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The inner membrane complex (IMC), a series of flattened vesicles at the periphery of apicomplexan parasites, is thought to be important for parasite shape, motility and replication, but few of the IMC proteins that function in these processes have been identified. TgPhIL1, a Toxoplasma gondii protein that was previously identified through photosensitized labeling with 5-[125I] iodonapthaline-1-azide, associates with the IMC and/or underlying cytoskeleton and is concentrated at the apical end of the parasite. Orthologs of TgPhIL1 are found in other apicomplexans, but the function of this conserved protein family is unknown. As a first step towards determining the function of TgPhIL1 and its orthologs, we generated a T. gondii parasite line in which the single copy of TgPhIL1 was disrupted by homologous recombination. The TgPhIL1 knockout parasites have a distinctly different morphology than wild-type parasites, and normal shape is restored in the knockout background after complementation with the wild-type allele. The knockout parasites are outcompeted in culture by parasites expressing functional TgPhIL1, and they generate a reduced parasite load in the spleen and liver of infected mice. These findings demonstrate a role for TgPhIL1 in the morphology, growth and fitness of T. gondii tachyzoites.
Citation: Barkhuff WD, Gilk SD, Whitmarsh R, Tilley LD, Hunter C, Ward GE (2011) Targeted Disruption of TgPhIL1 in Toxoplasma gondii Results in Altered Parasite Morphology and Fitness. PLoS ONE 6(8): e23977. https://doi.org/10.1371/journal.pone.0023977
Editor: Georges Snounou, Université Pierre et Marie Curie, France
Received: January 4, 2011; Accepted: August 1, 2011; Published: August 25, 2011
Copyright: © 2011 Barkhuff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grants AI01719 (GEW) and AI42334 (CH), www.nih.gov. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The Phylum Apicomplexa contains a number of medically important parasites including Cryptosporidium spp., which cause diarrheal illness in children and immunocompromised patients, Plasmodium spp., which are the causative agents of malaria, and Toxoplasma gondii, which causes life-threatening disease in immunocompromised people and the developing fetus. Other apicomplexan organisms, including Neospora caninum and Eimeria spp., cause severe disease in domestic animals. Despite the significant morbidity, mortality and economic loss caused by these pathogens, information is lacking regarding many aspects of their basic biology. T. gondii is a powerful model system for studying conserved aspects of apicomplexan biology, due to the ease with which T. gondii can be cultured and genetically manipulated [1], [2].
Apicomplexan parasites contain a number of unusual subcellular structures and organelles [3], [4], including the inner membrane complex (IMC), a series of flattened vesicles that are tightly apposed to the cytosolic face of the plasma membrane [5], [6]. Together, the plasma membrane and IMC are referred to as the pellicle. Underlying the IMC is the subpellicular network, a meshwork of intermediate filament-like proteins [3], [7], [8]. Additional cytoskeletal structures associated with the pellicle include the conoid, which is a cone-shaped structure composed of a novel polymeric form of tubulin [9], a pair of polar rings located at either end of the conoid [9], [10], and 22 microtubules radiating posteriorly from the lower polar ring [10].
Various functions for the IMC, subpellicular network, and subpellicular microtubules have been proposed [3], [4], [8], [11]. During parasite replication, a process known as endodyogeny, the IMC forms early and may provide a scaffold upon which daughter parasites are assembled, as well as mechanical stability for the mature tachyzoites [3], [7], [12]. Additionally, some components of the myosin motor complex, the proteins that provide the mechanochemical force for parasite motility and host cell invasion, are anchored in the IMC. Localization of the motor complex between the outer membrane of the IMC and the plasma membrane is thought to be critical for motility and invasion [13]–[15]. While one member of the myosin motor complex, GAP45, appears to physically connect the IMC to the plasma membrane [16], and the recently identified GAPM proteins may function to anchor the subpellicular network to the flattened IMC vesicles [17], the detailed mechanism(s) by which the IMC, subpellicular network and subpellicular microtubules associate with each other and are organized at the parasite periphery have yet to be elucidated.
TgPhIL1 (Photosensitized INA-Labeled protein 1) was previously identified through photosensitized labeling with 5-[125I] iodonapthaline-1-azide (INA) and localized to the parasite periphery, concentrated at the apical end directly posterior to the conoid [18]. TgPhIL1 is highly conserved among members of the Phylum Apicomplexa but shows little homology to proteins outside of the phylum. When parasites were treated with Clostridium septicum alpha-toxin, which causes the plasma membrane to swell away from the IMC [19], TgPhIL1 remained associated with the IMC and/or associated cytoskeletal structures [18]. Although TgPhIL1 labeling with INA suggests this protein is membrane associated, TgPhIL1 is also highly insoluble and extracts much like a cytoskeletal protein [18].
In order to learn more about the function of TgPhIL1, we generated parasites containing a TgPhIL1 deletion. Phenotypic analysis of the knockout parasites revealed that TgPhIL1 is necessary for maintaining the distinctive shape of T. gondii tachyzoites, and parasites lacking TgPhIL1 show both a growth defect in culture and reduced fitness in a mouse model of infection.
Materials and Methods
Ethics statement
All mouse experiments were performed with approval of the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania (Protocol # 801490; "Regulation of the early response to Toxoplasma gondii").
Culture of parasites
Wild-type RH strain T. gondii was maintained by serial passage in confluent primary human foreskin fibroblast (HFF) cells in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1% (vol/vol) heat inactivated fetal bovine serum (FBS), 10 units/ml penicillin G, 10 µg/ml streptomycin sulfate, and 10 mM HEPES buffer, as previously described [20].
Generation of the TgPhIL1 knockout parasite line (RHΔTgPhIL1)
All PCR reactions were performed with Pfx polymerase (Invitrogen, Carlsbad CA) unless otherwise indicated. All primers were synthesized by Sigma Genosys (The Woodlands TX) and restriction enzymes were purchased from either New England BioLabs (Ipswich MA) or Invitrogen.
In order to generate the TgPhIL1 knockout construct, PCR was performed on T. gondii genomic DNA using primers PhIL15′KOfor (5′GCTGAGGAGGGGAAGAAGATCGAG3′) and PhIL13′KOrevXbaI (5′TGCTCTAGATATCCGCAAATGCTCGCTCGCC3′) to generate a 6.5 kb amplicon containing 2.9 kb of TgPhIL1 5′ flanking sequence, the 1.3 kb TgPhIL1 open reading frame (ORF), and 3.1 kb of TgPhIL1 3′ flanking sequence. The amplified product was cloned into pCR-Blunt TOPO (Invitrogen) and inverse PCR was performed with primers KOinvfor (5′AGGAGTGTTCATGTTGTTTGCTG3′) and KOinvrev (5′TGAATTCGTCTAATCCAGAGTCTGTC3′) to remove the TgPhIL1 ORF. The ble cassette was digested from pGRA1/ble [21] with HindIII and XbaI and the ends were filled in with Klenow, followed by blunt ligation to the inverse PCR product in order to generate the knockout construct pΔPhIL1/ble. The plasmid was linearized with Not1 and transfected into RH parasites. The transfected parasites were added to confluent HFF cells and selected with phleomycin as described previously [21]. Individual clones were isolated by limiting dilution and clones lacking TgPhIL1 expression were identified by immunofluorescence with anti-TgPhIL1 [18].
Cloning TgPhIL1 under its endogenous promoter for complementation
PCR was performed on T. gondii genomic DNA using the primers PhIL-Endofor -XbaI (5′GGGGTCTAGATGAAAGACTGGAGCATTTCG-3′), corresponding to sequence 2 kb upstream of the TgPhIL1 ORF, and PhIL1-Endorev-BglII (5′GGGGAGATCTTCACCGAGAGAAGTCGAGTG-3′) containing the 3′ end of the TgPhIL1 ORF, followed by a stop codon. After digestion with XbaI and BglII, the product was ligated into ptubIMC1-YFP/sagCAT [22] which had also been digested with XbaI and BglII in order to remove the tubulin promoter and TgIMC1 ORF, generating pTgPhIL1Endo/sagCAT. Complemented parasites were maintained in 20 µM chloramphenicol.
Western blot analysis
Western blot analysis of parasite lysates was performed as described previously [23], using either a rabbit polyclonal anti-TgActin antibody generously provided by Dr. David Sibley [24] or rabbit polyclonal anti-TgPhIL1 [18], at dilutions of 1∶10,000 and 1∶5,000 respectively.
Parasites were attached to glass coverslips with BD-CellTak (BD Biosciences, San Jose CA) as previously described [23], fixed in phosphate-buffered saline (PBS) containing 4% (vol/vol) paraformaldehyde for 10 minutes at 23°C and permeabilized for 30 minutes at 23°C in PBS containing either 10 mM deoxycholic acid (for anti-α-tubulin and anti-TgPhIL1) or 0.25% (vol/vol) Triton X-100 (for anti-ISP1 and anti-T. gondii polyclonal serum). The permeabilized parasites were incubated for 15 minutes in PBS containing 2% (wt/vol) BSA (PBS-2% BSA) and one or more of the following primary antibodies: anti-TgPhIL1, diluted 1∶500; mouse monoclonal anti-α-tubulin (Sigma-Aldrich, St. Louis MO), diluted 1∶400; mouse monoclonal anti-ISP1 (monoclonal 7E8; [25]), diluted 1∶2000; and rabbit polyclonal anti-T. gondii (catalog #90700556; AbD Serotec, Raleigh, NC), diluted 1∶2000. Immunofluorescence with anti-TgMORN1 ([26]; 1∶500), and anti-TgIMC1 ([23], 1∶100) was performed after fixation of infected HFF monolayers on glass coverslips in 100% MeOH on ice for 5 minutes. After incubation with primary antibodies, the samples were incubated with a 1∶1000 dilution of Alexa488- and/or Alexa546-conjugated secondary antibody (Invitrogen).
Morphometric analysis
Parasites resuspended in Hanks Buffered Salt Solution (HBSS; Invitrogen) containing 10 mM HEPES, pH 7.2, were attached to either glass coverslips or 8-well chambered coverglasses (Nunc, Rochester NY) using BD-CellTak, incubated for 15 minutes at 23°C with or without various concentrations of mercuric chloride (HgCl2), and visualized by differential interference contrast (DIC) microscopy. The widest point along both the long and short axes of the parasite was determined using ImageJ (http://rsbweb.nih.gov/ij/).
Electron microscopy
Parasites were pelleted at 1,000 xg, resuspended in Karnovsky's reagent (1% [wt/vol] paraformaldehyde, 2.5% [vol/vol] glutaraldehyde) and incubated for 60 minutes at 4°C. The fixed cells were rinsed 3 times (4 minutes each) in Millonig's phosphate buffer [27] and embedded in 2% (wt/vol) SeaPrep Agarose (Cambrex BioScience Rockland, Rockland ME), for 15 minutes at 4°C. The samples were then incubated in Karnovsky's reagent for 15 minutes at 4°C, followed by washing in Millonig's buffer and trimming of the agarose blocks. 1% (wt/vol) osmium tetraoxide (OsO4) was added and the blocks were stored in Millonig's buffer overnight. The following day, the blocks were sequentially dehydrated in 35%, 50%, 70%, 85%, 95%, and 100% (vol/vol) ethanol. They were further dehydrated in propylene oxide, infiltrated with and embedded in Spurr's resin, and allowed to polymerize for 12 hours. Ultrathin sections were cut, placed on nickel grids and contrasted with 2% (wt/vol) uranyl acetate in 50% (vol/vol) ethanol for 6 minutes, followed by lead citrate for 4 minutes, before being analyzed on a JEOL 1210 transmission electron microscope (JEOL, Peabody MA).
Growth competition
Freshly egressed wild-type RH, RHΔTgPhIL1, and TgPhIL1 complemented clones (RHΔTgPhIL1/TgPhIL1-C5 and RHΔTgPhIL1/TgPhIL1-C7) were filtered through a 3-µm Nuclepore syringe filter (Whatman, Piscataway NJ). 5×105 parasites of each strain were added pairwise to a 25 cm2 flask of confluent HFF cells, in the combinations indicated, for a total of 1×106 parasites per experiment. Immediately following the lysis of the monolayers (approximately 36 hours post-infection), 0.3 ml of parasite-containing culture supernatant was added to a fresh monolayer. After every third such passage, immunofluorescence was performed on the released parasites with rabbit polyclonal anti-TgPhIL1. A total of 500 parasites/sample were scored as either negative or positive for TgPhIL1. The medium was replaced 12 hours after each infection to ensure that parasites that failed to invade were not counted in the analysis.
Mouse infections and analysis
Female 6 to 8 week old C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained under specific-pathogen-free conditions in accordance with institutional guidelines. 5 mice per group were infected intraperitoneally with 200 µl of PBS containing 1×104 RH or RHΔTgPhIL1 tachyzoites. Peritoneal cells were harvested on day 7 by lavage with cold PBS, spun onto coverslips and stained using the HEMA-3 kit (Fisher Scientific) as directed by the manufacturer. Parasite DNA levels were measured by real time PCR on DNA isolated from liver and spleen samples 7 days post-infection using the High Pure PCR Template Purification Kit (Roche, Indianapolis IN). The tandemly arrayed, 35-fold-repetitive T. gondii B1 gene [28] was amplified by real-time PCR (Forward 5′- TCCCCTCTGCTGGCGAAAAGT-3′; Reverse 5′- AGCGTTCGTGGTCAACTATCGATTG-3′) using the AB7500 fast real-time PCR thermal cycler and Power SYBR green reagents (Applied Biosystems, Foster City CA). Quantitect primers for the mouse housekeeping gene β-actin (Qiagen, Valencia CA) were used as a normalization control. The cycle numbers were normalized relative to their individual actin signals and these were averaged among the 5 mice per group; RHΔTgPhIL1 was set to 1.
Laser Scanning Cytometry (LSC)-based invasion assay
Syringe-released parasites were filtered through a Nuclepore filter, washed in HBSS by centrifugation (4 minutes, 1,000 xg) and resuspended at a concentration of 1×106 parasites/ml in HBSS containing 1% FBS. Parasites were added to confluent monolayers of HFF cells grown on 25 mm2 round glass coverslips in 6-well plates (3×106 parasites added/well). The coverslips were incubated for 1 hour at 37°C, and processed and analyzed by LSC as described previously [29].
Motility assay
Freshly egressed and filtered parasites were harvested, centrifuged at 1,000 xg for 4 minutes, and resuspended in HBSS at a concentration of 1.5×107 parasites/ml. 100 µl of the parasite suspension was added to BD-CellTak-coated 8-well chambered coverglasses (Nunc, Rochester NY) and incubated at 37°C for 45 minutes. The buffer was removed by gentle aspiration, and the samples were fixed in 2.5% (vol/vol) paraformaldehyde for 15 minutes at 23°C, then blocked with PBS-2% BSA for 15 minutes. Trails were stained with 5 µg/ml mouse monoclonal anti-TgSAG1 (Argene, North Massapequa NY) in PBS-0.5% BSA for 15 minutes, washed three times in PBS, incubated for 15 minutes with Alexa 488-conjugated goat anti-mouse antibody, washed again and visualized by fluorescence microscopy.
Conoid extension assay
Conoid extension assays were performed as described previously [30]. Briefly, freshly egressed parasites were incubated for 5 minutes at 23°C in HBSS containing 1 µM ionomycin (Sigma) or an equivalent volume of DMSO, fixed for 1 hour at 23°C in 1% (vol/vol) glutaraldehyde, smeared onto a glass slide, and air-dried. 100 conoids per sample were scored as either extended or retracted by phase contrast microscopy.
Generation of a TgPhIL1 knockout parasite line
TgPhIL1 was targeted for disruption by a construct encoding ble, which confers resistance to the drug phleomycin [21], flanked by 5′ and 3′ sequences from the TgPhIL1 genomic locus (Fig. 1A). Following transfection and three rounds of selection, 6% of the population lacked TgPhIL1 staining by immunofluorescence (Fig. 1B). Parasites in this population were cloned by limiting dilution, and individual clones lacking TgPhIL1 were identified by immunofluorescence. Immunoblotting with anti-TgPhIL1 antibody confirmed the lack of TgPhIL1 expression in several of the clones, one of which (RHΔTgPhIL1; Fig. 1C) was chosen arbitrarily for all subsequent experiments.
(A) A knockout construct containing a ble cassette, flanked by 3 kb of upstream and downstream noncoding sequence from the TgPhIL1 genomic locus was linearized and transfected into wild-type RH parasites, where it was predicted to integrate into the TgPhIL1 locus by homologous recombination. (B) After three rounds of selection with phleomycin, immunofluorescence with anti-TgPhIL1 antiserum (red) revealed that 6% of the parasites were negative for TgPhIL1, indicating successful disruption of the open reading frame. A combined phase contrast and immunofluorescence image is shown. Scale bar = 5 µm. (C) Western blotting with anti-TgPhIL1 confirmed the absence of TgPhIL1 expression in the knockout parasites (RHΔTgPhIL1) and restoration of TgPhIL1 expression to approximately wild-type (RH) levels in the complemented clones (RHΔTgPhIL1/PhIL1-C5, C6, C7). The blot was simultaneously probed with anti-actin as a loading control. As seen here, TgPhIL1 sometimes resolves by SDS-PAGE as a tightly spaced doublet, possibly reflecting the presence of an as yet uncharacterized posttranslational modification. Numbers on the left indicate molecular mass in kDa.
For complementation studies, we reintroduced TgPhIL1 into the RHΔTgPhIL1 parasites using a construct containing 2 kb of TgPhIL1 upstream genomic sequence, the TgPhIL1 ORF, and a chloramphenicol acetyltransferase cassette for selection. The construct was transfected into RHΔTgPhIL1 parasites, which were then subjected to three rounds of selection with chloramphenicol. Immunofluorescence with anti-TgPhIL1 antibody showed that stable transfectants had been generated and that the expressed TgPhIL1 localizes properly (data not shown). Three clones positive for TgPhIL1 by immunofluorescence were isolated and analyzed by Western blot with the anti-TgPhIL1 antibody. Each of the complemented clones showed levels of TgPhIL1 expression comparable to that of wild-type parasites (Fig. 1C). Two clones, RHΔTgPhIL1/PhIL1-C5 and RHΔTgPhIL1/PhIL1-C7, were chosen arbitrarily for subsequent experiments.
Morphology of the TgPhIL1 knockout parasites
In the course of generating the RHΔTgPhIL1 parasites, it was obvious by light microscopy that these parasites had an altered morphology compared to wild-type (RH) parasites (Fig. 2, compare left and middle panels). To quantify this difference, parasites were measured along their long and short axes at their widest point, confirming that the RHΔTgPhIL1 parasites are indeed shorter and wider than the RH parasites from which they were derived (Fig. 2). Importantly, the shape of the complemented clones appeared similar to that of wild-type parasites, and morphometric analysis confirmed that complementation of TgPhIL1 had indeed restored the length and width of the mutant to wild-type values (Fig. 2, compare left and right panels).
The maximum length and width of individual wild-type (RH), RHΔTgPhIL1, and RHΔTgPhIL1/PhIL1-C5 parasites were measured in differential interference contrast images such as those shown. The experiment was done in triplicate, and numbers indicate the average measurements from 100 parasites/experiment, in µm, plus or minus standard error. The RHΔTgPhIL1 parasites are shorter and wider than either the wild-type or complemented clones (unpaired student's t-test, p<0.05; n.s. = not significant). Scale bars = 5 µm.
To determine whether the morphological effects of the knockout are further accentuated under conditions of osmotic stress, the parasites were treated with various concentrations of HgCl2, which disrupts water balance across the plasma membrane by inhibition of aquaporin channels [31], [32]. Both the RHΔTgPhIL1 and wild-type parasites swell following treatment with 0.5 µM HgCl2, resulting in each case in a decrease in the length to width ratio of ∼15% (Fig. S1). Thus, the already squat-shaped RHΔTgPhIL1 parasites become even shorter and wider under conditions of osmotic stress.
Ultrastructure of the TgPhIL1 knockout parasites
Given their striking difference in shape, we examined the RHΔTgPhIL1 parasites by transmission electron microscopy for ultrastructural changes, particularly in the IMC and/or the region of the pellicle just posterior to the conoid, where TgPhIL1 is known to be concentrated ([18]; see also Fig. S3B). No morphological differences in the IMC, the conoid, or the spacing between the IMC and the plasma membrane were observed (Fig. 3).
The ultrastructure of the conoid (C), IMC, and spacing between the IMC and plasma membrane is indistinguishable in (A) wild-type (RH) and (B) RHΔTgPhIL1 parasites. Scale bars = 1 µm. Inset shows an enlarged portion of the RHΔTgPhIL1parasite from the lower left corner of panel B; the normal trilaminar structure of the pellicle is evident (arrowheads). Scale bar = 0.1 µm.
Conoid extension, motility and invasion of the TgPhIL1 knockout parasites
Since TgPhIL1 localizes in part to a ring just posterior to the conoid, its absence might have an effect on conoid extension. To test this hypothesis, wild-type and RHΔTgPhIL1 parasites were treated with 1 µM ionomycin, which induces conoid extension [30]. In response to ionomycin treatment, 86±6.3% of wild-type parasites extended their conoid compared to 14±0.35% of untreated parasites. Similarly, 83±1.8% of the RHΔTgPhIL1 parasites extended their conoids in response to ionomycin treatment, compared to 17±2.5% of untreated parasites (Fig. S2), demonstrating that PhIL1 does not play a significant role in conoid extension.
Since the shape of T. gondii is thought to be important for gliding motility [33], [34], the alteration in morphology caused by PhIL1 disruption might be expected to affect parasite motility. However, analysis of trails deposited by gliding parasites revealed that the RHΔTgPhIL1 parasites were able to undergo both circular and helical gliding, and the number and shape of trails deposited by the knockout parasites was qualitatively indistinguishable from the number and shape deposited by wild-type parasites (Fig. 4A).
(A) Motility assays were performed on wild-type (RH) and RHΔTgPhIL1 parasites. The density and shape of the trails deposited by RHΔTgPhIL1 parasites are qualitatively indistinguishable from those deposited by RH parasites. Scale bars = 10 µm. (B) Laser-scanning cytometry (LSC)-based invasion assays were performed on wild-type (RH), RHΔTgPhIL1, and RHΔTgPhIL1/PhIL1-C5 parasites. Samples were analyzed in duplicate and the results shown are the average from two independent experiments, plus or minus standard error. No significant difference was seen in the ability of the RHΔTgPhIL1 parasites to invade host cells when compared to either RH or RHΔTgPhIL1/TgPhIL1-C5 parasites (unpaired student's t-test, p>0.05).
To determine whether RHΔTgPhIL1 parasites were defective in their ability to invade host cells, quantitative LSC-based invasion assays [29] were performed. Wild-type, RHΔTgPhIL1, and RHΔTgPhIL1/PhIL1-C5 parasites were again indistinguishable in their ability to invade host cells (Fig. 4B). Taken together, these data indicate that PhIL1 plays no detectable role in conoid extension, motility, or invasion.
Other IMC-associated antigens
To determine whether the absence of TgPhIL1 affects the subpellicular microtubules, RHΔTgPhIL1 parasites were stained with anti-tubulin and visualized by immunofluorescence microscopy. As shown in Fig. S3A (panel a), the subpellicular microtubules in RHΔTgPhIL1 parasites are of similar length and show a similar distribution to those of wild-type parasites. Similarly, no differences were observed in the distribution of: TgIMC1, a major component of the subpellicular network (Fig. S3A, panel b); TgMORN1, which localizes in part to ring-shaped structures associated with the growing ends of the IMC cisternae during daughter cell formation ([26]; Fig. S3A, panel c); and ISP1, a protein associated with the most apical plate of the IMC ([25]; Figs. S3A, panel d). These data, together with the ultrastructural analyses, suggest the IMC, subpellicular network, and microtubules are not significantly altered in the knockout parasites.
Growth competition assays
To assess how well the knockout parasites grow in vitro, wild-type and RHΔTgPhIL1 parasites were added to confluent monolayers of HFF cells and the number of parasites per vacuole was counted at various times (0–36 hours) post infection. No difference in growth rate between the wild-type and knockout parasites was detected (data not shown). To look for a more subtle growth defect over a longer time period, equal numbers of wild-type and RHΔTgPhIL1 parasites were mixed and used to infect the same flask of HFF cells. Each time the parasites lysed the host cell monolayer, culture supernatant containing the released parasites was added to a fresh host cell monolayer. After every third passage, anti-TgPhIL1 immunofluorescence was performed on the released parasites to determine the relative number of wild-type and knockout parasites present. As shown in Fig. 5, wild-type parasites start to outgrow the RHΔTgPhIL1 parasites by passage 4 and continue to do so at passages 7 and 10. This same experiment was performed with pairwise cultures of the knockout parasites and each of the complemented lines (i.e., RHΔTgPhIL1 vs. RHΔTgPhIL1/TgPhIL1-C5 and RHΔTgPhIL1 vs. RHΔTgPhIL1/TgPhIL1-C7); like the wild-type parasites, the complemented lines outgrow the PhIL1 knockout parasites at all time points (Fig. 5).
Equal numbers of parasites from the different lines were mixed pairwise and serially passaged through monolayers of HFF cells. After every three passages, immunofluorescence was performed and 500 parasites were counted and scored as either positive or negative for TgPhIL1. Each color (red, green, and blue) indicates a pair of parasite lines passaged together. Wild-type (RH) parasites (green triangles) and each of the complemented clones (RHΔTgPhIL1/TgPhIL1-C5 [red triangles] and RHΔTgPhIL1/TgPhIL1-C7 [blue triangles] outgrew the RHΔTgPhIL1 parasites (squares) at all time points (passages 4, 7 and 10). Each experiment was performed in duplicate, and the results shown are the average of the three experiments, plus or minus standard error. The difference between the growth of each of the pairs was significant at all passage numbers >1 (unpaired student's t-test, p<0.05).
Decreased survival of TgPhIL1 knockout parasites in vivo
Since the RHΔTgPhIL1 parasites were outgrown by wild-type parasites in vitro, we tested for a growth difference in vivo. Mice were infected intraperitoneally with 1×104 wild-type or RHΔTgPhIL1 tachyzoites. Seven days post-infection, large numbers of extracellular and intracellular wild-type parasites were observed in the peritoneal fluid. Significantly fewer free and intracellular parasites were detected in the peritoneal fluid of mice infected with the RHΔTgPhIL1 parasites (Fig. 6A-C). The spleen and liver were also harvested seven days after infection, and quantitative PCR (qPCR) was performed to determine the amount of parasite DNA present. As shown in Fig. 6D, less parasite DNA was present in the spleen and liver of mice infected with RHΔTgPhIL1 parasites compared to wild-type parasites; this difference was particularly evident in the spleen, where five-fold less RHΔTgPhIL1 parasite DNA was observed. These data demonstrate that the PhIL1 knockout parasites exhibit a reduced ability to survive and/or disseminate in mice when compared to wild-type parasites.
Mice were infected intraperitoneally with 1×104 wild-type (RH) or RHΔTgPhIL1 tachyzoites. (A-C) Peritoneal exudate cells (PECs) were harvested seven days post-infection, spun onto coverslips and examined for the presence of parasites. Panel A shows that PECs from RH-infected mice carry a significantly higher parasite load than those from RHΔTgPhIL1-infected mice (unpaired student t-test, p<0.05). Results shown are the average from five mice, plus or minus standard error. Panels B and C show representative examples of the coverslips from which the data in panel A were derived (Panel B, RH-infected PECs; Panel C, RHΔTgPhIL1-infected PECs). Black arrows indicate extracellular parasites and white arrows intracellular parasites. Scale bars = 20 µm. (D) DNA was harvested from spleen and liver samples seven days post-infection, and qPCR was performed to determine the amount of parasite DNA present. Results shown are the average parasite loads from five mice, plus or minus standard error. The liver and spleen each contained a higher load of RH than RHΔTgPhIL1 parasites, although only the differences observed in the spleen were statistically significant (unpaired student t-test, p<0.05).
TgPhIL1, which was originally discovered in photolabeling experiments designed to identify novel proteins of the T. gondii pellicle [18], is conserved among members of the Phylum Apicomplexa but exhibits very little sequence similarity to non-apicomplexan proteins currently in the sequence databases. Formulating a hypothesis on the function of a protein that contains no identifiable protein domains or homologs of known function is challenging. Based on its localization to the IMC and properties suggestive of both a cytoskeletal and membrane-associated protein, TgPhIL1 could serve to organize or physically connect elements of the parasite cytoskeleton with the membranes of the IMC [18].
As a first step in elucidating the function of TgPhIL1, we generated a TgPhIL1 knockout parasite line. The knockout parasites have a conspicuously altered morphology, being shorter and wider than the wild-type parasites from which they were derived. Given that the shape of T. gondii is thought to be important for motility [33], [34], it was surprising that the RHΔTgPhIL1 parasites were capable of normal motility in vitro, although we cannot rule out some more subtle effect on in vitro motility or a defect that would only be apparent in an infected organism, where the parasites are required to travel longer distances and through various biological barriers [35]. Because PhIL1 localizes to a ring-like structure at the posterior end of the conoid, we also hypothesized that the protein might play some role in conoid extension; however, conoid extension was unaffected by disruption of TgPhIL1.
Although indistinguishable from wild-type parasites in a number of respects, the knockout parasites showed significantly slower growth in culture. The growth defect observed in culture translated into a significant decrease in parasite fitness in a mouse model of infection. The number of RHΔTgPhIL1 parasites detected in the liver, spleen and peritoneal fluid of infected mice was consistently lower with RHΔTgPhIL1 parasites than with wild-type parasites. No difference was observed in the survival of mice infected with wild-type and RHΔTgPhIL1 parasites, with all mice dying around day 8 (data not shown). However, these experiments were done with a single dose of 1×104 parasites; a more extensive analysis, using a range of parasite inocula, would be required to establish whether the decreased fitness of the knockout parasites affects host survival.
An alternative approach to identifying the biological function of TgPhIL1 would be to identify proteins with which it interacts. However, TgPhIL1's highly insoluble nature [18] makes coimmunoprecipitation studies to identify interacting proteins technically challenging. In P. falciparum, a genome-wide yeast-two-hybrid screen has been performed, with the results available online (www.plasmodb.org). In this screen, the P. falciparum homologue of TgPhIL1 (PFA0440w) was shown to interact with another P. falciparum protein (PFF0325c), and a homolog of this P. falciparum PhIL1-binding protein exists in T. gondii (38.m01070). While PFF0325c shows no homology to non-apicomplexan proteins, it was found to interact in the yeast-two-hybrid screen with five proteins in addition to PfPhIL1 (PFA0110w, PFA0285c, PF10_0378, PF08_0137, PFD0320c). Two of these are DnaJ-domain containing proteins, one is predicted to be exported, and several have homologs in T. gondii. As additional functional annotation of genes from other organisms becomes available, new insights into the function of the potential PhIL1-binding proteins and of PhIL1 itself may become apparent.
If one of the primary functions of TgPhIL1 is to provide structural stability to the parasite, as suggested by the data reported here, a reduced ability of the knockout parasites to tolerate osmotic or mechanical stresses encountered during in vivo infection might be an underlying cause of the parasite's reduced fitness. Intriguingly, disruption of the IMC-associated proteins IMC1a and IMC1b in Plasmodium berghei also alters the shape and reduces the mechanical stability of sporozoites and ookinetes, respectively [36], [37]. The rate of parasite proliferation early during infection may affect parasite loads later, as the parasite has a limited time window in which to proliferate before the immune system becomes fully activated. The development of drugs that target components of the IMC and cytoskeleton may therefore be a potentially useful approach to disease management.
In the context of a population of parasites, even relatively subtle growth defects such as the one reported here are likely to present a significant selective disadvantage over time, since parasites exhibiting such a defect will be outcompeted during infection by parasites whose growth is not impaired. Several other T. gondii proteins have recently been identified that are non-essential, but make a clear contribution to parasite growth and fitness (e.g., [38]–[40] and GEW, unpublished data). With the recent development of highly efficient methods for gene replacement in T. gondii [41], [42], many more such genes are likely to be identified. In these cases, it may be that we simply do not know the relevant assay, host, or environmental conditions to reveal a clear phenotype when the gene is disrupted. The overall fitness of a given parasite strain will be determined by both its essential genes and the additive effect of genes which may not be essential but provide some degree of fitness benefit to the parasite in the context of particular host organisms or environmental conditions. A broader experimental context is likely to be required for elucidating the biological function of genes such as TgPhIL1, which are beneficial to the parasite but not strictly essential.
Supporting Information
Figure S1.
Osmotic stress causes an equivalent amount of swelling in RH and TgPhIL1 knockout parasites. Wild-type (RH) and RHΔTgPhIL1 parasites were incubated for 15 minutes in the presence or absence of varying concentrations of HgCl2. The maximum length and width of individual parasites, in µm, was determined as described in Materials and Methods and expressed as the length to width ratio (LW ratio). The experiment was done in duplicate, and numbers indicate the average measurements from 100 parasites/experiment plus or minus standard error. Statistical significance was calculated using an unpaired student's t-test.
Figure S2.
Conoid extension in RH and TgPhIL1 knockout parasites. Wild-type (RH) and RHΔTgPhIL1 parasites were incubated for 5 minutes in the presence (+) or absence (-) of 1 µM ionomycin, and the percentage of parasites with extended conoids was scored by phase microscopy. The results shown are the average of two experiments +/- standard error. The two parasite lines showed no significant difference in ionomycin-induced conoid extension (unpaired student's t-test, p>0.05).
Figure S3.
Localization of α-tubulin, TgIMC1, TgMORN1 and ISP1 in TgPhIL1 knockout parasites. (A) The distributions of α-tubulin, TgIMC1, TgMORN1 and ISP1 were examined in wild-type (RH) and RHΔTgPhIL1 parasites by immunofluorescence microscopy. (a) The splayed subpellicular microtubules of the parasite are indistinguishable in the wild-type and RHΔTgPhIL1 parasites. (b) TgIMC1 localizes to the periphery of the mother cell and the growing daughters during endodyogeny in both parasite lines. (c) TgMORN1 localizes to the basal end of the parasite, the centrocone, and a pair of rings around the dividing nucleus during endodyogeny in both parasite lines (red = TgMORN1; green = TgSAG1, a plasma membrane marker). (d) ISP1 is found in an indistinguishable apical cap-like distribution in the two parasite lines (red = ISP1; green = polyclonal serum directed against total tachyzoite antigens). Scale bars = 5 µm. (B) The localization of PhIL1 is shown for reference: as previously described [18], PhIL1-YFP localizes to the parasite periphery and is concentrated at both the basal end and the apical end just posterior to the conoid (YFP fluorescence, middle panel). The corresponding DIC and merged fluorescence/DIC images are shown in the upper and lower panels, respectively. Scale bar = 5 µm.
We thank the UVM Statistics lab, Drs. Douglas Johnson and Chris Huston and members of the Ward laboratory for helpful comments on the manuscript. We thank Marc-Jan Gubbels and Peter Bradley for generously providing antibodies and Nicole Bishop and Michele von Turkovich of the University of Vermont Microscopy Imaging Center for assistance with electron microscopy.
Author Contributions
Conceived and designed the experiments: WDB SDG RW LDT CH GEW. Performed the experiments: WDB SDG RW LDT. Analyzed the data: WDB SDG RW LDT CH GEW. Wrote the paper: WDB SDG RW LDT CH GEW.
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text:
Ipswich: Woman appeals for kind-hearted man to come forward after she was bitten by dog
PUBLISHED: 16:30 22 February 2013 | UPDATED: 16:30 22 February 2013
AN injured woman has appealed for a kind-hearted man to come forward after he came to her rescue when she was attacked by a dog.
Sandra Gonta, of Birkfield Drive, Ipswich, suffered puncture wounds to her arm after a German Shepherd jumped up and bit her.
Shaken and in severe pain, the 53-year-old was helped by a man who took her into a nearby shop after the incident and called for the help of paramedics.
Today, Ms Gonta and her daughter Melissa thanked the caring stranger who took the time to offer a helping hand.
Police and paramedics were alerted to the drama which took place at 12.30pm on Tuesday, while Ms Gonta walked to the shops in Cambridge Drive.
She told The Star that as she attempted to cross the road with her shopping trolley in tow a black German Shepherd suddenly bit her arm.
She said: "There were two girls walking with the dog which was on a lead. But the dog bit me as I started to cross the road.
"They tried to pull the dog off me but it seemed to take a long time. She was really trying to get it off me. There was a lot of blood and it was very painful.
"A man who had seen the whole thing came up to help me when the two girls walked off with the dog. I just want to thank him for what he did because he called the ambulance."
Ms Gonta was taken to Ipswich Hospital where her wounds were treated. She also had a tetanus jab.
Her daughter Melissa said: "It sounded awful, the more the girls tried to pull the dog off the more it dug its teeth in.
"The wound was still bleeding the next day. I just think it would be nice to thank the man who helped her when she was all on her own."
A police spokeswoman encouraged anyone who witnessed the incident to contact officers at Ipswich police station on 101.

subset: commoncrawl


text:
As the cold Canadian winter sets in, Project Winter Survival has launched its first-ever PSA to raise awareness about homelessness.
The 30-second spot, titled "Road Rage" and created by Toronto agency Blammo Worldwide, shows two men arguing and getting physical with each other near their parked cars. The video, which is made to look like it was filmed by a passerby on a cellphone, has a surprising twist that draws attention to homelessness.
Project Winter Survival was founded in 2000 by husband-and-wife team Jody Steinhauer, founder and president of Toronto-based The Bargains Group, and Lorne Simon, chief of PR at Michael Communications PR Group. The grassroots organization creates survival kits with donated items such as sleeping bags, toques and personal care products. The kits are handed out to the homeless in the Greater Toronto Area through agencies such as The Salvation Army, Red Cross and the Scott Mission. This year, 200 volunteers put together 3,000 kits, which were handed out on Jan. 19.
"The message [of the PSA] is that every single day we're walking by homeless people who are potentially freezing to death, and we're not even noticing it… or we do see them, but we still walk by," said Steinhauer. "I think there's a huge subliminal message that we should look around a little bit more and [ask] 'what are we missing?'"
Andrew Simon, chief creative officer at Blammo Worldwide, said the agency "wanted to do something that would be very provocative and attention-getting, but also remind people that to a lot of Canadians, the homeless are invisible."
The campaign was borne out of a meeting Simon had with Lorne Simon. The two had worked together on Crimestoppers campaigns while Andrew was at DDB Toronto.
"We had a lot of success together and he told me about this initiative… which sounded fantastic," said Andrew Simon, who also worked with Untitled Films on the PSA. "It was just a question of, 'It's late in the game, can we get anything together to launch in January?' That was our biggest challenge, but right from the bat it was wonderful."
The campaign also includes radio spots. Project Winter Survival is working on getting donated airtime.


text:
Pradaxa – Management of Major Bleeding
Major and life-threatening bleeding is expected to occur in some patients treated with Pradaxa® (=Dabigatran). The question will then urgently arise how to best treat such catastrophic bleeding. As there have been only very limited data published on this topic in the peer-reviewed medical literature, we don't know how to best manage a patient with major and life-threatening bleeding on Pradaxa®, even though suggestions have been made in the absence of such data [ref 1,2]. There (a) is no direct reversal agent available for Pradaxa®, and (b) have not been reversal strategies (backed up by clinical study data). It is key that data will be published in peer-reviewed journals indicating how major bleedingwhile on these drugs can be treated effectively. In the event of bleeding complications in patients receiving Pradaxa®, management should, of course, be individualized according to the severity and location of the bleed.
Treatment options
- Activated charcoal to prevent residual drug in the stomach to be absorbed
- Consideration of 1-2 sessions of hemodialysis.
- Recombinant factor VIIa (NovoSeven®) i.v.
- Antifibrinolytic therapy (Amicar®=aminocaproic acid or Lysteda®=tranexamic acid)
- Activated prothrombin complex concentrates (aPCCs; FEIBA®) i.v.
- Possibly not effective are: Prothrombin complex concentrates (PCCs; in the U.S.: Bebulin®, Profilnine®) intravenously with or without additional fresh frozen plasma (FFP) [ref 3]
- Unlikely effective is: Fresh frozen plasma (FFP)
Published Data about management of bleeding on new oral anticoagulants
- Pradaxa® (Dabigatran): Treatment of 12 healthy volunteers with Pradaxa,150 mg twice daily, led to an expected prolongation of certain clotting tests (aPTT, Ecarin clotting time, thrombin clot time) and this prolongation was NOT reversed by giving an unactivated PCC (Prothrombin Complex Concentrate; Cofact; 4-factor concentrate; 50 U/kg x 1), suggesting that PCCs might also NOT be effective in patients who bleed on Pradaxa [ref 3].
- Xarelto® (Rivaroxaban): In the study just mentioned above [ref 3], treatment of the 12 healthy volunteers with Xarelto 20 mg twice daily, led to an expected prolongation of the PT and to an inhibition of the ETP (endogenous thrombin potential), and these abnormalities were reversed immediately by giving an unactivated PCC (Prothrombin Complex Concentrate; Cofact; 4-factor concentrate; 50 U/kg x 1), suggesting that PCCs might be effective in patients who bleed on Xarelto. However, a lot of additional (patient) data are still needed to understand how to manage Xarelto®-associated bleeding.
- Edoxaban (not FDA approved): this drug also does not have a reversal drug. A small, non-human ex vivo plasma-mixing study presented at the Dec 2010 annual meeting of the American Society of Hematology (ASH) shows that the anticoagulant effect of Edoxaban can be reversed by PCCs and by NovoSeven® (ref 2). The maximum recombinant factor VIIa dose used in this study was equivalent to 50 mcg/kg. A lot of additional data are still needed to understand how to manage Edoxaban-associated bleeding.
Personal Approach
At this point, not knowing what the most effective and safest treatment is, I would give the patient who has a life-threatening bleed on Pradaxa®:
- Charcoal to prevent residual drug in the stomach to be absorbed.
- Consider hemodialysis.
- Recombinant factor VIIa (NovoSeven; 90 mcg/kg x1). It is important to realize that it is not clear (a) whether recombinant factor VIIa is effective in this situation and (b) what VIIa dose might be appropriate. In addition, rVIIa is prothrombotic, and that needs to be kept in mind when considering use of the drug.
- I would consider fibrinolytic drugs (Amicar or tranexamic acid) as adjuncts.
- One could consider FEIBA® (activated prothrombin complex concentrate). But I would worry some about its prothrombotic effect, if the patient had had a recent thrombotic event or was quite hypercoagulable by history.
- Based on reference 3, I would NOT give Prothrombin complex concentrate (PCC; in the U.S.: Bebulin® or Profilnine®.
- I would NOT give FFP.
Hospital Guideline
Management of major bleeding on Pradaxa® and other treatment related issues may best be addressed in a health care system by the establishment of a structured treatment algorithm/guide/help for the whole hospital or medical practice. As an example, here is the document that we have established for our institution, the University of North Carolina (UNC) Health Care System. The reader is welcome to (a) take the document and modify it to fit his/her institution/practice's need or (b) use as a clinical reference for management issues.
- van Ryn J. Dabigatran etexilate – a novel, reversible, oral direct thrombin inhibitor: Interpretation of coagulation assays and reversal of anticoagulant activity. Thromb Haemost 2010; 103: 1116–1127.
- Crowther MA. Managing bleeding in anticoagulated patients with a focus on novel therapeutic agents. J Thromb Haemost 2009; 7 (Suppl 1):107–110.
- Eerenberg ES et al. Reversal of rivaroxaban and dabigatran by Prothrombin Complex Concentrate: A randomized, placebo-controlled, crossover study in healthy subjects. Circulation 2011,124: prepublished on the web.
- Morishima Y et al. Anti-Inhibitor Coagulant Complex, Prothrombin Complex Concentrate, and recombinant factor VIIa reverse prothrombin time prolonged by Edoxaban in human plasma (abstract 3319; ASH annual meeting Dec 4-7, 2010, Orlando, FL).
- A well balanced and well explained audio-powerpoint presentation on Dabigatran, its action and reversal option of bleeding can be found here. However, Dabigatran is consistently pronounced incorrectly (correct pronounciation is on 2nd syllable) and vitamin K is not a reversal option.
For patients
This same information, written for patients and other non health care professionals, can be found here.
Disclosure: I have consulted for OrthoMcNeil, the company developing Xarelto (Rivaroxaban) in the U.S.
Disclaimer: ClotConnect.org, its contributors, authors, advisors, members and affiliate organizations do not assume any liability for the content of the website, blog and educational materials. Medical information changes rapidly. While information is believed to be correct, no representation is made and no responsibility is assumed for the accuracy of information contained on or available through this web site and blog. Information is subject to change without notice.
Last updated: Oct 6th, 2011
Tags: bleeding, charcoal, Dabigatran, FEIBA, NovoSeven, PCCs, Pradaxa, prothrombin complex concentrates, recombinant factor VIIa, Rivaroxaban
3 Responses to "Pradaxa – Management of Major Bleeding"
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text:
Pom & Olive Mediterranean Opens in Costa Mesa
A new restaurant has opened in the space that used to be a Chinese vegan restaurant called Happy Family, and before that, May Garden, which was a long-running establishment that peddled sweet-and-sour-kung-pao Chinese food to the masses the same time it offered a secret Taiwanese menu for those who knew to ask for it. I still miss May Garden.
The new place isn't another Chinese joint, but a Mediterranean one, which is a broad enough category that they can offer Greek dolmades on the menu but also Persian fesenjan it sells as "pomegranate stew". The huevos rancheros I saw on the breakfast menu is harder to reconcile.
Pom & Olive is located at 1400 Bristol Street, Building C in Costa Mesa, and is actually within walking distance from OC Weekly World Headquarters.
The menu, with that contradictory Mexican breakfast dish among the shawarmas and koobidehs, can be checked out on their website.
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text:
It Wasn't Exactly Rascal Flatts Live At The ACM Awards
(AP) – Rascal Flatts thought it would be better to fake it than not show up.
After viewers of Sunday's Academy of Country Music Awards accused the band of lip-syncing their performance of "Rewind," the band has come clean and admitted they did.
Rascal Flatts say on their Facebook page singer Gary LeVox (leh-VOH') lost his voice after doing several shows earlier in the week. They say they made a last-minute decision to lip-sync at the ACMs instead of canceling.
In their words, "We've never done it before, and we're obviously not very good at it."


text:
Tour Itinerary:
Day 1: Take the overnight flight to Lima, Peru.
Day 2: Upon arrival, transfer to your hotel. Afternoon city tour of Lima. Lima has always been one of the most important cities in South America since it's founding in 1535. Journey through the historic center of the city. Admire the beauty of San Martin and Major squares, the Cathedral, the Government Palace and visit San Francisco Convent, religious building from the end of the XVI century and its catacombs. Also drive through the residential districts of San Isidro and Miraflores.
Day 3: Morning transfer to the airport to take the flight to Cuzco. Upon arrival in Cuzco, transfer to your hotel. Afternoon city tour of Cuzco, visiting the Korikancha temple or Temple of the Sun, the Cathedral and its Art Museum and the artisans quarter of San Blas and San Blas church. Continuing to the nearby water shrine of Tambomachay (the Inca Fountains), Puca Pucara (the Red Fortress), amphitheater of Kenko and finally the Sacsahuaman Fortress. Return to your hotel.
Day 4: Morning transfer to the train station. Train takes us through the Sacred Valley of the Incas up to the town of Aguas Calientes. From here, we will board a bus to the Lost Citadel of Machu Picchu. We will then have a guided tour of the ruins. Machu Picchu, the Lost City of the Incas, was a flourishing ceremonial and agricultural site, probably the gateway to the jungle marches of the Inca Empire. The central buildings were erected with the polished stone works of the Cusco Imperial style, though the site was probably occupied well before the Incas started their expansion. It was associated with the earth cult revolving around the Coca leaf, which was a privilege of the Inca Royal family and priests. You will have time to enjoy a light lunch or snack, and return to your hotel at your leisure. In the evening we will enjoy a wonderful dinner at the hotel or in the village of Aguas Calientes.
Day 5: Day free. You may choose to return to Machu Picchu or relax in the village of Aguas Calientes and its hot springs. Afternoon return to Cuzco by train, upon arrival transfer to your hotel.
Day 6: Day at leisure. You may choose to return to Machu Picchu, hike Huayna Picchu, or relax in the village of Aguas Calientes and its hot springs. In the afternoon, we will board the train to return to Cuzco. Upon arrival transfer to your hotel.
Day 7: Transfer to the airport to take the flight back home or overlap with another excursion.
Price Includes:
Also see tour packages in:
South America Peru Local Culture Cultural Journey Archeology/History


text:
Marilyn Manson, 'mOBSCENE' – Sexiest Rock Music Videos
Marilyn Manson has arguably the greatest videography of any modern rock musician. Along with chilling and controversial imagery, sex has always been a constant theme throughout Manson's videos. Along with clips for 'Heart Shaped Glasses,' 'Tainted Love' and the x-rated '(s)aint,' Manson's video for 'mOBSCENE' showcases creatively carnal scenes. Among burlesque and military 'Rockette' dancing, 'mOBSCENE' features modern pin-up girl and Manson's former wife Dita Von Teese for a pants-tightening exhibition in a giant martini glass.
Marilyn Manson, 'mOBSCENE'
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text:
Download Pocket Frogs iPhone iPad iOS
NimbleBit , the publisher behind many iOS games (Scoops - Ice Cream Fun For Everyone ,Mega Panda ,Pocket Frogs ,Tiny Tower ,Dizzypad HD ,Textropolis), brings Pocket Frogs with a number of new features along with the usual bug fixes. Pocket Frogs games has been update to version 1.2.2 with several major changes and improvements. Games release that improves performance, provides several new options.
Overall Satisfaction94
Pocket frogs is one of my favorite games and it's very entertaining.
one of the best free games that I play when ever I down spare time.
Just an amazing game everybody should have it seriously.
I would recommend this game to anyone who loves frogs.
It's the best game I have ever played on the iPod I totlaly luv it.
Wonderful game --it's probably my all-time favorite.
I love breeding and making awesome combinations.
This game is the best game in the world I have the coolest frogs.
The worst game in the world.
Fun & Engaging94
Awesome game with tons of types of frogs.
Very addictive and easy gameplay with zen visuals.
Soo relaxing and super fun breeding different frogs.
Awesome game I play it all the time it is so addictig.
Fun little game that'll keep you entertained for weeks on end.
Pocket frogs is a really fun game and it keeps ya hoppin.
You will get addicted to this fun and relaxing game.
Fun to try and make specific mixes for the requests.
I play it every day almost nonstop I just love it.
I play it every day for like 3 hours straight.
I love it I play it everyday keep up the good work.
I play it everyday and it keeps me entertained.
Family Friendly93
Great elementary genetics teaching tool for kids and adults too.
Fun for the whole family when you need to kill some time.
Fun and engaging - my whole family loves this one.
This is the greatest game ever good for little kids.
Value for Money82
especially potions and stamps which are also available for real money.
This game tries to sell imaginary game products for real money.
in game real money purchases without a warning.
Replay Value88
This game has provided hours of fun for my family.
Motivated to get higher levels & more decorations for each background.
every collection I open has frogs from higher levels.
I've been playing for a while and it never gets old.
espessially when you get to new levels and unlock new things.
It's lame now that there's no new levels.
There are endless possibilities and hours of entertainment.
Nice but gets boring after awhile.
the game gets a little repetitive after a while.
Social Aspects88
Great value for a FREE game that you can play with friends.
Also a great game to play with friends around the country.
Production Values92
The graphics are great and this game just plays great.
Catching the flies is relaxing with great soothing sound effects.
This game has beautiful graphics that keep you wanting more frogs.
Ease of Use92
A deceptively simple game that is unbelievably addicting.
It's a simple game that have hours of fun.
Simple concept that's well executed and surprisingly addictive.
Such a simple concept & enjoyable to play.
Security & Privacy78
Add me on your Plus account - my username is Jibboolie.
The option to have a plus account is nice.
Updates & Support75
This game has beautiful graphics that keep you wanting more frogs. found in 33 reviews
Just an amazing game everybody should have it seriously. found in 252 reviews
I love watching the little baby frogs get bigger. found in 11 reviews
Pocket frogs is definitely the coolest app ever. found in 4862 reviews
This frog game is top calass in my book. found in 127 reviews
This is a very original and interesting game. found in 25 reviews
The best game Eva take my advise and get it so good. found in 25 reviews
Love dis game addicted to it u gotta try it. found in 14 reviews
Not normally a game player this one has me totally hooked. found in 18 reviews
It's totally addicting and a great time passer. found in 37 reviews
Great elementary genetics teaching tool for kids and adults too. found in 29 reviews
Awesome game with tons of types of frogs. found in 998 reviews
And it's a great stress reliever to take frogs to the pond. found in 27 reviews
Extremely addictive with endless possibilities and striking colors. found in 17 reviews
This game is the best frog game eva. found in 33 reviews
one of the best free games that I play when ever I down spare time. found in 255 reviews
This game has provided hours of fun for my family. found in 80 reviews
There are endless possibilities and hours of entertainment. found in 17 reviews
You will get addicted to this fun and relaxing game. found in 97 reviews
Very addicting fun for all ages even 3 and a half year olds. found in 49 reviews
could you please let us purchase more habitats. found in 13 reviews
Needs more game to find presents and earn money. found in 23 reviews
NEEDS A NEW UPDATE. found in 7 reviews
Great game but sometimes gets a little boring after a while. found in 43 reviews
I literally haven't stopped playing since I got it. found in 27 reviews
I think it needs a new update with some fun and exciting ideas. found in 6 reviews
Please add new award levels. found in 11 reviews
Needs something new to update the game. found in 10 reviews
It needs more sets and awards to collect. found in 9 reviews
But I think there should be more ways to get coins. found in 81 reviews
and I don't want to sell because I like them all. found in 15 reviews
It's a great way to waste time when you don't want to do something. found in 70 reviews
I would give 5 stars if habitats weren't so expensive. found in 10 reviews
I also wish you could have more than 8 things in the mailbox. found in 10 reviews
Kool game except it takes to long to level up. found in 19 reviews
A little boring after awhile but I keep coming back. found in 13 reviews
If I sell the doubles the game crashes more. found in 16 reviews
It won't let me place my endangered frog in my habitat. found in 8 reviews
Would love to see more activities for my frogs. found in 13 reviews
Wish I could buy /sell directly from other. found in 15 reviews
It's the worst game in the whole world. found in 6 reviews
Why can't you get plus network back. found in 12 reviews
Any time I click on them the game crashes. found in 16 reviews
iPhone 320x480 1
iPhone 320x480 2
iPhone 320x480 3
iPhone 320x480 4
iPad 360x480 1
iPad iphone 360x480 2
iPad 360x480 3
iPad 360x480 4
Icon 175x175 1
iOS Pocket Frogs 1.2.2 Mobile
Skulls of the Shogun
Ace Flight Warrior - Ultimate Survival Rush
Santa Tracker - Santa on Map
Island: The Lost Medallion HD
Word Join
Wayang Jump
Scoops - Ice Cream Fun For Everyone
Mega Panda
Pocket Frogs
Tiny Tower
Dizzypad HD
R:4.5 - 225383 Ratings
Analysis of ratings from user comments
App Popularity (trend)
App Average Rating (trend)
289,703 total
This app is popular with:
collectorsanimal loversfamilies


text: On Thu, Feb 12, 2009 at 00:15, David Cournapeau <firstname.lastname@example.com> wrote: > Gael Varoquaux wrote: >> >From a programmer's perspective, because, IMHO, openmp is implemented >> using pthreads. > > Since openmp also exists on windows, I doubt that it is required that > openmp uses pthread :) It is implemented using threads, with Windows native threads on Windows. I think Gaël really just meant "threads" there. -- Robert Kern "I have come to believe that the whole world is an enigma, a harmless enigma that is made terrible by our own mad attempt to interpret it as though it had an underlying truth." -- Umberto Eco


text:
Rajnath Singh faces bitter experience, becomes emotional - Exclusive
Wed, Dec 23, 2015, 04:09 PM
Union Home Minister Rajnath Singh faced bitter experience and at the same time he was seen emotional when he arrived at Delhi's Safdarjung Airport on Tuesday. The union home minister tried to console the family members of the 10 paramilitary personnel, who died in a plane crash . A grieving daughter of the Sub Inspector Ravinder Kumar, one of the 10 personnel, who was killed yesterday, was seen confronting the home minister. She asked, "Why do the soldier's family have to cry every time ?" The family members of Shivrain, who also died in the said incident, has alleged that the aircraft was too old.


text:
BBB Warning: Watch Out For Tax Scams
Started your taxes yet? It's that time of the year. And Uncle Sam isn't the only one trying to get hold of your money. There are a bunch of 'fraudsters' out there as well. And with a look at some of those scams...and best ways to protect yourself, we're joined by David Quinlan with the Seattle Better Business Bureau.


text:
Nike 6.0 BMX Tunnel Jam
Nike built this incredible underground skate park in a tunnel in the UK. Heres what went down. Nike 6.0s Tunnel Jam Results: Best Team: Nike 6.0 Global (Dennis Enarson, Garrett Reynolds, Maxime Charveron, Bjorn Bommel Mager) Second place: DK Bikes (Brett Banasiewicz, Drew Bezanson, Josh Cox, Anthony Watkins) Third place: Federal (Max Vincent, Ty Morrow, Dan Lacey, Mark Love) Fourth place: Fly Bikes (Kevin Kalkhoff, Daneil Naran Penafiel, Stefan Lantschner, Dylan Smith) Best Rider (As judged by the riders): Baz Keep with his crazy pocket transfers and the highest airs of the weekend. Best Line: Dan Lacey for a huge over vert Ruben wallride to 180 gap across the course. Best Trick: Jason Phelan tailwhip launch to double peg grind.
Video Rating: 4 / 5
Check out highlights from the BMX action that went down at the Gatorade Free Flow Tour's stop at Incline Club in New Jersey. Filmed and edited by CJ Capozzoli. Featured riders include Big Daddy, Joe Huelette, Anthony Malmone, Tommy Kopplinger, Cameron Eader, Michael Taber, Eddie Rovi, Erik Nilson, Jordan Prince, Courtney Freeman, Chris Aul, Charlie Quiqley, Matty Cranmer, Dominick Simoncini, Billy Pery, Josh Lane, Brandon Kitson, Brad Kaminskim, Gary Nilsson, Stephan Salley, Glen Purdy,
Video Rating: 5 / 5


text:
Recent Posts
- 1 What is Open Church? (8)
- 2 Scriptures (20)
- 3 Being Church (15)
- 3 Body of Christ (22)
- 3 Gatherings (12)
- 3 Relationships (8)
- 4 Disciples Apostles Mission (10)
- 5 Spirit-led Participation (11)
- 6 Beginning (6)
- 7 institutional church (14)
- jmw (9)
- 1 Corinthians 12 1 Corinthians 14 7 institutional church Acts 2 agree being church Berry St Berry Street biblical books booths burden character Christian church churchless community dale desert design different disciples ekklesia encourage Exodus 20 fellowship follow following Frank Viola freedom Galatians 5 Galatians 6 gather gathering God Gospel hebrews 10 Holy Spirit house church imitate institutional church instruct Isaiah 43 james choung Jesus Mark 2 Matthew 17 matthew 18 meeting Milpitas Nashville new zealand nz oikos old wineskins open church orderly order of worship organic church out of church participatory past perspective preach reddick relating rutz sabbath Salvation Army spirit-led Steve Sims strengthen together viola worship
Monthly Archives: August 2012
Being Church 24/7
Being Church 24/7, by Molong Nacua Jesus never intended for Christianity to become a religious sect. He did however want His followers to follow His footsteps in how He lived life, as designed by God, on this earth. Watching what … Continue reading
Posted in 3 Body of Christ, 3 Relationships Tagged being church
Out-Of-Church Christians
When Christians Quit Church by Andy Butcher, Charisma Mag. http://www.charismamag.com/site-archives/146-covers/cover-story/1477-when-christians-quit-church- The "Out of Church" Christians by Andrew Strom (NZ) Why are tens of thousands of devoted Christians leaving the churches? Is it a 'movement'? What is causing this world-wide phenomenon? http://homepages.ihug.co.nz/~revival/00-Out-Of-Church.html http://homepages.ihug.co.nz/~revival/out-of2.html A … Continue reading
Posted in 3 Body of Christ, 7 institutional church Tagged churchless, new zealand, nz, out of church
Open Church – Books Worth Reading
The Open Church by James Rutz The goal is to enable everyone to be a participant, not just a spectator. The church can quickly be restored to everyone because, contrary to popular belief, laypersons are eager to share their hearts, … Continue reading
Posted in 1 What is Open Church?, 8 RESOURCES Tagged books, dale, open church, reddick, rutz, viola
Do not let yourselves be burdened again
It is for freedom that Christ has set us free. Stand firm, then, and do not let yourselves be burdened again by a yoke of slavery. ~ Galatians 5:1 Sometimes we make rules when none are needed. Choosing to live … Continue reading
Posted in 2 Scriptures, 5 Spirit-led Participation Tagged burden, freedom, Galatians 5 2 Comments
Old Wineskins
… "What's wrong with institutions as a context for missional community? … The problem is not a "people-issue"; it is a "system-issue". Too often, the modern church depends on institutional systems that askew our foundational dependence on the Lord Himself; … Continue reading
Do not dwell on the past
Isaiah 43: 18 "Forget the former things; do not dwell on the past. 19 See, I am doing a new thing! Now it springs up; do you not perceive it? I am making a way in the desert and streams in the wasteland. … Continue reading
Posted in 1 What is Open Church?, 2 Scriptures, 7 institutional church Tagged desert, Isaiah 43, past, perspective, worship
Telling the Jesus Story
If James Cameron can spend $500 million, invent a new camera and new 3D techniques in order to produce the most visually stunning film ever recorded, and you can't remember a single character's name, do you really think your 'church' … Continue reading
Posted in 3 Body of Christ, 7 institutional church Tagged Gospel, Jesus


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2009Q3 Reports: Local Arrangements Chair
Local Chair Report (Li, Haizhou)
The Local Organizing Committee has completed most of the logistics arrangement for ACL-IJCNLP 2009 by July 3. They are summarized as follows:
1) Conference venue The rooms for ACL-IJCNLP 2009 main conference, tutorials, workshops, EMNLP, exhibition, software demos, and other ACL meetings have been confirmed with A/V and power facilities. They have been published at http://www.acl-ijcnlp-2009.org/main/conferencevenue.html.
2) Banquet, reception, ACL Exec++, ACL Board meeting, CL Editorial and other functions have been confirmed and announced at http://www.acl-ijcnlp-2009.org/main/program_glance.html
3) Onsite logistics Wireless internet, A/V and projectors, poster and lecture presentation facilities have been confirmed and announced at http://www.acl-ijcnlp-2009.org/main/infoparticipants.html
4) Publicity and Printing - Handbooks and conference proceedings are printed in Singapore, to be ready by 25 July, 2009 - Onsite publicity and press conference are being arranged - Conference blog is established in May 2009
5) Registration - Student travel grants have been awarded on 20 June - Student volunteers have been awarded on 2 July - Conference bags, souvenirs, T-shirt, videographer, photographer are ready on 30 June
6) Local sponsors By 1 July, local team has secured 4 local sponsors providing in-kind contributions to the conference as follows: - minimum 50 paid registration from Singapore - sponsor the entertainment performances in welcome reception and banquet - sponsor duty-paid wine to conference banquet - souvenirs for conference participants - travel grants
7) Hotel arrangement - Participants have taken up most of rooms offered by conference hotels in a package deal, which in general is a better deal than the regular rate. - The local committee has also negotiated with the hotels to release more rooms in June. - It should be noted that hotel adjusts room rate all the time, while our conference rate is fixed. Participants are advised to compare the room rates before making reservation.
8) Website ACL-IJCNLP 2009 promises a timely publication of information on the official website. All technical programmes and venue arrangement, time table, travel information can be found at http://www.acl-ijcnlp-2009.org/index.html. The local committee also provide a conference blog for people to exchange travel and conference related information.


text:
Release Date: 2012 |
no EMERITUS (5)...DoofusWainwright CONTRIBUTOR (4.5)Experimental indie types now legitimate big beasts...Ian Hammons (4.5)Shields is the work of a more mature band that finally is able to use their composition sk...BrianE (4.5)Grizzly Bear continue to push their sonic boundaries....
LudditeStereo (4)Chamber pop giants Grizzly Bear deliver more chamber and less pop with Shields...
Album Rating: 4.5nice review dude. kinda reads like a 4.5-5. but still, good job.
great album.
Album Rating: 5.0Awesome album. Awesome review.
Bands: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z


text:
As 2013 marks the year of the snake according to the Chinese zodiac, Stussy has prepped a corresponding capsule collection to kick off the new year. Featuring a trio of graphic tees alongside a hooded sweatshirt, the collection sees metallic snakeskin takes on Stussy's N°4 branding as well as a collaborative design by artist Mark Ward that reads "In This Great Future You Can't Forget Your Past." The entirety of the New Year Snake Collection is set to be available in the days ahead at select Stussy Japan stockists and chapter stores.
Source: honeyee.com


text:
user banned (girl); TTC since May 2008; Pounding Mill, VA, United States 3612 postsJun 8th '13
Quoting madison.jade:" My sisters wants to name her baby girl Hunter. Opinions ?"
That's my nephews name.
Devil Duckie 3 kids; New York 48700 postsJun 8th '13
Quoting lolajessup:" I personally wouldn't but I have a family where I work that has a daughter named hunter. Their girls are hunter and autumn and their boys are Remington and gauge."
That's just stupid!


text:
For 40 years my act consisted of one joke," George Burns was fond of saying. "Then she died." The woman in question, as anyone within earshot of a radio or television in the 1950s would know, was his wife, Gracie Allen—and the feminine side of a showbiz team whose ditzy banter in an era of idealized domesticity made it one of the most beloved and successful comedy acts in history.
Both onstage and off, as Burns himself was always the first to acknowledge, Gracie, the perfectly honed not-so-Dumb Dora to his long-suffering straight man, was more than half an act. "Next to Gracie, I was wonderful," he wrote in an affectionate biography, 1988's Gracie: A Love Story. "All I had to do was stand next to her and imagine some of the applause was for me."
When Burns first met the 17-year-old daughter of Edward Allen, a San Francisco song-and-dance man, George and Gracie were both aspiring vaudevillians. Glass fragments from an exploding hurricane lamp had left the Irish lass with one eye that appeared green and the other blue; another childhood accident had scalded and permanently scarred her left arm. But Burns, the former Nathan Birnbaum, a New York City clothier's son who was divorced and 10 years her senior, saw a partnership. "She could sing and she could dance and she was willing to work cheap," he wrote. "Who cared how old she was?"
Before long, his interest was more than professional, and he bought her a $20 wedding band—"very special," he recalled. "The metal band actually changed colors as it aged in my pocket." The ring, of course, did not do justice to the marriage. The couple stuck together through their steamer-trunk years on the vaudeville circuit, and 17 more on the nation's most popular radio show before moving to Hollywood in 1950 and brightening television's nascent horizon with The George Burns and Gracie Allen Show. True, their marriage did have its rough spots. One oft-repeated story has it that whenever Gracie suspected George of philandering, he would buy her an expensive gift. "I wish George would find another girlfriend," she once told a friend. "I could use a silver-fox jacket."
But they always managed to ride over the bumps. Once, in the middle of the night, Gracie elbowed George and asked him to make her laugh. Half-asleep, he mumbled, "Googie, googie, googie." It became his pet name for her, and as a couple, Googie and her Natty were indestructible. "Sometimes after dinner when Gracie needed to relax, she would say, 'George, sing what you sang to get me to sleep last night,' " recalls their close friend Carol Channing. "And sometimes he'd put his arm out for a tango, and Gracie would jump up and join him in these long Groucho Marx steps back and forth across the living room."
In 1934 they adopted a daughter, Sandra, and the following year, a son, Ronnie. With a family, thriving career and a stream of friends like Jack Benny and Fred Astaire coursing through their comfortable custom-built house at 720 North Maple Drive, George hoped that it all would go on forever. But in the early 1950s, Gracie had a mild heart attack, and in 1958, exhausted and suffering from the chest pains that had plagued her ever since, she made good on her threat to retire. On June 4 of that year, George and Gracie filmed their final TV show. Six years later she was dead. When George went into her hospital room for their last goodbye, she was still wearing the $20 wedding band that he had given her 38 years before. "For the first time in 40 years I was alone. So I did the only thing there was to do," he remembered. "I leaned over and I kissed her on her lips and whispered, 'I love you, Googie.' "
Burns never made a secret of the tough time he had dealing with his loss. "When I miss her a great deal, I crawl in on her side of the bed, in the middle of the day even," he told Carol Channing. "I stay there until I feel warm and good, and then I go on about my business." He also became something of a fixture at Hollywood's Forest Lawn Cemetery, where every month he would go to the mausoleum to talk to Gracie. "I don't know if she hears me," he said. "But after speaking to her, I feel better."
That their chats should continue beyond the grave didn't really seem so odd. Throughout his life, whenever people asked Burns how to make a marriage work, he had a standard response: "I tell them the answer's easy—marry Gracie." Taking his own advice, he never married again.


text:
Wednesday night's debate in Rio Rico featured candidates for Arizona House District 2 Chris Ackerley, Andrea Dalessandro and Rosanna Gabaldón. Here are some highlights.
All the candidates said they would vote no on Prop. 121, the open primaries proposition, and yes on Prop. 204, the sales tax proposition.
The candidates also agree the Legislature spends too much time on issues that don't help most of us, such as bills to allow guns on campus and Bibles studies in public schools.
Phoenix lawmakers have a habit of forgetting Southern Arizona. What's your plan to make sure we're heard?
Dalessandro and Gabaldón, both Democrats, said they'll be full-time legislators. Ackerley, a Republican who has a full-time job as a high school teacher, said he has an advantage because he's in the majority party and he'll be invited to key discussions about bills while his opponents won't have a seat at that table.
Should the Legislature return swept funds to the State Parks system?
Gabaldón said recreation facilities are an important part of economic development and help the tourism industry.
Ackerley said we need a health State Parks system, but there are other priorities right now. But he called fund sweeps "troubling."
Dalessandro said sweeps fly in the face of voters. Parks are important to industries including tourism, real estate, restaurants and retail, she said.
On abortion
Gabaldón is pro-choice, Ackerley is pro-life and Dalessandro said abortion should be legal.
Stay tuned to the Pueblo Politics blog throughout 2012 for news, updates and information about Arizona politics. You can follow Arizona Daily Star reporters Brady McCombs, Becky Pallack and Tim Steller on Twitter.


text:
Artist description:
I was bored. Ignore the bad background.
Flash Draw Created Mar 14, 2008 470 x 338px Unless noted Copyright 2008 xXToxic_KittenXx.
We encourage you to submit a comment and let the artist know what you think of this drawing. Please log in or join to post a comment.
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tishypoo Jan 18, 2009
she great!
sasukeluver101 Mar 24, 2008
maybe add water to "water" nypmh backgrpound 10 XD
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text:
Cal Expo RV Park, Sacramento, California
Date of Stay:
December, 2012 -
Overall Rating:
Positives: this park is relatively quiet, considering that it is next to a Harness Racetrack. The internet was OK, the water pressure was good. The close proximity to the American River Bike Trail allowed for many recreation options. Negatives: part of the park is little more than a gravel parking lot, with rigs positioned next to each other even though there were many empty spots. Additionally, there were enough security lights to make one feel that it was the middle of the day, all night long. The section that is paved, is unlevel. We camped at Cal Expo RV Park in a Fifth Wheel.
Fernley RV Park, Fernley, Nevada
Date of Stay:
October, 2012 -
Overall Rating:
We stayed here as part of the Amazon Camper Force, working the holiday season at the Fulfillment Center. This park has sites in the front, on pavement and close together. There are additional sites in the back, with the permanent modular homes. Neither Wi-Fi or TV work in the back section of the park. The staff helped connect our sewer and water, and were very helpful throughout our 2 month stay. Propane is available on site. There is significant train traffic that rocks your rig, at all hours. This is not a destination resort, but certainly filled our needs for the duration of our Amazon commitment. We camped at Fernley RV Park in a Fifth Wheel.
Meadowbrook Seniors RV Park (55+ park), Perris, California
Date of Stay:
August, 2012 -
Overall Rating:
This park is one of the quietest parks we have stayed in. The office staff is helpful and the laundry room is clean. Wi-Fi works well in the clubhouse, which was air conditioned and comfortable. There is a hiking trail around the park, affording nice views for the effort of the climb. Nightly rate reflects monthly rate paid. We camped at Meadowbrook Seniors RV Park (55+ park) in a Fifth Wheel.