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Prevalence of the "High-Pathogenicity Island" of Yersinia Species among Escherichia coliStrains That Are Pathogenic to Humans
S. Schubert, A. Rakin, H. Karch, E. Carniel, J. Heesemann
S. Schubert
Max von Pettenkofer Institut, 80336 Munich, 1 and
A. Rakin
H. Karch
Institut für Hygiene und Mikrobiologie, Universität Würzburg, 97080 Würzburg, 2 Germany, and
E. Carniel
Unité de Bactériologie Moléculaire et Médicale, Laboratoire des Yersinia, Institut Pasteur, 75724 Paris Cedex 15, France 3
J. Heesemann
The fyuA-irp gene cluster contributes to the virulence of highly pathogenic Yersinia (Yersinia pestis,Yersinia pseudotuberculosis, and Yersinia enterocolitica 1B). The cluster encodes an iron uptake system mediated by the siderophore yersiniabactin and reveals features of a pathogenicity island. Two evolutionary lineages of this "high pathogenicity island" (HPI) can be distinguished on the basis of DNA sequence comparison: a Y. pestis group and a Y. enterocolitica group. In this study we demonstrate that the HPI of the Y. pestis evolutionary group is disseminated among species of the family Enterobacteriaceae which are pathogenic to humans. It prevails in enteroaggregativeEscherichia coli and in E. coli blood culture isolates (93 and 80%, respectively), but is rarely found in enteropathogenic E. coli, enteroinvasive E. coli, and enterotoxigenic E. coli isolates. In contrast, the HPI was absent from enterohemorrhagic E. coli, Shigella, and Salmonella entericastrains investigated. Polypeptides encoded by the fyuA,irp1, and irp2 genes located on the HPI could be detected in E. coli strains pathogenic to humans. However, these E. coli strains showed a reduced sensitivity to the bacteriocin pesticin, whose uptake is mediated by the FyuA receptor. Escherichia strains do not possess thehms gene locus thought to be a part of the HPI of Y. pestis. Deletions of the fyuA-irp gene cluster affecting solely the fyuA part of the HPI were identified in 3% of the E. coli strains tested. These results suggest horizontal transfer of the HPI between Y. pestis and some pathogenic E. coli strains.
The pathogenicity of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica is determined by a 70-kb virulence plasmid, pYV (11, 38). Y. pestis carries two additional plasmids which are required for full virulence: a 100-kb plasmid encoding capsular antigen fraction 1 and mouse exotoxin and a 9.5-kb plasmid coding for the bacteriocin pesticin and a plasminogen activator (35, 36). Y. pestis, Y. pseudotuberculosis serotype O1, and Y. enterocoliticabiotype 1B strains possess a chromosomal determinant that is involved in iron uptake mediated by the siderophore yersiniabactin (7, 22,23). This iron acquisition determinant, recently designated the "high-pathogenicity island" (HPI), is absolutely necessary for expression of the trait of virulence to mice (9, 16).
In Y. pestis, the HPI is known as the pgm locus, which comprises about 102 kb of chromosomal DNA and includes the following genes involved in iron storage and uptake: (i) the hemin storage (hms) locus (37); (ii) theirp1 and irp2 genes coding for the iron-repressible high-molecular-weight proteins HMWP1 and HMWP2, which presumably are involved in the production of yersiniabactin (28); and (iii) the fyuA or psn gene (for ferric yersiniabactin uptake or pesticin sensitivity) coding for the yersiniabactin receptor FyuA that also serves as a receptor for pesticin (9, 10, 23, 40). The 102-kb HPI of Y. pestis is flanked by single copies of the IS100insertion element. Homologous recombination between these two IS100 copies may be responsible for the deletion of the intervening 102-kb DNA segment. In contrast to Y. pestis, highly pathogenic strains of Y. enterocolitica and almost all Y. pseudotuberculosis strains lack the hmslocus and therefore are pigmentation negative but do carry a 45-kb stretch of chromosomal DNA comprising the irp1-irp2 andfyuA genes (fyuA-irp gene cluster [Fig.1]) (4, 8, 34).
Physical map of the HPI of Y. enterocoliticaO8 WA-C with the presumed 5′ and 3′ borders of the HPI. Horizontal lines below the restriction map represent different subcloned DNA fragments (E6, E8.8, E3.5, and Cl8) which were used as probes in hybridization assays. Arrows and bars within the restriction map indicate the locations of the identified genes and repeated sequence (RS) elements, respectively. E, EcoRI; C, ClaI; p12H2 and p17A11, cosmid clones. The locations of the 5′-HPI probe and the adjacent asnT tRNA are indicated. The asterisk indicates an additional internal EcoRI site found only in theirp2 genes of Y. enterocolitica biotype 1B strains.
The irp1-irp2-fyuA fragment is unstable in Y. enterocolitica and Y. pseudotuberculosis. In Y. enterocolitica, this instability may be due to the IS1328 insertion element or to other insertion elements adjacent to fyuA which have been recognized recently (8, 41). DNA sequencing of irp2 andfyuA revealed a G+C content of about 60 and 56 mol%, respectively, much higher than the G+C content of the wholeYersinia chromosome (46 to 50 mol%). These data suggest that pathogenic Yersinia spp. have acquired their HPIs from an organism with a higher G+C content by horizontal transfer.
Recently, we have shown that some colicin-producing E. colistrains carry genes with sequences nearly identical to those of theirp2 and fyuA genes of Yersinia spp. (41). This observation prompted us to investigate the possible dissemination of the HPI of Yersinia spp. (Yersinia HPI) among species ofEnterobacteriaceae pathogenic to humans. In this study, we used PCR and Southern hybridization assays to screen for the presence of irp2 and fyuA sequences within a panel of five established pathotypes of E. coli(enterotoxigenic E. coli [ETEC], enteroaggregativeE. coli [EAEC], enteropathogenic E. coli[EPEC], enterohemorrhagic E. coli [EHEC], and enteroinvasive E. coli [EIEC]) as well asShigella species and certain serotypes of Salmonella enterica. Additionally, we screened E. coli strains isolated from blood cultures of inpatients and stool samples of healthy individuals.
Surprisingly, the irp2-fyuA gene cluster was detected in more than 93% of EAEC strains, about 27% of EIEC strains, and 5% of ETEC and EPEC strains but in none of the EHEC, Salmonella, or Shigella strains studied.
Bacterial strains.We analyzed 220 E. colistrains, 24 S. enterica strains, and 14 Shigellastrains of different species (Table 1). The E. coli strains included 135 strains of the five established enteric pathotypes, EPEC (serotypes O111, O119, O127, and O157), EIEC (O124, O143, and O145), ETEC (O6, O25, O128, and O148), EHEC (O157:H7, O26, O55, and O111), and EAEC (O44, O86, O119, and O125), which were obtained either from E. coli strain collections of the national reference centers of Salmonellaand other enteric bacteria (Robert Koch Institut, Berlin, and Hygienisches Institut, Hamburg, Germany) or from children with diarrhea from different countries, partially listed in reference 44. The entericE. coli pathotypes were confirmed by PCR amplification with specific primers for virulence genes as described previously (17,18, 27, 33, 43). Additionally, all EAEC strains were proved to be enteroaggregative by the HEp-2 cell assay (12, 45, 47). EAEC 17-2 has been described previously (29, 31, 48) and was kindly provided by J. P. Nataro (Center for Vaccine Development, Baltimore, Md.). The toxin production of ETEC and EHEC strains was tested by enzyme-linked immunosorbent assay, and the invasive phenotype of EIEC strains was confirmed by invasion assays (5).
Presence of fyuA and irp2 genes in different pathogenic species of Enterobacteriaceae isolated from humans
In addition, 60 E. coli strains were isolated from blood cultures of inpatients at the university hospitals of Würzburg and Munich, Germany (serotypes of blood culture isolates were O2, O4, O6, O11, O25, O40, O54, and O125). Twenty-five E. colistrains were isolated from stool samples of healthy individuals. It was shown by PCR that the E. coli isolates from both inpatient blood cultures and stool samples of healthy individuals did not belong to any of the established E. coli enteric pathotypes.
Isolation of plasmid and genomic DNAs.Plasmid DNA for large- and small-scale preparations was extracted by the alkaline lysis method (1). Large-scale plasmid preparations were further purified with anion-exchange resin columns (Macherey-Nagel, Düren, Germany). For EAEC strains, it turned out to be advantageous to wash the bacterial cell pellet twice in phosphate-buffered saline before starting the alkaline lysis. Bacterial genomic DNA was isolated by a lysozyme-sodium dodecyl sulfate (SDS)-proteinase K method (1) and further purified by phenol and chloroform extractions.
Hybridization and DNA probes.Restriction enzyme-digested genomic and plasmid DNA fragments were resolved through 0.8% agarose gels, and DNA was transferred to Zeta-Probe BT blotting membranes (Bio-Rad Laboratories) with a vacuum blotter (Pharmacia, Freiburg, Germany), modifying the method of Southern (46). In order to generate DNA probes of the Yersinia HPI, two overlapping cosmids of a genomic library from Y. enterocolitica O8 WA-C (40) covering the entire Yersinia HPI (Fig. 1) were subjected to endonuclease digestion. Three EcoRI fragments and one ClaI fragment were subcloned into the pBluescript KS vector, resulting in plasmids E3.5, E8.8, E6, and Cl8, respectively (Fig. 1). These cloned fragments of theYersinia HPI were used as the HPI probes in the hybridization assays. Digoxigenin labeling of the probes and hybridization were performed with a DNA labeling and detection kit (Boehringer, Mannheim, Germany). After prehybridization at 68°C for 2 h and addition of heat-denatured probe, the blots were incubated overnight at 68°C in the absence of formamide. The detection was performed according to the manufacturer's instructions.
PCR.Different sets of primers used for PCR amplification were synthesized by Carl Roth (Karlsruhe, Germany). PCR amplifications were performed in an automated thermal cycler (TRIO Thermoblock; Biometra, Göttingen, Germany) as described by Saiki et al. (42) with TaqI polymerase and different pairs of oligonucleotide primers. The initial denaturation step (94°C, 5 min) was followed by 35 cycles of denaturation (94°C, 1 min), annealing (at the annealing temperature [Tm ], 1 min), and extension (72°C, 1 min), with one final extension step (72°C, 8 min). The sequences of the forward (FP) and reverse (RP) primers used for PCR reactions, the size of the amplified fragment (S), andTm were as follows: (i) irp2 (FP), 5′-AAGGATTCGCTGTTACCGGAC-3′; irp2 (RP), 5′-TCGTCGGGCAGCGTTTCTTCT-3′ (S, 280 bp;Tm , 61°C); (ii) irp2-P242 (FP), 5′-AAGGATTCGCTGTTACCGGAC-3′; irp2-P505 (RP), 5′-TCGTCGGGCAGCGTTTCTTCT-3′ (S, 264 bp;Tm , 58°C); (iii) fyuA (FP), 5′-GCGAC GGGAAGCGATTTA-3′; fyuA (RP), 5′-CGCAGTAGGCACGATGTTGTA-3′ (S, 780 bp;Tm , 60°C); (iv) hmsR (FP) 5′-TAAAGAAAGACCCCACCAATC-3′, hmsR (RP), 5′-ATCATCGGCATCAAGCAAATC-3′ (S, 730 bp;Tm , 56°C); entF (FP), 5′-TATCAGCGTTATCACCATTTG-3′, entF (RP), 5′-CCAGTTCCGGCAGCGTTTCTT-3′ (S, 511 bp;Tm , 55°C); and Ec-chrom (FP), 5′-TTTATTCCGTTGCGTGAGGTT-3′, HPI-5end (RP), 5′-TAGGATACCTTCACGCTGCTGTCGCGC-3′ (S, 900 bp;Tm , 52°C).
Sequence analysis of the fyuA gene of EAEC 17-2.In order to sequence the fyuA genes of E. colistrains, overlapping PCR fragments were generated with primers which were derived from the Y. enterocolitica fyuA gene as described previously (41). The PCR products were purified with anion-exchange resin columns (Quiagen, Hilden, Germany). A total of 100 ng of purified PCR product was subjected to Taqcycle-sequencing reactions with the Prism Ready Reaction dye dideoxy terminator cycle-sequencing kit (Applied Biosystems, Darmstadt, Germany) according to the manufacturer's instructions. Electrophoresis of sequencing products was performed on 7% polyacrylamide gels with an automated sequencer (model 373A; Applied Biosystems). The nucleotide sequences were analyzed with the ANALYSIS program version 1.2 (Applied Biosystems) and the DNASIS program version 2.0 (Hitachi Software Engineering Co., Tokyo, Japan).
Detection of FyuA, HMWP2, and HMWP1.Whole cells ofYersinia and E. coli strains growing under iron-deficient conditions were incubated for 1 h with35S-labeled amino acids (Amersham, Braunschweig, Germany) (15). Labeled proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE). Dried gels were exposed to Kodak BioMax MR film at room temperature.
For immunoblotting, ultrasonicated bacterial cell pellets were treated with 2% Triton X-100 and the insoluble membrane material was purified and subjected to SDS-PAGE as described previously (20, 23). After electrotransfer to polyvinylidene difluoride membranes (Millipore, Eschborn, Germany), FyuA was detected by antiserum (anti-FyuA) raised against FyuA of Y. enterocolitica O8 Y1852 in rabbits (23). HMWP1 and HMWP2 were detected with an antiserum raised against purified HMWP1 and HMWP2 of Y. enterocolitica O8 Ye 8081 in mice (7). Goat anti-rabbit and goat anti-mouse antibodies conjugated to alkaline phosphatase (Sigma Chemical Co., St. Louis, Mo.) were employed as second antibodies, and the reaction was developed with Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (21).
Pesticin sensitivity test.The pesticin assay was performed as described elsewhere (13, 23). Briefly, the pesticin-producing strain Y. pestis EV76 was grown overnight at 26°C, and pesticin production was induced by an additional incubation of 16 h in the presence of 0.3 μg of mitomycin C ml−1. The cells were collected by centrifugation, and the supernatant was used as a crude pesticin preparation after sterilization with 0.1% chloroform (25). Sensitivity to pesticin was monitored by serial dilutions (1:2) of crude pesticin preparations on mid-log-phase bacterial cultures by a double-layer technique. The plates were incubated at 37°C for 16 to 18 h.Y. enterocolitica Y8081 and Y. pseudotuberculosisY-P-I were used as reference pesticin-sensitive strains (25).
Survey for the presence of irp2 and fyuAgenes among E. coli strains pathogenic to humans.The results of the survey with PCR to detect the irp2 andfyuA genes in E. coli strains pathogenic to humans are shown in Table 1. The irp2 gene was detected in 93% and the fyuA gene was detected in about 92% of EAEC strains. Among EIEC strains, 20% yielded an amplified irp2fragment and 13% yielded a fyuA fragment. Five percent of EPEC and ETEC strains harbored the irp2 and fyuAgenes. In contrast, no irp2 or fyuA gene was detectable in EHEC, Salmonella, or Shigellastrains tested in this study. Additionally, E. coli strains isolated from blood cultures of inpatients were subjected to the same PCR assays. Surprisingly, in 83% of the isolates from blood cultures the irp2 gene was detectable by PCR (Table 1). In contrast, the irp2 and fyuA genes were detectable in only 32 and 28%, respectively, of E. coli strains isolated from stool samples of healthy individuals (n = 25).
DNA hybridization assays were subsequently performed with chromosomal DNA of E. coli strains in which a fragment ofirp2 was amplified by PCR.
To determine the location of the E. coli fyuA-irp gene cluster, both plasmid and chromosomal DNAs of EAEC, ETEC, EIEC, and EPEC strains were purified, digested with EcoRI endonuclease, and used for Southern hybridization with probes E3.5 and Cl8 (Fig. 1). Corresponding DNA restriction fragments were detectable only in E. coli chromosomal DNA preparations.
Probes E6, E8.8, and E3.5, covering the central part of theYersinia HPI (Fig. 1), were used to evaluate the degree of conservation of the fyuA-irp gene cluster among differentE. coli strains. Forty-seven of 57 E. colistrains which were positive for irp2 and fyuA by PCR displayed Southern hybridization patterns identical to those ofY. pestis and Y. pseudotuberculosis but different from that of Y. enterocolitica O8 (WA-314, biotype 1B). This difference is due to an additional internal EcoRI restriction site present in the irp2 gene of Y. enterocolitica biotype 1B strains but absent from otherirp2-positive Yersinia strains (Fig. 1). TenE. coli strains positive for irp2 andfyuA by PCR showed a slightly different hybridization pattern, which is apparently due to an additional EcoRI restriction site in the DNA fragment corresponding to E8.8 (Fig. 1 and2). Interestingly, this hybridization pattern was shared by E. coli strains which belonged to different pathotypes.
Southern hybridizations of EcoRI-digested genomic DNA of Y. enterocolitica and different E. coli strains with four DNA fragments, E6, E8.8, E3.5, and Cl8 (as shown in Fig. 1), representing the central part of theYersinia HPI. Lane 1, E. coli DH5α; lane 2,Y. enterocolitica O8 WA-C; lane 3, Y. pseudotuberculosis O1; lane 4, EAEC 17-2 (HPI positive); lane 5, EPEC 12-1; (HPI positive); lane 6, EIEC E12860 (HPI positive); lane 7, ETEC H488/84 (HPI positive); lane 8, E. coli 14094/96 from blood culture; (HPI positive); lane 9, EAEC DMI 155 (HPI positive); lane 10, EIEC H823/88 (HPI positive); lane 11, EHEC 1249/87 (HPI negative); and lane 12, EAEC 4827/94; (HPI negative).
Five E. coli strains which have been shown to beirp2 positive and fyuA negative by PCR gave no hybridization signal with E3.5 and E8.8 probes located in thefyuA region (i.e., the 3′ part of the fyuA-irpgene cluster) but revealed a hybridization signal with an E6 probe, indicating that all of the deletions affected the fyuAregion. To examine possible variations of the irp2 region (i.e., the 5′ region) of the fyuA-irp gene cluster, a hybridization analysis was performed with a probe derived from a 0.9-kbBglII-SacII DNA fragment which was previously shown to be located at the 5′ border of the Yersinia HPI in the Y. enterocolitica O8 strain Ye 8081 (8) (Fig.1). This probe hybridized to a single EcoRI chromosomal fragment in Y. enterocolitica WA-C and allirp2-positive E. coli strains used in this study (data not shown). The sizes of these fragments varied from 4 to 12 kb, suggesting a different position of the EcoRI restriction site located outside the fyuA-irp gene cluster. No signal was detectable in irp2-negative E. coli strains used as controls.
As mentioned above, the size of the HPI of Y. pestis differs from those of other Yersinia spp., and this HPI is thought to be composed of two parts, the fyuA-irp gene cluster and the hemin storage (hms) locus, respectively. In order to determine whether irp2-positive E. colistrains possess genes homologous to hms of Y. pestis, hybridizations with a probe derived from thehmsR gene of Y. pestis were performed. This probe did not hybridize either with the irp2-positive E. coli strains or with Y. enterocolitica O8 WA-C but recognized the corresponding fragment in Y. pestis (data not shown).
Sequence analysis of the fyuA-irp gene cluster inE. coli. In order to determine the nucleotide sequence conservation of the fyuA gene of E. coli, PCR-amplified fyuA DNA fragments of EAEC 17-2 were sequenced and compared with Yersinia fyuA sequences. ThefyuA gene of strain 17-2 has 99.6, 99.8, and 98.8% identity with those of Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, respectively. Comparing the nucleotide substitutions of the fyuA genes from EAEC andY. pestis, only five of nine nucleotide substitutions caused amino acid mismatches. The G+C content of the fyuA gene was 56.2 mol%, which is much higher than the overall G+C content ofE. coli (48 to 52 mol%) or Y. enterocolitica (46 to 50 mol%) chromosomes.
To investigate the conservation of the irp2 gene, a small fragment which, in Y. enterocolitica, is located at the beginning of the irp2 gene (bp 242 to 526) was sequenced in EAEC 17-2 and EIEC E12860. The sequences of the EIEC E12860 and EAEC 17-2 irp2 fragments were identical and had 99.7, 99.7, and 98.6% identity with those of Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, respectively.
In addition, the linkage of the Yersinia HPI toasnT tRNA in E. coli was demonstrated by PCR analysis. We have generated a PCR primer derived from the E. coli chromosome adjacent to the asnT tRNA gene. This FP (Ec-chrom) was used for PCR in combination with a RP (HPI-5end) located at the 5′ end of the Yersinia HPI. This RP was derived from the 0.9-kb BglII-SacII DNA fragment which was previously used as a 5′-border probe. By means of this 5′-border PCR, we screened a panel of 10 HPI-positive E. coli strains. All of the strains revealed single PCR products of the same size. We have sequenced these PCR fragments and found the Yersinia HPI to be directly linked to the asnT tRNA gene in these E. coli strains (data not shown).
Presence of HMWP1, HMWP2, and FyuA proteins in E. colistrains.By immunoblot analysis with anti-FyuA rabbit serum, FyuA was detectable in the outer membrane fraction offyuA-positive E. coli strains (Table 1 and Fig.3). Since FyuA has been shown to function as a pesticin receptor (23), the sensitivity of FyuA-positive E. coli strains to pesticin was tested. A moderate degree of pesticin sensitivity was detected in some FyuA-positive E. coli strains. However, the diameter of the inhibition zone was smaller and the halo appeared more turbid than those obtained with the pesticin-sensitive strain Y. pseudotuberculosis Y-P-I. The majority of FyuA-positive and allfyuA-negative E. coli strains turned out to be insensitive to pesticin under the test conditions.
Immunoblot of outer membrane proteins probed with anti-FyuA rabbit serum. Lane 1, Y. enterocolitica O8 WA-C; lane 2, E. coli 14094/96 from blood culture (HPI positive); lane 3, EAEC 17-2 (HPI positive); and lane 4, EAEC 4827/94 (HPI negative).
Additionally, cell lysates of EAEC cultivated under iron-restricted conditions were analyzed by SDS-PAGE and immunoblotting with mouse anti-HMWP1 and anti-HMWP2 antiserum (7). E. colistrains harboring irp1 and irp2 genes were shown to express the corresponding iron-repressible HMWP1 and HMWP2 (Fig.4). However, in some E. colistrains, HMWP1 and HMWP2 were not detected, even though theirp1 and irp2 genes were detected by PCR and DNA hybridization.
(A) Immunoblot of outer membrane proteins probed with anti-HMWP1 and anti-HMWP2 mouse serum. Lanes: 1, Y. enterocolitica O8 WA-C; 2, Y. pseudotuberculosis O1; 3,E. coli DH5α; 4, EAEC 4827/94 (HPI negative); 5, EPEC 2403/85 (HPI negative); 6, EAEC 17-2 (HPI positive); 7, EPEC 12-1 (HPI positive); 8, EIEC E12860 (HPI positive); 9, ETEC H488/84 (HPI positive); 10, E. coli 14094/96 from blood culture (HPI positive); and 11, EHEC 1249/87 (HPI negative). (B) Fluorographs of a polyacrylamide gel containing 35S-labeled proteins fromE. coli DH5α (lane 1), Y. enterocolitica O8 WA-C (lane 2), EPEC 2403/85 (HPI positive) (lane 3), EIEC E12860 (HPI positive) (lane 4), EAEC 17-2 (HPI positive) (lane 5), and EAEC 4827/94 (HPI negative) (lane 6).
The fyuA-irp gene cluster identified primarily in yersiniae meets the basic criteria of so-called pathogenicity islands, such as (i) atypical G+C content of the 45-kb chromosomal DNA region, (ii) requiring a cluster of genes for virulence, (iii) the presence of "mobility" genes (e.g., insertion elements) as well as association with a tRNA gene at one boundary, and (iv) instability (8, 14,39). Moreover, we recently demonstrated that thefyuA-irp gene cluster comprises two evolutionary lineages, one assigned to Y. enterocolitica biotype 1B strains and the other to Y. pestis and Y. pseudotuberculosis. Surprisingly, the fyuA-irp gene cluster of the latter lineage could also be detected in four pesticin-sensitive E. coli strains, indicating that it might be transmissible (41). Therefore, we investigated the distribution of thefyuA-irp gene cluster among some pathogenic species of the family Enterobacteriaceae.
We demonstrated that the fyuA-irp gene cluster is absent inShigella, S. enterica serotypes Enteritidis and Typhimurium, and Shiga toxin-producing E. coli (EHEC). The gene cluster is infrequently detected in E. coli strains of EPEC, ETEC, and EIEC pathotypes. Strikingly, the EAEC pathotype, which causes acute and chronic diarrhea in infants (26), frequently harbors a chromosomal fyuA-irp gene cluster. Nearly 93% of the 60 EAEC strains studied were irp2positive. This result prompted us to examine the prevalence of thefyuA-irp gene cluster in clinical E. coliisolates from blood cultures of hospitalized patients. Most of theE. coli blood culture isolates (80%) carried thefyuA-irp gene cluster. In contrast, less than one-third ofE. coli strains isolated from stool samples of healthy individuals possessed the fyuA and irp genes.
This high prevalence of the fyuA-irp gene cluster among isolates of E. coli from humans raises the question of why, so far, pesticin-sensitive E. coli strains have been considered rare. By immunoblotting, we were able to demonstrate thatirp1, irp2, and fyuA are expressed by almost all E. coli strains carrying the fyuA-irpgene cluster. However, only some fyuA-positive strains showed low-level sensitivity to pesticin, while the majority of FyuA-HMWP-positive E. coli strains were insensitive to pesticin. This may be due to point mutations of the fyuAgene or other, e.g., posttranslational, modifications of the FyuA protein in these E. coli strains. On the other hand, the pesticin receptor FyuA may be not accessible to the 40-kDa pesticin polypeptide due to masking of the receptor by surface components, e.g., the capsule or pili. Nevertheless, one can assume that E. coli strains carrying the fyuA-irp gene cluster are able to produce the siderophore yersiniabactin, which might contribute to the virulence of these E. coli isolates. This attractive hypothesis has to be verified by comparing the virulence of the parental strain and of the isogenic fyuA and irpmutants in a suitable infection model.
Sequencing of the fyuA gene of EAEC 17-2 and internal fragments of irp2 of various E. coli strains indicated that the highest homology was found with the fyuAand irp2 genes of the Y. pestis-Y. pseudotuberculosis lineage group. This observation was supported by Southern hybridization of EcoRI-digested chromosomal DNA from E. coli strains of different pathotypes, using probes covering the entire fyuA-irp gene cluster (Fig. 2). Moreover, we could demonstrate that, as with Yersinia, the HPI in E. coli is linked to the asnT tRNA gene. From this, we conclude that the fyuA-irp gene cluster might be a mobile genetic element which has spread by horizontal transfer through different genera of the family Enterobacteriaceaebut could be only maintained in certain pathotypes, such asYersinia species virulent in mice and enteroaggregative and septicemic E. coli isolates, which probably colonized the intestines of these patients.
As with Y. enterocolitica, we could not detect thehms locus of Y. pestis infyuA-irp-positive E. coli isolates. This is not surprising, since the hms locus was shown to be important for Y. pestis to block the flea proventriculus (24). Therefore, one possible explanation would be that the original HPI of Y. pestis, composed of the hmslocus and the fyuA-irp gene cluster, spread by horizontal transfer into the family Enterobacteriaceae, followed by a deletion of the hms region. However, a more likely hypothesis is that the fyuA-irp gene cluster forms a mobile element that can spread independently of the hms region. The different G+C contents of the two loci (47 mol% for hmsversus 56 to 59 mol% for fyuA-irp) and the distinct codon usage argue for the latter hypothesis (16).
Certain E. coli strains (up to 3% ofirp2-positive strains) carry a truncated fyuA-irpgene cluster, with deletions proceeding from fyuA or bothfyuA and irp1 to the 3′ end of the HPI. Such spontaneous deletions involve only the 3′ part of thefyuA-irp gene cluster and abrogate expression of the yersiniabactin receptor. These E. coli isolates may have lost the receptor after adaptation to a new environment. AllE. coli strains used in this study possess the highly efficient enterobactin siderophore system (detected by PCR [data not shown]) (3, 32). Therefore, it seems unlikely that iron acquisition is the primary reason for the wide dissemination of yersinia genes among E. coli strains pathogenic to humans. It is rather tempting to suggest that other effects of gene products of the fyuA-irp gene cluster are of benefit for certainE. coli pathotypes. The possible cytotoxic effect on T cells of the siderophore desferrioxamine B (2) could be an attractive alternative to the yersiniabactin function. Interestingly, it has been shown that the yersiniabactin-like siderophore pyochelin ofPseudomonas aeruginosa promotes damage to endothelial cells by formation of free radicals (6). On the other hand, theirp2 gene product, HMWP2, has extensive similarity to a superfamily of adenylate-forming enzymes involved in the nonribosomal peptide synthesis of not only siderophores but also peptide antibiotics (19). Therefore, it is also conceivable that other products of the putative enzyme HMWP2 might be of benefit to the E. coli strains that carry the fyuA-irp gene cluster.
It remains unclear whether multiple insertion elements located within the fyuA-irp gene cluster are responsible for the observed deletions. However, genetic instability of the HPI is well known inY. pestis, Y. pseudotuberculosis, and Y. enterocolitica, causing spontaneous loss of iron acquisition in vivo and consequently virulence attenuation. Thus, strong selective pressure is necessary for the maintenance of HPIs. By analogy with yersiniae, we can suggest that the fyuA-irp gene cluster may also contribute to the virulence of certain clinically importantE. coli pathotypes, such as EAEC and septicemia-causingE. coli strains. The reason for the high-frequency distribution of the fyuA-irp gene cluster among EAEC strains remains unknown. The genes located on this cluster are apparently not involved in the EAEC phenotype, since we could detect EAEC strains that exhibit the aggregative adherent (AA) phenotype in HEp-2 cell assay and lack the fyuA-irp gene cluster. Moreover, Nataro et al. (30) have shown that this phenotype is closely associated with the presence of a 60-MDa plasmid which encodes bundle-forming fimbriae. It has been shown that cloning of two distinct parts of this plasmid is sufficient to express the AA phenotype in an E. coli HB101 strain that lacks the fyuA-irp gene cluster.
Taken together, these results indicate that (i) the fyuA-irpgene cluster of Yersinia spp. is largely conserved among different HPI-positive E. coli strains even though some distinct changes of the hybridization patterns exist; (ii) deletions of the fyuA-irp gene cluster in E. coli are of different sizes and involve the right-hand side of the HPI, including the fyuA locus; and (iii) the fyuA-irp gene cluster of E. coli strains is more closely related to the HPIs of Y. pestis and Y. pseudotuberculosis than to the HPI of Y. enterocolitica serotype O8. However, the exact function of the fyuA-irp gene cluster in E. coli strains still remains unclear, and our future work will focus on the effect of irp2 and fyuA mutations on the virulence of pathogenic E. coli strains.
We thank Daniela Fischer for excellent technical assistance.
This study was supported by a grant from the Deutsche Forschungsgemeinschaft to J.H. (HE 1297/2-3).
Received 28 August 1997.
Returned for modification 15 October 1997.
Accepted 25 November 1997.
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Q: onClick anchor tag a current div display using jquery I'm working on Image gallery here I want onClick anchor tag  current image display in .gallery-container is not working. Previously on image img click, I got the output .gallery-column img.
$('.gallery-column img').on('click', function(){
    var expandImg = document.getElementById('expandedImg');
    expandImg.src = this.src;
    expandImg.parentElement.style.display = "block";
    var imageCaption = $(this).next("div").html();
    $("#expandedImgCaption").html(imageCaption);
});

This code is working fine. Now I have added anchor  it is not working.
 I have tried but unable to achieve.
Can anyone suggest me?

$(document).ready(function() {
  var carouselImg = document.getElementsByClassName('carousel-img');
  $('a.post-carousel').on('click', function() {
    var expandImg = document.getElementById('expandedImg');
    var carouselImg = document.getElementsByClassName('carousel-img');
    expandImg.src = carouselImg.src;

    expandImg.parentElement.style.display = "block";
    var imageCaption = $(this).next("div").html();
    $("#expandedImgCaption").html(imageCaption);
  });
});
* {
  box-sizing: border-box;
}

.gallery-wrap {
  width: 50%;
  height: 1066px;
  margin: 0 auto;
  display: flex;
}

.gallery-row {
  width: 52%;
  max-height: 497px;
}

.gallery-column {
  position: relative;
  padding: 10px;
}

.img-caption {
  position: absolute;
  bottom: 10%;
}

.gallery-column img {
  width: 100%;
  opacity: 0.8;
  cursor: pointer;
}

.gallery-column img:hover {
  opacity: 1;
}

.gallery-container {
  position: relative;
  width: 90%;
  height: 500px;
}

#expandedImg {
  width: 100%;
  height: 100%;
}

Still more than 2 Millions+ people using

Still more than 2 Millions+ people using

olor sit amet, consectetur adipiscing elit. Donec

sit amet, consectetur adipiscing elit. Donec

nsectetur adipiscing elit. Donec

A: Try using this like the following way:

$(document).ready(function() {
  var carouselImg = document.getElementsByClassName('carousel-img');
  $('a.post-carousel').on('click', function() {
    var expandImg = document.getElementById('expandedImg');
    expandImg.src = $(this).find('img').attr('src');

    var imageCaption = $(this).find('.img-caption > h3').text();
    $('.gallery-container').find('.img-caption > h3').text(imageCaption);
  });
});
* {
  box-sizing: border-box;
}
.gallery-wrap {
  width: 50%;
  height: 1066px;
  margin: 0 auto;
  display: flex;
}

.gallery-row {
  width: 52%;
  max-height: 497px;
}

.gallery-column {
  position: relative;
  padding: 10px;
}

.img-caption {
  position: absolute;
  bottom: 10%;
}

.gallery-column img {
  width: 100%;
  opacity: 0.8;
  cursor: pointer;
}

.gallery-column img:hover {
  opacity: 1;
}

.gallery-container {
  position: relative;
  width: 90%;
  height: 500px;
}

#expandedImg {
  width: 100%;
  height: 100%;
}

Still more than 2 Millions+ people using

Still more than 2 Millions+ people using

olor sit amet, consectetur adipiscing elit. Donec

sit amet, consectetur adipiscing elit. Donec

nsectetur adipiscing elit. Donec

A: That could be simply achieved using the this keyword to target the related .carousel-img.
NOTE 1: You've missed to attach the id expandedImgCaption to the img-caption of expandedImg:

NOTE 2: You don't need the next() since the div is inside the anchor not next of it :
$("#expandedImgCaption").html($('.img-caption', this).html());

$(document).ready(function() {
  $('a.post-carousel').on('click', function() {
    $('#expandedImg').attr('src', $('.carousel-img', this).attr('src')).parent().show();
    $("#expandedImgCaption").html($('.img-caption', this).html());
  });
});
* {
  box-sizing: border-box;
}

.gallery-wrap {
  width: 50%;
  height: 1066px;
  margin: 0 auto;
  display: flex;
}

.gallery-row {
  width: 52%;
  max-height: 497px;
}

.gallery-column {
  position: relative;
  padding: 10px;
}

.img-caption {
  position: absolute;
  bottom: 10%;
}

.gallery-column img {
  width: 100%;
  opacity: 0.8;
  cursor: pointer;
}

.gallery-column img:hover {
  opacity: 1;
}

.gallery-container {
  position: relative;
  width: 90%;
  height: 500px;
}

#expandedImg {
  width: 100%;
  height: 100%;
}

Still more than 2 Millions+ people using

Still more than 2 Millions+ people using

olor sit amet, consectetur adipiscing elit. Donec

sit amet, consectetur adipiscing elit. Donec

nsectetur adipiscing elit. Donec

redpajama_set_name: RedPajamaStackExchange


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One of comics' rock 'n' roll superstars, Simon Bisley's incredible artwork changed the industry forever in the late 1980s with his work on Slaine: The Horned God and Judge Dredd, producing a myriad imitators and launching a decade-long fad for painted comics art. A unique and irrepressible talent, Simon's work has reached far beyond the page to influence mainstream culture, and while he has always evolved, his style remains timelessly recognizable. Simon sits down with Mike Molcher from 2000 AD to discuss his work, career, and philosophy.


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The lawyer of the U.S. President Donald Trump Rudolf Giuliani in an interview to Chuck Todd on NBC has said a phrase which the American edition Mashable suggests to consider "ideal for designation of the events of a nightmare on the Internet in 2018". Todd tried to achieve the answer to a question why Trump was allowed from Giuliani to communicate with Office of the special adviser regarding participation in alleged intervention of Russia in an election of the president of the USA. Trump's representative then expressed fear that the president can appear in "a perjury trap". "When you speak to me about it, you know that he has to testify, and he has nothing to worry because he will tell the truth, but it is silly because it is only someone's version of the truth, but not the truth" — Giuliani has said. "The truth is the truth" — Todd has interfered. "No, it is not the truth. The truth is not the truth" — the lawyer.Mashable has insisted reminds that Trump who long time attacked media, accusing them of distribution of counterfeit news about him, in February, 2017 declared journalists enemies of the people. It is more than hell and strange news in Tape of a Bottom Telegram-channel. Subscribe!


Aaron Paul, who got his starmaking turn as meth manufacturer Jesse Pinkman in Breaking Bad, said that he is in "serious talks" with show creator Vince Gilligan to appear in the upcoming Breaking Bad spinoff Better Call Saul, according to The Associated Press.
The actor was promoting his upcoming action film Need for Speed when Jesse Pinkman came up, as it presumably will in every interview Paul will give for years.
Last year, Gilligan revealed that the spinoff was originally conceived as a half-hour sitcom before he and his team settled on an hour-long format. "There's obviously a danger inherent in doing a spinoff, but I just love the character of Saul Goodman so much, and part of me doesn't want to say no to this world," Gilligan said.
Better Call Saul features Bob Odenkirk as shady lawyer Saul Goodman and is set to premiere on AMC in November.


text:
Olympic champion (Retired)
Born: 1986 Jamaica
Annual: US$0.00
Monthly: US$0.00
Weekly: US$0.00
Daily: US$0.00
Earnings overview: - Express Oct 2018: Bolt retired from sprinting in 2017. According to Forbes, Bolt has an estimated net worth of $31million (£23.5m). He has more than a dozen sponsors. He won nine Olympic gold medals and set multiple world records. Bolt opened his third 'Tracks & Records' restaurant in Jamaica this year and has a contract in place to roll out up to 15 restaurants in the UK. - Forbes June 2017: $34.3 million - Forbes July 2016: $32.5 million - Forbes June 2015: $21 million - Forbes June 2014: $23.2 million - Forbes June 2013: $24.2 million - Forbes June 2012: $20.3 million - DailyMail: May 2012: "Although his finances are a closely guarded secret, he is believed to earn £8m-£10m a year from sponsorship, most of which are five-year deals that tie him up until the 2017 World Athletics Championships in London."
Text: Express
Text: Dailymail.co.uk
Text: Forbes


FIC director Dr. Roger Glass (c) receives the 2018 Geoffrey Beene Builders of Science Award from Beene Foundation executives Tom Hutton and Mara Hutton during the recent Research!America advocacy awards dinner.
Glass was cited for his leadership of NIH efforts to transform African medical education, establish research training programs in West African countries that saw widespread Ebola infection, expand Fogarty's Scholars and Fellows program for early-career scientists and help found the Global Alliance for Chronic Diseases. He was also recognized for his research on rotavirus, norovirus and cholera, and the recent WHO approval of a new low-cost rotavirus vaccine he helped develop over the last three decades.
The award was presented by Mara Hutton, vice president of the Geoffrey Beene Foundation, which was established to honor the late fashion designer and to support research in cancer and Alzheimer's disease. Hutton noted that Fogarty is the most leveraged organization at NIH, with nearly 90 percent of its grants receiving co-funding from other institutes or centers. She thanked Glass for his commitment to improving the health of the world's most vulnerable people.
Accepting the award, Glass acknowledged NIH director Dr. Francis Collins and other NIH leaders in the audience for their support of Fogarty's cross-cutting global health research and training programs.
NIAMS director Dr. Stephen Katz (r) receives the highest AAD award from the academy's immediate past president Dr. Henry Lim at AAD's 2018 annual meeting.
Katz has been director of NIAMS since August 1995 and was also a senior investigator in NCI's Dermatology Branch from 1974 to 2014. He has focused his research on immunology and the skin.
His work has demonstrated that skin is an important component of the immune system, both in its normal function and as a target in immunologically mediated disease.
Katz and his colleagues have studied Langerhans cells and epidermally derived cytokines, in addition to adding considerable new knowledge about inherited and acquired blistering skin diseases.


172 air-conditioned rooms. 1 king or 2 double beds. Bathroom with shower. Hair dryer. TV.
155 rooms located in 1 six-storey building. 1 king or 2 double beds. Bathroom. Hair dryer.
616 air-conditioned rooms located in 2 eight-storey buildings. Full bathroom. Hair dryer.


text:
<?php

/**
 * Konfig
 *
 * Yet another simple configuration loader library.
 *
 * @author  Xeriab Nabil (aka KodeBurner) <kodeburner@gmail.com>
 * @license https://raw.github.com/xeriab/konfig/master/LICENSE MIT
 * @link    https://xeriab.github.io/projects/konfig
 */

namespace Exen\Konfig\Test\FileParser;

use Exen\Konfig\FileParser\Php;

/**
 * Tests for Exen\Konfig\FileParser\Php.
 *
 * @package Test
 * @subpackage FileParser
 */
class PhpTest extends \PHPUnit_Framework_TestCase
{
    /**
     * @var Php
     */
    protected $php;

    /**
     * Sets up the fixture, for example, opens a network connection.
     * This method is called before a test is executed.
     */
    protected function setUp()
    {
        $this->php = new Php();
    }

    /**
     * Tears down the fixture, for example, closes a network connection.
     * This method is called after a test is executed.
     */
    protected function tearDown()
    {
        // Nothing to put here
    }

    /**
     * @covers Exen\Konfig\FileParser\Php::getSupportedFileExtensions()
     */
    public function testGetSupportedFileExtensions()
    {
        $expected = ['php', 'inc'];
        $actual = $this->php->getSupportedFileExtensions();
        $this->assertEquals($expected, $actual);
    }

    /**
     * @covers Exen\Konfig\FileParser\Php::parse()
     * @expectedException Exen\Konfig\Exception\UnsupportedFileFormatException
     * @expectedExceptionMessage PHP file does not return an array
     */
    public function testLoadInvalidPhp()
    {
        $this->php->parse(KONFIG_TEST_MOCKS . 'fail/error.php');
    }

    /**
     * @covers Exen\Konfig\FileParser\Php::parse()
     * @expectedException Exen\Konfig\Exception\ParseException
     * @expectedExceptionMessage PHP file threw an exception
     */
    public function testLoadExceptionalPhp()
    {
        $this->php->parse(KONFIG_TEST_MOCKS . 'fail/error-exception.php');
    }

    /**
     * @covers Exen\Konfig\FileParser\Php::parse()
     */
    public function testLoadPhpArray()
    {
        $actual = $this->php->parse(KONFIG_TEST_MOCKS . 'pass/config.php');
        $this->assertEquals('localhost', $actual['host']);
        $this->assertEquals('80', $actual['port']);
    }

    /**
     * @covers Exen\Konfig\FileParser\Php::parse()
     */
    public function testLoadPhpCallable()
    {
        $actual = $this->php->parse(KONFIG_TEST_MOCKS . 'pass/config-exec.php');
        $this->assertEquals('localhost', $actual['host']);
        $this->assertEquals('80', $actual['port']);
    }
}

// END OF ./tests/FileParser/PhpTest.php FILE

redpajama_set_name: RedPajamaGithub


Parents or guardians who would like a Parent Portal account should complete the Parent Portal Registration Form and return it to their child's building office, or to the Registrar at 15 Mountain Road in Ravena. Please be prepared to show your ID to the school staff receiving the form. If you have additional questions about the Parent Portal, contact Carol Eckl (518-756-5200, ext. 6014).
The RCS School District is committed to ensuring student privacy in accordance with local, state and federal regulations and district policies. To this end and pursuant to U.S. Department of Education regulations, the district is providing the following Parents' Bill of Rights for Data Privacy and Security.
PBIS is a team-based systematic approach in teaching behavioral expectations throughout the school. It is based on a proactive model which teaches the behaviors, reinforces and recognizes students who are able to model these behaviors and has systems in place to support students who have a difficult time or may present with more challenging behaviors. Read more about PBIS efforts at RCS.
Want a better understanding of Common Core Standards in New York?
Take a look at EngageNY, a helpful webpage aimed at giving you a better understanding Common Core.
The New York State Education Department releases School Report Cards on every public school and district in the state. Based on some key assessments taken by students in New York public schools, the report cards provide achievement data on districts and schools, as compared to their past years' performances and to other similar schools. Report cards are released each spring for the previous school year.
NYS mandates that all public school districts offer Academic Intervention Services (AIS) to any student who is falling behind in meeting state standards. Students are identified for AIS according to their performance on the state ELA, math, and science exams in grades 3-8. RCS students who score in Performance Levels 1 and 2 are eligible to receive AIS in the designated subject area.
RCS offers English language learners appropriate programming in English as Second Language (ESL) classes, at all grade levels K-12.
The RCS School District provides individualized instruction, accommodation of alternative learning styles, appreciation for diversity and promotion of healthy lifestyles. The district has specialized programs to assist and support all children in meeting their highest potential.
For information regarding special education, visit the Special Education page.


Canada has close to 900,000 km of roads, which are key to supporting the Canadian economy as they enable for efficient transportation of goods and services. The Canadian road network should be well maintained and perform to a high standard to ensure the function and growth of the economy and the Country. It is also important to consider sustainability, and more specifically, recycling, when designing the roads. In order to improve on the performance while being sustainable, research is being conducted on the feasibility of using recycled materials in asphalt pavements. One of these materials is crumb rubber, obtained from recycled rubber tires, used as an additive to conventional Hot Mix Asphalt (HMA) to produce Rubber Modified Asphalt (RMA). These tires would otherwise end up in landfills, which is undesirable when considering sustainability. The Ontario Tire Stewardship (OTS), along with the Centre for Pavement and Transportation Technology (CPATT), the Ontario Ministry of Transportation (MTO), and the Ontario Hot Mix Producers Association (OHMPA) have combined their efforts in order to carry out a pilot study on paving roads in Ontario with crumb tire rubber. Three highways, Highway 7, Highway 35, and Highway 115, have been paved using RMA, along with a section paved with conventional HMA to conduct a comparative study on the performance of the RMA sections. During the paving, samples were collected from the pavers in order to conduct laboratory testing on the materials to determine their performance. The material was used to test the performance using the Thermal Stress Restrained Specimen Test, which introduces a stress in the sample material by cooling it to the point of failure while restraining the specimen, preventing it from relaxing. The specimen is cooled until failure, and the temperature at failure, along with the stress in the specimen is recorded for comparison. This test was conducted to determine the ability of the mixes to withstand the low temperatures that pavements in Ontario would have to endure. A structural evaluation was also carried out on the pavement mixes using the AASHTOWare software (formerly the Mechanistic Empirical Pavement Design Guide, MEPDG). Although there are limitations to the software, it was still important to determine the structural capacity of the mix within a pavement structure to withstand the stresses of traffic and climate. The results of the AASHTOWare software determines the performance of the rubber mixes, as compared to the conventional mixes, to ensure that utilizing the rubber mixes does not hinder the performance of the pavement.


A separate cabinet with fuel test and recycling equipment is offered for optimal environmental control and safe operation. The cabinet is connected to the fuel storage tank and dispenser unit by tubing. The fuel can be sampled in a closed system, avoiding spill and unsafe operation. After a sample has been taken and controlled, the sample fuel will be stored in a tank in the cabinet. The jet fuel will be filtered and cleaned before it is transferred to a jet fuel storage tank.
Our basic aviation fuel recycle system design is based on a stainless steel drip tray/base frame and enclosed with an aluminium shelter of high offshore quality, equipped with sliding door. This gives the unit a minimal need for maintenance and the operations will be performed in a dry, sheltered and illuminated area.


TOTAL Defense & Jacksonville Brazilian Jiu-Jitsu offers Kids Jiu Jitsu classes with a unique and individualized curriculum so your child gets the training and attention they need to succeed.
Our well-known and experienced Jiu-Jitsu instructors teach our students the basic skills up to expert level Jiu-Jitsu and Judo techniques as they become the best martial artists they can be and conquer their fitness and martial arts goals.
TOTAL Defense's Mixed Martial Arts/Combatives program offers a real deal MMA program that can turn anyone, regardless of age, gender or previous experience into a quick and powerful fighting machine. We teach the most effective stand up and ground fighting techniques in the area.
My kids participated in the FREE Kids on Guard workshop here and it was an excellent program. The instructors were nice and they learned all about stranger danger and bully prevention. They offer it once a month and I can't recommend it enough.
First Coast Center for the Arts is a facility that provides activities for every family member no matter what age. We offer Dance, Karate, Music, Yoga, Pilates, Aerobic Dance.
JAX Cooking Studio offers hands-on cooking classes for adults, children, and teens. We combine nutrition and culinary skills to give you an experience you're sure to remember! Register at jaxcookingstudio.com/classes.
Titus Taekwon-Do is strictly here for the moral character development of the community and its children.
Zone Martial Arts is a fun and positive atmosphere where kids and adults can learn martial arts and get in shape while learning valuable life skills.
Yates Family YMCA is committed to youth development, healthy living and social responsibility. We offer several programs for both adults and children, including swim lessons, martial arts, personal training and group fitness at our gym.


text:
Q: How to solve $\arccos (\sin \frac{\pi}{8})$? I can't solve it, despite applying the method of solving using a right triangle as in $\cos (\arctan \frac {3}{ 5})$. The question asks for calculation.

A: I think it's good for beginners to always think of arc functions as angles.  I'd set
$$\arccos \sin \frac{\pi}{8} = \theta,$$  so that
$$\sin \frac{\pi}{8} = \cos \theta.$$
Now you can just realize that the "co" in "cosine" is short for "complementary", so $\theta$ is the complementary angle to $\frac{\pi}{8}$, that is $\theta = \frac{3\pi}{8}.$

A: For any $\theta\in\left[0,\frac{\pi}{2}\right]$ we have
$$ \arccos\sin\theta = \frac{\pi}{2}-\arcsin\sin\theta = \frac{\pi}{2}-\theta.$$

A: Hint:
$\sin\alpha=\cos(\frac12\pi-\alpha)$


text:
A Vegetarian Abroad: Gili Trawangan, Indonesia
Posted on June 28, 2017 by Becca
I've been vegetarian for most of my life. And while I can't speak about the specifics of meat production in other countries, I can speak with a semblance of knowledge about how it goes down in the ole U-S-of-A.* In America, the vast majority of meat comes from factory "farms," where animals are commodities: means to an end. As such, their lives are spent in space-efficient pens, and they're fed what's cheap: byproducts of subsidies like soy and corn, or worse: recycled parts of their fallen comrades. (If you think I'm exaggerating, check out the documentary "Food, Inc.") They are given antibiotics and hormones to ensure their meat stays juicy despite unnatural diet and unnatural living conditions.
More than this, factory farms contribute about 10% of our carbon emissions, and the number is growing as natural habitats–from rainforest to desert–are converted into pastures.** What's more, consuming meat from these animals posits dangers to our own health. Of course, large government subsidies to these farms means their affairs are kept largely hush-hush, a whisper drowned in boisterous campaigns advocating Meat! It's What's for Dinner, Goddammit! [Emphasis my own.] In fact, compared to every other world nation, American meat consumption ranks highest or second highest across the board (in the categories poultry, pork, veal, and beef).*** That's more than the [former] European Union combined. Thanks, propaganda!
But I digress. Believe it or not, the purpose of this post is NOT to mount my soap box nor to scold the omnivores in the house. My mission is quite the opposite, actually…
Strange & tasteless, the snake fruit
A week ago I arrived in Lombok, an island in Indonesia that gets largely overshadowed by its neighbor, Bali. Especially in the coastal towns and offshore islands, the native people have a distinct cuisine based on native crops: coconut, spicy pepper (and I mean spicy), tropical fruits, green beans, rice and, of course, the sea. I visited several markets where fish and seafood is brought in daily and offered to customers, charged per 100 grams and barbecued over a fire pit for you to eat on the spot–often whole. But instead of recoiling from such a display (as I might in America, scurrying past the meat aisle as though the butcher had her wily eye on me, next), I found it inspiring (aside from the Giardia I'm still coping with). Without the ocean, the locals wouldn't be able to survive, period. They rely on it for food, transport, supplies, diversion and livelihood. Though Muslim, they still show gratitude towards the gods of the sea and the creatures within it. And although they are grateful for the sea, they don't hesitate to consume its inhabitants. It would be a foreign concept.
Yeah, this just wouldn't fly in America.
A big disconnect in American culture is lack of appreciation for our food. In Japan, too, (where I was a few weeks ago, and where eating from the sea is like breathing air), food and the ocean and nature at large are shown respect–a bow before and after eating, before and after swimming, climbing a mountain, et cetera. In America, there seems to be a sense of entitlement for food, potentially due to a disconnect from our food sources. Have you ever seen an animal carcass at the grocery store? What about even a picture of the animal on the label of a meat package? Don't think, just do.
I wouldn't argue when someone says humans are "designed" to eat meat. Our ability to eat widely is what helped us to the top of the food chain. And, when animals are treated with respect, killed with compassion, and consumed for subsistence rather than desire, it becomes a very natural process. All things in moderation, of course.
Humans, however, are capable of subsisting without meat. We also are endowed with a conscience which allows us to choose what we will and will not eat. Such has been the practice of Buddhists, Jains, Hindus, Jews, and many other religions around the world for centuries. Ultimately this is the choice that I've made, too. So no, I did not eat the market meats, but only because years of vegetarianism-for-a-cause has lent me sympathy for the critters who happen to sit below us on the food chain. It was a personal choice more than a boycott-the-man one. Because despite my emotions, the process in these Indonesian markets feels very…natural.
To reiterate, I'm not preaching vegetarianism. Everyone has the right to choose what they eat and how they live. But until America in particular cleans up its act and learns some respect (which I believe will come hand in hand), I can only hope more people will cut back–in any small way–in their support of a tasteless industry. That, or move to Indonesia.
Contemporary Indonesian farming methods?
*Experience gained from extensive book-reading, documentary-watching and lecture-listening on the subject.
**Statistics from EPA 2015 official report.
***Statistics from OCED 2016 official report.
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Funding public education fairly isn't easy, and it has become harder with the introduction of school choice options that spread public education allocations to voucher programs and privately managed charter schools. School choice has made it downright difficult to ensure every child gets the same educational opportunities.
Milwaukee, Wisconsin has had years of experience with the kind of school choice reformers advocate. A state program has offered its students tuition vouchers for over 25 years; currently, 26,000 students use these public funds to pay their private school tuitions. The Milwaukee School District operates both traditional public schools and district-managed charter schools. Independently managed charter schools are also an option for Milwaukee students.
Yet even affluent public schools have fewer resources than many private schools. Raising money from parents and other outside donors allows them to do a better job than they otherwise would, resulting in better educations for some children in the public system than for others.
The Milwaukee School District plans for philanthropy to help make up for this inequity in funding. It is reinvigorating the school district's Foundation (formed in 1998) to solicit city businesses, residents, and former students for support. A newly appointed member of the MPS Foundation Board, Tina Chang, spelled out their vision of the challenge to the Milwaukee Journal Sentinel.
"This is all about making sure MPS children get the same opportunities as the most successful choice and charter schools," [Chang said,] which all have robust fund-raising arms. It's also about keeping up with suburban districts, many of which raise private funds to fill the gaps when state and local funding fails to keep pace with costs for staff, facilities and programming.
With a new board and staff in place, the Foundation hopes it can raise $850,000 this year. If they are successful, those funds will provide needed programs and services for the district's students. The district budget is nearly $1 billion dollars with "projected revenue down $1.3 million compared to the 2015-16 amended adopted budget." The new fundraising plans are as necessary as they are modest, given the funding gap.
Are parents and school districts willing to address the economic inequality of school fundraising?


Flowers are a timeless way to express your sympathy after the passing of a family member, friend, or colleague. We can create a wide range of sympathy flower arrangements, memorial tributes or supply even a single rose; appropriate to honouring the memory of your loved one.
We understand the importance of such a day for all involved, and we are devoted to assisting you at this difficult time. We offer a timed delivery service, and can deliver to a funeral directors of your choice, a home address or to the funeral service itself.
Please contact us at your convenience to discuss a fitting tribute.


text:
HomeClub NewsManchester United handed away FA Cup tie in Round Five
Manchester United handed away FA Cup tie in Round Five
January 29, 2018 Jordan Street Club News 0
Manchester United have been drawn away to either Huddersfield Town or Birmingham City in the FA Cup fifth round, the club has confirmed.
Jose Mourinho's men progressed comfortably into the next round after beating Yeovil Town 4-0; a game that was Alexis Sanchez's debut.
Now we're into the last sixteen and the draw is opening up nicely for the Red Devils and whoever wins the replay between Huddersfield and Birmingham, you'd have to fancy United to book their place in the last eight.
#MUFC will face Birmingham City or Huddersfield Town in the fifth round of the @EmiratesFACup. The tie will be played on the weekend of 16-19 February 2018. pic.twitter.com/zscm9UeyKX
— Manchester United (@ManUtd) January 29, 2018
If I were choosing, I'd love to go against Birmingham. It has been a few years since we've played the Championship side and it'd be an interesting tie to watch. However, taking revenge on Huddersfield Town in their own backyard would also be fun.
I love the FA Cup. It's a great tournament that is always excellent to win and no doubt United will be amongst the favourites to go all the way this season. Yes, they'll probably have to overcome Manchester City at some point, but you've got to beat the best to be the best…
The tie will be played over the weekend of 16-19th February.
Player Ratings: Sanchez stars in comfortable Manchester United 4-0 cup win
4-3-3: Manchester United predicted XI vs Spurs – Sanchez starts
Manchester United drawn at home in FA Cup third round
December 7, 2015 Jordan Street Club News 0
It feels as though every year I always find myself saying 'this year I'd love a good cup run', and shortly after that dream gets shattered. The draw for the third round of the FA […]
Player Ratings: West Ham 1-2 Manchester United
April 13, 2016 Jordan Street Player Ratings 0
David de Gea – Made a brilliant save from a West Ham corner, and some stunning saves in the closing moments of the game. 7/10 Tim Fosu-Mensah – What an outstanding talent this lad us. Absolutely unreal […]
Manchester United drawn away to Blackburn in the FA Cup Fifth Round
Well, Manchester United earned their place in the FA Cup draw, thanks to an impressive victory at home to Wigan Athletic. Jose Mourinho's men won the match 4-0, with Bastian Schweinsteiger getting himself on the […]


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text:
Valley Vibrations: Nation-touring band settles locally
Bobby Shipman
Be My Doppelganger was shocked when fans began to sing along to the lyrics of their songs at a show in New York City.
On their first cross-country tour, the "melodic rock" foursome had managed to attract fans from all over the country and even across seas.
At the first New York City gig, they met a group of Austrians who had planned their vacation to the U.S. around the show.
"Going to New York our first time was cool because there were people there to see us," said Matt Glick, vocalist and guitarist for Be My Doppelganger. "That was real surprising."
The band has played big city venues from San Diego to Seattle and Baltimore to Chicago on several national tours.
Be My Doppelganger got together in 2005 with intentions to stay solely in the studio. But after they began to play together, they decided to start doing shows.
After their start, Be My Doppelganger underwent a few member changes before they reached their present partnership.
Ryan Dunn plays guitar, Thomas Poole drums and Glick's brother Bradley plays bass and assists vocals.
Fans have referred to Be My Doppelganger's sound as "pop-punk."
"Our influences are a lot more broad than a lot of bands that are considered pop punk," Glick said. "We have roots in punk rock for sure. There's not much we are not influenced by."
Early to mid-90s alternative—nowadays considered classic—rock plays a major role in their inspiration; bands like Nirvana, Elastica or Dinosaur Jr. British invasion bands and psychedelic sounds from the 60s have also influenced the band's music.
Poole said they might be the only "pop punk" band that doesn't listen to pop punk.
After numerous failed band attempts prior to Be My Doppelganger, Poole said he loves the freedom he has now.
"I think every other band that I've done before, we always had this goal of, 'We are going to do this–sound like this.'," Poole said. "With (Be My Doppelganger) we were just kind of like, 'F*ck it. We are going to do what we want to do.' We never put any sort of limit on what we wanted to do."
"Our last record is probably the one recording we are most proud of," Glick said. "It just kind of turned out perfect, I think. Except for the release of it, which was a nightmare."
Glick said everything that could possibly go wrong with the release of its third album "Artless" did.
Be My Doppelganger's members said the high points of their musical career, so far, consist of when they toured with their childhood idols.
"We played some really cool festivals with bands we grew up idolizing, which has been pretty cool," Glick said.
His favorite memory is playing with The Dead Milkmen, who were a huge underground punk rock band in the 90s, Glick said.
In previous years, the band would only play shows occasionally in Evansville, but they've been performing locally more frequently, due to their busy schedules. Glick recently relocated his restaurant Bagelrama from Henderson, Kentucky to downtown Evansville.
"It's like a party," Poole said. "We promote a lot of heavy drinking–but responsibly–we like to have a good time and we like to make other people have a good time and interact with us."
The members of Be My Doppelganger said their show consists of a definite punk rock vibe.
"We take playing seriously," Poole said. "But we don't take ourselves seriously at all."
Be My Doppelganger will perform Friday at Cecil's Bar & Grill located on Virginia Street.
Tags: Be My Doppelganger, Cecil's, pop-punk, Punk Rock, Valley Vibrations
Bobby Shipman, Editor-in-Chief
Fulfilling a lifelong dream
English student stresses diversity through teaching research
A little fun for a big cause
"Friends Facing Fears" creates unity, promotes friendship
Educating for a better tomorrow
"First Date" to feature contemporary love
Supporting the influencers of tomorrow
Professor to present poetry at Griffin Center
Finding new ways to offer help
Student uses written word to speak out


Elegant Kerala kasavu saree with peacock design from Malabarshopping collection. It has a perfectly ..
Be tradition with your Kerala traditional hand painted saree of pure cotton fabric with a variety am..
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Kerala traditional Krishna embossing kasavu tissue saree depicts the popular golden design of krishna in its pallu and fleets in the body of the bottom. The golden colored pallu is woven into the fabric with a nice blend of golden colors which gives the saree a royal look. The body of the saree has delicate Kasavu works that add elegance to the saree.


Forecasters predict up to three inches of new snowfall in Lansing Sunday.
LANSING – Sunny, springtime weather could be short-lived.
Forecasters predict up to 3 inches of snowfall during the day Sunday near Lansing, and another inch overnight.
Areas north of Lansing into Clinton County could see a total of 5 inches.
Saturday will be breezy and partly sunny with a high near 50 degrees, according to the National Weather Service in Grand Rapids. Sunday will be colder, however, with temperatures in the mid-30s.
A mixture of snow and rain is set to hit the Lansing-area before 7 a.m. Sunday and to continue throughout the day.
The expected wet, heavy snow could lead to slick driving conditions, especially under overpasses, said Wayne Hoepner, a meteorologist with the weather service in Grand Rapids.
Some of the snow may stick to grass and elevated areas Sunday, but is likely to melt by Monday afternoon, Hoepner said.
"You may not need to shovel, but, if you do, be careful because the snow is going to be heavy," Hoepner said.
Monday will be mostly sunny with a high near 49 degrees, forecasters say.
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It took less than three minutes for Floyd Mayweather to knock down his Japanese kickboxer opponent Tenshin Nasukawa three times in a totally one-sided bout of exhibition boxing on New Year's Eve. And believe it or not, Mayweather made cool USD 12.8 Million for his new year's eve effort, almost USD 91,428 for every second of time spent in the ring against the Japanese.
It was a quick and definitive victory for the 41-year-old American. Mayweather was gracious in victory, hugging the weeping Nasukawa and calling him "still a great champion". Nasukawa, however, barely landed a clean punch against the vastly more experienced Mayweather.
Mayweather declared his earnings for the night via social media. According to the American, who posted on Instagram indicating he was receiving $12.8 million.
"What if I told you I was making $9,000,000 for 9 minutes of sparring in Tokyo Japan would you do the same if you were me?" Mayweather posed the question to his 22.4 million followers.
"I like to call it a 9 minute walk thru #Tokyo #Japan".
"It's all about having fun," Mayweather told reporters after the fight, praising Nasukawa as a "young hungry lion".
The Mayweather-Nasukawa bout wrapped up an evening of more than a dozen fights.


Anyone here going to St. John's in August?
yes. I look forward to seeing you Mr. Dark Prince. a/s/l. I think we will have some fun in Jamaica!
Yesss the kid is 99% almost officially in (Unless Fordham waitlist actually comes through). Leavin Beantown and coming back to NY. Sick.
I drove by the beautiful campus a couple of days ago and almost cried That's how much I want to join you guys this fall.
olesya154 wrote: I drove by the beautiful campus a couple of days ago and almost cried That's how much I want to join you guys this fall.
LoL SJU has a beautiful campus?
You should take an online tour of Cornell.
Well, since I am not applying to Cornell why would I want to look at their campus?
I went to st johns for UG and the campus is nice, especially in the spring when you can really enjoy it. However bear in mind , especially the UG, is really a commuter school.
Agreed! This school is perfect for me since I live just a short-drive distance away in Forest Hills. St. John's pleeease take meeee!!!
Eh to give you a true sense of beauty.
SJU is just a big piece of grass. Once you get passed the great lawn, there really isn't that much else to see.
Eh just trying to give you a sense of the campus since a "drive by" can only tell you so much.
Plus you usually don't leave the law school building if you go to law school there [food is so much better than the UG food and the library is not half bad].
Unlike you, I don't make assumptions.
The campus is nice, let's hope the quality of instruction is just as nice if not nicer. The only problem is that it's not close to any subway stations, so even from where I live in Forest Hills, it'll take me around 40 minutes.
BTW, are any of you guys coming to the Admitted Students Day on March 28?
Already committed to BLS, but Im going to vent about SJU. I went complete in November. Still no response. I wanna go and demand my money back and/or a decision pls!!!!
Congrats to all of you who got in and are going to SJU!
ResIpsaLoquitur wrote: Already committed to BLS, but Im going to vent about SJU. I went complete in November. Still no response. I wanna go and demand my money back and/or a decision pls!!!!
Anyone planning on living in campus owned housing? I think it's a few blocks from the school.
Also, do you think I'd need a car?
If you live in Brooklyn, then you'll def. need a car. If you live in Queens or Manhattan then probably not. I live in Forest Hills, so I'll be taking the train and the bus.
you need a car less living in brooklyn . if you live in jamaica estates a car is more necessary.
Needless to say the quality of the UG (as shown by MSFly) is quite low.
It is the second time I've seen you tout the fact that sjl is your safety to people who are happy to go there. This says nothing about sjl or sj ug, and just shows you to be a sad prick.
I don't understand why someone would shell out the cash for SJ UG when Queens College a few minutes away is just as good (if not better) and costs maybe 1/10 as much, if even.


A new clinic by John Parker on 10 key items for keeping your layout running smooth.
An article taken from our Dispatch newsletter on Modeling Rivers by Rick Inglis.
A step-by-step instruction on Making Realistic Trees by Craig Linn.
How to build a Trackside Shed by George Franke.
Some good suggestions on detailing in Six Easy Diesel Details by Bob Sobol.
A pdf file version of a presentation on Layout Automation containing lots of good ideas and resources, given at a club meeting on September 22, 2016.
A pdf file of a presentation by Wally Muscott, on Junk Box Modelling, showing how one can use a variety of things around the house and workbench for tools and materials.


Yottawatts | Watts Up With That?
But its actually about powers of ten and electrical power (watts). I was researching the amount of solar insolation the earth receives from the sun (174 Petawatts) and ran across this page in WikiPedia full of variations on my namesake.
By the way, Petawatts has nothing to do with my disdain for the sometimes crazy tactics of the animal rights group.
systems is overwhelmed by thermal fluctuations.
1.39 kW (12.2 MWh/year) – Geo: per capita average power use in the U.S.
1 kW to 2 kW – Tech: heat output of a domestic electric kettle.
would be needed to draw 1 megawatt. Also, 1 MW equals approximately 1341 horsepower.
power plant produces on the order of 500–2000 MW peak output.
Jupiter and its moon Io due to Jupiter's tremendous magnetic field.
December 8, 2006 in Uncategorized.
And all named after James Watt.
An interesting thing I learned in engineering and math classes in college was that many of our units are named after scientists and engineers. This is especially true in electonics.
Some examples… volts (Volta), amps-ampere (Ampere), farads-charge capacity (Faraday), coloumbs- a unit of charge (Coloumb), just to name a few.
You often run across the same names when learning both higher math and physics, which shows how brilliant many of these guys were.
BTW James Watt invented the steam engine amongst other things.
And a few others names like Roentgens, for radiation.(some bunker humor going on there) Roentgen discovered the X-ray.
On the bunker, are you still "sheltering in place"? Do you need an air-drop of Celebration Ales?