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Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1β cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF.
Citation: Cigana C, Curcurù L, Leone MR, Ieranò T, Lorè NI, Bianconi I, et al. (2009) Pseudomonas aeruginosa Exploits Lipid A and Muropeptides Modification as a Strategy to Lower Innate Immunity during Cystic Fibrosis Lung Infection. PLoS ONE 4(12): e8439. https://doi.org/10.1371/journal.pone.0008439
Editor: Edgardo Moreno, Universidad Nacional, Costa Rica
Received: August 28, 2009; Accepted: November 28, 2009; Published: December 23, 2009
Copyright: © 2009 Cigana et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported to AM and MLB by Fondazione per la ricerca sulla Fibrosi Cistica (FFC#8/2007) with the contribute of Furla Spa, Grazia Valentini Scagliarini Spa, Delegazione FFC di Bologna and to AB by HEALTH-F7-2009-223670. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The strategy of innate immune recognition is based on the detection of constitutive and conserved products of microbial metabolism called pathogen-associated molecular patterns (PAMPs) or alternatively microbe-associated molecular patterns (MAMPs), since they are also common to all microbe and not only to the pathogen version [1]. These molecular signatures are recognized by the host through a family of pattern recognition receptors (PRRs), which includes Toll-like (TLRs) and nucleotide binding and oligomerization domain-like receptors NLR (Nod-Like receptor) [2]. For example, lipid A contained in bacterial lipopolysaccharide (LPS) acts as a PAMP and is sensed by the cognate PRR TLR4-MD2, while different motifs contained in peptidoglycan (PGN) of Gram-positive or Gram-negative bacteria are recognized by the intracellular PRRs Nod1 and Nod2 [3], [4]. Interaction of PAMPs with PRRs results in activation of antimicrobial responses [1], such as production of antimicrobial peptides and secretion of pro-inflammatory cytokines, necessary for pathogen's eradication. However, many pathogens have evolved adaptive strategies for subverting the host innate immune system by evading detection by PRRs and/or impairing the downstream cellular signalling pathway [5]. In this study, we present evidence that Pseudomonas aeruginosa exploits PAMPs modification as a strategy to lower innate immune system detection and signalling during chronic stages of lung infection in fibrosis cystic (CF) patients.
CF lung disease is characterized by transient airway P. aeruginosa infections and excessive neutrophil-dominated inflammation early in life followed by permanent chronic infection that causes persistent respiratory symptoms and decline in lung functions [6]. The long term colonization of CF airways selects pathoadaptive variants with several features which differentiate late P. aeruginosa isolates from the initially acquired strain [7], [8]. P. aeruginosa strains that initiate infections are characterized by a large arsenal of virulence factors, such as many toxic factors, like pyocyanin, a type III secretion system (T3SS), several proteases, lipases and phospholipases, rahmnolipids and other factors [9]. In contrast, these invasive functions are selected against in CF chronic infection leading to less virulent but more persistent phenotypes including alginate producing mucoid strains [7]. However, besides all these relevant features, which contribute to either forms of infection, the LPS modification appears to be one of the main factors in the adaptation of this pathogen during chronic infections. It is already known that P. aeruginosa acute infection also implies a consistent change in LPS lipid A structure [10] even though no conclusive information are present on the putative LPS lipid A changes in the acute to chronic evolution of the infection. In general, production of fully hexa-acylated lipid A is associated with a more strong TLR4 mediated inflammatory response while lipid A with lower levels of acylation triggers reduced cellular responses [11]. However, lipid A isolated from clinical strains of P. aeruginosa often results in a blend of different species [10] with a resulting biological activity that might be different when compared to single species bioactivity. Lipid A modifications are regulated by the environmental sensor-kinase transcriptional regulatory system PhoP-PhoQ and catalysed by PagP, PagL and LpxO which lead to acylation, deacylation and hydroxylation of the molecule, respectively [12], [13], [14].
As for PGN, an essential cell wall component, no data have been produced so far on the structure itself and eventual alterations in infection by P. aeruginosa. PGN changes may even more advantage bacterial life within the host and contribute to establish successful chronic infection in CF patients. In fact, in these last years a growing body of studies are highlighting the role played by muropeptide receptors Nod1 and Nod2 in the development of both antibacterial responses and chronic inflammation, such as Crohn's disease and asthma [15]. To fill the gap in the context of CF disease mediated by P. aeruginosa infection we analysed, at chemical and biological level, the LPS and PGN from three clonal strains of one P. aeruginosa lineage. P. aeruginosa strains were isolated at the onset of infection and after years of chronic colonization from a CF patient with a severe course of the airways infection [8]. Our results showed that the biological activity of LPS and PGN is consistent with a reduced immunostimulatory potential in accordance with the need to evade immune system and favour survival during the course of the chronic P. aeruginosa infection.
P. aeruginosa Lipid A Chemical and Genetic Modifications in Clonal Strains of Early Colonization and Late Chronic Infection of CF Patient
The LPS of P. aeruginosa sequential strains AA2, isolated at the onset of chronic colonization, and AA43 (mucoid) and AA44, isolated before patient's death (Figure S1), was extracted by conventional methods (see Supporting Information File S1) and showed the typical ladder like pattern by SDS-PAGE electrophoresis (data not shown). The O-repeating unit resulted identical in all three strains and the structure is already reported elsewhere [16].
The structure of LPS lipid A of the three P. aeruginosa strains (Figure 1) was determined by chemical analyses and MS spectrometry (see Supporting Information File S1 for the whole structure determination). As shown in Figure 1, MALDI negative ion spectra revealed that LPSs extracted from AA2 and AA43 presented the same lipid A species even though with a different relative abundance. In both cases, MALDI mass spectra showed a main peak that matched with a penta-acylated lipid A constituted by a bis-phosphorylated disaccharide backbone carrying a 10∶0 (3-OH) in ester linkage on GlcN II and two 12∶0 (3-OH) in amide linkage on both GlcN residues. Furthermore, both 12∶0 (3-OH) were substituted by a secondary fatty acid, a 12∶0 on GlcN II and a 12∶0 (2-OH) on the GlcN I. Both lipid A blend also contained a tetra-acylated lipid A deriving from the previous by loss of the only 10∶0 (3-OH) in ester linkage on GlcN II. An asymmetric hexa-acylated lipid A bearing the extra 16∶0 that esterifies the 10∶0 (3-OH) on GlcN II was present almost exclusively in the lipid A blend of strain AA43. Minor species carrying two secondary 12∶0 (2-OH) fatty acids and others lacking a phosphate group were also present.
MALDI MS spectra of lipid A blend obtained by acid hydrolysis of LPS from P. aeruginosa clinical strains isolated at the onset of chronic colonization (AA2) (A) and after years of chronic infection (AA43 and AA44) from a CF patient (B and C). A difference of 238 Da corresponds to a 16∶0 fatty acid residue whereas 170 corresponds to a 10∶0 (3-OH) residue and 80 Da are indicative of a phosphate group. The 16 Da difference is relative to the presence of a Hydroxy group at C-2 of the secondary 12∶0 fatty acids. The non indicated ion peaks are relative to the species already indicated and bearing sodium or potassium counter-ions.
As for AA44 completely different lipid A were found, in which the main difference with those above was the presence of a further 10∶0 (3-OH) on GlcN I. The most prevalent one was a symmetric hexa-acylated lipid A constituted by a bis-phosphorylated disaccharide backbone carrying two 10∶0 (3-OH) in ester linkage and two 12∶0 (3-OH) in amide linkage. Further, both amide chains were substituted by a secondary fatty acid, a 12∶0 and a 12∶0 (2-OH) on the GlcN II and I, respectively. In addition, hepta-acylated lipid A bearing the additional 16∶0 on GlcN II species was present.
Lipid A modifications in AA2, AA43 and AA44 were stable as they were not lost after serial passages in vitro, which would suggest the presence of genetic mutations. Sequence analysis and multiple alignment of phoP, phoQ, pagL, lpxO1 and lpxO2 genes responsible for modifying lipid A, revealed pagL mutation in AA44 but not in AA2 and AA43 strains (Table S1 in Supporting Information File S1 and Figure S2). In particular, insertion of four nucleotides (CCTG) at position 502 lead to a frameshift mutation.
LPS of P. aeruginosa Clinical Isolates Stimulates Different Inflammatory Response in Human Cells (HEK 293-hTLR4/MD2-CD14, C38 and THP-1) Including Those of CF Origin (IB3-1)
HEK 293-hTLR4/MD2-CD14 were exposed to different LPSs concentration (10, 50 and 100 ng/mL) of P. aeruginosa AA2, AA43, AA44 and PAO1 for 4 h and NF-kB activation was evaluated through the assessment of luciferase activity (Figure 2A). LPS of P. aeruginosa AA2 induced a significantly higher NF-kB activation with respect to cells exposed to LPSs of AA43 and AA44 (LPS AA2 vs AA43 and AA44 p<0.01) in a dose dependent manner. IL-8 expression was measured through real time quantitative (q)-PCR after 4 h stimulation, as above (Figure 2B). In accordance with NF-κB activity, the level of IL-8 expression increased higher after stimulation with LPS from AA2 with respect to LPSs from AA43 and AA44 (LPS AA2 vs AA43 and AA44 p<0.01). For ELISA assay, HEK 293-hTLR4/MD2-CD14 were stimulated for 24 h with P. aeruginosa LPS as above (Figure 2C). The level of IL-8 release induced by LPS from AA2 was significantly higher than that induced by LPSs of AA43 and AA44 (LPS AA2 vs AA43 and AA44 p<0.05). Under all conditions, stimulation with LPS from PAO1 was considerably higher than those induced by LPSs from the clinical isolates (LPS PAO1 vs AA2, AA43 and AA44 p<0.05) (Figure 2A, B and C).
A) Fold of activation of NF-kB after 4 h of stimulation with different concentrations of LPS; commercial LPS of P. aeruginosa was used as a control. B) IL-8 mRNA induction after stimulation with 100 ng/mL of LPS for 4 h. C) IL-8 secretion after stimulation with 100 ng/mL of LPS for 24 h. Commercial LPS of PAO1 was used as a control. *p<0.05, **p<0.01, ***p<0.001 in the Student's t-test.
Next, we tested CF respiratory cells (IB3-1) and the isogenic corrected cells (C38) after LPS stimulation. When tested for the pro-inflammatory markers, in IB3-1 cells LPS from AA2 induced significantly more IL-8 and TNF-α expression, assessed by real time q-PCR, when compared with LPSs from AA44 and AA43 (LPS AA2 vs AA43 and AA44 p<0.05) (Figure 3A and C). IL-8 protein release, measured by ELISA, was consistent with expression (LPS AA2 vs AA43 and AA44 p<0.05) (Figure 3B). Similar results were obtained in C38 cells, even though these cells were generally less responsive when compared to IB3-1. LPS from PAO1 was used as positive control and stimulated both IB3-1 and C38 cells (Figure 3A, B and C).
A) Fold of induction of IL-8 and C) TNF-α mRNA in IB3-1 and C38 cells after stimulation with 100 ng/mL of LPS for 4 h. Commercial LPS of PAO1 was used as control. The values represent the expression levels relative to untreated IB3-1 (means±SD). B) IL-8 secretion from IB3-1 and C38 cells after stimulation with 100 ng/mL of LPS for 24 h. LPS of PAO1 was used as control. D) TNF-α secretion from THP-1 after stimulation for 6 h with 100 ng/mL of LPS derived from the clinical isolates AA2, AA43 and AA44. LPSs from P. aeruginosa serotype 1022 and from E. coli serotype OIII:B4 (Sigma) were used as controls. *p<0.05, **p<0.01, ***p<0.001 in the Student's t-test.
As levels of TNF-α secretion were undetectable in epithelial cell lines, we evaluated LPS stimulation in differentiated macrophagic THP-1 cells (Figure 3D). TNF-α protein release was significantly higher after treatment with LPS from AA2 in comparison to that induced by LPSs from AA43 and AA44 (LPS AA2 vs AA43 and AA44 p<0.05). LPSs from PAO1 and E. coli OIII:B4 were used as positive controls and both stimulated THP-1 cells.
Altered LPS Structure of P. aeruginosa Late Strains Induces Low Leukocyte Recruitment and Cytokine Patterns In Vivo
To address the question whether the differences in the lipid A structures could affect the inflammatory response in vivo, we analyzed leukocytes recruitment in the bronchoalveolar lavage fluid (BALF) of C57Bl/6 mice exposed for 16 h to different LPS structures of strains AA2, AA43 and AA44 by means of nasal instillation. While there was no significant difference in monocytes and lymphocytes number (data not shown), neutrophils profile showed striking differences in total differential cell counts (Figure 4A). Significant higher recruitment of neutrophils was observed in mice exposed to LPS from AA2 strain in comparison to those treated with LPSs from AA43 and AA44 (LPS AA2 vs AA43 and AA44 p<0.001). Cytokines levels were tested in the murine lung homogenates. MIP-2 level in lungs treated with LPS from AA2 strain was significantly higher than those treated with LPSs from AA43 and AA44 strains (LPS AA2 vs AA43 and AA44 p<0.001) (Figure 4B). Similar trends were obtained with IL-1β and KC (Figure 4C and D).
C57Bl/6 mouse were exposed for 16 h to 10 mg/mouse of LPS derived from a P. aeruginosa reference strain (PAO1) and three clinical isolates (AA2, AA43 and AA44). A) Total cells were recovered from the BALF and quantified. B, C and D) MIP-2, IL-1β and KC secretions were quantified by ELISA in lung homogenates. *p<0.05, **p<0.01 in the Student's t-test.
Murine lung histopathology showed that the LPS from AA2 strain (Figure 5A and B) caused more severe lesions and leukocytes recruitment in the airways than LPSs from AA43 and AA44 strains did (Figure 5C, D, E and F) indicating a reduced detection for LPS isolated in the late P. aeruginosa strains.
Mice exposed for 16 h to 10 mg/mouse of LPS derived from three P. aeruginosa clinical isolates (AA2, AA43 and AA44). After H&E staining, extensive recruitment of inflammatory cells is visible in the bronchial lumen (asterisk) of animals treated with LPS of AA2 strain (A and B), whereas treatment of mice with LPS of AA43 (C and D) and AA44 (E and F) showed limited accumulation.
Structural Analysis of P. aeruginosa PGN Fragments and Muramyl Peptides in Clonal Strains of Early Colonization and Late Chronic Infection
PGNs from strains AA2, AA43 and AA44 were digested by the muramidase mutanolysin to generate the entire spectrum of muropeptides. The generated PGNs fragments were reduced with sodium borohydride, then identified by RP-HPLC and LC-MS (Figure S3 and S4) [17], [18]. The composition is reported in Table 1 (see Supporting Information File S1 for the whole structure determination).
Despite the presence of common fragments in the muropeptide blend deriving from the bacterial PGNs, the relative ratio among the peaks sensibly changed among strains AA2, AA43 and AA44 and, furthermore, distinctive chemical features were found in the structure of some minor constituents of the muropeptides mixture (Table 1).
PGN of P. aeruginosa Early Strain Induces Higher Inflammatory Response than Those of Late Strains in Human Cells via Activation of Nod1
HEK293 cells were transfected with hNod1 or hNod2 and stimulated with different concentrations of PGN of AA2, AA43 and AA44 (0.5, 1 and 10 µg/mL) during 18 h. γTriDAP and MDP were used as positive controls. In HEK293hNod1 cells, PGN from AA2 strain induced a major NF-kB activation with respect to PGNs from AA43 and AA44 in a dose dependent manner (PGN AA2 vs AA43 and AA44 p<0.05) (Figure 6A). In contrast with these results no NF-κB activation was found in HEK293 cells expressing hNod2 under the same experimental conditions applied for Nod1 (Figure 6D). IL-8 expression, measured by real time q-PCR in HEK293hNod1 cells treated as above, was significantly higher after stimulation with PGN of AA2 when compared to stimulation with PGNs from AA43 and AA44 (PGN AA2 vs AA43 and AA44 p<0.05) (Figure 6B). IL-8 secretion was measured at 24 h post-transfection. PGN from AA2 strain induced a higher IL-8 production compared to PGNs from AA43 and AA44 strains (PGN AA2 vs AA43 and AA44 p<0.05) (Figure 6C). Transfection of HEK293-hNod1 cell monolayers with a plasmid encoding siRNA for Nod1 prevented the PGNs of the three P. aeruginosa strains to activate NF-κB (Figure 6A and B). Likewise, IL-8 expression and production was abolished in the presence of Nod1 siRNA (Figure 6C). None of PGN tested activated IL-8 expression and secretion in HEK-293hNod2 (Figure 6E and F).
A, D) Fold of activation of NF-kB after 18 h of stimulation with different concentration of PGN (0,5 µg/mL; 1 µg/mL; 10 µg/mL); triDAP and MDP were used as controls. B, E) IL-8 mRNA induction after stimulation with 1 µg/mL of PGN for 18 h. C, F) IL-8 secretion after stimulation with 1 µg/mL of PGN for 24 h. *p<0.05; **p<0.01 in the Student's t-test.
Next, Nod1 and Nod2 expression were confirmed in IB3-1 and C38 cells in the absence or presence of stimuli by real time q-PCR (data not shown). TNF-α and IL-8 expression by real time q-PCR was measured at higher levels in IB3-1 cells treated with PGN from AA2 in comparison to PGNs from AA43 and AA44 (PGN AA2 vs AA43 and AA44 p<0.05) (Figure 7A and B). In agreement with these results, levels of IL-8 proteins of cells stimulated with PGN from AA2 were higher than those elicited by PGNs from AA43 and AA44 (PGN AA2 vs AA43 and AA44 PGN p<0.05) (Figure 7C). Similar results were obtained in C38 cells even though these cells were generally less responsive when compared to IB3-1, as showed above for LPS.
A) Fold of induction of TNF-α and (B) IL-8 mRNA after stimulation with 1 µg/mL of PGN for 4 h. The values represent the expression levels relative to untreated IB3-1 (means±SD). C) IL-8 secretion after stimulation with 1 µg/mL of PGN for 24 h. *p<0.05, **p<0.01, ***p<0.001 in the Student's t-test.
In this work, we analysed the PAMPs chemical modification as a strategy of P. aeruginosa to hijack genes involved in innate immune responses and to favour survival in patients with CF. A major question, derived from previous reports [7], [8], was whether P. aeruginosa could establish life-long chronic infections in CF hosts according with strategies used by a number of other bacterial pathogens. Persistence is normally established after an acute period of infection involving activation of both the innate and acquired immune system. While the acute infection is usually fully resolved by eliminating the invading bacteria, some bacteria including Salmonella enterica sv typhi, Helicobacter pylori, Mycobacterium tuberculosis and others survive and cause persistent life-long infection by evading immune surveillance [19].
Chemical structure of LPS and PGN were determined for three P. aeruginosa clones isolated from airways of a CF patient during a period of 7.5 years. Our previous study showed that these P. aeruginosa strains, which were isolated at the late stage of CF chronic infection, have better capacity to persist in the murine lung and to cause chronic infection when compared to early strain [8]. Overall, in all three strains LPS lipid A were consistent with the results reported previously though this molecule displayed much more heterogeneous composition than other case-studies [10], [11], [20]. Among the three strains LPS lipid A diversity was observed in the number and location of fatty-acid side chains. Early AA2 and late AA43 mucoid P. aeruginosa strains synthesized a LPS blend essentially composed by tetra-, penta- and hexa-acylated species lacking 10∶0 (3-OH) primary fatty acid. In contrast, the late non-mucoid AA44 strain was constituted by homologue lipid A species which further carried a 10∶0 (3-OH) residue, i.e., hexa-acylated and hepta-acylated moieties. These findings are in accordance with previous observations [10], [11], [20] that P. aeruginosa synthesizes more highly acylated (hexa- and hepta-acylated) LPS structures during adaptation to the CF airways. Furthermore, our data might be particularly significant as here we compare LPS of a pair of serial P. aeruginosa strains, including mucoid and non-mucoid phenotypes. In mucoid AA43 strain the capsule may act as a physical barrier thus partially preventing invasion by host phagocytes and can also mask the LPS molecules to prevent complement deposition, as observed in other microorganisms, such as E. coli, Haemophilus influenzae and Klebsiella pneumoniae [21], [22], [23]. By contrast, for the non-mucoid AA44 the absence of capsule may make this strain more susceptible to phagocytosis. This issue could underline a major selective pressure on AA44 to drastically reduce the immunostimulatory activity of its LPS, promote intracellular survival and resistance to antimicrobial peptides through relevant structural changes in lipid A.
As previously described, P. aeruginosa strains with severe CF lung disease lacked deacylated lipid A structures, suggesting that loss of deacylase enzymatic activity (PagL) can occur during long-term adaptation to the CF airway [10], [24]. Retention of the 3-hydroxydecanoic acid at the lipid A 3-O position is presumably due to lack of expression of PagL, a recently identified 3-O position lipid A deacylase [13]. However, genetic data in support of biological activity have not been provided yet. Here, sequence analysis and alignment of the three clonally related P. aeruginosa strains demonstrated for the first time pathoadaptive mutation in pagL of late AA44 strain. These results indicate that P. aeruginosa modified lipid A upon pagL inhibition contributes to reduce TLR4-signalling correlating with data previously obtained for other gram-negative bacteria as Salmonella typhimurium [12] and Bordetella pertussis [13].
In contrast to AA44, the early AA2 and the late AA43 strains were equipped essentially by penta- and hexa-acylated lipid A but differed in their ratio. The lipid A isoforms have profound implications for human disease, owing to altered recognition by the TLR4 complex [14]. A variety of studies in different bacterial species indicated that the acylation state of lipid A can alter TLR4-mediated responses [25]. In general, production of fully hexa-acylated lipid A is associated with a more strong TLR4 mediated inflammatory response while lipid A with lower levels of acylation triggers reduced cellular responses [25]. This notion has been also demonstrated in P. aeruginosa when single species of penta- or hexa-acylated lipid A were tested [11], while in Salmonella hepta-acylated isoforms display significantly lower stimulatory activity as compared to the hexa-acylated species [26]. Interestingly, our results suggested that, in addition to the isoforms, also the ratio of penta- and hexa-acylated lipid A had an impact on the biological activity. As shown previously, lipid A under-acylated with respect to hexa-acylated is not only reduced in its ability to stimulate host cellular immune responses, but can also act as antagonist capable of blocking the immunopotential of hexa-acylated E. coli LPS lipid A [27], [28], [29], [30], [31]. In particular, penta-acylated lipid A of P. aeruginosa could antagonize TLR-4 dependent responses of the human cells to hexa-acylated lipid A from E. coli [32].
On the basis of the previous notions it was not surprising that in our study LPS of the late AA43, harbouring a mixture of hexa- and under-acylated lipid A, and AA44, presenting hexa-acylated and hepta-acylated lipid A moieties, displayed a reduced immunomodulatory TLR4-mediated activity compared to LPS of early AA2. In particular, the LPSs of AA43 and AA44 showed weak NF-κB and IL-8 inflammatory response when tested in HEK293 cells expressing TLR-4. Similar results were confirmed in macrophage-like THP-1 cells, in a CF airway epithelial IB3-1cell line and in its isogenic non-CF C38 cells, even though the latter were reported as generally less responsive to stimuli when compared to IB3-1 [33]. In the lung of mice neutrophils recruitment was higher after stimulation with lipid A from early AA2 strain with respect to lipid A from late strains AA43 and AA44. A pool of cytokines including MIP-2, KC and IL-1β were found decreased confirming a reduced inflammation for the LPSs of late strains. Altogether these results emphasize the reduced immunopotential of LPS extracted from late colonizer P. aeruginosa strains, in line with other reports that demonstrated the lost of large arsenal of virulence factors during chronic infection [7].
We supported the findings described above on LPS by analysing PGN, as additional structure, in our P. aeruginosa strains. The concept that bacteria could modulate PGN composition to escape the immune surveillance has been recently reported for pathogens and commensals but not in P. aeruginosa [34], [35], [36], [37], [38]. In accordance with data of lipid A composition, structural analysis of PGN fragments derived by muramidase digestion of PGN from the three clonal strains of P. aeruginosa showed a different relative amount of muropeptides.
Despite these differences and in agreement with the biological impact of lipid A of the three clinical isolates, muropeptides of the late AA43 and AA44 strains displayed a dramatic reduced ability to stimulate the Nod1 receptor in HEK293 and CF cells, with respect to PGN of the early AA2 strain. Nod1 and not Nod2 is involved in the NF-κB and IL-8 mediated response to P. aeruginosa PGN, as shown in HEK293 cells expressing alternatively these two PRRs or silenced for Nod1. This result is consistent with another previous study [39] that reported the peculiar ability of P. aeruginosa to elicit a Nod1-mediated response. At our knowledge, this is a first report that shows an immune evasion strategy involving the in vivo selection of pathogenic morphotypes harboring PAMP variants able to modulate recognition by different PRRs.
The minimal structure recognized by Nod1 is the dipeptide D- γ-glutyamyl-meso-diaminolimelic acid (iEDAP) [40] while it is evident that the intact macromolecular PGN does not stimulate Nod1. It was initially described that the Nod1 recognition system requires the exposure of the DAP moiety [40]. Then, other reports, employing synthetic compounds, demonstrated that an exposed DAP moiety is not essential for sensing by Nod1 [41], [42]. However, the identity of major Nod1 stimulatory molecule(s), as well as that of putative antagonistic molecules, remains still unknown.
Therefore, as the molecular details of the PGN-Nod1 relationship are not yet elucidated, we are not able to clarify the mechanisms underlining the reduction of Nod1 activation observed with the PGN of the P. aeruginosa strains isolated from the chronic stage of the infection. Difference in muropeptide composition suggests that the enzymatic mechanisms accounting for PGN modifications in AA2, AA43 and AA44 are different or at less differently regulated in these strains. For example, we may observe a reduced amount of GlcNAc2MurNAc(Anh)1MurNAc1Ala4Glu2DAP2 in the PGN of AA44 strain with respect to the other two strains. This might indicate that the activity of the P. aeruginosa lytic transglycosylases already identified on the P. aeruginosa genome [43] is different in AA44 with respect to the other strains.
However, we might address the question of how the reduced activation of TLR4 and Nod1 could help the persistence of the P. aeruginosa strains associated to late stage of CF infection. Actually, the reduced production of inflammatory mediators following activation of TLR4 by LPS of late colonizer strains could account for an impaired ability of the host to mount an adequate reaction aimed at P. aeruginosa clearance. Likewise, the role of Nod proteins, and in particular Nod1, has been recently demonstrated in the lung infections sustained by bacterial pathogens, such as Chlamydophila pneumonia [44], [45]. In particular, it has been reported that in a murine model of infection the absence of Rip2, which acts as an adaptor molecule in Nod signalling, favors significant delay in C. pneumonia clearence from the lungs and promotes more severe and chronic lung inflammation.
Taken together our results, obtained by comparing sequential strains isolated from one CF patient, showed that the synergy of the action of the two modified P. aeruginosa PAMPs, PGN and lipid A, could drastically impair the CF host immune detection system thus preventing the eradication of the infection with this pathogen. This failure occurs despite the deleterious consequence of the persistent inflammatory reaction that leads to the decline of lung function and likely to fatal outcome of the disease. These findings emphasize further studies to determine the generality of this mechanism with additional P. aeruginosa clonal lineages or to establish whether other bacterial ligands play a critical role in evading immune system thus promoting survival and establishing favourable conditions for chronic persistence in CF patients.
Materials and Methods
Ethics Statement
Animal studies were conducted according to protocols approved by the San Raffaele Scientific Institute (Milan, Italy) Institutional Animal Care and Use Committee (IACUC) and adhered strictly to the Italian Ministry of Health guidelines for the use and care of experimental animals.
Research on the bacterial isolates from the individual with CF has been approved by the responsible physician at the CF center at Hannover Medical School, Germany. All patients gave informed consent before the sample collection. Approval for storing of biological materials was obtained by the Hannover Medical School, Germany.
Bacterial Strains and CF Patient
P. aeruginosa clinical isolate AA2 was obtained from sputa or throat swabs from CF patient homozygous for ΔF508 cftr mutation attending the Medizinische Hochschule of Hannover, Germany at the onset of chronic colonization. AA43 and AA44 were collected 7.5 years after acquisition and before patient's death. Two intermediate P. aeruginosa strains, AA11 and AA12, were also isolated after one year of infection from this patient [8]. Additional clinical data were reported in the online data supplement (Figure S1). Genotypic and phenotypic data of P. aeruginosa strains were published previously [8], [46], [47]. In particular, to establish the mucoid phenotype quantitative determination of alginate production by P. aeruginosa strains was carried out by carbazole assay as already reported elsewhere [46], [8]. PAO1 was used as reference strain [48]. P. aeruginosa was cultured in Pseudomonas isolation agar (PIA) or Trypticase Soy Broth (TSB) at 37°C.
LPS and PGN Extraction and Lipid A and Muropeptide Identification
Sequence Analysis
One loop of a single P. aeruginosa colony, grown on PIA, was processed for DNA extraction using a commercial DNA isolation kit (Qiagen) according to the instructions of the manufacturer. PCR amplifications of the entire pagL, phoP, phoQ, lpxO1 and lpoO2 genes were carried out using Taq DNA polymerase (Qiagen). The primers used were detailed in the Online data supplement (Table S1 in Supporting Information File S1). The amplified DNA samples were sequenced by standard automated DNA sequence technology employing the primers described above. The sequence results were compared with the PAO1 sequence (www.pseudomonas.org) by the BLAST programme at the NCBI database (www.ncbi.nlm.nih.gov/blast/) and within the strains of the AA lineage in order to determine the occurrence of sequence variants.
Cell Cultures and Transfections
The human embryonic Kidney epithelial cell line HEK 293 was grown in D-MEM supplemented with 10% of FBS (both by Cambrex Bio Science). Stably transfected cell line HEK 293-hTLR4/MD2-CD14 (InvivoGen) was cultured in DMEM with 10% FBS and 10 µg/mL Blasticidin-S and 50 µg/mL HygroGold® (both by InvivoGen). HEK293 cells were transiently transfected with PolyFect Transfection Reagent (Qiagen) according to the manufacturer's instructions. For NF-kB studies, the cells were transfected overnight with a reaction mix composed by 1 µL of PolyFect Transfection Reagent (Qiagen), 150 ng of Firefly luciferase reporter constructs, pGL3.ELAM.tk (containing NF-kB promoter sequences), and 15 ng of Renilla luciferase reporter plasmid, pRLTK (as an internal control).
To study Nod(s) activity, HEK293 cells were transfected with 10 ng of hNod1 (pcDNA3-Nod1-FLAG) or 1 ng hNod2 (pUNO-hNOD2a – InvivoGen). Vector plasmids (pcDNA3) were used as controls in all transfection experiments.
siRNA Assay
siRNA inhibition of Nod1 was carried out as previously described [49] by using psiRNAh-Nod1(InvivoGen). Briefly HEK293 were transfected with 1 ng of plasmid expressing psiRNA or vector control (containing a scramble sequence) together with the plasmid expressing hNod1. Nod1 inhibition was evaluated through western blot analysis with mouse mAbs anti-DYKDDDK (Clone 2EL-1B11, Chemicon International) as previously reported [49].
Cell Cultures of CF Origin
IB3-1 cells, an adeno-associated virus-transformed human bronchial epithelial cell line derived from a CF patient (ΔF508/W1282X) and C38 cells, the rescued cell line which expresses a plasmid encoding a copy of functional CFTR, have been obtained from LGC Promochem [50], [51]. Cells were grown in LHC-8 media (Biosource) supplemented with 5% foetal bovine serum (FBS) (Cambrex Bio Science). All culture flasks and plates were coated with a solution of LHC-basal medium (Biosource) containing 35 µg/mL bovine collagen (BD Biosciences), 1 µg/mL bovine serum albumin (BSA, Sigma) and 10 µg/mL human fibronectin (BD Bio Science) as described [50].
Cells were seeded at the density of 8×104 cells/cm2 for 4 h experiments and 4×104 cells/cm2 for the 24 h experiments. The day after cells were treated with 100 ng/mL of LPS derived from the clinical isolates AA2, AA43 and AA44. LPS from P. aeruginosa serotype 1022 (source strain ATCC 27316) (Sigma) was used as control at the same concentration. In the presence of each LPS for 48 h at 100 ng/mL the cell viability was >95% as determined by Tripan Blue exclusion test. Cells seeded at the density described above were also transfected with 0.5% of PolyFect Transfection Reagent and 1 µg/mL of PGN derived from the clinical isolates AA2, AA43 and AA44. γTriDAP (L-Ala-D-Glu-meso-DAP), chemically synthesized by AnaSpec (San Jose, CA), and MDP (MurNAc-L-Ala-D-isoGln) (InvivoGen) were used as positive controls at the concentration of 1 µg/mL. Growth media were collected at the end of incubation, centrifuged and stored at –80°C, while cells were lysed.
Culture of a Macrophagic Cell Line
The non-adherent human myelomonocytic THP-1 cells, obtained from LGC Promochem, were grown in RPMI-1640 supplemented with 10% FBS. Cells were seeded at the density of 8×104 cells/cm2 and allowed to differentiate to macrophage like cells by induction with 60 ng/mL of phorbol myristic acid (PMA) for 36 h, as described previously [52]. The differentiated cells were treated for 6 h with 100 ng/mL of LPS derived from the clinical isolates AA2, AA43 and AA44. LPSs from P. aeruginosa serotype 1022 and from E. coli serotype OIII:B4 (Sigma) were used as controls. Growth media were collected at the end of incubation, centrifuged and stored at –80°C.
LPS and PGN Stimulations and NF-κB Activity Assessment
For LPS, the day before stimulation HEK 293-hTLR4/MD2-CD14 cells were transiently transfected with the NF-kB reporter constructs as above. Twenty-four hours post-tranfection, cells were left untreated or incubated during 4 (to measure NF-κB and IL-8 mRNA) or 24 (for ELISA) h with different concentration of LPS purified from the clinical isolates AA2, AA43, and AA44. Commercial LPS of P. aeruginosa PAO1 (10–50–100 ng/mL) (Sigma) was used as positive controls.
For PGN, HEK 293 cells were simultaneously transfected with (i) hNod1 or hNod2 (ii) NF-kB reporter constructs and (iii) muramidase mutanolysin-digested PGN (Sigma) purified by the three P. aeruginosa clinical isolates AA2, AA43, and AA44, at different concentrations (0,5–1–10 µg/mL). γTriDAP and MDP were used as positive controls at the concentration of 1 µg/mL.
NF-kB-dependent luciferase activation and IL-8 mRNA evaluation was measured at 18 h of post-transfection while IL-8 ELISA was performed at 24 h post-transfection.
To measure NF-kB activity, cells stimulated with LPS or PGN, were lysed in 30 µL of passive lysis buffer, and 10 µL of lysate were assayed for Firefly and Renilla activity according to the manufacturer's instructions. The data shown represent the mean values±S.D. of three separate experiments performed in duplicate. Results are reported as fold induction of relative luciferase units (RLU) over unstimulated cells. Relative luciferase activity is calculated as the ratio between the value of the NF-κB inducible Firefly luciferase and that of the constitutive Renilla luciferase reporter.
RNA Quantification
Total RNA was extracted from lysed cells with the Total RNA Isolation kit (Roche), converted to cDNA with High Capacity cDNA Archive Kit (Applied Biosystems) and random primers and finally amplified using the Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen) as described [53]. Primer sets for TNF-α and IL-8 have been reported previously (Sigma-Genosys) [54], [53].
IL-8 and TNF-α Secretion
Released IL-8 and TNF-α were determined in supernatants collected from the cell cultures using ELISA kits (Biosource Europe and R&D Systems). According to the manufacturer the sensitivities of the assays are less than 0.7 pg/ml for IL-8 and 11 pg/ml for TNF-α. Measurements were performed at least in duplicate. Values were normalized to 106 cells; results were expressed as mean±SD.
C57Bl/6 mice (20–22 gr) were purchased by Charles River. Mice were housed in filtered cages under specific-pathogen conditions and permitted unlimited access to food and water. Mice were exposed to intranasal injection of LPS (10 mg/mouse) derived from P. aeruginosa. Control animals were exposed to PBS. Sixteen hours after exposure, mice were sacrificed by CO2 administration. Bronchoalveolar lavage (BAL) was performed by washing the lungs three times with 1 ml of RPMI (Euroclone) with proteinase inhibitors. Lungs were removed and homogenized in 1 ml of PBS with ions containing proteinase inhibitors. BAL cells were pelletted by centrifugation and counted in a hemacytometer, and differentials were determined by examination of cytocentrifuge slides stained with May Grunwald Giemsa staining (Sigma). The lung homogenates and BALF were centrifuged at 13000 rpm for 30 minutes at 4°C, the supernatants were stored at −20°C for cytokine analysis.
Measurement of Murine Cytokines
IL-1β, KC and JE concentration in lung homogenates were determined by ELISA (R&D Systems), according to manufacturer instructions using antibody pairs and recombinant standards from R&D System.
For histopathology, lungs were removed en bloc and fixed in 4% paraformaldehyde/PBS, at 4°C for 24 h, and processed for paraffin embedding. Longitudinal sections of 5 µm taken at regular intervals were obtained using a microtome from the proximal, medial and distal lung regions. Sections were stained with H&E according to standard procedures.
Supporting Information
Figure S1.
P. aeruginosa sequential isolates from patient AA.
Figure S2.
Sequence alignment of pagL in sequential P. aeruginosa isolates.
Figure S3.
RP-HPLC analysis of PGNs fragments.
Figure S4.
MS-MS analysis of dimer muropeptide.
Supporting Information File S1.
Results concerning full structural assignments of lipid A and muropeptides; experimental procedures; list of primers used for P. aeruginosa gene sequences (Table S1).
The authors thank B. Tummler, Klinische Forschergruppe, OE 6710, Medizinische Hochschule Hannover, Hannover, Germany, for supplying the P. aeruginosa strains from CF patients.
Author Contributions
Conceived and designed the experiments: AM MLB AB. Performed the experiments: CC LC MRL TI NIL IB. Analyzed the data: CC LC MRL TI NIL IB AS FC RL. Contributed reagents/materials/analysis tools: AS FC RL AM MLB AB. Wrote the paper: AM MLB AB.
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text:
The artisanal online marketplace Etsy took devotees by surprise last week when it announced that in July it will jack up the fee it charges sellers for a transaction, from 3.5% to 5%.
If you ask Brian Hoffman, founder of the decentralized online marketplace OpenBazaar where users pay in cryptocurrency, the Etsy move is a reminder of the power that centralized companies hold over users.
"That is something that centralized services can do," Hoffman said on stage last week at Yahoo Finance's All Markets Summit: Crypto in San Francisco. "They go public, they need to demonstrate profits, they start adding new rules and restrictions. A marketplace like OpenBazaar can never do that overnight. The protocol is completely free, you don't pay for any part of it except for the transaction fees associated with cryptocurrency. And you have a choice of cryptocurrencies, so if fees go up on bitcoin, you can switch over and accept something else."
OpenBazaar is more software protocol than company. It launched in 2014 at a hackathon in Toronto and was originally called DarkMarket. Hoffman forked the project code on GitHub and renamed it OpenBazaar. It has since raised more than $4 million in venture funding from big names like Digital Currency Group and Andreessen Horowitz.
On OpenBazaar, sellers are offering all manner of goods, from vintage comic books, to bitcoin-themed clothing, to digital cameras and handheld video game consoles, to handmade jewelry and pottery. Buyers can pay in a number of different cryptocurrencies, but not in fiat currency—"out of principle," Hoffman says.
So, why would a seller of hand-crafted goods list their items on OpenBazaar instead of, say, Etsy or eBay? Hoffman says it's an intimate and easy entry for the crypto-curious. "The people that are using it are trying to figure out, What's a way that I can offer a service or something I have for sale in order to get into crypto? It's a more organic way, rather than just going to Coinbase and buying it directly," he says. "There's a garage-sale feel to it… I think the same type of people that use Etsy are willing to try a new platform, and if they have something very unique to offer, that people are willing to part with their cryptocurrency for, that's the target audience."
Etsy, eBay, and any other online marketplace with centralized power will get disrupted by decentralized platforms that operate using blockchain, Hoffman predicts. "What's important about cryptocurrencies that I think starts to gets watered down as we get more mainstream is that we're trying to disintermediate the existing infrastructure, the huge players like Facebook, and Google, all these big players that are dominating their space."
Watch Yahoo Finance's All Markets Summit: Crypto here.
Daniel Roberts covers bitcoin and blockchain at Yahoo Finance. Follow him on Twitter at @readDanwrite.
Read more:
Ripple exec: XRP is 'Crypto 2.0'
Coinbase exec: 'Adding more assets is a very big priority for us'
Warren Buffett on buying bitcoin: 'That is not investing'
Lightning Labs CEO: We are back to a 'bitcoin, not blockchain' world

subset: commoncrawl


text:
Like everyone else, I have spent the last two weeks trying to wrap my head around the results of the presidential elections.
Without a doubt, Donald Trump's election is a huge setback for this country's efforts to come to grips with our changing climate and threatened natural environment.
Among my colleagues at NYER, there is a range of political opinions, but we are clear on the primacy of science, and everyone's need for a healthy environment. The vast majority of the scientific community has been sounding an alarm for years that if our planet is to support future generations, we have to change course now, especially when it comes to fossil fuels.
For the time being, this country's incoming leadership refuses to acknowledge the profound importance, and compromised state, of our environment. In light of that, here are five things that I am personally taking to heart as we head into 2017.
To be clear, these are my opinions, based on what I've learned as a reporter and as a person.
I really hope you'll send us your feedback. And we'll do our very best to keep covering the environmental issues — like air and water quality, trash management & recycling, energy supply, and climate resiliency — that impact readers in the metro area.
1.) We are not alone — there is a global environmental movement
There is not enough media coverage of the fact that people of all backgrounds are engaged in important environmental work across the world. You can hear their voices and stories from organizations like Greenpeace International, and news outlets like Democracy Now, which reported directly from the U.N. climate talks in Morocco last week.
There are a myriad of important and useful ways we can support — and be a part of — the global environmental movement in the next year.
For starters, citizens of this country can contact incoming members of Congress, and the new administration, to voice their opinion on whether the U.S. should remain an active participant in the U.N. Convention on Climate Change, and its 2015 Paris Agreement.
"Will that accomplish anything?" a friend said to me the other day. Well, the alternative is that we remain silent as the Trump administration tries to pull the U.S. out of the global climate accords. Consider this: 48 nations — including Bangladesh, Ethiopia and the Philippines — promised to "rapidly move to 100% renewable power" at the UN climate summit last week, the Guardian reported.
It's worth noting that significant public resistance to the Keystone Pipeline paved the way for the Obama administration to squash it, and, yes, this battle may very well be fought again.
(There are more ideas on what we can do below.)
2.) The majority of the American people accept the reality of climate change, and want to address it.
According to a Gallup Poll earlier this year, 65 percent of Americans now say that increases in the earth's temperature over the last century are primarily attributable to human activities, rather than natural causes.
This represents a "striking" 10-percentage-point increase in the past year and is four points above the previous high of 61 percent in 2007, Gallup reports.
64 percent of U.S. adults told Gallup they are worried a "great deal" or "fair amount" about global warming — the highest reading since 2008.
3.) The facts, and science, will have the last word.
According to an analysis released this month by the World Meteorological Organization, the planet just had its hottest five-year period on record, with 2015 claiming the title of hottest individual year, which will be beat by 2016.
"The effects of climate change have been consistently visible on the global scale since the 1980s," the WMO reported, pointing to "rising global temperature, both over land and in the ocean; sea-level rise; and the widespread melting of ice. It has increased the risks of extreme events such as heatwaves, drought, record rainfall and damaging floods."
The WMO singled out Superstorm Sandy as one of several "high-impact" global weather events whose likelihood was increased by climate change.
The October 29th, 2012 storm caused the deaths of 43 New York City residents and created $19 billion in economic damage in the five boroughs. Sandy had a 'storm tide' over 14 feet above Mean Low Water at the Battery. Fifty-one square miles of New York City flooded during the storm, 17 percent of the city's total land mass.
4.) Local action is going to matter — a lot.
Some of this country's most populous states — like California and New York — are moving ahead now to cut carbon emissions, and transform their energy supplies. How much will it matter? I heard a participant at the U.N. climate talks last week argue that local governments in the U.S. could accomplish half of our carbon reduction commitments, as per the Paris Agreement, without federal support.
In the next 14 years, New York State is planning to cut its greenhouse gas emissions by 40 percent, relative to 1990 levels. And the State says that half of New York's electricity will come from renewable energy sources by 2030.
The State's long-term goal is to decrease total carbon emissions 80 percent by 2050. The City of New York has similar goals, and says it is looking even further ahead to a 100 percent carbon free future, along with zero waste to landfills by 2030.
Undoubtedly, there are many hard questions to be asked about how, for example, the State is reconfiguring our energy markets, and whether New York City can get to a zero waste future. But, we are arguably on the road.
5.) Building an environmentally sustainable society will be a long, challenging process, but we already knew that.
Building a truly sustainable society — which is not a net drain on the planet — could take generations. That was true before November 8th, and remains so.
And as quixotic as it may seem, we know that it's worth it. Every child — and every adult — deserves a fighting chance at a decent life, which will not be possible on a degraded planet.
How can we participate? Here are just a few suggestions that show the wide range of actions (personal, and as part of a group) that we can take:
- call your senators and congresspeople and tell them what you think about retaining the U.S. Environmental Protection Agency, along with the Clean Air and Clean Water Acts, and the Clean Power Plan.
- support candidates at all levels of government who share your views on clean energy, waste reduction, and strong protections for air and water
- better yet, run for public office yourself!
- get involved with and/or donate funds to national environmental advocacy organizations like the Natural Resources Defense Council, the Environmental Defense Fund, and 350.org; and local groups like Environmental Advocates of New York and the NY League of Conservation Voters.
- talk with your friends, neighbors and co-workers about climate change, and share fact-based information
- participate in community meetings with local officials about issues like cleaning up polluted waterways and climate resiliency planning. If you live in NYC, these meetings are often sponsored by your local community board
- learn about ways to reduce energy and water use, and generate less trash at home
- participate in a neighborhood clean-up day
- talk with the children in your life about environmental issues
- you tell us — what can people do?
Finally, here are some interesting thoughts from Randy Cohen, who used to write The Ethicist column for The New York Times Sunday Magazine. In November, 2008, a Texas woman wrote to Cohen for advice because her neighbors had decided to lease their land for gas drilling, and she was under pressure to join them.
"For environmental reasons, we strongly oppose this drilling," the woman wrote on behalf of herself and her partner. She asked Cohen if holding out, while all her neighbors went ahead, was a futile, meaningless gesture.
Cohen responded, in part:
"It is understandable that you feel powerless in the face of community-wide sentiment…but you should not sign the lease…
To fail to resist what you see as injustice simply because you fear that you cannot win the fight assures the very defeat you dread.
If nothing else, this is a short term view. Political struggle is long. Even if you lose the first battle, you fight on, and by resisting from the outset, you shape the conditions of that struggle.
The most potent argument for your declining to sign what you regard as a devil's bargain is this: It violates your own principles…Ethics concerns our actions, not just our arguments."
And so this next chapter in our history begins. As this post was being finished, President Obama moved to prohibit any new oil drilling in the Arctic Ocean, one of his last actions before leaving office.


text:
Words For Websites
On your marks, get set, go!
When someone visits your website you only have a moment to let
them know that they've reached the right place
- Balance is key. When writing for a website you need to bear in mind that you have two audiences to satisfy at the same time:-
- The website visitor (your potential or existing customers)
- The search engines
It is important to get the visitor to the site. It is equally important that your visitors see a great message once they have arrived. Don't put search engines before well written text.
- Usual rules apply. Although writing for websites is different to writing off-line, many of the general rules of copywriting still apply. For instance: know your audience, talk about them, not you. Benefits, not features. Set objectives. Tell your visitors what you want them to do.
- Visitors don't read, they scan. Keep it simple. Use white space, short paragraphs, headings and bullet points.
- Key words and phrases. Your copy is one of your search engine optimisation (SEO) tools, i.e. one way you can help the search engines to find your site. Key words and phrases are the terms your customers are searching on in order to find your product or service. When visitors reach your site and start scanning it, it is these key words and phrases they are looking for.
- Research your keywords using a tool such as Google Keyword Estimator.
- Use the Keywords or phrase in the title and then two or three times throughout the page.
- Ensure your web developer includes the same phrases in the relevant 'behind the scenes' places.
- Don't spoil effective copy by hijacking it with keywords. It still needs to be readable, fluent and clear, not stilted and confusing.
- Refresh, Re-write, Review. Your website content should not be static. Update it regularly. Use Google Analytics to see which pages work and which don't. Give visitors a reason to return.
- Take my hand. Lead your visitor from one page to the next. Use internal links. Guide them through your site. Think layers. The deeper into the site your visitors get, the more detailed information they expect to find.
- Why am I here? What is the purpose of your website? In most cases, it is to capture something. If you have an e-commerce site, you want to capture the sale. If you're not selling on line, you should aim to capture email addresses so that you can start to build a relationship with potential customers. Offer a free report, newsletter or a product sample in exchange for an email address and permission to contact them. Give them loads of ways to interact with the site and register their email address. Then keep in touch. Make it easy for your visitors to do what you want them to do.
- . . . and they're off! "Web users are impatient, task-focused and extremely disloyal: if they can't find what they're looking for easily, they're off" says online expert, Catherine Tool. Bear this in mind when writing your copy. Start off by keeping it simple and to the point and explaining the benefits, then lead them deeper into the website to add the details.
"That's all great, but I just haven't got time"
If this sounds like you, Fill the Gap Marketing Ltd can help. We provide professional copywriting services from £50 – leaving you free to concentrate on your core business.
We work in partnership with specialists who advise on Search Engine Optimisation, pay per click advertising campaigns and social media marketing. All potentially great ways to drive traffic to your website.
Contact us for a complimentary consultation or website review. We can help you to grow your business profitably.


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The West Indies regular Twenty20 cricket captain Darren Sammy has been ruled out of the Twenty20 series against New Zealand.
Sammy is still bothered by a hamstring injury he sustained prior to the start of the third One-day International.
His place in the squad will be filled by left-arm spinner Nikita Miller and ODI captain Dwayne Bravo will lead the side in his absence.
The joining T20 players - Samuel Badree, Andre Fletcher and Andre Russell - arrived in New Zealand on Monday.
Russell will be considered for selection for the final ODI against the Black Caps in Hamilton tomorrow.
Neither Chris Gayle nor Kieron Pollard was considered for selection to the T20I squad due to injury.
Gayle continues his recovery from a hamstring injury sustained during the tour of India last November, and Pollard continues his rehabilitation following a knee injury around the same time.
The first T20 international is in Auckland on Saturday with the second in Wellington next Wednesday.


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Coral Strand Smart Choice Hotel
- Free WiFi and free parking
- Great for families
- Collect nights
Coral Strand Smart Choice Hotelfrom £156
- Standard Room
- Classic Room, Ocean View
- Superior Room
- Superior Room, Ocean View
More great choices
What's around Coral Strand Smart Choice Hotel
Top spots nearby
Recommended by this property
- Beau Vallon Beach (2-minute walk)
- Beau Vallon Market (2-minute walk)
- Victoria Clocktower (26-minute walk)
- Seychelles National Botanical Gardens (32-minute walk)
- Morne Seychellois National Park (35-minute walk)
- Stade Linite (3.2 mi / 5.2 km)
- Baie Ternay (4.1 mi / 6.6 km)
Getting around
- Victoria (SEZ-Seychelles Intl.) 14-min drive
- Airport shuttle (surcharge)
At a glance
Key facts
Hotel size
- This hotel has 160 rooms
- This hotel is arranged over 5 floors
- Check-in time 2 PM-11:30 PM
- Check-out time is 11:00 AM
- Express check-in/out
Required at check-in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Children stay free when occupying the parent or guardian's room, using existing bedding
- Babysitting/childcare *
- Pets not allowed
- Free WiFi in public areas
- Free WiFi and wired Internet access in rooms
- Airport shuttle (available 24 hours) *
- Free extended parking
- Free nearby parking
Payment types at the property
In the hotel
- Buffet breakfast daily (surcharge)
- 3 restaurants
- Bar/lounge
- Beach bar
- Poolside bar
- 24-hour room service
- Barbecue grills
- Fishing on site
- Kayaking on site
- Personal motorised watercraft on site
- Parasailing on site
- Sailing on site
- Volleyball on site
- Bicycle rentals nearby
- Ecotours nearby
- Hiking/biking trails nearby
- Scooter/moped rentals nearby
- Scuba diving nearby
- Snorkelling nearby
- Surfing/boogie boarding nearby
- Waterskiing nearby
- Windsurfing nearby
- 24-hour business centre
- Number of meeting rooms - 2
- Conference space
- Conference space size (feet) - 1292
- Conference space size (meters) - 120
- Conference centre
- 24-hour front desk
- Concierge services
- Tours/ticket assistance
- Limo or Town Car service available
- Dry cleaning/laundry service
- Laundry facilities
- Free newspapers in lobby
- Luggage storage
- Wedding services
- Multilingual staff
- Porter
- Number of buildings/towers - 1
- Year Built – 1972
- Lift
- Designated smoking areas
- Garden
- Terrace
- Library
- Television in common areas
- Accessible bathroom
- In-room accessibility
- Roll-in shower
- Wheelchair-accessible path of travel
- Wheelchair-accessible parking
- Arabic
- Chinese
- English
- French
- German
- Italian
- Russian
- Spanish
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Minibar
- Coffee/tea maker
- Slippers
- Blackout drapes/curtains
- Furnished balcony or patio
- Private bathroom
- Rainfall showerhead
- Shower only
- Free toiletries
- Hair dryer
- 32-inch plasma TV
- Satellite TV channels
- Free WiFi
- Phone
- Fridge
- Free bottled water
- Daily housekeeping
- In-room safe (laptop compatible)
- Connecting/adjoining rooms available
- Access via exterior corridors
Also popular with travellers from the UK
Special features
DiningIsland Trader – Overlooking the ocean, this buffet restaurant specialises in Halal dishes and serves breakfast and dinner.
Mahek – Overlooking the ocean, this restaurant specialises in Indian cuisine and serves lunch and dinner. Open daily.
Ocean Deck Pub – This beach pub specialises in international cuisine and serves brunch, lunch, dinner and light fare. A children's menu is available. Happy hour is offered. Open daily.
Coral Asia – This poolside sushi bar specialises in sushi and serves brunch, lunch and dinner. Open daily.
On Site
- Fishing on site
- Kayaking on site
- Personal motorised watercraft on site
- Parasailing on site
- Sailing on site
- Volleyball on site
- Bicycle rentals nearby
- Ecotours nearby
- Hiking/biking trails nearby
- Scooter/moped rentals nearby
- Scuba diving nearby
- Snorkelling nearby
- Surfing/boogie boarding nearby
- Waterskiing nearby
- Windsurfing nearby
Coral Strand Smart Choice Hotel's small print
Also known as
- Coral Strand Smart Choice Hotel Mahe Island
- Coral Strand Smart Choice Hotel Hotel Mahe Island
- Coral Strand Smart Choice
- Coral Strand Smart Choice Hotel Hotel
- Coral Strand Smart Choice Hotel Mahe Island
- Coral Strand Smart Choice Hotel Hotel Mahe Island
- Coral Strand Smart Choice Hotel
- Coral Strand Smart Choice Mahe Island
- Coral Strand Smart Choice
- Hotel Coral Strand
- Coral Strand Beau Vallon
- Coral Strand Smart Choice
- Coral Strand Smart Choice Hotel Hotel
- Coral Strand Smart Choice Hotel Mahe Island
Deposit of 50% is payable for all booking from the 22 December until 15 January. To complete the reservation process and obtain additional information, please contact the property using the information on the reservation confirmation received after booking.
The property has connecting/adjoining rooms. These are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
Only registered guests are allowed in the guestrooms.Please note that cultural norms and guest policies may differ by country and by property. The policies listed are provided by the property.
For transfers, guests must contact the property 24 hours prior to arrival on the number on the booking confirmation.
The name on the credit card used at check-in to pay for incidentals must be the primary name on the guestroom reservation.
Mandatory fees
You'll be asked to pay the following charges at check-in or check-out:
- Christmas Eve (24 December) Gala Dinner per adult: EUR 120
- Christmas Eve (24 December) Gala Dinner per child: EUR 60 (up to 12 years old)
- New Year's Eve (31 December) Gala Dinner per adult: EUR 150
- New Year's Eve (31 December) Gala Dinner per child: EUR 75 (up to 12 years old)
Optional extras
Early check-in can be arranged for an extra charge (subject to availability, amount varies)
Late check-out can be arranged for an extra charge (subject to availability, amount varies)
Rollaway beds are available for EUR 30.0 per night
Buffet breakfast is offered for an extra charge of EUR 16 for adults and EUR 0 for children (approximately)
Babysitting/childcare is available for an extra charge
Airport shuttle service is offered for an extra charge of EUR 60 per vehicle (one-way)
Airport shuttle service for children costs EUR 60 (one-way)
We have included all charges provided to us by this property. However, charges can vary, for example, based on length of stay or the unit you book.
Recent reviews
Good 7.2 From 231 reviews
We were married at the Coral Strand 30 years ago. It was a beautiful hotel with fantastic service and we were very well looked after so we always vowed to return. We did return and were very disappointed. The first room we were given stank of cats pee, the shower squirted more water on the ceiling than on me, sink taps weren't attached properly, the bathroom door was mouldy as were the curtains. We complained and were immediately moved to another room which was a big improvement. We met two other couples there that had exactly the same problems (cat pee smell etc) and had to be moved. Some staff couldn't be nicer and yet some others were quite sullen. The food was average, the main lift only worked sometimes and when we checked out nobody offered to help with our baggage and, guess what, the lift wasn't working so we had to carry them on our own down three floors. Overall we thought the hotel was looking very tired. The location is fantastic!
Nice hotel. The only one in the area right on the beach. Good pool. Room overlooked pool and ocean. Friendly, helpful and efficient staff. Nice breakfast in restaurant overlooking beach and ocean. Not much to do in the area but there are restaurants nearby. Most seem to close around 10pm. Would recommend Gecko Bar at the Savoy (about 2 mins walk from the hotel). Nice food or just have a drink plus you get a 10% discount for being Coral Strand resident. Wednesday evening the "strip" livens up with music and food stalls otherwise very quiet. The Boathouse Bar is best for just having a drink and that's less than 10 min walk away. Overall very nice if you're ok with quiet life and beach holiday. Would recommend Teddy's Glass Bottom Boat trip to St Anne's Island for snorkelling island tour. Great boat crew and really nice lunch. One of our best days out. You can book at hotel or from one of the many vendors on the strip.
The hotel is situated on the beach in a perfect location. The Beau Vallon beach is regarded as one of the best in the Seychelles. There are some excellent water sport activities at the location. The hotel food is good and reasonably priced although the alcoholic drinks incl local beers are very expensive. Some parts of the hotel need a bit of decoration. The staff are very helpful. Our sea view room was double booked and we were compensated with free breakfast for the duration of our stay, although we did get a 'sea view' room after a couple of days. We went on Teddy's glass bottom boat trip arranged via one of the salesmen who frequent the beach in front of the hotel. That was excellent and I suggest a must do. The adjacent savoy hotel does excellent food which is discounted by 10% when staying at coral strand
Chaos on arrival, staff shouting at each other on the telephone, then they couldn't find our reservation. Finally got a room but not in the booked higher room category! Ended up in a room with damaged wardrobe, messy shower cabin and an A/C unit that couldn't be controlled apart from on/off... Breakfast is average, area behind the bar minging! This place is old, rundown, overpriced and run by non professional staff. Stay away!
The hotel is old and bad maintained. We moved right away out of this hotel.
Is the description of this property not correct? Tell us


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Every month this year I've signed up to join 'Nothing But UFO's in 2013', hosted by Carrie at A Passion for Applique. Every month we list three UFO's to work on and at the end of the month we have to finish at least one of the UFO's to be in the draw for a prize.
I am one of the lucky winners for March 2013 draw! (Yes, I know, a long overdue post!)
Two pieces of beautiful fabrics, two skeins embroidery floss from The Gentle Arts (which I love) and a gorgeous sewing companion from KaHolly.


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Choosing Text Fonts for Data Visualizations
Choosing the perfect text font for any given data visualization can be a tricky process. Although increasing the size of a word may convey emphasis or importance, it's not always the best route as it may overshadow the visuals.
Font sizes and formats
Here are some ideas to consider when you choose font sizes and formats:
Make important text a little bigger. A general rule of thumb is that low-hierarchy text should be in a smaller font than text of high-hierarchy text. The following figure shows how the font sizes of various labels in a hierarchy may vary based on level.
Consistency is key. If you decide that all your main titles will be in a size 30 font, for example, you must ensure that all your main titles are exactly the same size.
The key factor is to have consistency for each individual label type. Consistent label sizes for each level are easy for users to grasp, and the sizes help users identify certain areas of your data visualization at a glance.
Avoid using all caps. The era of text messages and social media has created a new set of rules for using text. Today, using all caps indicates that someone is shouting, which usually evokes hostility and defensiveness. All caps are also harder to read, as all the letters are the same size. In addition, readers may gloss over or skip all-caps words.
It may be fine to use a simple all-caps title such as SALES EXECUTIVE DASHBOARD, but don't use all caps in labels throughout your data visualization. In general, you have very little to gain from using all caps, so it's best to avoid the practice.
Font types
Thanks to web browsers such as Internet Explorer and Google Chrome, most people have become accustomed to a few standard fonts, such as Verdana, Garamond, Arial, Helvetica, and Times New Roman.
These fonts are somewhat boring, but it's highly recommended to stick to one or more of them, for two reasons:
Each user's web browser may be different. Data visualizations are commonly hosted on company sites or portals that must be viewed in a web browser (unless, of course, you're developing a static image like an infographic). No matter what fonts you use to create a data viz, each user's web browser automatically defaults displaying any text using one or more of browser-safe fonts mentioned earlier.
If you use unusual fonts, your visualization may look distorted or become unreadable when your fonts are replaced with browser-safe fonts. It's safer to design your visualization with the common fonts.
One way to get around having to use browser-safe fonts is by posting your visualization in an Adobe PDF or Microsoft PowerPoint format. These file types are not affected by the browser, but these options are not available in all tools.
Avoid fancy or custom fonts. Using fancy or custom fonts may make it harder for someone else to understand or edit your data viz, especially if you use nonstandard or specialized fonts that need to be individually downloaded and installed on a desktop. Yes, custom fonts may look great, but they are simply not worth the risk of having the user not being able to view them.
We recommend using Verdana and Garamond, both of which are on the extended list of web browser-safe fonts. They add a little bit of flair without having the straight military feel of Arial.


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[Originally published on Chowk.com]
Time and time again we have been asked why we protest? Why do we choose to spend half our Sundays making posters, and the other half carrying them around in 40 degree Celsius heat, shouting till our throats give out, in Ramadan? It is a waste of time, a waste of effort, a waste of money. What have our rallies solved? They haven't brought world peace, nor have they saved lives in Swat, nor have they resulted in true democracy in Pakistan. So why?
Why not to protest?
Reason # 1: "I'm going to get beaten up and arrested"
Following are the two articles of the constitution of the Islamic Republic of Pakistan that legitimize peaceful protest demonstrations:
Article 16 – Freedom of Assembly: "Every citizen shall have the right to assemble peacefully and without arms, subject to any reasonable restrictions imposed by law in the interest of public order."
Article 19 – Freedom of speech, etc: "Every citizen shall have the right to freedom of speech and expression, and there shall be freedom of the press, subject to any reasonable restrictions imposed by law in the interest of the glory of Islam or the integrity, security or defence of Pakistan or any part thereof, friendly relations with foreign States, public order, decency or morality, or in relation to contempt of court, commission of or incitement to an offence."
Hence, as long as you stick to peaceful and legal means of demonstrating, you will be safe. In all the protests we have organized and attended, not once have been attacked by the police or arrested. Of course there are exceptional circumstances where there is a very real danger of injury or arrest. What we must realize here is that the danger that individuals face from coming out on the streets is negligent in contrast with the danger Pakistan faces if they do not.
Reason # 2: "There is no point, it is not worth it, change will never come!"
These were the very arguments that Mao Zedong, Martin Luther King, and Nelson Mandela all were faced with. They were dismissed as idealistic, and told that they could not make a difference at all. Imagine what would have happened had they given in to the criticism they faced? There still would be apartheid in South Africa, African Americans in the USA would not be allowed to vote, let alone one of them being the president, and China would not be a trading giant.
Now lets look a little closer to home. Benazir Bhutto spent eight years in prison, including solitary confinement to become Prime Minister in 1988 and try to bring about change.
The Indians of the Subcontinent rose in an uprising to get their country back. Had they not protested against British rule, had they decided it was too scary or there was no point, then we would not be where we are.
Today, if the entire country mobilizes against injustice then a government that claims to be popularly elected and democratic will ultimately have to give in. Change will take place. Not overnight, or in a matter of hours. It will talke time. We just have to keep fighting until it takes place.
Reason # 3: "Pakistan has no future."
Last week, one of our friends escaped to Canada, citing security concerns as the reason. This is just one example of the many families that have fled Pakistan to enjoy the stability that other nations have achieved through their own struggle. That's the difference between Pakistan and other nations. The people in other countries have struggled to get where they are. Pakistanis, on the other hand, run to those countries, rather than getting their own nation up to the desired level. We as a nation are not willing to fight, to swim. We prefer to sink or to get on somebody else's boat, rather than making our own.
If we leave, who will improve the country for people to not want to escape from it?
Skeptics will say that there is no future left for Pakistan. They are wrong. Pakistan certainly has a future; it is every single one of us. It is you, and it is me.
Frustrated with the undemocratic actions of the present government and the apathy of the youth alike, on March 7, 2009, some students of Islamabad decided to hold a protest demonstration against the imposition of governor rule in Punjab. They held posters expressing their dissatisfaction with the present government and shouted slogans such as "Hum sab kya chahtay hein? Jamhuriat! Jamhuriat!" ("What do we all want? Democracy! Democracy!"), "Adliya ki bahali tak Jang rahi gi! Jang rahay gi!" ("We will fight till the freedom of Judiciary!"), and "Give Punjab it's CM back!". It was particularly inspiring for all the students to have passer-by's join them. Others drove by, flashing victory signs, pumping fists, and giving thumbs up. "Hum mulk bachanay niklay hein, aao hamaray saath chalo!" (We are out to save our country, come join us!) shouted the students back..
This recent demonstration by patriotic and concerned students is a prime example of how each and every one of us can contribute to betterment in Pakistan, to its future, and to its present. If there is continuous expression of dissatisfaction by the people, the government will have to give in to the demands of the public. To save this country you do not need money, contacts, a degree, or a position. All you need is passion.
Why are demonstrations like these worth it?
Reason # 1: The feel-good factor:
Ask anybody who went to the Long March last year, and they will tell you how amazing it felt. They will tell you about the spiritual electric charge in the air, the feeling of being part of one body, of one entity. A good protest can often have a spiritual effect on people, inspiring them to continue the fight for a good cause tomorrow.
After a protest, you will feel satisfied with having played a small but effective part in contributing to a positive change in the system of your country.
Reason # 2: Get attention to your cause:
Protest demonstrations are very effective in getting attention to your cause. You get noticed by the media, by passersby, by politicians, by other activists. People become aware of issues, rethink them, and develop their on perspective on them. By speaking out, you make sure that your cause is not forgotten.
Reason # 3: Silence is consent:
If you are silent about injustice that is taking place, you are in effect consenting to it. Hence, coming out and speaking up against it will show that you disagree with what is going on. We must stand up for our rights, and fight till we get them.
Before 1947, our forefathers shed blood to give us Pakistan. It is now our duty to sustain it well, and to save it from becoming a failed state that many international quarters have already claimed it to be. It is our duty to work for its very survival. Edward R. Murrow famously said: "A nation of sheep will beget a government of wolves". Let us not be sheep anymore, and let us get rid of the wolves. Once and for all.
(Usama Khilji and Humna Bhojani are student activists from Islamabad. If you wish to contact them or join in with them in their future protests, email at humnabhojani AT hotmail DOT com; Usamakhilji AT hotmail DOT com)


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Those who blush will never break taboos. And neither will those whose heads lower at the thought of shame. But those who don't go red in the face from wearing what nobody else would wear will knock taboos over as if they were made of crumbling substances. And rebels will break things, too, and criminals as well, even if they aren't criminals at all but rather just the changers, the innovators, the first walkers on untrodden ground. Although the laughers will not break taboos, those who are laughed at might have already broken many of them.
Lovers similarly have a tendency to leap over restrictions, love even more so. Remember love doesn't just choose us; it strangles right and wrong out of us instead of air, it cripples our minds, making us do what others say not to. Love whispers sweet defiance into our ears, tells us to be the next Romeo and Juliet, to be the next Adam and Eve. Love may even make you forget that some loves are forbidden.
But believers will pray for a host of unbroken things. Conformists will ask for conformity, unaware that the established practices today were once unestablished. Yet the future won't believe in anything, won't conform to anything other than change. Yes, the future will wear change for clothes; it will push boundaries
Somewhere they have never been pushed before so that even the past may be revised until it is a past nobody can forget.
Artists will help transform this. Many will erase structures and paint other structures in its place. And the new becomes old, waiting to copulate with another new, which will again become old after it births another new. And at some point, all taboo breakers will no longer be breakers but rather makers of what we consider common.


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ENID, Okla. — The bright, eye-catching packets are labeled "incense," "potpourri," "bath salts" or "spice." The labels read, "aromatherapy use only" and "not for human consumption."
But the substances inside the packets are far from the innocent labels and disclaimers make them sound.
Using them produces a marijuana-like, amphetamine-like or LSD-like high, but their adverse effects are powerful.
Enid Police Det. Zeke Frazee is well aware of the frightening aspects of "spice." He pointed to common side effects, including hallucinations, seizures, extreme paranoia, nausea and dangerously accelerated heart rate. Yet because the products are used simply to get high, people won't admit using it.
"They don't tell anyone what they've smoked," Frazee said.
Michelle Hennedy, director of the emergency department at St. Mary's Regional Medical Center, agrees.
Patients have come to the emergency room numerous times with symptoms that make the medical staff suspect they are high on "spice" or "bath salts."
"A lot of what we're seeing is patients whose behavior is not appropriate," Hennedy said. "They can be confused, combative, they're very angry."
Hennedy said they often are brought in because they have fallen, passed out or some similar incident. She estimates most are between 18 and 40 years old.
But not every user is 18. Amber Fitzgerald, human resources and communications director for Enid Public Schools, said a student at Enid High School was arrested twice in April on charges of public intoxication and possession of drug paraphernalia. The arrests were 19 days apart. A second student also was arrested for public intoxication.
When patients are brought to the hospital with symptoms of drug overdose, physicians can order drug tests – but the tests don't disclose synthetic drugs.
When patients are asked what they've used, "they're not honest with us," Hennedy said.
Likewise, there is no standard field test for law enforcement to use in order to find out if a packet of "incense" is a formula already illegal or a newer formula.
"It must be sent to OSBI lab and the result comes back in two to three weeks," Frazee said.
Frazee said product reviews and instructions for use of "incense" are easy to find online.
"If you go on YouTube, there are kids talking about how to smoke this," he said.
Some of the online videos Frazee has watched warn that synthetic marijuana is far more potent than marijuana from a plant.
"It's more like acid or LSD," Frazee said.
Frazee said synthetic marijuana was first developed in the early 1990s as part of legitimate medical research being done by John Huffman, organic chemistry professor at Clemson University. After Huffman's research papers were published, entrepreneurs copied his formula and began marketing it.
Gas stations, gift stores and convenience stores sell it in Enid. Sometimes it is kept out of sight and the customer has to ask for it. Other times, it is displayed in plain sight behind the counter, Frazee said. After all, despite its dangerous qualities, it's a legal product.
Some of the earlier formulas were outlawed in Oklahoma last year, but the people making "spice" tweaked the formula and quickly were back in business.
Store owners Frazee has talked to, said that since it's not illegal and they make good money off it, they'll continue to sell it. He said he believes the stores pay $3 to $4 per bag for it.
"When you see it in plain view, they are selling it as incense," Frazee said. "They know what it does. I've even spoken with those store owners and operators. They said it's legal and they make good money off it, and until it's illegal, they would continue to sell it. If you have to hide it and it's selling for $30 a bag, shouldn't that tell you something?"
Maine Street Mini-Mart is one of the many businesses in Enid that sells "spice." Store owner A.J. Marand, contacted by telephone on Wednesday, hung up when asked if he would talk about why his business sells the product.
Glen Kuhn, charged with possession of a controlled drug with intent to distribute because his business, Your Quality Store, allegedly stocked illegal "spice" for sale, also declined to comment for this article.
"I've talked to people who sell this and they've told me a million stories about people who've bought this and had bad trips," Frazee said. "They sell it anyway. It's about money."
Because the envelopes are marked "not for human consumption," and "for aromatherapy use only," the Food and Drug Administration does not regulate the formulas and no consistency exists from one batch to the next. Additionally, there is no age limit to buy it.
"This stuff is new, so it doesn't have long-term studies yet as for the effects," Frazee said.
Hennedy said the more someone uses the product, the more their judgment is skewed, and they can put themselves in danger because they are not thinking clearly. But, while Hennedy worries about the safety of the patients who are using "incense," she also worries about the safety of the emergency room staff when rendering treatment to them.
"It's a concern because you never really know what you're treating," Hennedy said. "You always want to do the best for the patient. I wonder if they are going to go home and do it again or introduce someone else to it — someone younger."
"Smoking synthetic marijuana is playing Russian roulette, because you don't know what it's going to do to you," Frazee said.


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ARE the New Bible Versions produced since 1881 REALLY an attempt to restore an "easier to understand" English Bible to the world, or are these "New" Versions part of a Satanic conspiracy to destroy the KJV and the true Christian Faith!
Find out the answer to this question in this video by Bryan Denlinger. You will see many of the New Versions up-close and in detail. Unlike other documentary films, this video actually shows the proof on camera. You will see new "Bibles" that contain profanity, as well as known OCCULT symbols!
You will also hear from a medical doctor named Dr. Laurence Dunegan, who was told in a meeting in 1969 that the Bible was going to be re-written to form a "new religion"!! This 1 hour long video is packed with shocking information that will change the way you think about the "New" Versions, and give you a love and appreciation for God's true word, the King James Version!
In this new section you will see the shocking "Illuminated Bible". This new perVersion contains many horrible images, and even states that "God will bring in the new order"!!! You truly have to see it to believe it.


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My Dog Tulip (2009)
- Studio: Independent
- Release: October 12, 2009
- Director: Paul Fierlinger, Sandra Fierlinger
- Writer: Paul Fierlinger
- Cast: Christopher Plummer, Lynn Redgrave, Isabella Rossellini
Video archive for the film My Dog Tulip, which has a domestic theatrical release in the year of 2009. There are currently four videos available for the film, of which one and only one is a trailer, as listed below. Happy viewing!
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