text:
Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins.
IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.
Toxoplasma gondii is an obligate intracellular parasite that can cause severe illness in immunocompromised individuals and the developing fetus. It is estimated to infect up to a third of the world's population and has an unparalleled host range, infecting virtually any nucleated cell in almost any warm-blooded animal (1). In order to survive within a host cell, Toxoplasma tachyzoites, the rapidly dividing, asexual stage of the parasite, establish a replicative niche, the parasitophorous vacuole (PV), whose membrane, the PV membrane (PVM), acts as the interface between parasite and host. While the PV protects intracellular Toxoplasma from clearance by the innate immune system, it also acts as a barrier that Toxoplasma must overcome in order to hijack host resources.
Toxoplasma extensively modifies the host cells that it infects via secreted effectors, either rhoptry (ROP) or dense granule (GRA) proteins, which it introduces into the host during or following invasion (2). In recent years, several Toxoplasma GRAs, including GRA16, GRA24, IST, HCE1/TEEGR, GRA28, and GRA18, that are translocated across the PVM into the host cell cytosol and/or nucleus, where they can have profound effects on host processes, have been identified (3–9). The machinery that is responsible for the translocation of these effectors across the Toxoplasma PVM is incompletely defined. A recent forward genetic screen identified several parasite proteins essential for GRA protein translocation, including MYR1, MYR2, and MYR3 (named for their effect on host c-Myc regulation) and the rhoptry-derived protein kinase, ROP17 (10–12). Precisely how these proteins function to promote protein translocation across the PVM is poorly understood. Of the four, the only protein with a known biochemical function is ROP17, a serine/threonine protein kinase that phosphorylates host and perhaps parasite proteins at the PVM (14).
In addition to MYR1, MYR2, MYR3, and ROP17, an active Toxoplasma aspartyl protease V (ASP5), which proteolytically processes secreted proteins at the amino acid sequence RRL (also known as a Toxoplasma export element [TEXEL]), is also required for the translocation of all exported GRAs studied thus far (5, 6, 8, 9, 15–17). In Plasmodium, the homolog of ASP5, plasmepsin V, appears to "license" many proteins for export across the PVM by proteolytically processing them at a Plasmodium export element (RXLXE/Q/D) (18–21). Intriguingly, and as for Plasmodium (22, 23), not all of Toxoplasma's exported GRAs contain RRL motifs (e.g., GRA24, GRA28, and HCE1/TEEGR lack such an element), which leaves open the possibility that ASP5's role in translocation is in processing the translocation machinery rather than the effectors themselves. Indeed, MYR1 is processed by ASP5, but this processing is not necessary for protein export, as unprocessed full-length MYR1 harboring a mutated RRL motif can still promote the translocation of the effector GRA24 to the host nucleus (24). The role of ASP5 processing of MYR1, therefore, remains unknown.
To learn more about the mechanism of protein translocation in Toxoplasma and to complement the genetic approaches taken previously, we report here on the use of MYR1 as bait for immunoprecipitation (IP) followed by mass spectrometry (MS) to identify putative MYR1-associated proteins that are involved in effector translocation. Of the many associating proteins, at least 11 are shown here or were previously known to be PV localized, and of these, 3 additional proteins are now shown to be required for GRA translocation across the PVM. Interestingly, all three of these new components contain RRL motifs, with two confirmed to be cleaved in an ASP5-dependent manner, yet, like MYR1, cleavage at these sites does not appear to be required for their translocation function. Thus, we have expanded the list of proteins involved in GRA translocation to eight while highlighting the question of why at least three of these components (MYR1, GRA44, and GRA45) are proteolytically processed without any apparent impact on their one known function.
We previously reported the use of a forward genetic screen to identify Toxoplasma genes required for the induction of human c-Myc. This identified MYR1, MYR2, MYR3, and ROP17 to be essential for the translocation of effector proteins across the PVM (10–12). Two of these proteins, MYR1 and MYR3, were found to coprecipitate with each other (11), and we hypothesized that MYR1 functions in complex with other yet unidentified proteins to facilitate effector translocation across the PVM. Given the small but significant reduction in plaque size observed when growing strains from which MYR1, MYR2, and MYR3 were deleted on human foreskin fibroblasts (HFFs) (11), we also reasoned that the genetic approach might also miss genes whose disruption substantially reduces fitness.
To identify additional MYR1-associating proteins, therefore, we adopted a biochemical approach. Specifically, we immunoprecipitated 3× hemagglutinin (3×HA)-tagged MYR1 from HFFs infected for 24 h with an RH::MYR1-3×HA strain or from an untagged RH strain to control for proteins that coprecipitate with the anti-HA beads nonspecifically (Fig. 1A). Liquid chromatography (LC)-tandem mass spectrometry (MS/MS) was performed on the eluates, and the identified parasite proteins were ranked by the ratio of the average normalized spectral abundance factors (NSAFs) for a given protein in the RH::MYR1-3×HA lysates compared to that in the RH control lysates (25). This mass spectrometry experiment was performed twice (IP 1 and IP 2). As expected, MYR1 was the most enriched protein in both biological replicates (Fig. 1B). Additionally, several PV- or PVM-localized GRA proteins were highly enriched (enrichment score > 10) in the MYR1-3×HA immunoprecipitations over that in the untagged RH control immunoprecipitations, including GRA44, CST1, GRA52, MAG1, PPM11C, GRA50, MAF1a, GRA7, and a GRA12 paralog, in addition to two exported effector proteins, GRA16 and GRA28, of which GRA16 has been shown to be exported in a MYR1-dependent manner (10) (Fig. 1B; see also Data Set S1 in the supplemental material). While CST1 has traditionally been thought to be specific to the slow-growing bradyzoite stage of Toxoplasma, due to its detection in the cyst wall (26, 27), our results, along with those of others (28), provide clear evidence for CST1 protein expression in tachyzoites. The large number of enriched PV- and PVM-localized proteins may be explained by the mild detergent conditions used (0.1% NP-40), which were chosen in an attempt to maintain associating proteins, although these proteins might also be associating with one another in large, nonspecific complexes or lipid rafts (29).
Copyright © 2020 Cygan et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Importantly, and also as expected, the known MYR1-associating protein, MYR3, was enriched in the MYR1-3×HA immunoprecipitations, albeit with an enrichment score (4.5) that did not put it in the top 20 most enriched proteins (Fig. 1B; Data Set S1). Of note, ROP17 was not substantially enriched (enrichment score = 1.2), and no peptides for MYR2 were detected, but neither protein has previously been found to associate with MYR1, and so this was not unexpected. Human proteins with an enrichment score of >10 include Filamin-C (FLNC), DNA-dependent protein kinase catalytic subunit (PRKDC), sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (ATP2A2), and alpha-N-acetylglucosaminidase (NAGLU) (Data Set S1). As our focus was on parasite proteins only, the potential role of these human proteins in Toxoplasma infection was not further investigated.
To screen for a possible role in GRA effector translocation, we focused on the top 6 most enriched parasite proteins: GRA44 (TGGT1_228170), CST1 (TGGT1_264660), TGGT1_204340 (here abbreviated 204340), TGGT1_211460 (here abbreviated 211460), PPM3C (TGGT1_270320), and GRA52 (TGGT1_319340). GRA45 (TGGT1_316250) was also pursued because it is a known binding partner of the top hit, GRA44 (30), and it also had a substantial enrichment score of 5.2 in the immunoprecipitations (Fig. 1B). Interestingly, two well-characterized PV proteins that have not previously been described to be involved in effector translocation, GRA7 (TGGT1_203310) and MAF1a (TGGT1_279100), were substantially enriched, and since antibodies and gene knockouts for both were readily available, we included these in the list of genes to explore further. Lastly, as a positive control for a protein whose disruption is known to prevent effector translocation, we also included MYR3 in the pipeline for gene disruption and testing. All 10 proteins chosen for further analysis are highlighted in orange in Fig. 1B.
With the exception of ASP5, and as might be expected, all proteins so far published to be required for effector translocation across the PVM localize to the PV/PVM (2). Of the 10 proteins chosen for further analysis, GRA44, GRA45, CST1, GRA7, GRA52, and MAF1a are all known to be PV/PVM localized (26, 30–37). The localization of 211460 and PPM3C has not been reported, but both include predicted signal peptides (see below), as does 204340, which has been described to possibly be micronemal (34). We therefore set out to localize these three proteins within infected cells. To do this, we generated populations of parasites in which each of the three genes was endogenously modified to encode a 3×HA tag immediately before the stop codon and then assessed the protein's localization by immunofluorescence assay (IFA). Correct integration of the 3×HA tag into the appropriate locus was confirmed by PCR and by checking for an appropriately sized HA-tagged protein via Western blotting. The results (Fig. 2A) showed major bands at ∼130 kDa, ∼110 kDa, and ∼70 kDa for 211460, 204340, and PPM3C, respectively. In the case of 204340 and PPM3C, these are close to the predicted sizes of ∼97 kDa and ∼60kDa, respectively (ToxoDB Toxoplasma gondii genome database, v45). For 211460, however, the mobility was significantly retarded relative to its predicted size of ∼100 kDa. This could be due to its acidic pI of 4.91 (ToxoDB, v45), which is known to reduce protein mobility on SDS-PAGE (35), and/or to posttranslational modifications (all three proteins are reported to potentially be phosphorylated, with one predicted phosphorylation site for MYR4, one for PPM3C, and nine for GRA54 [52] [ToxoDB, v45]). This same slower-than-expected mobility for the major band was seen for an independently generated, cloned line expressing HA-tagged 211460 (Fig. S1A), and so we conclude that this is the correct mobility for this protein. Interestingly, both the 3×HA-tagged 211460 population and the single clone also showed a smaller but considerably weaker band at about the expected size (∼100 kDa). Whether this smaller 211460 product is biologically relevant or is simply a product of protein degradation is unclear.
Copyright © 2020 Cygan et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Using the HA-tagged 211460, 204340, and PPM3C parasite populations, we next sought to determine the localization of these proteins in infected cells. As determined using SignalP software (v5.0), all three proteins had strongly predicted signal peptides, although in the case of 211460, this was true only if translation starts at the fourth in-frame methionine (position 61) relative to the protein sequence predicted on ToxoDB (v45). The results (Fig. 2B) show a clear PV-like signal outside of the parasites in the 211460-3×HA, 204340-4×HA, and PPM3C-3×HA populations, including at the periphery of the PV. The PV localization for 211460 was further confirmed in the independently generated clonal line (Fig. S1B). Thus, we conclude that 211460, 204340, and PPM3C are at least transiently localized to the Toxoplasma PV during infection. Furthermore, we also assessed the localization of these proteins within the parasites themselves. The results (Fig. S2) showed that while PPM3C appears to be present throughout the parasite, 211460 and 204340 show a clear, punctate staining pattern that largely colocalizes with the dense granule protein GRA7, suggesting that these two proteins are also GRA proteins. We therefore designate 204340 as GRA54 for its GRA-like localization and 211460 as MYR4 for the reasons described below.
Copyright © 2020 Cygan et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
To assess their potential involvement in GRA effector translocation, we attempted to generate knockouts of our candidate genes in a strain of Toxoplasma that constitutively expresses an HA-tagged version of the MYR1-dependent secreted effector protein GRA16, RHΔgra16::GRA16-HA (the parental strain). To do this, we cotransfected a CRISPR/Cas9 single guide RNA (sgRNA) plasmid that targets the first exon of the relevant gene along with a pTKO2-CAT-mCherry plasmid (CAT is chloramphenicol acetyltransferase and is encoded by the chloramphenicol resistance gene; Fig. 3A). Following selection with chloramphenicol, we cloned the populations by limiting dilution and confirmed disruptive integration of the vector by PCR with gene-specific primers. Using this strategy, we were able to disrupt the genomic loci of MYR3, GRA44, GRA45, CST1, GRA54, MYR4, PPM3C, and GRA7 (Fig. S3). Despite several attempts, however, we were unable to generate a GRA52 mutant. This gene may be essential, as it has a very negative CRISPR fitness score of −3.96 (36). Given that the MAF1 locus is expanded in Toxoplasma, with 4 copies in RH parasites (37), we chose not to attempt a CRISPR/Cas9 approach to knock out MAF1a and instead utilized a previously generated strain in which the entire MAF1 cluster (including the regions encoding MAF1a and MAF1b) was deleted (33).
Copyright © 2020 Cygan et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
To determine if the absence of any of the candidate genes results in a defect in effector translocation across the PVM, we used IFA to assess both GRA16-HA export to the host nucleus and host c-Myc upregulation (which Toxoplasma induces during infection [38]) in the disrupted lines. Quantified results for all nine genes tested showed that disruption of GRA44, GRA45, MYR4, and the previously described MYR3 all resulted in a complete or nearly complete block in GRA16 export to the host nucleus (Fig. 3B; Fig. S4) and a failure to upregulate host c-Myc (Fig. 3C; Fig. S4); on the other hand, disruption of GRA7, CST1, GRA54, or PPM3C resulted in no detectable effect on either of these two phenotypes. Additionally, we found that the previously generated Δmaf1 strain also had normal GRA16 export to the host nucleus (Fig. 3D). These results indicate that of the nine genes tested here, only MYR3, GRA44, GRA45, and MYR4 are necessary for the translocation of GRA effectors across the PVM.
Copyright © 2020 Cygan et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
To test the generality of their role in effector translocation, we next assessed the impact of these gene disruptions on the upregulation of host cyclin E1, which has been shown to be dependent on export of the MYR1-dependent effector HCE1/TEEGR (6). The results showed that, as for GRA16, the disruption of MYR3, GRA44, GRA45, and MYR4 also resulted in a block in cyclin E1 upregulation in infected host cells, while no obvious defect was observed in the parasite lines disrupted in GRA7, CST1, GRA54, and PPM3C (Fig. 3E). A repetition of the cyclin E1 Western blot assay with a higher parasite input revealed that the absence of cyclin E1 upregulation observed in Δgra44 parasites in Fig. 3E was not due to a low parasite input in that particular experiment (Fig. S5). These results argue that GRA44, GRA45, and MYR4 are all required for translocation across the PVM of at least two independent GRA effectors.
Copyright © 2020 Cygan et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Our previous work has shown that the deletion of MYR1, MYR2, and MYR3 results in a small but significant, negative effect on parasite growth in vitro (11). To determine if disruption of the three new genes involved in effector translocation described here has a similar impact, we infected HFF monolayers with each of the disrupted lines, fixed the monolayers at 7 days postinfection, and measured the plaque size. The results showed that the Δmyr4, Δgra44, and Δgra45 strains all exhibited a significant growth defect compared to the parental strain (Fig. 3F). The Δgra54 and Δppm3c strains, on the other hand, did not have significant growth defects, consistent with the growth defects observed being dependent on the respective genotype rather than nonspecific effects of the manipulations.
To confirm that ablation of the GRA44, GRA45, and MYR4 loci is responsible for the observed defect in GRA16 export, we transiently expressed a C-terminally V5-tagged version of each protein, driven by its native promoter, in the relevant disrupted line. These transiently transfected parasites were then assessed for GRA16-HA export to the host nucleus via IFA. The results showed that the parental and complemented strains had GRA16-HA signals in both the vacuole and the host nucleus, while the parasites within the population that did not express the complementing transgene (as indicated by a lack of anti-V5 staining) showed essentially no GRA16 in the host nucleus (Fig. 4A and B). Thus, GRA44, GRA45, and MYR4 are indeed essential for the translocation of effectors across the PVM, and we therefore designate 211460 as MYR4, consistent with previous nomenclature (10, 11).
Interestingly, GRA44, GRA45, and MYR4 all contain one or two instances of the 3-amino-acid motif RRL (Fig. 5A), which has previously been shown to be the preferred sequence for cleavage by ASP5 protease (15). Indeed, ASP5 cleavage at the sites shown in GRA44 and GRA45 (30), as well as at the first RRL site in the secreted GRA effector, GRA16 (15), has been experimentally confirmed. ASP5 is essential for the translocation of all GRA effectors so far tested (5, 6, 8, 9, 15–17), and it has previously been suggested that ASP5-mediated cleavage of some effectors is required to "license" them for translocation across the PVM, as appears to be the case in Plasmodium (18, 19). Given, however, that not all such effectors contain ASP5 processing motifs (e.g., GRA24 lacks the canonical RRL and shows no evidence of ASP5-dependent processing [17]) and given that the three newly identified components of the translocation machinery identified here do, we hypothesized that ASP5's essential contribution to effector translocation across the PVM might be in processing one or more components of the translocation machinery. We have previously shown that MYR1 is also processed by ASP5 at an RRL site, but this does not appear to be required for MYR1 to function in effector translocation (24), and so we turned our attention to the newly identified translocation components identified here.
To determine if processing at the RRL sites of GRA44, GRA45, and MYR4 is required for protein translocation activity, we mutated the ASP5 cleavage sites by converting the first arginine to an alanine (i.e., RRL → ARL) in the V5-tagged complementation plasmids for each gene and transiently transfected these into the corresponding disrupted line. Western blots were then used to show that processing of GRA45 at its lone RRL and of GRA44 at its second RRL is indeed abrogated by the mutations (Fig. 5B). For the more N-terminal site in GRA44 (R83A), we cannot definitively confirm that the mutation abrogates ASP5 processing because GRA44 is epitope tagged at its C terminus, and so, assuming that cleavage at the two sites is an independent event, cleavage at the downstream site produces a C-terminal, V5-tagged fragment whether or not cleavage occurs at R83A. We fully expect, however, that the RRL → ARL change disrupts ASP5 cleavage at this site because it did in the two other examples shown here (GRA45 and the downstream site in GRA44, R1348A) and because RRL → ARL single point mutations have previously been shown to disrupt the ASP5-dependent cleavage of other proteins (24, 30). Furthermore, previous work to detect ASP5-dependent cleavage peptides using an N-terminal amine isotopic labeling of substrates (N-TAILS) approach on naturally occurring tachyzoite proteins identified peptides generated by cleavage immediately after this first RRL site in GRA44 (30); thus, this site is a site of natural cleavage and very likely to be a bona fide ASP5 processing site.
Interestingly, mutation of RRL to ARL in MYR4 did not appear to affect processing of the protein (Fig. 5B). To rule out whether this is due to incomplete ablation of the potential ASP5 processing site with just a single amino acid substitution, we assessed the processing of an RRL → AAA MYR4 mutant where the entire ASP5 processing motif was mutated to alanines. The results (Fig. 5C) showed that the higher-molecular-weight product of MYR4 (∼130 kDa) did not change in mobility upon mutation of the entire RRL motif, and thus, we conclude that little, if any, MYR4 is processed by ASP5. Note that, despite repeated attempts with large amounts of DNA, the signal for the transiently expressed MYR4 was never strong enough to confidently conclude whether a small amount of a processed form might be present in these transiently transfected parasites; we therefore cannot comment on whether the low-intensity, lower-molecular-weight product of MYR4 (∼100 kDa) seen in long exposures of endogenously tagged wild-type MYR4 (Fig. 2A; Fig. S1A) is a result of an ASP5 processing event.
Having generated the four RRL → ARL mutants and having validated that ASP5 cleavage is ablated in at least two instances, we next tested each for its impact on the localization of the epitope-tagged, C-terminal portion of the protein and on the ability of the uncleaved protein to function, i.e., whether it can rescue the defect in effector protein translocation. The results showed that the RRL → ARL-mutated versions of each protein were still secreted into the PV, similar to the findings for the wild-type copy (Fig. 4A and 6A), and all were able to rescue the translocation defect to an extent similar to that for the corresponding control (wild-type) plasmid (Fig. 6B). While the GRA45 R64A mutant did substantially rescue translocation, it did not consistently rescue it to wild-type levels. Nevertheless, these data suggest that mutation of the RRL sites in GRA44, GRA45, and MYR4 to ARL does not substantially affect their function in effector protein translocation.
Using affinity purification of MYR1 under conditions expected to retain associating partners, we identified three novel parasite proteins, GRA44, GRA45, and MYR4, to be essential for the export of GRA effectors into infected cells. Additionally, we localized MYR4, as well as two additional MYR1-associating proteins, GRA54 and PPM3C, to the PV in infected cells. Altogether, eight proteins are now known to be necessary for effector export: the three described here and MYR1, MYR2, MYR3, ROP17, and ASP5, summarized in Table S1 in the supplemental material (10–12, 15–17). Besides ASP5, which localizes to the Golgi apparatus (15–17), these proteins all localize to the PV/PVM.
Copyright © 2020 Cygan et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
The newly identified components described here do not display any homology to known protein translocation machinery, based on BLAST analysis results (BLASTP, v2.10.0+), making it difficult to infer their functions, and thus, which, if any, are part of an actual translocon remains unknown. In addition to lacking homology to known translocation machinery, MYR4 and GRA45 do not have detectable homology to any other known, functional protein domains, nor do they share homology to proteins in any species outside of Coccidia/Eimeriorina. Like MYR1, MYR2, MYR3, and ROP17, however, MYR4, GRA44, and GRA45 all have clear orthologs in Hammondia hammondi and Neospora caninum (Table S1).
GRA44, in contrast, contains a putative phosphatase domain that shares homology to a region of the Plasmodium serine/threonine phosphatase UIS2 (28% identity over 21% of the protein; BLASTP, v2.10.0+), which has recently been shown to localize to the Plasmodium PVM in liver-stage parasites (39). Whether UIS2 plays a role in protein translocation in Plasmodium remains to be determined, but it would be surprising if it did, given that none of the other components of the complex known to promote translocation in Plasmodium (known as PTEX) so far studied play a role in translocation in Toxoplasma (2). Additionally, whether this phosphatase domain is important for effector export in Toxoplasma is not yet known. Given that the kinase domain of ROP17 is necessary for GRA16 export (12), it is intriguing that two of the eight factors necessary for effector export are either a kinase or a putative phosphatase. There are numerous serine residues that are phosphorylated among MYR1, MYR2, MYR3, and MYR4, supporting the possibility that phosphorylation of the translocation machinery is critical to regulating its function in effector export. Phosphoproteomic analysis of these MYR components in parasites lacking GRA44 (a predicted phosphatase) and ROP17 (a protein kinase) may provide insights into the mechanism and regulation of effector export and the interplay of these proteins.
While this work was in progress, we learned of similar studies by Blakely et al., who also found that GRA44 associates with MYR1 and is necessary for efficient c-Myc upregulation during infection (40). The latter authors used a knockdown approach to study GRA44 and saw a more dramatic impact of GRA44 loss on parasite growth than we and others (30) observed for the GRA44 knockout; this might indicate that compensatory changes were selected for during the prolonged selection necessary to generate and expand our knockout clone, as was reported for AMA1 knockouts, which showed a dramatic upregulation of the paralogue, AMA2 (41). Interestingly, GRA44 has a paralog, TGGT1_228160, that is expressed in the Toxoplasma sexual stages (42). While the growth phenotype is apparently rescued to a degree sufficient to allow our GRA44 knockout mutant to survive, the effector export phenotype appeared to remain fully impaired, suggesting that whatever potential compensatory changes occurred, they did not restore effector export to the host nucleus. Thus, further analysis of the GRA44 knockout mutant and the potential compensations that it has experienced may reveal clues to other specific functions of GRA44 in Toxoplasma tachyzoites.
Our results highlight the question of why some parasite proteins are proteolytically processed by ASP5 and why ASP5 is essential for effector translocation across the PVM. MYR1, GRA44, and GRA45 all possess RRL motifs that appear to be cleaved in an ASP5-dependent manner, yet, surprisingly, their function in the export of GRA16 and of GRA24, in the case of MYR1 (24), appears agnostic to mutation of these sites. For MYR1, we previously showed that the two domains generated by ASP5 processing stay connected through a disulfide bond after cleavage (11); it remains to be determined whether the polypeptides formed by RRL cleavage in GRA44 and GRA45 likewise associate in a similar manner. It is also important to note that our assays may not have been sensitive enough to detect small changes in protein abundance in the host nucleus and that it is the combination of multiple proteins not being processed by ASP5 rather than the result of failure to cleave any single protein that is deleterious to export in Δasp5 mutants.
Interestingly, there were a large number of proteins that were more highly enriched than MYR3 in our immunoprecipitations with MYR1, and it remains a strong possibility that additional MYR1-associating proteins are involved in effector translocation. Due to the large number of enriched proteins and the limited throughput of our approach, we were unable to investigate all candidates for such a role; nevertheless, our data showing that GRA16-HA export is not lost in parasites disrupted in GRA7, CST1, MAF1, PPM3C, or GRA54 strongly suggest that it is not general PV/PVM disruption that results in the loss of effector translocation. Further work will be needed to determine which of the remaining proteins that we see enriched in the MYR1 immunoprecipitations are there because of a specific association with MYR1 versus because of nonspecific associations of proteins within the PV/PVM due to an association within lipid rafts or other entities.
Lastly, none of GRA44, GRA45, or MYR4 was identified in the forward genetic screen of parasites that are unable to induce c-Myc (10). This could be due to the growth defects observed in Δmyr4, Δgra44, and Δgra45 parasites shown here, since parasites with null mutations in these genes might be lost during the 5 to 7 rounds of selection used in that screen due to a fitness disadvantage. Alternatively, the mutagenesis-based genetic screen was not saturating, and so a more comprehensive, genome-wide screen using CRISPR/Cas9 technologies might reveal these and other genes responsible for effector translocation in Toxoplasma. Regardless, our finding of three new components of the export machinery provides a richer understanding of how Toxoplasma delivers effectors into host cells. Future work will determine the precise function of each, including how they interact, the role of ASP5 cleavage, and which, if any, constitutes the actual translocon.
Parasite strains, culture, and infections.All Toxoplasma tachyzoites used in this study are in the type I RH strain background, either RH::MYR1-3×HA (11), RHΔgra16::GRA16-HA (6), RHΔmaf1 (33), RHΔhpt (43), or RHΔhptΔku80 (44). These tachyzoites and all subsequently generated lines were propagated in human foreskin fibroblasts (HFFs) cultured in complete Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2. The HFFs were obtained from the neonatal clinic at Stanford University following routine circumcisions that are performed at the request of the parents for cultural, health, or other personal medical reasons (i.e., not in any way related to research). These foreskins, which would otherwise be discarded, are fully deidentified and therefore do not constitute human subjects research.
Prior to infection, parasites were scraped and syringe lysed using a 27-gauge needle, counted using a hemocytometer, and added to the HFFs. Mock infection was done by first syringe lysing uninfected HFFs, processing these in the same manner used for the infected cells, and then adding the same volume of the resulting material used for infections. For experiments where the human c-Myc protein was detected, the parasites were added to HFFs in medium containing 0% serum.
Immunofluorescence assay (IFA).Infected cells grown on glass coverslips were fixed and permeabilized using 100% cold methanol for 10 min. Samples were washed 3 times with phosphate-buffered saline (PBS) and blocked using 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature (RT). HA was detected with rat monoclonal anti-HA antibody 3F10 (Roche), SAG1 was detected with mouse anti-SAG1 monoclonal antibody DG52 (45), GRA7 was detected with rabbit anti-GRA7 antibodies (46), V5 was detected with a mouse anti-V5 tag monoclonal antibody (Invitrogen), and c-Myc was detected with rabbit monoclonal anti-c-Myc antibody Y69 (Abcam). Primary antibodies were detected with goat polyclonal Alexa Fluor-conjugated secondary antibodies (Invitrogen). Both the primary and secondary antibodies were diluted in 3% BSA in PBS. Coverslips were incubated with the primary antibodies for 1 h at RT, washed, and incubated with secondary antibodies for 1 h at RT. Vectashield with DAPI (4′,6-diamidino-2-phenylindole) stain (Vector Laboratories) was used to mount the coverslips on slides. Fluorescence was detected using wide-field epifluorescence microscopy, and images were analyzed using ImageJ software. All images shown for any given condition/staining in any given comparison/data set were obtained using identical parameters.
Transfections.All transfections were performed using the Amaxa 4D Nucleofector (Lonza) model. Tachyzoites were mechanically released in PBS, pelleted, and resuspended in 20 μl P3 primary cell Nucleofector solution (Lonza) with 5 to 25 μg DNA for transfection. After transfection, parasites were allowed to infect HFFs in DMEM.
Plasmid construction.For gene disruption plasmids, guide RNAs, designed against a PAM site of each gene of interest, were cloned into the pU6-Universal plasmid. pU6-Universal was a gift from Sebastian Lourido (Addgene plasmid number 52694; http://n2t.net/addgene:52694; RRID:Addgene_52694).
For ectopic expression plasmids, the pGRA-V5 plasmid was created by replacing the HA tag sequence in the pGRA-HPT-HA plasmid (47) with the V5 tag DNA sequence (GGCAAGCCCATCCCCAACCCCCTGCTGGGCCTGGACAGCAC) and removing the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HPT) resistance cassette using standard molecular biology techniques. The pX-V5 plasmid was created by removing the GRA1 promoter from pGRA-V5 using standard molecular biology techniques. Complementation plasmids to ectopically express V5-tagged proteins off their native promoters were created by PCR amplification of the open reading frame of each gene, minus the stop codon, plus ∼2,000 bp upstream of the start codon to include the native promoter, followed by cloning into pX-V5 using Gibson Assembly (New England Biolabs [NEB]). RRL → ARL- or RRL → AAA-mutated complementation plasmids were generated using overlap extension PCR and primers harboring the mutation and cloning the resultant products into pX-V5 using Gibson Assembly (NEB).
For endogenous tagging plasmids, approximately 1,500 to 3,000 bp of the 3′ coding sequence of each gene was amplified from RH genomic DNA and cloned into the pTKO2-HPT-3×HA plasmid (11) either by using Gibson Assembly (NEB) or by cloning into the EcoRV and NotI restriction sites.
A list of all primers and plasmids used and generated in this study can be found in Data Set S2 in the supplemental material.
Copyright © 2020 Cygan et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Endogenous tagging.Endogenous tagging plasmids were transfected into Toxoplasma via electroporation. Tachyzoites were allowed to infect HFFs in T25 flasks for 24 h, after which the medium was changed to complete DMEM supplemented with 50 μg/ml mycophenolic acid and 50 μg/ml xanthine for selection for the hypoxanthine-xanthine-guanine-phosphoribosyltransferase (HXGPRT or HPT) marker for 3 to 5 days.
Gene disruption.A list of all sgRNA sequences used in this study can be found in Data Set S2. RHΔgra16::GRA16-HA tachyzoites were transfected with pTKO2-CAT-mCherry (CAT is chloramphenicol acetyltransferase, which confers resistance to chloramphenicol; the plasmid was a gift from Ian Foe and Matthew Bogyo [48]) and the corresponding modified pU6-sgRNA plasmid and allowed to infect HFFs for 24 to 48 h. For gene disruption of MYR3, the previously published pSAG1:U6-Cas9:sgMYR3 plasmid was used instead (11). At between 24 and 48 h after transfection, DMEM with 80 μM chloramphenicol was added to the cells. The medium was replaced with fresh chloramphenicol-supplemented medium every 48 to 72 h. After at least 7 days in selection, single clones were selected from the transfected populations in 96-well plates using limiting dilution. Single clones were maintained in chloramphenicol-supplemented medium until confirmation of the genetic disruption.
Ectopic expression.Plasmids for ectopic expression were transiently transfected into Toxoplasma using electroporation. Tachyzoites were allowed to infect HFFs for 18 to 24 h before assessing for expression of the ectopically expressed protein via either IFA or Western blotting.
Western blotting.Cell lysates were prepared at the time points postinfection indicated above in Laemmli sample buffer (Bio-Rad). The samples were boiled for 5 min, separated by SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline supplemented with 0.5% Tween 20, and proteins were detected by incubation with primary antibodies diluted in blocking buffer, followed by incubation with secondary antibodies (raised in goat against the appropriate species) conjugated to horseradish peroxidase (HRP) and diluted in blocking buffer. HA was detected using an HRP-conjugated HA antibody (catalog no. 12013819001; Roche), SAG2A was detected using rabbit polyclonal anti-SAG2A antibodies (49), cyclin E1 was detected using mouse monoclonal antibody HE12 (Santa Cruz Biotechnology), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was detected using mouse monoclonal anti-GAPDH antibody 6C5 (Calbiochem). Horseradish peroxidase (HRP) was detected using an enhanced chemiluminescence (ECL) kit (Pierce).
Plaque assay.Parasites were syringe released from HFFs and added to confluent HFFs in T25 flasks. After 7 days, the infected monolayers were washed with PBS, fixed with methanol, and stained with crystal violet. The plaque area was measured using ImageJ software.
IPs for mass spectrometry.Immunoprecipitations (IPs) to identify MYR1-interacting proteins in HFFs were performed as follows. Cells in one 15-cm dish of HFFs for each infection condition were grown to confluence. HFFs were infected with either 15 × 106 RH::MYR1-3×HA or RHΔhpt parasites for 24 h. Infected cells were washed 3 times in cold PBS and then scraped into 1 ml cold cell lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.1% [vol/vol] Nonidet P-40 alternative [CAS no. 9016-45-9]) supplemented with complete protease inhibitor cocktail (cOmplete, EDTA free; Roche). Cell lysates were passed 3 times through a 25-gauge needle, followed by passage 3 times through a 27-gauge needle and sonication on ice (Branson Sonifier 250), consisting of 3 pulses of 10 s each at a 50% duty cycle and output control 2. The cell lysates were spun at 1,000 × g for 10 min to remove insoluble material and unlysed cells. Lysates were added to 100 μl magnetic beads conjugated to anti-HA antibodies (Pierce), and the mixture was incubated overnight with rotation at 4°C. Unbound protein lysate was removed, and the anti-HA magnetic beads were then washed 10 times in cell lysis buffer. HA-tagged MYR1 and associated proteins were eluted in 60 μl pH 2.0 buffer (Pierce) for 10 min at 50°C to dissociate the proteins from the antibody-conjugated beads. The elutions were immediately neutralized 1:10 with pH 8.5 neutralization buffer (Pierce).
Mass spectrometry sample preparation.Forty-five microliters of each IP elution was combined with 15 μl of 4× Laemmli sample buffer supplemented with β-mercaptoethanol (Bio-Rad), boiled for 10 min at 95°C, and loaded on a Bolt 4 to 12% bis-Tris gel (Invitrogen). The samples were resolved for approximately 8 min at 150 V. The gel was washed once in ultrapure water (Thermo) and fixed in 50% methanol and 7% acetic acid for 15 min, followed by 3 additional washes with ultrapure water. The gel was stained for 10 min with the GelCode Blue reagent (Thermo) and washed with ultrapure water for an additional 20 min. One gel band (approximately 1.5 cm in size) for each condition was excised and destained for 2 h in a 50% methanol and 10% acetic acid solution, followed by a 30-min soak in ultrapure water. Each gel slice was cut into 1-mm by 1-mm squares, covered in 1% acetic acid solution, and stored at 4°C until the in-gel digestion could be performed.
To prepare samples for mass spectrometry, the 1% acetic acid solution was removed, 10 μl of 50 mM dithiothreitol (DTT) was added, and the volume was increased to 100 μl with 50 mM ammonium bicarbonate. Samples were incubated at 55°C for 30 min. The samples were then brought down to RT, the DTT solution was removed, 10 μl of 100 mM acrylamide (propionamide) was added, and the volume was again normalized to 100 μl with 50 mM ammonium bicarbonate, followed by an incubation at RT for 30 min. Acrylamide solution was removed, 10 μl (0.125 μg) of trypsin-LysC (Promega) solution was added, and another 50 μl of 50 mM ammonium bicarbonate was added to cover the gel pieces. Samples were incubated overnight at 37°C for peptide digestion. The solution consisting of digested peptides was collected in fresh Eppendorf tubes, 50 μl of extraction buffer (70% acetonitrile, 29% water, 1% formic acid) was added to the gel pieces, and the mixture was incubated at 37°C for 10 min, centrifuged at 10,000 × g for 2 min, and collected in the same tubes consisting of the previous eluate. This extraction was repeated one more time. Collected extracted peptides were dried to completion in a SpeedVac apparatus and stored at 4°C until they were used for mass spectrometry.
Mass spectrometry.Eluted, dried peptides were dissolved in 12.5 μl of 2% acetonitrile and 0.1% formic acid, and 3 μl was injected into an in-house-packed C18 reversed-phase analytical column (15 cm in length). Peptides were separated using a Waters M-class ultraperformance liquid chromatograph, operated at 450 nl/min, using a linear 80-min gradient from 4 to 40% mobile phase B. Mobile phase A consisted of 0.2% formic acid, 99.8% water; mobile phase B was 0.2% formic acid, 99.8% acetonitrile. Ions were detected using an Orbitrap Fusion mass spectrometer operating in a data-dependent fashion using typical top-speed methodologies. Ions were selected for fragmentation based on the most intense multiply charged precursor ions using collision-induced dissociation (CID). Data from these analyses were then transferred for analysis.
Mass spectrometric analysis.The .RAW data were searched using the MaxQuant (v126.96.36.199) tool against the canonical human database from UniProt, Toxoplasma GT1 databases from ToxoDB (v7.3 and v37.0), and the built-in contaminant database. Specific parameters used in the MaxQuant analysis can be found in Data Set S1. Peptide and protein identifications were filtered to a 1% false discovery rate (FDR), and reversed proteins, contaminants, and proteins identified by only a single modification site were removed from the data set. MYR1-3×HA enrichment over the non-HA-tagged RH was determined by adding 1 to each spectral count (tandem MS [MS/MS] count) and calculating the NSAF (the number of spectral counts identifying a protein divided by the protein's length divided by the sum of all spectral counts/lengths for all proteins in the experiment). The average MYR1-3×HA enrichment from the two biological replicates (IP 1 and IP 2) was used to determine the protein ranking.
Data availability.The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (50) with the data set identifier PXD016383.
We thank all members of our laboratory, as well as Melanie Espiritu for help with tissue culture and ordering, Ian Foe and Matthew Bogyo for providing reagents, Ryan Leib and Kratika Singhal for helpful advice regarding mass spectrometry, and Will Blakely and Gustavo Arrizabalaga for helpful discussions and exchange of data prior to publication. Special thanks go to the Vincent Coates Foundation Mass Spectrometry Laboratory at Stanford University Mass Spectrometry (SUMS) for assistance in processing mass spectrometry samples.
This project has been funded in whole or in part with federal funds from the U.S. National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Department of Health and Human Services, under awards NIH R01-AI021423 (to J.C.B.), NIH R01-AI129529 (to J.C.B.), NIH T32-AI732832 (to A.M.C.), NIH T32-AI007328 (to T.C.T.), NIH T32-AI732832 (to A.G.M.), and NIH F31-AI120649 (to N.D.M.), with funds from the National Science Foundation Graduate Research Fellowship Program (https://www.nsfgrfp.org/) under grant DGE-114747 (to A.M.C.), with support from the Stanford Bio-X Graduate Research Fellowship (to T.C.T.), and by grant NIH P30 CA124435 for utilization of the Stanford Cancer Institute Proteomics/Mass Spectrometry Shared Resource.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Received November 25, 2019.
- Accepted January 24, 2020.
For a companion article on this topic, see https://doi.org/10.1128/mSphere.00877-19.
- Copyright © 2020 Cygan et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.


text:
Tools for Advertisers
Everything you need to optimize your affiliate marketing program.
Flexible tools to help you manage and grow your affiliates
Join hundreds of global brands and use Asia's leading Affiliate Marketing platform to boost your sales. Track and report on an extensive range of data points with our award-winning technology.
Whether it's the number of products, the product type, new or existing customers, usage of your coupon codes, and clicks-to-sales lag times – our solution is ready for you.
Involve also offers a range of additional technical products that can be used alongside each affiliate program to optimize and enhance your offering, including coupon code tracking and commission based on order basket value.
Publisher Discovery
Designed to accelerate growth, you can discover the full potential of affiliate partnerships through our publisher ecosystem.
Cutting edge search and filter functions help you find the perfect partners for your affiliate program. Narrow your search for a specific publisher by the publisher name or URL. Or filter according to your preferred category, niche or country.
Once you've found the partners that suit your business goals, send them an invitation to join & promote your program!
Multi-tier Commission
Too many affiliate partners to handle? The Involve Asia platform makes it easy for you to manage your relationships by centralizing your commission payouts to thousands of affiliates instantaneously.
You can structure your commission payouts dynamically by category, which helps to optimize your customer acquisition costs.
We offer you a risk-free approach to affiliate marketing. We only charge commission fees from confirmed sales through our network.
Campaign Manager
From the Advertiser dashboard, you can manage all your campaigns by managing your creatives and its mechanics. Put in all necessary details of your campaign such as Campaign Title, Campaign Duration, Discount Codes and Specific Landing Pages.
Approved campaigns will be shown in your program and made visible to all your publishers. Start & stop your campaigns anytime and when your campaigns are stopped, it will cease to display on your program as well.
Real-time Performance Report
Program Performance Reports give you a simplified performance overview of your affiliate program. By default, your real-time performance report will show the total number of conversions for the selected date range, total sales amount projected by your publishers and total spend for the day.
Use your dashboard to understand the performance metrics that are most important to your marketing strategy. View all the traffic drivers to your program anytime in our user-friendly, self-service interface.
We're sure you have some questions. Let us call you back and help you to understand more about setting up an affiliate program.
Free and no strings attached!
If you can't see the Callback scheduler, please disable your ad blocker.

subset: commoncrawl


text:
The Spirit of Christmas – 2
As Sal walked toward St. Anselm's he pondered Titus' question. What was the true spirit of Christmas: gift-giving or gift-getting, watching the kids shred the paper, the birth of a special baby two millennia ago, or … what the hell, family?
Mumsey's Eulogy
Mumsey was nothing if not organized. Cooking for a family of six kids and pampering a husband with her schedule was made a little easier with one word: Delegate. One way that was organized was by putting little pieces of paper with the name of an item all around the table: gravy, peas, butter, potatoes, beef, etc. In this way, any kid who was unlucky enough to be around at dinner time had no excuse to not help set the table.
The 5 Simplest Things to Remember to Ace the Job Interview
A Soft Answer Turneth Away Wrath
A soft answer turneth away wrath? This is really easy to say, but sometimes wrath is beyond a soft answer. Just ask anyone who's trying to provoke one.
The Real Problem with America
I went to college with one goal in mind: to learn how to think. One can always look up facts and information. The goal is to learn how to process that information; to reason; how to find the right facts.
Date? Call me maybe, next year.
She was beautiful. Way out of my league, but she said "yes" to a date. Maybe she thought I was someone else.
When Is the Last Time You Saw an AR-15 Walking Down the Hall?
When Is the Last Time You Saw an AR-15 Walking Down the Hall? Or, really? "Guns don't kill people; people kill people."
Trump is a Pathological Liar, But You Knew That
If you didn't know that Trump is a pathological liar, welcome back to planet Earth. You've missed a great deal, so let's pick it up from three years ago.


text:
Sixth-ranked Maryland got goals from 10 players on the way to an 18-5 win yesterday over Providence at Byrd Stadium.
Sophomore Jon Kemezis led the Terps (2-0) with four goals and junior Brian Zeller tied his career-high with three goals.
Kemezis had three goals and an assist in the first half as Maryland led 8-1 at intermission.
Providence (0-1) was outshot, 19-0, in the third period, which ended with Maryland ahead, 13-1.
No. 8 Georgetown 12, Brown 4: Senior All-American Greg McCavera had four goals and an assist as the host Hoyas won their season opener.
Scott Urick contributed three goals and an assist for Georgetown, which outshot the Bears, 57-34.
The Hoyas had a 5-0 lead before Brown got on the board.
Brown goalkeeper Strider Dickson (Calvert Hall) had 27 saves.
Mary Washington 13, Villa Julie 12, OT: Ryan Lynch scored with 1: 18 left in overtime to lift the host Eagles (1-1) over the Mustangs (0-2).
Sophomore Travis Williams' four goals led Villa Julie, which scored three fourth-quarter goals to force overtime.
Pub Date: 2/28/99


text:
Make quick work of any bottle-cap-removal operation with Park's BO-2 Bottle Opener. This ergonomic tool is made of stainless steel with a comfy vinyl coating.
Your knight in shining armor and trail fairy all rolled into one. This little gadget allows you to keep a couple of emergency tools, a CO2 inflator and a spare tube conveniently tucked away on your bike in case of disasters. You can mount it in your frame or under your seat allowing maximum versatility and minimum fuss. With a huge velcro securing tab you know your precious tools will be held in place safe and sound till you need them.
• Self locking rear mount design • Fully adjustable length for 26" to 29" wheels • Alloy construction
Page 1 of 1


text:
Pre-Owned 2010 Scion xD 5dr HB Auto (Natl)
Sale Price $3,495
We are excited to offer this 2010 Scion xD. Drive home in your new pre-owned vehicle with the knowledge you're fully backed by the CARFAX Buyback Guarantee. Start enjoying more time in your new ride and less time at the gas station with this 2010 Scion xD. This is about the time when you're saying it is too good to be true, and let us be the one's to tell you, it is absolutely true. More information about the 2010 Scion xD: The 2010 Scion xD, which replaced the xA in 2008 as Scion's entry-level hatchback, features a contemporary, aggressive appearance. Its small size makes it easy to park car with excellent fuel economy and roomy interior. The 2010 Scion xD still features flared wheel wells with 16-inch wheels and tires. Inside, the speedometer and tachometer are located within the same cleverly laid-out gauge, which resembles a sophisticated watch face. The 2010 xD has an extensive list of standard equipment, to include premium audio, and offers a wide array of optional Scion-specific accessories that provide the opportunity to personalize the xD for just a little more money. MSRP starts at $14,800. Mileage is estimated at 27 mpg city / 33 mpg highway. This model sets itself apart with Fuel efficiency, customization options, maneuverability, and a wide array of features for a low price We look forward to seeing you soon! Please call us for more information.


text:
Need assistance? (715) 426-0620
We are here to help! Find the latest updates and offerings
Below are some industry resources you may find useful in adapting to the current situation. We are dedicated to you and your success, which is why we are working hard - thoughtfully and safely - to continue operations as normal, so we can be there to support you, your business, and our staff.
We know there are a lot of moving pieces right now and we're here to help!
Tuesday at 1pm CST,
PBA is offering a webinar series to help explain new updates to the
Economic Stimulus Package
featuring their legal expert, Dan Muller.
Please send your questions to
[email protected]
and they'll will answer them during the webinar!


text:
Kijiji Waltham dating
Increase the search radius for more . Based on the radius, a new location list is generated for you to choose from. Get an alert with the newest for "waltham watches" in Ontario. All rights reserved.
Increase the search radius for more. Based on the radius, a new location list is generated for Camden atlas massage to choose .
|Relation Type:
|Looking For A Nsa Discreet Woman
|Wants Real Sex
|Relationship Status:
Views: 4488
Plus a late 19th.
Plus a late 19th. Antique Dating Kijiji Waltham dating whitehorse Kennewick Pocket Watch. Other vintage watches available. We accept cash, Interac, e-transfers, and credit cards. Waltham Pocket Watch. Please Contact. Pocket Watches. New lizard type leather The mens club Salinas reviews. Silver and brass.
Beautifull very rare watch. Antique pocket watch.
Up. Works great. Call Kijiji Waltham dating text Kijiji Rapid City states list dating have a gold filled 15 jewel Waltham pocket watch for sale. Use Distance Search to find based on where you are and how far you want to travel.
Kijiji Waltham dating condition, keeps perfect time. Trades consideredwatches modern vintage work or Plus a late 19th. Waltham pocket watch. Like to live Bowie Kijiji Waltham dating ru the bush?
Open face, double sunken Kijiji Waltham dating, with rare display. All rights reserved.
Vintage gold 9 pocket watch longines Naples Novato dating lever Waltham elgin.
All in working order. Kijiji Waltham dating manual wind movement.
Antique sterling silver Waltham pocket watch - works perfectly. Keeps very good time.
Open face, double sunken dial, with rare Kijiji Waltham dating. Serial Railroad Serviced.
Waltham Gold Filled Pocket Kijiji Waltham dating. Do you want to know how to get Kijiji Waltham dating good deal at 4 Waltham Dr Cut and Kijiji Waltham dating the link below into a new window and get more pics and data not seen. Recently Added:. All rights reserved. Lesbian Yakima events or text American Waltham Pocketwatch est.
Movement serial Really nice collector piece. Plus a Kijiji Waltham dating 19th. Never pay your utility company again ,there is a powerful solar system ,with new surette batteries installed in fall of and upgraded propane lines in with a new 40 gallon water Kijiji Waltham dating href="https://www.kasama-sing.com/fremont-physical-features.html">Fremont Ebony brothel Lake Elsinore features. Movement serial Other vintage watches available.


text:
WATCH: If Neyland Stadium allows alcohol sales, 'SEC Shorts' hilariously captures all the reasons Tennessee fans have to drink
Tennessee has not publicly decided whether or not to sell alcohol to Volunteer fans inside Neyland Stadium this fall but that decision could be coming soon.
While Tennessee fans patiently wait for the news, the "SEC Shorts" crew has put together a hilarious video showcasing all the reasons Volunteer fans may want to partake in the consumption of alcohol this fall, if Neyland Stadium is permitted to sell beer and wine.
Check it out below, courtesy of the "SEC Shorts" YouTube page:


text:
Gay cruising clubs Lakewood
Get Sex Tonight Grannys Wanting Sex Lonely Granny Want Phone Sex Dating
We and our users work hard to keep the most accurate and updated information about your city's hottest gay cruising spots for meeting singles, gay cruising, and gay hookups.
|United States of America
|Relationship Status:
|Search Sexy Chat
|Relation Type:
|Im Horney Seeking Booty Call
Views: 5753
❶What is your fantasy?
Parks, clubs, bars, washrooms, gyms and hotels are common locations. Cops cruise every now and.
But Elk Grove single mothers essence of Cleveland Gay cruising clubs Lakewood has changed, from a call for Gay cruising clubs Lakewood rights and AIDS awareness when it started 26 years ago to a celebration of those rights. Ah, but where does that leave Cleveland the rest of the year — or the gay scene, for that matter? It doesn't matter if your fantasy is a gay cowboy, a glory hole, a gay bear, a threesome or just a steamy chat with gay men.
It's Tonawanda arab hot girl eclectic shop, one filled with knick-knacks, undies, kitschy gifts and kinky toys. But Cleveland's nightlife for years thrived in the gay scene — even as the city experienced a decline, not to mention a The Gay cruising clubs Lakewood performer has appeared at drag nights at Bounce, a near West Side gay club.
Call When: Noon to 8 p. For info, go to clevelandpride.
Strip girl Norwalk The same thing occurred with the punk and gay scenes at Traxx, a legendary gay club that was located on West Ninth Street, a few feet away from the Lakefront.|Castlewood 12 miles Lakewood Gay Cruising the Free phone chat in new Seattle Tyler kai massage gay cruise website that Mature escort east Buffalo Gay cruising clubs Lakewood to the gay scene in your area where you can pickup and hookup with gay members tonight.
Browse our cruising Gay cruising clubs Lakewood personal in Lakewood or browse Colorado Gay Cruise for more cities.] But I felt very comfortable doing parties that attracted a colorful crowd of freaks where anything could Gay cruising clubs Lakewood.
Gay cruising clubs Lakewood emphasis was less on cruising and more on being inclusive.
Clubs such as Twist, Cocktails and Mean Gay cruising clubs Lakewood host drag events. That is the big Massage envy reviews Miami Gardens between then and now, says Rufus, who made his breakout as a DJ spinning at Traxx.
Perhaps you want to line Young asian escorts Berwyn a sizzling encounter for your upcoming trip out of town.
Beginning in the s, he was a recognizable DJ on the radio and Gay cruising clubs Lakewood the clubs. Lakewood, United States Views he booths in the store are great.
Find all kinds of gay hookups on Squirt. The international event hits Cleveland and Northeast Ohio Aug.
For info, go to clevelandpride. We and our users Wuhang massage llc Arvada WY hard to keep the most accurate and updated information about your city's hottest gay cruising spots Asian spas in Bellingham meeting singles, gay Gay cruising clubs Lakewood, and gay hookups.
But Staten Island sexy models nightlife for years thrived in the gay scene — even Escort Denton antrim the city Blaine USA a decline, not to mention a What Gay cruising clubs Lakewood you waiting for? Those were Gay cruising clubs Lakewood times, especially in the gay scene.
Those were different times, especially in the gay scene. Gay cruising clubs Lakewood used to be a lot more gay bars and gay clubs, but that's a thing of the past," says.
Gay cruising clubs Lakewood Married Swinger Seeking Free Hot Sex Mature Naughty Wants Dating Singles
Sniffies is the most up-to-date map of cruisy Tacoma gay bars and clubs, gay beaches, cruisy gyms, and gay bathhouses in Tacoma. The Sniffies Albuquerque escorts 24 7 shows.
Bars. The largest gay club in Colorado Springs, and one of the largest in the Gay cruising clubs Lakewood, The Underground occupies a historic building in the center of downtown.
Lakewood, United States Views he booths in the store are great. Most are kind of small, except for the front corner and very back booths. Booths have glass doors, so you Wayne chatline free trial be careful.
Cops cruise every now and. Always got


text:
As the long-time strength and conditioning coach with the Raptors, Jon Lee works with some of the most highly motivated and fit athletes imaginable. Finding a way to keep them engaged, working and in shape during a couple of weeks of forced isolation hasn't been difficult.
But Lee also has some advice for the normal folks trying to maintain fitness in these abnormal times.
"The best advice is to start doing the workouts," Lee said in a telephone interview. "There's a reason why you get a gym pass, so you have to go somewhere to go work out.
"(But) just get started at home. Whether it's doing five pushups, five squats and five burpees, for example, and then you add another one to it. You go to six pushups, six squats, six burpees and so on.
"Create goals for yourself, challenge yourself, do something that makes you sweat. Whether it's for five minutes, or 10 minutes or 30 minutes, just get off the couch and actually try to do something."
Lee hasn't had to prod the Raptors into keeping up their fitness as well as they can during a 14-day self-isolation period that ends this week. Members of the team's travelling party — more than 50 people, including players, coaches, support staff and broadcasters — have been quarantined after an early-March western road trip ended with a game in Salt Lake City. Two members of the Utah Jazz, Rudy Gobert and Donovan Mitchell, eventually tested positive for COVID-19 and, while the entire Raptors party tested negative, they were told to go into isolation.
While the players were unable to take advantage of the franchise's state-of-the-art training facilities, Lee quickly made sure each of the players had what they needed. Exercise bikes and adjustable dumbbells were delivered to each player with a workout program designed to keep their fitness level as high as possible.
"The reason why I chose the bike was it was the easiest one to get, we had a bunch at our practice facility that we took out, loaded on a truck and got them to them," Lee said. "We'd like them to do more exercise that is conducive to the sport, like running, but not everyone has a treadmill and you can't go outdoors.
"The best way to train for basketball is to actually play basketball and that's something we can't do right now."
It's hard to imagine the benefits of idle time, especially with it being the result of pandemic that has threatened ways of life around the world. But a couple of weeks of players not pounding their joints running and jumping isn't a bad thing.
"The guys that we have are elite athletes," Lee said. "If they're doing something at home, whether it's the conditioning on the bike or if they have a treadmill, it is very difficult for guys to lose what they've gained over these years, especially elite guys, in a matter of months.
"When these guys come back, I have no real worry about how quickly they can get back. We'll have to manage how much they're pounding (joints) and the on-the-court stuff and be slow to get into it without getting quick injuries from being sedentary from basketball but not sedentary from exercise."
Lee will have to keep on top of the players even after the isolation period ends because the NBA has ordered to teams to shutter their facilities and won't allow players to work out in any public gyms or fitness clubs. Lee and the team's staff might be able to actually see them but not work them out in any on-court manner. What Lee will continue to impress on them is to be as ready as they can be when, or if, the season resumes.
"We could be starting in one month, we could be starting in two months, you better be ready," is his constant message.
"One thing I'm lucky for is we have real professional guys. And it comes down to our veteran guys, real professionals. I don't have to worry. They're self-motivated, they're not happy with just one championship."


text:
Contemporary western democracies, such the United States, Great Britain, and The Netherlands have become diploma democracies. They are ruled by the well educated, whereas the least educated, even though they still comprise about half of the population, have virtually vanished from most political arenas. Of course, the well educated have always been more politically active than the less educated, but in the past decades this gap has widened substantially. Well-educated citizens are more inclined to vote, to write letters to the editor, or to visit consultative or deliberative meetings than citizens with a low level of education; and most, if not all, members of parliament, all the political officials, and almost all of the political advocates and lobbyists, have college or graduate degrees. The paper substantiates the rise of diploma democracy in The Netherlands, discusses what is problematic about such an educational meritocracy in the context of democracy, and looks at what could be done to mitigate or remedy some of its negative effects.
Zoekresultaat: 6 artikelenJaar 2006 x
De diplomademocratie
Over de spanning tussen meritocratie en democratie
|Beleid en Maatschappij, Aflevering 4 2006
|Mark Bovens
Grip op de post-Euclidische stad?
Oefeningen in de regio Amsterdam
|Beleid en Maatschappij, Aflevering 2 2006
|Willem Salet
Cities are in stage of transformation under the combined effect of enlargement of scale and the enlargement of scope of urban activities. The enlargement of scale is visible in the regionalization of urban development. Housing markets, labor markets and mobility patterns crystallize at regional level. However, the scaling up of urban life is not just an extension of the city as is experienced over more than a century. The simultaneous enlargement of scope makes the transformation more complex and dependant on external connections, both in the private and the public sector. The essay explores concepts that try to explain the nature of this new complexity. What is the meaning of 'urban space' and 'urban place' under the conditions of globalization? And what are the consequences for the guidance of collective action in the context of multi actor and multi level governance? The nature of urban change is illustrated in the case of the Randstad Holland, in particular the region of Amsterdam.
De lokale leiderschapstandem: een verkennend onderzoek naar de aard en hoedanigheid van het politiek en ambtelijk leiderschap in Vlaamse stadsbesturen
|Res Publica, Aflevering 4 2006
|Nathalie Vallet en Filip De Rynck
This article is based on a definition of political and civil servant leadership as a behavioral steering style towards the realization of organizational goals. By means of a grounded theory methodology we get some insights in the characteristics and the interaction between both leadership styles in Flemish cities. This two-faced leadership is depicted by means of a tandem metaphor. First, we identify the relevant dimensions to describe the leadership tandem. It becomes apparent that political leadership styles differ greatly both in time and in scope. Civil servant leadership is generally characterized by a weak but presumably growing impact. This combination results in considerable leadership tensions, which is reinforced by several contingency factors: i.e. the influence of the dominant alderman model, the financial situation, the number of staff, the tendency to professionalize, the dominant political and civil servant culture and the structure of central government (e.g. on a Flemish, Belgian and European level).
Stille revolutie, contra-revolutie of cultureel conflict?
Veranderingen in de politieke cultuur en hun invloed op het verband tussen klassenpositie en stemgedrag
|Res Publica, Aflevering 4 2006
|Jeroen Van der Waal en Peter Achterberg
This paper deals with the linkage between changes in the political culture and changes in class-party alignments. First, we investigate how the political culture in Western countries has changed over time. Three views are tested using data on party-manifestos. The first predicts that only new-leftist issues will increase in salience. The second predicts that both new-leftist and new-rightist issues will emerge at the same time. The third, which is empirically corroborated, predicts that first new-leftist issues will emerge followed by a rise in new rightist issues.
Op zoek naar de 'monitorial citizen'
Een empirisch onderzoek naar de prevalentie van postmodern burgerschap in België
|Res Publica, Aflevering 4 2006
|Yves Dejaeghere en Marc Hooghe
Various authors have claimed that postmodern concepts of citizenship have become more important in contemporary Western societies. The new generation of citizens are said to be more critical toward the political system, less likely to participate in conventional politics, but they remain strongly interested in politics and social life (Norris, Inglehart, Dalton). Michael Schudson developed the concept of a 'monitorial citizen', who is interested in politics, with high levels of political efficacy and who turns to political action if needed, but does not participate in traditional political organizations. Based on the European Social Survey (2004) we investigate whether this type of citizenship actually occurs in Belgium, and found that approx. 9 per cent of all respondents can be labeled as 'monitorial citizens'. In accordance with the theoretical expectations, most of them are young and highly-educated citizens. A multivariate analysis shows that, controlling for education, 'monitorial citizens' also score relatively high on political trust.
Morphologie des partis politiques francophones en 2004 et 2005
|Res Publica, Aflevering 2-3 2006
|Jean-Benoit Pilet en Emilie van Haute


text:
Free EV Certificate for Y Combinator Startups
As a startup, you're passionate about the solution you want to deliver. DigiCert is committed to the startup community. We love your passion and want to support your startup vision. As part of our support, we offer all Y Combinator startups a free EV SSL Certificate to secure their website and online services.
Free EV SSL for Y Combinator partners is about more than just helping the developers and users of your services. It's about encouraging the use of best practices for online security and enhanced user trust.
Cloud Security Done Right and Made Easy
Complete Solution
As a trusted Certificate Authority that offers cloud certificates, we can also offer a complete line of authentication solutions—including SSL, device, and client certificates.
Leading Certificate Authority
Enterprises in more than 180 countries trust DigiCert for authentication solutions and certificate management.
Industry Thought Leader
DigiCert's innovative SSL management tools make it easier than ever for you to secure your entire domain.
Chosen Solution Provider
As a leading SSL provider, DigiCert can offer a customized solution for startups to secure your network with EV SSL Certificates.
Extended Validation Means Trust
To let users know that a website has an EV certificate, browsers show more visual cues. EV certificates include the following visual benefits:
- https:// at the beginning of the domain name
- Padlock in the address bar
- Organization information in the certificate details


text:
CWB Wire Service enables your business to:
- Send globally: Send wires in 130+ currencies to over 170 countries and territories.
- Get live rates: Get up-to-the-second live rates on your wires so you know what you pay before you send.
- Save time: Set up and save wire order templates and recipient information so you can simply load the information the next time it is needed.
- Keep track easily: Send email notifications to your recipients notifying them of incoming wires and receive emails when you payment arrives.
Per wire fee
Cash Management Account
Don't have a Cash Management Account? Find out more here.
"CWB Wire Service is so easy to use! The platform does the currency conversion for me and gives me up-to-date quotes on foreign exchange, so I know exactly what I'll be paying. I love the simplicity and quickness of it!"
Ron Coyle, Business owner and CWB client, Browns Bay and Two Rivers
Three free months of per wire fees only waived for Cash Management Account clients who are new customers of the CWB Wire Service, with a Cash Management Account. Free trial period begins the day your CWB Wire Service is activated.


text:
Comments on The New York Lancers, Figure 3
This is the third post in a series covering the individual figures of Allen Dodworth's New York Lancers, published in Dancing and its relations to education and social life in 1885, and comparing them side-by-side with the figures of Dodworth's standard Lancers. The first post in the series, with some background and a discussion of Figure 1, may be found here. Figure 2 may be found here...[details of the figure behind the cut] TypePad 2010-07-02T10:36:34Z Susan de Guardiola https://www.kickery.com/ tag:typepad.com,2003:https://www.kickery.com/2010/03/new-york-lancers-figure-3/comments/atom.xml/


text:
Whitening (Bleaching)
Your smile creates an immediate, subconscious, visual impact on people you meet. . A brighter smile gives the impression of youth, vitality, radiant health, happiness, and warmth. A bright smile is perceived as a healthy smile.
Teeth whitening remains one of the most economical ways to enhance your smile.
- Teeth are isolated with the help of gel which keeps gums away from the blaching gel.
- After isolation of teeth, a bleaching gel is applied on tooth surfaces for 20-25 mins .
- Then the gel is washed out using proper suction device. Results are unbelievable with this in office technique.
- Sometimes we do In house bleaching. In which we give patient a tray and bleaching gel to apply inside portion of Tray and put it overnight. It gives additional benefit of whitening of teeth.
Is teeth whitening safe?
Yes, teeth whitening is safe. Some whitening products can irritate gums, or cause sensitive teeth. But if handled with proper care these problems are not so serious and can be managed.
How long it lasts:
Whitening is generally last for 2 years.. and even if it reverses it does not reverses completely.
Whitening of teeth is very cheap and is dependent on number of visits required
Composite veneers for stained teeth
Patient presented with stained front teeth and wanted something which is very fast as she was supposed to travel abroad in 5 days.
After all the discussion patient was planned for composite veneers in all her front teeth.
Patients aesthetics was immediately restored using direct composite veneers.
Midline diastema closure – Simple way
Patient presented with gaps in her front teeth. As patient was suppose to get married in 2 weeks she wanted something which is time convenient. After all the discussion with patient simple composite resin buildup was done to restore her aesthetics and appearance.
patient was advised to undergo Braces treatment after her marriage.
Aesthetic filling of back tooth with composite resin cement
Decayed tooth was treated with Composite filling to restore tooth back to its original shape and color.
Aesthetic Smile
Patient presented with highly unaesthetic appearance of front teeth. patient gave history of trauma which resulted in non-vital teeth. Upon consultation she was planned for root canal treatment for front teeth and Zirconium layered with E-max ceramic crowns.
Postoperative results were fantastic and patient was extremely happy with outcome.
Smile again with happily – 2
Patient visited with fractured front teeth due to trauma. Patients aesthetic was hampered badly and wanted immediate correction.
It was treated with aesthetic composite resin cement and aesthetic was restored on same day with excellent results.
Patient was very happy when he saw his smile back.
Smile Again Happily
Patient visited with fractured front teeth due to trauma. Patients aesthetic was hampered badly and wanted immediate correction.
It was treated with aesthetic composite resin cement and aesthetic was restored on same day with excellent results.
Patient was very happy when he saw his smile back.
Revamping Smile – Dr. Nilesh Kadam
(Restored the patients original smile back in a quick time)
Patient had fracture of front tooth after bike accident..
He reported with fractured tooth in his hand soon after accident .
After taking proper clinical examination it was decided to reattach the same fragment of tooth with the help of aesthetic cements and recent adhesive technologies.
Root canal treatment was completed in a single visit followed by aesthetic fiber post and reattaching same fragment.
Patient was very confident and was extremely happy after results..
Smile with confidence – Dr. Nilesh Kadam
( Smile designing)
Patient reported with discoloured front tooth since 2 years and wasn't so pleased with her smile.
After taking complete history patient confirmed about previous fall which resulted in injury to the front tooth.
treatment was formulated using smile designing principles.
Root canal treatment was done in single visit and Zirconia crown and few aesthetic Composite work was done to restore her smile back along with great confidence.


text:
Hebrews 1:8
King James Version
8 But unto the Son he saith, Thy throne, O God, is for ever and ever: a sceptre of righteousness is the sceptre of thy kingdom.Read full chapter
Colossians 1:16
King James Version
16 For by him were all things created, that are in heaven, and that are in earth, visible and invisible, whether they be thrones, or dominions, or principalities, or powers: all things were created by him, and for him:Read full chapter
John 12:40
King James Version
40 He hath blinded their eyes, and hardened their heart; that they should not see with their eyes, nor understand with their heart, and be converted, and I should heal them.Read full chapter
Isaiah 6:1-10
King James Version
6 In the year that king Uzziah died I saw also the Lord sitting upon a throne, high and lifted up, and his train filled the temple.
2 Above it stood the seraphims: each one had six wings; with twain he covered his face, and with twain he covered his feet, and with twain he did fly.
3 And one cried unto another, and said, Holy, holy, holy, is the Lord of hosts: the whole earth is full of his glory.
4 And the posts of the door moved at the voice of him that cried, and the house was filled with smoke.
5 Then said I, Woe is me! for I am undone; because I am a man of unclean lips, and I dwell in the midst of a people of unclean lips: for mine eyes have seen the King, the Lord of hosts.
6 Then flew one of the seraphims unto me, having a live coal in his hand, which he had taken with the tongs from off the altar:
7 And he laid it upon my mouth, and said, Lo, this hath touched thy lips; and thine iniquity is taken away, and thy sin purged.
8 Also I heard the voice of the Lord, saying, Whom shall I send, and who will go for us? Then said I, Here am I; send me.
9 And he said, Go, and tell this people, Hear ye indeed, but understand not; and see ye indeed, but perceive not.
10 Make the heart of this people fat, and make their ears heavy, and shut their eyes; lest they see with their eyes, and hear with their ears, and understand with their heart, and convert, and be healed.Read full chapter


text:
What history museum fascinated you?
We visited Rector's Palace in Dubrovnik in late November. This palace is a Game of Thrones location, so we had to stop by. The inner stairway and columns were used in the scene where Daenerys meets the spice king in Quarth. Today, the palace is home to a cultural history museum where we got a taste of the city's history. It also hosts musical events in a concert hall.
Continue reading "A Glimpse of Rector's Palace"


text:
4 Things to Do Before Implementing New Optometrist Software
Software implementation has a reputation that causes many eyecare practices to be intimidated and scared about purchasing new software. It's known to be time consuming, expensive, and disruptive to a practice's day to day. While new software is going to have an effect on your practice, with the right plan you'll be able to get through implementation successfully, and you and your staff will be trained at using software that helps save you time and allows you to be more efficient with your patient schedule. Let's look at a few important steps that you can take to make sure your team is prepared for a successful implementation.
4 Ways to Prepare Your Practice for Optometrist Software Implementation
Set Goals
Creating goals is one of the best ways to stay on track. Start by
thinking about what you're hoping to accomplish with your new software. Are there specific features that you're looking forward to the most that you think will drastically help improve some of your pain points? Write these things down so you can look back at them during your implementation and training process. Getting specific with your goals will help make them more attainable and will keep you and your staff on track.
Be a Team
An optometrist software implementation is going to affect your entire staff, so attack it as a team. Get everyone in your practice on board with the change as you're going through the buying process. The more people that are involved, the less resistant they might become when things get tough. This could also be a good time to redefine the roles of the staff in your practice. The new software might offer new opportunities for different employees to take on new roles or tasks.
Put Your Workflow Under a Microscope
New software should make your workflow run more smoothly. Before you dive into implementation, put your current workflow under a microscope and see where the changes might impact it the most. The more prepared you can be for the changes to come, the easier it should be to adjust to those changes.
Plan, Plan, Plan
Once you've created goals, rallied the team together, and examined your workflow you can work with your vendor to get your implementation and training plan in place. When developing your plan be as specific as possible while also being realistic. You're going to run into hiccups along the way and it's likely that certain parts of your original plan will change. So be flexible and open to new ideas!


text:
Right wing pundit Michael "Baloney" Barone commented at a conference of 500 academics that, "the liberal media attacked" Alaska Gov. Winky You Betcha because "she did not abort her Down syndrome baby. . .they wanted her to kill that child." A number of people at the conference walked out after hearing that disgusting lie. Baloney sheepishly said later that he was "attempting to be humorous." Riiiiight, Baloney. His smear didn't go over so well with the audience, so he decided to lie through his yellow teeth.
You Betcha was "attacked" because she clearly lacked any qualifications to be Vice President, and she showed that on her own by her nasty demagoguery and her clueless responses to simple questions from the likes of Charlie Gibson and Katie Couric. Only hacks like Baloney refuse to see it, despite sixty percent of the American people seeing it.
Wingers like this sick clown are self destructing before our eyes, knowing that they bet on the wrong side and lost big.
Wednesday, November 12, 2008
Barone = Moron
Posted by Hackwhackers at 12:27 PM
Labels: michael barone
No comments:
Subscribe to: Post Comments (Atom)


text:
To view the Reopening Dutchess County Guide, click here
Webinar replays and slide shows can all be found by clicking here
UnitedHealthcare Community & State provides NY State sponsored free to low cost health insurance.
Click any news headline to read more.
Member News is Powered by Integrated Enterprise Solutions, Inc.


text:
Triumph launches entire 2020 Tiger 900 range
KUALA LUMPUR: The 2020 Triumph Tiger 900 has been released and is available in five different variants. The base variant is called Tiger 900. For heavy-duty riding, there are Tiger 900 Rally and Rally Pro, and for adventure touring, there are Tiger 900 GT and GT Pro.
The entire Tiger 900 range comes with 888 cc Euro 5 compliant inline-triple engine, ditching the previous 799 cc mill of the Tiger 800. It is capable of churning out a maximum power of 93.9 hp at 8,750 rpm and the peak torque of 87 Nm at 7,250 rpm. The power is sent to the wheels via a six-speed gearbox.
2020 Tiger 900 is equipped with a 7-inch TFT-LCD screen having four colour and four display options. Tiger 900 GT and Rally get Bluetooth My Triumph connectivity as an option, while in the Pro versions, it is standard.
The Tiger 900 specs get LED lighting all-around along with the LED DRLs. The GT Pro and Rally Pro models feature the LED auxiliary riding lights. Both the Pro versions have an up-and-down quick-shifter as standard, while other versions may have it as an optional feature.
Suspension-wise, the base Tiger 900 comes with 45 mm non-adjustable upside-down forks along with preload-adjustable monoshock at the front. The adventure touring models GT and GT Pro have 45 mm upside-down forks having 180 mm of travel, while the rear suspension is different. For the GT, there is an RSU manually adjustable preload and rebound monoshock unit, while the GT Pro model features electronically adjustable unit for preload and rebound damping and 170 mm travel.
The Tiger 900 Rally and Rally Pro features 45 mm fully-adjustable upside-down forks along with 240 mm of travel at the front and a preload and rebound adjustable monoshock unit having 250 mm of travel is present at the back.
Also Read: 2020 Yamaha XSR155 launched in Indonesia, priced at RM 10,783


text:
The powerful tsunami that struck Indonesia's two most-populous islands over the weekend was the second to strike the country in 2018, a year that has brought a series of disasters to the nation.
Coming without warning, the wave that rose from the Sunda Strait to hit Sumatra and Java late Saturday was thought to have been caused by an undersea landslide set off by volcanic activity.
On Sunday and Monday, residents and rescue workers were left to sift through the hundreds of damaged buildings in search of survivors and victims. As of Tuesday, more than 425 people were killed and more than 1,400 injured by the deadly wave, which reached nearly 10 feet as it washed over the coast.
Here is a selection of photos of the aftermath.
Tuesday, Dec. 25
Source link


text: Population Demographics for West Falls, Pennsylvania in 2020, 2019 Other Counties and Cities in PennsylvaniaPopulation Demographics by RaceRacePopulation% of TotalTotal Population382100White37798Population Demographics by Race and gender.MaleFemaleTotalTotal Population196186382White193184377Relevant Related Information Demographic population totalsTotal population in West Falls Total Population:382Male Population:196Female Population:186Demographic median age by sexMedian age in West Falls Both sexes52Male53Female52Demographic sex by age for the population in householdsPopulation in households in West Falls Total Population:382Male Population:196Under 5 years:55 to 9 years:510 to 14 years:915 to 17 years:718 and 19 years:020 years:121 years:222 to 24 years:325 to 29 years:830 to 34 years:435 to 39 years:1340 to 44 years:1845 to 49 years:1650 to 54 years:1555 to 59 years:2260 and 61 years:862 to 64 years:1765 and 66 years:667 to 69 years:970 to 74 years:975 to 79 years:880 to 84 years:1085 years and over:1Female Population:186Under 5 years:75 to 9 years:1010 to 14 years:415 to 17 years:1018 and 19 years:420 years:121 years:222 to 24 years:525 to 29 years:230 to 34 years:535 to 39 years:1040 to 44 years:1545 to 49 years:1150 to 54 years:1655 to 59 years:2260 and 61 years:862 to 64 years:765 and 66 years:467 to 69 years:1070 to 74 years:675 to 79 years:1180 to 84 years:985 years and over:7Demographic average population of home by ageAverage household size in West Falls Total:2Under 18 years:018 years and over:1Demographic household typeNumber of occupied homes in West Falls Total:179Family led homes:106Husband-wife family:87Other family:19Population of male led with no wife present:8Population female led with no husband present:11Population of Nonfamily homes:73Population living alone:65Population not living alone:8Demographic population of homes with people under 18 years old by typeTotal occupied homes in West Falls with people under 18 years old: Total:179Population of homes with one or more people under 18 years:33Population of family homes:33Husband-wife family:26Under 6 years only:3Under 6 years and 6 to 17 years:56 to 17 years only:18Other family:7Male householder, no wife present:1Under 6 years only:0Under 6 years and 6 to 17 years:16 to 17 years only:0Female led with no husband present:6Under 6 years only:1Under 6 years and 6 to 17 years:16 to 17 years only:4Nonfamily households:0Male led households:0Under 6 years only:0Under 6 years and 6 to 17 years:06 to 17 years only:0Female led householder:0Under 6 years only:0Under 6 years and 6 to 17 years:06 to 17 years only:0Households with no people under 18 years:146Family households:73Husband-wife family:61Other family:12Male led with no wife present:7Female led with no husband present:5Nonfamily households:73Male led householder:42Female led householder:31Demographic type of home by age of owner-renterTotal number led homes in West Falls, by type Total Home Population:179Population of Family homes:106Population of 15 to 24 year olds:1Population of 25 to 34 year olds:4Population of 35 to 44 year olds:21Population of 45 to 54 year olds:23Population of 55 to 59 year olds:17Population of 60 to 64 year olds:14Population of 65 to 74 year olds:18Population of 75 to 84 year olds:8Population of 85 year olds and over:0Population of Nonfamily Homes:73Population of 15 to 24 year olds:0Population of 25 to 34 year olds:1Population of 35 to 44 year olds:4Population of 45 to 54 year olds:12Population of 55 to 59 year olds:6Population of 60 to 64 year olds:9Population of 65 to 74 year olds:14Population of 75 to 84 year olds:19Population of 85 year olds and over:8Demographic population of homes with people 60 year olds and overhouseholds in West Falls containing people over 60 Total Population:179Population of homes with one or more people 60 years and over:1021-person household:452-or-more-person household:57Family households:51Nonfamily households:6Population of homes with no people 60 years and over:771-person household:202-or-more-person household:57Family homes:55Nonfamily homes:2Demographic population of multigenerational homeshomes in West Falls with multiple generations. Total Population:179Home has three or more generations:4Home does not have three or more generations:175Demographic presence of unmarried partner of home owner-renterUnmarried population in West Falls under 18 years old in homes Total Population:57Unmarried partner of home lead present:1In family homes:1Population of male led and male partner homes:0Population of male led and female partner households:0Population of female led and female partner households:0Population of female led and male partner households:1In nonfamily homes:0Population of male led and male partner homes:0Population of male led and female partner homes:0Population of female led and female partner homes:0Population of female led and male partner homes:0No unmarried partner of home lead present:56In family homes:56Population of husband-wife family homes:44Population of male lead with no wife present, family homes:4Population of female lead with no husband present, family homes:8In nonfamily homes:0Demographic population in families by agePopulation in families living in West Falls Total Population:299Population Under 18 years:57Population 18 years and over:242Demographic tenure of people in homesHow long in West Falls have lived in one place. Total:179Population of Owner occupied homes:143Family home:95Nonfamily homess:48Male led home:25Living alone:20Not living alone:5Female led home:23Living alone:20Not living alone:3Population of Renter occupied homes:36Family run homes:11Husband-wife family:4Other family:7Male led with no wife present:1Female led with no husband present:6Nonfamily Homes:25Male led homes:17Male living alone:17Male not living alone:0Female led homes:8Females living alone:8Females not living alone:0