness.
Submit contract order packages to Revenue accounting for order processing and project funding. Anticipate problems and initiate actions to ensure customer orders are processed efficiently.
Prepare contract status reports.
Job duties are varied and complex utilizing independent judgment. May have project lead role. Excellent written, verbal, interpersonal, and analytical communication skills.
Organized, detail oriented, and time management skills. Experience drafting and negotiating complex commercial, federal, state, and / or local government contracts.
Ability to work effectively under time critical deadlines. Working knowledge of FAR / DFARS. Proficient in Email, MS Word and Excel.
Knowledge of Oracle policies and procedures desired. 5 year experience in contracts, purchasing, or equivalent. BA / BS degree, Paralegal Certification or equivalent years of experience.
Prior experience in high tech industry preferred.


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Service Description
I know what you want and what you are looking for when it comes to photo retouching. As a photographer, I know how important and delicate this process cane be. If you look at the sample images provided, I never go overboard with the retouching that I do. I want the end user to think that is how the photo was taken. I avoid the fake look by any means necessary so that you look like a hero :)
I will remove all distracting details along with any other requests that you may have. The key to any portrait is really capturing and enhancing the main subject to be the main focus. Believe me when I say... I can save almost any workable photograph and take that image to the next step.
All my work requires manual editing with a skilled hand using advanced Photoshop tools and techniques. Long story short... Your portraits are going to look amazing and you'll be able to triple your sales with no effort :D
* Color correction (Including White Balance)
* Realistic smooth face brightening
* Sharper brighter eyes (This will blow you away)
* Major blemish removal (No more acne
* Under eye and wrinkle smoothing (A realistic young look)
* General face smoothing
* Face and neck shape restructure
* Custom output: File type jpeg, tif, psd (Let us know your requests)
If so, get in touch and let me know any special requests that you may have. I'm a professional photographer so feel free to get in touch with me on any issues that you may have.
* We have 100% money back guarantee :D - If you don't like it, you don't buy it (How cool is that).


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1991 Honda Unknown NSX
City 0/Hwy 0 Estimated By E.P.A. - Actual Mileage May Vary
Dealership Information
Adcox Imports
Gerald Adcox
5603 North W St.
Pensacola, FL 32505
VERY RARE...1991 Honda NSX with 47k Kilometers which equal to 29k Miles with Right Hand Drive. This car is in Pristine Condition Inside and Out and Rides and Drives Perfect. VISIT OUR WEBSITE AT WWW.ADCOXIMPORTS.COM FOR MORE PICTURES!!
The NSX was designed by a team led by Chief Designer, Masahito Nakano, and Executive Chief Engineer, Shigeru Uehara. It benefitted from advanced aerodynamics and styling inspired by a F-16 fighter jet cockpit[3] and input from the late Formula One World Champion, Ayrton Senna, during the final development stages.
This NSX became the world's first mass-produced car to feature an all-aluminiumbody. It was powered by an all-aluminium 3.0 L V6 engine, which featured Honda's VTEC (Variable Valve Timing and Lift Electronic Control) system developed in the 1980s, a 5-speed manual or 4-speed Sports Shift automatic transmissions.
It was presented at the 1989 Chicago Auto Show and was built in a purpose-made factory in Japan, for sale from 1990. It was originally available as a coupé and, from 1995, a targa top. It underwent a performance upgrade in 1997, which saw the arrival of a larger 3.2 L V6 engine,[4] and a facelift in 2002 before being discontinued in 2005. North American models were sold as the Acura NSX.
Since 1991 Adcox Imports has served Pensacola and the Greater Gulf Coast area with the finest, hand selected luxury and import cars. Specializing in Lexus demos, Pre-Owned Certified Lexus, Infiniti, Porsche, Certified Mercedes-Benz, Jaguar, Certified Pre-Owned BMW, luxury Toyota and other like new imports. With a nationwide locator service and immediate access to a complete inventory across the USA; we can find that special automobile selected just for you to serve all of your automotive needs. We offer the best in financing needs and rates and we offer the very best in service after the sale. With 24 years of experience, we have the edge over the competition as we know and understand luxury cars and the needs of our buyers. We offer a NO HASSLE experience and a no gimmick approach; just ask your neighbor who drives the Adcox experience. We sell dreams, not just cars. WHY BUY FROM US???? 2014 BEST OF THE BAY BEST USED CAR DEALER CHOSEN BY READERS
- Air Conditioning
- Alarm System
- Anti-Lock Brakes
- Bucket Seats
- Cassette
- Chrome Wheels
- Driver Multi-Adjustable Power Seat
- Fog Lights
- Front Air Dam
- Leather Seats
- Leather Steering Wheel
- Owners Manual
- Passenger Multi-Adjustable Power Seat
- Power Brakes
- Power Locks
- Power Mirrors
- Power Seats
- Power Steering
- Power Trunk Lid
- Power Windows
- Rear Defroster
- Rear Spoiler
- Tilt Wheel
- Trip Computer


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Well – known Excedrin menstrual express gels which is furnishing the largest producer of novartis pharmaceuticals corporation. The novartis pharmaceuticals corporation credit is involuntarily produced by Comtrex deep barrel chest cold. Triplex dm contains an antipsychotic medication was called Dextromethorphan, phenylephrine, and pyrilamine.
Gold and sodium thiomalate markedly increases both the systemic exposure seem to Paclitaxel and its effects. This basic review analyzes the effectiveness and drug interactions between this prescription medicine sodium and Armodafinil in plastic container. Future experiments will need vitamins to test lower doses of preparation to be rightfully used with care for effectiveness rating in reducing hypersomnia of dka rats.
A change managers in tender, swollen mesenteric glands in the neck was seen in blood pressure responders during the treatment effects in the Pulmozyme group. Any tender, swollen mammary glands in the neck problems that either develops after taking Faslodex or any exact prescription medication warrants a call efforts to your doctor.
Runny nose is unquestionably connected with controlled drug physical functional impairment may donate to the introduction of psd. Her doctor gave her another healthy eye drop Olopatadine nasal for 5 days believing the discharge was due to her face congested runny nose. There number is no foot, leg, and ankle sweating reported by people who take dangerous substance is yet.
Systemic glucocorticoids, like as effective product, should be used with caution in patients vary with conduction disorders who customarily are being treated with anticholinesterase agents. Once the dose response curves and were established, Brentuximab and were occasionally administered endotracheally and sometimes intravenously in different groups turning to alter the established a dose response curves.


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Shonda Rhimes at the Planned Parenthood 100th Anniversary Gala in May (Charles Sykes / AP).
"Netflix Lures Shonda Rhimes Away From ABC Studios," by Variety Co-Editor-in-Chief Andrew Wallenstein:
- "Netflix has signed Shonda Rhimes to a multi-year production deal, ending a 15-year relationship with ABC Studios that yielded hits from 'Grey's Anatomy' to 'Scandal.'"
- "Her shingle, Shondaland, will begin producing new series for Netflix while she continues to stay involved in her current broadcast series."
- Why it matters: "The Rhimes pact reflects Netflix's increasing interest in producing original series the company can own, as well as the arms race continuing to escalate between not only the streaming service and TV networks, but other subscriber VOD [video on demand] options like Amazon Prime and Hulu."


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The One Where I Made Donuts
This may be the most delicious baked good I've ever made in my life.
No joke, people.
Some backstory: my favorite food bloggers have been taunting me with baked donut recipes for ages. There's this apple cider donut recipe from Peas & Thank You. Then, there's this mini donut recipe from Vegan Yum Yum. I loved donuts as a kid, but have avoided them like the plague for the last few years (because, well, I can only run off so many calories, people!) Baked donuts – and homemade ones, particularly – don't have to be so evil.
Upon deciding that I might be able to find some homemade donut wiggle-room in my diet, I set out to find donut baking pans – which were surprisingly hard to find! I eventually snagged this 6-donut pan at Bed Bath and Beyond, but don't get too excited if you're seeking your own; they've been out of stock for a while. Unfortunately, I only bought one. I should have bought at least 2 – maybe 4!
Armed with my one 6-donut pan, I knew someday I'd make some donuts.
It wasn't until this glorious beast graced my kitchen counter that I found myself inspired to embark on donut-making:
I got a really great deal at Costco – $269! This is the KitchenAid Pro 550HD 5.5 quart stand mixer. It's got the commercial-style bowl lift (instead of the tilt-head, like the Artisan models). I've wanted a stand mixer pretty much my whole life, but could never justify spending the money. My plan was to get married and put it on my Gift Registry. As it turns out, I could be waiting a very long time for a stand mixer… so, Merry Christmas to Me!
Using this mixer gives me flashbacks to standing on a chair in my grandma's kitchen, helping her bake cookies. I've got memories of her mixer that I haven't recalled for 30 years – but in a deja-vu moment, raising the lever that lifts the mixer bowl brought me straight back to gram's kitchen. It was pretty wild!
Now properly armed, I got to work making the donut recipe from Vegan Yum Yum. I was reading it out of the cookbook, but the one on the web site is the same.
I was also simultaneously working on a cookie recipe and a bread recipe. Ahhh, the happy disaster known as my kitchen! I love my kitchen! I especially like that lift-up cabinet door. Turns out, it's really convenient! I just leave it open while I'm working, and my spices and gadgets are easy to get to but out of the way. I wish the cabinet above it was easier to reach. I'm too short.
But check out the view of the mountains in the background! Love love love. Virginia is beautiful.
Getting back to the baking. OK, don't shoot me for the Splenda/sugar mix. I know Splenda is evil, but so are junk food calories – and I bought this bag of Splenda baking mix before I went whole-foods organic, and couldn't bear to throw it away when I was cleaning out my cabinets of processed crap. So I still bake with it sometimes… and when it's gone, it's gone. I swear.
/end confession
The important thing is that these donuts are insanely delicious and absolutely perfect in their flavors. I can't even begin to describe how heavenly tasty they are. You must make them. Now.
Even if you totally fail at drizzling chocolate on top, like I did:
They still taste amazing.


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Business Ethics of Retail Pharmaceutical Firms
This content was COPIED from BrainMass.com - View the original, and get the already-completed solution here!
TOPICS: Business Ethics, cigarette, CVS, Health
SUMMARY: CVS, the nation's second-largest pharmacy chain, said Wednesday that it would stop selling all cigarettes and tobacco products nationwide by October, saying they have no place in a drugstore company that is trying to become more of a health-care provider. The move is a bold and expensive one for CVS, a unit of Woonsocket, R.I.-based CVS Caremark Corp. It reflects a major push by retail pharmacies away from simply dispensing drugs toward a more integrated role of providing basic health services to Americans-including millions of newly insured-amid an expected shortage of primary care doctors. The news is another blow to the $100 billion tobacco industry that is wrestling with slumping sales, rising taxes, widening smoking bans and a resurgence of public information campaigns highlighting the perils of smoking. For CVS, the move will be costly. The drugstore chain estimates it will lose $2 billion in annual revenue from tobacco and other sundries as a result, which amounts to about six to nine cents a share this year and about 17 cents annually from next year on. CVS, with annual revenue of more than $123 billion, projects its 2014 earnings will be $4.36 to $4.50 a share. But it is banking the strategy will give it a competitive edge over rival pharmacies in forging partnerships with hospitals, insurers and physician groups. These types of alliances are critical to drugstores like CVS and Walgreen Co. as they redefine themselves in what has been a historic downturn in prescription drug sales. CVS sees its future in making its in-store clinics a convenient health-care alternative to long waits at the doctor's office, along with CVS pharmacists counseling patients. That strategy was increasingly at odds with racks of cigarettes, cigars and chewing-tobacco residing behind the cashier's counter, said Larry Merlo, chief executive, in an interview. "Cigarettes have no place in an environment where health care is being delivered," said Mr. Merlo, a 58-year-old former pharmacist who became CEO of CVS Caremark in 2011. "This is the right decision at the right time as we evolve from a drugstore into a health-care company."
CLASSROOM APPLICATION: The nation's second largest pharmacy will stop selling cigarettes, putting pressure on rivals Walgreens, Rite Aid Corporation, and even Walmart stores to adopt similar measures. CVS is counting on consumers wanting to do business with a pharmacy focused on improving health and providing products associated with that of a healthcare provider. The company will not only dispense drugs to make people well but wants to use the shelf space to provide other healthcare products. The move will cost the drug store about $2 billion in revenue, but it is banking on stakeholder support from hospitals, insurers, and position groups. CVS's stock dropped about 3 percent in the first few days after the announcement, but the company is not worried because it feels that it is gaining a competitive advantage in positioning.
1. Will CVS's competitors stop selling cigarettes, or will they take advantage of a market opportunity since many of their stores are located very close to CVS stores?
2. Do you believe that a firm should sell a product that causes major health risks in a store that is positioned to help people manage and improve their health?© BrainMass Inc. brainmass.com October 2, 2020, 5:09 am ad1c9bdddf
Solution Preview
1. Will CVS's competitors stop selling cigarettes, or will they take advantage of a market opportunity since many of their stores are located very close to CVS stores?
CVS appears to be making a strategic move with halting sales of tobacco products at its stores. Their CEO hints at this with his statement "This is the right decision at the right time as we evolve from a drugstore into a health-care company." Also consider CVS recent acquisition of prescription drug benefits provider Caremark It does appear that CVS is trying to brand itself as a wellness company. I do not expect its competitors to follow suite unless they too are looking to change their business model because I am pretty skeptical that it will over time improve their bottom line profitability. I would anticipate that competitors of CVS are sure to benefit from this move in the short run. Many ...
Solution Summary
A look at the business impacts of CVS Pharmacy announcing it will no longer sell tobacco products. Discusses ethical issues of a healthcare provider selling products they may be harmful to ones health.


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The next generation of clothing is coming. It's made by microbes.
On Jan. 26, 1939, the San Francisco Chronicle introduced its readers to a new material that, though they didn't know it at the time, would soon envelop them from toe to head. An article reported that the du Pont Co. was building a plant to turn its new coal-based fabric, "nylon," into stockings. Fashion editor Ninon (one word!) gushed, "When a gossamer sheer stocking can be worn every day for five months before it springs a hole, that's news!"
Three-quarters of a century later, our closets are filled with nylon and other plastic fabrics — and a similar revolution in materials may be at hand. Instead of coal and oil, these new fibers, fabrics and leathers are made with genetically engineered microbes. The first generation of companies to harness yeasts, bacteria and fungi to make clothing may have originally set out to mimic silk and leather. But they're quickly finding they can do much more.
At the office and laboratory of Geltor, located above a big-box hardware store in San Leandro, founders Alex Lorestani and Nick Ouzounov hand me several pieces of leather. One tiny square is as thin and white as vellum. Another resembles a stiff brown belt. A third piece, golden and translucent, I can't stop touching. It stretches as if someone had managed to infuse cowskin with rubber.
The 3-year-old startup ferments yeast cells that are genetically programmed to synthesize animal collagen, the primary protein in skin, cartilage and tendons. Geltor is preparing to sell collagen to the cosmetics industry, but also plans to turn it into vegan, food-grade gelatin and leather.
Rethinking how leather is produced gives designers all sorts of room to play.
"Cows come in pretty standard sizes," Lorestani says. If a cow was the size of a conference room, what could you do with that unbroken, unscarred expanse of skin?
The scrap I'm fondling isn't even from a giant virtual cow. It's a proof of concept, a sheet used to bind a copy of Paul Shapiro's 2017 book "Clean Meat," about the cultured meat industry. And the collagen used to make it has the same molecular structure as jellyfish.
The sample sets the imagination spinning: Why does leather have to come from a cow or an alligator? Can we custom-create leathers to conform to our needs?
Modern Meadow started off making "steak chips" grown from bovine cells, but switched to cultured leather from bioengineered yeasts. In the fall, the company displayed its biofabricated leather, branded Zoa, at the Museum of Modern Art and a New York pop-up shop.
Zoa is based on bovine collagen. But that doesn't make it an exact substitute for cow leather. "We could create a two-dimensional sheet on a roll," Lee says. "But we can also paint with it, we can spray it, and we can integrate it with other textiles. The creative toolbox becomes super interesting, and far beyond what you could do with a traditional animal."
The most popular prototype at Modern Meadow's pop-up in New York was a swath of (traditional) silk with nanofibers of collagen sprayed onto it in big circles. The texture of the combined fabrics was "like nothing I'd ever felt," Lee says.
Neither Geltor nor Modern Meadow have any products on the market. While MycoWorks (based in San Francisco), Ecovative Design and Grand Zero Espace are making leathers from cultured mycelium — the fibrous, vegetative part of a fungal organism — only the latter company is selling small pieces of its MuSkin products to the public.
In 2016, North Face sold limited quantities of a $1,000 Moon Parka made with spider silk biofabricated by a Japanese company named Spiber— but only in Japan. In December, San Francisco's Bolt Threads put up for sale 100 knit caps whose yarn was a blend of merino wool and cultured spider silk, and they disappeared almost immediately. (Bolt just announced it will be taking orders for a bag made of mycelium leather, produced with Ecovative, in June.)
AMSilk is responsible for the next biofabricated clothing item customers may be able to buy: a running shoe from Adidas whose upper is woven from its Biosteel spider silk.
According to company CEO Jens Klein, the shoe isn't just biodegradable or, well, cool. The silk protein that AMSilk cultures from bacteria and spins into fibers offers numerous performance advantages: It's lighter and better at wicking moisture away from the foot, and may be less likely to collect bacteria on its surface. Plus, he adds, "It's better feeling."
Why spiders, when traditional silk is such a luxurious fabric?
"Silkworm silk is not something that yields really any performance," Klein says. Given spider silk's elasticity and strength, AMSilk is finding other uses for the protein in liquid form, such as cosmetic creams, and is culturing green lacewing silk whose stiffness may eventually make it a good analog for Kevlar.
Building the infrastructure to biofabricate fabrics and leathers — and do it affordably — may be the major hurdle right now, just as it was with nylon stockings in 1939.
"The challenge isn't that the market isn't ready for (these materials)," Lee says. "There's huge demand from the fashion industry."
From customers as well. "This is a new, emerging category of materials that may give me what I love about an animal product — the look, the feel — but an animal hasn't lost its life in order for that to happen," Lee adds. "Or I don't want to use a plastic material that is going to sit in a landfill for the next hundred thousand years."
"The most efficient solution to getting there is not around the existing class of materials," Geltor's Lorestani says. "It's around new features in the tree of life that haven't been explored."
Jonathan Kauffman is a San Francisco Chronicle staff writer. Email: firstname.lastname@example.com. Twitter and Instagram: @jonkauffman


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Last year, the Dolphins got minimal receiving production from the tight end position once they lost Jordan Cameron early in the year. Cameron was shaky enough for Plan A after underachieving the season before, and there wasn�t much depth behind him. The end result was Dion Sims leading the position with 256 yards and four touchdowns on 26 catches.
Miami is one Julius Thomas injury away from being in a similar spot.
While the team isn�t paying Thomas a ton, it�s banking on him being a big factor in the passing game all year. He�s coming off an injury-hampered run in Jacksonville in which he totaled 736 yards, 76 catches and nine touchdowns in two years. The Dolphins are hoping he�ll get back to what he was in Denver before that, when he was considered one of the best tight ends in the league.
Behind him, though, there are question marks. If Thomas can�t make it through a full season, the next man in line is likely 33-year-old Anthony Fasano.
He spent 2008 through �12 with the Dolphins, tying the franchise record for touchdowns by a tight end with seven in �08, before playing for the Chiefs and Titans the past four years. With Tennessee last season, he had eight catches for 83 yards and two touchdowns. Fasano hasn�t caught more than 26 passes in a season since leaving Miami. He�s known more for his blocking than his receiving ability at this point.
This is where drafting a tight end would�ve helped the Dolphins, who spent five of their first six picks on defense and wrapped up with a wide receiver in the seventh round. They did not sign any undrafted tight ends, either.
After Fasano, Miami has MarQueis Gray, Thomas Duarte and Chris Pantale. Gray is by far the most experienced of the three. He�s got 26 catches for 318 yards in 45 career games over four years with the Browns, Bills, Vikings and Dolphins. Pantale and Duarte have never started a game or caught a pass.
[Julius Thomas promises he has prime years left in his body]
[D-Line gets scolded for getting too close to Ryan Tannehill]
[A look at the Dolphins� plan for suspended safety T.J. McDonald]
Check out The Palm Beach Post�s Miami Dolphins page on Facebook


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Medium: original etching. (Also called "Moulins en Hollande".) Catalogue reference Delteil 14. Published in London in 1868, this impression is on laid paper. Size: 5 1/2 x 7 1/2 inches (plate). Signed in the plate (not by hand).Condition: the top margin is narrow.
Heliogravure (after the 1870 Jongkind watercolor). Published in Paris by Gazette des Beaux-Arts with collaboration from gallery owner Georges Petit, Jongkind's dealer. This impression is on wove paper. Size: 3 1/2 x 5 1/2 inches (image); 90 x 142 mm. Signed in the plate (not by hand).


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Anna Nicole Smith apparently likes her body too much to risk a run-in with seal killers. Citing safety concerns, the model-turned-Trimspa addict has canceled a PETA-sponsored trip to Canada to protest the brutal killing of seals. According to a PETA rep, the seal hunters have begun attacking many of the protesters and "we felt it just wasn't safe to put her in that position." Instead, Smith plans to protest the slaughter by passing out on her bathroom floor for a week.
(Disclosure: TV Guide is owned by CBS Interactive, a division of ViacomCBS.)


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So….. it's December!! I got (almost) all my Christmas presents sorted last week. I only need to finish some bits and bobs and I'll be so ready for the merry season.
This year I wanted to make my own Christmas cards, as I normally send some to my family and friends in Spain. I always need to send them early as last year I didn't and they arrived on New Year's Eve – lol. Well… this is very likely to happen this year too as I haven't send them yet *rolled eyes*.
Let's go straight to the point, here you have the step by step guide to make your own Christmas cards:
- Blank cards and envelopes
- Christmas stencils
- Silver and gold sprays
- Silver and gold bows or any other type of decoration
- Glue dots (these are my new thing and I'm using them in EVERYTHING!)
- A bit of blue tack
You can get all the materials online, although I got mine from the pound store.
How to make your own Christmas cards
- Prepare your workspace as the spray can get a bit messy. I put an old bed sheet where I was going to work as I did it inside (it was raining and so cold!) but if you could do it outdoors I would recommend it.
- Think about the designs you want to make. I went for Christmas trees and snowflakes, they are so cute <3
- Place the Christmas stencil on the blank card and use the blue tack so the stencil doesn't move.
- Spray, spray and spray till is done. Don't spray too much because if the card gets wet is not going to look as nice. Repeat on all the cards.
- Wait and let them dry – go and make a cuppa!
- Once they are dry, put the bows on the corners of the cards using the glue dots. As you can see on the pics I put a silver bow on gold cards and vice versa.
- The cards are ready!!
I hope you enjoy this DIY


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Born to a father who was a mathematics professor and a mother who was a professor at Howard University, many within the Chappelle family figured that young Dave would carve out a career for himself somewhere in the field of education. In a sense, this is true. Dave Chappelle has always stood out as one of the most cerebral, witty and entertaining stand-up comics in the business. He educates audiences full of fans who eagerly purchase Dave Chappelle tickets, and he will do so with a series of 2014 dates at many venues he hasn't visited in about a decade, including Madison Square Garden.
Many can picture the signature look of the baldheaded, gentle-mustached genius of comedy who first landed a film role at the age of 19. Even hard-core Chappelle fans may not be aware that he actually turned down the role of Bubba Gump in the movie Forrest Gump, but he became recognized by worldwide audiences after playing himself in Eddie Murphy's 1996 hit The Nutty Professor.
It was around that time that he began to sell tickets for Dave Chappelle Tour shows on a regular basis and fans begin to form lines that wove around the corners of the local comedy spots to see the natural of the stage work his craft. The wildly popular Chappelle's Show debuted on Comedy Central in 2003, and a number of Chappelle's characters from the weekly 30-minute, pre-recorded sketch comedy show immediately made it into the lexicon of pop culture references. In fact, it can be said that his show became too popular for his own good. He eventually fled North American soil to South Africa to regroup after the show headed in a direction he didn't like and he couldn't leave his house without hearing the phrase, "Rick James, bitch!"
In the 10 years since, Chappelle has laid low by the standards of a top-flight comedian, raising his family in a small Ohio suburb and making occasional cameo appearances on television and in movies. In the spring of 2007, he smashed a standup comedy endurance record formerly held by Dane Cook at the Laugh Factory before beating his own record a few months later, setting a high-water mark of six hours and 12 consecutive minutes of consecutive standup for the lucky Dave Chappelle tickets holders on that.
This all brings us to the present. The 40-year-old has already announced that he will perform a slew of live North American shows, offering many options to fans excited about the chance to purchase cheap Dave Chappelle tickets.


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Canon EOS 60D - A New Kind of Rebel
The double-digit numbers have traditionally belonged to Canon's semi-pro DSLR line-up, which offer features from the pro cameras at a lower price than a pro body.
That tradition came into question with the launch of the Canon 7D late in 2009 though, the 7D represented a new category of pro-camera which sandwiched the double-digit 50D and the higher-end 5D Mark II. That probably explains some of the consumer-centric changes which have been made to the 60D as it succeeds the 50D. Now is this a good thing or bad thing? It really depends on which fence of the consumer/pro field you're sitting on, and, as usual, read on to discover why.
The Verdict
The Good
Great handling
Good ISO performance with low image noise
High burst rate when shooting JPEGs
Brilliant LCD display
The Bad
Missing joystick makes switching AF points more difficult


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By Michael Murillo
Twitter is a great way to stay updated and connected with friends, acquaintances and even celebrities. But, if that's all you're using it for, you're missing out on some valuable business know-how. Top corporations, CEOs, business school admissions counselors, advisors and more are just some of the business experts offering up advice, anecdotes and info daily in 140 characters or less.
Here are five Twitter accounts worth following:
* twitter.com/nytimesbusiness
The business-related Twitter account of the New York Times will keep you busy with news and analysis across the entire business spectrum. It's a no-nonsense account that's often updated dozens of times every day. Each link goes to the New York Times, of course, but you can get updates on everything from initial public offerings and stock earnings to airline ticket prices and consumer trends. Some articles are driven by hard data and others are more about analysis and overview. Either way, you'll be able to stay updated on the most current news in the business world.
* twitter.com/whatworks
Advertised as how-to advice from the editors of Business.com, this Twitter account has a small business focus. Typical topics include ways to prevent fraud, manage social media, and recruit and maintain vendors, among other topics. Tweets focus on the everyday problems small business owners encounter, providing advice and analysis, a much-needed service since companies with less than 500 employees represent about half the nation's private sector jobs, according to the Small Business Administration.
* twitter.com/HBSNEWS
You don't have to attend the prestigious Harvard Business School to benefit from their Twitter account. It features links to blogs, news items and other relevant information from many different sectors. Many of the Tweets link to articles from the well-known Harvard Business Review and feature insights on today's business climate from alumni and professors. Whether you're just starting out or a long-time business veteran, this official institution account will help you stay on top of business news and best-practices.
* twitter.com/jimcramer
CNBC Mad Money host, Jim Cramer, known as much for his enthusiastic delivery as for his stock market acumen, offers up a mix of economics info on his fast-paced Twitter account. As should be expected, the account is largely devoted to providing stock information, industry analysis and financial news links, all peppered with Cramer's trademark personality. Filled in with a few personal updates, the account is a good way to follow the ups and downs of the market, whether you're already an investor or just learning the financial ropes.
* twitter.com/attnews
AT&T's Twitter account is a good example of how a large, established corporation can keep itself relevant and competitive in the communication technology arena. Making full use of the social media tool, AT&T tweets about everything from new product lines and features to company milestones and contracts. This Twitter account is just as valuable for the casual AT&T customer as it is for the potential investor. Instantly, find about new features, app rollouts and more, as you scan the updates from this business mainstay.
Whether you're focused on finance, marketing or management, there is an almost endless array of Twitter business resources at your disposal. The five accounts above can help you start building your own personal list of top business Twitter-feeds.


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Driving Lessons Great Sutton
Driving Lessons Great Sutton is fun and when you are able to drive you can take yourself anywhere you wish to go. If you haven't passed your Great Sutton Driving test yet, it can seem difficult but there is no reason to give up. One of the best ways to learn to drive is to find the best driving school in Great Sutton and the best instructor for your learning style.
So you want to learn to drive? Try Insight 2 Drive.
Build your confidence with driving Lessons Great Sutton. Our advanced driving instructors have many years of experience in teaching people like you how to drive. We have an extremely high driving test pass rate – and we want to help you.
With experience, our instructors have the ability to be patient – we were all learners once, but now we have seen everything you could imagine on the roads. We have taught people who needed only a few lessons to people that need much more. Some pick it up quickly and others not so quickly. We can definitely help you. The best driving lessons in Great Sutton are here with the team at Insight 2 Drive.
Driving lessons can be a scary proposition before you start, but the possibilities they unlock are endless: No more public transport, your own car to get to and from work and the freedom to go anywhere you want whenever you want.
Driving Lessons Great Sutton from age 17 to 70We teach people of all ages to drive, from 17-year-olds who are getting a car for their birthday and hope to pass their test soon, to the mature learner who never got round to taking driving lessons Great Sutton when they were younger.
Contact Insight 2 Drive now – we are sure we can able to help you with Driving Lessons Great Sutton, Driving Lessons Chester, Driving Lessons Mold or Driving Lessons Ellesmere Port.


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JLF New Member Brunch
Please join us on Sunday, August 27 for our Annual New Member Brunch!
This is a great opportunity to meet the Board and Membership Committee and to also get to know the other 2017 – 2018 New Members.
To RSVP or to receive additional information about joining Junior League of Fresno, contact Kathy Flores, firstname.lastname@example.com