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FppA, a Novel Pseudomonas aeruginosa Prepilin Peptidase Involved in Assembly of Type IVb Pili
Sophie de Bentzmann, Marianne Aurouze, Geneviève Ball, Alain Filloux
Sophie de Bentzmann
Laboratoire d'Ingénierie des Systèmes Macromoléculaires, CNRS-IBSM-UPR9027, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
Marianne Aurouze
Geneviève Ball
Alain Filloux
For correspondence: filloux@ibsm.cnrs-mrs.fr
Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Whereas molecular mechanisms of type IVa pilus assembly have been well documented for Pseudomonas aeruginosa and involve the PilD prepilin peptidase, no type IVb pili have been described in this microorganism. One subclass of type IVb prepilins has been identified as the Flp prepilin subfamily. Long and bundled Flp pili involved in tight adherence have been identified in Actinobacillus actinomycetemcomitans, for which assembly was due to a dedicated machinery encoded by the tad-rcp locus. A similar flp-tad-rcp locus containing flp, tad, and rcp gene homologues was identified in the P. aeruginosa genome. The function of these genes has been investigated, which revealed their involvement in the formation of extracellular Flp appendages. We also identified a gene (designated by open reading frame PA4295) outside the flp-tad-rcp locus, that we named fppA, encoding a novel prepilin peptidase. This is the second enzyme of this kind found in P. aeruginosa; however, it appears to be truncated and is similar to the C-terminal domain of the previously characterized PilD peptidase. In this study, we show that FppA is responsible for the maturation of the Flp prepilin and belongs to the aspartic acid protease family. We also demonstrate that FppA is required for the assembly of cell surface appendages that we called Flp pili. Finally, we observed an Flp-dependent bacterial aggregation process on the epithelial cell surface and an increased biofilm phenotype linked to Flp pilus assembly.
Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that is responsible for human infections in hospitalized and cystic fibrosis (CF) patients. Bacterial attachment is a major virulence trait required for chronic infections such as for P. aeruginosa acquiring a biofilm lifestyle in CF lungs (7). Formation and maturation of biofilm by P. aeruginosa are well documented at the molecular level and involve a large arsenal of cell surface-associated fibrils, including flagella, type IVa pili (TFP), or putative fimbrial structures called Cup (34, 51). Among these structures, the type IVa pili (TFP) are known to contribute at several stages of biofilm development, including initial attachment to surfaces, surface colonization, and development of mushroom-like structures through a mode of movement called twitching motility (25). The P. aeruginosa TFP are members of the type IVa family, which is characterized by a number of features including the size of the prepilin (9). In P. aeruginosa, the prepilin PilA is cleaved by the prepilin peptidase PilD (33), before it is assembled into a fiber structure exposed at the bacterial cell surface. The atomic resolution structures of four pilin subunits, combined with electron microscopy (EM) images of the pili, have provided information to describe the helical symmetry of the pilus fibers (8, 9, 24). Accessory components including a traffic ATPase (PilB), a secretin (PilQ), and a polytopic inner membrane protein (PilC) are required for TFP assembly (4, 32).
Another subclass of TFP, called type IVb, has also been described. Type IVb prepilins form a subgroup characterized by the 15- to 30-residue length of their leader peptide, the 190-amino-acid (aa) average length of their mature pilin, the nature of the N-terminus residue after the cleavage site, and the average length of the exposed D region in the assembled pili (9). One member of this class is the bundle-forming pilus (BFP), which is found in enteropathogenic Escherichia coli (EPEC) (11). The Bfp fibrils have a tendency to aggregate into a rope-like bundle and are responsible for bacterium-bacterium interaction and microcolony formation (6). The 14-gene bfp operon encodes proteins that are sufficient for BFP biogenesis in EPEC (39). Among these genes, bfpA encodes the bundlin, the major structural subunit of BFP (5), which is assembled with the help of typical TFP assembly components. Among these components, one can find a prepilin peptidase (BfpP), two traffic ATPases (BfpD and BfpF), BfpD involved in assembly (1), and BfpF required for retraction (3), a secretin (BfpB), a PilC-like protein (BfpE), and minor pilins (BfpI, BfpJ, and BfpK). Additional components not classically found in TFP systems are also required for BFP assembly and are a lytic trans-glycosylase (BfpH) and four proteins of unknown function, BfpG, BfpC, BfpU, and BfpL (1). The class IVb of TFP also includes the longus pilus of enterotoxigenic E. coli (14) and the toxin-coregulated pilus (TCP) of Vibrio cholerae (49).
Within the type IVb pilin family, there is a clear monophyletic Flp prepilin subfamily, which has been initially described in Actinobacillus actinomycetemcomitans, a gram-negative bacterium responsible for localized juvenile periodontitis (38, 58). The Flp prepilin subfamily shares unique features, including a long leader peptide, a short size for the mature pilin ranging from 50 to 80 aa, and a shared Flp motif of 20 hydrophobic residues at the N terminus of the mature pilin with adjacent glutamate and tyrosine residues in its center (38). In A. actinomycetemcomitans, the Flp pilin requires for its assembly a subset of components called Tad and Rcp, which includes a secretin (RcpA), a traffic ATPase (TadA) (2), and PilC-like proteins (TadB and TadC), as well as several other components that have no homologues in other type IV pilus assembly systems. The presence of thick fibrils composed of bundled thin Flp pili at the surface of A. actinomycetemcomitans is associated with the formation of extremely tenacious biofilms on a variety of solid surfaces and more particularly the teeth. Flp pili have also been described in Caulobacter crescentus (44) and Haemophilus ducreyi (31). Genome mining of the P. aeruginosa genome (45) revealed several genes encoding type IV pilins; one of these genes encodes an Flp-like protein. In this study, we further analyzed the assembly of the P. aeruginosa Flp pilin into a cell surface appendage and we characterized the role of a novel prepilin peptidase in this process.
Bacterial strains and growth conditions.The strains and plasmids used in this study are listed in Table 1. Strains were grown at 37°C in L broth or on LB agar. The E. coli TG1 and TOP10F′ strains were used for standard genetic manipulations. Recombinant plasmids were introduced into P. aeruginosa using E. coli S17-1 or the conjugative properties of pRK2013. Transconjugants were selected on Pseudomonas isolation agar supplemented with antibiotics. Antibiotics were used at the following concentrations (μg/ml): for E. coli, ampicillin, 50; streptomycin, 50; tetracycline, 15; and kanamycin, 50; for P. aeruginosa, carbenicillin, 500; streptomycin 2,000; tetracycline, 200; and kanamycin, 1,000.
Construction of P. aeruginosa mutant strains.Overlapping PCR was used as previously described (52) to generate a fragment that linked 500-bp-long DNA fragments corresponding to regions located upstream and downstream from the gene of interest, thus linking in frame the start and stop codons of the gene. The oligonucleotides used are listed in Table 2. The resulting DNA fragment was cloned into the suicide vector pKNG101. The recombinant plasmid was then mobilized into P. aeruginosa, and double recombination events were selected on LB plates containing 5% sucrose as previously described (22). In the mutant strain, this recombination event resulted in a nonpolar in-frame deletion of the gene of interest.
Oligonucleotides used for gene cloning and P. aeruginosa mutation engineering
Cloning of P. aeruginosa genes.The flp and fppA genes were PCR amplified using the oligonucleotide couples Flp11 Up/Flp12 Down and FppA Up-1/FppA Down-1, respectively. PCR products were cloned into the PCR2.1 vector. The flp and fppA genes were subsequently cloned into the broad-host-range plasmids pMMB190 and pBBR1MCS-2, respectively. The rcpA and tadA genes were obtained from a comprehensive P. aeruginosa gene collection (26) cloned into an entry vector of the Gateway system (Invitrogen). These genes were moved into a pDEST14 destination vector by LR recombination according to the manufacturer's instructions. The rcpA and tadA genes were further subcloned into the broad-host-range cosmid pLAFR3 at BamHI/HindIII restriction sites, yielding pLAFRrcpA and pLAFRtadA, respectively.
Site-directed mutagenesis of the fppA gene.The mutations in the fppA gene, which resulted in the replacement of aspartate residues (at position 17 or 78) with alanine residues, were generated by overlapping PCR as previously described (13). Briefly, oligonucleotides were designed to change the aspartate codons (D17/GAC and D78/GAC) into alanine codons (A17/GCC and A78/GCG) (Table 2). The 5′ and 3′ DNA regions from these codons were amplified using oligonucleotide pairs such as FppA-Up-2 and FppAD17A-Down, FppAD17A-Up and FppA Down-2, FppA-Up-2 and FppAD78A-Down, or FppAD78A-Up and FppA-Down-2. The 5′ and 3′ DNA fragments of the fppA gene were then reassembled by mixing and reamplifying with the oligonucleotide pair FppA-Up-2 and FppA-Down-2. The double mutation (D17A and D78A) in the fppA gene was generated by using the single mutated gene fppA(D17A) as a DNA matrix.
Bioinformatic analysis.The genome sequence of P. aeruginosa PAO1 and its annotation are available at http://www.pseudomonas.com . The flp-tad-rcp gene deduced amino acid sequences were subjected to analysis using different softwares, such as TMHMM, SignalP or PSORT, ScanProsite, BlastP or MAXHOM, PSI-BLAST, Ssearch, and T-COFFEE. These programs are available at http://embl-heidelberg.de/predictprotein/predictprotein.html .
Production of antibodies directed against Flp.A strategy for peptide-based antibody production was developed (Eurogentec). A peptide containing the 15 amino acids (DGVGEKVGGLAPTAN) at the C-terminal end of Flp was synthesized. Preimmune sera of two rabbits were checked for absence of cross-reactivity. The rabbits were inoculated with the designed peptide at a concentration of 200 μg/ml, followed by three boosters spaced by 15 days, 1 month, and 2 months, respectively. After that period, rabbits were sacrificed. Collected sera were further purified against the initial peptide.
Tris-glycine gel electrophoresis and Western blot analysis.Production of Flp was analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For enhanced resolution of the small-size Flp pilin (Flp maturation), Tris-glycine gel electrophoresis was performed with cathode and anode buffers containing 0.1 M Tris, 0.1 M Tricine, and 0.1% SDS at pH 8.25 and 0.2 M Tris at pH 8.9, respectively. Bacterial cells were grown to an A 600 of 0.5, induced for at least 3 h with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), and collected at 0.025 A 600 equivalent unit per μl in SDS-PAGE loading buffer. The samples were boiled for 10 min, and the proteins were separated by electrophoresis on a 16.5% polyacrylamide gel. Proteins were blotted onto nitrocellulose membranes. Flp was immunodetected using the Flp rabbit polyclonal antibody at a dilution of 1:150. A peroxidase-conjugated goat anti-rabbit immunoglobulin G at a dilution of 1:5,000 in Tris-buffered saline containing 10% milk and 0.1% Tween was used to reveal the Flp protein.
Transmission electron microscopy.For transmission electron microscopy (TEM), bacterial cells were scraped from LB plates containing appropriate antibiotics and 5 mM IPTG after overnight growth. Bacteria were collected in an Eppendorf tube containing 50 μl of 10 mM phosphate-buffered saline (PBS). A drop of the bacterial suspension was placed on Formvar- and carbon-coated copper grids and left for approximately 5 min. Grids were further fixed with 4% paraformaldehyde for 5 min and rinsed two times with 10 mM PBS for 5 min. Grids were then incubated with a 5% bovine serum albumin (BSA) solution in 10 mM PBS for 10 min and incubated with the Flp antiserum at a 1:150 dilution in 0.5% BSA, 10 mM PBS for 45 min. Grids were finally incubated with a 10-nm colloidal gold protein A solution for 30 min in 0.5% BSA, 10 mM PBS. After repetitive washes in 10 mM PBS and in water, grids were immersed in a drop of 1% uranyl acetate for 1 min. Grids were examined in a JEOL 1200EX transmission electron microscope operating at 80.0 kV.
Twitching motility assay.The presence of functional, retractile pili was assessed by picking a single colony with a toothpick and stabbing it to the bottom of a 3-mm-thick 1% LB agar plate. Plates were incubated overnight at 37°C, agar was removed, and the twitching zones were stained with crystal violet for 5 min and then rinsed with tap water to remove unbound dye.
Adherence assay on inert surfaces.The adherence assay was performed in 24-well polystyrene microtiter dishes as previously described (3). Attached bacteria were stained with 100 μl of crystal violet at 1% for 15 min and washed twice with water. Staining was extracted with treatment with 400 μl 95% ethanol. Subsequently, 600 μl water was added and A 570 was measured. All quantification assays were performed in triplicate.
Adherence assay on bronchial epithelial cell surface.Briefly, 16HBE14o- human bronchial epithelial cells were inoculated in Labteck chambers, in minimal essential medium supplemented with 10% fetal calf serum and antibiotics for 24 h at 37°C in a 5% CO2 atmosphere. Four hours before infection, the medium was exchanged with a medium free of serum and antibiotics. The epithelial cells were infected with bacteria at a multiplicity of infection of 30 for a period of 1 or 4 h at 37°C. The samples were rinsed twice with PBS, fixed with 4% formaldehyde, stained with 0.1% crystal violet for 5 min, washed twice with water, dried, sealed with Eukitt mounting medium (EMS), and observed with an Axioscop 40 microscope (Zeiss).
Characterization of the PAO1 flp-tad-rcp gene cluster.Mining the P. aeruginosa PAO1 genome identified a series of genes homologous to the flp-tad-rcp genes previously characterized in A. actinomycetemcomitans (20). The PA4306 open reading frame (ORF) was identified as an flp-like gene (21). This ORF is transcribed in a reverse orientation as compared to a cluster of nine genes, designated by locus tags PA4297 to PA4305, encoding Tad-Rcp homologous proteins (Fig. 1A and Table 3). The P. aeruginosa flp gene encodes a protein of 72 aa. The P. aeruginosa Flp precursor has a long leader peptide of 21 aa and an alanine residue instead of a phenylalanine residue at position +1 after the putative cleavage site (Fig. 1B). These features are characteristic of type IVb prepilins. The presence of a tyrosine residue at position +6 and the short C-terminal domain (smaller than 90 aa) (Fig. 1B) further supported that it belongs to the Flp subfamily (COG3847) (20). In addition, most of the contiguous genes are homologous to the flp-tad-rcp genes of A. actinomycetemcomitans (Fig. 1A). The names and characteristics attributed to the PA4297-to-PA4305 ORFs are given in Table 3. Most strikingly, PA4302 encodes a protein (421 aa) from the traffic NTPase family (37), involved in processes such as type IV pilus assembly, type II protein secretion, or T-DNA transfer from Agrobacterium tumefaciens (12, 35). The close homology with A. tumefaciens VirB11 ATPase (40) led to the annotation of PA4302 as HvbA (homolog to VirB11) at http://pseudomonas.com . However, it is now clear that the PA4302 product is homologous to the TadA ATPase (COG4962) from A. actinomycetemcomitans (2, 37). PA4304 encodes a protein (416 aa) from the secretin family, previously annotated XqhC (XcpQ homolog C) and here renamed RcpA (COG4964) (15) in accordance with the Tad-Rcp nomenclature. Type IV pilus assembly requires in all cases a traffic ATPase, a secretin, and an inner membrane protein known as PilC in P. aeruginosa. In the tad-rcp gene cluster from PAO1, it is remarkable that two ORFs encode PilC homologues, namely PA4300 (303 aa) and PA4301 (294 aa). The proteins encoded by these ORFs were renamed TadC and TadB, respectively, according to the Tad-Rcp nomenclature. The ORFs PA4297 (556 aa), PA4299 (245 aa), PA4303 (394 aa), and PA4305 (303 aa) encode homologues of TadG, TadD, TadZ, and RcpC, respectively. Finally, no obvious homology could be found between the protein encoded by PA4298 (94 aa) and a member of the Flp-Tad-Rcp system. It is notable that rcpB, tadE and tadF, which are found in the A. actinomycetemcomitans tad-rcp gene cluster, have no obvious homologues in the P. aeruginosa cluster.
The flp-tad-rcp gene cluster. (A) Genetic organization of the flp-tad-rcp (ORFs PA4297 to PA4306) gene cluster in P. aeruginosa. The P. aeruginosa (Pa) cluster is compared with the A. actinomycetemcomitans (Aa) cluster. Each arrow represents a gene (not to scale) and indicates the transcription orientation. The original nomenclature of the A. actinomycetemcomitans gene cluster is indicated above each gene and the homologous genes in the P. aeruginosa cluster have been named similarly. In the P. aeruginosa cluster, the original annotation number is also indicated underneath. The P. aeruginosa fppA gene is homologous to the A. actinomycetemcomitans orfB gene and encodes a prepilin peptidase. The PA4293 and PA4296 ORFs (hatched) encode a histidine kinase sensor, PprA, and a response regulator, PprB, respectively. (B) Part of the amino acid sequence of the P. aeruginosa Flp pilin (72 amino acids). The positively charged residues within the leader peptide (underlined with dotted line) are represented by italics. The conserved residues within the Flp family of pilins are boldace and in larger letters (G, −1; A, +1; E, +5; and Y, +6). The hydrophobic domain downstream from the FppA cleavage site (indicated by an arrow) is represented by bold letters. The peptide that was used for raising Flp antibody has been underlined.
Characteristics of the PAO1 flp-tad-rcp genes
The PAO1 tad-rcp cluster is required for Flp assembly.The production of Flp protein from P. aeruginosa strain PAO1 was tested at different growth stages when bacteria were grown at 37°C in L broth. Analysis of whole-cell extracts by Western blotting using antibodies directed against Flp indicated that Flp production may occur at a late stage during the growth but was not strictly reproducible from one culture to the other (data not shown). The flp gene was thus cloned under the control of the IPTG-inducible tac promoter of pMMB190, yielding pMMBflp. This plasmid was mobilized into the PAO1 strain, and Flp production was analyzed by Western blotting using antibodies directed against Flp. Whole-cell extract analysis clearly showed that Flp was produced in a large amount upon induction. The export and assembly of Flp pilin subunits into filaments at the bacterial cell surface were then investigated by TEM coupled with immunogold labeling. Filaments labeled with gold particles were seen emerging from the bacterial cell surface (Fig. 2A to C and E to G), which could appear as bundled filaments (Fig. 2E). Enlargement and Fourier transformation (Fig. 2D) revealed a filament with a twisted appearance and with regularly spaced gold particles. Whether these Flp structures could be assembled in mutants affected in the tad-rcp locus was further examined. We engineered tadA or rcpA gene deletion in the PAO1 strain, as described in Materials and Methods, yielding PAO1ΔtadA and PAO1ΔrcpA, respectively. The tadA and rcpA mutants were transformed with pMMBflp and analyzed for Flp assembly. No immunogold-labeled filaments could be identified by TEM using these mutants. Introduction in trans of the tadA and rcpA genes cloned in the pLAFR3 cosmid, yielding pLAFRtadA and pLAFRrcpA, respectively, in the appropriate P. aeruginosa tadA or rcpA mutant, restored Flp pilus assembly. These data suggested that the P. aeruginosa flp gene encodes an Flp pilin whose assembly into a pilus structure required the components encoded by the tad-rcp gene cluster.
Electron micrographs of P. aeruginosa Flp pili. (A to C and E to G) TEM views at magnifications of ×50,000 (A and B), ×80,000 (C), or ×140,000 (E to G) after immunogold labeling of Flp-producing PAO1 strains (PAO1/pMMBflp). In panel E, a nonlabeled flagellar structure is clearly visible next to a labeled Flp fiber with bundled filaments (see arrows). (D) Fourier transformation and ×4 enlargement of a small region of the labeled Flp filament.
Characterization of the Flp prepilin peptidase, FppA.Type IV prepilins are processed by prepilin peptidases which belong to a novel family of bilobed aspartic proteases called type IV prepilin peptidases (TFPP) (27). Downstream from the identified PAO1 tad-rcp gene cluster, one ORF transcribed in a reverse orientation to the tad-rcp locus encodes a homologue to CpaA from C. crescentus (44) and OrfB from A. actynomycetemcomitans (Fig. 1A and Fig. 3A). The protein encoded by PA4295 presented 45% similarity to OrfB and 42% similarity to CpaA (Fig. 3A). The PA4295-encoded protein is 160 residues long and has four predicted hydrophobic transmembrane domains (Fig. 3B). This protein corresponds to the C-terminal domain of the well-characterized PilD prepilin peptidase from P. aeruginosa (36). It thus lacks the N-terminal domain, or domain 1, that contains conserved cysteine residues. However, it still contains the two aspartate residues, which are conserved within the TFPP family (27) and which have been reported to be absolutely required for protease activity. Moreover, it clearly lacks the putative methyltransferase box, LGGKCS, which has been identified in PilD, including the critical glycine residue, G96 (36). We named the PA4295-encoded protein, FppA, for Flp prepilin peptidase A, suggesting that FppA could be the prepilin peptidase responsible for maturation of the Flp precursor and for Flp pilus biogenesis.
Characterization of the FppA prepilin peptidase. (A) T-COFFEE multiple alignment of FppA and its homologues, OrfB from A. actinomycetemcomitans and CpaA from C. crescentus. Portions inconsistent between the combined alignments are in green and blue, and consistent ones are in yellow, orange, or red. The conserved aspartate residues (catalytic site) between the three aligned prepilin peptidases are marked with an asterisk. (B) Membrane topology model of the FppA protein. Each circle represents one of the 160 FppA residues. Black circles are located within each of the four predicted transmembrane domains of FppA. Gray residues indicate the position of the two conserved aspartate residues of the TFPP active site. (C) Production and maturation of Flp prepilin from PAO1/pMMBflp (lane 1), PAO1ΔfppA/pMMBflp (lane 2), and PAO1ΔpilD/pMMBflp (lane 8). The arrows on the left indicate the precursor (p) and mature (m) forms of the Flp pilin. Flp processing was also monitored in PAO1ΔfppA/pMMBflp containing pBBR1MCS-2 (lane 4), pBBRfppA (lane 3), or one of the fppA(D17A) (lane 5), fppA(D78A) (lane 6), or fppA(D17/78A) (lane 7) mutated genes. Proteins were separated by electrophoresis on a Tris-glycine gel.
We investigated whether FppA is required for maturation of the Flp pilin subunit. For that purpose, we engineered a deletion mutant for the fppA gene, as described in Materials and Methods, yielding PAO1ΔfppA. The pMMBflp plasmid was introduced in PAO1 and PAO1ΔfppA, and whole-cell samples were analyzed by Western blotting using antibodies directed against Flp. When Flp was produced in the PAO1 parental strain, two bands were identified (Fig. 3C, lane 1). However, only the upper band was identified in the fppA mutant (Fig. 3C, lane 2), indicating that it corresponds to the precursor form, whereas in the PAO1 parental strain the lower band corresponds to the mature Flp form. Maturation of the overproduced Flp prepilin could be fully achieved by the introduction of a plasmid containing the fppA gene, namely pBBRfppA (Fig. 3C, lane 3). This observation also indicated that the level of FppA prepilin peptidase could be limiting for processing large amounts of Flp prepilin. The occurrence of the two Flp forms thus results from the FppA-dependent processing of the Flp precursor. Furthermore, similarly to the tadA and rcpA mutants, no Flp-containing appendages were assembled at the surface of an fppA mutant when assessed by TEM coupled with immunogold labeling of Flp pili, but assembly was restored when pBBRfppA was introduced in the fppA mutant (data not shown).
We further investigated whether the other TFPP encoded in the PAO1 genome, namely PilD, is able to process the Flp prepilin (Fig. 3C). No Flp processing occurred in the fppA mutant that contained an intact pilD gene (Fig. 3C, lane 2), whereas comparable Flp maturation occurred in a pilD mutant (Fig. 3C, lane 8) or the wild-type strain (Fig. 3C, lane 1), suggesting an FppA specialization in Flp maturation.
The aspartate residues are part of the FppA active site.Since the aspartate residues identified at positions 17 and 78 were designated as critical for the activity of FppA, we mutagenized the fppA gene to substitute these aspartate residues (D) for alanine residues (A). The genes encoding FppAD17A, FppAD78A, and the double mutant FppAD17/78A were obtained by performing overlapping PCR mutagenesis as described in Materials and Methods. The level of mature Flp pilin produced from pMMBflp was subsequently analyzed in an fppA mutant complemented with the wild-type FppA or the various FppA derivatives, FppAD17A, FppAD78A, and FppAD17/78A (Fig. 3C, lanes 5 to 7). Only the production of wild-type FppA could restore maturation of Flp, whereas with any of the FppA aspartate mutants, only the Flp precursor form could be identified. These results clearly indicated that both aspartate residues are necessary for catalysis and that FppA belongs to the TFFP family of proteases.
Role of Flp pili in motility and attachment.The twitching motility of P. aeruginosa is supported by type IV pilus assembly and retraction. A P. aeruginosa pilD mutant, which lacks type IVa pili, is thus nonmotile (Fig. 4A). Overproduction of the Flp subunit (pMMBflp) in the pilD mutant could not restore motility even though the bacteria assembled Flp pili on their surface. Overproduction of the prepilin peptidase FppA (pBBRfppA) in the pilD mutant could also not restore motility. We further investigated whether Flp pili could be associated with attachment and biofilm formation on plastic surfaces or on respiratory epithelial cell surfaces. In addition to the previously constructed fppA, tadA, and rcpA mutants, we engineered an flp mutant in the PAO1 strain and tested all strains for their biofilm formation capability in comparison to the wild-type strain. No significant alteration of the phenotype was observed (data not shown), suggesting that either the flp gene is not expressed at the chromosomal level under these growth conditions or the Flp-dependent phenotype is masked by the contribution of other appendages, such as the type IVa pili or the flagella. A strain (PAO1ΔfliCΔpilA) devoid of flagella and type IVa pili, which are major determinants of adherence in P. aeruginosa, was thus used. The plasmids allowing concomitant production of Flp and FppA, pMMBflp and pBBRfppA, were introduced into PAO1ΔfliCΔpilA. Wells of a microtiter plate were inoculated with bacteria, and after 6 to 8 h of incubation, the culture medium was removed and the remaining attached bacteria were stained with crystal violet. The strain containing both pMMBflp and pBBRfppA forms a thicker biofilm on a plastic surface (Fig. 4B), as compared to the strain carrying the cloning vectors. Quantification indicated that the PAO1ΔpilAΔfliC strain containing both pMMBflp and pBBRfppA forms a ring with a fivefold-higher efficiency than that of the PAO1ΔpilAΔfliC strain (Fig. 4C). Whether Flp assembly could also influence attachment to respiratory cell surfaces was examined. After 4 h of contact, the PAO1ΔfliCΔpilA strain containing pMMBflp binds efficiently to bronchial epithelial cells, forming substantial bacterial aggregates at the cell surface, whereas the PAO1ΔfliCΔpilA strain containing the cloning vector (pMMB190) binds poorly (Fig. 4D).
Functional analysis of P. aeruginosa Flp pili. (A) Twitching motility was tested for various P. aeruginosa strains. The PAO1 strain presents a clear zone of motility, whereas the pilD mutant is nonmotile despite overexpression of the flp (pMMBflp) or fppA (pBBRfppA) gene. (B) Attachment to polystyrene was analyzed in PAO1ΔfliCΔpilA/pMMBflp/pBBRfppA and PAO1ΔfliC ΔpilA/pMMB190/pBBR1MCS-2. Rings of attachment could be observed in strains overexpressing the flp and fppA genes, but not in strains containing the cloning vectors. (C) Quantification of crystal violet staining is presented. Error bars correspond to standard deviation from three distinct experiments. (D) Attachment to bronchial epithelial cells was examined with PAO1ΔfliCΔpilA/pMMBflp and PAO1ΔfliCΔpilA/pMMB190. Upon expression of the flp gene (pMMBflp), bacteria formed substantial aggregates at the cell surface.
Several studies have revealed that P. aeruginosa can assemble a large number of appendages on the cell surface. Those appendages are involved in processes such as motility, attachment, or both. This is the case for flagella and type IVa pili. More recently, three fimbrial gene clusters, called cupA, cupB, and cupC, have been identified as being involved in biofilm formation (51). Whereas these clusters encode components of the chaperone-usher pathway described in E. coli (50), no cup-associated fimbrial structures have been described to date in P. aeruginosa. In this study, we used functional genomic approaches to further mine the P. aeruginosa genome to identify genes involved in the assembly of cell-surface appendages that could be relevant for bacterial biofilm development. We identified a cluster whose genes encode components previously reported in A. actinomycetemcomitans as being involved in the assembly of Flp pili (17) that support tight adherence and biofilm formation (18).
The A. actinomycetemcomitans Flp pili are made following the packing of subunits encoded by the flp-1 gene (21). Next to this gene another flp homologue, flp-2, could be identified. However, neither has flp-2 been shown to be required for Flp-assembly nor could its product be found in the pilus structure. In the P. aeruginosa genome, only one flp gene (ORF PA4306) could be identified. The Flp pilins belong to the so-called type IVb family and, more precisely, to a subfamily that possesses a tyrosine at the +6 position after the putative cleavage site and that are rather short polypeptides (smaller than 90 aa) (20).
The leader peptides of the type IV prepilins are cleaved by TFPP. TFPP have been shown to process not only the type IV prepilins but also the so-called pseudopilins involved in the type II protein secretion process (12). In some cases, as in P. aeruginosa, a single TFPP is involved in type IVa pilin and pseudopilin processing, namely PilD (47). In other cases, such as in V. cholerae, one TFPP is involved in the type IV piliation process (TcpJ) (23), whereas another is required for type II protein secretion (VcpD) (30). Processing of type IVa and IVb prepilins has been shown in many cases. However, no demonstration of the functionality of a TFPP required for Flp prepilin processing has been reported, even though it has been proposed that the Flp prepilin peptidases from A. actinomycetemcomitans or C. crescentus are encoded by the orfB and cpaA genes (20, 44), respectively. In both cases, the gene product corresponds to the C-terminal domain of classical TFPP, but lacks the large cytoplasmic domain 1, which includes the four highly conserved cysteine residues (27). It was shown that replacement of these cysteine residues with alanine or serine residues in PilD of P. aeruginosa did not abolish its peptidase activity (46). In vitro and in vivo studies performed with TcpJ from V. cholerae indicated that only the conserved aspartate residues (position 125 and 189) were absolutely required for protease activity (27). We identified an ORF, PA4295, located next to the flp-tad-rcp gene cluster of P. aeruginosa, that encodes a homologue of CpaA and OrfB. The gene product is predicted to have four transmembrane segments and the conserved aspartate residues of the TFPP family are found at position 17 in the cytoplasmic N-terminal domain and at position 78 within the cytoplasmic loop (Fig. 3B). We showed in this study that FppA is able to process the P. aeruginosa Flp prepilin, revealing for the first time that naturally truncated versions of the TFPP family, such as CpaA, OrfB, and FppA, are functional peptidases. This observation confirms that cysteine residues from the cytoplasmic domain 1 are not essential. Furthermore, site-directed mutagenesis of both aspartate residues yielded an inactive FppA, as recently shown in PibD from the archaeon Sulfolobus solfataricus (48). It thus confirmed that they are part of the active site of this truncated peptidase. FppA appears to be specific for Flp prepilins, since no processing of the Flp subunit occurred in the absence of FppA, indicating that PilD cannot substitute for FppA function, and reciprocally, no functional type IVa pili were detectable in the absence of PilD. Moreover, overproduction of FppA in a pilD mutant did not allow restoration of type IVa pilus-associated phenotypes such as twitching motility. It is not clear whether particular residues or domains of FppA impart specificity for Flp or whether the additional domains found in PilD contain Flp avoidance regions. It is also not known whether FppA, just like PilD, possesses a methyltransferase activity that allows methylation of the N-terminal residue of the mature Flp pilin. However, the putative methyltransferase box LGGKCS described for PilD is clearly absent in FppA, including the glycine residue, which is essential for the N-methyltransferase activity of PilD (36). The issue of methylation of the Flp-like subunit was previously addressed with CpaA (44), and the strong homology between CpaA and FppA suggests that these peptidases have no methyltransferase activity. It is also known that N methylation of pilin, which occurs onto the phenylalanine residue of mature PilA, does not seem to be essential for type IVa pilus assembly (36).
In A. actinomycetemcomitans, Flp subunits are assembled into a pilus structure that is involved in attachment to surfaces. Upon overexpression of the flp gene of P. aeruginosa, we were able to identify by immunogold labeling cell surface structures that contain Flp. Only overexpression of the flp gene is required under our growth conditions to observe Flp pili, indicating that basal expression of the tad-rcp gene cluster is sufficient for assembly. Since the flp gene and the tad-rcp genes are divergently transcribed, their expression might be differentially controlled. One could argue that the long and thick bundle-like Flp structures that we observed do not resemble the ones that could be assembled when Flp protein is expressed at the chromosomal level; however, the structures looked quite similar to the ones observed in A. actinomycetemcomitans (21). The P. aeruginosa Flp pili appeared to be involved in surface attachment both on abiotic surfaces (polystyrene) and on biotic surfaces (human bronchial epithelial cell line 16HBE14o-). The processing and assembly into a pilus structure of the overproduced Flp subunits are limited by the amount of FppA. Upon co-overproduction of Flp and FppA, increased levels of bacteria remained attached on the surfaces. Moreover, under these conditions, bacterial aggregates could be seen, which indicates that not only are Flp pili involved in bacterium-surface or bacterium-cell attachment, but they also seem to be involved in bacterium-bacterium contact and aggregation.
Type IVa pili are assembled with the help of a complex molecular machine, the core of which consists of a traffic NTPase, a polytopic inner membrane protein, and an outer membrane channel or secretin, which are in P. aeruginosa PilB, PilC, and PilQ, respectively. Type IVb pilus assembly, such as Bfp from enteropathogenic E. coli or TCP from V. cholerae, requires a subset of additional components that are mostly cell envelope located (29, 39). In this study, we have shown that the P. aeruginosa tad-rcp gene cluster encodes a functional system that is responsible for the assembly of Flp pili at the cell surface. Indeed, no Flp pili could be seen at the surface of tadA (PA4302) and rcpA (PA4304) mutants, which have had the genes encoding the traffic NTPase and the secretin, respectively, deleted. Moreover, two ORFs encode PilC homologues, tadB (PA4301) and tadC (PA4300). PilC and other members of this family have been proposed to form a platform within the cytoplasmic membrane through which pilins and pseudopilins are driven to the periplasmic side of the cytoplasmic membrane thanks to the energy provided by the traffic NTPase (12). The extrusion of the pilus structure through the outer membrane is then mediated by the secretin (56). It is likely that PilC homologues are assembled as homomultimers, whereas in the Tad system, TadB and TadC could form heteromultimeric complexes.
The Flp assembly machinery seems to require an additional subset of components, which are conserved between C. crescentus and A. actinomycetemcomitans and have been found in P. aeruginosa. It includes rcpC (PA4305), tadZ (PA4303), tadD (PA4296), and tadG (PA4297). The rcpB, tadE, and tadF genes from A. actinomycetemcomitans could not be clearly found in the P. aeruginosa cluster. However, PA4298 encodes a protein (94 aa) with a putative signal peptide, such as RcpB, which is larger with 167 residues. Outside of the tad-rcp cluster but next to fppA, PA4294 encodes a 168-aa-long polypeptide with a putative N-terminal transmembrane domain and presenting slight homology with a domain of TadG. Whether the PA4294 protein could be related to TadE or TadF, which are both proteins presenting a predicted N-terminal transmembrane domain, is unclear. Finally, even though slight differences could be found between the tad-rcp gene clusters of A. actinomycetemcomitans and P. aeruginosa, they are conserved and are both involved in Flp pilus assembly.
Analysis of the PAO1 genome revealed how rich this organism is in terms of systems that assemble various cell surface appendages. The assembly of these different structures may respond to particular environmental conditions in order to promote the adaptation of the microorganism to a novel ecological niche. ORFs encoding a two-component system have been identified adjacent to the tad-rcp locus (Fig. 1A): PA4293 encodes a histidine kinase sensor, and PA4296 encodes a response regulator that belongs to the NarL family. This two-component system was reported as PprA and PprB (55). A mutant strain affected in the pprA gene shows an altered outer membrane profile. Whether expression of the flp-tad-rcp operon is under the control of that particular two-component system needs further investigation. Interestingly, the PprB (PA4296) response regulator has been described recently as a quorum-sensing (QS) modulator but apparently does not control any of the tad genes (10). Moreover, many studies dedicated to QS regulon showed that two or more genes of the tad cluster are QS regulated (43, 53, 54). Additional data showed that whereas flagellin down-regulation occurred in response to CF airway fluid independently of QS, flp-tad genes can be slightly up-regulated under these conditions (57; supporting table available at www.pnas.org ), suggesting that the bacteria may develop alternative strategies such as Flp-mediated adherence to colonize (42) and survive in muco-purulent environments such as those encountered in CF lungs during chronic infection. Moreover, other conditions may also account for potential signals that could control the expression of the flp gene, such as exposure to hydrogen peroxide (41). Finally, it has been shown that several tad genes are VqsR regulated (19) but not AlgR regulated (28). It should be noted that we additionally tested several growth conditions, including anaerobic environment, oxidative stress, or addition of subinhibitory concentrations of aminoglycoside antibiotics (16), but none of them appeared to be relevant for a detectable level of Flp production (data not shown). Obviously, it appears that control of production and assembly of the Flp pili is highly complex and environment dependent, which supports the ubiquitous nature of P. aeruginosa, which may adapt and develop a biofilm in many distinct environments.
We thank A. Bernadac for technical assistance and A. Fox for her helpful comments.
The work of A.F. and S.D. is supported by the French cystic fibrosis foundation (VLM) and the foundation Bettencourt-Schueller. M.A. has been supported by the French research and technology ministry as well as by the VLM.
Received 10 March 2006.
Accepted 21 April 2006.
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Journal of Bacteriology Jun 2006, 188 (13) 4851-4860; DOI: 10.1128/JB.00345-06
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Home / News / Media Watch / Media outlets ignore death threats to black GOP Senator Scott
Media outlets ignore death threats to black GOP Senator Scott
News networks ABC, CBS, and NBC have all turned a blind eye to a New York Times report detailing recent death threats and racial slurs being left on the office voicemail of Republican Senator Tim Scott (SC), who's the only black Republican and one of only three in the chamber.
None of them have reported on the outrage over comments by House Speaker Nancy Pelosi (D-CA) who told CBS that Scott and the Republican Party were "trying to get away with murder, actually, the murder of George Floyd."
READ: CBS forced to correct false story about nurse crying for masks
In a New York Times article published this week, reporter Catie Edmondson detailed how "Mr. Scott shocked his colleagues when he spoke at Republicans' weekly luncheon about death threats he and his aides had received in recent days…"
The Times acknowledged that Scott had shared two of the audio recordings with them:
The calls, filled with profanity and racist slurs, included one diatribe addressed to "Uncle Tim," where the caller, after cruelly and profanely condemning Mr. Scott, his family and some of his Republican colleagues, told Mr. Scott to "take your one-way ticket straight to hell."
"One caller discussed putting the senator 'in his crosshairs,' Mr. Scott said," Edmondson added.
READ: CNN settles lawsuit over lies about high school student
In his interview with The Times about the messages, the Senator blamed the media for how his police reform legislation was being portrayed. "It's interesting that we are on the right side of the police reform conversation, yet we're on the wrong side according to the people, based on the way that we are characterized in the national press," he said.
And he was right. ABC and NBC skipped the official rollout of Scott's Justice Act, while CBS downplayed it in a 22-second mention.
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Just as they didn't have time for him, the broadcast networks didn't have time to report on outrage to Pelosi's vile smears. CBS Evening News had time to promote Bolton's book (which they failed to mention they benefited from since they and the book's publisher were owned by the same company), but they refused to cover the comments made to their own West Coast correspondent, Steve Futterman.
Nancy Pelosi says with their police reform proposals, Senate Republicans are "trying to get away with murder, actually — the murder of George Floyd."
Hear it in this @CBSNewsRadio interview with my colleague @sfutterman:https://t.co/DAsGYssPzD
— Steven Portnoy (@stevenportnoy) June 23, 2020
Pelosi made her comment on CBS News Radio (here's the link, at the 2:30 mark) after Futterman asked: "Is there any hope that two sides can reach some compromise [on police reform]? (…) Is there some way that the two sides can reach some compromise to at least get something passed?"
After Pelosi rambled on for a little bit, suggesting the GOP wasn't serious about reforms, she capped off by declaring: "But so far, they were trying to get away with murder, actually, the murder of George Floyd."
Futterman was unfazed by Pelosi's incendiary and false comment. He followed up by asking if she understood why people wanted to defund the police. There was no fact check about how the Republican Party had no hand in the killing of George Floyd.
Metro Voice Note: Nicholas Fondacaro writes for Newsbusters, where this originally appeared.
abc bias brutality cbs death threats democrats george floyd legislation media murder nbc networks new york times news blackout pelosi police reporting republicans senator tim scott 2020-06-25
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Enjoy a day trip to Shrewsbury Flower Show, which attracts the best floral exhibitors from all over the world (admission to show is extra).
Shrewsbury is known for being a pretty and floral town, however, possibly the biggest and most important events in the region's gardening calendar is the Shrewsbury Flower Show. The show is a flourishing institution, running every August since 1875, and this is your chance to see over 3 million flowers in one go! It attracts the best floral exhibitors from all over the world, and visitors flock in their thousands each year. The reputation of the show ensures that it attracts the most expert and horticultural judges in the UK. And it's not just the floral displays that provide the attraction - all the family will find something to enjoy. Visitors can enjoy live music, horse-jumping, falconry and other arena events. Admission to the show not included in price.
Return transport by luxury air conditioned coach.
Once you have boarded our luxury coach at your selected pick up point we travel to Shrewsbury Flower Show, with an expected arrival time of 10.30am. After enjoying a full day at the show, the coach will pick you up in the late afternoon for your return journey home.
To make an online booking, please first select the number of passengers required in the dropdown menu below, then click the shopping trolley icon in the Book Now column. If the shopping trolley icon is unavailable, online booking is not currently available for this departure. Please call our reservations team on 01286 675175 or use the Call Back facility and a member of our team will be happy to assist.


text:
5 złotych wzór 1994 – moneta pięciozłotowa bimetaliczna, wprowadzona do obiegu w dniu denominacji z 1 stycznia 1995 r., zarządzeniem z 29 listopada 1994 r.

Pięciozłotówka jest bita od 1994 r.

Awers
W centralnym punkcie, na wewnętrznym rdzeniu umieszczono godło – orła w koronie, pod łapą orła, z prawej strony, znak mennicy – litery M W, dookoła rdzenia otok, na zewnętrznym pierścieniu z zewnętrznym otokiem dookoła napis: "RZECZPOSPOLITA POLSKA", a na samym dole rok bicia.

Rewers
Na tej stronie znajdują się: centralnie na rdzeniu wypukła cyfra 5 nominału, tło monety z wypukłym karbowaniem – poziomym z prawej strony i pionowym z lewej, w tle duża, płaska cyfra 5, z obu stron nominału wklęsłe gałązki, na samym dole, na pierścieniu zewnętrznym napis: "ZŁOTYCH".

Nakład
Monetę jest bita w na krążku bimetalicznym o średnicy 24 mm, o wewnętrznym 16-milimetrowym rdzeniu z brązalu (CuAl6Ni2) i zewnętrznym pierścieniu z miedzioniklu MN25, masie 6,54 grama, z rantem moletowanym nieregularnie, według projektu Ewy Tyc-Karpińskiej, w Mennicy Państwowej / Mennicy Polskiej S.A. Do roku 2022 nakłady monety w poszczególnych latach przedstawiały się następująco:

Opis
W 2005 r. Mennica Polska, z okazji 10 lat w obiegu, wyemitowała zestawy kwadratowych klip okolicznościowych, w skład których wchodził również numizmat przedstawiający w centralnej części po obu stronach rysunki awersu i rewersu pięciozłotówki wzór 1994. Na awersie w miejscu cyfr określających rok bicia umieszczono napis "1995–2005". Numizmaty te powstały na metalicznych kwadratach  w czterech wersjach:

 krążek wewnętrzny w brązalu, część zewnętrzna w miedzioniklu (tak samo jak w przypadku monety wprowadzonej do obiegu) – masa 18,2 grama, nakład 5000 sztuk,
 w miedzi – masa 19,5 grama, nakład 2000 sztuk,
 w srebrze Ag925 – masa 20,81 grama, nakład 1000 sztuk,
 w złocie Au900 – masa 39 gramów, nakład 50 sztuk.

Ponieważ emitentem zestawów okolicznościowych była Mennica Polska a nie Narodowy Bank Polski numizmaty te nie są monetami w dosłownym tego słowa znaczeniu. Mimo to, w niektórych polskich katalogach wymieniane są na równi z monetami obiegowymi.

22 maja 2018 r. została wprowadzona przez Narodowy Bank Polski do obiegu, w nakładzie  sztuk, moneta okolicznościowa o nominale 5 złotych z identycznym wzorem awersu jak pięciozłotówka wzór 1994, rokiem emisji 2018 oraz rysunkiem rewersu monety obiegowej uzupełnionym o napis w otoku: "100-LECIE ODZYSKANIA PRZEZ POLSKĘ NIEPODLEGŁOŚCI •2018•".

27 lutego 2019 r. zostały wprowadzone przez Narodowy Bank Polski do sprzedaży (obiegu) wersje rocznika 2019 wybite w srebrze (5000 sztuk) i złocie (1000 sztuk) będące częścią zestawów kolekcjonerskich upamiętniających stulecie wprowadzenia złotego.

Wersje próbne
Istnieje wersja tej monety należąca do serii próbnych w niklu z roku 1994, z wypukłym napisem PRÓBA, wybita w nakładzie 500 sztuk.

Zobacz też
 1 grosz wzór 1990
 2 grosze wzór 1990
 5 groszy wzór 1990
 10 groszy wzór 1990
 20 groszy wzór 1990
 50 groszy wzór 1990
 1 złoty wzór 1990
 2 złote wzór 1994
 5 złotych
 monety obiegowe III Rzeczypospolitej

Przypisy

Linki zewnętrzne
 awers
 rewers

500 1994

redpajama_set_name: RedPajamaWikipedia


What is Healthy Ketosis and Who Can Benefit?
Most of you have probably heard of the KETO diet, short for the Ketogenic diet, a dietary pattern and lifestyle characterized by low consumption of carbohydrates and high consumption of healthy dietary fat. KETO has been making headlines across many health forums due to its benefits and positive health outcomes for certain disease states. If you have pre-diabetes, type 2 diabetes, obesity, high blood pressure, cancer, PCOS, migraines, Alzheimers, Parkinson's or are wanting to lose weight, the KETO diet could be highly beneficial for you.
So what happens on KETO? By consuming high amounts of fat and very low carbs, the body goes into a state known as ketosis. Ketosis is a metabolic state that happens when you consume a low carb, high healthy fat, and moderate protein diet. It is a way of eating that causes your body to switch from using glucose (sugar) as its primary source of fuel to running off of ketones. So what are ketones? Ketones are produced when the body burns fat, and they are primarily used as an alternative fuel source in place of sugar. Your body changes from being a sugar-burner to a fat-burner! Once you transition your diet from the Standard American Diet to a KETO Diet, it can take the body a few days to a few weeks to become keto adapted. Once KETO adapted, the body burns stored fat for energy which not only promotes weight loss but also increases insulin sensitivity which can improve blood sugar and in some cases reverse pre-diabetes and type 2 diabetes.
Another huge benefit of ketosis for anyone with insulin resistance, pre-diabetes or type 2 diabetes is that the body is using fat for fuel instead of sugar (glucose). Fat is a much more efficient fuel source for individuals with the aforementioned conditions because their cells are not resistant to fat as they are sugar for fuel. With insulin resistance, the cells cannot uptake glucose effectively for fuel because the cells are insulin resistant. Our cells cannot become resistant to ketones. So, the cells are well fed, so to speak, and complications associated with those conditions are reduced and/or healed. This is why ketosis improves outcomes and health status for individuals with Alzheimer's and dementia - because the brain is being fed by an alternate fuel source and insulin sensitivity is increased. Much more to come on insulin sensitivity and resistance in a future blog post!
A ketogenic way of eating combined with Intermittent fasting (I.F.) is a great way to improve your overall health, especially if you suffer from a chronic illness. I.F. is time restricted eating - restricting your daily caloric intake to a small window of time, and "fasting" for the rest of that time. There are many different schedules that could be implemented with I.F., the key is consuming your daily nutritional intake in a short timespan, consistently. I.F. allows insulin levels to lower which causes the body to burn stored energy (which can lead to weight loss and enable sustained weight loss). Then the body will start breaking down its own stored body fat for energy. This also enables autophagy, a process in which old or damaged cells are broken down or recycled in the body - an internal cleansing process. I.F. allows the body and the pancreas to rest, which again allows insulin levels to lower promoting insulin sensitivity. Insulin is also our fat storage hormone, so as we lower the level, the less fat we will store in our body and the less complications we have from elevated insulin levels.
To name a few of the benefits associated with this way of eating are stabilization of blood sugar levels, improved insulin sensitivity, use of stored body fat as fuel, appetite control and hunger control, regulation of hormones, lower blood pressure, lower levels of inflammation in the body, decreased mood swings, decreased depression and anxiety, weight loss, improved overall sense of well-being, improved immune system and improvements in blood work!
Stay tuned for future blog posts that will discuss more about the benefits of I.F., the risks associated with elevated insulin levels as well as more information about the KETO diet and allowable foods!


text:
from twisted.internet.defer import Deferred, DeferredList
from twisted.python.failure import Failure
from twisted.trial.unittest import TestCase

import smartanthill.litemq.exchange as ex
from smartanthill.exception import LiteMQResendFailed

class LiteMQCase(TestCase):

    g_resent_nums = 0

    def test_declare_exchange(self):
        for type_, class_ in {"direct": ex.ExchangeDirect,
                              "fanout": ex.ExchangeFanout}.items():
            self.assertIsInstance(
                ex.ExchangeFactory().newExchange("exchange_name", type_),
                class_
            )

        self.assertRaises(
            AttributeError,
            lambda: ex.ExchangeFactory().newExchange("exchange_name",
                                                     "unknown-type")
        )

    def test_queue_ack_success(self):
        message, properties = "Test message", {"foo": "bar"}

        def _callback(m, p):
            self.assertEqual(m, message)
            self.assertEqual(p, properties)
            return True

        def _resback(result):
            self.assertIsInstance(result, bool)
            self.assertEqual(result, True)

        q = ex.Queue("queue_name", "routing_key", _callback, ack=True)
        d = q.put(message, properties)
        self.assertIsInstance(d, Deferred)
        d.addCallbacks(_resback)
        return d

    def test_queue_ack_fails(self):
        self.g_resent_nums, resend_max = 0, 3

        def _callback(m, p):
            self.g_resent_nums += 1
            # test exception
            if self.g_resent_nums == 1:
                return 1/0
            # test "ack-invalid" that is equl to False
            else:
                return False

        def _errback(result):
            self.assertIsInstance(result, Failure)
            self.assertTrue(result.check(LiteMQResendFailed))
            self.assertEqual(resend_max, self.g_resent_nums)

        q = ex.Queue("queue_name", "routing_key", _callback, ack=True)
        q.RESEND_MAX = resend_max
        q.RESEND_DELAY = 0
        d = q.put("Test message", {"foo": "bar"})
        self.assertIsInstance(d, Deferred)
        d.addBoth(_errback)
        return d

    def test_queue_nonack(self):
        self.g_resent_nums, resend_max = 0, 3

        def _callback(m, p):
            self.g_resent_nums += 1
            return 1/0

        def _errback(result):
            self.assertNotIsInstance(result, Failure)
            self.assertIsInstance(result, bool)
            self.assertEqual(result, False)
            self.assertEqual(self.g_resent_nums, 1)

        q = ex.Queue("queue_name", "routing_key", _callback, ack=False)
        q.RESEND_MAX = resend_max
        q.RESEND_DELAY = 0
        d = q.put("Test message", {"foo": "bar"})
        self.assertIsInstance(d, Deferred)
        d.addBoth(_errback)
        return d

    def test_exchange_direct(self):
        message, properties = "Test message", {"foo": "bar"}

        def _callback(m, p):
            self.assertEqual(m, message)
            self.assertEqual(p, properties)

        myex = ex.ExchangeFactory().newExchange("exchange_name", "direct")
        myex.bind_queue("queue_name", "routing_key", _callback)

        empty_result = myex.publish("invalid_routing_key", message, properties)
        self.assertEqual(empty_result, [])

        result = myex.publish("routing_key", message, properties)
        self.assertIsInstance(result, list)
        self.assertEqual(len(result), 1)
        d = result[0]

        def _resback(result):
            self.assertEqual(result, None)
            myex.unbind_queue("queue_name")
            self.assertEqual(len(myex._queues), 0)

        self.assertIsInstance(d, Deferred)
        d.addCallbacks(_resback)
        return d

    def test_exchange_fanout(self):
        self.g_resent_nums = 0
        message, properties = "Test message", {"foo": "bar"}

        def _callback(m, p):
            self.g_resent_nums += 1
            self.assertEqual(m, message)
            self.assertEqual(p, properties)

        myex = ex.ExchangeFactory().newExchange("exchange_name", "fanout")
        myex.bind_queue("queue_name", "routing_key", _callback)

        result = myex.publish("invalid_routing_key", message, properties)
        self.assertIsInstance(result, list)
        self.assertEqual(len(result), 1)
        d1 = result[0]

        result = myex.publish("routing_key", message, properties)
        self.assertIsInstance(result, list)
        self.assertEqual(len(result), 1)
        d2 = result[0]

        self.assertIsInstance(d1, Deferred)
        self.assertIsInstance(d2, Deferred)

        dl = DeferredList([d1, d2])

        def _resback(result):
            self.assertEqual(result, [(True, None), (True, None)])

        dl.addCallbacks(_resback)
        return dl

redpajama_set_name: RedPajamaGithub


text:
« Hull v Arsenal – the great Hull / Arsenal competition, and an Untold wedding
Hull v Arsenal, Saturday 17 September 2016. The Match Officials »
So the marauding Twatters have been busy this week, calling for the manager to go, and it was nice to see Untold fighting back with #CurseTheStar #CurseJackWilson. I await news of the demise of both the paper and the journalist.
In August it was reported that Hull City could soon be yet another of the clubs in the League that are under Chinese control as the Allam family were in advanced talks concerning flogging the club off to the highest bidder.
In this case the bidder was Dai Yongge and his sister Hawken Xiu Li. The couple are said to have made an offer to buy Hull but I am not sure if matters are progressing.
Certainly Ehab Allam has been trying to sell the club for a while, and the ups and downs of the club led to Steve Bruce resigning as manager and the club fail to sign a single player.
The manager is now Mike Phelan, who was said to have only 13 fit senior players available for the opening games of the season.
So where are we now on the injury table…
The injury table from physioroom this season seems to be taking on a slightly odd form, since it is now including injuries which will be cleared up by the time of the next match. Here's the list
Club Injured players
1 Stoke City 7
2 Sunderland 7
3 Arsenal 6
4 Hull City 6
5 Crystal Palace 6
6 Everton 5
7 West Ham United 5
8 Middlesbrough 5
9 Manchester City 4
10 Liverpool 4
Two of the seven that they have on the Arsenal list (Giroud and Theo) are said to be ready to return this weekend, Olivier from a toe injury and Theo from a knee injury.
Beyond that Ramsey is one week away, Gabriel a few weeks, and Jenkinson about six or seven weeks away, leaving Mert, and Welbeck scheduled for January and February respectively.
Guessing the team to play however is getting harder with the surprise exclusion of Granit Xhaka from the starting line up on Tuesday. Arsène Wenger talked about him needing time to adjust to the pace of the Premier League. (But then we also have Santi Caz and Coquelin – not to mention Elneny.)
Speaking of Granit he said, "It's not about his quality at the moment as he adapts to the pace of the English game. And for me it's about pairs as well who work together. Coquelin and Cazorla have worked well together. He will play games and every week he is stronger and better. He will have a huge impact as a player.
"He has the stature, he has the power, he has the strength. What we want is to use that in a very efficient way. We are in September, we started in August so it's one month. Many players take time to get into the team, but he will do that without any problem.
"I personally prefer him as a box-to-box player, because he has the engine, he has the power, he has the long pass. He likes to come deep and distribute the game, but he has the engine to have the impact with his runs."
Meanwhile Olivier Giroud has been getting a bit agitated vis a vis his red card against PSG – although of course this doesn't affect him in the Premier League. The press are also still talking about the "controversial dropping" of Cech for the PSG game, but then I suppose they have to write about something to cover up the embarrassment that they would feel if they ever could be embarrassed about how wrong their stuff is. At least a few people have now realised just how good Ospina is – which they could have realised had they been watching the summer internationals.
Theo may or may not be ready for the game against Hull, and with people generally thinking the Ox has lost a bit of his pre-season flair this might be the moment for Lucas Perez to take centre stage.
Wenger added that Xhaka and Mohamed Elneny, are "top, top quality" but "there's big competition in midfield at the moment".
So picking a team is going to be a bit of guesswork. But we could try
Iwobi Ozil Alexis
Cazorla Coquelin
Monreal Koscileny Mustafi Bellerin
That line up could give us a beach incorporating Opsina, Holding, Gibbs, Granit, Walcott, Giroud, Elneny. Or maybe Debuchy. Or maybe the Ox.
As for Hull… while Arsenal's league record has been loss, draw, win, win, Hull's record has been win, win, loss draw.
Man City 4 4 0 0 11 4 7 12
Chelsea 4 3 1 0 9 4 5 10
Everton 4 3 1 0 7 2 5 10
Man Utd 4 3 0 1 7 3 4 9
Tottenham 4 2 2 0 7 2 5 8
Liverpool 4 2 1 1 9 7 2 7
Arsenal 4 2 1 1 8 6 2 7
Hull 4 2 1 1 5 3 2 7
Middlesbrough 4 1 2 1 4 4 0 5
Watford 4 1 1 2 7 8 -1 4
They have got Dieumerci Mbokani in the squad for the first time, with players such as Mason and Keane ready to make home debuts.
Hull famously have a poor record against Arsenal, not only having but one victory that since the first world war, but also historically a bizarre attitude to Arsenal, claiming on occasions that Cesc Fabregas entered the pitch area after the match improperly dressed, among other things.
Here is the record since the first world war: Arsenal having lost just one of these games…
22 Mar 1930 Arsenal v Hull City D 2-2 FA Cup
26 Mar 1930 Arsenal v Hull City W 1-0 FA Cup
29 Nov 1977 Arsenal v Hull City W 5-1 League Cup
28 Sep 1988 Hull City v Arsenal W 1-2 League Cup
12 Oct 1988 Arsenal v Hull City W 3-0 League Cup
27 Sep 2008 Arsenal v Hull City L 1-2 Premier League
17 Jan 2009 Hull City v Arsenal W 1-3 Premier League
19 Dec 2009 Arsenal v Hull City W 3-0 Premier League
13 Mar 2010 Hull City v Arsenal W 1-2 Premier League
20 Apr 2014 Hull City v Arsenal W 0-3 Premier League
17 May 2014 Arsenal v Hull City W 3-2 FA Cup
18 Oct 2014 Arsenal v Hull City D 2-2 Premier League
04 Jan 2015 Arsenal v Hull City W 2-0 FA Cup
04 May 2015 Hull City v Arsenal W 1-3 Premier League
20 Feb 2016 Arsenal v Hull City D 0-0 FA Cup
08 Mar 2016 Hull City v Arsenal W 0-4 FA Cup
There will be more thoughts (if I have any more thoughts) Saturday morning.
#CurseTheStar #CurseJackWilson. It is time for fans to fight back.
September 16th, 2016 | Category: Arsenal stories | 5 comments - (Comments are closed)
5 comments to Hull v Arsenal: a guess at the team, injury news, bits and stuff…
Post press conference…
Wenger said that Ramsey won't travel (still not full fit), Giroud has an injury on the big toe, and Gabriel is ready for Tuesday cup game.
I wish Perez well in his first real test as a central striker.
Our away supporters also have an important role to play. Bags of support throughout the game both for Lucas AND the whole team. 😉
Yes, choosing a team has become a little harder now, but it's all good eh? 🙂
There have been occasional snippets on Arsenal.com, some in Wenger interviews, stating that Gibbs, Debuchy and Sanogo are or have been injured.
I've started some commentaries to follow for the game, and in a couple of them is mention of a young former Hull player who recently died. The different "stories" have different ages for the person when he died (24 and 26). How can people writing this stuff make that kind of a mistake?


JAMESTOWN, NORTH DAKOTA – One year to the date of her arrest at Standing Rock, Red Fawn Fallis was transferred to a half-way house at a undisclosed location in North Dakota.
On October 27, 2016, Red Fawn Fallis was one of more than 140 people arrested in a violent clash with law enforcement led by the Morton County Sheriff's Department protesting the Dakota Access Pipeline near the Standing Rock Sioux Indian Reservation.
Of those arrested that day, Fallis faced the most serious charge–attempted murder. The attempted murder charge was dropped, but she has been held since her arrest pending a trial on a charge of possessing a firearm.
Witnesses deny she had a weapon.
On October 27, 2017 – the anniversary date of her arrest – Red Fawn Fallis was released from the Stutsman County Jail to a half-way house in North Dakota.
Red Fawn, a Lakota water protector originally from the Pine Ridge Reservation, is scheduled to stand trial in federal district court on January 29, 2018 on multiple charges stemming from her participation in the protests surrounding construction of the Dakota Access Pipeline. The path of the pipeline lay in close proximity to the Standing Rock Sioux Reservation and its water source and traversed lands of historic and cultural significance to the Standing Rock Sioux Tribe.
Red Fawn has plead Not Guilty to all charges. Her trial was recently moved from Bismarck to Fargo, North Dakota due to the publicity that has surrounded both the opposition to the construction of the pipeline and to Red Fawn's arrest.
Red Fawn is well-known and respected for her role as a medic and community leader at the Oceti Sakowin camp during fall 2016.
"While she was moved to a half-way house is great, people have to know she still faces a serious charge and people need to keep her in their prayers," her sister, Red Dawn Foster, told Native News Online on Friday evening.
I am so very sorry that RedFawn is still under scrutiny for the police riot last year. The government is corrupt from the top to the bottom and I don't know what else to do than to offer support to our brave water protectors. Bless you all. This is a story that can't be stopped.
Please support our heroes , like Red Fawn , a member of this Native community, that risked physical harm and even death at the hands of the enforcers that where against these water protectors, at standing rock.
I am glad for this news, it is a sorry system that drags it's feet when they want to.
Continued prayers for Red Fawn.


text:
Q: Determining solvability of system of equations I have a bizarre system of 4 equations I'm trying to solve. I'm pretty sure they're unsolvable but I'm not sure how to prove it mathematically. I've tried using Matlab's "Solve" function and received back some empty symbols but I'm not sure if that's definitive proof that the system is unsolvable.
Here are the equations:
$5.25 = a/b$
$9.0781= a/b + a/d$
$10.1875 = a/b+a/c$
$11.1875 = a/b+a/c+a/d$
Is this system unsolvable? How can you prove unsolvability in a case like this?
Thanks in advance!

A: Let us divide all equations by $a$ and group terms in a same side of each equation ; the resulting system can be given the following expression :
$$\left[\begin{array}{cccc}-\alpha&1&0&0\\
-\beta&1&0&1\\
-\gamma&1&1&0\\
-\delta&1&1&1\end{array}\right]\left[\begin{array}{c}1/a\\1/b\\1/c\\1/d\end{array}\right]=\left[\begin{array}{c}0\\0\\0\\0\end{array}\right]$$
(where $\alpha=5.25$, $\beta=9.0781$, etc.)
As the determinant of the square matrix is $-\alpha+\beta+\gamma-\delta=2.8281 \neq 0$, the only solution to such a system would be the null vector. But this is impossible to have for example $1/a=0$ with a real number $a$.
Conclusion : the initial system hasn't any solution.

redpajama_set_name: RedPajamaStackExchange


text:
Q: Which one should I use for dimension reduction with PCA in MATLAB, pcacov or eigs? I'm trying to reduce my training set dimension from 1296*70000 to 128*70000.
I wrote Below code:
A=DicH;
[M N]=size(A);
mu=mean(A,2);%mean of columns

Phi=zeros(M,N);
C=zeros(M,M);
for j=1:N
    Phi(:,j)=A(:,j)-mu;
    c=Phi(:,j)*(Phi(:,j))';
    C=C+c;
end

C=C/N;%Covariance Dictionary
[V,landa] = eigs(C,128);%Eigen Vectors & Eigen Values
E=V'*Phi;%Reduced Dic
%*******************Using Pcacov*****************
%S=zeros(M,1);
%[U,landa] = pcacov(C);%Eigen Vectors & Eigen Values
% for k=1:128;
%     S=V(:,k)+S;
%     U(:,k)=S;
% end
%E=U'*Phi;%Reduced Dic

I get two different answers! Which one should I use "eigs" or "pcacov"??

A: You should take advantage of the built-in functions in Matlab, and use the pca function directly, or even the cov function if you want to compare eigs to pcaconv.
Now to answer your question, both return the same eigenvectors but not in the same order. See the following example:
>> load hald
>> covx = cov(ingredients);
>> [COEFF,latent] = pcacov(covx)

COEFF =

   -0.0678   -0.6460    0.5673    0.5062
   -0.6785   -0.0200   -0.5440    0.4933
    0.0290    0.7553    0.4036    0.5156
    0.7309   -0.1085   -0.4684    0.4844

latent =

  517.7969
   67.4964
   12.4054
    0.2372

>> [V, D] = eigs(covx)

V =

    0.5062    0.5673    0.6460   -0.0678
    0.4933   -0.5440    0.0200   -0.6785
    0.5156    0.4036   -0.7553    0.0290
    0.4844   -0.4684    0.1085    0.7309

D =

    0.2372         0         0         0
         0   12.4054         0         0
         0         0   67.4964         0
         0         0         0  517.7969

>>

In your code, you overwrite the result of pcavconv in the commented-out section with a transformation of the result of eigs so it is not clear what your are comparing at this point. When using pcacov, you just need to extract the 128 first columns of U.


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Additional credentials: M.P.A Health Care Management, May 2000, Reiki Master, Oct 2010, Ordained Minister, May 2013, Psychoenergetic Healing Certification, May 2008.
Additional credentials: Theta Healing 2007.
Additional credentials: BSA Art, Viginia Commonwealth University, 1981; Reiki Master, 1999; Akashic Records Integration Process Modual A: 2010, Modual B: 2012, Emotion Code Certification, 2013.
Additional credentials: Akashic AllianceTM Co-Founder, Facilitator & Teacher (Akashic Records); Certified Emotion Code Practitioner; Certified Past Life Regressionist; Ordained Priest of Melchizedek; Masters in Metaphysical Education.
Additional credentials: Doctor of Philosophy, Ph.D., specializing in Holistic Life Coaching, University of Sedona 2012; Ordained Minister since 2009 International Metaphysical Ministry.
Rev. Dr. Olympia Freeman, C. Ht.


text:
Q: Javascript parser to add percentages to non-percentages Looking for a consistent way to write (or extend) a parser to do math equations. I'm using mathjs for most of this, and it works great. But it does not handle percentages. I'm trying to write rules that will handle adding percentages to percentages, percentages to non-percentages, etc..
1 + 6 + 7% = 7.49 – correct .
1 + 6 + 7% = 7.07 – incorrect .
56.47 + 15% = 64.9405 – correct .
56.47 + 15% = 56.62 – incorrect .
etc..
Any tips or suggestion welcome.

A: You could do something like this:

      Number.prototype.plusPecentage = function(n){
        return this+(this*n/100);
      }

      console.log((1 + 6).plusPecentage(7))

Or this:

Math.plusPecentage = function(n,p){
  return n+(n*p/100);
}

console.log(Math.plusPecentage(7,7))

You could also do the same but extending mathjs library doing math.plusPecentage = function () {//code}


National Liberal Party (PNL) urges president Traian Basescu to clarify the assertion related to the support he may have provided in 2005 for the establishment of the required majority in the election of Moldavian president Vladimir Voronin. The Liberals regard the affirmation "made with easiness" by the head of state in an interview granted some days ago to Romania Actualitati radio channel is "a huge error of foreign policy." "If it is true that Traian Basescu intervened directly in the election of Vladimir Voronin by the Moldavian Parliament, then he should be held liable both for the unacceptable degradation of the relationship between Romania and Republic of Moldova, and for the decline of the democratic process in the neighbor country, a decline that started after the Communist power took the office in 2005. By his statements, Traian Basescu does nothing else than confirming and legalizing the anti – Romanians attacks of the Communist power from Chisinau.


With their most recent record, "Melting Sun", German outfit LANTLÔS separated themselves from the post-black-metal-scene they emerged from. Their stylistic change took them into a post-rock setting that doesn't need any blastbeats or screaming. Fortunately fans and critics welcomed this change of direction and even praised it. The band's European tour is supported by Irish post-rockers FALLOCH. Today's show in Oberhausen also bills German black metallers HALLIG.
After waiting for some time in the venue, HALLIG finally take the stage. This four-piece play melodic but old school black metal. I don't know if the lyrics are German or English, because you just don't understand them. The music itself is not very interesting or special, but the band's drummer is quite good. HALLIG play for almost half an hour, but they fail to impress the audience. There are about 50 people in front of the stage and only two or three bang their heads, while the rest is rather drinking a beer.
FALLOCH are far better. The band's post-rock is able to find a good balance between post-rock and songwriting. The songs are rather short for post rock standards and include lot of singing and even real choruses. Sometimes the British band seems like a post-rock version of old BIFFY CLYRO. Now the venue is more crowded than before and FALLOCH seem to attract more people than HALLIG did before. For about forty minutes the quartet deliver a solid show, without many high, but definitely no lows.
Then it's time for German five piece LANTLÔS, whose show includes tracks of their last two black metal records, as well as four of six songs taken from "Melting Sun". 'Intrauterin and 'Bloody Lips and Paper Skin', both from "Agape" open the set. Especially the slow but heavy opener might be a whole new level of LANTLÔS than the fans of the band's new direction of post-rock. But these new blend of fans won't be disappointed because what's following are the first four songs taken from "Melting Sun". Live these tunes are a lot more trough than on record, which fits these compositions very well. The band's performance is more than solid, but there isn't a lot of action on stage. The guys play their instruments very well, but stay focused on playing instead of posing. Even the faster songs from the group's past are played in such manner. After little more than 60 minutes and the great 'Coma' from ".Neon" the band wants to leave the stage, but the crowd begs for more. So they just play 'Melting Sun I: Azure Chimes' one more time before they say goodbye at last. LANTLÔS definitely have the potential to attract far more people than just about 100 as today. I'm pretty sure than "Melting Sun" will appeal to many people more in the following months. They'd deserve it.


OPELOUSAS, La. - A state judge Monday ordered Holden Matthews held without bond after prosecutors argued he was a flight risk and offered evidence that he captured video on his phone of burning three predominately black churches in St. Landry Parish.
Twenty-Seventh Judicial Court Judge James Doherty of Opelousas denied Matthews' bail, citing concerns about releasing the 21-year-old and a pending investigation by the FBI and Bureau of Alcohol, Tobacco, Firearms and Explosives.
Under Louisiana law, the hate crimes could apply to various protected groups including race, age, gender and religion. The District Attorney's Office did not specify a basis for the hate-crime charges against Matthews.
Matthews, the son of a St. Landry Parish Sheriff's deputy, is accused of setting the churches on fire in a 10-day span. His parents, Roy and Angela Matthews, were present at the hearing. Matthews appeared stoic with his court-appointed attorney, Quincy Cawthorne, via a video feed from the jail.
Matthews was arrested in connection with the fires Wednesday. Authorities are investigating Matthews' involvement in the music genre of black metal, whose followers have been tied to church fires and extreme acts in other parts of the country.
State Fire Marshal Butch Browning said Matthews may face federal hate crime charges from the FBI. He also testified that Matthews intentionally set the fires, used gasoline as an accelerant and documented his actions through videos found on his cellphone.
Once Matthews was arrested, investigators found photos of the fires on his phone after they were set and possibly before 911 was called, Browning said. There were videos on Matthews' phone of firefighters attempting to extinguish them and photos of the churches after the fires had been extinguished.
Investigators also found news reports of the fires on Matthews' phone. He had superimposed himself on those reports claiming he was responsible, Browning said. There was also a video on Matthews' phone that showed him talking with a friend about burning churches in St. Landry Parish, Browning said.
Browning also mentioned Matthews' ties to black metal. Matthews told the FBI that the movie "Lords of Chaos," which loosely documents a Norwegian black metal band, may have been a motivation, though he maintained his innocence, Browning said.
Cravins argued that Matthews was a flight risk, which Cawthorne contended wasn't possible because Matthews didn't have the resources. Browning, while on the stand, said, "you don't have to have money to run."
An arrest affidavit showed investigators tied him to the crimes through surveillance video that showed a truck registered to his father at the fires, as well as the purchase of a gas can and cellphone tower data.
In surveillance footage from Walmart, Matthews is seen getting into a light-colored pickup with a bed cover, according to the affidavit. A beige Ford is registered to Matthews' father, Roy.
Surveillance footage collected from homes and businesses near each church shows a light-colored, extended cab pickup around the area at the time of the fire, according to court records.
St. Mary Baptist Church, which is in Port Barre, was the first church burned, on March 26/ The Greater Union Baptist Church in Opelousas burned a week later. On April 4, a fire destroyed Mount Pleasant Baptist Church on Highway 182 south of Opelousas.
A pretrial hearing is set for July. Jury selection will begin Sept. 10.


Glamorous weather greeted an impressive turnout of 37 Moths for the first Grand Prix of 2014 sponsored by Data to Value Ltd. at Queen Mary Sailing Club. With a range of abilities from Olympic medallist to complete newbie battling it out in shifty conditions on a crowded reservoir, the event promised to provide some close racing and some surprising results. GPS tracking for the event was ably provided by Trac Trac meaning that the vital statistics from each race could be captured and visualised for those keen to analyse what happened during the races.
The 4 races on Saturday featured sunshine and a solid breeze of 6 to 16 knots (most of the time anyway except when you found a big hole and sat there while everybody sailed by). With 7 laps per race at times there was a need for an abacus but the racing was fast and furious with plenty of place changing. The breeze pulsed in and out leaving the fleet at times low riding and at times with a lot on making the turn at the leeward mark!
Race 1 started with Ed Chapman showing the fleet how to start in style hitting the line exactly on the buzzer at max speed in his Ninja. Unfortunately for Ed the water temperatures of the reservoir are still very low which led to his boat ventilating and dramatically cartwheeling in front of a swarm of eager moths. He claimed his GoPro footage became corrupted although we suspect it may just be a reluctance to feature in the post racing videos! Picking out the shifts and sailing consistently, Paul Goodison proved his mastery of the moth to take victory, closely followed by fellow Weymouth sailor Chris Draper and Tom Offer. Who claims to be from Rock in order to regularly win the furthest travelled prize at events. See the race replay here.
The second race saw an exciting start including contact between a certain unnamed America's Cup helm and a regular mothie. It was however Jason Belben who pulled out a significant lead during the first lap on the competition. The next leg saw Belben however choose the wrong side downwind to be overhauled by James Phare, Ricky Tagg and Chris Draper. Phare held onto the lead to be closely followed home by Simon Hiscocks, Ricky Tagg and Chris Draper. See the race replay here.
Race 3 saw most of the fleet head for the far left hand side of the beat to make the most of the stronger breeze with Ricky, Simon and Jason rounding first. Ricky lead until the last few hundred meters of the next downwind when gybing into some light air allowed Simon past into first. Simon then dominated the remained of the race to be followed home by Chris Draper and Jason Belben. See the race replay here.
Race 4 saw Ed Chapman and Chris Draper port tack the fleet to great effect leading Simon Hiscocks and Leigh Albrecht round the windward mark. It was however again Simon Hiscocks that dominated passing Chapman and Draper to gain a considerable lead from Draper and Belben. See the race replay here.
The fleet then headed home for some complimentary tasty pasta dishes and a refreshing beverage to chat through the days action while watching the paddy's unfortunately win the rugby. BenPatonRacing.com headed to shore early after only completing two races. Possibly this was to check the trac trac being a true professional that he is or maybe just to get Tinder up and running early?! I hear there may be some video diaries to follow up on his site shortly. Ben I'm not sure missing races should be happening at this level! Sunday brought some unseasonal sunny conditions with one of the main concerns being the sun cream, especially us redheads of the fleet. There wasn't that much wind but the fleet headed out for some marginal foiling action.
The first race saw some major place changes. Chris Draper won the low riding race to the windward mark before things got very interesting with boats popping and falling off the foils left right and centre. It was John Clifton who figured it out best to take the win from Neil Baker. See the race replay here.
Chris Draper took out the second race with the Exocets of Tommy (special) Offer and Ricky Tagg in hot pursuit. I'm sure a lot of people can say 'they were winning' at some point but it was never over until you got through the finish line. If you're not foiling its not much fun, especially if others are as they are sailing past! Nobody was quite sure what was happening overall with the big dog Simon Hiscocks struggling a bit in the lighter winds and a mixed bag for the other contenders. The wind had however filled in a bit for the final races. See the replays here and here.
Tommy special stamped his authority on the event sailing really well to win race 7 and race 8 and take out the event convincingly from Draper, Goodison, Tagg then Hiscocks.
Final results interactive visualised results and statistics are available at www.datatovalue.co.uk/mothopen. Big thanks to the event sponsors Data to Value, the series sponsors Noble Marine, Magic Marine for the prizes and Alex Irwin from Sportography who got some pretty cool pictures and video of the racing.
The next stop Parkstone Yacht Club on 5/6 April, unfortunately I can't make it, good luck to who ever is 3rd to take this on! Look forward to reading it.


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