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We have recently identified the enzyme NMN deamidase (PncC), which plays a key role in the regeneration of NAD in bacteria by recycling back to the coenzyme the pyridine by-products of its non redox consumption. In several bacterial species, PncC is fused to a COG1058 domain of unknown function, highly conserved and widely distributed in all living organisms. Here, we demonstrate that the PncC-fused domain is endowed with a novel Co+2- and K+-dependent ADP-ribose pyrophosphatase activity, and discuss the functional connection of such an activity with NAD recycling. An in-depth phylogenetic analysis of the COG1058 domain evidenced that in most bacterial species it is fused to PncC, while in α- and some δ-proteobacteria, as well as in archaea and fungi, it occurs as a stand-alone protein. Notably, in mammals and plants it is fused to FAD synthase. We extended the enzymatic characterization to a representative bacterial single-domain protein, which resulted to be a more versatile ADP-ribose pyrophosphatase, active also towards diadenosine 5′-diphosphate and FAD. Multiple sequence alignment analysis, and superposition of the available three-dimensional structure of an archaeal COG1058 member with the structure of the enzyme MoeA of the molybdenum cofactor biosynthesis, allowed identification of residues likely involved in catalysis. Their role has been confirmed by site-directed mutagenesis.
Citation: Cialabrini L, Ruggieri S, Kazanov MD, Sorci L, Mazzola F, Orsomando G, et al. (2013) Genomics-Guided Analysis of NAD Recycling Yields Functional Elucidation of COG1058 as a New Family of Pyrophosphatases. PLoS ONE8(6): e65595. https://doi.org/10.1371/journal.pone.0065595
Editor: Luis Eduardo Soares Netto, Instituto de Biociencias - Universidade de São Paulo, Brazil
Received: February 7, 2013; Accepted: April 29, 2013; Published: June 12, 2013
Copyright: © 2013 Cialabrini 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 research was partly supported by the Italian Ministry of Foreign Affairs, "Direzione Generale per la Promozione del Sistema Paese" to NR; by the U.S. Department of Energy (DOE), Office of Biological and Environmental Research (BER) Genomic Science Program (GSP) as part of the Pacific Northwest National Laboratory (PNNL) Foundational Scientific Focus Area to ALO; and by The Ministry of Education and Science of Russian Federation, projects 8135 and 8049 to MDK. 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.
NAD is an ubiquitous and essential coenzyme involved in a huge number of redox reactions in all forms of cellular life. In addition, NAD is utilized as a co-substrate in a variety of non redox reactions playing an important role in DNA replication, DNA repair, RNA ligation, cell differentiation, and cellular signal transduction [1], [2], [3]. Specific enzymes catalyze the transfer of the NAD ribonucleotidyl moiety, either in the form of AMP or ADP-ribose (ADPR), to different functional groups of proteins and nucleic acids, thereby modulating their function. NAD-dependent ADP-ribosylation and deacetylation of various target proteins, as well as NAD-dependent dephosphorylation of tRNA are well known in eukaryotes. In contrast, non redox NAD-dependent processes in bacteria are still relatively unexplored. Only few bacterial NAD-consuming enzymes have been so far characterized: i) the NAD-dependent DNA ligase, which uses the AMP moiety of NAD to activate the 5′-phosphate of nicked DNA ends; ii) the NAD-dependent deacetylase CobB of the Sirt2 family, which catalyzes protein deacetylation by transferring ADPR from NAD to the acetyl group, with the release of O-acetyl-ADPR [4]; iii) various mono ADP-ribosyltransferases, which catalyze the covalent attachment of single ADPR units to both endogenous and host proteins to regulate their function [5], [6]; iv) an ortholog of the yeast tRNA 2′-phosphotransferase, able to catalyze tRNA dephosphorylation by transferring ADPR from NAD to the 2′-phosphate group of tRNA, with the release of 1,2 cyclic phosphate ADPR [7]. The occurrence of an intensive NAD consumption in bacteria is suggested by the remarkably rapid turnover of the intracellular NAD pool, which also emphasizes the importance of the continuous replenishing of the dinucleotide [8]. Notably, all the products of NAD consumption can contribute to its regeneration (Figure 1). In particular, the pyridine by-products, nicotinamide mononucleotide (NMN) and nicotinamide (Nm), can be recycled back to NAD through various recycling pathways that, in recent years, have begun to be elucidated in the majority of bacterial species, thanks to genomics-guided approaches [9], [10], [11], [12]. In turn, the ADPR moiety released from ADP-ribosylated proteins, or deriving from O-acetyl-ADPR and 1,2 cyclic phosphate ADPR, can be further hydrolyzed by ADPR pyrophosphatases (ADPRP) of the Nudix family, yielding AMP and ribose-5-phosphate, which may be reused in NAD biogenesis via conversion to ATP and PRPP, respectively, as already proposed in eukaryotes [13], [14], [15]. In this view, the occurrence in some bacterial species of a Nudix ADPRP domain fused to the enzyme NMN adenylyltransferase (NadM) provides evidence of the strict link between NAD consumption and regeneration (Figure 1) [16], [17].
Reactions described in this study, numbered from 1 to 3, are catalyzed by: 1) NMN deamidase (PncC); 2) NMN adenylyltransferase of the NadM family; 3) ADPR pyrophosphatase. In several bacterial species PncC and NadM occur in fused forms with COG1058 ADPRP and Nudix ADPRP, respectively, as discussed in this work. Abbreviations: Nm, nicotinamide; NMN, nicotinamide mononucleotide; NaMN, nicotinate mononucleotide; NaAD, nicotinate adenine dinucleotide.
Very recently, combining comparative genomic analysis, metabolic pathways reconstruction, and experimental characterization, we have identified the enzyme NMN deamidase (PncC), which converts NMN to nicotinate mononucleotide (NaMN), thus channeling the mononucleotide towards the deamidated NAD biosynthetic pathway (Figure 1) [10]. This enzyme plays a key role in NMN and Nm recycling back to NAD in the majority of bacterial species. Very often, PncC is found fused to a domain of unknown function, which belongs to the family of proteins classified as COG1058 in the Clusters of Orthologous Groups database, currently annotated as "predicted nucleotide-utilizing enzyme family, related to molybdopterin-biosynthesis enzyme MoeA" (PF00994). Members of this family share a high similarity with bacterial MoeA and its eukaryotic orthologues (i.e. the E-domains of mammalian gephyrin and plant Cnx1), which are involved in the last step of Molybdenum Cofactor (MoCo) biosynthesis [18]. In particular, these enzymes bind both molybdate and the adenylated form of cyclic pyranopterin monophosphate (MPT-AMP), and catalyze MPT-AMP hydrolysis, releasing AMP, with the concomitant insertion of molybdenum into MPT, yielding the active product MoCo [19]. Based on the catalyzed reaction, they have been considered to act like the enzyme ADPRP of the Nudix hydrolase family [19] (Figure 2). This suggestion, together with the observed tendency of ADPRP to associate with enzymes of NAD biosynthesis, led us to hypothesize that members of COG1058 might be endowed with ADPRP activity.
Both substrates share an adenosine group linked to two different moieties through the pyrophosphate bridge which is cleaved during the enzyme-catalyzed reaction.
In this work, we demonstrated the ADPRP activity of the bacterial COG1058 domain, and provided evidence that COG1058 represents a novel pyrophosphatase family.
Materials and Methods
Cloning, Expression, and Protein Purification
The COG1058 gene of A. tumefaciens was amplified by polymerase chain reaction (PCR) from genomic DNA and cloned into the pET100/D-TOPO vector (INVITROGEN Champion™ pET Directional TOPO® Expression Kits) according to manual's instructions. Sequences of the synthetic oligonucleotides used as primers are reported in Table S1. The construct was sequence-verified for accuracy and used to transform E.coli BL21(DE3) cells for protein expression. Cells were grown at 37°C in Luria Bertani medium supplemented with 0.1 mg/ml ampicillin. After reaching an A600 of 0.6, expression was induced with 1 mM isopropyl β-D-thiogalactopyranoside. After 3 h induction at 37°C, the cells were harvested by centrifugation at 5,000×g for 10 min, at 4°C. All subsequent steps were performed at 4°C. Induced cells were resuspended in one-twentieth of the original culture volume with buffer A (50 mM TRIS/HCl buffer, pH 8.0, 1 mM MgCl2, 0.2 mM EDTA, 10 mM Imidazole) containing 1 mM phenylmethylsulfonyl fluoride and 0.002 mg/ml leupeptin, antipain and chymostatin. The suspension was sonicated for 3 min at 50 watt, with 30 sec intervals, and centrifuged at 15,000×g for 30 min. The supernatant deriving from 40 ml culture was applied to a 1-ml HisTrap HP column (GE Healthcare), equilibrated with buffer A. The column was washed with 30 mM Imidazole in buffer A, and elution was performed with an Imidazole gradient from 30 mM to 500 mM in buffer A. Fractions containing the recombinant protein (eluted at about 100 mM Imidazole) were pooled and purity of the preparation was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis [20]. The pool (0.2 mg/ml protein concentration, as determined by using bovine serum albumin as the standard [21]), resulted to be stable for several months at 4°C, and was used for the catalytic characterization.
S. oneidensis COG1058/PncC protein was obtained as described in [10].
Site-directed Mutagenesis
Site-directed mutagenesis of A. tumefaciens COG1058 was carried out using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). Sequences of mutagenic primers are reported in Table S1. pET100/D-TOPO recombinant plasmid was used as the template for the PCR mutagenesis reactions, by following kit's instructions. The mutants were sequenced to verify incorporation of the desired modification and to ensure the absence of random mutations. For mutants expression, the mutagenized plasmids were transformed into E.coli BL21(DE3) cells and expression and purification of the mutated proteins were performed as for the wild-type protein.
Pyrophosphatase Activity Assays
Activity was assayed by measuring the formation of the mononucleotides deriving from the pyrophosphate bond hydrolysis of the tested compounds. Nucleotides were separated by HPLC on a system equipped with a diode-array detector. The reaction mixtures contained 0.8 µg/ml S. oneidensis or 0.08 µg/ml A.tumefaciens purified recombinant protein, in 100 mM TRIS/HCl buffer, pH 7.5, 100 mM KCl, 0.1 mg/ml bovine serum albumin, 1 mM CoCl2, 0.5 mM substrate. After incubation at 37°C, reactions were stopped and subjected to HPLC analysis, using different procedures depending on the tested substrate. In particular, when FAD was tested as the substrate, reactions were stopped by adding formic acid (1∶20 of final assay volume). When NADH and NADPH were tested, reactions were stopped with 0.12 M NaOH; after 10 min on ice the samples were centrifuged for 6 min at 12,000×g and the supernatants were neutralized with 0.01 M HCl. In both cases, the samples were loaded onto a Phenomenex C18 Kinetex column (2.6 µm, 4.6×150 mm). For FMN determination, elution conditions were as follows: 5 min at 100% buffer A (100 mM potassium phosphate, pH 3.0, 10% methanol), 15 min up to 100% buffer B (100 mM potassium phosphate, pH 3.0, 30% methanol), holding at 100% buffer B for 5 min, returning to 100% buffer A in 1 min, and holding at 100% buffer A for 12 min. Flow rate was maintained at 0.5 ml/min, and temperature was fixed at 25°C. For NMNH determination, column was eluted as described above using buffer A consisting of 100 mM potassium phosphate, pH 6.0 and buffer B consisting of buffer A, containing 20% methanol. For the determination of the mononucleotides produced from all other tested substrates, reactions were stopped with 0.6 M HClO4; after 10 minutes on ice the samples were centrifuged for 6 min at 12,000×g and the supernatants were neutralized with 0.8 M K2CO3, kept on ice for 10 min and centrifuged as described above. Nucleotide separation was performed on a Supelco LC-18-T column (5 µm, 4.6×250 mm) at 18°C. Elution conditions were as described in [22]. In all assays, the amount of enzyme used ensured a substrate consumption below 5% of the initial concentration after a 10 min incubation. In addition, withdrawals from the assay mixtures at two different incubation times were always performed to ensure a linear time frame. Controls without enzymes were always processed in parallel to correct for the non-enzymatic, metal-ion catalyzed hydrolysis of several substrates. All measurements were performed in duplicate. Kinetic values were determined by fitting initial velocity data to the standard Michaelis-Menten equation using GraphPad Prism software package.
Bioinformatics Tools and Resources
The COG1058 protein sequences in available complete genomes were taken from The SEED comparative genomics database [23]. Due to the large number of sequences retrieved, a special procedure had to be used for the construction of multiple sequence alignment: i) an approximate phylogenetic tree was built by the FastTree tool [24]; ii) all sequences were divided into fifteen clusters corresponding to the separate branches of the tree; iii) multiple alignment of sequences belonging to the same cluster was obtained using Clustal Omega [25]; iv) poorly aligned regions were cut from the cluster alignments; v) the final alignment was constructed using the profile-to-profile alignment option of the Clustal Omega algorithm. The phylogenetic tree was built by RAxML [26]. The species tree was taken from the Superfamily database [27]. Visualization of protein three-dimensional structures and structure comparison were performed using Chimera [28]. Multiple sequence alignment figures were prepared using TeXshade [29]. Genome context analysis was performed in The SEED environment.
Bacterial Members of the COG1058 Family are Endowed with ADP-ribose Pyrophosphatase Activity
Both Shewanella oneidensis (So) COG1058/PncC protein, in which the COG1058 domain is fused with the NMN deamidase (PncC) domain, and A. tumefaciens (At) COG1058 protein (gi 159184889), which comprises only the COG1058 domain, were assayed for the ADPRP activity. Both proteins were found to possess such activity in HEPES/KOH buffer, pH 7.5, 1.0 mM Mg+2. The ADPRP activity of the At enzyme was further characterized in order to determine the optimal conditions for the reaction. Catalysis resulted to be metal-dependent (Figure 3A). Among the tested divalent cations, Co+2 was the most effective in supporting the enzyme activity, with Ni+2, Mg+2 and Mn+2 being about seven-fold less efficient; 1 mM Ca+2, Cu+2 and Zn+2 did not sustain the activity at all (Figure 3A). Mg+2 and Co+2 titration experiments showed that the enzyme-catalyzed ADPR hydrolysis was optimal at 0.5 mM Co+2 (Figure 3B), while 10 mM Mg+2 was needed to reach the maximum activity, corresponding to about 30% of the optimal Co+2-dependent activity (not shown). We found that the presence in the reaction mixtures of a monovalent cation was also essential for the catalytic activity; replacement of HEPES/KOH buffer with TRIS/HCl abolished the enzymatic activity unless a monovalent cation was present (Figure 3C). Among the cations tested, K+ was the most effective (Figure 3C). The K+-dependence of the reaction velocity in the presence of either TRIS/HCl or HEPES/KOH buffers at pH 7.5, is shown in Figure 3D. Maximum activation was reached at different K+ concentrations depending on the buffering species: in TRIS/HCl buffer, 100 mM K+ was the most effective, whereas in HEPES/KOH buffer, maximum activity was reached at about 200 mM K+ concentration. As shown in Figures 3D and 3E, ADPR hydrolysis is significantly affected also by the buffering species present in the reaction mixture. Among the buffers tested at pH 7.5, in the presence of 100 mM K+, TRIS buffer was the best at sustaining activity, followed by MOPS, HEPES, and Phosphate. In the presence of Imidazole and TRICINE, a very low activity was measured (Figure 3E). Optimal pH was determined by measuring ADPR hydrolysis in a 50 mM BIS-TRIS/TRIS buffer system at pH values ranging from 5.5 to 8.5. Activity was optimal in a narrow range around pH 7.5 (Figure 3F).
Enzymatic assays were performed in the presence of 0.5 mM ADPR and 10 ng of pure protein. Reaction mixtures were incubated for 10 min at 37°C in: A) 100 mM HEPES/KOH, pH 7.5, in the presence of different divalent cations at 1 mM concentration (all ions were added as chloride salts); B) 100 mM HEPES/KOH, pH 7.5, with different concentrations of MgCl2 or CoCl2; C) 100 mM TRIS/HCl buffer, pH 7.5, 1 mM Co+2, in the presence of 10 mM and 100 mM of the indicated monovalent cations (added as chloride salts); D) 100 mM TRIS/HCl, pH 7.5 and 100 mM HEPES/KOH, pH 7.5, 1 mM Co+2, in the presence of different K+ concentrations (K+ ions were added as KCl); E) different buffer species at 100 mM concentration, pH 7.5, 1 mM Co+2, 0.1 M K+; F) 100 mM BIS-TRIS buffer at varying pH values, 1 mM Co+2, 0.1 M K+. One Unit of enzyme activity represents the amount of enzyme catalyzing the formation of 1 µmol of product per min, under the specified conditions.
The same Co+2-dependence and K+-activation, as well as pH optimum and buffering species dependence, were displayed by the SoCOG1058/PncC enzyme (not shown).
Substrate Specificity Screening Reveals that ADP-ribose is the Preferred Substrate of Bacterial COG1058 Enzymes
To get a deeper insight into the COG1058 domain substrate specificity, we performed a detailed in vitro screening of several compounds containing a pyrophosphate bond as potential substrates of AtCOG1058 and SoCOG1058/PncC enzymes, by using the assay conditions previously optimized towards ADPR. The results of the screening performed with the two enzymes are shown in Figure 4. Both enzymes display a Co+2-dependent pyrophosphatase activity towards a limited set of substrates, with ADPR being the preferred among the tested compounds. The stand-alone domain also hydrolyzes diadenosine 5′-diphosphate (Ap2A) to a significant extent (75% rate with respect to ADPR substrate), and shows some activity with FAD (30% rate), NADH and nicotinate adenine dinucleotide (NaAD) (14% rate). A very low, but still detectable activity is displayed by this enzyme towards NADPH and NAD, while NADP is not a substrate. The SoCOG1058/PncC bifunctional enzyme is more strictly specific for ADPR; in fact it hydrolyzes Ap2A at about 10% rate with respect to ADPR, while FAD is not a substrate. On the other hand, it behaves similarly to the AtCOG1058 enzyme towards the pyridine dinucleotides. For both enzymes, replacement of the ADP-linked ribose with mannose significantly decreases the activity, which is further decreased or completely abolished when ribose is replaced by glucose (Figure 4). A very low activity is displayed by the AtCOG1058 enzyme towards GDP-mannose, while GDP-glucose is not recognized by either protein. The presence of a phosphate group on the adenine-linked ribose significantly decreases or fully abolishes the activity. As to the ApnA series, for both enzymes the activity falls off when n >2. ATP is hydrolyzed to AMP only by the AtCOG1058 protein and to a very low extent (Figure 4). Neither enzyme is able to hydrolyze the pyrophosphate bond in ribonucleoside diphosphates (not shown).
The pyrophosphatase activity of the pure recombinant enzymes was assayed as described in "Materials and Methods", in the presence of the listed compounds at 0.5 mM concentration each. Abbreviations: Ap3A, diadenosine triphosphate; Ap4A, diadenosine tetraphosphate; Ap5A, diadenosine pentaphosphate; NGD, nicotinamide guanine dinucleotide; NHD, nicotinamide hypoxanthine dinucleotide.
Results of the kinetic analyses for the preferred substrates are reported in Figure 5. Although the AtCOG1058 enzyme hydrolyzes ADPR and Ap2A at similar rates, the catalytic efficiency (kcat/Km) towards ADPR is about 14 fold higher. In addition, the bifunctional enzyme is about seven-fold less efficient towards ADPR than the stand-alone pyrophosphatase.
Plots of the initial velocities of the catalyzed reactions versus substrate concentrations. Kinetic parameters, calculated as described in Materials and Methods, are reported in the table.
Neither enzyme is able to remove a phosphate group from ribonucleoside mono- and diphosphates or from NADP, nor to hydrolyze the phosphodiester bond in 2′, 3′- and 3′, 5′-cyclic nucleotides (not shown).
COG1058 Phylogenetic Analysis Reveals a Wide Distribution and Fusion with Different Catalytic Domains
Analysis of the phylogenetic distribution of COG1058 members in the three kingdoms of life revealed that they are widely distributed, occurring in approximately half of the eukaryotic, bacterial, and archaeal genomes (Figure 6). The COG1058 domain can be found either as a stand-alone domain, or in a fused form with NMN deamidase or FAD synthase (that catalyzes FMN adenylylation to FAD). In particular, in archaea, in α- and some δ-proteobacteria, the COG1058 domain occurs only as a single domain, while in remaining bacterial taxonomic groups it is mostly found fused with PncC (Figure 6A–C). Eukaryotes have both single- and two-domain proteins, the latter being composed of the COG1058 domain fused to FAD synthase (Figure 6B). While the single-domain form is mostly present in fungi, the fused form is widely distributed among plants and animals. Notably, in plants the COG1058 domain is located at the N-terminus, whereas in animals it occurs at the C-terminus. The large-scale topology of the COG1058 phylogenetic tree (Figure 7, and Figure S1) is largely consistent with the top-level topology of the species tree, i.e. the bacterial, archaeal and eukaryotic proteins form distinct clusters. This implies the absence of the horizontal transfer of the COG1058 domain between the three main kingdoms. The analysis of the tree also shows that the proteins in the α-proteobacterial group are evolutionarily closer to the eukaryotic and archaeal proteins than to their bacterial counterparts. In addition, the δ-proteobacterial group of enzymes is present in the eukaryotic branch, suggesting that it likely represents the ancestor group of the eukaryotic proteins. As to the domain composition, the two-domain bacterial proteins cluster into a homogenous group, well separated from all other forms, i.e. the bacterial, archaeal and eukaryotic single-domain enzymes and the eukaryotic two-domain enzymes. In this view, the COG1058 domain fused to NMN deamidase is evolutionarily distant from both the stand-alone and the FAD synthetase-fused form, the latter two forms being closely related to each other.
Schematic representation of bacterial (A), eukaryotic (B) and archaeal (C) species trees showing COG1058 genes mapping. Green circle designates the COG1058 gene; the FAD synthase gene is represented by a red circle; the fused COG1058/pncC gene is shown as a blue square. Numbers within squares represents the number of gene copies per genome.
Schematic representation of the COG1058 phylogenetic tree (full version is in Fig. S1). The stand-alone COG1058 gene and the gene fused with FAD synthetase and pncC genes are depicted as green, red and blue circles, respectively. The Shewanella oneidensis and Agrobacterium tumefaciens COG1058 proteins, experimentally characterized in this work, are marked by red stars. Thermoplasma acidophilum COG1058 protein, whose 3D structure is available, is highlighted.
Mutagenesis Guided by Multiple Sequence Alignment and Structural Analysis Reveals the Identity of Catalytic Residues
A multiple sequence alignment using a collection of 361 bacterial, 36 archaeal, and 34 eukaryotic completely sequenced genomes, annotated in The SEED database [23], revealed that the COG1058 domain is highly conserved (Figure S2). A multiple alignment of the domain in the most divergent sequences, including the AtCOG1058 and the SoCOG1058/PncC proteins characterized in this work, is depicted in Figure 8. A total of eight residues (highlighted in magenta in Figure 8) were conserved among over 95% of the COG1058 proteins in all sequenced genomes, suggesting their likely role in protein's function or stability. The conserved residues appear to define two putative signature motifs, namely GXEX3G and GGL/IGPX3D. In addition, five residues (highlighted in green in Figure 8) were found to be conserved in all COG1058 sequences, with the exception of the plant subfamily of proteins (Figure S2). To get an insight into the possible functions of the conserved residues, we performed a structural homology search by using as the query the available high-resolution (2 Å) crystal structure of the Thermoplasma acidophilum COG1058 protein (PDB ID: 3KBQ), as determined at the Midwest Center for Structural Genomics (http://www.mcsg.anl.gov/). Notably, a DALI search revealed high structural similarity (Z score >10 ) with the fold of proteins from the E. coli MoCo biosynthetic pathway (MogA, the domain III of MoeA, and MobA), as well as with domains of mammalian gephyrin and plant Cnx1, which are also involved in MoCo biosynthesis [30], [31], [32]. The superimposition of the 3KBQ three-dimensional structure and domain III of E. coli MoeA (PDB ID: 1G8L) is shown in Figure 9A. Based on both the significant overall structure homology and the similarity of the reactions catalyzed by the two proteins, we speculated that their active site might be at least partially conserved. Indeed, a high degree of structural conservation of the residues important for MoeA catalysis and highly conserved in COG1058 was evident (Figure 9B). They comprise the MoeA acidic triad Glu188, Asp228 and Asp259, which has been predicted to be involved in the catalysis by coordinating the divalent cation required for the MoeA-catalyzed reaction, as well as Gly251 and Gly252 of the SSGGVS motif, which in the MoeA-MPT model is located in proximity of the phosphate group of MPT [30], [31], [33]. As shown in Figure 9B, these residues have the same structural location as Glu11, Asp44, Asp75, Gly67 and Gly68 of the 3KBQ structure. With the exception of Asp44, which is replaced by an asparagine in the plant proteins, the superimposed 3KBQ residues are highly conserved in all COG1058 members (Figure 8 and Figure S2). The importance of the charged conserved residues was experimentally validated by performing site-directed mutagenesis on the A. tumefaciens enzyme. Three mutants were generated by replacing the At COG1058 residues Glu21, Asp54 and Asp 85 with alanine. A D54N mutant was also obtained, to determine whether such substitution, which occurs in all plant proteins, would affect the catalytic activity. All four mutants were purified and assayed for the ADPR pyrophosphatase activity (Figure S3 ). None of them resulted to be endowed with a detectable enzymatic activity, confirming their essentiality for catalysis, and suggesting that the plant COG1058 subfamily is devoid of ADPR pyrophosphatase activity.
Multiple alignment of representative members of the COG1058 family (full version is available in Figure 2). Positions of residues conserved in all members of the family are highlighted at the top of the alignment in magenta. The residues highlighted in green are conserved in all proteins, with the exception of the plant subfamily. Residues are numbered according to the T. acidophilum protein. Proteins experimentally characterized in this work are marked by red stars. Residues mutated in the A.tumefaciens protein are marked with black asterisks.
A) Ribbon representation of superposed T. acidophilum COG1058 (blue) and E. coli MoeA (cyan) structures. The sulfate ion found in the COG1058 structure, likely indicative of the position of the active site, is shown as ball and stick; B) Superposed COG1058 and MoeA structures viewed from the top. The MoeA acidic residues predicted to be involved in catalysis and the two glycines of the conserved motif proposed to interact with the phosphate moiety of the MPT substrate are highlighted in orange, and their superposition to identical residues in the COG1058 structure is shown.
In this work, we identified the bacterial members of COG1058 as novel ADPRPs, endowed with structural and catalytic properties clearly distinct from those of the ADPRPs belonging to the Nudix hydrolase family. Besides possessing a completely different fold, COG1058 ADPRPs show unique Co+2- and K+-dependence, with an optimum pH at 7.5, whereas Nudix ADPRPs are either Mg+2- or Mn+2-dependent, with a more alkaline optimum pH [34]. Nevertheless, COG1058 ADPRPs display a catalytic efficiency comparable to that of characterized bacterial Nudix ADPRPs [35], [36]. In addition, both types of ADPRPs exhibit a peculiar tendency to occur in a fused form with enzymes involved in the recycling to NAD of its by-products, suggesting a common functional connection with NAD regeneration (Figure 1). Our discovery of a novel bacterial ADPRP family reinforces the relevance of ADPR in bacteria and suggests the existence of sustained ADPR-producing processes. This is also in keeping with the finding that in bacteria the overall intracellular ADPR content, including both the free and the protein-bound forms, is in the low millimolar range [22]. It is well known that free ADPR is a highly reactive molecule that causes non-enzymatic glycation of proteins, leading to loss of function [37]; on the other hand, recent evidence in both eukaryotes and bacteria shows that ADPR acts as a cellular signal [13], [38], [39]. In particular, in bacteria, it functions as a signal of NAD pool consumption promoting transcription of NAD biosynthetic genes through its binding to the NrtR repressor [39]. In this view, ADPRPs represent not just "housecleaning enzymes", merely scavenging potentially toxic ADPR, but also important players in ADPR signaling. In addition, their fusion to NAD-synthesizing enzymes suggests their direct contribution to NAD regeneration.
The genomic context analysis of the ADPRP(COG1058)/pncC gene shows that it is most frequently associated in predicted operons with the recombinase gene recA and genes coding for enzymes involved in various aspects of DNA/RNA metabolism, including RNA repair. Interestingly, in some δ-proteobacteria and in the Deinococcus/Thermus group, ADPRP(COG1058)/pncC and recA are associated in a polycistronic operon with the gene ligT, encoding the enzyme 2′-5′ RNA ligase, which is capable of joining in vitro yeast tRNAs splicing intermediates to form internal 2′-5′ linkages, and whose role in bacterial RNA repair has been recently postulated [40], [41]. In the Deinococcus/Thermus group, such operon is further extended to include a gene encoding a protein of the YgfZ family, known to participate in the repair of iron/sulphur clusters, hence involved in maintaining the activity of several Fe-S enzymes, including the enzyme MiaB responsible of the methylthiolation of tRNA [42]. The presence of the ADPRP(COG1058)/pncC gene in such operons might be indicative of the requirement of NMN deamidase and ADPRP activities during RecA-dependent processes likely involved in DNA/RNA repair. The former would scavenge NMN, a potent inhibitor of the NAD-dependent DNA ligase, ensuring, at the same time, NAD supply to the ligase reaction [10]; the ADPR activity, in turn, might be involved in the scavenging of free ADPR. Indeed, the occurrence in bacteria of ADPR-producing processes during nucleic acids repair has been recently supported by bioinformatic predictions [43]. In addition, in Deinococcus radiodurans cells a marked induction of the operon ADPRP(COG1058)/pncC-recA-ligT has been observed after irradiation or desiccation [41], [44], and a Streptococcus mutans strain deleted of ADPRP(COG1058)/pncC shows a significant increase in the sensitivity to DNA damaging agents [45]. Finally, the proposed role of the bacterial (COG1058)ADPRP in the scavenging of ADPR during DNA/RNA repair processes is in keeping with the marked increase of the ADP-ribosyltransferase activity observed in several bacterial species in response to DNA damage [46].
In this work we have identified as ADPRPs both the SoCOG1058 domain fused to NMN deamidase and the stand-alone AtCOG1058 domain. These results, together with those derived from the multiple sequence analysis performed on the whole COG1058 and confirmed by the mutagenesis experiments, enabled us to extend the assignment of the pyrophosphatase function to all COG1058 members, with the apparent exception of plant proteins that lack conservation of catalytic residues. COG1058 pyrophosphatase activity is in keeping with the high structural similarity of the proteins with the bacterial enzyme MoeA and its eukaryotic orthologues of the MoCo biosynthetic pathway, which catalyze a complex reaction also involving the cleavage of a pyrophosphate bond. In particular, the structural conservation of identical catalytic residues in T. acidophilum COG1058 and MoeA suggests a common catalytic mechanism. Given that the sequence conservation is limited to a stretch of 60 residues (30% identity), it can be hypothesized that the two families might have evolved by divergent evolution from a common ancestor [47].
The phylogenetic analysis of COG1058 showed that the eukaryotic members are evolutionarily closer to the more versatile AtCOG1058 ADPRP than to the strictly ADPR-specific SoCOG1058 enzyme, suggesting that the eukaryotic pyrophosphatases might have evolved a distinct substrate specificity. Considering that in higher eukaryotes the COG1058 domain occurs in a fused form with FAD synthase, it will be worth to investigate whether COG1058 eukaryotic pyrophosphatases might hydrolyze FAD as the preferred substrate.
Supporting Information
Figure S1.
Phylogenetic tree of COG1058. Color notations are the same as in Figure 7.
Figure S2.
Multiple alignment of COG1058 sequences.
Figure S3.
At COG1058 mutants characterization. SDS-PAGE (upper panel) of 8 µg and 0.8 µg of each purified protein. HPLC chromatograms (lower panel) of the reaction mixtures prepared as described in Materials and Methods, incubated for 10 min in the presence of 0.08 µg/ml of each protein. A control mixture, in the absence of protein, was also analyzed (thin gray line). AMP and ADPR standards were subjected to HPLC analysis in the same conditions (thin black line).
Table S1.
Sequences of oligonucleotides used as primers for cloning and mutagenesis.
Author Contributions
Conceived and designed the experiments: NR ALO. Performed the experiments: LC MDK LS FM GO. Analyzed the data: SR NR ALO. Contributed reagents/materials/analysis tools: LC SR LS MDK FM GO ALO NR. Wrote the paper: NR SR MDK ALO.
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text: On Thu 12 Mar 1998, Mark Baker wrote: > On Thu, Mar 12, 1998 at 11:01:22AM +0100, Paul Slootman wrote: > > > > > An exim-UUCP-Howto is IMO badly needed. > > > > > > If you want to write one I'll include it in the package. > > > > Maybe it would be sufficient to provide a UUCP option when configuring > > exim? > > Maybe. If anyone wants to supply a diff to eximconfig I'll consider > including it. Hmmm, I'll have a look (although I gave up my uucp link a couple of months ago). > The initial menu from eximconfig already only just fits a 25 line screen, so > I don't really want to add another main entry. Actually, the introductory paragraph scrolls off the screen... (see the wishlist bug I reported today). > > I myself have tried out exim recently after being a smail user for a > > long time (9 years, I believe). It's great! I.e., another vote for > > making it the standard debian MTA. > > Did you use eximconfig? If so, how did you get on with it? As I already have > a working, and moderately customised, configuration, I haven't actually run > it myself except to test, so I'd very much like to hear of any problems with > it, particularly if exim is going to be the standard in 2.0. Hmmm, I think it went OK; I had to hack the generated config file anyway because I needed to do some rewriting. AFAIK the "normal" stuff didn't need any extra hacking. I'll be upgrading my main system at work today or tomorrow (1.3.1 -> hamm), so I'll see how that goes. Paul Slootman -- home: firstname.lastname@example.com | work: firstname.lastname@example.com http://www.wurtel.demon.nl | Murphy Software, Enschede, the Netherlands Support Randal Schwartz! See http://www.lightlink.com/fors/ or send empty email to email@example.com -- E-mail the word "unsubscribe" to email@example.com TO UNSUBSCRIBE FROM THIS MAILING LIST. Trouble? E-mail to firstname.lastname@example.com

subset: commoncrawl


text: Lets say we end up trading out 3rd to Washington and draft Quinn. (though im not sold on the fact we would have to if he were there at 6) Teddy Ginn keeps sliding down the first round. Lets say Ginn is still on the board in the ~25 or later range. Would you trade both 2nd rounders to move up and grab him? So our whole first day draft would be Brady Quinn & Ted Ginn? ** I didnt look at draft chart numbers I personally wouldn't mind that case. However, if we don't get Quinn I'd like to see 1 Levi Brown 2a Ryan Kalil 2b Drew Stanton 3 Scott Chandler (maybe too early?)


text:
BISHOP - Top-ranked Georgia won all four events to post a 16-3 win over Tennessee-Martin at the Georgia Equestrian Center.
Three Georgia riders posted MVP rides, including Haylie Jayne in Equitation Over Fences, Lauren Love in Western Horsemanship and Sarah Locker in Reining. Most impressive was the Bulldogs' 5-0 sweep in Horsemanship.
In addition to Love's winning ride, other scorers included Kelley Mundrick, Alicia Shrum, Katelyn Van Allen and Dana Kennedy.
Georgia next competes in the Southern Equestrian Championships on Friday against South Carolina, and the winner will face top-seeded Auburn on Saturday.
© 2017. All Rights Reserved. | Contact Us


text:
This final note from Athens, Ga., today. It's worth a mention that there are a bunch of other Athenses in the United States. Athens, Ala., Ohio and N.Y., to name just a few.
Couple of years ago, the original Athens helped fly the mayors of all those places to come for a visit. Heidi Davison was the mayor of Athens, Ga., then. She spotted trouble right away.
Heidi Davison: We started walking around and there were all these big huge black bags on the streets. And we were like 'what is all this stuff?' And we went back to the desk clerk and asked what is all this stuff and she said, 'oh we've had a garbage strike for three weeks.'
She says the trip was an enlightening experience.
Davison: We've never had a garbage strike. I don't think we've ever had a strike, which is good, I'm glad. Because I don't think our police are set up for riots. I don't remember riot gear being in their budget.
Davison also told us, by the way, the plan was for a Greek delegation to make a return trip here. Never happened, Davison says.


text:
National League Standings, May 7, 1972
Bob P
June 1, 2004
Tony, you are correct! Agee's two-out homer in the bottom of the tenth after Bud Harrelson reached on an error gave the Mets an 8-6 win in this game.
The Mets were down 6-0 going to the bottom of the eighth but they scored five in that inning and one more in the ninth before Agee won it in extra innings. The Padres made six errors in the game, including four over the last three innings when the Mets scored all eight of their runs.
San Diego OF Johnny Jeter had two homers in the game.
Example One
The Mets suck! They need to trade Smith and get somebody like
This comment is off-topic and will be removed. It has nothing to do with
any specific game. But here's an acceptable alternative:
Example Two
The Mets suck! Smith made three errors in this game, and hit into a
double play, and the Mets blew a 5-0 lead. They need to get rid of Smith
and get somebody like Jones.
See the difference? Here you're getting the same point across, but it's in
the proper context. We wouldn't consider this message to be off-topic, and
we would let it remain.
We do appreciate anybody who takes the time to share their thoughts on our
site, and we hate to remove anybody's postings. But if we didn't take
steps to ensure that only on-topic messages were retained, The Ultimate
Mets Database would become a confusing jumble of unrelated comments,
and would thus be less enjoyable to visitors like you.
Thank you!


text:
This post brought to you by White Cloud. All opinions are 100% original of the Handmade Home.
Hey guys, happy weekend! Jamin here again, and today I want to share with you something really cool.
White Cloud is teaming up with Children's Miracle Network(CMN) to create miracles in the lives of children receiving treatment in its 168 U.S. member hospitals. These kids have put their creativity and imaginations to work by illustrating a template that may become a new White Cloud Facial Tissue box design!
As a person who spent some time in a hospital as a child I love this contest, simply because it gives kids something to dream towards. A way to get outside the walls of the hospital with their imagination, to create and maybe even inspire, not to mention how cool will it be for them to see their design on a tissue box in Walmart. (Find a store near you!)
I guess you can say I have a soft spot for children in hospitals. I remember the nurses and volunteers that found ways to focus me on things outside of my sickness, to help me find a happiness outside of my immediate surroundings. We had wheelchair races, art projects and even attempted baseball in the hallways. I truly believe they played a great part in the healing process.
So here's how you can participate in this cool aspect of the healing process. Simply go to White Cloud's Facebook page and use the Voting App to vote for the design you like the most and White Cloud will make donations to their hospitals to help the CMN continue the amazing work they are doing.
Also, fans who vote for the designs and partner with White Cloud and the CMN will be offered a chance to enter into a sweepstakes. Three drawings will be held for $25 Walmart gift cards throughout the voting period and at the end of the voting period, three grand prize winners will be selected to win a one-year supply of White Cloud products. {Fans can gain additional entries into the sweepstakes by sharing a link to the contest with their friends.}
We urge you guys to go check out this great cause!
Happy Voting and Happy weekend everyone!
Abbreviated rules : No purchase necessary. Void where prohibited. The Children's Miracle Network Contest and Sweepstakes are sponsored by Kruger Products (USA), Inc. The Contest is open to legal residents of the 50 United States and the District of Columbia, between the ages of 4 and 14, who are eligible patients at one of the 168 participating Children's Miracle Network Hospitals. The entry period for the Contest begins on November 29, 2012 and ends on December 28, 2012. The Voter Sweepstakes is open to legal residents of the 50 United States and the District of Columbia, 18 years of age or older. The Voter Sweepstakes begin at 6:00 p.m.ET on January 3, 2013 and end at 11:59 p.m. ET on January 30, 2013. For official rules, visit https://mywhitecloud.com/tissue…
Read our full disclosure here.


text:
If you haven't heard of Redbox, it's essentially Blockbuster's worst nightmare. It is a little kiosk that you might see around town, in grocery stores, outside pharmacies, etc., that offer $1 a night movie rentals. Plus they have a decent selection, making them a key player in media distribution, but also a threat to DVD sales.
Paramount and Redbox have just announced that all of Paramount's upcoming movie releases will be offered on Redbox the same day as they go on sale in retail stores. The reason for this deal? Dennis Maguire, Paramount's Home Entertainment's worldwide president had this to say on the subject:
"By granting Redbox day-and-date availability we are allowing the consumer a choice of how to consume our movies while maximizing the profitability of our releases in the home entertainment window,"
And when asked about the cannibalization of sales, he claimed that Redbox "had minimal impact on our DVD sales," thanks in part to a 10-month trial period where Paramount watched the sale of DVD's versus the growth of Redbox.
Read [Business Week] Via [Mashable]


text:
TTC journal- Starting in Nov. 2011
I realized that I was inundating thread with all the gritty details of ttc so I thought I'd just start something here to keep track.
Stats (as of 11/11/12):
Today is CD14. We are ttc#2 but this is our first REAL cycle.
I am taking prenatals and EPO. We have been Bding every day since CD8.
My cm has been creamy and getting more plentiful over the past several days. Today I finally found the first traces of EWCM. It's still mixed with lots of creamy cm (or semen?) but it's definitely EWCM so I will prob be Oing today or tomorrow which means lots of BDing.
Today I had a significant BM (I know, ew...) so I don't know if that's linked to Oing or not as I have the same thing happen right before my period almost every cycle.
CD16- So now I'm thoroughly confused. Each cycle I usually have a ton of EWCM to tell mw when I O but this time I only had the teeniest amount of mucus looking substance- just a couple smudges worth. And now it seems like my cervix is slowly getting firmer. If I Oed already, then I think I'm golden because we BDed almost every day, but just in case I'm Oing late, we're going to BD every other day for a few days. I definitely don't want to miss it after all that "work". :)
CD19(5dpo)- I have accepted at this point that I have Oed. My cm has become much less abundant and very tacky. The good thing about that is that DH and I "did our duty" to get a tiny baby bean here, now it's up to the universe. That being said, my body is doing some odd things but I know that it can't possibly be anything real because I'm only like 4 or 5 dpo. I'm getting little pokes and tugs in my abdomen and my breasts are sore already (they usually get sore right before AF, but not before). FXed
CD20(6dpo)- I got some spotting today. A swipe of red and some pink behind. No panty liner or anything needed.
CD21/22(7-8dpo)- Gassy. Feeling very positive about pregnancy. I'm sure that I am, with the IB. Not too many people actually believe me.
CD23(9dpo)- I've had some cramps all day. They weren't awful most of the day but starting at about 4ish pm they started getting pretty intense. I thought at first that they were gas pains but now I'm sure they're not. The worst ones are located right beneath my c/s incision scar (right above the pubic bone).
Re: TTC journal- Starting in Nov. 2011
hey seamama have u got ur bfp??
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text:
Carb Cleaner Increase the Performance and Power associated with Your Nissan
Typically the Mass Air Flow (MAF) sensor inside your car motor is one of those pieces that remain hidden coming from sight, tends to be ignored during the program of a normal providing procedure, and yet performs an integral part within ensuring that your vehicle performs optimally. The MAF sensor is in charge of figuring out the volume of atmosphere that is being taken into the car engine. When it becomes dirty, mostly from microscopic pollen particles and soot, it leads to the gearbox to failure, error codes being thrown up, and loss of the fourth gear. Therefore , when you notice the drop in gas distance or your car using less smoothly, you could be almost certain that you will certainly need to clean the mass air flow sensor.
Removing the Sensor
To clean the mass air movement sensor, you will need isopropyl alcohol consumption in 70/30 proportion in addition to a zip lock bag. You might also need a sizable crosshead or a flat blade screwdriver (depending on the model of your car) to remove the MAF sensor. The sensor will be found nestled away inside air outlet trunk. To clean typically the mass sensor, remove this completely from the container taking extra care since you detach the hoses attached to it. Deal with the sensor carefully since its internal wires are extremely fragile and may break in case you accidentally drop it on some hard area (such as your garage's) or knock it against any object. Remove virtually any bolts attached to it before you embark on your own task to clean MAF sensor.
Cleaning this Automobile Part
Now put typically the sensor inside zip lock bag and douse that with the contents of a full bottle of isopropyl alcohol. Then gently swish the bag to make sure the alcohol gets directly into every hole and break of the sensor. Maintain the sensor seated in typically the alcohol for about 20 minutes or till a person are absolutely sure that every parts of it are completely clean. Afterwards, take out there the sensor and spot it on a dry out piece of clean cloth. Once you clean MAF before you re-order it, you must make sure this engine part is absolutely dry. If it is usually not, water and/or alcoholic beverages can seep into some other parts of the engine in addition to wreak havoc with other sensitive vehicle components.
An additional Method
There is work out clean MAF sensor. You may use a brake or carb cleaner on the cables of the sensor. Just apply several drops of typically the cleaner on these surfaces and before long, the wire connections will look squeaky clean. The particular cleaner of course, evaporates. Make sure it is dry nevertheless before installing this sensor back in to your car.
When you clean the MAF and before you re-order it, also squirt a few drops of oil about the rubber O-ring, merely to make sure that the component can function smoothly plus without creaking and groaning.
Attention to Fine detail
Thus, now that you understand how easy it is to clean MAF, make it a point to once in a new while open the hood and peer inside to be able to check that it is usually clear of dirt and muck.
According to : www.carcareproducts.cn.com which is a carb cleaner supplier


text:
Notes: My first batch tasted too heavily of vanilla (and alcohol) so I halved the teaspoon that is called for and changed the vanilla type. As I was using the Enjoy Life Mega Chunks, I only put in one and a half cups; they are just so big and chunky you don't need two and a half cups as the original recipe on Food52.com calls for. I tried a batch with cinnamon; I liked the depth they created, but didn't love the heat that lingered. I tried a batch without and then realized that it does need something, otherwise it's just straight chocolate flavor. I might try cayenne next time, or adding half chocolate half dried fruit. But that's the extent of my substitutions. You can see brand names I used below; use whatever works best for your food allergic needs.
Here is the result right out of the oven to cool - don't touch!
|Food52.com Divine Gluten-Free Chocolate Cookies
Fallen Meringue Chocolate Cookies: Tree Nut-Free, Gluten-Free
(Recipe originally posted on Food52.com by Jestei further adapted by me)
2.5 cups Domino's powdered sugar
½ cup Hershey's unsweetened cocoa powder
½ teaspoon cinnamon
¼ teaspoon kosher salt
3 egg whites (Organic Valley)
½ teaspoon vanilla extract (Nielsen Massey is nut-free and gluten-free)
1.5 cups Enjoy Life Mega Chunks
Preheat the oven to 350F and line baking sheet with parchment paper.
In a large bowl of standing mixer or large bowl with hand held mixer, mix powdered sugar, cocoa powder and salt on low. Stir in the egg whites with a stand or hand mixer until the batter is well mixed. The batter will look shiny and silky. Stir in the vanilla extract and chocolate chips by hand.
Using a teaspoon, blob batter onto the baking trays, about an inch apart. They will spread a bit.
Bake until the cookies are cracking on the surface, about 15 minutes.
Let cool for ten minutes on the trays then carefully remove to cooling rack. This is a crucial step, you cannot move them if you don't let them cool first, they will just break apart and you will have to eat the broken cookies.
Yield: 24 - 36 cookies depending on the size spoon you use scoop raw batter.


text:
Artist description:
Hi there! I worked on this all afternoon. I wanted to thank all of you at RMD (artists and behind the scenes people) for being so encouraging and friendly! RMD is a really fun place to be!!! ((((Hugs All)))) :-D
Java Draw Created Aug 05, 2010 338 x 470px Unless noted Copyright 2010 AuroraSkye.
We encourage you to submit a comment and let the artist know what you think of this drawing. Please log in or join to post a comment.
Post a comment
I love all your eggs!
You deserve it :D 5/5
Nice u deserved to win :)
So colorful so nice! ^_^ 5h
idk u but thats real sweet :D thx
hugzzzzzzzzzzzzzzzzzzzzzzzz back at you
awesome egg! and congrats! thoroughly deserved!! :D 5/5
Very nice!
a very thoughtful draw,and really colorful.5/5


text:
Memorial Day observances on Monday in Cheyenne
Two ceremonies on Monday will help local residents observe Memorial Day.Members of St. Mark's Episcopal Church will read the names of coalition forces killed in Operation Enduring Freedom and Operation Iraqi Freedom.
The reading starts at 7:30 a.m. Monday and will end around 5:15 p.m. at the church, 1908 Central Ave.
The reading will honor more than 6,400 men and women who have died in the two conflicts.
Listings of the war dead will be hung on the interior walls of the church.
"We do it as a way to honor the fallen," said John Peacock of St. Mark's. People can drop by the church to listen.
The annual Memorial Day wreath-laying ceremony will begin at 11 a.m. Monday at Beth El Cemetery on Pershing Boulevard.
The wreath-laying ceremony is open to the public. Veterans of Foreign Wars Post 1881 and American Legion Post 6 sponsor the event.
Monday's ceremony marks the 54th year for the joint event, said Phil Harris of VFW Post 1881.
"We just feel that it is a way we can honor our friends and neighbors in Cheyenne who have died during war and peace time and are buried in the cemetery," Harris said.
The ceremony is a way of "taking a few minutes of the day to pay tribute to the men and women who are no longer with us," he said.
The ceremony also honors those who are willing to stand ready today, he added.
"This is the moment that we take to remind ourselves as veterans and the public that there was a sacrifice for these freedoms we enjoy," he said.
Representatives from area organizations — like veterans and community service groups — will place wreaths at a memorial at the cemetery.
Lt. James Washington will be the guest speaker. He is the commanding officer of the Navy Operational Support Center in Cheyenne.
U.S. Sen. Mike Enzi, R-Wyo., also will speak.
The National Guard Museum will be open from 9 a.m. to 2 p.m. Monday, as well. The museum is located next to Beth El Cemetery on Pershing Boulevard.
(Becky Orr firstname.lastname@example.com)


text:
The vision at Van Michael Salon is to create a "raving fan experience for each and every client.
"Pure energy" describes the atmosphere of the Van Michael Salon where design and business have merged to create one of the most successful salons in the country. Van Michael Salon offers three different salon experiences; Van Michael Salon, New Talents Salon and The Aveda Lifestyle Store.
Van Michael Salon is a full service Aveda Concept Salons that offers the latest in haircuts,.. styling, and color. Services include signature Aveda treatments such as the Aveda herbal scalp massage; stress relieving neck massage, hand massage & finishing touchup.
The salon also offers personal color consultations, the full line of Aveda complimentary value-added services, and the entire Aveda retail line of products. The highly trained Van Michael Artistic Team provides all services.
Van Michael Salon offers two different experiences: The Van Michael Salon, which is home to the senior staff or New Talents Salon for emerging artists fresh to the industry.
New Talents, very successful in its own right is Van Michael Salon's training salon. New Talents, a name trademarked in 1992 are quality, value oriented sister locations of Van Michael Salon. The name says it all; New Talents are where the newest team members hone their craft. Voted one of Atlanta's best salons and most affordable salon by Atlanta Magazine.
I've been a Van Michael customer for 15 + years. Nice atmosphere. Friendly and professional service.
Posted: 5/15/2008 • Link to this review
Highly recommend this spa and salon. The service is above any I have been to.
Posted: 4/06/2008 • Link to this review
Went to Terry at Van Michaels on Sandy Springs Circle to get my hair highlighted. She put very dark brown 3 inch stripes in my blonde hair. It was such a disaster, they couldn't fix it and they refunded my money. I went to Jamison Shaw and over the course of 6 weeks and they were able to fix it.
Posted: 2/05/2008 • Last edited: 1/09/2009 • Link to this review
Reviews: 9
Madison just did a fantastic job on my hair. I wanted to go lighter and still look natural and she did a wonderful job. So accomodating and friendly! Highly recommended! Hardin is great for cuts as well. My hair is very fine and hard to really "style." She cut 3 inches off the last time and my hair never looked so healthy!
Posted: 1/16/2008 • Link to this review
I get my hair colored by Carol and she is the best! I'd definitely recommend her!
Posted: 10/29/2007 • Last edited: 1/02/2008 • Link to this review
Robin always does a great job, and she keeps notes of your prior visits so she knows what you like and don't like. The scalp massage is to-die-for.
Posted: 10/20/2007 • Last edited: 2/12/2009 • Link to this review
I went to Van Michael for the first time recently and had a wonderful experience. I just switched to Van Michael after going to a different salon for over 5 years. Van Michael is GREAT! I saw Kat. She really listens to what you want your hair to look like.
Posted: 10/09/2007 • Last edited: 1/02/2008 • Link to this review
Reviews: 15
Great haircut for a reasonable price. I love the little extras like the scalp and hand massages. I have been to several different stylists over the years and Ian is my all time favorite!
Posted: 10/08/2007 • Last edited: 1/02/2008 • Link to this review
Reviews: 55
I have seen Len since I moved here, and yes his cuts cost $70 but he is one of the most experienced guys in the place! The traffic around this location can be a nightmare, but where in Atlanta isn't traffic an issue? I love AVEDA so I will be a regular at this salon, although the receptionists can be snotty. I called them out and the manager was quick to make it up to me.
Posted: 10/08/2007 • Last edited: 1/02/2008 • Link to this review
I've been a client of Van Michael (Sandy Springs) for more than 10 years and would not get my hair cut anywhere else. Harry and his team are consummate professionals, and will do whatever it takes to ensure that you are satisfied with your haircut. It is not inexpensive, but their skillfulness, professionalism, and personal touch make it very worthwhile.
Posted: 9/24/2007 • Last edited: 1/02/2008 • Link to this review
Reviews: 1
Great value for the services you receive. Call early for an appointment!
Posted: 9/24/2007 • Last edited: 1/02/2008 • Link to this review
if you are into snobbery, this is a great place for you
Posted: 6/21/2007 • Last edited: 1/02/2008 • Link to this review
I must confess that I would not have come back to kudzu but i think that it would not have been fair of me to not tell the truth. for the amount of money that I paid for what I recieved well I was shocked with the attitude. If I had to return I would rather have a more mature hair stylist than the MEW TALENT.
Posted: 6/17/2007 • Last edited: 1/02/2008 • Link to this review
Reviews: 4
Christopher is your go-to guy if you need a haircut from someone who understands wavy hair. Curly haired girls know, that is hard to find. Christopher is a keeper!
Posted: 6/16/2007 • Last edited: 1/02/2008 • Link to this review
I love this salon and they did wonders for my very curly hard to maanage hair. I would recommend them to anyone!
Posted: 6/02/2007 • Last edited: 1/02/2008 • Link to this review


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Behringer SD8 Digital Stage Box
Behringer SD8 Digital Stage BoxDetails
The Behringer SD8 Stage Box provides 8 MIDAS-designed mic preamps, 8 balanced analog XLR returns at the stage end, plus an integrated 2-port Ultranet distribution hub. Designed to be the perfect solution for connecting onstage performances with the Front of House console and the rest of the world, the SD8 is extremely compact and is ideal for both permanent installations and mobile applications.
Product Ref: 67043View Full Description
Buy on finance from £10.89 per month Finance from £10.89 per month
Please note: This is only available where all items in the order are dispatched in a single delivery.
To make it as easy as possible for you to get the gear you want, we offer a range of instant finance options. If you spend £250 or more online, you can choose to pay over 48, 36 or 24 months. The majority of decisions are given instantly, so you could get your new gear sooner than you thought!
Representative Example
To help you understand how our finance options work, the examples below are based on a total order value of £250:
|Deposit to Pay
|Total Payable
|Classic Credit 48 Months 14.9%
|No Deposit
|48 months: £6.83
Final payment: £6.83
No settlement fee
|Classic Credit 36 Months 15.9%
|No Deposit
|36 months: £8.65
Final payment: £8.65
No settlement fee
|Classic Credit 24 Months 18.9%
|No Deposit
|24 months: £12.41
Final payment: £12.41
No settlement fee
How to Apply
Applying for finance couldn't be easier - if your order totals £250 or more, simply proceed through the checkout as normal, then select Pay by Finance on the payment page, then click the orange APPLY FOR INSTANT FINANCE button. This will start your application with V12 Retail Finance, our finance partner, and completing this form should take approximately 10 minutes.
You should get an instant answer on whether your application has been successful, though in some rare cases V12 may need to look at the application in more detail.
Please note: This is only available on orders placed via the website consisting of a single delivery, and is not available in store or via phone currently.
Full DescriptionView Full Description
MIDAS - The Legend in Sound Quality
Ever since its formation in the 1970s, MIDAS has had a long history of innovation and leadership in the world of audio mixing consoles. MIDAS has earned their impeccable reputation due to their no-compromise approach to both audio and build-quality, and in particular for their Award-winning Mic Preamps. Behringer is proud to incorporate MIDAS designed preamps to ensure the ultimate in high-quality audio reproduction in both live and studio environments.
Convenient Connectivity
When used as part of a complete X32 system, up to three SD8 Stage Boxes can be combined via the built-in AES50 ports to create up to 24 bidirectional channels and 24 balanced XLR outputs. The built-in Ultranet distribution hub has the ability to provide power and 16 channels of audio to 2 Behringer POWERPLAY P16-M personal monitoring mixers per SD8, all via CAT5e cabling - and without the need for any additional hardware. The SD8 also features two programmable inputs that are compatible with Hi-Z sources, allowing you to connect keyboards, acoustic guitars and more - no DI box required.
Remotely Controllable Preamps
Setting up the SD8 is extremely easy, with virtually all parameter control taking place at the console - including the award-winning MIDAS-designed mic preamps, +48V phantom power, destination routing and more. All you have to do is place the SD8 in your desired position, run several CAT5e cables along with mic and instrument cables - and do the rest from the FOH console.
Klark Teknik SuperMac
Klark Teknik SuperMac - AES50-Compliant - technology delivers the high-capacity, ultra-low latency digital connectivity. Klark Teknik engineers worked hand-in-hand with Behringer to ensure the SD8 meets, and exceeds, the requirements demanded by todays discerning musicians and professional audio engineers.
- 8 MIDAS-designed, fully programmable mic preamps deliver audiophile sound quality
- 8 analog, servo-balanced XLR outputs
- AES50 network ports featuring Klark Teknik's SuperMac networking capability for ultra-low jitter and latency
- 2-port Ultranet distribution hub for powered connection of 2 Behringer P-16M personal monitoring mixers
- Digital audio and control connectivity for Turbosound speaker systems with Ultranet networking
- Rack ears and protective bumpers included for installed and mobile applications
- Dual AES50 ports for cascading multiple units - no merger or router required
- Remote operation via shielded CAT5e cable up to 330ft / 100m length
- USB connector for system updates via PC
- Ultra-compact and roadworthy, all-steel chassis
- Designed and engineered by Behringer Germany
The product price includes 4 years' warranty and 60 day money back guarantee.
However, the Gear4music extended warranty scheme provides an enhanced service, and is designed to offer a higher level of protection than many 'free' warranties offered by some retailers and manufacturers.
The highlights of this enhanced warranty service are:
- Warranty is extended to 4 years from the date of delivery.
- Our Money Back guarantee is extended from 30 days to 60 days (terms apply after 30 days)
- Collection and delivery costs are included for the first 2 years
- Claim 50% cash back on the cost of the policy after 4 years if it has not been used!
- Warranty is transferable if the item is re-sold
- Loan or replacement equipment will be provided if the product isn't repaired within 30 days
- If an item can not be repaired, a similar alternative product or cash discount (as defined in this policy) will be offered
- The policy can be cancelled within 30 days, and at any time after that for a pro-rata discount
Please note that this extended warranty is currently only available on products sold within the EU or EEA, and is subject to the terms and conditions as detailed on the extended warranty certificate provided.
For customers in outlying areas of the country, the price of the Extended Warranty may be marginally higher, due to the potential for higher collection and redelivery transport costs.
This enhanced warranty is in addition to the standard Gear4music and manufacturer warranty policies, and the Gear4music terms and conditions of trade. No statement or condition of sale contained within these terms affects or restricts your statutory rights as defined under the Consumer Contracts Regulation 2014.
Once the product is delivered, you have 60 days to decide that you definitely want to keep it. If you decide it is not suitable for any reason, you can return it back to us for replacement or full refund, including standard delivery charges.
All Products are new unless stated, and include a minimum of 2 years warranty.
Some items are only eligible for money back guarantee if they are returned to us unused and sealed in the original packaging, and in the case of software, unregistered. These items include:
- Software
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We are unable to accept returns of any custom made items designed to work together as a unique package, such as computer system built to a customer's personal specifications. Exceptions may only be possible with prior agreement of the vendor, and may be subject to a cancellation fee.
If you also purchase items that do not qualify for free delivery, some delivery charges may be applied to your order which will be shown in your shopping basket.


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Where to buy sequin dresses? If this is a question of you, i will suggest you eDressit.com New sequin dresses, prom dresses there, are elegant and affordable. They look fabulous and sparkling with A-line, sheath, mermaid, trumpet or vintage ball types. Browse our large selection of sparkly evening dresses for your perfect party. An elegant sequin evening dress can be worn to several events including prom, homecoming, graduation, summer parties, etc. This section is full of fashion sequin long dresses, vintage ball gowns, and short cocktail dresses in several colors and styles. Whatever the event you are looking for, we have your perfect dress with sequins for you.
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PALM BAY, Fla. (AP) -- A 4-year-old boy heard cries for help coming from Turkey Creek as he played in his backyard, but his parents didn't believe him at first.
Clay Uhing made a couple of trips inside the house before his parents finally went outside long enough to hear a distinct cry of ''Call 911!''
Clay's persistence paid off Tuesday and he is credited with aiding a woman and her 13-year-old grandson whose canoe capsized.
Beverly and Mike Huttinger of Palm Bay had managed to swim to shore but were soaked, shivering and unable to walk to safety because of the thick woods. They might have been stranded for hours if not for the tenacity of the pint-sized hero.
''It really was my son's insistence,'' said Clay's father, Jim Uhing. ''He kept saying somebody out there needed help.''
Members of the Palm Bay Fire Department retrieved Huttinger and her grandson by boat a short time later. Neither needed medical attention.
Huttinger, who was not wearing a life jacket when the boat capsized in the 50-foot-wide creek, cried when she learned a 4-year-old was responsible for their rescue.
She called Clay on the telephone and thanked him.
''I just thought it was so sweet,'' she said. ''I told him I thought he was really a smart boy to do that.''
© 2017. All Rights Reserved. Contact Us


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An 'amphitheatre of the modern world' is how this vast venue describes itself, although the action that takes place within the walls of its (52,000 capacity) outdoor stadium tends to be more musical than gladiatorial. Everyone from the late Pavarotti to U2 has played here at one time or another. But don't be surprised to find that Vanessa Paradis or Zucchero have been booked to play for the weekend that you're in town. It's nothing if not a mixed bag.
Area Nice
Telephone 04 92 29 31 29
Open Box office 1-6pm Mon-Fri.


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Community images
Bats & Balls
published 21/10/2011
ProGreat fun, great quality
published 05/05/2012
ProAlmost endless hours of fun, great value for money...
ConsThere are very few different games you can play wi...
published 17/08/2011
ProA good childrens game
ConsCan get a little tedious after a while
published 21/06/2010
ProCheap and fun
published 23/05/2011
Profun for all family, cheap
Consnot fantastic quality
published 04/04/2011
Profun for children and adults
Consball can be hard to remove, sometimes velcro pulls...
published 07/05/2011
ProEasy to play, inexpensive, amusing for a wide age ...
ConsSet is not very robust, difficult for younger chil...
published 26/07/2010
ProCan be used by anyone, anywhere.
ConsDoes slip on small hands.
published 16/07/2009
ProGood for those with poor co-ordination
Type: Bats & Balls
Age: 3 Years +
Listed on Ciao since: 15/07/2009


text: On Sun, Jul 28, 2013 at 12:04:55PM +0200, Magnus Therning wrote: > On Jul 27, 2013 6:51 PM, "Nicola Squartini" <tensor5 at gmail.com> wrote: >> >> Hi, >> >> For those of you interested in Haskell for the web, I created using >> cblrepo a new repository containing all the latest happstack, >> clckwrks and gitit packages. If you want to use it, add this to >> your pacman.conf >> >> [haskell-happstack] >> SigLevel = Never >> Server = ftp://noaxiom.org/$repo/$arch >> >> I believe that some of the dependencies now in haskell-happstack >> should rather go in haskell-core. Here is a minimal list: >> >> base16-bytestring >> haskell-src-meta >> syb-with-class >> time-compat > > Would you mind putting this request in a bug on github/habs instead > please? It reduces the risk that I forget to deal with it ;-) I noticed you did what I asked, which is great :) There is just one thing that I feel I need to point out before I add the packages, and I think it's better to highlight and keep any possible ensuing discussion here rather than bury it in a ticket on github. By adding these package in [haskell-core] you are relieving yourself of some of the work with packaging happstack, but you are also handing over some of the control. If any of the packages added to [haskell-core] is frequently updated on hackage and the happstack developers are a bit slow with reacting to new versions of its dependencies then you are likely to find that at times [haskell-happstack] isn't usable -- [haskell-core] has moved on, but [haskell-happstack] can't be rebuilt on top of it yet. We've had discussions in the past on how to deal with this, but nothing's really materialised (probably my fault). We've also not run into this particular problem terribly often with the only other auxiliary repo, [haskell-web]. So maybe it's just good that [haskell-happstack] comes along and forces the issue ;) Anyway, this isn't a big deal and I'm sure we can work something out if it becomes a recurring issue. However, I think you really should know up front what you are getting yourself into ;) /M -- Magnus Therning OpenPGP: 0xAB4DFBA4 email: magnus at therning.org jabber: magnus at therning.org twitter: magthe http://therning.org/magnus Perl is another example of filling a tiny, short-term need, and then being a real problem in the longer term. -- Alan Kay -------------- next part -------------- A non-text attachment was scrubbed... Name: not available Type: application/pgp-signature Size: 230 bytes Desc: not available URL: <http://www.haskell.org/pipermail/arch-haskell/attachments/20130729/09d60206/attachment.pgp>


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Erik Compton sees himself as more dreamer than role model, more of a grinder than an inspiration.
In reality, this 30-year-old golfer from Miami fits all those descriptions.
He's a 5-foot-9, 150-pound father who lives life like he's never heard the word "quit."
He's a survivor of two heart transplants who wants to make a living playing golf.
He's one of 156 players in possession of what he calls a Golden Ticket — a tee time this week at Pebble Beach to play in the U.S. Open.
Some of those tickets, of course, are more golden than others.
"I'm a dreamer," Compton said Monday after playing a practice round with Nick Watney and Ben Crane. "So I have dreamed that I could get another heart and I could come back out and play."
He is a little more than two years removed from his second heart transplant, both operations necessitated by a disease called viral cardiomyopathy, which inflames the heart and leaves it unable to pump as hard as it needs to.
When Compton was first diagnosed with the disease at age 9, he was the fastest kid at school, the best athlete, the one who played pitcher and shortstop and got picked first in the neighborhood football games.
His first transplant came when he was 12, but instead of settling for a sedentary lifestyle for their son, Peter and Eli Compton encouraged him to get back out there and try something.
Golf was the answer.
"To our surprise, he kept getting better and better," Peter Compton said.
The tenor of the conversations with other parents started to change. From, "Oh, isn't it great that Erik's out there doing things," to "When's your kid gonna stop beating our kid?"
Compton kept winning — won his way to a spot on the team at University of Georgia and a place on the Nationwide Tour and Canadian Tours in the early 2000s. Won the heart of his wife, Barbara, who knew nothing about golf when they met.
Three years ago, as Compton was wrapping up a day of fishing, he started feeling pain in his chest. His left main coronary artery — the so-called "Widow Maker" — was failing. He was having a heart attack. He drove himself through the detour-riddled streets of Miami and made it to Jackson Memorial Hospital, where doctors saved his life.
About seven months later, he had his second heart transplant. Shortly after, the Comptons had a daughter.
"When I was laying there in the ICU and after the transplant, I pretty much had come to grips that I wasn't ever going to play golf again," Compton said. "I sold all my golf equipment. I didn't have any status anywhere. I did not know that things were going to turn out, that I would be getting a heart as strong as I did as quick as I did."
Within weeks of the second heart transplant, Compton was training for Q-School, trying to find his way back to the PGA Tour. He fought for the right to play using a golf cart. But at U.S. Open qualifying earlier this month, he walked, which made the success that much sweeter.
The final day of sectional qualifying is 36 holes, which for Compton equaled a 10-hour trek across the Springfield Country Club in Columbus, Ohio. It marked the first time he'd done such a thing since his second heart transplant. He won one of three spots there in a playoff, and all that came one day after finishing four rounds at the Memorial, where he'd made the cut.
He called it one of his top three golf moments.
"Just to make it walking when I was in a fight to get a golf cart a few years ago was pretty special," he said. "To do it walking and competing against other guys that don't have the same issue."
According to the American Heart Association, Compton had one of 2,163 heart transplants in the United States in 2008, and the five-year survival rate for men is 73.1 percent. As of June 2009, there were 2,791 patients on the transplant list.
So, while he doesn't want to necessarily be viewed as a role model, Compton knows his presence at the U.S. Open is a great advertisement for what is possible. His message is that becoming an organ donor is a more than worthy cause.
"It gives people faith to know that if something like this could happen to him, and he could be here, then anything is possible," Peter Compton said. "It's a legacy. He's an inspiration for people."
At 2:31 p.m. Thursday, Compton will put the tee in the ground on the 10th hole and start what he hopes will be a four-day journey that might lead to bigger things. Golf, he freely admits, is his life. Life, as he has long known, is a gift.
"I'm 30 years old and the rest of the year is wide open," said Compton, who is depending on sponsor's exemptions to get into tournaments. "Obviously this is a great week, so I'm going to focus on this week. And I don't know where I'll be the week after next week. Hopefully, I play well here and I have a 10-year exemption."
He has overcome the odds before.


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Lind back in cleanup spot against Niemann
Lind back in cleanup spot against Niemann
Lind has spent the majority of the year hitting behind Jose Bautista in the No. 4 hole, but was recently demoted to eighth following a period of extended struggles.
Blue Jays manager John Farrell said he likes what he has been seeing from Lind in recent days, but the decision for a higher spot in the order ultimately came down to the veteran's past performance against Rays starter Jeff Niemann.
"He's had good success against Niemann," Farrell said of Lind, who has hit .464 (13-for-28) with three homers against Tampa Bay's right-hander. "I wanted to split up the right-handers as well as [Niemann] has been tough on right-handers throughout the course of the season, so that's where we're at today."
Lind began the season hitting just .186 with two homers and eight RBIs in 27 games. The was a slight improvement since dropping to eighth in the batting, but Lind still seems to be struggling to find his previous form.
The 28-year-old entered play on Monday hitting .214 with a homer and three RBIs in his past five games.
"I've seen a little bit better rhythm particularly in his setup where he has a little bit [of] movement in his hands," Farrell said. "Hopefully some RBIs on the West Coast, has a chance to build on it from there. I can tell you, we need him."


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" Brand: Avon / Type: Mascara / Suitable for: Eye "
I bought this mascara a few months ago specifically for us going to Glastonbury as I wanted a reasonably inexpensive mascara that was waterproof (incase of the usual downpours which never happened this year) and that would not irritate my eyes - it was on the recommendation of a friend and for around £8 I figured it was worth a try
It comes in several colours but I wanted plain old black! Firstly, yep - its waterproof... VERY waterproof... and VERY VERY sweatproof! If you are partial to going to the gym in full make up, then this baby isn't going to shift! I wore it in temperatures of 30 degrees+ and it was still in place after 14 hours of dancing. My lashes looked thicker, longer and most importantly for me, I didn't get that horrid 'clumped together' look that some of the cheaper mascara brands achieve.
The only downside is trying to get the stuff off!! You need ALOT of eye make-up remover and even when I thought it was all off, I could still feel traces of it on my lashes... Its best to put a remover pad on each eye for 30 seconds or so to encourage the product to melt before you start rubbing away! It also felt very gentle to wear - my eyes didn't water or itch so I should expect it would be good for contact lens wearers or people with sensitive skin
Its certainly something I shall buy again when this tube has ran out and is a bargain at the price, especially as Avon regularly have promotions on their Perfect Wear line


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Tete de Veau salad, pheasant in calvados cream sauce, a creme caramel the size of a soup plate and a complimentary tipple of apple liqueur all for 17 euros! What's not to like about that!!


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This year is the 20th anniversary of Dining Out For Life, the national fundraiser for AIDS service organizations. This year's event on Thursday, April 25, involves a variety of local eateries that will donate 25 percent or more of your bill to Saint Louis Effort For AIDS (SLEFA). More than 100 area restaurants are participating. For the full list, visit diningoutforlife.com/stlouis/restaurants. Since 1990, Dining Out For Life has raised more than $4.3 million for SLEFA.
Chef WIL FERNANDEZ-CRUZ, who recently left The Restaurant at The Cheshire after a short tenure, has landed the job of chef de cuisine of Ibby's, located on the Danforth campus of Washington University.
Piccione Pastry, an Italian-style pastry purveyor from the folks behind Butler's Pantry, is set to open in the U. City Loop at the corner of Delmar and Skinker boulevards next month, with an array of traditional treats on the menu.
Destination Desserts, a new gourmet bakery with a social conscience, has begun operations in Maryland Heights in the Center for Head Injury Services at 11786 Westline Industrial Drive. The bakery is designed to train, employ and advance the lives of people with head injuries and other cognitive learning conditions. Start-up costs were covered by a variety of grants, and all the proceeds from the bakery will go to support the center. Look for a Destination Desserts food truck to hit the streets sometime next month. Visit destinationdesserts.org to learn more.
Mike Shannon's Grill, the latest from the folks who brought us Mike Shannon's Steaks And Seafood downtown, opened its doors last week, just across the river in Edwardsville, Ill., where Cardinals broadcasting legend and co-owner MIKE SHANNON has had a home for more than 30 years.
The Wild Card Wine Trail will take place May 4 and 5 on the Hermann Wine Trail, featuring fine food and drink at seven wineries along the scenic 20 miles between Hermann and New Haven. This year's menus will feature bacon-inspired food and wine pairings. Tickets are $30 (hermannwinetrail.com).


text:
Close Up
On August 5, 2001, the Gloucester Fishermen's Wives Association (GFWA) dedicated a monument to honor the women who have been – and are – the soul of fishing communities.
As Angela Sanfilippo, GFWA president, so eloquently said at the dedication ceremony:
"The memorial serves as a testimonial to what wives, mothers, sisters, and children of fishermen of the world have endured because their men chose to be on the water. They had no choice but to stand on rock, to be on land."
Envisioned by the wives, mothers, daughters, and sisters of Gloucester fishermen, the memorial honors not only the faith, diligence, and fortitude of the wives of fishermen and mariners everywhere but also honors all women for their unselfish contribution to the well being of their families and their communities.
Check out the Gloucester Fishermen's Wives Association Website for more information: http://www.gfwa.org/memorial/memorial.html


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If you wish you can now relocate your glow plug ignitor to a new more convenient location allowing for re-starts without the need to remove body shells or reach inside with extended reach ignitors.
This special patent designed adaptor is suitable for all model engines used in car, boat, air planes and helicopters. The connector locks easily onto the glow plug from any angle and makes for a convenient and practical way to ignite your glow engine model.
Royal mail at £3.50, or Express at £6.95.
Full details at checkout.
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To see shipping costs simply add the item to your basket and choose your country from the drop down list on the view cart page.
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Our system will drop you an automated e-mail as soon as this product comes back into stock.


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Here's What Your Candidates Said About Home Schooling 2016
Click on your district on the map, below. Then click on the names of your candidates to see how they responded to the ICHE Candidate Survey on home schooling.
The Candidate Letter can be downloaded in MS Word format here.
District: 4
Follow the links below to see how individual candidates answered the questions.
Dem - Kristi Milan STATE SENATOR
Rep - Mary Souza STATE SENATOR
Dem - Patrick P Mitchell STATE REPRESENTATIVE, POSITION A
Rep - Lucas Luke Malek STATE REPRESENTATIVE, POSITION A
Dem - Tom Hearn STATE REPRESENTATIVE, POSITION B


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Leeds General Infirmary child heart treatment transfers claims looked into
A heart unit under threat of closure has been accused of failing to send some children to another centre for life-saving, complex surgery.
The Care Quality Commission (CQC) said it was looking into claims Leeds Infirmary resisted sending patients to Newcastle's Freeman Hospital.
Leeds Teaching Hospitals Trust has strenuously denied the claims.
A spokesman said it had not restricted referrals and was holding talks with the health watchdog.
A review of child heart surgery in the UK is currently taking place with some units facing closure, including Leeds.
'Best outcomes'
A statement from the Leeds trust said: "We strongly refute any suggestion that we would act improperly - either by restricting referrals or by failing to carry out surgery - where either of these actions was the right thing to do.
"Our principal concern has always been, and continues to be, a patient's health.
"Every decision we take in relation to children's heart surgery treatment or referral is based upon clinical judgement about the best outcomes."
The claims have highlighted cases in which families have said their children have received palliative care - treatment to relieve pain but not cure, which is often given to terminally ill patients - when they believed they could have recovered.
Nicola Garbutt, whose one-year-old grandson George Hall was born with an under-developed heart, claims the family had to fight to have him transferred by ambulance and treated in Newcastle.
The family, who live in Skipton in North Yorkshire, claimed doctors in Leeds told them little could be done for George but said after being treated in Newcastle he was doing well.
Ms Garbutt said: "We lived with a year being told he needed palliative care and if we had trusted Leeds it would still be palliative care with a life restriction.
"But now we've got George back and he is going to live a normal life."
'Protect reputation'
BBC Look North's health reporter Sharon Barbour has also learned two charities have raised concerns about the transfer of children from Leeds for complex heart surgery.
In a letter to the NHS, the charity HeartLine said: "The problem is that where a parent wishes to refer a child away from the care of Leeds General Infirmary they are being treated in a wholly inappropriate manner."
The letter mentioned four families who raised concerns after they requested their children be transferred out of Leeds.
One parent claimed she was told by staff in Leeds her child was inoperable but she said her child was later operated on elsewhere.
The letter adds: "Leeds is trying to protect its reputation as a safe surgical centre.
"It is sending complex surgeries to other centres - rather than Newcastle, or not risking surgeries - which may be unsuccessful."
Another charity, the Children's Heart Federation, has written to the CQC watchdog.
Its letter said: "We are currently aware of much concern regarding the paediatric cardiac care service at Leeds General Infirmary... with chilling information on specific current cases.
"We seek assurances that there are no unsafe practices at Leeds General Infirmary and that all children and their carers are being treated appropriately."
A statement from the CQC said: "We are aware of the concerns that have been raised and we are currently considering whether any regulatory action is required.
"CQC's priority is to ensure the safety and welfare of patients is being met at all times and we will not hesitate to take appropriate action to ensure this."
Last month the High Court ruled the consultation over changes to children's heart surgery in England and Wales - which recommended the Leeds unit close - was flawed.
Legal action was brought by campaigners trying to save operations at Leeds General Infirmary but the ruling could affect other units.