The propanediol utilization (pdu) operon ofSalmonella enterica serovar Typhimurium LT2 contains genes needed for the coenzyme B12-dependent catabolism of 1,2-propanediol. Here the completed DNA sequence of the pduoperon is presented. Analyses of previously unpublished pduDNA sequence substantiated previous studies indicating that thepdu operon was acquired by horizontal gene transfer and allowed the identification of 16 hypothetical genes. This brings the total number of genes in the pdu operon to 21 and the total number of genes at the pdu locus to 23. Of these, six encode proteins of unknown function and are not closely related to sequences of known function found in GenBank. Two encode proteins involved in transport and regulation. Six probably encode enzymes needed for the pathway of 1,2-propanediol degradation. Two encode proteins related to those used for the reactivation of adenosylcobalamin (AdoCbl)-dependent diol dehydratase. Five encode proteins related to those involved in the formation of polyhedral organelles known as carboxysomes, and two encode proteins that appear distantly related to those involved in carboxysome formation. In addition, it is shown that S. enterica forms polyhedral bodies that are involved in the degradation of 1,2-propanediol. Polyhedra are formed during either aerobic or anaerobic growth on propanediol, but not during growth on other carbon sources. Genetic tests demonstrate that genes of the pdu operon are required for polyhedral body formation, and immunoelectron microscopy shows that AdoCbl-dependent diol dehydratase is associated with these polyhedra. This is the first evidence for a B12-dependent enzyme associated with a polyhedral body. It is proposed that the polyhedra consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell that is related to the shell of carboxysomes. The specific function of these unusual polyhedral bodies was not determined, but some possibilities are discussed.
Salmonella entericaserovar Typhimurium LT2 degrades 1,2-propanediol by a pathway that requires coenzyme B12, adenosylcobalamin (AdoCbl) (29). Several lines of evidence indicate the importance of this process to the Salmonella lifestyle. 1,2-Propanediol is produced by the fermentation of the common plant sugars rhamnose and fucose (31, 34). Fucose is also found in the glycoconjugates of intestinal cells, where it is involved in host-parasite interactions (12). In vivo expression technology has indicated that 1,2-propanediol utilization (pdu) genes may be important for growth in host tissues, and competitive index studies with mice have shown that pdu mutations confer a virulence defect (14,25). The pdu genes are contiguous and coregulated with the cobalamin (cob) (B12) biosynthetic genes, indicating that propanediol catabolism is the primary reason for de novo B12 synthesis in S. enterica (2, 8,37, 41). If one includes the cob genes, S. enterica maintains 40 to 50 genes primarily for the transformation of propanediol. Moreover, nearly all natural isolates ofSalmonella tested synthesized B12 de novo and degraded propanediol (30). Some of these aspects ofSalmonella biology have been reviewed recently (40).
The pathway of 1,2-propanediol degradation has been investigated (34, 54). It initiates with the conversion of 1,2-propanediol to propionaldehyde by an AdoCbl-dependent diol dehydratase (1). Subsequently, propionaldehyde is catabolized to propionic acid and propanol, presumably by coenzyme A (CoA)-dependent aldehyde dehydrogenase, phosphotransacylase, propionate kinase, and alcohol dehydrogenase. This pathway provides a source of ATP, an electron sink, and carbon compounds that can be diverted to central metabolism via known pathways (27, 56). In addition,S. enterica can carry out the anaerobic respiration of 1,2-propanediol with tetrathionate as terminal electron acceptor (9). However, 1,2-propanediol respiration is not supported by the more common anaerobic electron acceptors, nitrate, fumarate, trimethylamine-N-oxide (TMAO), or dimethyl sulfoxide (DMSO) (9).
The genes required for 1,2-propanediol degradation cluster at thepdu locus on centisome 44 of the S. entericachromosome (29). This locus includes the pocR andpduF genes, as well as the genes of the adjacent and divergently transcribed pdu operon (10, 13). ThepocR and pduF genes encode a positive transcriptional regulatory protein and a 1,2-propanediol diffusion facilitator, respectively (10, 13, 37). The pduoperon is estimated to include about 20 genes (9). Those identified thus far are pduABCDEGHJ (10, 13, 58). The pduA and pduB genes encode a close and a distant relative of carboxysome shell proteins (13). ThepduCDE genes encode an AdoCbl-dependent diol dehydratase, and the pduG gene encodes a putative cobalamin adenosyltransferase (10, 58). The pduHJ genes were identified by genetic tests, but neither their DNA sequence nor their specific function is known (58). The DNA sequences of the pocR, pduF, and pduABCDE genes and a portion of the pduG genes were determined, and analyses of these sequences indicated that the pdu locus was acquired by a horizontal gene transfer (10, 13, 41). The regulation of the pdu operon has also been investigated. It is coinduced with the adjacent cob operon in response to 1,2-propanediol, and its induction is influenced by cyclic AMP levels, the redox state of the cell, iron, magnesium, pH, and perhaps the growth phase (2,8, 25, 37-39). In addition, recent electron microscopy (EM) studies have shown that S. enterica forms polyhedral bodies similar in size and appearance to carboxysomes during anaerobic growth on 1,2-propanediol (46). However, neither the composition of these polyhedra nor their role in 1,2-propanediol degradation has been investigated.
Here the completed DNA sequence of the pdu operon and its analysis are presented. In addition, results establish that polyhedral bodies are involved in AdoCbl-dependent 1,2-propanediol degradation. The first evidence for the association of a coenzyme B12-dependent enzyme with a polyhedral organelle is presented, and we propose that these organelles consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell that is related to the shell of carboxysomes. The specific function of these unusual organelles is not determined, but some possibilities are discussed.
Chemicals and reagents.Fumaric acid, tetrathionate, vitamin B12, MgSO4, CaCl2 · 2H2O, Na2SeO4, MnSO4 · H2O, FeSO4 · 7H2O, and sodium cacodylate were from Sigma Chemical Company, St. Louis, Mo. Formaldehyde, (r,s) 1,2-propanediol, pyruvic acid, Na2MoO4 · 2H2O, and CoCl2 were from Fisher Scientific, Pittsburgh, Pa. Glutaraldehyde was from Tousimis, Rockville, Md. Uranyl acetate was from E.M. Sciences, Ft. Washington, Pa. Osmium tetroxide and LR White resin were from Ted Pella, Inc., Redding, Calif. Yeast extract and Luria-Bertani (LB) broth were from Difco Laboratories, Detroit, Mich. Powdered milk was from Nestle, Glendale, Calif.
Bacterial strains, media, and growth conditions.The bacterial strains used in this study are derivatives of S. enterica serovar Typhimurium LT2, formerly S. typhimurium LT2 (Table 1). The minimal medium used was NCE (6, 57) supplemented with 0.01% yeast extract and DB minerals, which consist of 4.5 μM CaCl2, 2 μM Na2MoO4, 2 μM Na2SeO4, 2 μM MnSO4, 2 μM FeSO4, and 5 μM CoCl2. LB medium was the rich medium used (32). 1,2-Propanediol was used at 82 mM, tetrathionate was used at 10 mM, pyruvate was used at 40 mM, fumarate was used at 20 mM, and vitamin B12 (CN-Cbl) was used at 0.15 μM. Cultures were grown for about 24 h at 37°C in 5 ml of minimal medium supplemented with the appropriate growth substrates and inoculated with 0.1 ml of rich medium culture that had been grown about 16 h at 37°C.
Cloning and DNA sequencing.Two pdu clones, EM55 and TA417, were used as a template for completing the DNA sequence of the pdu operon. These were obtained by screening for clones that complemented a pdu mutation as follows. An S. enterica gene library was prepared by using strain BE26 (vector pEG5005) and the in vivo cloning method of Groisman and Casadaban (24). A transducing lysate was prepared from this library by using P22HT105/1int-201 and cells that were grown at 30°C on LB medium supplemented with 0.2% glucose and 0.02% galactose (18, 43). Plasmid clones were transferred to S. enterica RT822 (pdu-12::mudA) by transduction with all incubations carried out at 30°C. Kanamycin resistance was selected and transductants were screened for their Pdu phenotype on MacConkey–1,2-propanediol–B12 indicator medium (29). A red color indicated complementation of thepdu-12::mudA mutation by the plasmid clone. The procedure was performed twice. About 5,000 plasmid-containing transductants were screened, and two complementing clones were identified, pTA417 and pEM55.
Plasmid DNA was purified from strains containing plasmids pTA417 and pEM55 by using Qiagen tip 100 columns (Qiagen, Inc., Chatsworth, Calif.). Purified DNA was used as template for obtaining newpdu DNA sequence by primer walking. DNA sequencing was carried out by the University of Florida Interdisciplinary Center for Biotechnology Research DNA Sequencing Core Facility by using Applied Biosystems, Inc., automated sequencing equipment (Perkin-Elmer, Norwalk, Conn.).
DNA sequence analysis.Several DNA sequence analysis programs were employed, and, except where noted, default parameters were used. Genes were identified by using Genemark software with theS. typhimurium species option selected (11). BlastP and Ψ-Blast softwares were used to search the nonredundant (nr) database of the National Center for Biotechnology Information (NCBI) for protein sequences related to those encoded by thepdu genes (4, 5). ProDom was used for the identification of homologous protein domains (15). ClustalW and Blast2 were used for sequence alignments (4, 50). Phylogenetic trees were constructed by using the PHYLIP package of Felsenstein (21). Codon usage bias and G+C composition were analyzed as previously described (45). Edited multiple sequence alignments were obtained as follows: whenever a gap character was present in one or more sequences, that region of the alignment was deleted. In addition, overhangs at the ends of alignments were deleted. This editing procedure removes nonhomologous protein regions and generally improves phylogenetic comparisons (19).
EM.Two fixation protocols were used. In the standard protocol, cells were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 30 min at room temperature and then in 1% osmium tetroxide in the same buffer for 1 h at 4°C. The samples were then dehydrated through a graded ethanol series followed by absolute acetone and embedded in Spurr's low-viscosity resin. The modified protocol differed only in that samples were held at room temperature overnight in 75% ethanol containing 1% uranyl acetate during the alcohol dehydration series; this modification imparted extra contrast to the polyhedral bodies.
Specimens were thin sectioned on an LKB Nova or an RMC MT-6000-XL ultramicrotome, collected on Formvar-coated copper grids, post-stained with lead citrate, and observed and photographed with a Zeiss EM-10CA transmission electron microscope.
For immunogold localization of diol dehydratase, cells were fixed in 0.5% glutaraldehyde–4% formaldehyde on ice for 20 min (3). They were then dehydrated in a graded series of ethanol to absolute ethanol. Samples were embedded in LR White resin and polymerized at 50°C for 5 days (3). Thin sections were placed on Formvar-coated nickel grids, blocked for 20 min on 1% powdered milk in phosphate-buffered saline (PBS) at pH 7.2, and floated overnight at 4°C on rabbit polyclonal antibody to diol dehydratase from Klebsiella oxytoca diluted 1:1,000 with PBS. After washing on high-salt Tris-Tween buffer (3), grids were floated for 1 h on goat anti-rabbit antibody conjugated with 12-nm-particle-diameter colloidal gold (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.). Samples were washed with buffer and deionized water, and then sections were poststained with 0.5% uranyl acetate and lead citrate.
Nucleotide sequence accession number.The sequence reported here has been assigned GenBank accession no. af026270.
Cloning and sequencing of pdu DNA.Plasmids pTA417 and pEM55 were needed to complete the DNA sequence of thepdu operon, (see Materials and Methods). Plasmid pTA417 was used to determine the pdu DNA sequence through bp 18,781, and plasmid pEM55 was used to complete the DNA sequence of thepdu operon (bp 19,215). DNA sequence was determined for both strands, and all ambiguities were resolved by additional sequencing reactions with different primers. A total of 11,624 bp of previously unreported pdu DNA sequence was determined.
Identification of pdu genes.Analysis of previously unreported pdu DNA sequence with GeneMark software allowed the identification of 16 hypothetical genes,pduGHJKLMNOPQSTUVWX. The pduGsequence completed a previously reported partial open reading frame (ORF), ORF1 (10). In addition, the codon adaptation indices and the G+C contents for each codon position of the proposedpdu genes were consistent with those of expressed sequences (Table 2).
The S. enterica pdu locus on centisome 44 now includes 23 genes, pocR, pduF, andpduABCDEGHJKLMNOPQSTUVWX. We propose that the pduoperon ends at bp 19,215 and that the terminal gene of the operon ispduX. Seventy-eight base pairs downstream of thepduX gene is the end of a long ORF transcribed in the opposite direction. It is related to an Escherichia coligene of unknown function, yeeX. However, experimental evidence that the pduX gene is the terminal gene of thepdu operon has not yet been obtained.
With one exception, the pdu genes previously identified by genetic tests, but of unknown DNA sequence, can be correlated to thepdu genes identified here. The pduG genes identified here and previously were shown to be the same by complementation analysis (7). The previously identifiedpduH gene represented a region in the pdu operon identified by deletion endpoints (58), and hence can correspond to the pduH gene identified here. However, thepduJ gene previously identified was not correlated with thepduJ gene identified here, and this possible lack of correspondence will need to be addressed in the future.
Analyses of previously unpublished pdu DNA sequence supported previous work indicating that S. enterica acquired the pdu locus and the adjacent cob operon by a single horizontal gene transfer (10, 30, 41). The G+C content of the 16 putative coding sequences identified here averaged about 59%, which is significantly higher than the typical 54% average for native S. enterica coding sequences (Table 2). In addition, the G+C contents of the first, second, and third codon positions varied from that of native S. enterica coding sequences. On the other hand, the G+C composition of pdugenes is similar to that of the adjacent cob genes, for which there is considerable evidence of horizontal gene transfer (41).
Sequences similar to those of the PduGHJKLMNOPQSTUVWX proteins.BlastP software and Ψ-Blast software were used to search the nr database of the NCBI for protein sequences related to those encoded by the pduGHJKLMNOPQSTUVWX genes. Table 3 summarizes the results of those analyses and also includes information on previously sequencedpdu genes (10, 13, 41).
1,2-Propanediol utilization pathway genes.The hypothetical PduP protein identified here is related to a number of CoA-dependent aldehyde dehydrogenases. It is most closely related to the EutE protein of S. enterica. These two proteins are 45% identical in sequence over 465 amino acids. The EutE protein is a putative CoA-dependent aldehyde dehydrogenase proposed to function in the AdoCbl-dependent pathway of ethanolamine degradation (49). Accordingly, it seems likely that the PduP protein is a CoA-dependent aldehyde dehydrogenase used in the pdu pathway for the conversion of propionaldehyde to propionyl-CoA.
The sequence of the PduQ protein was found to be related to those of many alcohol dehydrogenases. PduQ is most closely related to the AdhE enzyme of E. coli (23); these proteins are 35% identical in sequence over 330 amino acids. The PduQ protein aligns with the carboxy-terminal portion of the bifunctional AdhE enzyme, which has both alcohol dehydrogenase and aldehyde dehydrogenase activity. In addition, the PduQ protein is closely related to a number of monofunctional alcohol dehydrogenases, including the hypothetical alcohol dehydrogenase involved in ethanolamine degradation in S. enterica, the EutG protein (49). Thus, we propose that the PduQ protein functions as a propanol dehydrogenase in the pathway of 1,2-propanediol degradation.
The PduW protein was found to be related to acetate kinases. It is 87% identical in sequence, over 400 amino acids, to the ackgene product of S. enterica serovar Typhimurium LT2. Thus, the pduW protein is likely a propionate kinase whose role in 1,2-propanediol degradation is the conversion of propionyl-phosphate to propionate.
Genes for the reactivation of AdoCbl-dependent diol dehydratase.The PduGH proteins appear to be involved in the reactivation of diol dehydratase, and the PduG protein may also be involved in the adenosylation of B12. The PduG protein is 92% identical in sequence, over 611 amino acids, to the DdrA proteinK. oxytoca, and the PduH protein is 87% identical in sequence (99 of 124 amino acids) to the DdrB protein of K. oxytoca. The DdrAB proteins are involved in the reactivation of AdoCbl-dependent diol dehydratase (33, 55). Inactivation of diol dehydratase occurs due to the breakdown of AdoCbl to an inactive form that has an upper ligand other than an adenosyl group (26,53). The DdrAB proteins are proposed to reactivate diol dehydratase by removing the inactive cofactor and replacing it with AdoCbl (33, 55). The PduGH proteins may have a similar function. In addition, the PduG protein has been proposed to be an adenosyltransferase on the basis of genetic tests (58), and it has high sequence similarity to a proposed cobalamin adenosyltransferase from Citrobacter freundii (17,44). Hence, the PduG protein may be bifunctional.
Pdu proteins of unknown function.The PduO protein may have resulted from the fusion of two genes. The sequence of the N-terminal 170 amino acids of this protein is 35% identical to that of ORFW ofC. freundii and is also similar to those of unknown ORFs from Clostridium, Pyrococcus,Bacillus, and Mycobacterium. The sequence of the C-terminal 121 amino acids of PduO is 37% identical to that of ORFY from C. freundii and also has similarity to ORFs fromClostridium, Klebsiella, andPseudomonas. ORFW and ORFY are arranged with genes involved in the AdoCbl-dependent degradation of glycerol by C. freundii (17, 44). Thus, the PduO protein appears to have a function that is common to AdoCbl-dependent degradation of 1,2-propanediol and glycerol.
The PduS protein was found to be 30% identical in sequence over 72% of its length to the glucose repression mediator protein of E. coli. However, the sequence of this E. coli protein is unpublished. It was submitted to GenBank by the E. coligenome sequencing project being conducted in Japan, and it is unclear how this function was assigned. The PduS protein was also found to be distantly related to a number of membrane oxidoreductases, and of these, it was most closely related to the RnfC protein ofRhodobacter capsulatus (42). The PduS and RnfC proteins are 28% identical (90 of 318 amino acids).
The PduL protein is distantly related to phycocyanobilin lyase (CpcE). Ψ-Blast reiteration 1 gave an Expect value of 2 × 10−24. Phycocyanobilin lyase catalyzes the covalent attachment of phycocyanobilin (a linear tetrapyrrole) to phycocyanin via a thioether linkage (20). Since cobalamins are tetrapyrroles, perhaps the PduL protein includes a pyrrole binding site.
The PduV protein was compared to the Prosite database and was found to have a domain closely related to the ATP and GTP binding motifs. Analyses of the other Pdu proteins of unknown function did not allow the identification of motifs similar to those of the Prosite dictionary.
Pdu proteins related to carboxysome proteins.Protein sequence similarity analyses showed that five Pdu proteins (AJKNT) are clearly related to those involved in the formation of polyhedral organelles known as carboxysomes and that two Pdu proteins (BU) are tentatively related to carboxysome proteins.
The PduAJKT proteins are clearly related to a group of proteins that includes the major shell proteins of carboxysomes (Table4). The percent identities shown in Table4 were determined from an edited multiple sequence alignment (see Materials and Methods). The edited alignment consisted of sequences 85 amino acids in length that aligned to the following PduA amino acids: ALGMVETKGLTAAIEAADAMVKSANVMLVGYEKIGSGLVTVIVRGDVGAVKAATDAGAAAARNVKAVHVIPRPHTDVEKILPKEL.
The PduN protein is closely related to the CcmL-CchB family of proteins, which are needed for the proper assembly and function of carboxysomes (36, 48). A phylogenetic tree constructed from an edited multiple sequence alignment indicated that the PduN protein is more closely related to homologous proteins fromSynechococcus, Synechocystis, and E. coli than to homologous proteins from Thiobacillus. The percent identities between the PduN amino acid sequence and sequences derived from other CcmL-CchB family members ranged from 28 to 53%. The tree was constructed from 1,000 bootstrap replicates, and the high bootstrap values (626 to 999) indicated a statistically significant branching order. The edited multiple sequence alignment (see Materials and Methods) used for tree construction consisted of a group of sequences 77 amino acids in length that aligned to the following PduN amino acid sequence: MHLARVTGAVVSTQKSPSLIGKKLLLVRGDEV AVDSVGAGVGELVLLSGGSSARHVFSGPNEAIDLAVV GIVDTLSC.
The PduB and PduU proteins were found to be distantly related to proteins involved in polyhedral body formation. The PduB protein was shown to be 29% identical in sequence (53 of 178 amino acids) to the EutL protein of S. enterica and to the Eut b2439 protein ofE. coli by BlastP analysis. The PduB protein is also distantly related to the carboxysome shell proteins shown in Table 4. When a Ψ-Blast search was conducted starting with Yrb2 protein (accession no. P46205), the 20 most closely related proteins identified were carboxysome shell proteins listed in Table 4. Reiteration with these 20 shell proteins indicated that they are related to the PduB protein, with an Expect value of 2 × 10−4.
The PduU protein was determined to be 57% identical to the EutS protein of S. enterica and 56% identical to the Eut b2462 protein of E. coli by BlastP analysis. The EutS protein is a putative carboxysome structural protein distantly related to the carboxysome proteins listed in Table 4.
Formation of polyhedral bodies by S. enterica. S. enterica formed polyhedral structures similar in size and appearance to carboxysomes during aerobic growth on minimal 1,2-propanediol–B12 medium (Fig.1A), but not during aerobic growth on glucose (Fig. 1C). Polyhedra were 100 to 200 nm in cross-section and appeared to consist of a proteinaceous shell and interior. The bodies formed by S. enterica are somewhat irregular in shape compared to the carboxysomes of Thiobacillus neapolitanus, suggesting some differences between these structures (Fig. 1A and B).
Electron micrographs of S. enterica grown aerobically on minimal, 1,2-propanediol, vitamin B12 medium (A), Thiobacillus neapolitanus grown autotrophically (B), S. enterica grown aerobically on minimal glucose medium (C), and immunogold localization of the S. entericaAdoCbl-dependent diol dehydratase (D). Bars, 100 nm.
Similar polyhedra were also formed during anaerobic growth on 1,2-propanediol–tetrathionate, but not during anaerobic growth on glucose or pyruvate-tetrathionate, indicating that polyhedra are specifically involved in the catabolism of 1,2-propanediol. It was expected that polyhedra would be formed during anaerobic growth on glycerol-tetrathionate. Although S. enterica does not degrade glycerol in a B12-dependent manner, glycerol induces expression of the pdu operon to high levels (8,37). However, polyhedra were observed in very few cells grown anaerobically on glycerol-tetrathionate. This suggests that 1,2-propanediol plays a role in formation of polyhedra in addition to induction of the pdu operon. Tetrathionate was used as a terminal electron acceptor for these experiments, because other terminal electron acceptors, such as fumarate, nitrate, DMSO, and TMAO, do not support anaerobic growth on 1,2-propanediol. Vitamin B12 supplementation did not affect polyhedral body formation either aerobically or anaerobically.
Role of pdu genes in polyhedral body formation.EM studies showed that pdu mutants either failed to produce polyhedral bodies or produced aberrantly shaped structures. During either aerobic or anaerobic growth in the presence of 1,2-propanediol, strain BE25 (pdu-12::mudJ) failed to produce polyhedra, whereas an otherwise isogenic strain formed these structures under similar growth conditions. These results show that genes of the pdu operon are necessary for polyhedral body formation, and strongly indicate that polyhedra have a role in the catabolism of 1,2-propanediol. Thepdu-12::mudJ mutation is a polar mutation that was previously shown to be located in either thepduD or pduE genes, both of which encode subunits of diol dehydratase (58).
An additional pdu mutant (strain RT818,pdu-8::mudA) was found to produce bodies that appeared to have a proteinaceous shell and interior, but were aberrantly shaped (Fig.2). Many were highly elongated and spanned the entire length of the cell. This shows that genes on either side of the pdu-8::mudA insertion are required for polyhedral body formation. Thus, multiple pdugenes are required for the formation of polyhedra.
Electron micrograph of the aberrant polyhedra (arrowheads) formed by S. enterica pdu mutant RT818 (pdu-8::mudJ). Bar, 100 nm.
Neither the catabolism of propanediol nor genes of thecob operon are needed for polyhedral body formation.Strains unable to synthesize AdoCbl de novo (Cob−) produced polyhedral bodies in the presence of 1,2-propanediol both aerobically and anaerobically regardless of vitamin B12supplementation. This showed that B12-dependent catabolism of 1,2-propanediol is not required for formation of polyhedra. Results also showed that the genes of the cob operon (which are coregulated with those of the pdu operon) are not needed for polyhedral body formation. Both strains BE43 and TT18117 formed polyhedra. BE43 contains a polar mutation in the first gene of thecob operon that prevents expression of the first 17 of 20cob genes, and TT18117 contains a deletion of the 17 terminal genes of the cob operon. In the above experiments, pyruvate or succinate was used as a carbon and energy source because expression of the pdu operon is relatively high during growth on these compounds (2, 8).
Association of B12-dependent diol dehydratase with polyhedral bodies.Immuno-EM indicated that the AdoCbl-dependent diol dehydratase of S. enterica is associated with polyhedral bodies (Fig. 1D). In the micrograph, antibody-conjugated gold particles (solid black circles) indicate the location of diol dehydratase. Clustering of the gold particles in the interior of the polyhedral bodies shows that the AdoCbl-dependent diol dehydratase is associated with the polyhedra and is consistent with the encasement of this enzyme within a protein shell. Because of differences in the fixation procedures, polyhedra are somewhat less distinct in the immuno-EM pictures than in the standard thin sections (Fig. 1). Immunogold labeling, similar to that described above, was also carried out with pdu mutants unable to express diol dehydratase. Little to no labeling occurred; the very small amount of labeling observed probably resulted from nonspecific antibody binding.
Visualization of polyhedral bodies by EM.The polyhedral structures were easiest to observe in older cells that had begun to lyse. Early-log-phase cells contained the structures, but they were obscured by the cytoplasmic contents of the cell. The modified EM fixation procedure with an overnight stain of uranyl acetate in alcohol enhanced the visibility of the structures. The cytoplasm of the fixed cells in the modified fixation process was less dense, and it was easier to visualize the sharp edges of the more darkly stained polyhedral bodies.
To better understand the physiology and molecular biology of AdoCbl-dependent processes, 1,2-propanediol degradation by S. enterica serovar Typhimurium LT2 was investigated. The DNA sequence of the pdu operon was completed and analyzed, and evidence was presented that polyhedral organelles are involved in AdoCbl-dependent 1,2 propanediol degradation. Analyses of previously unpublished pdu DNA sequence substantiated previous studies indicating that the pdu and cob operons were acquired by a single horizontal gene transfer event (30) and allowed the identification of 16 hypothetical genes. In all, 23pdu genes are proposed, and these genes fall into six classes: pathway, diol dehydratase reactivation, unknown, polyhedral body formation, transport, and regulation (Fig.3 and Table 3).
The pdu locus of S. enterica with a summary of the encoded functions. Genes thought to be involved in the formation of polyhedra are indicated above the genetic map. Genes thought to be involved in the pathway of 1,2-propanediol degradation and in the conversion of vitamin B12 (CN-Cbl) to coenzyme B12 (AdoCbl) are indicated below the genetic map. Arrows above pocR, pduF, and pdu indicate the direction of transcription.
With one exception, pdu genes corresponding to each enzyme of the proposed 1,2-propanediol degradative pathway have now been identified (Fig. 3). A pdu gene corresponding to a proposed phosphotransacylase was not identified. This enzyme might be encoded by one of the six pdu genes of unknown function, or perhaps the proposed CoA-dependent aldehyde dehydrogenase (PduP) is bifunctional or a kinase; a Blast-ProDom search showed that the PduP protein shares a domain with ProA proteins, enzymes that catalyze the reduction of glutamate-5-semialdehyde to gamma-glutamyl-5-phosphate.
Six hypothetical Pdu proteins of unknown function (PduLMOSVX) were also identified. Genetic tests have shown that the pdu operon encodes functions for the conversion of vitamin B12(CN-Cbl) to AdoCbl, the active cofactor of diol dehydratase. The PduG protein has been implicated in this process, but additional Pdu proteins might also be involved. Studies with several different systems have indicated that a decyanase, one or two cobalt reductases, and an adenosyltransferase are needed (22, 28). The PduL protein is a possibility, since related proteins are found in an operon that encodes an AdoCbl-dependent glycerol dehydratase (17, 44). Other possible functions for Pdu proteins are suggested by physiological studies. S. enterica grows via the anaerobic respiration of 1,2-propanediol with tetrathionate as a terminal electron acceptor (9). Hence, some Pdu proteins might be specific to the 1,2-propanediol–tetrathionate respiration. The PduS protein is a possibility, since it is related in amino acid sequence to several membrane-bound oxidoreductases. In addition, previous studies have indicated that the pdu operon encodes a protein involved in regulation of the prpBCDE operon (56). Hence, one of the Pdu proteins of unknown function may fulfill this regulatory role.
The polyhedral bodies formed by S. enterica during growth on 1,2-propanediol were also investigated. Based on results reported here and previous results, we propose that S. enterica forms polyhedral organelles involved in 1,2-propanediol degradation that consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell related to the shell of carboxysomes. During growth on 1,2-propanediol, S. entericaforms polyhedra that are proteinaceous in nature and that have sharp edges indicative of a shell (this study and reference46). Immunogold labeling indicated that diol dehydratase is associated with the polyhedra and that it is localized to the interior of these structures. DNA sequence analyses showed that the pdu operon encodes five to seven proteins that are related to those involved in the formation of carboxysomes (this study and reference 13), and genetic tests showed that genes of the pdu operon are required for polyhedral body formation. In addition, a pdu mutant was found that produced aberrantly shaped polyhedra. Thus, some pdu genes are required for proper organelle shape, while others apparently encode its basic structural components. Analogous Synechococcus mutants (that produce aberrantly shaped carboxysomes) were previously identified (35).
Although the polyhedral bodies involved in 1,2-propanediol degradation are apparently related to carboxysomes structurally, a functional relationship is uncertain. Carboxysomes are proposed to play a role in concentrating CO2 for RuBisCo, since mutations in shell genes result in strains that require high CO2 for autotrophic growth (36, 47, 48). On the other hand, the polyhedra of S. enterica function in AdoCbl-dependent catabolism of 1,2-propanediol, and this process has no known association with CO2. Previous reports, which identified the carboxysome shell protein gene homologues in the eut andpdu operons of S. enterica, discussed some possible functions for polyhedral bodies in the AdoCbl-dependent catabolism of ethanolamine and 1,2-propanediol (13, 40, 49). It was suggested that polyhedral bodies could be used to sequester toxic aldehydes formed both during 1,2-propanediol and ethanolamine degradation and channel them to subsequent pathway enzymes. It was also suggested that polyhedra might be used to protect diol dehydratase and ethanolamine ammonia-lyase from oxygen, a molecule to which both are sensitive (13). The finding reported here, that AdoCbl-dependent diol dehydratase is associated with polyhedra, is consistent with both of these hypotheses.
Although their precise function is unknown, the size of the polyhedral organelles and the number of genes involved attest to the substantial resources devoted to AdoCbl-dependent 1,2-propanediol degradation. Formation of these bodies must play an important role in S. enterica survival and niche establishment among the competitive flora of natural environments.
This work was supported by grant GM59486 from the National Institutes of Health, by the Florida Agricultural Experiment Station, and by a Research Project Enhancement Award from the Experiment Station.
We thank Tetsuo Toraya for providing the antibody used in the immunogold labeling studies.
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(1999) Biochemistry of coenzyme B12-dependent glycerol and diol dehydratases and organization of the encoding genes. FEMS Microbiol. Rev. 22:553–566.
(1995) Growth temperature-dependent activity of glycerol dehydratase in Escherichia coli expressing the Citrobacter freundii dha regulon. FEMS Microbiol. Lett. 100:281–286.
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(1980) In situ reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase. J. Bacteriol. 143:1458–1465.
(1997) Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon. J. Bacteriol. 179:928–940.
(1982) Biosynthesis of the cobalamin coenzymes. in B12. ed Dolphin D. (John Wiley and Sons, New York, N.Y), pp 145–168.
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(1997) Integration host factor is required for 1,2-propanediol-dependent transcription of the cob/pdu regulon in Salmonella typhimurium LT2. J. Bacteriol. 179:3797–3800.
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Type of face mask
A cloth mask blocks only some of virus particles. Yet, it does protect from droplets, e.g., from coughs or sneezes.
✔ This is the face covering recommended by the CDC for the general public.
I need to cover my face for the next...
How long during the day?
hrs/day
Price of one mask
$/mask
🔎 You need
mask(s)
💸 Total cost
💡 Remember to change the mask every 8 hours.
💰 Five masks are enough, even over a longer period of time, provided that you wash and reuse them.
Check out 40 similar coronavirus calculators (covid-19)
Vaccine production
Mask vs. no mask
Coronavirus Mask Calculator
By Dominika Miszewska, PhD candidate and Maria Kluziak
Why is wearing a mask important?
How does the mask calculator work?
How to wear a mask safely?
Are masks controversial?
We're not going to lie - the situation is serious. Experts claim that over 20 million people in the US might have been infected with the novel coronavirus, as the CDC estimates that the actual number is 10 times higher than what's reported. The same type of estimation goes for Canada, where their COVID-19 Immunity Task Force started a series of antibody tests to show a more detailed picture to show how many cases are actually there, on top of the reported 105 thousand. With a possible second wave looming on the horizon, we need to take action! Luckily, we have tools to mitigate the spread of the virus with surprising efficacy - face coverings!
The benefits of wearing masks in public spaces are unquestionable; many people go through COVID-19 with little to no symptoms, and thus can unknowingly spread it even by the simple act of talking. That's why wearing a mask in public is so important! Recent studies report that face coverings reduce the transmission of the novel coronavirus by 50%, though some mention numbers even higher than that. Depending on the type of mask, they can stop up to 99% of viral matter (N99 filters). Even a regular, homemade cloth mask (which is what the CDC recommends) is enough to significantly reduce the risk of spreading the infection. A recent model shows that public mask wearing could save up to 33,000 lives over the next three months!
We have prepared this calculator in hopes that it will help you make an informed decision about the kind of mask that you need to protect yourself and others. With its assistance, you can estimate how many masks or filters you will need for a given period of time, and how much money they will cost you. Read on if you're interested in the scientific background of the importance of face masks in this difficult time, and check if you've been wearing your mask correctly!
The main problem with the novel coronavirus is its high infection rate - we have reasons to believe that the number of infected people might surpass the reported cases as many as 10 times. With no precautions undertaken, the virus would spread exponentially, eventually overwhelm healthcare systems, and lead to a grim situation where hospitals simply cannot house anymore people in need of medical help. Luckily, we now know that there are a number of things we can do to effectively slow this process enough to let medics treat everyone and even, in the long run, allow scientists to find a way to beat COVID-19. These include:
washing your hands thoroughly with soap;
frequently using hand sanitizer;
practicing social distancing;
avoiding public gatherings in crowded spaces, for example by stockpiling food every once in a while instead of frequent grocery shopping; and
Last, but not least, wearing masks in public spaces.
In light of more and more research on the benefits of wearing masks during the COVID-19 pandemic, the CDC has updated its policy regarding face coverings, strongly recommending wearing masks in public spaces, especially when maintaining a distance is difficult or impossible. It's important to note that, provided that you don't suffer from a preexisting respiratory system condition, you should wear a mask in public even if you're feeling fine. Research conducted so far suggests that a vast number of people infected with the novel coronavirus do not experience any symptoms, and could unknowingly spread the disease further.
Luckily, studies show that masks allow for a decrease in the spread of the virus, with a surprisingly high effectiveness. Even a regular, homemade cloth facemask can provide protection against airborne viral matter. In the case of N95 and N99 masks with filters, the efficacy can reach even 99.98% of viral matter. A recently published paper provides strong evidence that implementing mask-wearing policies has the potential to drastically decrease infection rates, making the spread of COVID-19 manageable. Combined with the other protective measures, such as increased hygiene and distancing, it may be just enough to get the pandemic under control!
In essence, the message to take away here is this: a mask protects both you and the people around you. While it may be impossible to pinpoint exact percentages, this infographic provides a quite accurate perspective on the benefits of wearing a mask during the COVID pandemic.
For most of us, face masks are a novelty, and we don't usually take them into account in our budgets. What's more, with all the new kinds available, working out which mask is the best for you and how long it can last might get confusing. Our coronavirus mask calculator is designed to make your life a little easier by estimating the costs of face masks, as well as telling you how many masks (or filters, depending on the kind) you need to get to meet your needs.
The calculator takes the data about the kind of mask you're using and combines it with the information you provide to tell you several things. While using it, you will learn:
How effective the mask you're using is at protecting you. You can click through the different types and compare their efficacy in order to make the best possible choice regarding your safety;
How many masks or filters you will need for a given period of time. While getting your hands on a mask is no longer the struggle it was at the start of the pandemic, it's still important to plan ahead;
How often you need to change your mask (or its filter). It's important to remember that masks don't work indefinitely! To stay protected, you need to make sure your mask is fresh and maintained properly; and
How much will the masks and filters cost you. With this information, you can set up a mask budget to last you for as long as you need!
The results vary depending on the type of mask you choose. Our calculator allows you to choose from the most common kinds:
Cloth masks are typically cheap, widely accessible, and can easily be made on your own. As far as we know, they block only some of virus particles present in exhaled air, although their overall efficacy in protection might be higher. Main reason is that they effectively stop fluids from coughs and sneezes. One of the biggest pros of this kind of a mask is that they're reusable. Provided that you wash your masks regularly, as little as five of them can suffice on a daily basis. The CDC recommends cloth masks as the best choice for everyday purposes.
Surgical masks, similarly to cloth mask, filter only some of viral matter. They again do, however, block droplets (from sneezing or coughing), which is also an effective way to decrease the risk of infecting someone. They require frequent changing, and as they're not reusable, they are the least eco friendly option. Still, a surgical mask is better than no mask. If you use surgical masks, make sure to put a new one on every two hours.
N95/N99 RESPIRATORS
N95/N99 respirator masks block up to 99% of viral matter! If you're using N95/N99 masks with no changeable filter, make sure to change them after every 16 hours of use.
MASKS WITH N95 FILTER
N95 mask filters can block up to 95% of viral matter. Remember to exchange the filter once a week.
OTHER FFRs (including N99)
Masks with other FFRs (Filtering Facepiece Respirators) can block up to 99% of viral matter! It depends on the filter type and how often you need to change it, but once a week is a good rule of thumb.
Wearing a mask is easy enough, but it's important to keep in mind the way you wear it determines how well it will work. Most importantly, remember that a mask should always cover both your mouth and your nose. A common mistake is wearing a mask over only your mouth, only your nose, or even just your chin. In all these cases, it essentially defeats the purpose of putting the mask on in the first place, as it allows potential viral matter to go through undisturbed!
When putting your mask on and taking it off, it's important to follow some simple rules.
PUTTING YOUR MASK ON SAFELY:
Clean your hands thoroughly with soap and water, or an alcohol-based hand sanitizer, before you touch the mask you're about to put on.
In the case of a surgical mask, figure out its top and its front. The side with a stiff bendable edge is the top, and the colored side is usually the front, while the white side should touch your face.
If your mask has:
Earloops: hold it by the loops. Place a loop behind each ear.
Ties: bring the mask to your nose level, place the ties over your head and tie them into a bow.
Mold or pinch the edge of the mask to fit your nose. The more fitting it is, the better the effect. If you wear glasses, they should not fog over if the mask is fitted correctly.
Pull the bottom of the mask over your mouth and chin.
REMOVING YOUR MASK SAFELY:
Clean your hands thoroughly with soap and water, or hand sanitizer, before starting. Avoid touching the front of the mask, as it could be contaminated!
Hold the ear loops or untie the ties and gently lift them to remove the mask.
If you're using a disposable mask, throw it in the trash carefully. If your mask is reusable, wash it as soon as possible. Clean your hands thoroughly again once you're done with the process!
If you'd rather see how it's done, we encourage you to check out this helpful instructional video provided by the World Health Organization.
It's hard to argue that the new normal isn't inconvenient. It's true that masks are not the most comfortable thing to wear, especially in the warm summer months. Unfortunately, some people go as far as considering wearing masks a limitation on their personal freedom. Anti-mask movements have appeared in the United States, and even some authority figures have voiced anti-mask sentiments, making this a political issue. But are masks really that controversial?
Face masks work. New research confirming this statement appears frequently. According to scientists, countries in which face coverings were used widely during the early stages of the pandemic are more likely to not only have significantly lower death rates, but also to have shorter outbreaks than in areas with no mask-related policies. A study focused on the same thing in the USA's context found that the decline of daily COVID-19 cases was faster in states with mask mandates, and estimates that face coverings most likely prevented as many as 450,000 cases of COVID-19 by May 22, 2020.
These numbers speak for themselves. Even though wearing a mask might be inconvenient, it's one of our strongest lines of defense in the fight against COVID-19. Wearing a mask does not take away your personal freedoms - it ensures your right to stay healthy, and shows that you care for the well-being of yourself and others!
Dominika Miszewska, PhD candidate and Maria Kluziak

redpajama_set_name: RedPajamaCommonCrawl


text:
Japanese unicorn Mercari's market cap seen reaching $3.6bn
Flea market app operator raises IPO target price on firm demand
Investors at home and abroad are bullish on Mercari's growth prospects. (Photo by Satoshi Morotomi)
Nikkei staff writers June 2, 2018 01:40 JST | Japan
TOKYO -- Japanese flea market app operator Mercari has raised the estimated price for its initial public offering this month amid keen investor interest in a Japanese unicorn, bringing its market valuation as high as $3.64 billion.
The startup said Friday it has set the indicative prices for book-building at between 2,700 yen and 3,000 yen, up from the originally planned 2,200 yen to 2,700 yen. A debut at the top end of the new range would put Mercari's market cap at more than 400 billion yen, or $3.64 billion -- far above the threshold for unicorns, which are startup companies valued in excess of $1 billion.
Investors attending pre-IPO briefings inside and outside Japan praised Mercari's growth potential, particularly in its home market, according to the company. Underwriters will solicit interest next week based on the tentative terms and set the official price on June 11 ahead of Mercari's June 19 debut on the Tokyo Stock Exchange's startup-laden Mothers market.
The company plans to issue as many as 21 million new shares. An additional 22.5 million shares will be sold by existing investors, including CEO Shintaro Yamada, trading house Mitsui & Co., mobile advertising company United and venture capital firms.
Mercari is set to raise as much as 63 billion yen through the listing. It plans to spend about 11 billion yen on advertising to grow its user base in Japan and the U.S., with the remaining funds to go toward paying developers and expanding its services.


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RIVALRY REVENGE: Stewart, Hall help struggling Temple charge back to beat cold-shooting Belton 53-43
SENIOR MOMENT: Temple senior guard/forward Joseph Stewart scored 13 points against rival Belton on Tuesday night as the visiting Wildcats rallied from a 21-10 deficit early in the second quarter to defeat the Tigers 53-43 in a District 12-6A game at Tiger Gym. Stewart, who made two of Temple's six 3-point baskets, did not play in the Wildcats' 53-31 home loss to the Tigers on Dec. 22 because of COVID-19 contact tracing involving his father. Sophomore guard Jaylon Hall led Temple with 14 points in the rematch with Belton. (Photo by Greg Wille, TempleBeltonSports.com)
BELTON – The Belton Tigers entered Tuesday night as prime contenders for the District 12-6A championship, and they were playing at home against a seventh-place Temple team they dominated 53-31 on Dec. 22 at Wildcat Gym.
Meanwhile, the Wildcats came into Tiger Gym with only one district win to their credit and weren't even particularly close to 12-6A's fourth and final playoff position.
However, there is a reason that competitive basketball games are played on a hardwood court instead of on paper. Anything can happen on any given evening, especially when a rivalry is involved.
Belton led 21-10 early in the second quarter, but the cold-shooting Tigers made only eight field goals in a frustrating final 22 minutes and Temple's Joseph Stewart and Jaylon Hall came up with key plays in crunch time to help the resilient Wildcats control the second half and handle the pressure to earn a stunning 53-43 victory.
It was a sweet and much-needed win for third-year head coach Michael Thomas and Temple (8-12 overall, 2-7 in 12-6A), whose only other district victory was 51-45 at home over last-place Killeen on Jan. 15.
Hall scored 14 points and Stewart had 13 for the Wildcats, who made six 3-point baskets to Belton's two and shot 9-for-11 on free throws in the fourth quarter after not attempting a foul shot through three periods. Temple trailed throughout the first half and took its first lead at 29-27 on Stewart's 3-pointer with 2:25 remaining in the third quarter, putting the Wildcats ahead to stay.
"I've been showing the guys a lot of film and just trying to get them to understand that you've got to play basketball. Quit worrying about the crowd saying, 'Shoot it! Jack it up!' You've got to play for the team and play disciplined basketball," Thomas said. "We did a good job tonight, man. Belton has a good team and I'm proud of the guys. They fought. It's my birthday, so they played well for my birthday."
The number of candles on Thomas' birthday cake – 39 – was nine more than the number of points his squad allowed in the final three quarters against Belton (15-5, 6-3). The Tigers got 19 points from standout junior forward TJ Johnson – who entered averaging 25.8 per game – but no more than eight from anyone else in their lowest-scoring game this season by eight points.
It's common for Johnson, his freshman brother Trap Johnson and senior guards Kayden Downs and Ben Jones to make a combined eight to 12 – or more – 3-point shots in a game for Belton's let-it-fly attack, but against Temple they had an uncharacteristically rough performance from long distance. In more than 20 total shots from behind the arc, Jones made two and the Johnson brothers and Downs came up empty.
The Wildcats limited Downs, Trap Johnson and Jones to a combined seven made field goals and 22 points. The Tigers also were hindered by 9-for-17 accuracy on free throws and committed seven turnovers during the fourth quarter while striving to catch up to the increasingly confident Wildcats.
"We didn't shoot the ball worth a . . . it was pretty obvious. Senseless turnovers at times, but for the most part we just didn't shoot the ball well at all," Belton third-year head coach Jason Fossett said. "I thought we got on a little run there early in the second quarter and we got ahead 21-10, and then you can't shoot it much worse than we did after that point.
"Midway through the third quarter I thought we were still OK, but then you start missing, the game starts turning and you're taking shots you usually make and they don't go in. You start pressing a little bit. We just didn't play well, bottom line."
MAN-TO-MAN: Temple junior forward Leon Hudson (left) defends Belton junior forward TJ Johnson as Trap Johnson (23) and Joseph Stewart look on during the Wildcats' 53-43 victory over the Tigers on Tuesday night at Tiger Gym. The 6-foot-6 TJ Johnson came in averaging 25.8 points but was limited to 19 by Temple, which allowed only two 3-point baskets by the Tigers. Aided by the 6-5 Hudson's defense and rebounding, the Wildcats permitted just 22 points in the final 22 minutes as Temple (8-12) improved to 2-7 in District 12-6A. TJ Johnson blocked four shots for Belton (15-5, 6-3 in district). (Photo by Greg Wille, TempleBeltonSports.com)
A free throw by TJ Johnson gave the Tigers their final lead at 27-26 with 3:58 left in the third. Temple then closed the period with a momentum-shifting 10-2 run and outscored Belton 27-16 in the final 11-plus minutes to grab a win that has the potential to spark a late-season surge for the Wildcats.
"I think honestly we've just been having more fun with each other, so we've been coming together. In practice, we've been enjoying the grind with each other," said Stewart, a 6-foot-1 senior guard/forward who made two 3-pointers overall and scored five of his 13 points in the fourth quarter. "It's not like we're coming to practice and dreading it. Instead, we're coming to practice and we're happy to be with each other, so getting better is not hard anymore."
Stewart did not play in Temple's 22-point home loss to Belton five weeks ago because of COVID-19 contact tracing involving his father. After watching that disappointing defeat from home on his computer, Stewart played a pivotal role – with his now-recovered dad present to see it – as the Wildcats got their revenge against the Tigers.
"Belton's always motivation, because it's Belton," Stewart said.
Said Thomas about Stewart: "That's a quality kid, a straight-A student. He's going to (the University of Texas) for finance, I believe. He's a thinker. He helps us on the floor."
Temple sophomore guard Jaydon Hall made two 3-pointers and scored all eight of his points in the second half, and 6-5 junior forward Leon Hudson provided rugged defense and rebounding on the interior along with scoring six points.
"No doubt, give Temple a lot of credit. They did a good job and came in here and did what they needed to do to get a win," Fossett said. "Hat's off to Coach Thomas. He had his kids ready to play. We've got to do a better job the next time."
The 6-6 TJ Johnson was Belton's dominant player in the first quarter, producing 10 points on five close-range field goals. Temple trailed 9-4 early but stayed in it as Stewart and Jaylon Hall each scored five points in the opening period. Jaylon Hall's 3 from the right side made it 11-10 before TJ Johnson grabbed junior Seth Morgan's inbound pass and powered in for a basket and a 13-10 Tigers advantage.
Belton then produced a scoring flurry in the second quarter's first 2 minutes. Downs penetrated and made a difficult jump shot in the lane; Jones sank a 3-pointer from the left wing while drawing a foul and then completed the rare 4-point play; and Trap Johnson hit two free throws for a 21-10 Tigers lead at the 6:10 mark.
But as Fossett pointed out afterward, Belton's offensive highlights after that juncture were few and far between. The Tigers' only other field goal in the remainder of the first half was TJ Johnson's putback with 18.2 seconds left.
Meanwhile, it was Jaylon Hall's banked-in 3 from the right wing that sparked Temple's big comeback. Hudson made a fast-break floater, junior Lawrence Auston hit a tough banker on the break and 6-5 junior Quinton Anderson scored inside off a deft pass from senior Aundra Jackson to slice Belton's lead to 22-19 2:53 before halftime. In the closing seconds, Jaylon Hall dished to Anderson underneath for a buzzer-beating bucket and a 24-21 game at intermission.
Trap Johnson opened the third with a hard-driving layup from the left wing to push Belton's lead to 26-21, but unfortunately for the Tigers it was not a sign of things to come. Jaydon Hall answered with a right-side 3-pointer, then Hudson scored inside for a 26-26 deadlock at the 5:28 mark and blocked TJ Johnson's attempted layup out of bounds, a defensive highlight that seemed to energize the Wildcats.
"Leon Hudson did a good job tonight," Thomas said.
A free throw by TJ Johnson – who was an uncharacteristic 3-for-7 from the foul line – gave Belton a 27-26 edge with 3:58 remaining in the third, but Temple took control of the game from that point on. Stewart's right-corner 3 with 2:25 left propelled the Wildcats into the lead for good at 29-27, igniting a game-ending 27-16 run.
Jackson converted his steal into a layup, Hudson made a driving bucket off the glass and Jaydon Hall buried a right-wing 3 to complete a 10-0 spurt before Downs' left-handed runner with 4.5 seconds left finally ended the Tigers' 6½-minute field goal drought. Temple outscored Belton 15-5 in the third quarter to seize a 36-29 lead.
Although the Tigers' misfires from 3-point territory continued to mount, TJ Johnson kept Belton within striking distance with a driving lefty hoop across the lane and a one-handed dunk off his steal to go along with the junior's stellar rebounding and four blocked shots.
However, Temple couldn't be denied. Jaylon Hall scored from the baseline and Auston collected a loose ball and put in another bucket for a 44-35 lead with 3:20 left. Belton used a tough-angle, left-corner 3-pointer by Jones – its first 3 since Jones made one early in the second quarter – and a TJ Johnson free throw to get within 44-39 with 2:05 left, but the Tigers' fouls piled up and Jaylon Hall and Stewart each went 2-for-2 on free throws for a seven-point lead at the 1:20 mark.
After a driving basket by Belton senior guard Luke Bramlett made it 48-43 with 1:16 left, Jaylon Hall shook off missing the front end of a 1-and-1 situation by making two free throws with 44.6 seconds remaining, followed by two Jackson foul shots that sealed the outcome after Downs fouled out with 28.1 left.
Temple, now four games behind third-place squads Belton and Bryan with five district games remaining, will shoot for a two-game winning streak when it hosts Bryan at 7 p.m. Friday. The Wildcats lost 76-55 to the host Vikings on Jan. 5.
"It's a good win for us and hopefully it gives us a little momentum," Thomas said. "Like I told the kids, to have a chance (to make the playoffs) you've got to win out basically. We're going to keep fighting."
Now two games behind district-leading, No. 13-ranked Killeen Ellison (21-1, 8-1) and a game behind second-place Harker Heights (7-2 in 12-6A), Belton will seek to bounce back when it hosts fifth-place Copperas Cove at 7 p.m. Friday. The Tigers grabbed an 82-67 road win over the Bulldawgs on Jan. 5.
"You've got to protect your home court, and we didn't do a very good job of that tonight," Fossett said. "We better come out with some different energy level Friday against Cove or this thing can spiral in a hurry."
DISTRICT 12-6A
Temple 53, Belton 43
Temple 10 11 15 17 – 53
Belton 13 11 5 14 – 43
Temple (8-12, 2-7) – Jaylon Hall 14, Joseph Stewart 13, Jaydon Hall 8, Leon Hudson 6, Quinton Anderson 4, Lawrence Auston 4, Aundra Jackson 4.
Belton (15-5, 6-3) – TJ Johnson 19, Kayden Downs 8, Trap Johnson 7, Ben Jones 7, Luke Bramlett 2.
3-point field goals – Temple 6 (Jaydon Hall 2, Jaylon Hall 2, Stewart 2); Belton 2 (Jones 2).
Free throws – Temple 9-11; Belton 9-17.
Junior varsity – Belton 62, Temple 44.
Freshman A: Belton Red 54, Temple Blue 48.
Freshman B: Temple White 45, Belton White 32.
#Temple #TempleWildcats #TempleBasketball #TempleHighSchool #Belton #BeltonTigers #BeltonBasketball #BeltonHighSchool #TXHShoops #TempleBeltonSports
FAMILIAR FOES MEET AGAIN: No. 10 Academy battles No. 14 Franklin for trip to Region III tournament
READY FOR ROUND 2: No. 10 Academy boys battle Clifton, seek return trip to Region III quarterfinals
By GREG WILLE TempleBeltonSports.com gwille2@hot.rr.com GATESVILLE – After charging past Fairfield in its Class 3A playoff opener, the Academy boys basketball team is set for Round 2 action tonight. T
FULL STEAM AHEAD: Johnson, District 12-6A co-champion Belton begin playoff charge tonight vs. DeSoto


KOTA KINABALU: Dried prawn and fish products are exempt from Sales and Services Tax (SST), said Finance Minister Lim Guan Eng.
Lim said the five per cent sales tax supposedly imposed on essential food products such as 'ikan jeruk' and 'ikan kering/masin' under the Fish and Sea Creature category was untrue.
"If there is, maybe it is overlooked. Perhaps, the tax imposed was based on the previous proposal list.
"There is a new list, which everyone should check. Dried fish and prawn, among other items, are exempt from SST," he told reporters at a press conference after attending a SST briefing and forum here today.
However, he said people should propose to the ministry should they think there are more items, which should be exempted from SST.
"We will review. Perhaps there is a situation requiring us to review and we will decide by year end. The ministry has the power to make amendments," said Lim.
Also present was customs director-general Datuk Seri Subromaniam Tholasy.
To ensure traders would not take advantage from SST, Lim said the Domestic Trade and Consumer Affairs Ministry (KPDNHEP) would go to the ground to ensure there was no profiteering.
Meanwhile, Subromaniam said customs would work with KPDNHEP to look into setting recommended retail price to stabilised prices.
Earlier in his speech, Lim warned telco companies against deducting certain percentage from customer's prepaid top-up card as the item was exempt from SST.
"When a customer purchases a RM10 prepaid top-up card, they should get RM10. We will assure customers get the same prepaid value.
"There should be no deduction (in prepaid card value). It should not be 'we take first and rebate later'. We'll sort out (this matter) with all telco companies," he said.


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Official sketch hints at C8 Chevy Corvette Z06's interior
Viknesh Vijayenthiran November 12, 2019 Comment Now!
We may have just received our first official look at the next Chevrolet Corvette Z06. Well, at least its interior.
Roadshow recently received a handful of design sketches of the C8 Corvette direct from Chevrolet, and one of those sketches, shown below, depicts a design marked with a "Z06" label on the steering wheel.
While the sketch doesn't necessarily depict the design we'll see in the new Z06, it at least shows what the design team are dreaming up for the next generation of the attainable track superstar.
C8 Chevrolet Corvette Z06 interior sketch - Photo credit: Roadshow/Chevrolet
The layout of the dash matches the design used in the Stingray, which adds to the sketch's credibility, but the controls on the steering wheel are different and includes a giant red button with "Spec Mode" written on it. There's also the layout of a racetrack, specifically Italy's Mugello, in the instrument cluster, and carbon fiber on many of the surfaces instead of the usual leather.
The new Z06 is currently thought to be arriving with a naturally aspirated version of the 5.5-liter V-8 fitted to the C8.R race car. This is a high-tech engine with dual overhead cams and a flat-plane crank, and in the Z06 is expected to spit out around 600 horsepower. A twin-turbocharged version with closer to 800 hp is expected to power the new ZR1. Sadly, there's no word whether a manual transmission will be offered, and there's a good chance one won't make it at all, though it's hard not to love Chevy's brilliant 8-speed dual-clutch transmission that debuted in the Stingray.
Chevy also hasn't indicated when the new Z06 will arrive. If past Corvette generations are any indication, we should see the car debut in the next 12 to 24 months.
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"the quality of the work is high (and)... indeed compares favourably with the much larger BP Portrait Awards"
Duncan Macmillan, The Scotsman, 2 December 2017
All media enquiries should be sent to Sara Cameron McBean | phone +44(0)7784567254 | sara@scottishportraitawards.org
Highlands artist wins £5,000 prize
Winner of the SPA in Fine Art, Daniel Murray (right) with SPA Director, Gordon Mitchell
Winner of the Richard Coward SPA in Photography, Lucas Chi-Peng Kao (right) with Mira Knoche, the subject of his work.
Winner of the SPA Young Fine Artist, Eve Woehrling (right) with Helen Wilson, winner of the 2017 SPA in Fine Art.
Winner of the SPA Young Photographer, Eliza Coulson, with her portrait Fragile.
Edinburgh, Wednesday 31 October 2018 The winners of the Scottish Portrait Awards 2018 were announced on Wednesday night (31 October 2018) at the Scottish Arts Club in Edinburgh with the top fine art prize of £5,000 going to Daniel Murray from Poolewe for his portrait, Jané Seated. In the portrait, Murray's friend Jané appears to float before layers of stylized blue hills that Murray says were inspired in part by the landscape of the place where they both grew up, and partly by Jané's love of tapestry. The judges, who included John Byrne and Imogen Gibbon, Chief Curator of Portraiture at the National Galleries of Scotland, said that they 'admired the painting's ethereal quality and well as its execution'.
The fine art judges also included Guy Peploe, owner of the Scottish Gallery, award-winning artist Jennifer McRae and contemporary artist Gordon Mitchell who chairs the panel and is the SPA Director. They picked Edinburgh-artist Alan McGowan for the £1,000 second prize, praising his portrait of Lydia for the way he 'layered the paint in an almost abstract way to generate remarkable qualities of light and shade'.
Thirty-four-year-old Lucas Chih-Peng Kao, an award-winning film-maker and photographer, won the £2,000 Richard Coward SPA in Photography with his portrait of artist Mira Knoche. Born in Taiwan, Lucas has lived in Edinburgh for many years. The photograph, shot on 8x10 film, was taken on a cold February evening while Knoche played with the palette knife she often uses in her paintings to 'scape away at the façade and get to the core of things.' Kat Gollock won second prize with her poignant photograph, Oyster Boys, which features the late Scott Hutchison. The SPA Photography Panel is chaired by Siobhán Coward, and includes Ben Harman from Stills Gallery, Edinburgh, Malcolm Dickson of Street-Level Photoworks in Glasgow and photographer Robin Gillanders.
The inaugural SPA Young Fine Artist award was won by seventeen-year-old high-school student Eve Woehrling for her watercolour painting of her brother, Portrait of Saul while twenty-year-old Eliza Coulson won the SPA Young Photographer award for her self- portrait Fragile. Eliza is currently studying at Glasgow School of Art.
This is the second year of the Scottish Portrait Awards which was founded by the Scottish Arts Club Charitable Trust with the aim of creating a vibrant new platform for celebrating exceptional Scottish talent in portraiture. The competition is open to anyone born, living or studying in Scotland over the age of 16 years. Entries for the fine art competition can be in any 2D or 3D medium. Entries for the SPA Photography must be in black and white.
In 2018, hundreds of entries were received from across the country and beyond. The judges selected works for the quality of their story-telling, and the diversity of styles and materials used, as well as skill in execution. According to the SPA Director, Gordon Mitchell, 'The result is an exhibition that is as varied as it is entertaining; some works reflecting humour, courage and character, others conveying grief, loneliness and love and still others an exploration of the connection between the paint, the marks and identity itself.'
The SPA exhibition is on at the Scottish Arts Club, 24 Rutland Square, Edinburgh from 3 November to 1 December at 2.30-5.30pm daily except Mondays. It will transfer to Glasgow Art Club where it will be on from 21 January to 9 February 2019. Entries for the 2019 Scottish Portrait Awards are open from 2 April to 30 June 2019. See the website for details: www.scottishportraitawards.com
Helen Wilson stands in fron of her winning portrait, Jonah of Kittyfield Farm, with SPA Judge John Byrne and Jonah Gaskell, the subject of her painting.
Scottish Portrait Award winners stand out in talented field
Edinburgh Thursday 2 November The winners of the Scottish Portrait Awards (SPAs) 2017 were announced last night at the opening event of the Scottish Portrait Awards exhibition at the Scottish Arts Club in Edinburgh. Glasgow artist, Helen Wilson took the £5,000 SPA in Fine Art while Robin Gillanders of Edinburgh received the £2,000 Richard Coward SPA in Photography. Commendations were awarded to Ewan McClure and Virginia Colley for fine art and Robert Andrew Mercer and Oscar Pereira Beiroa for photography.
Robin Gillanders receives the Richard Coward SPA in Photography from Siobhán Coward, chair of the SPA Photography Panel.
Helen Wilson, known for her paintings of ballerinas, musicians and other performers won with her painting of Jonah of Kittyfield Farm. Commenting on the work, the Glasgow based artist said, 'I am fascinated by people in their natural working environment, especially if they are passionate about what they do, be they dancers, chefs, singers, fishmongers… or indeed farmers. Jonah's family, who run Kittyfield Farm, are just such people, and while the farm occupies an idyllic setting, with views over the River Tweed to the Eildon Hills, their everyday reality is… muddy!'
Well-known Edinburgh photographer Robin Gillanders says he had not entered a photographic competition since he was 18 years old, but the Scottish Portrait Awards 'seemed made for me.' He took his rich-toned photograph of Shetlandic poet and author, Robert Alan Jamieson, especially for the competition. Inviting Jamieson to his home-studio, he asked the writer to bring something of symbolic value to him. Jamieson came with the eagle's feather that appears in the photograph. Gillanders used a traditional large format 8x10 camera complete with dark cloth draped over his head and took only five shots to find his winning masterpiece.
The judges praised Wilson's uncompromising honesty and direct engagement through the work, while Gillander's portrait won because of it strong composition, tonal range and sense of narrative for the character. Both the fine art and photographic winners were praised for their story-telling quality and for capturing the subject very well. In both cases the judges' decision was unanimous.
Press coverage 2017 SPAS
The Scotsman Review, by Duncan Macmillan https://www.scotsman.com/lifestyle/culture/art/art-reviews-hidden-gems-at-edinburgh-city-art-centre-the-scottish-portrait-awards-2017-1-4627243
Winners go head to head in Scottish Portrait Awards – Mike Wade, The Times www.thetimes.co.uk/article/artists-go-head-to-head-in-scottish-portrait-awards-lsjjp0wl9
Art judged head and shoulders above rest – Mike Wade, The Times www.thetimes.co.uk/article/scottish-portrait-awards-art-judged-head-and-shoulders-above-rest-03bxdz9nl
Scottish Portrait Awards in Fine Art and Photography, BBC News Online, http://www.bbc.co.uk/news/uk-scotland-41830674
Winners announced at the first Scottish Portrait Awards, BBC News Online: http://www.bbc.co.uk/news/uk-scotland-edinburgh-east-fife-41818551
Established photographer wins first Scottish Portrait Award, Amateur Photography http://www.amateurphotographer.co.uk/latest/photo-news/scottish-portrait-awards-112509
Numerous other stories in local newspapers and online sites including a full page in Edinburgh Life and major feature in Scottish Art Scene scottish-art-scene.com/Scottish.Portrait.Awards.pdf
Cover photo credit: Carolyn Scott
A motivational poem and comic strip by George McBean to inspire fine artists and photographers across Scotland to enter the Scottish Portrait Awards


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Le  HMS Swiftsure est un vaisseau de ligne de troisième rang de , construit pour la Royal Navy par l'architecte naval  aux chantiers de Harwich et lancé en 1673. En 1685, le navire est transformé en .

Description
À l'origine le Swiftsure est un navire de  et de , mesurant  de long,  de large et ayant un tirant d'eau de .
Sa modification de 1696 voit une réduction du nombre de canons, désormais , et de son déplacement, désormais . Il mesure alors  de long,  de large et a un tirant d'eau de .

Une nouvelle modification intervient en 1718 suivant les . Le Swiftsure retrouve alors ses  et voit son déplacement augmenter à . La longueur est portée à , la largeur demeurant à , alors que le tirant d'eau est porté à .

Enfin, en 1742, les  font du vaisseau un navire de , long de , large de , au tirant d'eau de .

Histoire
Le , le Switsure participe à la bataille de la Hougue, bataille navale pendant la guerre de la Ligue d'Augsbourg, qui voit la victoire de la flotte anglo-hollandaise sur la flotte française du vice-amiral de Tourville, au large de la pointe du Cotentin.

Le navire est réduit à  par l'architecte naval Snelgrove aux chantiers de Deptford en 1696.
Il fait partie de la flotte de l'amiral Cloudesley Shovell en 1707 et prend part cette année-là à la défaite britannique de Toulon et au désastre naval de Sorlingues, qui, outre la mort de Shovell, déplore côté britannique la perte de   et de près de .
Le Swiftsure ayant subi peu de dommages durant ce dernier affrontement, il parvient à rejoindre Portsmouth.

Le , le vaisseau est engagé sous le commandement de Robert Wynn dans la bataille de Vigo, au large des côtes de la Galice ; cet affrontement met aux prises une flotte anglo-hollandaise dirigée par l'amiral George Rooke, secondé par les amiraux hollandais Philips van Almonde et Philips van der Goes, avec la Flotte des Indes, un convoi franco-espagnol commandé par les amiraux François Louis Rousselet de Châteaurenault et Manuel de Velasco.

Il subit une deuxième modification aux chantiers navals de Woolwich et est relancé le  avec à nouveau  et prend désormais le nom de Revenge.

Une nouvelle modification intervient par ordre du  aux chantiers de Deptford. Le nouveau lancement a lieu le .

Le , sous les ordres de John Storr et dans l'escadre commandée par Edward Hawke, il prend part à la bataille des Cardinaux.

Le Revenge est finalement vendu en 1787.

Notes et références

Notes

Références

 Autres sources

Voir aussi

Bibliographie

Navire de ligne de troisième rang de la Royal Navy
Trois-mâts
Bateau des années 1670
Bateau britannique de la bataille des Cardinaux

redpajama_set_name: RedPajamaWikipedia


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Q: JQuery Mobile site causing odd URL outcomes I am working on a JQuery Mobile site generated through Dreamweaver using one of their templates.  On the main index page, I have some pages going to subindexes, and I have several direct URLs.
Here are two URLs in the list:

Learn More About Us

Meet Our Staff

The first one, when clicked on the actual site
http://www.morningsiderecovery.com/mobile-05-08-12/

leads to the proper destination
http://www.morningsiderecovery.com/mobile-05-08-12/about-morningside-recovery/

The second one, when clicked on, leads to the following bogus url, which won't resolve and sends me to the default home page:
http://www.morningsiderecovery.com/mobile-05-08-12/#/mobile-05-08-12/morningside-staff/

I'm getting the exact same behavior in IE, Chrome, and Firefox (PC) and Safari (both Mac and iPhone versions).
Does anyone have an idea why this may be happening, and how I can make it stop?  This one's got me completely perplexed.
Thanks so much,
Jim Y.

redpajama_set_name: RedPajamaStackExchange


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RBS forced to sell Citizens ending the most successful UK retail banking foray into US market
British businesses don't have a great track record in breaking into the US retail market. You only have to look at the disastrous foray that the Marks & Spencer acquisition of Brooks Brothers was, Tesco's humiliating and expensive attempt with the Fresh & Easy brand and, most recently, the failure of Yo Sushi! to realise how difficult it is for firms with strong brands in their domestic markets to make it across the pond.
The retail banking track record is no better with Barclays, Lloyds and Natwest all quitting the US in the late 1980s and 1990s. Losses from the acquisition of Crocker drove Midland Bank into the arms of HSBC. Even HSBC has not been immune to the problem with the disastrous acquisition of subprime Household continuing to hurt the bank to this day.
It is quite ironic then that RBSG is being forced to exit the one reasonably successful move into retail and commercial banking that British banks have made in the US. Whilst Fred Goodwin, the former CEO of RBSG, has been criticised for much of the way that he ran the global banking group (particularly paying over the odds for ABN Amro just as the wholesale markets were closing down) his strategy for building a presence in the US retail and commercial banking sector should be heralded as one his smarter moves.
Rather than trying to take on the large US retail banks where they were, at that time, competing aggressively with each other in New York, California, Texas and Florida, Goodwin decided to build his beachhead in the Mid-Atlantic by the acquisition of Citizens Financial Group. A series of small but strategically significant acquisitions followed that expanded it into New England and the Midwest. Citizens is now the 15th largest commercial banking organisation in the US. Whilst there have been challenges including writedowns following the acquisition of Charter One and recent issues with the way that capital is planned, overall Citizens is a highly capitalised and profitable bank. Yes its capital is under deployed but that is addressable. Indeed its reputation with its customers is far better than RBS' in its own domestic market.
It is a great shame then for RBSG that due to having to take state intervention and becoming largely nationalised, primarily due to the acquisition of ABN Amro and the disastrous business in Ireland, that RBSG is being forced by the EU to dispose of its ownership of Citizens by the end of 2016.
As the first step of moving towards this in January 2014 Citizens sold off 103 branches in the Chicago area to US Bancorp.
It has been announced that the next step will be to float or sell 20-25% of its share of Citizens. A flotation is more likely as there have been few signs of interest from potential buyers. However for Canadian, Japanese or Spanish banks that want to significantly grow their presence particularly in the Midwest and given that it is a forced sale it could be an interesting opportunity.
The flotation will help to rebuild its balance sheet, but the sale is what is really needed as that could release more than $3bn of capital, which would help RBSG reduce the government holding in the bank.
This is all a sad ending to what could have been had RBSG scaled back its ambition to be global investment bank.
As a footnote, British banks should not give up on being able to build a presence in the US retail and commercial banking market. RBSG has shown that it can be done. Barclays is having success with its Barclaycard US operation building scale to take on the other cards providers, however this is a monoline not a full service retail banking offering.
The British banks can also look to the Spanish banks, Santander and BBVA which with respectively the acquisition of Sovereign Bank and Compass Bank, are demonstrating that it is possible for Europeans banks to build a presence in the US retail banking market. It takes time, patience and recognition that whilst both the US and European markets have the words 'retail banking' in their names that they are quite different.
Labels: Banking, Barclaycard, Barclays, BBVA, Citizens, Citizens Bank, Compass Bank, Household, HSBC, RBS, RBSG, retail banking, Santander, Sovereign Bank, US Bancorp
New NAB CEO faces challenge of what to do with Yor...
RBS forced to sell Citizens ending the most succes...


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House Subcommittee on Oversight and Investigations Examines Security of Bioresearch Labs
Tannaz Rasouli, Sr. Director, Public Policy & Strategic Outreach
trasouli@aamc.org
The House Energy and Commerce Oversight and Investigations Subcommittee April 20 held a hearing to discuss the security of U.S. biomedical research labs.
In his opening remarks, Subcommittee Chair Tim Murphy (R– Pa.) expressed his concerns with the Federal Select Agent Program and the Federal government's high-containment laboratories. Rep. Murphy also highlighted a recent Government Accountability Office (GAO) report that evaluated "the biosafety, biosecurity, and oversight policies for the 8 departments and 15 component agencies that own and operate the Federal government's high-containment laboratories," adding that the GAO found that "while the departments and agencies have improved on their biosecurity policies in recent years, comprehensive policies and better oversight of the labs are still needed."
House Energy and Commerce Chair Fred Upton (R-Mich.) reiterated Rep. Murphy's concerns over security at bioresearch labs, adding that "[O]ne safety lapse is too many, and we have seen a disturbing trend of late that must be fixed. This is not a game of gotcha - we all want our researchers and lab workers to be safe."
Ranking Member Kathy Castor (D – Fla.) highlighted the work bioresearch labs contribute to the biodefense of the country, and added that "labs that handle these dangerous pathogens must be held to the highest standards." However, she voiced her concern over the recent security lapses, adding that "these recent incidents raise questions about whether or not we can trust high-containment labs to safely handle select agents and other dangerous pathogens."
National Institutes of Health (NIH) Principal Deputy Director Lawrence Tabak, D.D.S., Ph.D., highlighted how NIH has taken steps to address security lapses at bioresearch labs, noting "NIH identified and inventoried all potentially hazardous biological materials stored in all owned and leased facilities included all infectious agents, non-regulated toxins, poisons, and venoms." Tabak then discussed NIH's long-term strategy to address safety at bioresearch labs, adding "NIH developed the Potentially Hazardous Biological Materials Management Plan, which addresses accountability at all levels of NIH and has been fully implemented."
Other witnesses included:
Major General Brian C. Lein, Commanding General, U.S. Department of Defense;
Steve Monroe, Ph.D. Associate Director for Laboratory Science and Safety, Centers for Disease Control and Prevention;
John Neumann, Director, Natural Resources and Environment, Government Accountability Office; and
Segaran Pillai, Ph.D., Director, Office of Laboratory Science and Safety, Food and Drug Administration


Accidentally pushed the drum-tray back in without the drum? The green light won't show, and the tray won't budge - all you need to do is to carefully push a tiny lever below the tray, as pictured.
Thoroughly cleaning a drum is a complicated but not impossible task. Use the following images to guide you through the complete teardown and cleanup of your RP color drum.
All photos by Rob Brown.


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Disney Buttons & Pins
Walt Disney World pin
In September 1929, less than a year after Steamboat Willie, the first successful Mickey Mouse cartoon, was released, the first Mickey Mouse Club was formed by the Fox Dome Theatre in Ocean Park, California. Disney quickly saw the potential of such a Club and supplied theatre managers with starter packs containing a manual, song sheets, badges and so on. The original Mickey Mouse Club reached its peak around 1932 and buttons from this period are highly collectable.
The first Disneyland theme park opened in July 1955 and The Mickey Mouse Club TV show, originally intended to promote the amusement park, began in October 1955. Buttons and pins featuring Disney characters, TV show cast members, Disneyland and its attractions were produced in large numbers. Many different Disneyland pins and buttons continued to be produced through the 1960s and 1970s. Disney cast member buttons are among the rarest and most valued from this period.
The 1984 Olympic Games in Los Angeles sparked a new pin-trading craze. Disney joined in with 30th birthday promotional pins at Disneyland in 1985 and 15th anniversary pins at Disney World in 1986.
In 1999, pin collecting was given another boost when pin trading kiosks were opened at the Disney theme parks as part of the Disney Millennium Celebration.
Disney buttons & pins available now
Maleficent Disney trading pin *
AU $8.00End Date: Friday Aug-16-2019 17:40:05 ESTBuy It Now for only: AU $8.00Buy It Now | Add to watch list […]
Disney Mickey & Minnie Mouse Black & White Collector Pin
Disney Green and Gold large Pirate Ship trading Pin badge 2016 Limited to 300
AU $10.95 (0 Bids)End Date: Sunday Jul-21-2019 12:47:12 ESTBuy It Now for only: AU $14.95Buy It Now | Bid now | Add to watch list […]
Frozen Olaf Disney Pin 121225 Store 30th Anniversary Week 9 LE Flawed
Disney Stitch,hidden mickey,white rabbit lot of 4 pins Disney pin
AU $15.00 (0 Bids)End Date: Saturday Jul-20-2019 9:08:15 ESTBid now | Add to watch list […]


The girl of your dreams: Smart, talented, and accomplished. Find her for $14,455! The Society of Single Professionals—yes, it really exists—has launched mingling events with hefty price tags to help the 1 percent find love.
But when it comes to catching a lady, the 99 percent could use a hand too: Women don't just outpace men in college enrollment rates—almost two-thirds rated a lucrative career as a top priority compared to 59 percent of guys, according to a Pew Research Center study.
Skip the fancy gifts. You save up for months and buy her a pricey watch. She doesn't appreciate the gesture. Why? "Just because she's used to money doesn't mean it's what she values," says Tracey Steinberg, dating and flirting expert. Choose gifts that remind you of your shared relationship—she's with you because you make her laugh, not because of your salary.
Dress up. Focus on quality when it comes to the clothes on your back. Think J.CREW, Banana Republic, or better, Steinberg suggests. She's going to be more attracted to you if you look good. And while you may not be able to tell where she bought her dress, she can read the fit of your suit.
Act like an anthropologist. As Wedeman says, "Don't assume that what's stylish in your background will be stylish in another." Maybe your big Italian family screams to communicate, and that doesn't fly in her house. Next time you're in her environment, sit back and watch how those close to her act so that you can behave appropriately, says Wedeman.
Put yourself in her world. Love tennis? Grab a few racquets and play together at her courts, not yours. "If you spend time doing something you like in her environment, you'll feel more comfortable there." She'll appreciate the effort, and you'll be enjoying yourself. If her world may be yours some day, you've got to make it a little bit your own, Wedeman says.


Also exciting, it's your first time!
I love New York. There is just something about the #feels of it that really makes you feel alive. Okay, maybe that's a bit too much, but hopefully you'll get what I mean 14 days from now.
Seeing as it is your first time, I feel obliged to give you some tips or rules you should follow.
How many times have you bought a New York Cheesecake Gu pot? Now you can have the reeeeeeeal one. And the one from Juniors is the best.
Levain Bakery – yes, that is a cookie.
Right in the heart of all the big lights and billboards, is one of the most Instagrammable spot of the city that never sleeps. We've all seen this, the one with a yellow cab in the shot and a sign of some broadway show in the background. However tempting to just stand in the middle and not care as you're taking selfies, please be mindful of surroundings.
You know I love my musicals! And when it comes to great musicals, New York is spoilt for choice. (But so are we in London, really.) Last time in the city, my best friend and I splashed out to watch Lion King and it was amazing! I wonder which show I should drag you in to?
Well unless you're a museum buff, then by all means. I mean, it's always hard to decide which one to go.But if you are going to have to pick one, the iconic Met Museum is the way to go.
I will start sounding like a broken record but please go on free walking tours. They are the best way to explore any city. New York has plenty to choose from! I also booked myself in a couple of Viator tours that were amazing and great for value. I did about four tours on my own the last time and each time would end up having a group to have drinks/pizza with after.
It is a great way to see the city in a different way!
Like the Eiffel Tower is to Paris and Big Ben to London, the Statue of Liberty is that to the Big Apple. You would never go to Paris and not see the Eiffel Tower, would you? Even if you have been there twice.
P.S. If you forget how to hail a cab, just look at this beaut.
Don't: Think that the Flat Iron is a Steak house.
I love this park!! Jazz music playing everywhere, paintings displayed by artists, the different sections–there is just so much to it.
I can go on and on and on but I would like to leave some element of surprise for you.