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Most of the Leishmania genome is reported to be constitutively expressed during the life cycle of the parasite, with a few regulated genes. Inter-species comparative transcriptomics evidenced a low number of species-specific differences related to differentially distributed genes or the differential regulation of conserved genes. It is of uppermost importance to ensure that the observed differences are indeed species-specific and not simply specific of the strains selected for representing the species. The relevance of this concern is illustrated by current study.
Methodology/Principal Findings
We selected 5 clinical isolates of L. braziliensis characterized by their diversity of clinical and in vitro phenotypes. Real-time quantitative PCR was performed on promastigote and amastigote life stages to assess gene expression profiles at seven time points covering the whole life cycle. We tested 12 genes encoding proteins with roles in transport, thiol-based redox metabolism, cellular reduction, RNA poly(A)-tail metabolism, cytoskeleton function and ribosomal function. The general trend of expression profiles showed that regulation of gene expression essentially occurs around the stationary phase of promastigotes. However, the genes involved in this phenomenon appeared to vary significantly among the isolates considered.
Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics. Results obtained on an individual strain are not necessarily representative of a given species. Therefore, extreme care should be taken when comparing the profiles of different species and extrapolating functional differences between them.
Author Summary
Leishmania is a group of parasites (Protozoa, Trypanosomatidae) responsible for a wide spectrum of clinical forms. Among the factors explaining this phenotypic polymorphism, parasite features are important contributors. One approach to identify them consists in characterizing the gene expression profiles throughout the life cycle. In a recent study, the transcriptome of 3 Leishmania species was compared and this revealed species-specific differences, albeit in a low number. A key issue, however, is to ensure that the observed differences are indeed species-specific and not specific of the strains selected for representing the species. In order to illustrate the relevance of this concern, we analyzed here the gene expression profiles of 5 clinical isolates of L. braziliensis at seven time points of the life cycle. Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics: one Leishmania strain is not necessarily representative of a given species.
Citation: Adaui V, Castillo D, Zimic M, Gutierrez A, Decuypere S, Vanaerschot M, et al. (2011) Comparative Gene Expression Analysis throughout the Life Cycle of Leishmania braziliensis: Diversity of Expression Profiles among Clinical Isolates. PLoS Negl Trop Dis 5(5): e1021. https://doi.org/10.1371/journal.pntd.0001021
Editor: Elodie Ghedin, University of Pittsburgh, United States of America
Received: October 29, 2010; Accepted: February 21, 2011; Published: May 10, 2011
Copyright: © 2011 Adaui 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 the European Commission (INCO-DEV contracts LeishNatDrug-R - ICA4-CT-2001-10076, and LeishEpiNetSA - INCO-CT2005-015407), and the Directorate-General for Development Cooperation of the Belgian Government (framework agreement 02 - project 95501, and framework agreement 03 - project 95502). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Leishmania are digenic Protozoan parasites endemic worldwide and causing a spectrum of diseases in humans collectively referred to as leishmaniasis. As part of their life cycle, Leishmania alternate between the alimentary tract of the sandfly vector (where they grow as extracellular flagellated promastigotes and differentiate into infective non-dividing metacyclic forms) and the phagolysosome of the vertebrate host macrophages (where parasites differentiate into aflagellated replicative amastigotes). Important morphological and biochemical changes underlie the differentiations involved in the life cycle and are most likely the result of regulated changes in gene expression in response to environmental signals (e.g. temperature change and pH shift) [1], [2], [3]. A recent study assessing gene expression profiles throughout the life cycle of a single strain of L. infantum showed that most of the Leishmania genome is constitutively expressed and that consequently, regulated genes are a minority [4]. Interestingly, most significant events of regulation were shown to occur during promastigote development, with a dramatic down-regulation during the transition from promastigotes to amastigotes, hereby supporting the hypothesis of pre-adaptation of the parasite to intracellular survival in the macrophage [4].
In parallel to these longitudinal studies of gene expression throughout the life cycle of a given strain, another critical dimension needs to be explored, i.e. the gene expression diversity within natural populations of Leishmania. Indeed, these parasites successfully colonized a large range of hosts in various ecological niches, hereby determining different transmission types in which humans play a variable role (from dead-end host to reservoir). Not surprisingly, the parasites are characterized by high phenotypic diversity: infectivity, tissue tropism, clinical pattern or drug susceptibility, among others. This polymorphism is reflected at the taxonomic level, with more than 20 described species. A first comparative transcriptomic study throughout the life cycle of 3 strains belonging to the species L. major, L. infantum and L. braziliensis was recently reported: this provided in the 3 species a similar picture of a mostly conservative gene expression, and a minority of species-specific differences related to differentially distributed genes or the differential regulation of conserved genes, either of which are subject to translational and/or post-translational controls [5]. This type of research being undertaken among other reasons for a better understanding of the differences in virulence and pathogenicity of the respective species, it is of utmost importance to ensure that the observed differences are indeed species-specific or simply specific to the strains selected as representative of the species.
In a previous study targeting a limited number of genes, we analyzed the expression profile of 21 L. braziliensis clinical isolates during in vitro promastigote growth [6]. A fraction of genes were up-regulated during differentiation, but the set of regulated genes varied between isolates, providing a picture of intra-species gene expression mosaic. We aimed here to continue our assessment of gene expression diversity within a single species (L. braziliensis) by extending it to the whole life cycle of the parasite. This type of study is complicated by the extreme sensitivity of the amastigote stage to disturbance of the intracellular parasite's environment during harvesting, hence extreme care is needed to 'freeze' gene expression levels instantly [7]. Our previous work also demonstrated the importance of time-course analyses to better observe the dynamics of gene expression changes during in vitro growth and to allow more reliable comparisons between different strains [6], [7]. These considerations currently impede high-throughput experiments involving the clinically relevant amastigote stage. We surveyed here 5 clinical isolates of L. braziliensis, characterized by their diversity of clinical and in vitro phenotypes. We applied a standardized and reproducible biological protocol [6], [7] and used real-time quantitative PCR to assess gene expression profiles at seven time points covering the whole life cycle. We tested 12 genes encoding proteins with roles in transport, thiol-based redox metabolism, cellular reduction, RNA poly(A)-tail metabolism and housekeeping functions.
Materials and Methods
Ethical statements
The five parasite isolates used here were obtained from patients with cutaneous leishmaniasis at the Instituto de Medicina Tropical A. von Humboldt, Lima, Peru. Protocol and informed consent were approved by the Research Ethic Committees of Universidad Peruana Cayetano Heredia (Lima, Peru) and Institute of Tropical Medicine (Antwerp, Belgium). Written, informed consent was obtained from all participating subjects or their legal guardians. Animal experimentation concerned only the use of peritoneal macrophages obtained from mice. Our animal protocol adhered to the guidelines at the Universidad Peruana Cayetano Heredia, Lima, Peru and was in agreement with the Peruvian and Belgian regulations for the protection and welfare of laboratory animals. Mouse care and experimental procedures were performed under approval of the Ethic Committee of the Universidad Peruana Cayetano Heredia as well as the Animal Ethic Committee of the Institute of Tropical Medicine Antwerp (PAR-018/2).
The five parasite isolates were obtained from Peruvian patients with confirmed cutaneous leishmaniasis and originating from 4 regions of the country (Table 1). The selected isolates were previously characterized as L. braziliensis by PCR-RFLP assays targeting a range of markers [8], but we retyped them here to obtain a more precise perception of their genetic diversity. Therefore, we sequenced part of the coding region of the hsp70 genes, a locus recently shown to be phylogenetically very informative [9] and more and more used for species identification of Neotropical Leishmania species [10]–[12]. Sequencing concerned the isolates of this study [GenBank accession numbers: FR715986 (isolate PER002), FR715987 (isolate PER006), FR715988 (isolate PER182), FR715989 (isolate PER104), FR715990 (isolate PER163)] and the additional Peruvian L. braziliensis reference strain LEM2222 (http://www.parasitologie.univ-montp1.fr/cnrl.htm; GenBank accession number: FR715991). The sequences were aligned with reference strains recently published by our group [9] and available GenBank entries, and compared by Neighbor-Joining analysis of p-distances, using the software package MEGA4 (http://www.megasoftware.net/). Data on the in vitro SbV and SbIII susceptibility of the studied isolates, as tested by the intracellular amastigote-macrophage model [8], and of the clinical treatment outcome of respective patients, were available (see Table 1 for summary of isolates' features). Isolates were used here for gene expression analysis within a maximum of 25 in vitro passages post-isolation from patients.
In vitro promastigote production
The in vitro conditions for promastigote generation have been described previously [6]. Briefly, growth curves were initiated by inoculating 3×106 parasites/mL in 5 mL medium 199 (M199; Sigma) containing 20% heat-inactivated fetal bovine serum (FBS; Lonza Bioscience), 25 mM Hepes (pH 7.4), 100 units/mL penicillin and 100 µg/mL streptomycin (Lonza). Two independently grown cultures and corresponding harvests at 24 h (early-log phase), 72 h (late-log phase), 120 h (early-stationary phase) and 168 h (late-stationary phase) time points were performed in parallel for each isolate (biological replicates).
In vitro intracellular amastigote production
Intracellular amastigotes were obtained after infection with axenic amastigote-like forms, as this was reported to enhance infectivity in species of L. (Viannia) subgenus [13]. Briefly, stationary-phase promastigotes were incubated in M199-20% FBS, at pH 5.5 and 34°C, during 96 h [14]. Following this incubation, parasites were washed, resuspended in M199-pH7.4–10% FBS (pre-warmed at 32°C), and then used to infect murine peritoneal macrophages (collected from BALB/c mice) at a parasite to macrophage ratio of 10∶1. Infected cultures were incubated for 4 h at 32°C in a humidified 5% CO2/95% air environment, then washed with pre-warmed medium to remove free extracellular parasites, and further incubated for 3 days. Intracellular amastigotes were co-harvested with macrophages at 24 h, 48 h and 72 h post-infection using a protocol that has shown to 'freeze' L. donovani gene expression levels instantly [7]. Infection rates were monitored using control plates, as described elsewhere [15]; up to 200 cells were counted in order to determine the percentage of infected macrophages and the average number of amastigotes by infected macrophages. The whole experiment was done in 2 biological replicates.
RNA isolation and cDNA synthesis
RNA sampling protocols, RNA isolation and quality control were performed as described elsewhere [16]. First strand cDNA was synthesized from 150 ng total RNA of promastigote samples and from 1 µg total RNA of mixed amastigote-macrophage samples, using a 18mer oligo(dT) and Transcriptor Reverse Transcriptase, according to the manufacturer's instructions (Roche Applied Science). The resulting cDNA was diluted 10-fold with DEPC-treated water (Ambion) for further use.
Real-time quantitative PCR
From the cDNA diluted samples, 2 µL was used as template in 25 µL SYBR Green-based quantitative PCR (qPCR) reactions on the iCycler (Bio-Rad), as previously described [16], with the only modification that amplification was done for 34 cycles [6]. We analyzed 12 Leishmania-specific genes, with predicted function in transport (LbAQP1, MRPA), thiol-based redox metabolism (GSH1, GSH2, ODC, TRYR), cellular reduction (ACR2, TDR1), RNA poly(A)-tail metabolism (PABP, PAP14), cytoskeleton function (Actin) and ribosomal function (S8). Primer sequences, parameters and reproducibility of the respective qPCR assays have been described previously [6].
Data analysis and statistics
Analysis was performed on duplicate biological samples that were each assayed in triplicate. The arithmetic average threshold cycle (Ct) was used for data analysis. The Ct values of each qPCR run were imported as Excel files into qBasePlus 1.3 (Biogazelle NV, Zulte, Belgium), a software for real-time PCR data analysis based on the geNorm method [17] and qBase technology [18]. Three genes (ACR2, GSH2, PAP14) showed the most stable expression through parasite life stages in our sample panel (geNorm stability mean M-value and mean coefficient of variation lower than 0.45 and 20%, respectively) and data were normalized to their geometric mean. The use of multiple control genes allows more accurate and reliable normalization of gene expression data [17].
The linear component of the variability of the expression level of each gene during in vitro growth was modelled independently for each isolate using linear regression. The main predictor was the development time of the parasite during in vitro promastigote or intracellular amastigote growth/differentiation. The time point 24 h was considered as the baseline time in the regression models, in order to prevent negative values for gene expression levels, a biological impossibility. The constant terms of the regression models were used as measure of the baseline expression of a given gene, with the criterion of significance that the constant terms differed by at least 2-fold from the lowest constant term in the series.
In addition, the fold change (FC) of gene expression of (i) stationary versus logarithmic phase promastigotes (time points 120 h and 168 h versus 24 h and 72 h of the growth curves, respectively); (ii) late-differentiating phase amastigotes versus early-differentiating phase amastigotes (time points 72 h versus 24 h and 48 h post-infection macrophages, respectively); and (iii) stationary phase promastigotes (time points 120 h and 168 h of the growth curves) versus early-differentiating phase amastigotes (time points 24 h and 48 h post-infection macrophages) was determined for each parasite isolate. FC were considered significant if they satisfied a 2-fold cutoff and P<0.01, as recommended elsewhere [19]. All analyses were performed using the statistical software Stata 10 (StataCorp).
Molecular typing of the tested isolates
Out of the 5 isolates, 4 (PER002, -006, -104 and -182) clustered in a well-supported group (bootstrap value of 92%) constituted by several reference strains of the L. braziliensis complex (Fig. 1). The fifth isolate, PER163, has 100% sequence identity with the MLEE typed L. braziliensis reference strain LEM2222, and they cluster with a bootstrap support of 78%. Even though PER163 has more sequence similarity (99.64%) with the main L. braziliensis group than with L. naiffi (99.49%), it clusters with the latter without bootstrap support.
The total alignment contains 1380 nucleotides. Bootstrap support of the branches was inferred from 2000 replicates, and is shown in percentages at the internodes when exceeding 70%. The tree is drawn according to the scale on the left, expressed as distance per nucleotide. Different species are depicted on the right, whereby species of the L. (Leishmania) subgenus are condensed. The tree was rooted on the branch leading to L. (Viannia). Isolates are indicated with their WHO code wherever possible, with accession numbers between brackets. Accessions starting with XM are derived from a contemporary annotation of full genome sequences, and are GenBank specific.
In vitro phenotypes
A series of parameters were measured during the growth of promastigotes and the macrophage infection by amastigotes for phenotypic comparison of the 5 isolates. At the promastigote level, our observations led us to the empirical description of 3 main phenotypes (Table 1): (i) slow growth, but high density at stationary phase in PER163 and PER006, (ii) rapid growth and high density at stationary phase in PER104 and PER182, and (iii) slow growth and lower density at stationary phase in PER002. At the amastigote level, 2 main phenotypes were observed (Table 1): (i) high percentage of infected macrophages and high number of amastigotes per macrophage in PER163 and PER104, and (ii) low values for both parameters in PER002, PER006 and PER182.
Gene expression profiling through in vitro life stages
We obtained highly reproducible gene expression measurements between biological replicates, in both the promastigote and intracellular amastigote developmental time series (Fig. 2, 3, 4), thereby confirming that parasites were manipulated in a standardized way. Visual inspection of the graphs revealed notable differences between life stages in general and between genes according individuals. For instance, LbAQP1 showed in PER163 and PER006 a clear up-regulation during promastigote development, followed by a 5-fold down-regulation during the transition to amastigotes (Fig. 2). In sharp contrast, TRYR showed a similar expression during the 4 time points of promastigote growth curve in PER163, while in PER006 a clear up-regulation was observed (Fig. 3). A last example concerns the expression of Actin shown to be down-regulated from promastigote to amastigote in PER163 and PER006, whereas it appeared to be clearly constitutive and at relative low levels in PER182 through the 7 time points analyzed here (Fig. 4).
Time scale in promastigotes (PRO): 24 h, 72 h, 120 h and 168 h of growth. Time scale in intracellular amastigotes (AMA): 24 h, 48 h, and 72 h post-infection macrophages. White and grey bars at each time point represent two experimental biological replicate series (A and B). Normalized expression levels of each gene were rescaled relative to the sample with the lowest expression. Results are expressed as means (± standard errors) of triplicate measurements from one quantitative experiment.
In order to summarize and better visualize the gene expression profiles during the life cycle, we examined 5 parameters in the 5 isolates: the baseline expression levels (given by the constant term of the linear regression models) in (1) promastigotes and (2) amastigotes, and the fold change in mRNA abundance (3) from logarithmic to stationary phase promastigotes (further called FC-PRO), (4) during the transition from stationary phase promastigotes to early-differentiating phase amastigotes (further called FC-PRO-AMA), and (5) from early- to late-differentiating phase intracellular amastigotes (further called FC-AMA). Results are schematized in Figures 5 and 6.
(A) Gene expression analysis among promastigotes: PRO constant term, measure of the baseline expression of a given gene in the promastigote stage (highlighted in black if significantly higher –by at least 2-fold– than the lowest constant term in the series). (B) Gene expression analysis among amastigotes: AMA constant term, measure of the baseline expression of a given gene in the amastigote stage (highlighted in black if significantly different –by at least 2-fold– from the lowest constant term in the series).
Modulation of gene expression can be in the form of increased expression (up-regulation) or decreased expression (down-regulation); a 2-fold cutoff was used here. (A) FC-PRO, measure of gene expression modulation during development of promastigotes (highlighted in black if up-regulation –i.e. at least 2-fold increase– is present). (B) FC-PRO-AMA, measure of gene expression modulation during transition from promastigotes to amastigotes (highlighted in black if modulation, always down-regulation –i.e. at least 2-fold decrease–, is present). (C) FC-AMA, measure of gene expression modulation during development of amastigotes (highlighted in black if modulation is present).
First, when analyzing all parameters together, we identified 3 genes that were constitutively expressed during the whole life cycle and in the 5 isolates: ACR2, GSH2 and PAP14. Secondly, analysis of the baseline expression in promastigotes (Fig. 5A) discriminated 2 groups of parasites: (i) PER002 and PER182, which showed the lowest baseline levels for the 12 genes studied here and (ii) PER163, PER006 and PER104 which showed significantly higher baseline levels for 2 to 5 genes (out of the following set: Actin, LbAQP1, MRPA, PABP, S8 and TRYR). Thirdly, analysis of the baseline expression in amastigotes (Fig. 5B) also discriminated 2 groups of parasites: (i) PER163 and PER006 showed the lowest baseline for the 12 genes at the amastigote stage, which is in sharp contrast to promastigote data, and (ii) PER002, PER104 and PER182 which showed significantly higher baseline expression of 4 to 6 genes (out of the following set: Actin, LbAQP1, GSH1, ODC, PABP, TDR1 and TRYR). Fourthly, up-regulation of gene expression during promastigote differentiation (FC-PRO ≥2, Fig. 6A) was observed for up to 3 genes out of the following set: LbAQP1, GSH1 and TRYR. As did other parameters, the FC-PRO also discriminated PER163 and PER006 from other isolates, as the former showed the lowest number of up-regulated genes. Fifthly, down-regulation during the promastigote-amastigote transition (FC-PRO-AMA ≥2, Fig. 6B) was observed in all isolates, but to a very different extent: (i) involving 7 and 8 genes in PER163 and PER006 respectively, (ii) 3 genes in PER002 and (iii) one gene only in PER104 and PER182. Sixthly, and in stark contrast to previous parameters, we did not observe up-regulation of any gene in any isolate during amastigote development (FC-AMA <2, Fig. 6C).
Most of the Leishmania genome is reported to be expressed constitutively [4], [5], a small fraction of it only (estimated at 5,7% in L. infantum and 9% in L. braziliensis; [4], [5]) being regulated during the life cycle. In our attempt to explore the diversity of gene expression profiles within a single species -L. braziliensis in our case- we thus focused our analysis on a set of genes that were more prone to show variation in gene expression in L. braziliensis when assaying promastigotes [6].
Overall, the fine monitoring of the expression of these 12 genes over seven time points of the promastigote and amastigote stages supported previous data on the dynamics of up-/down-regulation during the life cycle. When considering the present sample of isolates as a single pool and looking at the general trend of the expression profiles, regulation appeared to be concentrated around the stationary phases of promastigotes: (i) up-regulation during promastigote growth (3 genes in total), (ii) down-regulation during the promastigote-amastigote transition (9 genes in total) and (iii) no gene showing any up-regulation during the period of monitoring (24 to 72 hours) of amastigote growth. Several reports on the expression profile of the intracellular amastigote stage compared with logarithmic or stationary phase promastigotes or both in several Leishmania spp. showed a highly significant predominance of down-regulated over up-regulated genes in amastigotes [4], [20], [21]. Altogether, our results are in agreement with the previously formulated hypothesis that Leishmania are pre-adapted for intracellular survival [4], [5].
However, when considering each isolate separately, our results clearly illustrate the unique character of the different parasite populations in terms of gene expression dynamics. Indeed, major differences were observed between the isolates studied here. This concerned essentially the baseline expression levels in promastigotes and amastigotes and the degree of down-regulation during the transition from promastigotes to amastigotes. In terms of similarity of gene expression patterns, 2 groups of parasites could be distinguished: PER163 and PER006 on one hand, and PER002, PER104 and PER182 on the other hand. This grouping was not dependent of the genetic relationships among isolates as inferred from the Hsp70 sequences (Fig. 1) nor confirmed by other genetic markers (MLMT and AFLP, unpublished results). Strikingly, the isolates PER163 and PER006, which have a higher baseline expression level for several genes in promastigotes than the other isolates and the highest number of down-regulated genes during the transition from promastigotes to amastigotes, were the 2 isolates showing the lowest constitutive levels of baseline expression in amastigotes: the coherence between the different parameters studied here further supports the validity of our methodology. Reciprocally, isolates PER002, PER104 and PER182 call the attention by their low baseline expression levels in promastigotes, the few down-regulated genes during the transition from promastigotes to amastigotes, and the higher level of baseline expression in intracellular amastigotes compared to the isolates PER163 and PER006.
Gene expression diversity in the present sample is not a surprise, considering the phenotype diversity of the analyzed L. braziliensis isolates: growth rate at promastigote stage, in vitro infectivity for macrophages, in vitro susceptibility to SbV and SbIII or the treatment outcome in patients. The choice of genes likely also played a role in the observed diversity, as they encode proteins with roles in transport, thiol-based redox metabolism, cellular reduction and RNA metabolism, and were previously shown to undergo significant changes in expression in some of the phenotypes present here [7], [22]. However, our aim was not to search any correlation between the gene expression patterns and the main phenotypes observed. This would require another experimental set-up, including (i) a larger sample size, (ii) a broader coverage of the transcriptome and (iii) complementary studies at other 'omic levels. Proteomic approaches are very relevant, considering the importance of post-transcriptional regulation in Trypanosomatids [23]. But, even more important, the metabolome should be explored, because of its closest position to the phenotype [24].
In conclusion, our results show that the gene expression pattern of a strain is not necessarily representative of a given species. They remind us of an essential feature of natural populations of Leishmania: diversity. We recommend to keep this dimension in mind in any future work and to take extreme care when comparing the profiles of different species represented by single strains and extrapolating functional differences between them.
Accession Numbers
The accession numbers for the genes analyzed in this study are as annotated at L. braziliensis GeneDB database (http://www.genedb.org/Homepage/Lbraziliensis). LbAQP1 (GeneDB ID LbrM.31.0020) encodes an aquaglyceroporin. MRPA (LbrM.23.0280) codes for an ABC-thiol transporter. GSH1 (LbrM.18.1700) encodes a putative gamma-glutamylcysteine synthetase (γ-GCS). GSH2 (LbrM.14.0880) codes for a putative glutathione synthetase (GS). ODC (LbrM.12.0300) codes for a putative ornithine decarboxylase. TRYR (LbrM.05.0350) encodes trypanothione reductase. ACR2 (LbrM.32.2980) encodes a putative SbV/AsV reductase according to the orthologous ACR2 sequence in L. major (GeneDB ID LmjF.32.2740; GenBank accession number AY567836.1). TDR1 (LbrM.31.0550) encodes a thiol-dependent reductase 1. PABP (LbrM.30.2560) encodes a putative RNA-binding protein. PAP14 (LbrM.14.1350) codes for a putative poly(A) polymerase. Actin (LbrM.04.1250) codes for the actin protein. S8 (LbrM.24.2160) encodes a putative 40S ribosomal protein S8.
Author Contributions
Conceived and designed the experiments: VA JA J-CD. Performed the experiments: VA DC SDD KS. Analyzed the data: VA MZ AG MV IM GVdA J-CD. Contributed reagents/materials/analysis tools: MZ AL-C LM AG SD JA J-CD. Wrote the paper: VA MZ JA J-CD.
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- 13. Puentes F, Diaz D, Hoya RD, Gutíerrez JA, Lozano JM, et al. (2000) Cultivation and characterization of stable Leishmania guyanensis complex axenic amastigotes derived from infected U937 cells. Am J Trop Med Hyg 63: 102–110.F. PuentesD. DiazRD HoyaJA GutíerrezJM Lozano2000Cultivation and characterization of stable Leishmania guyanensis complex axenic amastigotes derived from infected U937 cells.Am J Trop Med Hyg63102110
- 14. Teixeira MC, de Jesus Santos R, Sampaio RB, Pontes-de-Carvalho L, dos-Santos WL (2002) A simple and reproducible method to obtain large numbers of axenic amastigotes of different Leishmania species. Parasitol Res 88: 963–968.MC TeixeiraR. de Jesus SantosRB SampaioL. Pontes-de-CarvalhoWL dos-Santos2002A simple and reproducible method to obtain large numbers of axenic amastigotes of different Leishmania species.Parasitol Res88963968
- 15. Gamboa D, Torres K, De Doncker S, Zimic M, Arevalo J, et al. (2008) Evaluation of an in vitro and in vivo model for experimental infection with Leishmania (Viannia) braziliensis and L. (V.) peruviana. Parasitology 135: 319–326.D. GamboaK. TorresS. De DonckerM. ZimicJ. Arevalo2008Evaluation of an in vitro and in vivo model for experimental infection with Leishmania (Viannia) braziliensis and L. (V.) peruviana.Parasitology135319326
- 16. Decuypere S, Rijal S, Yardley V, De Doncker S, Laurent T, et al. (2005) Gene expression analysis of the mechanism of natural Sb(V) resistance in Leishmania donovani isolates from Nepal. Antimicrob Agents Chemother 49: 4616–4621.S. DecuypereS. RijalV. YardleyS. De DonckerT. Laurent2005Gene expression analysis of the mechanism of natural Sb(V) resistance in Leishmania donovani isolates from Nepal.Antimicrob Agents Chemother4946164621
- 17. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034.J. VandesompeleK. De PreterF. PattynB. PoppeN. Van Roy2002Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol3RESEARCH0034
- 18. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8: R19.J. HellemansG. MortierA. De PaepeF. SpelemanJ. Vandesompele2007qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data.Genome Biol8R19
- 19. McCarthy DJ, Smyth GK (2009) Testing significance relative to a fold-change threshold is a TREAT. Bioinformatics 25: 765–771.DJ McCarthyGK Smyth2009Testing significance relative to a fold-change threshold is a TREAT.Bioinformatics25765771
- 20. Holzer TR, McMaster WR, Forney JD (2006) Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana. Mol Biochem Parasitol 146: 198–218.TR HolzerWR McMasterJD Forney2006Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana.Mol Biochem Parasitol146198218
- 21. Rochette A, Raymond F, Corbeil J, Ouellette M, Papadopoulou B (2009) Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum. Mol Biochem Parasitol 165: 32–47.A. RochetteF. RaymondJ. CorbeilM. OuelletteB. Papadopoulou2009Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum.Mol Biochem Parasitol1653247
- 22. Ashutosh , Sundar S, Goyal N (2007) Molecular mechanisms of antimony resistance in Leishmania. J Med Microbiol 56: 143–153.AshutoshS. SundarN. Goyal2007Molecular mechanisms of antimony resistance in Leishmania.J Med Microbiol56143153
- 23. Rochette A, Raymond F, Ubeda JM, Smith M, Messier N, et al. (2008) Genome-wide gene expression profiling analysis of Leishmania major and Leishmania infantum developmental stages reveals substantial differences between the two species. BMC Genomics 9: 255.A. RochetteF. RaymondJM UbedaM. SmithN. Messier2008Genome-wide gene expression profiling analysis of Leishmania major and Leishmania infantum developmental stages reveals substantial differences between the two species.BMC Genomics9255
- 24. t'Kindt R, Scheltema RA, Jankevics A, Brunker K, Rijal S, et al. (2010) Metabolomics to unveil and understand phenotypic diversity between pathogen populations. PLoS Negl Trop Dis 4: e904.R. t'KindtRA ScheltemaA. JankevicsK. BrunkerS. Rijal2010Metabolomics to unveil and understand phenotypic diversity between pathogen populations.PLoS Negl Trop Dis4e904


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Cuisinart CH-4 Elite Collection® 4-Cup Chopper/Grinder Reviews
Additional 8 CH-4 Elite Collection® 4-Cup Chopper/Grinder Cuisinart Reviews
- Most Helpful First
- Newest First
1 person found this review helpful
May 17, 2012
from houston tx
I would have preferred a grater and slicer attachment for this. I am sure it is not hard to make those attachments for a little price. This is helpful for all the salads.
1 person found this review helpful
April 19, 2012
from Northern California
I wanted something not quite as big as a food processor but big enough to make a batch of pesto--with ease of use and easy clean-up. I have a Vita-mix for bigger jobs. This little guy fit the bill. And, as always, Zappo service cannot be beat!
2 people found this review helpful
February 3, 2012
from Savannah, GA
This is an awesome little machine, and just the right size for a small household or small jobs. I like the fact that the blade stays put when you are pouring out ingredients into another container (my previous small processor did not do that). I also like the fact there are two holes in the lid for adding liquid (another nice feature for a small processor). It would be nice if there were a choice of base colors or materials such as stainless steel.
2 people found this review helpful
September 23, 2011
Mike F
from Panama City Beach FL
This is a great little machine. It is just right for the two of us. I got tired of chopping celantro.
3 people found this review helpful
June 20, 2011
Virginia t
from raleigh,north carolina
Ordered Cuisinart over the phone since I get confused when trying to do it online - it arrived next day - what wonderful service! I make my 7 pound dog's food. I needed a medium size, strong chopper/grinder. This was a perfect choice for a cooked chicken breast, applesauce, carrot, dash of seasoning and vitamin/mineral and turn on the Cuisinart for a few seconds and it's ready ! I put "finger pinches" on a plate -- result -- happy dog and doggie momma -- can make enough for couple days or a week - would be same basic idea for baby or toddler food... or for easy to eat nutritious for we elders who can't chew everything just grind/chop tuna, chicken salad, slaw or many other foods -- I know, I am 82.
Brands I'd also recommend: I read about all kinds --too big too heave - too little
2 people found this review helpful
January 13, 2011
Cathy f
from hoffman estates, ill
Cuisinart makes a great product. The first day I got the blender I tried to grind whole cloves; the Cuisinart did grind the cloves - not fine - but also when I stopped the grinding my new bowl was all scratched and fogged up. It's very quiet and works great, though.
Brands I'd also recommend: Joseph Sible
3 people found this review helpful
January 12, 2011
from Greensboro, NC
Got this for my girlfriend, who has a tiny kitchen but wanted a food processor. The build quality seems sturdy. Nice metal base. I like how it reverses the blade spin for grinding to save the edge sharpness. It sure doesn't take up much counter space, and is nice and light, so if it does get put in a cabinet it's not a pain in the tail to get back out when needed. It is on the verge of being too small, though. Two cans of garbanzo beans for making hummus, and it's about full. I wanted to get Cuisinart, because I know they are dependable. As is, there were only 4 and 12 cup versions available. A 6 or 8 cup version would have been perfect. Also, the lid has two holes that are only large enough to add liquid, not solids during chopping - unless you do one garbanzo bean at a time :) The ability to leave it on without pressing the button would be nice, but I guess that's why it's a "Chopper/Grinder" and not a "Food Processor" I bought it from Zappos because the have an awesome return policy, and I wasn't sure if my girlfriend would want to keep it because it was so tiny. I didn't hit a home run with this product like I did with the KitchenAid Stand Mixer I bought her (taking up 1/4 of her counter space, lol), but she did decide to keep it. Zappos shipped it to me lightning fast, too. Thanks!!
Brands I'd also recommend: KitchenAid, Wustof
3 people found this review helpful
October 17, 2010
Christine N
from Tucson, AZ
The Cuisinart 4 cup chopper/grinder is good looking and seems sturdy, but it's probably too small to be very useful for the kind of cooking I do. I wish I had ordered a food processor with a larger capacity - 7 or 9 cups would have been a lot more useful.

subset: commoncrawl


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Bella the Bunny Paperback
by Lily Small
Part of the Fairy Animals of Misty Wood series
Enter an enchanted world of animal friends! Perfect for fans of Holly Webb, Rainbow Magic, Secret Kingdom and Magic Kitten.
Each cute animal has its own pair of pretty fairy wings, and a special job to do in Misty Wood to make it a wonderful place to live.
In book 2, Bella the Bunny is opening flowers to make Misty Wood as pretty as can be.
There's a surprise inside one of the bluebells - Lexi the Ladybird!
Lexi is sad because she looks different to the other ladybirds.
But Bella knows just how to make her feel better...The Fairy Animals of Misty Wood are perfect books for girls.
They will fall in love with these gorgeous, magical kittens, puppies, bunnies and more - animal stories in an enchanted woodland setting.
- Format: Paperback
- Pages: 144 pages, black & white illustrations
- Publisher: Egmont UK Ltd
- Publication Date: 01/07/2013
- Category: Fantasy
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Is there a cover charge for the music?
Linda C. asked on May 18, 2013
They do charge a fee for some of their events. Please click here for the event calendar. You can click on each date for details and pricing.
Brette V. from Groupon replied on May 19, 2013


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PETER Norfolk did a Steve Redgrave after having to settle for silver in the quad doubles tennis.
The "Quadfather", individual champion in Athens and Beijing, told partner Andy Lapthorne he was quitting the sport following the 6-2, 5-7, 6-2, defeat to reigning champions Nicholas Taylor and David Wagner, of the USA.
But in an even quicker turnaround than Redgrave's famous retirement U-turn, minutes later Norfolk seemed ready to go back on the decision.
Norfolk, 51, said: "I've been playing a long time, I've achieved quite a lot, it's time to sit back and think what I'm going to do now. I have a young family.
"I'm sort of doing a Steve Redgrave – never get me in a boat, never get me on a court.
"It's been a hard road to get here this time. There have been some very tough challenges to get over.
"I have tried everything but I haven't succeeded. I still feel I've got it in me, the talent and the ability we'll see."
Londoner Lapthorne, 21, who had received good luck messages from Andy Murray and the players and staff of West Ham and Brentford football clubs, was in tears.
He said: "10 minutes ago, he said: 'That's me I'm done.'
"Knowing that's Pete's last ever match kills me, just kills me. I couldn't have tried any harder. I tried everything I know. I'm absolutely gutted, but also proud."


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4683 Bonnie Avenue SE, Kentwood MI, 49508
Sold Price:
3 beds , 2 Full baths
1,600 sqft
Status: Closed
Last Updated: 5/27/2017 2:17 PM
Description for 4683 Bonnie Avenue SE Kentwood, MI 49508
See virtual tour and floor plan under ''Photos''. Built in 1964 and is one of the first contemporary homes in Kentwood. Some of it reminded me of Frank Lloyd Wright - it has a spacious Double door formal slate entry, cathedral ceilings, great room with fireplace -unique floor plan - some renovation done this past year.New roof and some new drywall. A brand spanking New spacious deck. If you like something different you will love this house.
Sold Price:
Sold Date:07/10/2015
City Of Kentwood
2 Full
1,600 (approx)
Listing Information
City Of Kentwood
Public Water Service
Public Sewer, Storm Sewer
Cable TV, Electric, Natural Gas
School Information
Room Information
Family Room, Recreation Room
Eat-In Kitchen
Interior Features
Dishwasher, Garbage Disposal, Range, Refrigerator
Central Air, Ceiling Fan(s)
Gas Heat, Forced Air
Basement, Full
Insulated, Screened, Storm Window
1, Wood-burning
Exterior / Lot Features
Attached Garage, 2 Car Garage
Deck, Porch
401 res
Paved Road, Public Roads, Wooded
Driving Directions
44th West of Kalamazoo, South onBrooklyn and immediately East (left) on Bonnie. Or 48th East of Eastern toBrooklyn - North to Bonnie.
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Kentwood Real Estate
Kentwood is located in Michigan. Kentwood, Michigan 49508 has a population of 51,357.
The median household income in Kentwood, Michigan 49508 is $49,201. The median household income for the surrounding county is $52,716 compared to the national median of $53,482. The median age of people living in Kentwood 49508 is 33.9 years.
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Alejandro Rotter, MDAnti-Aging, Medical Spas & Medspas, Minimally Invasive Surgery (BOTOX®), Weight Loss Programs
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Alejandro Rotter, MD Reviews
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the office of dr rotter in doral is the worst office in the entire south florida i don't know how can that man call him self a dr. he us unethical, rude, obnoxious, and you can tell that all that so called dr cares about is money, you go to that office for him to treat you for an illness or medical complaint and he either ends up selling you some hormone treatment that supposedly he does, or a weight loss treatment, or his new thing now allergy testing, he ends up treating you for anything but what you are really there for, i have been there waiting to see him and i have heard his staff telling him to come see patients and he answers back very rudely let them wait, you hear him trying to sign medicare patients over to a manage care, of course to get more money in to his pocket, not because he cares about the patient, i was his patient but when i realized the kind of dr this man is and been my self in the medical field, i decided to change drs, to a real dr, a dr that cares about his patients, not about money, a dr that if i go see him for a headache, or a cold he is going to treat me for what i am there for, not going to try to sell me a hormone kit, and someone who is going to respect my time, not put me inside a room and tell his staff let them wait. besides he had a girl working there that was super nice and sweet to the patients very friendly and i called at the beginning of the year and i was told that she is no longer there, i am sure alot of patients were there cause of her, i have nothing against the other 2 girls they are also very nice, but thats about it. i will not recommend this dr to anyone i think he bought his title, just like he bought the diploma he has for his so called hormone replacement therapy. and everything else he does, do not let this man full you, or anyone in your family.
I became a patient of Dr Rotter
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Bioidentical hormone therapy
Body Composition Analysis
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Scar Revision / Scar Repair
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Weight Loss
Weight Loss Counseling
Weight Loss Injections
Weight Loss Program
Wellness Medicine
Wrinkle Treatments (BOTOX®, Restylane®, Juvedermtm)


text:
Lady About Town - Taking a cue from the past, Donna Karan's fall 2011 campaign follows Isabeli Fontana throughout an elegant New York day. Photographed by Inez van Lamsweerde and Vinoodh Matadin and styled by Nicoletta Santoro, Isabeli goes from day time office to a night out in Karan's sophisticated silvery hued designs. … [Read more...]
Raquel Zimmermann by Willy Vanderperre for Vogue China August 2011
Cyber Mod - Taking 60s' mod into a new decade, fashion editor Nicoletta Santoro pieces together a wardrobe of leather and black minis for Raquel Zimmermann in the August edition of Vogue China. Photographed by Willy Vanderperre, Raquel makes a stoic yet beautiful subject in the sleek designs of Lanvin, Prada, Marc Jacobs, Alexander Wang and many others. … [Read more...]
Donna Karan Resort 2012 Collection
Future Fantastic - Donna Karan's Resort 2012 collection looks towards the future with sculptured shapes and metal accents. Offering masculine tailoring with feminine frills, hoop skirts and the button-up t-shirt get reinvented for a sleekly chic uniform. Topped off with a color palette of red, white and black--the resort lookbook offers the perfect play between elegant and … [Read more...]
Natalia Vodianova for Vogue China June 2011 by Peter Lindbergh
Beijing Romance - Photographed by Peter Lindbergh, Natalia Vodianova stars in the cinematic June cover shoot from Vogue China. Alongside male model Zhao Lei, Natalia dons an elegant wardrobe featuring some of the season's most coveted looks from the likes of Balenciaga, Calvin Klein and Lanvin styled by Nicoletta Santoro. … [Read more...]
Karlie Kloss by Patrick Demarchelier for Vogue China May 2011
Graphic Play - Karlie Kloss' energetic personality takes center stage in the May issue of Vogue China. Donning bold graphic prints styled by fashion editor Nicoletta Santoro, Karlie poses for Patrick Demarchelier's lens in dynamic looks from labels such as Jil Sander, Moschino and Max Mara. … [Read more...]


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Rent Classic Cars
Do you feel like taking a trip down memory lane? Rent a classic car from Dockx Rental. All of our classic cars are in perfect condition. Would you like a stylish classic car for a wedding, anniversary, jubilee or just for a romantic trip? Every trip is a unique experience. Some models are only available with chauffeur.


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Civil Society Coalition: Support for the first European Games Baku-2015 has today held a round table meeting titled "Double standarts of the west: Democracy and human rights" in International Media Center.
SIA informs that MPs, representatives of NGOs and mass media representatives attended the meeting.
Chairman of the International Relations Research Center Rufiz Gonagov said that double standarts of the West increased on the eve of the First European Games Baku 2015: "It was expectable. Because this kind of "black campaigns" always led against Azerbaijan on the eve of such worthy and important events. It is a result of double standarts of the west. By joining together we will prevent "dirty campaign" led against our country".
"I suggest,the US and its authority has changed priority itself. Their proarmenian claims is an example of this fact. Actually, the United States has been losing its supporters. In consequence, it means that US is losing Azerbaijan. The US policy on democratic issues threatens the world." MP Zahid Oruj told in his speech.
At the meeting the head of Ses Media Group Behruz Guliyev, MPs Asim Mollazade, Fazil Mustafa, Musa Gasimli, President of APA Holding Vusala Mahirgizi and others stressed in their speeches that such kind of biased, grubby "campaign " against Azerbaijan on the eve of the First European Games is groundless, as well they condemned double standarts of the west against our country.


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Give What You're Good At: Go Pro Bono with Catchafire
Catchafire & NY Creative Interns
Wednesday, July 20, 2011 from 6:30 PM to 8:30 PM (EDT)
Event Details
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Wednesday, July 20, 2011 from 6:30 PM to 8:30 PM (EDT)
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The Hotel Malina is situated in the holiday resort Malinô Brdo in the Veľká Fatra mountain range at 960 m above the sea level above the centre of the Lower Liptov region – Ružomberok. The Hotel is located right in the Skipark Ružomberok ski resort – near the ski lifts with ...
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From day one of the occupation/liberation, everyone banged on about the importance of handing real power and eventually sovereignty to the Iraqis.
Every day in the pre-handover era, a mean-looking politician of one sort or another would come on TV and demand real power for the Iraqis (ie, his political group), especially in security matters. After all, "We Iraqis know better than the Americans how to deal with our fellow citizens and can do a better security job for our country," these characters would articulate.
Well, fair enough when it came to Abu Ghraib prison. After the way the US jailers conducted themselves there, it looked as if the Americans had caught the evil secret-police virus - medical name: the mukhabarat - which you can get from spending too much time under the sweltering sun of the Sunni triangle.
It has been two months since the occupation powers handed sovereignty to Iraqis and appointed a former Ba'athist as a prime minister. What we've had, from everyone from the president all the way down to police chiefs, are pledges that the Iraqi government will not tolerate anyone who is trying to destabilise the "democracy" in our country. And "will crush with hands of steel anyone who would use violence to oppose the state".
Sounds great. For all the Iraqis questioned in every single poll, security is the priority, and people have started to feel the difference. But what happens when you are squeezed between the need to establish security and democracy at the same time? The police/security agencies are feeling power coming back through their fingertips, and most of the people in these services were policemen in the good old days of the father of all democracies. It will take more than a three-week crash course in human rights to rehabilitate a Saddam-trained police force.
Three months ago - that is, before the handover - I and another journalist were sitting in the office of a senior police officer in charge of the anti-kidnapping department, when he got a phone call. Heard from my end, it went like this:
Officer: "You can't give me the suspect for 24 hours and expect me to get a confession."
Caller: "..."
Officer: "Ya habibi, put him in my custody for three days and I will show you the how electricity can work."
At the time this conversation took place, that officer's building was still guarded by three American APCs and Humvees; Italian forensic experts were going in and out of his office.
So what has happened since the handover on June 30? Nothing much, just a reinstallation of the 5,000-year-old tradition of human rights in this part of the world.
In Najaf police station, mid-August: cries of pain and thuds of people getting the shit beaten out of them were coming out of the hall where detainees from the anti-government Mahdi army were kept. A policeman carrying a thick electricity cable went running into the hall after being told that new suspects had just arrived. "I will smash your camera if you go close to that door," he shouted at me.
Ten days after that, with the fighting in Najaf still going on, the police figured it must be those journalists cramped in a particular hotel who were still fuelling the uprising by the Mahdi army under Moqtada al-Sadr. And when they realised that battering a cameraman every two days was not working, they went to plan B.
Plan B consisted of masked policemen storming the journalists' hotel, firing bullets into the ceiling, loading everyone into police and army pick-up trucks, threatening with pistols and shouting: "We will fuck your mothers." We were all delivered to the police chief, who calmly started telling us about the inhumane treatment the police are suffering from the militia. He and his men were trying to summon us for a press briefing.
Two days after plan B, the police started to open fire at any journalist moving around.
When everyone during the Najaf crisis was talking about the importance of ending the uprising of Moqtada and his group, and disarming the militias, uniformed militiamen with nicely trimmed beards and brand new SUVs carried on patrolling the streets - just that these guys, trained in a neighbouring country with a great record on Islamic human rights since late 70s, Iran - are affiliated with parties participating in the new Iraqi government.
But we have to admit things are different. At least detainees in police stations are just getting kicked and beaten and not asked to perform the famous Abu-Ghraib sex-pyramid dance.


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BEST WESTERN Hotel Erfurt-Apfelstaedt is located in the mid of Thuringia, near motorway A4 and A71 and close to the airport and trade fair (15km). Erfurt city centre can be reached within 20 km. The convenient location offers a great starting point for recreation and tours throughout Thuringia. ...
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• MPAYER is a mobile and web application that enables businesses to manage income and expense transactions of the business both Mobile Money(Mpesa, Airtel Money) and also cash. It also helps businesses keep track of customers, distinguish their different payments and generate reports on a real time basis through a mobile phone/ computer.
• Think of MPAYER as a tool that helps businesses to view their performance through a mobile phone or computer. This tool helps owners of business to manage and view activities of business including incomes and expenses, performance of various product/services and manages customer information.
• Our grand vision of MPAYER is that it will equip businesses with the correct tools that will make them grow in efficiency and grow their customers.
This venture has not initiated a round of financing on this platform. If they choose to, the summary of the fundraising round will appear to registered investors here.
Many businesses information on their income, expenses and customers is put to memory or written down. Mpayer enables businesses record their transactions, accept mobile payments, manage customers, generate reports, and back up information through their mobile phones.
We have a world class team and a pool of competent advisors and partners .Key to the team include: Kariuki Gathitu -founder and chief systems architect Stella Njoki- Co founder and Finance and Operations. Njoroge Kinyanjui-Mobile Developer We also have marketers.
The product has gone through iterations and we have a number of paying businesses that have been testing the software at its pilot phase. We are now ready to launch and fully scale the product and start enabling more businesses to grow.We hope to launch at Demo Africa.
Our revenue model is both subscription and commission based. We have different tiers for the subscription We charge a commission for the mobile payments transactions We charge for sms used through our system
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Kariuki GathituWorks at M-Payer
Stella NjokiWorks at M-Payer


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Brad Pitt in Gucci–Joining his partner Angelina Jolie to celebrate her latest movie Maleficent, actor Brad Pitt attended a special costume and props reception in London at Kensington Palace on May 8th. For the occasion, Pitt turned to Italian label Gucci for a snazzy head to toe look in black. Pitt completed his ensemble with sunglasses and a gold chain.
You are here: Home / Entertainment / Celebrity / Brad Pitt Wears Gucci to Maleficent Reception


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We had to go into town today to go to the store and while we were out we were shocked to see a big fire in the foothills to the north. Since we could tell that it was not close to where we live, we knew that we didn't have to rush home. By this evening the fire had gotten quite large and the winds are getting worse. I wonder what is going to happen to the people living there, we've been put through many close calls in the past and it scares you to death.
May 23, 2017


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Pooh Blanchard
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Pooh Blanchard is the victim that D'Angelo Barksdale is on trial for murdering in "The Target". Although D'Angelo is acquitted he later explains to his uncle, Avon Barksdale, that he felt forced to shoot Blanchard because Blanchard attacked him. Blanchard is an unseen character.


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Exploring Opera 9
Stable, fast, and complete: Opera 9 gives you everything you need for the Internet in a compact package.
Twelve years have passed since Norway's Opera Software ASA offered its first web browser to users, and every release since then has introduced revolutionary new features. Opera started life as a 1.3 MB competitor to the major players of the time, such as Internet Explorer and Netscape Navigator, but Opera has always offered many unique features. In fact, products by other vendors still lack many of Opera's special features, such as fast tiling of tabs and convenient arrow key-based scrolling. Although Opera is closed source, the browser runs on various operating systems, and Linux users have enjoyed the program for many years now.
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Red Hat Announces OpenShift.io
Red Hat takes its IDE online.
Microsoft Bakes Linux into Windows Server
Microsoft is slowly becoming a Linux vendor.
Fearless Coyote, Linux Kernel 4.11 is Out
The new kernel makes swap implementation more scalable, which will help cloud providers.
Docker Appoints Steve Singh CEO
The new CEO comes from a very strong SaaS background.
Docker Announces Linux Kit and Moby Project
Both projects help organizations build their own containerized systems.
Ubuntu Returns to Gnome as Its Mobile Plans Shatter
Mark Shuttleworth has resumed the position of CEO of Canonical.
Microsoft is Shutting Down CodePlex
Microsoft's open source code hosting platform CodePlex will come to an end after a more than 10-year stint.
Fedora 26 Alpha Released
Comes with Gnome 3.24
Old Linux Kernel Bug Discovered
The bug was introduced back in 2009 and has been lurking around all this time.
OpenSSH 7.5 Released
The new release deprecates the sshd_config UsePrivilegeSeparation option.


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This room can be further devided into Riverview A, Riverview B, Riverview C of came dimentions (648) and capacities ( 24'x27').
This room can be further devided into Seaside A, Seaside B, Seaside C of same dimention (648) and capacities (24'x27').


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Get in-depth analysis of current trends in the legal community, profiles of fascinating professionals, academic issues and lifestyle discussions for law school students, and a few out-of-the-ordinary goings on in the world delivered to your inbox.
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The Royal Brompton Hospital will argue proposals put forward by NHS bosses to end paediatric heart surgery are "flawed" and young patients will suffer.
It is the first time an NHS organisation has taken the health service to court.
On the first day of a judicial review of the plans, the hospital will argue they were made before a public consultation on them, that they fail to consider the knock-on effects, such as the closure of the children's intensive care unit and the cystic fibrosis unit, and that they are "irrational".
If the decision gets the go-ahead, it will leave only two specialist centres offering heart surgery for children in the capital, one at Great Ormond Street and the other at Guy's and St Thomas'. The High Court will also be told the decision-making committee included doctors from Great Ormond Street and Guy's and St Thomas' but that the Royal Brompton was not represented.
The plans were made in February as part of a "Safe and Sustainable" NHS review into child heart units across the country. A public consultation on the plans ended in July.
Teresa Moss, director of the national specialised commissioning team, said: "Safe and Sustainable do not accept the allegations made by the Royal Brompton. The public consultation shows there is widespread support for the Safe and Sustainable proposals.
"Independent experts have also challenged the allegations from the hospital...and found that the hospital's respiratory services would remain viable in the absence of a paediatric intensive care unit."
She added: "Many people will find it astonishing that taxpayers' money is being used so inappropriately at a time of financial austerity in the health service."
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- Healthcare
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Chapter FiveMature
The two teens whirled around to face the barn that was only a hundred yards away. It was shrouded in darkness from the tall oak trees hovering over it, with only the light of the moon lighting up the doorway. Cora's eyes darted all over the place, searching for the source of the frightened scream, looking for a sign of life besides her and Alex. In an instant, the boy had miraculously appeared by her side in seconds, his hand latching onto hers protectively. Everything about him was tense. There was something completely wrong, and he wasn't prepared to flee anytime soon.
Despite his apprehensive posture, Cora knew that this boy would run head-on into danger with no hesitation.
Maybe that's part of the story he's so careful to hide from me… Cora wondered while Alex evaluated the situation. She was pulled back to the present when another violent scream erupted from the hayloft of the rickety barn.
"Where did that come from?" Cora asked in a hushed, wavering voice. She crept closer to Alex.
"The barn, your parents went in there. I saw them."
Cora's blood went cold at his words. "Who else would be in there?"
"Luther's there. He's always there." Alex's voice went deep and husky, and his eyes were darting from Cora to the barn, back and forth.
Cora's mind instantly rewound back to earlier that night, when she had fought with her father about something that, now, seemed completely pointless. When Cora had gone exploring in the barn, she'd narrowly escaped Luther's clutches. Her parents might not be so lucky.
Not everyone can have a superhero.


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$42 for a body-type assessment and one personal-training session ($160 value)
Trainers focus on fat loss and muscle gain through high-impact workout incorporating kettlebells, strength training and metabolic exercise.
Led by a training staff with decades of experience, clients gather in groups or meet one-on-one to achieve personal fitness goals
From the merchant: Muscles are stretched and rejuvenated by Certified Personal Trainer and Active Isolated Stretch Practitioner Caroline
Trainers work one-on-one with clients, helping them with their personal weight-loss and fitness goals; sessions are twice a week
Personal trainers develop challenging workout plans designed to increase physical fitness and athletic performance
From the merchant: 45 minutes with your trainer in your own fully-equipped private training suite
Personal-training sessions are led by an expert in fitness, massage, and pain relief
An experienced trainer works with an individual during one-on-one sessions that help them reach their personal fitness goals
From the merchant: Five 45-minute training sessions with CT's best, and a 30-minute nutrition consultation with our expert nutritionist
From the merchant: In a nutshell I want to help you lose weight eat right and enjoy the person in the mirror. Come in today
From the merchant: Two or four one-hour personal training sessions at Sculpt Fitness
ACSM-certified personal trainer and owner Cliff Morton customizes workout routines to help clients reach their fitness goals
Personal trainers help clients meet their fitness and weight-loss goals with group training sessions and nutritional coaching
From the merchant: Get results with private training. Many choices such as cardio, strength, kickboxing, core, stretching & yoga
Certified personal trainers helps clients achieve fitness goals
From the merchant: Mobile in home and on site personal fitness training services for individuals, youth, groups,teams and businesses.
Two Personal Training Sessions for Corrective Exercise or Sports Performance
From the merchant: Personal Best 1 on 1 Fitness a 4,000 sq ft private training center in Branford helping you on your fitness journey
My own personal experience with a dissatisfied birth outcome and knowing there needs to be a better way than "do anything to lose the baby weight" led to the birth of Mind and Body Mama!....
Clients improve physiques and build stamina during one month of personal training sessions
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Local businesses like this one promote thriving, distinctive communities by offering a rich array of goods and services to locals like you
Four Weeks of 1:1 Personal Training Sessions (8 Sessions)
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From the merchant: A specialized in-home personal training service focused in the West Hartford and Farmington Valley area.
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Goriage Wool Blend Coat, Women's, Maroon
Sign up now to get your own personal style guide to what to wear and to see clothes recommendations from over 500 retailers.
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What the retailer says
Feminine, understated and bohemian are the three key words that define Parisian designer Judith Milgrom's luxury fashion label, Maje . Luxuriously crafted from a quality wool blend, the Goriage coat makes a sumptuous layering option for when the cold weather comes into play. Later it over everything from tailored separates to pared back denim. Maje wool blend coatExposed button fastening at frontStand collar, long sleeves, padded shoulders, one slip pocket at each side, contrasting trim at sides, fully lined70% wool, 20% nylon, 10% cashmere; lining 100% viscoseDry cleanTrue to sizeSize 8: Length 33"Model is 5ft 10" and wears a size 8 Color: Burgundy. Gender: Female. Age Group: Adult.. Goriage wool-blend coat, Women's, Size: 8, Maroon


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If I Had A Daughter…Would I Tell Her To Think Like A Man?
Recently, I had a conversation with a parent who asked about my sentiments towards the new Steve Harvey movie, "Think Like Man." She mentioned that she was raising a teenage daughter, and that she was confused about what values, morals, and behaviors she should convey to her daughter about self-respect, friendship, and romance.
She expressed that her daughter was a 17-year old senior who had recently been accepted into college and had a special friend whom she was dating. I thought for a moment, and then offered up some potential scenarios that might happen if she were to, per the book and the movie, teach her daughter to "think like a man," and to understand how masculinity is exercised in our culture: how being dominant, assertive, aggressive, independent, non-emotive, and maintain a sense of entitlement is, in many ways, encouraged in our society. I let her know that thinking like a man may enable her daughter to:
- Be less visible among her peers.
- Possibly make more money as an adult.


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Net Optics Receives Network Products Guide 2010 Product Innovation Award
Net Optics Director Xstream™ Selected a Winner in the Monitoring Access CategorySANTA CLARA, Calif. – March 23, 2010 – Net Optics, Inc., the global leader in intelligent network monitoring access solutions, has been honored by the Network Products Guide, the industry's leading information technology research and advisory guide, by naming Net Optics Director Xstream, a winner of the 2010 Product Innovation Awards. This annually venerated award recognizes and honors vendors, large and small, from all over the world with innovative and ground-breaking products that are bringing essential and incremental changes and are setting the bar high for others in all areas of information technology. Net Optics Director Xstream was selected the award winner for the Monitoring Access category.
Director Xstream enables the deployment of an intelligent, flexible and efficient monitoring access platform for 10G networks. Director Xstream enables a pool of 10G and 1G tools to be deployed across a large number of 10G network links, with remote, centralized control of exactly which traffic streams are directed to each tool. The Net Optics Xstream products enable organizations to see more traffic with fewer monitoring tools, relieve oversubscribed 10G monitoring tools, share tools and data access among groups without contention and centralize data monitoring in the network operations center. Unique to Director Xstream is TapFlow™ filtering technology, which enables organizations to select traffic of interest for each tool based on protocols, IP addresses, ports, and VLANs. Director Xstream enables customers to maintain the performance and security of applications and services while reducing Capex costs and improving operational efficiency. To read more about this product innovation, please visit http://www.networkproductsguide.com/innovations/2010/Net-Optics.html.
"Innovation is not just about new products alone," says Rake Narang, editor-in-chief at Network Products Guide. "To succeed companies need to see innovation not as a one-time errand but as something that has to be continuously evolved and improved upon over and over again. This requires putting greater focus on customer needs and making innovation the center of company's way of developing better solutions. Innovative products such as Net Optics Director Xstream are bringing improvements in intelligent network monitoring access by creating solutions that allow for 100 percent visibility."
"Network Products Guide's recognition of Net Optics Director Xstream further validates our products' excellence-in-class and ahead of the curve technology and innovations. High-performance 10G networks are being deployed more broadly, and businesses need monitoring and access solutions that can perform at 10G speeds," said Bob Shaw, president and CEO of Net Optics. "Product innovation is key to our continued commitment to meeting our customer needs and providing them with better solutions, such as Director Xstream."
About Network Products Guide Awards
As industry's leading technology research and advisory guide, Network Products Guide from Silicon Valley plays a vital role in keeping decision makers and end-users informed of the choices they can make in all areas of information technology. You will discover a wealth of information and tools in this guide including the best products and services, roadmaps, industry directions, technology advancements and independent product evaluations that facilitate in making the most pertinent technology decisions impacting business and personal goals. The guide follows conscientious research methodologies developed and enhanced by industry experts. To learn more, visit www.networkproductsguide.com.


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We need to focus on justice for refugees, not ignore it
By Kerry Fressard
In the wake of two days of non-violent peaceful protest at Parliament, some sensationalist and hyperbolic media outlets have failed to address the issue behind the protests. That is, the bipartisan support for human rights abuses, sexual and physical assault, and trauma occurring in Australia's offshore detention centres.
In order to refocus the discourse surrounding the protest it is important to focus on the issue at hand. Offshore detention is (intentionally) cruel, expensive, and does not effectively reduce suffering and loss of life.
Offshore detention is misguided as a deterrent. The argument put forward by the government is that detaining asylum seekers who attempt to come to Australia via boat on Manus Island, Christmas Island and Nauru saves lives and prevents suffering. As the government refuses to provide the public who elects them and pays their salary with information surrounding 'on water' matters we cannot say, or know, if it has been an effective deterrent. Furthermore, we certainly cannot make a strong case that it has in fact saved lives and reduced the suffering of asylum seekers. However, we can categorically confirm the abuse and torture of refugees and asylum seekers illegally held on offshore detention centres. Refugees have died in offshore detention, women have been sexually assaulted and raped, children have faced physical and emotional abuse, and a large proportion of those held in offshore detention suffer from emotional and psychological trauma as a result of their detainment. The suffering in offshore detention centres has been highlighted in the media, through Senate inquiries, in reports by NGOs and the United Nations Human Rights Commission, in accounts provided by doctors and teachers that have worked in the centres, and by the detainees themselves.
There are alternatives to the current policy of offshore detention. Community based processing is less harmful to asylum seekers and would cost a fraction of the $4-5 billion spent every year on offshore detention. Regional solutions such as processing refugees and asylum seekers before they attempt the journey by boat to Australia would most certainly act as a deterrent to risky boat journeys. Key to all alternatives is that they are timely, that asylum seekers are processed quickly so they can begin building their futures and moving on with their lives. It is important to ask why it is that these policies have not been implemented. Is it that the government and opposition are actually aiming to deter people fleeing war zones and persecution from seeking asylum in Australia at all? If so, I hope people reflect on the cruelty of such a move by politicians who consistently purport to be fighting to give people a fair go.
Significantly, the vast majority of the people being illegally detained offshore have been found to be refugees. These people deserve empathy and compassion and should be evacuated from the offshore detention centres immediately.
Media that does not address these issues and instead focuses on protester conduct is failing to report on the serious maltreatment and abuse of asylum seekers and refugees at the hands of Australian government policy. Surely, Australia's inhumane treatment of people fleeing from persecution is more important than the appearance of protesters or exposes on the number of protests that the members of the group have previously participated in. Because of the wilful ignorance and failure to focus on crimes committed in offshore detention, such outlets are in some part complicit in the abuse of asylum seekers and refugees.
A key role of the fourth estate is to investigate and report on the actions of the political elite that act in our names. The main story to come out of the two days of protest at Parliament House is not whether we restrict public access to the building, nor it is the personal lives of the human rights advocates involved, but rather that there remains to be bipartisan support for the abuse and torture of refugees held illegally on Manus Island, Christmas Island and Nauru. The camps remain open, people's lives hang in the balance, and their futures remain uncertain. Forget the spin, sensationalism and hyperbole, and let us focus on providing justice and freedom for refugees and asylum seekers.
Also by the author:
People ask us why we protested in parliament for refugees. We ask, why didn't you? (The Guardian)
We Are Witnessing The Unravelling Of The Cruel Offshore Detention System (The Huffington Post)
Help Support The AIMN
Please consider making a donation to support The AIMN and independent journalism.


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You're asking for trouble if you consider FMLA leave-related absences a negative factor when making employment decisions.
Courts view such decisions as direct evidence of retaliation—which makes it almost impossible for the employer to win a lawsuit. The employer would have to show that it would have made the same decision even if it hadn't wrongly used the FMLA leave as a factor.
Recent case: Eunice Hunter worked for the Valley View School District as a night custodian. She was involved in a serious car accident and had to have a series of operations. To minimize disruption to the school where she worked, Hunter made it a point to take intermittent FMLA leave during summers when kids were out of school.
After her final operation, her doctor cleared her to return to work, but with lifting restrictions.
That's when the school district decided to place her on involuntary medical leave. The superintendent wrote t...(register to read more)
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The last of Quinn's 2,163 domestic winners came on one of the biggest stages, when he rode Young Mick to a head victory in the Duke of Edinburgh Stakes at Royal Ascot 12 days ago. And though the abrupt ending of his 27-year riding career was a surprise to most, it was not to the man himself.
"I'd made up my mind a while back that I would finish this year, after the Royal meeting," said the 44-year-old Scot yesterday. "I wanted to ride on the new track, and it was a happy turn of fate that my last ride of the week was a winner. Win, lose or draw, it was always going to be my last ride of all, and it was a dream end to what has been a great part of my life.
"But it's just that it's time to move on to other things, a natural progression, and it's a decision I'm very comfortable with. I thoroughly enjoyed a day out watching some great tennis like an ordinary human being instead of scrambling off to Folkestone or wherever."
Stirling-born Quinn started with Herbert Jones in Malton in 1979 before moving south to join Paul Cole, who provided him with his first winner, Bolivar Baby at Kempton in October 1981, and guided him to the British apprentice championship three years later.
Cue another sporting comparison, this time with Colin Montgomerie. A senior title was never Quinn's and he could be the best man never to win one. He finished second in the table thrice, to Pat Eddery in 1996, to Kieren Fallon in 1999, the year when he notched his best-ever total of 151 winners, and to Kevin Darley in 2000. His 22 consecutive top 20 finishes on the table included 13 top ten spots and six in the top five.
Cole supplied the first of Quinn's three English Classic winners, Snurge in the 1990 St Leger, and also trained the best horse he ever rode, Generous. Ironically, Fahd Salman's brilliant chestnut may have been the catalyst that prevented his rider's transformation from star to superstar. Quinn lost the ride after finishing fourth in the 1991 2,000 Guineas and had to watch as his replacement, Alan Munro, went on to glory in the Derby, Irish Derby and King George VI and Queen Elizabeth Diamond Stakes.
Despite being rebuffed by the late Salman, Cole's principal patron, Quinn continued his association with the yard until he went freelance in 1999. A year later he became Henry Cecil's stable jockey, finished third in the Derby on Beat Hollow and took the Oaks on Love Divine.
Unfortunately, his move to Warren Place coincided with Cecil's downturn in fortune; his victory on Beat Hollow in the Grand Prix de Paris that year was also Cecil's most recent at Group One level.
"Right place, wrong time," said Quinn. "We had a good first year, then the horses were sick, the old-school owner-breeders were dying. The job could have kept me rolling but the firepower wasn't there. You've got to have a challenge and I seem to have exhausted my opportunities."
Circumstances may have conspired against Quinn at times, but he rejects any notion that he has retired as a nearly man. "Certainly not," he said. "I have ridden some wonderful horses and racing has taken me to 22 different countries and I've ridden winners in 20 of them. It's been a marvellous career, not a job, a way of life, and a great life. Basically I've earned a living doing my hobby."
Quinn's other top-level wins include an Irish Oaks on Knight's Baroness, Irish St Legers on Ibn Bey and Strategic Choice, a French 1,000 Guineas on Culture Vulture and a second St Leger, in 2000, on Millenary.
It was two rides last year on the last-named John Dunlop-trained veteran that were a reminder of Quinn's arguably under-appreciated qualities. In the Lonsdale Cup, and then the Doncaster Cup, he humoured the eight-year-old to victory with ice-cool, kid-glove seductions as he held up the sometimes moody old warhorse until the last possible stride.
Quinn has not yet formulated a second career, but it will definitely not be training. "I'll take some time off, then take a view," he said, "but I'd like it to involve horses and racing in some form."
Chris McGrath
Nap: Sir Arthur
(Epsom 8.55)
NB: Knot In Wood
(Haydock 3.20)
Yarmouth today
Hyperion's Selections
2.30 Majounes Song; 3.00 House Arrest; 3.30 Dr Synn; 4.00 Inch Lodge; 4.30 Soulard; 5.00 Granary Girl
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ED Magedson – Founder
Ripoff Report
simple fillingsInternet USA
simple fillings i order a ssn card lost to replace it for my son . waited to long and found out its a rip off . that is not nice . i lost $48 all i had in my pocket . i would off buy shoes for my son insted . i want my money back .. texas Internet
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I was charged $48 dollars . To get my son his new ssn card number that was lost . Then waited now fof more of the time they said i was going to get it 7 to 8 business day . It's been three weeks now . Called the company no answer . I could off use that money to pay my sons lunch for school .but he needs his ssn. Lilia ramos ,firstname.lastname@example.com
This report was posted on Ripoff Report on 08/11/2014 04:06 AM and is a permanent record located here: http://www.ripoffreport.com/reports/simple-fillings/internet/simple-fillings-i-order-a-ssn-card-lost-to-replace-it-for-my-son-waited-to-long-and-fo-1168908. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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Condition: Standard
All Product Details
Otherland the private, multidimensional universe created and controlled by The Grail Brotherhood, an organization made up of the world's most powerful and ruthless individuals was crumbling. The Brotherhood's plans for immortality within the network had been shattered by the monstrous intelligence that ran the network, a presence known only as the Other, and by an even more monstrous human being who called himself John Dread. Seizing control of the network from his employer, Felix Jongleur, Dread had now made himself the god of this virtual universe and was systematically turning the network's worlds into killing grounds.
As helplessly trapped as Renie Sulaweyo, !Xabbu, Sam Fredericks, Martine, Paul Jonas, and the rest of the small band who had entered Otherland in an attempt to save the many children held captive within this virtual reality, Jongleur was now forced to make common cause with his enemies. Yet even as they struggled through the maze of invented worlds, striving to reach the true heart of Otherland, time was growing shorter.
Caught in the surreal and deadly landscapes of the failing network, the desperate band battled against increasing odds to save the children, solve the bizarre mysteries of Otherland, and escape back to reality.
But before long there might not be a "real" world to return to for any of them. For destroying Jongleur's playground was merely a dress rehearsal for Johnny Dread. Dread's ultimate plan was to gain control of Jongleur's massive corporate holdings and spread apocalyptic destruction throughout the entire Earth....
Few science fiction sagas have achieved the level of critical acclaim-and best-selling popularity-as Tad Williams's Otherland novels. A brilliant blend of science fiction, fantasy, and technothriller, it is a rich, multilayered epic of future possibilities.


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Nemo Asashi 4 Vestibule Footprint - $29.95
Nemo Asashi 4 Vestibule Footprint DECENT FEATURES of the Nemo Asashi 4 Vestibule Footprint 70D abrasion-resistant nylon fabric tarp Sized to fit underneath a Nemo Asashi Vestibule Fabric is PU-coated for waterproofness Footprint converts the vestibule into extra sleeping space or a protected gear storage area The SPECS Area: 17 square feet Fabric: 70D abrasion-resistant nylon Fits Nemo Asashi vestibule Includes mesh bag Nemo products cannot be shipped to Japan. Please don't hate us. - $29.95