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The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect–infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.
Author Summary
Leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a poverty-related disease threatening 350 million people throughout the world. Drugs currently available to treat this disease are toxic to the patient and drug-resistant parasites are emerging. New therapeutics are therefore needed. Fortunately, interest in confronting the treatment challenges has grown and new technology has led to an increase in high-throughput screens conducted against Leishmania. In order to gain insight into the most efficient screening strategy, we compared two methods, one targeting the easily cultured insect-infective promastigote stage of the parasite, and the other, targeting the clinically relevant but more difficult to culture intracellular amastigote stage. We show that while a screen against promastigotes is amenable to automation, it fails to recognize all active compounds. These compounds revealed only by an intracellular assay might act on host cell pathways important for parasite development. Targeting such pathways is an emerging strategy in drug discovery against infectious diseases.
Citation: De Muylder G, Ang KKH, Chen S, Arkin MR, Engel JC, McKerrow JH (2011) A Screen against Leishmania Intracellular Amastigotes: Comparison to a Promastigote Screen and Identification of a Host Cell-Specific Hit. PLoS Negl Trop Dis 5(7): e1253. https://doi.org/10.1371/journal.pntd.0001253
Editor: Ana Rodriguez, New York University School of Medicine, United States of America
Received: March 14, 2011; Accepted: June 10, 2011; Published: July 19, 2011
Copyright: © 2011 De Muylder 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 Sandler Foundation, NIH grant AI090605, and the Belgian American Education Foundation (BAEF). 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.
Leishmaniasis is caused by protozoan parasites of the genus Leishmania. The disease is endemic in the tropics, subtropics and the Mediterranean basin. There are three main clinical syndromes caused by different species of Leishmania. Cutaneous and muccocutaneous leishmaniasis result in large, painful sores that can take many months to heal [1]. Visceral leishmaniasis results in fever, weight loss, and damage to internal organs such as the spleen and the liver and may be fatal if left untreated [2].
Leishmania parasites are transmitted to mammalian hosts through the bite of phlebotomine sandflies. The parasites that develop in the mid-gut of the flies, called promastigotes, are flagellated and extracellular. Upon injection in the bloodstream of a mammalian host, promastigotes are rapidly phagocytosed by macrophages where they differentiate into the amastigote form. Amastigotes multiply in the macrophage parasitophorous vacuole, leading to destruction of the host cell and release of free amastigotes into the bloodstream, where they are capable of infecting new phagocytic cells [3].
Current treatment for leishmaniasis relies on chemotherapy, as no efficient vaccine is available. Sodium stibogluconate and amphotericin B have been the first line treatment; however, they have significant side effects and unresponsiveness to sodium stibogluconate has been reported for many years [4]–[6]. A few new anti-leishmanial drugs have been recently released (miltefosine, paromomycin), but they also have drawbacks including cost and toxicity [7]. In addition, it has been shown in vitro that in some cases resistance can be easily induced [8].
New therapeutics are therefore urgently needed. Recognition of this need in recent years has led to partnerships between a number of foundations, agencies and universities to support the discovery of anti-parasitic agents, including anti-leishmanials. Lead discovery, one of the bottlenecks in the pipeline for novel anti-leishmanial drugs, would be facilitated by improved high-throughput technology allowing for the ability to screen large number of candidates [9], [10]. Several anti-leishmanial high-throughput screens have been reported [11]–[13]. Primary screens often target the parasite promastigote stage because of ease of culture and manipulation. Indeed, promastigotes from several Leishmania species are easily maintained as cell suspension in vitro. However, as the promastigote is the form of the parasite in the insect vector, it is not the appropriate target for an anti-leishmanial drug [14]. Culture conditions for axenic amastigotes have been developed in order to facilitate the study of this stage of the parasite [15], [16]. This has allowed amastigotes to be screened in a high-throughput manner [17]. However, expression arrays comparing L. infantum axenic amastigotes and amastigotes isolated from macrophages have shown differences in several cellular processes, including metabolism, intracellular transport and response to oxidative stress [18]. These observations highlight the importance of the host macrophage in driving the parasite to specific adaptations. The axenic amastigote model therefore has limitations as it does not encompass many aspects of intracellular parasite development [19]. Compounds active against axenic forms might be unable to reach the intracellular amastigote because of their inability to cross host cell membranes or maintain stability under low pH. Other compounds may need to be metabolized by the macrophage to gain activity. Finally, the macrophage itself might be directly targeted, thereby leading to parasite growth inhibition [20].
We have developed a host cell-based screening assay using a human macrophage cell line infected with L. donovani, one of the agents of visceral leishmaniasis. This assay format enables screening of compounds directly against the intracellular stage of the parasite. This assay was used to screen a library of 909 bioactive compounds consisting largely of FDA approved small molecules. In order to compare the efficiency of this screening method with traditional high-throughput screening assays, the same compound library was screened against free living promastigotes. A compound leading to sixty percent parasite growth inhibition at 10 µM was considered a hit in both assays. 59 hits were identified in the promastigote assay of which only 26 were also considered hits in the intracellular amastigote assay. Only one compound was specifically active against the intracellular amastigote stage. We conclude that the promastigote assay fails to identify all active compounds and leads to a rate of 56% false positives.
Materials and Methods
Cell culture
THP-1 cells (human acute monocytic leukemia cell line – ATCC TIB202) were grown in RPMI supplemented with 10% Foetal Bovine Serum (FBS) and 50 µM 2-Mercaptoethanol at 37°C in 5% CO2. L. donovani promastigotes [strain 1S, clone 2D (MHOM/SD/62/1S-cl2D)] were grown at 27°C in RPMI supplemented with 10% FBS and 10% Brain Heart Tryptose medium (BHT) [21]. Differentiation of promastigotes into axenic amastigotes was achieved by dilution of 5×105 promastigotes in 3 ml of low-pH axenic amastigote media (15 mM KCl; 136 mM KH2PO4; 10 mM K2HPO4·3H2O; 0.5 mM MgSO4·7H2O; 24 mM NaHCO3; 22 mM glucose; 1 mM glutamine, 1× RPMI 1640 vitamin mix, 10 µM folic acid, 100 µM adenosine, 1× RPMI amino acid mix, 5 µg/ml hemin, 50 U/ml of penicillin, 50 µg/ml of streptomycin, 25 mM MES and 20% FBS. The pH was adjusted to pH 5.66 at 22°C, yielding a final pH of 5.5 at 37°C) [22]. Axenic amastigotes were grown in ventilated flasks at 37°C in 5% CO2.
A library of 909 bioactive compounds was donated by Iconix Biosciences. These compounds were dissolved in DMSO at a stock concentration of 1 mM. Amphotericin B (Sigma) was used as a positive control.
Promastigote high-throughput assay
L. donovani promastigotes from an exponentially growing culture were diluted to 106/ml in RPMI containing 10% FBS and 10% BHT. The diluted culture (99 µl/well) was dispensed in sterile 96-well flat white opaque assay plates (Greiner Bio-One) using a WellMate multichannel dispenser (Matrix). 1 µl of 1 mM test compound dissolved in DMSO was added to the plates for a final concentration of 10 µM compound and 1% DMSO. Amphotericin B was added as a positive control (final concentration 2 µM, 1% DMSO) and as a negative control, 1 µl DMSO was added (1% final concentration). Compounds and controls were added to the assay plate with the robotic dispenser Biomek FXp liquid handler (Beckman Coulter). Promastigotes were incubated with the compounds for 72 h at 27°C. The parasites were then lysed by adding 50 µl of CellTiter-Glo (Promega) and placed on an orbital shaker for 5 min at room temperature. After lysis, the resulting ATP-bioluminescence was measured using the Analyst HT plate reader (Molecular Devices). Percentage inhibition of parasite growth was calculated for each well as [1-(RLUx-RLU+)/(RLU--RLU+)]*100 where RLUx, RLU+ and RLU- are respectively the Relative Light Units for each well, positive (amphotericin B) and negative (DMSO) controls. A screening window coefficient, denoted Z' factor, was used to evaluate the performance of the assay. The Z' factor, calculated as 1-(3σc++3σc−)/(µc+−µc−) where σc+, σc−, µc+ and µc− are respectively the standard deviation and mean values of positive and negative controls, is reflective of the assay signal dynamic range and the data variation associated with signal measurement [23]. For GI50 determinations (half maximal inhibitory concentration), compounds were serially diluted 3-fold in DMSO, with final assay concentrations ranging from 50 µM to 0.02 µM (1% final concentration of DMSO). GI50 curve fitting was carried out using GraphPad Prism 4 Software (GraphPad Software Inc., San Diego, CA).
Intracellular amastigote high-throughput assay
Sterile, black, 96-well, clear bottom plates (Greiner Bio-One) were seeded with exponentially growing THP-1 (5×105cells/ml). THP-1 were treated with 0.1 µM phorbol myristate acetate (PMA, Sigma) at 37°C for 48 h to achieve differentiation into adherent, non-dividing macrophages. Maturation of THP-1 cells towards monocyte-macrophage like cells is essential to avoid parasitized cells being overgrown by replicating cells. After activation by PMA, cells were washed and incubated with complete RPMI medium containing stationary phase L. donovani promastigotes at a macrophage/promastigote ratio of 1/15. After 4 h incubation at 37°C, non-internalized promastigotes were removed by 2–3 successive washes with RPMI containing 5% FBS and 5% horse serum. Test compounds (10 µM), positive control (2 µM amphotericin B) or negative control (1% DMSO) were then added to the cultures using a Biomek FXp liquid handler (Beckman Coulter). Cultures were incubated at 37°C for 72 h. Cells were then washed with phosphate-buffered saline (PBS), fixed for 30 minutes with 4% formaldehyde, rinsed again with PBS, stained for 2 h with 4′,6′-diamidino-2-phenylindole (DAPI 300 nM) and finally washed with PBS. For GI50 determination, compounds were serially diluted 3-fold in DMSO, with final assay concentrations ranging from 50 µM to 0.02 µM (1% final concentration of DMSO). Images were acquired with an INCell Analyzer 1000 automated epi-fluorescent microscope (G.E. Healthcare). The excitation and emission filters used to detect DAPI were 350/50 nm and 460/40 nm respectively. Eight image fields were acquired per well with a 20X objective. The proprietary INCell Developer Toolbox 1.7 software was used for image analysis. Segmentation parameters were set to identify host nuclei with a minimum area of 250 µm2 and parasite kinetoplast with an average area of 1 µm2. The intensity of parasite nucleus was too low to be detected with a 20X objective. A border, representing the boundary of the cell, was drawn around the nucleus (total area between 700 and 2000 µm2). Only parasites found within this area were included in the calculation to eliminate extracellular parasites. False positive parasite detection in the nucleus was also excluded from the calculation. Host cell nuclei and parasite kinetoplasts were counted and the ratio of parasites DNA to host nuclei was selected as the measurement output. Percentage inhibition of parasite growth was calculated as [1-(P/hcx-P/hc+)/(P/hc−-P/hc+)]*100 where P/hcx, P/hc+ and P/hc− are parasite per host cell ratio for every well, positive control (amphotericin B) and negative control (DMSO) respectively. Calculation of Z' factor and GI50 curve fitting were carried out as described above.
Dose response study against axenic amastigotes
L. donovani axenic amastigotes (5×105 cells/ml in axenic amastigote media) were dispensed in sterile 96-well flat white opaque assay plates (Greiner Bio-One) using a WellMate multichannel dispenser (Matrix). Compounds were serially diluted 3-fold in DMSO, with final assay concentrations ranging from 50 µM to 0.02 µM (1% final concentration of DMSO). 1% DMSO and amphotericin B (2 µM, 1% DMSO final concentration) were added as negative and positive controls respectively. Axenic amastigotes were incubated with the compounds for 72 h at 37°C with 5% CO2. Parasite viability was then measured using CellTiter-Glo as described above. Calculation of Z' factor, percentage of parasite growth inhibition and GI50 curve fitting were carried out as described above.
Development of an image-based high-throughput assay for drug screening against intracellular L. donovani
We developed a 96-well plate, cell-based assay simple to manipulate and reproducible, enabling screening of a high number of compounds against intra-macrophage L. donovani.
The human leukemia monocyte cell line THP-1 has been commonly used as a model for Leishmania infection and has been described as a suitable model for drug screening [24], [25]. In vitro infection of macrophages by Leishmania and analysis of intracellular parasite growth requires a method allowing for robust detection, discrimination and counting of parasites and host cells. In our setting, THP-1 cells infected with L. donovani were stained with the DNA marker DAPI (4′,6′-diamidino-2-phenylindole) allowing the visualization of host cell nuclei and parasite kinetoplasts. Images collected with an INCell Analyzer 1000 fluorescent microscope showed a significant size difference between host cell nuclei and parasite kinetoplasts. This feature was exploited for image segmentation and determination of the number of host cells and parasites (Figure 1A–D). The ratio between total number of parasites and total number of host cells was calculated for each well. In addition, counts of host cell nuclei were used as a quantitative measure of cell toxicity induced by the compounds. Incubation of L. donovani with THP-1 for 4 hours at a ratio of 15 parasites per host cell led to an average infection of 4.1 +/− 0.32 parasites per host cell after 72 h incubation, with an average of 30 +/− 9 percent of the cells infected and no change in the number of host cells (Figure 1E). Growth of parasite and host cells was not affected by 1% DMSO (Figure 2A–B). Amphotericin B, the first line drug used against leishmaniasis, was used as a positive control. At 2 µM amphotericin B did not affect THP-1 host macrophages (Figure 2A) but significantly inhibited growth of intracellular L. donovani (Figure 2B) with an estimated GI50 of 0.08 µM (Figure 2C). This is comparable to GI50 values from previous reports [11], [20].
A–D. Detection and segmentation of THP-1 host cell and L. donovani intracellular amastigotes. Images obtained with the INCell Analyzer 1000 (20X) of THP-1 cells infected with L. donovani and treated with 1% DMSO (A, B) or 2 µM amphotericin B (C, D). Insert shows the relative fluorescence of DAPI-stained parasite kinetoplast (k) and nucleic DNA (n) and host cell nucleus (N). Segmentation of host cell nuclei and parasite kinetoplast using INCell developer toolbox software (B, D). Red outline: parasite kinetoplast, blue outlines: host cell nucleus and border representing the boundary of the host cell. E. Evolution of the number of parasites and THP-1 host cells in a 72 h time course. THP-1 and L. donovani were counted at several time points after infection using the INCell 1000. White squares: average number of host nuclei per well (n = 8); black circles: average number of parasites counted per well divided by the total number of host nuclei per well (n = 8).
A. Number of infected THP-1 counted per well treated or not with 1% DMSO or 2 µM amphotericin B. B. Number of parasites counted per well divided by the number of host nuclei per field. C. Dose response curve for amphotericin B plotting the percentage of parasite growth inhibition. Values are mean from at least 3 independent experiments.
Screening of a bioactive small molecule library against L. donovani intracellular amastigotes and free living promastigotes
The intracellular amastigote imaging assay described above was used to screen a library of 909 bioactive small molecules (Iconix library). In the primary screen, compounds were assayed in duplicates at 10 µM. The average Z' value calculated per plate based on the positive and negative controls was 0.63, indicating a satisfactory robustness of the assay. Sixty percent parasite growth inhibition in at least one of the replicates was the cut-off arbitrarily determined for hit selection. This low threshold was purposely selected to evaluate sensitivity of the assay and guarantee identification of all active compounds. In addition, compounds toxic to the host cell, determined as inducing more than 20% reduction in THP-1 numbers, were excluded. A total of 27 compounds met these criteria and were selected for further analysis. This list of active chemicals included previously identified anti-leishmanials such as amphotericin B, pentamidine isothionate and tamoxifen citrate, thus validating the ability of the screen to identify molecules active against Leishmania.
The Iconix library was screened in parallel against L. donovani free-living promastigotes. Promastigote viability was determined after 72 hours incubation with the compounds, using an ATP-bioluminescence assay previously described for high-throughput screening against Trypanosoma brucei [26]. This assay measures luminescence produced by luciferase in presence of cellular ATP; the intensity of light is proportional to the amount of ATP released and correlates with the number of viable parasites (data not shown) [27]. Amphotericin B at 2 µM was used as a positive control and 1% DMSO as a negative control. In the primary screen, compounds were assayed at 10 µM. The assay was robust with an average Z' value of 0.72. Consistent with the image-based assay targeting intracellular L. donovani, 60% parasite growth inhibition was the cut-off used for active compound selection. Fifty-nine compounds were selected as hits for further validation.
The comparison of the results obtained for these screens showed that out of the 27 hits identified in the amastigote screen, 26 were also present in the promastigote screen. Only one compound, naloxonazine, showed complete specificity for the intracellular amastigote stage. Out of the 59 compounds identified in the promastigote screen, 19 were considered toxic to the THP-1 macrophage (Figure 3 and Table 1).
White bar: number of compounds identified in both screens. Light grey and black bars: number of compounds specifically active against promastigotes and intracellular amastigotes respectively. Hatched bar: number of compounds active against the promastigote stage but determined as toxic against THP-1 host cell in the intracellular amastigote screen.
GI50 values for these 60 hits (59 identified in the promastigote screen and one intracellular amastigote-specific hit) were then established for both stages of the parasite. 15 compounds (25% of the hits) were equipotent against both stages of the parasite. 14 compounds (23%) were more potent against the intracellular amastigotes while 13 compounds (22%) were more active against the promastigotes. The remaining compounds were toxic to the host cell (Table 1).
As axenic amastigotes have been considered to mimic the intracellular stage of the parasite, we analyzed their sensitivity to the 60 hits described above. This study indicated that compound activity against axenic amastigotes mostly correlated with promastigotes. The specific activity of naloxonazine against intracellular amastigotes was confirmed as this compound showed no activity against promastigotes or axenic amastigotes (Table 1).
Differential activity of naloxone and naloxonazine, two µ-opioid receptor antagonists, against L. donovani
The Iconix collection contained two opioid receptor antagonists, naloxone and naloxonazine. The first was not selected as a hit in any of the screens described above while the latter showed specific activity against the intracellular amastigote stage. To confirm these primary observations, the activity of both compounds was tested against promastigotes, intracellular and axenic amastigotes. Naloxonazine exhibited specific activity against intracellular amastigotes (GI50 intracellular amastigote: 3.45 µM; GI50 THP-1: 33.8 µM; GI50 promastigote: >50 µM; GI50 axenic amastigote: >50 µM), while naloxone was inactive against all parasite forms and not toxic to the host macrophage (Figure 4). At a curative concentration, the selectivity window of naloxonazine was reduced (GI90 intracellular amastigote: 12.5 µM; GI90 THP-1: 50 µM), limiting the possibility of using naloxonazine for treatment.
Lower panels: Dose response curve for naloxonazine (left) and naloxone (right) against intracellular amastigotes (black diamonds), promastigotes (black squares), axenic amastigotes (white diamonds) and THP-1 (white triangles) plotting the percentage of parasite growth inhibition.
Current chemotherapy for Leishmaniasis has several drawbacks, including cost, toxicities, route of administration, and the emergence of drug resistance. The pipeline for anti-leishmanial drugs therefore needs to be filled with new compounds. As the discovery of new and original leads suitable for optimization and drug development is dependent on the ability to screen many compounds, assays should be rapid, inexpensive and reproducible [14]. In addition, for pathogens displaying several life stages like Leishmania, there is a need to determine the best parasite stage to target. In the case of Leishmania there are three major options: first, targeting the extracellular living promastigote stage, second the axenic amastigote and third the intracellular amastigote stage. The first and second options meet the reproducibility, rapidity and low cost requirements for high-throughput screens, due to the ease in manipulating promastigotes or axenic amastigotes in vitro. This has been demonstrated by Siqueira-Neto et al. and Sharlow et al. who described screening 26,500 and 200,000 compounds against the Leishmania promastigote stage, respectively [11], [12]. These assays monitored parasite viability by measuring products from metabolically active cells (The Alamar Blue assay involves reduction of resazurin into fluorescent resorufin by live cells, while CellTiter-Glo luciferase catalyzes the production of luminescence in the presence of cellular ATP). The axenic amastigotes offer the ability to screen easily the relevant-like stage of the parasite and allow testing the potency of compounds under low pH conditions. However, the major disadvantage of these two approaches is the absence of the host cell in the assays: the natural niche of the parasite is not taken into account and aspects of parasite biology such as host-parasite interactions or accessibility of the target are ignored.
The intracellular amastigote stage has been logically designated as the more relevant target for primary screening against Leishmania, but previous methods were labor intensive and would not support automation [19], [28]. Methods traditionally used to detect and estimate the number of intracellular Leishmania include Giemsa staining or the use of reporter gene-expressing parasites (green fluorescent protein, luciferase or beta-galactosidase) [29], [30]. Giemsa staining is cumbersome as it needs manual counting. Reporter gene-expressing parasites are a powerful alternative, but stable recombinant parasite populations are required and they do not allow concomitant analysis of the host cell.
Here we describe a high content assay that allows the simultaneous visualization of both host cell nuclei and parasite kinetoplasts by using the DNA marker DAPI. The significant difference in the size of these two organelles facilitates discrimination between host cells and parasites, and thus accurate counting of both entities. Reduction in the number of kinetoplasts gives a measure of inhibition of parasite growth, while a reduction in the number of host cell nuclei is indicative of compound cytotoxicity. Thus, this image-based assay allows the identification of leishmaniocidal as well as leishmaniostatic compounds. All steps of this assay are amenable to automation and could be reduced to 384-well format resulting in a robust high-throughput screening methodology.
To evaluate what differences might be obtained from screening against the extracellular versus the intracellular parasite stages, we screened the same set of 909 compounds against both L. donovani promastigotes and intracellular amastigotes. We observed that the majority of the hits identified with the intracellular amastigote screen, defined as inducing 60% parasite growth inhibition at 10 µM, were also found in the promastigote screen. One compound showed specific inhibition of intracellular amastigote and was completely inactive against promastigotes. Fifty-six percent of the hits from the promastigote screen were not found in the intracellular amastigote screen. These results indicated that a promastigote screen failed to identify all active compounds and led to 56% of compounds being likely false positives. Thus, while the promastigote stage appears suitable for high-throughput screening, a fraction of the hits would be missed; furthermore, a high rate of false positives is characteristic of primary screens against promastigotes, underlying the importance of evaluating compound activity against intracellular amastigotes at least in a secondary screen. This is in accordance with the findings of Siqueira-Neto et al. who found that only 4% of their hits identified in a promastigote primary screen were active in an intracellular context [11]. Advantages of the intracellular amastigote assay include cell-health information, very low cost of consumables and a reduced necessity for secondary assays. The importance of the host cell in the assay was also demonstrated by the dose response study against axenic amastigotes (i.e. amastigote-like stage obtained from differentiation of promastigotes in vitro in the absence of a host cell); although this parasite form should mimic the intracellular stage, the activity of compounds against axenic amastigotes mostly correlated with promastigotes rather than intracellular amastigotes.
A similar assay was also successfully developed for screening drugs against the intracellular stage of the related parasite Trypanosoma cruzi [31]. Screening the Iconix library against intracellular T. cruzi identified 56 hits, among which 8 were also hits in the Leishmania screen presented here. Six of these were found to be more active against the intracellular amastigote stage of L. donovani compared to promastigotes, indicating inter-species activity of compounds only for the intracellular stages of these two different parasites.
Fifty percent of the compounds that were preferentially active against intracellular amastigotes are known to bind mammalian/eukaryotic G protein coupled receptors (opioid receptors, serotonin or dopamine receptors and adrenergic receptors). Heterotrimeric G proteins are absent in trypanosomatids [32] and we could not find convincing homologs of opioid receptors in the Leishmania genome. Compounds described as ligands of G protein coupled receptors may have different targets among parasitic proteins, leading to mechanisms of inhibition independent of the host cell. This is the case of a serotonin receptor agonist that interferes with P. falciparum growth by blocking a surface membrane channel [33], or a κ-opioid agonist active against T. brucei whose target remains to be identified as no homolog of κ-opioid receptor is found in the T. brucei genome [34]. However, the fact that the ligands of G protein coupled receptors identified in this study, showed a preferential activity towards the intracellular amastigote stage, also highlights the potential value of these host cell signaling pathways as targets. Previous reports demonstrated the involvement of such receptors in inhibition of infection by several intracellular pathogens including Leishmania [35]-[37]. Targeting host factors essential for parasite development is an emerging drug discovery paradigm. It is assumed to be less likely to induce drug resistant pathogens and offers the possibility to repurpose drugs by exploiting compounds currently used for diseases unrelated to microbial infection [38], [39].
Interestingly, one compound out of 909 was active against the intracellular amastigote stage but was completely inactive against promastigotes. This compound, naloxonazine, was also inactive against axenic amastigotes, indicating that its activity is dependent on a macrophage function. Naloxonazine is described as an irreversible µ1-opioid receptor antagonist [40]. There is evidence for the presence of opioid receptors on cells of the immune system [41], [42] and it is known that opioids are involved in modulation of host resistance to infectious diseases [43], [44]. The immune response of mice infected with L. donovani has been shown to be influenced by the opioid receptor agonist morphine, but the receptors involved and the mechanism leading to this immunomodulation remain unknown [45]. Loperamide, a µ-opioid receptor agonist, was also identified in this study as inhibiting parasite growth, and this compound was also more potent against the intracellular stage of the parasite. Another µ-opioid receptor antagonist, naloxone, also present in the Iconix library, did not show any activity against L. donovani. The differential selectivity of naloxone and naloxonazine for opioid receptor binding sites might explain their differential activity against intracellular L. donovani [46]. Naloxone is monomer-like while naloxonazine appears as an inverted dimer (Figure 4). The presence of the macrophage appears to be essential for the activity of naloxonazine against L. donovani, but the underlying cellular and molecular mechanisms remain to be elucidated. Although naloxonazine itself would not meet the requirement for a therapeutic drug due to the reduced selectivity window at a curative concentration, it would be interesting to analyze other compounds that target the same host cell pathway.
In summary, we report an automated screen against intracellular amastigotes of L. donovani. It has the advantage of screening against the relevant stage of the parasite, taking into consideration crucial aspects of its biology, and giving the opportunity to identify host factors critical for the establishment of infection. This is essential for the identification of new, original and diverse lead compounds for anti-leishmanial therapy.
We acknowledge G.E. Healthcare for the INCell Analyzer 1000, Stephanie Robertson, Benoit Vanhollebeke and Patricia Doyle Engel for critical reading of the manuscript.
Author Contributions
Conceived and designed the experiments: GDM JCE JHM. Performed the experiments: GDM KKHA SC. Analyzed the data: GDM KKHA SC JCE JHM. Contributed reagents/materials/analysis tools: MRA. Wrote the paper: GDM JCE MRA JHM.
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Newsletter Issue #812: Apple Addresses the Apple Music Royalty ProblemJune 22nd, 2015
Although Apple Music hasn't even debuted yet, it's already received plenty of publicity. In an example of good intentions possibly going bad, that publicity appears to be the result of the promised three-month free trial. As the term implies, it means you'll have a reasonable amount of time to sample the service before you decide if it's really worth $9.99 per month, or $14.99 for up to six users.
But in the goal of doing something that ought to have a positive impact, Apple apparently failed to consider the rights of the artists who create, produce and perform that music. So while over 70% of the revenue from Apple Music will be distributed to the music companies and, one hopes, to the composers and artists, the original plan called for no payment to be made during the free-trial periods.
Now from an accounting point of view, I suppose that this position made sense. Apple was giving up money to entice more people to subscribe to the service, thus improving the opportunity for artists to earn money. So, therefore, the artists ought to consider the reality of the situation and take a long view. Yes, I'm speculating here, but that's how it seems to be.
Print This Article

subset: commoncrawl


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Food styling with flour, dough & bread - Mary Valentin
Language: English
Duration: 3 hours
Topic: Food styling with bread
Educational objective: Food styling for photographers and food stylists on location
Required level: Photography knowledge
Equipment: Camera and a tripod
Location and timing: Brød&Co, Thursday 18 MAY, 14-17h
About the workshop
Though bread and other bakery items are beautiful, they can be some of the most difficult food items to style and shoot. Mary Valentin, baker, artist and experienced food stylist, was recently given this task. As an artist in residence, she styled and photographed bread and it's processes, from sourdough starters to seeded rye breads, dough, and finished breads, each with their own unique challenges. In this workshop, Mary will share her knowledge of styling and working with breads in the bakery setting. The location is a beautiful local artisan bread shop, considered to be making the best bread in Vejle.
About Mary
Mary Valentin has worked as a food stylist for print, packaging, TV and film for almost 20 years. Through her combination of experience in both the fine and culinary arts, Mary has developed a unique perspective and skill set that informs her work.
In her early career, while studying for her BFA in painting at School of the Art Institute in Chicago, Mary began to acquire valuable experience in the culinary arts through her work in Chicago-area restaurants, most notably as a sous chef at the Café du Midi from 1989 through 1992. Since then, Mary's commercial food styling clients have included Panera Bread, U.S. Foods, and Kraft. She has also done editorial work for Food Network, Chicago Magazine, Chicago Tribune and has art directed and food styled for cookbooks, such as Anupy Singla's "Vegan Indian Cookbook" and "Indian for Everyone."
While teaching at Kendall College in Chicago, Mary initiated a Food Styling and Photography course – the only one of its kind in the country at that time – designed for culinary students. Mary created the curriculum and taught the classes. Food Styling and Photography quickly became one of the most successful and popular classes at Kendall College, with waiting lists of students eager to add food styling and photography skills to their professional repertoires.
Along with her busy food styling career, Mary has presented at conferences, lectured at major corporations and conducted food photography tours including a photo-essay tour of Chicago hot-dog stands with Teri Campbell and Bruce Kraig. Mary has organized and curated photography shows and contests and is currently serving as Chair of the Food Photographers' and Stylists' Section of the IACP, International Association of Culinary Professionals.


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Homemade Bagel Dogs - Tasty Tuesday!
I love the Super Bowl. I haven't actually seen it yet. Or at least not 90% of it. Why? As much as I love football (and this is why I DVR it), I love hosting my annual Super Bowl party, more. This year, we had somewhere in the neighborhood of 25-30 people, and it was a blast. Hosting that many people of course means that I have to provide food, and I did my duty - cooking from 9am until the first guests were arriving.
Was it worth it? Absolutely. The surprise hit of the night appeared to be my homemade "bagel" dogs. I can't buy the real ones because they now have dairy (Little Miss is again off dairy), but these ones taste better and are super easy to make. I made 72 of them, and there were maybe eight left at the end of the night. So fun. But shhhh, don't tell me about the commercials because I haven't seen most of them yet.
Homemade Bagel Dogs
1 package all beef Kosher hot dogs (mine come in 8 packs)
1/3 c water
1 t yeast
pinch sugar
1 t olive oil
1/4 t salt
1 c flour (or less if it's not at all humid - flour needs to be adjusted based on the humidity in your house)
Place 1/3 c hot (but not steaming - just really warm to the touch) water into a bowl. Add the yeast, sugar and oil. Add 1/2 c flour and stir well to combine. Add the salt and stir again. Slowly add more flour until it comes together and doesn't stick to your fingers. Knead the dough for 10 minutes until it is smooth and nicely pliable.
Cover the bowl with a damp kitchen towel, and let it rise for an hour.
Cut the hot dogs into three pieces per hot dog (giving you 24 total, if you use my kind of hot dog).
Gently punch down the dough. Break off ping pong ball sized pieces, and roll them in your hand. Flatten slightly with your thumb. Pick up a piece of hot dog and roll the dough around the hot dog. Use your fingers to pinch it together at the seam and then to spread it so that it covers the entire length of the hot dog.
Repeat for all 24 hot dog pieces. As you finish each bagel dog, place it on a sil pat on a jelly roll pan. Once they are all completed, cover the pan with your kitchen cloth and again let them rise for an hour.
(See, much more puffy now - they're ready to be cooked!)
Preheat your oven to 450 degrees. Remove the towel and bake them for 10 minutes until golden brown and yummy. Serve warm - some people will want ketchup and/or mustard, but they're pretty darn good all on their own.
Enjoy this and more at Tempt My Tummy Tuesday with Blessed With Grace!


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That's right I'm Fired up and Ready to go! So I hope you are too!
It's about that time for the Fall Sports at our local high schools to be dwindlingdown…but instead of dwindling lets get improving for your next Sport season!
Starting Monday November 4th I am starting another amazing Sports Conditioning program.
Two Programs to choose from!
14+ year old program – Please fill out High School Winter 2013_registration form
9-13 Year Old Program – Please fill out Youth Winter 2013_registration form


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The real deal: Two nights' accommodations in two-person tents (one at a base camp, the other in the backcountry), all meals, entrance fees and the services of two naturalist-guides for $595 per person — all taxes included.
When: June 19, July 17, 31, Aug. 14, 28, Sept. 11, 2009.
The fine print: Airfare is not included. For REI members, the price is $550 per person. We recommend joining REI as membership costs only $20 per person and is good for life. Gratuities are extra. Group size maxes out at 10 people. A note on gear: Tents and kayak equipment are included, but a sleeping bag and a backpack are not. Read these guidelines before you book any Real Deal.
Book by: No deadline; based on availability.
Contact: REI Adventures, 800/622-2236, rei.com/adventures.
Why it's a deal: Consider that the package breaks down to $298 per person per night ($275 for members) and saves you the time and effort of coordinating professional hiking guides, kayaking gear, the campsite, the tent and meals. Novice hikers and kayakers should find such a package particularly helpful and valuable.
Itinerary highlights: The trip begins and ends in the Ozette campground in Olympic National Park, on the northern tip of Ozette Lake, which is just about three miles from the Pacific coast. After meeting your group, you'll start with a kayaking tour on the northern part of the lake and then make camp. Over the next 36 hours, you'll hike about nine miles total on the Ozette Loop, which will take you past the coast, tide pools, ancient rock art and an indigenous whaling village that has been noted as an important archaeological site. All your meals are included — enjoy sandwich spreads, fresh fruits and pasta dishes for dinners.
Congress designated 95 percent of this national park as wilderness, so you'll be seeing the largely untouched backcountry of this beautiful region (and very few other people). You could spot bald eagles, rhinoceros auklets, or any of the other 250 species of birds that call the park home; the Olympic torrent salamander can only be found here.
Before you go: Transfers to the campground aren't included; Ozette Lake is about 114 miles west of Seattle. Read more on Olympic National Park on the National Park Service's Web site.
Copyright © 2012 Newsweek Budget Travel, Inc.


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Thu, 11 Oct 2018, 6:00 PM –
Fri, 12 Oct 2018, 7:00 PMAdd to Calendar
Ross School of Business
Ann Arbor, MI United StatesView Map
Thursday, October 11th, 2018
Throughout Day
Coffee Chats
Ross School of Business
Welcome Reception 6:00 – 8:00 pm
Join us in the Ross Colloquium to kick-off an exciting weekend full of amazing women and advocates!
Friday, October 12th, 2018
Participant and Sponsor Check-In/Breakfast 9:30 – 10:00 am
Ross School of Business, Roberston Lobby
Event Kick-Off and Welcome 10:10 – 10:30 am
WILC Co-Chairs
Robertson Auditorium
Keynote Speech 10:30 – 11:10 am
Sarah Robb O'Hagan: CEO of Flywheel
Robertson Auditorium
Breakout Session #1: Visualize 11:20 am – 12:10 pm
- Lean on me: Building Your Personal Board of Directors
- Worldly Working Women: Understanding Unconscious Bias
- Power Couples: Navigating Dual Career Households
Lunch 12:20 – 1:20 pm
Speakers, Participants, and Sponsors
Ross Colloquium
Breakout Session #2: Pursue 1:30 – 2:20 pm
- Leaning In and Leaning Out: Strengths Spotting
- Males as Mentors
- Treat Yourself: Finding Self-Care and Resilience
Breakout Session #3: Defy 2:30 – 3:20 pm
- Watch Your Mouth: Eliminating Bossy and Apologetic Language
- Turning Lemons into Lemonade: How to Succeed in a Male Dominated Industry
- Women Who Hustle: From Entrepreneur to Intrapreneur, You Can Do It, Too!
Snacks 3:30 – 3:40 pm
Closing Keynote 3:40 – 4:10 pm
Closing Remarks and Cocktail Social 4:10 – 4:40 pm
Ross School of Business
Ann Arbor, MI United States
Sarah Robb O'Hagan
Former President of Equinox and Former Global President of Gatorade
Author of Extreme You: Step Up, Stand Out, Kick Ass, Repeat
Described by the media as everything from "Superwoman undercover" to "The Pied Piper of potential," Sarah Robb O'Hagan is an executive, activist, entrepreneur, and currently the CEO of fast-growing indoor cycling company Flywheel Sports. She formerly served as the president of luxury health club Equinox, the global president of Gatorade, a GM and marketing director at Nike, and the marketing director of the Virgin Entertainment Group. The founder of the "Extreme Living" movement, she is a rare blend of fierce businesswoman, cheer leading Mom, passionate women's advocate, and high-energy motivator. In her new book Extreme You, Sarah lays out a roadmap for becoming the most "extreme" version of yourself by operating at the edge of your potential and by building highly-collaborative, focused, and successful teams and corporations. Drawing on her decades experience at some of the world's most influential brands, she shares practical takeaways including how to make failure your fuel, discover your most competitive playing field, get out of line rather than always following conventional wisdom, and bring out the "extreme" in others.
Sarah personally oversaw the development of several breakthrough innovation efforts throughout her career, including: the Virgin Atlantic Airways-Austin Powers movie collaboration in which the airline was rebranded "Virgin Shaglantic," the roll-out of the game-changing Nike Plus technology developed alongside Apple, the transformation of Gatorade from a declining sports drink into an innovative sports performance company exemplified by the highly-successful G Series line, and Equinox's revolutionary shift from a bricks-and-mortar gym to an always-on, digital-first fitness business. She also experienced what she calls a "canyon of career despair" in her 20s, using major back-to-back failures to learn, to grow and to motivate herself forward to eventually lead a $5 billion global business by the age of 38. She shares first-hand accounts of these peaks and valleys—pairing stories of firings with a behind-the-scenes look at Gatorade's transformational turnaround under her stewardship—to illustrate what it truly takes to be successful, why you learn more when play for all or nothing, the importance of humility, and why you must always "play your own game."
Robb O'Hagan has been named twice to Forbes magazine's list of the "Most Powerful Women in Sports," named a "Woman to Watch" by Ad Age magazine, dubbed one of the "Most Creative People in Business" by Fast Company, and named to "40 Under 40" lists by Sports Business Journal, Crain's Chicago Business, and Sports Goods Business.
Sarah is a passionate advocate for an active lifestyle and believes the lessons learned from sports and fitness can be applied to improve performance in the work place. She has served on Hillary Clinton's US State Department Council to Empower Women and Girls through Sports, and is a trustee of the Women's Sports Foundation. She is an active member of the World Class New Zealand Network, as she remains committed to helping the country's development, international competitiveness and economic growth. In 2014, she was awarded the Sir Peter Blake Trust Award for outstanding leadership.
Hosted By
Contact the organizers


text:
# of watchers: 42
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|Wiki-page rating
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|Website reviews
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Comics and Manga Reviews
Miscellaneous reviews
2008-11-21 [Mikhul, the Bard]: Circus album review New album from Britney Spears -- it's great!
2008-11-21 [All_Most PUNK]: I think we need a music reviews section, Hedda.
2008-11-21 [Hedda]: Ehum... How did I miss that?
2008-11-21 [Rook.]: lol. Teh world complicatest hings... its easy to miss
2008-11-21 [Hedda]: Fixed as a category to the upcoming version. music reviews is added now.
2008-11-21 [Rook.]: alrighties!!
2008-11-21 [All_Most PUNK]: Maybe you are deaf?
2008-11-21 [Rook.]: No. I am legally blind though, without my glasses. LOL
2008-11-21 [All_Most PUNK]: I meant [Hedda] :P
2008-11-21 [Rook.]: i know :P ^^ lol
2008-12-06 [Hedda]: Sony Cybershot DSC-W120 ultracompact camera review This is a fast, cheap, good and small camera.
2008-12-13 [Doormat]: Am I allowed to post images of games and stuff if I take them myself? I'm going to assume no, but what if I source it properly? Kinda hard to write a review of a game without some pictures/scree
2008-12-13 [Hedda]: Using screenshots from a game in a review is fair use. You're not allowed to display such images in your house, but that's another issue.
2009-03-24 [Chimes]: ... It's not really an achievement... it's just using one of Elftown's features. If there was a reviewing contest maybe it would warrant a badge... but otherwise it's like rewarding people for breathing. :P
Buuuut, we should probably have a Reviews Group, so then people have people to go to with questions about the review feature and then we have a set group of people who put the reviews on the appropriate wikis. :]
2009-03-24 [SilverFire]: Pubky and I were planning to take care of that for at least the book reviews, possiblah all of it.
2009-03-24 [Chimes]: *shoos you to the empty wiki* :P Fancy building? :P
2009-03-24 [SilverFire]: Mind if I call it The Review Group instead? :P
2009-03-24 [Chimes]: Nope. :P Sounds better than my suggestion.
2009-03-24 [SilverFire]: nah, changed my mind to yours anyway. :P needs the plural to make it sound less like we are a group that asses people.
2009-03-24 [Chimes]: XD Fair enough.
2009-03-25 [All_Most PUNK]: I'm keeping the idea of reviewing people for the future >>
2009-05-31 [Chimes]: Uhuh...
2009-06-05 [NOOOPE]: Repo! The Genetic Opera review Are you a stupid tool? This is the perfect movie for you!
2009-08-04 [Closet Cowboy]: Yo gaurds its me, i am on elfpack and on this site you guys may remember me, you deleted me 10times, i am the guy whos spaming all of the wiki's, dont even bother banning me because i willbe back, hell I could even comeback as one of you, so go ahead delete me, i'll comeback.I always come back...but being deleted is such a bitch.
2009-08-04 [NOOOPE]: I know this might be hypocritical of me to say... but man, you really need to get a life.
2009-08-04 [Closet Cowboy]: i have one, i work, but i do this in my spare time
2009-08-04 [Triola]: Seriously. Take up knitting, please, for all our sakes.
2009-08-22 [Alexi Ice]: Dead Rising review The Video Game Dead Rising. I must say...it's amazing.
2009-08-22 [Alexi Ice]: Wasn't sure if this should go under Computer games or not, which is kinda stupid considering an X-box is like a giant computer. Lol...
2009-08-22 [Hedda]: Of course it's a computer game.
2010-01-07 [Easterling]: The Land of Green Plums by Herta Müller review Müller's style is poetic but doesn't suceed in capturing me.
2010-02-15 [All_Most PUNK]: The Wolfgand Amadeus Phoenix review was added to the corrsponding, music section (by Silvie, not by me).
2010-03-01 [Eyden13]: Graceling review
2010-03-07 [Akayume]: Touching Darkness; Scott Westerfeld review
2010-03-07 [All_Most PUNK]: Akayume, you need to choose book in the menu next time, so the review will be posted in the correct section :)
2010-03-07 [Akayume]: Ohh, I'm sorry. I've never written a review before. (: And I wrote this really quick before I went to drop of dinner at a friend's house.
2010-03-07 [All_Most PUNK]: No problem :D You did nothing wrong. It's just easier if you classify it.
2010-03-07 [Akayume]: I changed it (I believe). I'm sorry if I didn't work, I've never done this before. :/
2010-03-07 [All_Most PUNK]: Worry not, it's all fine.
2010-03-29 [another brick in the wall]: Bob Dylan; Blowin' in the Wind review Dylan answered by the gods of Gilgamesh
2010-04-04 [Jitter]: Doctor Who 5x01: The Eleventh Hour review
2010-04-11 [another brick in the wall]: if a review is for a consul game, would we still classify it as 'computer game' or as 'other'?
2010-04-11 [SilverFire]: Computer game. :)
2010-07-25 [*Phoenix*]: I added Doctor who to the page. :)
2011-04-20 [kians mummy]: sammie h wiki review wikis are a great way to interact with other people on here
2011-04-29 [Alexi Ice]: The link is empty, Sheep
2011-04-29 [sweet.tx.tea]: Working on it, Akane. I am counting all the reviews.
2011-04-29 [Alexi Ice]: Need any help?
2011-04-29 [sweet.tx.tea]: Not at this time, but I will keep you posted. :)
2011-04-29 [Alexi Ice]: KK <3
2011-05-03 [Alexi Ice]: The Vampire Knight anime show might fit on the Comics and Manga section since it's based on a manga?
2011-05-03 [SilverFire]: Er, no. :P Just like films based on books don't go in books...
2011-05-04 [Alexi Ice]: Just trying to figure out how to un-clutter this place. hahaha
2011-05-04 [Alexi Ice]: If I had known people posted Video Games with Computer games I would have shoved that one there. Sorry.
2011-05-04 [SilverFire]: You could have posted it on the page yourself, y'know. ¬_¬
2011-05-06 [sweet.tx.tea]: [SilverFire], I will have the count posted soon. Hopefully tonight. I am sorry for the delays. I have been out of town and have not had the internet connection I assumed I would have.
2011-05-06 [Alexi Ice]: Sorry Silver...fail.
<3 *Hugs Sheep*
2011-05-06 [kians mummy]: how come mine hasn't been put on
2011-05-06 [Akayume]: Have you tried looking under website reviews sammie? (:
2011-05-25 [Akayume]: Rosemary & White Cheddar Popcorn review
2011-05-27 [There Is No Cure]: http://www.asexuality.org/home/ review
2011-05-27 [kians mummy]: call of duty Black Ops for ps3 review this game is a shooting game and i thing it is the best
2011-05-28 [Alexi Ice]: Please make sure to place your reviews in the correct categories! If you need help with that please ask one of the Reviewers group! It makes it much easier for us to update these pages. Thank you.
2011-05-28 [djxmonster]: So there isn't any kind of guidelines as to what constitutes a review? So I can get a badge for this review: 'Cnn.com is awesome'?
2011-05-28 [Alexi Ice]: What makes a good review? - This page is probably going to be revamped soon and we have some rules though I don't know where they are posted. I'll have to ask one of the other bosses. Basically good spelling/gramm
2011-05-28 [Akayume]: I think that's pretty much it. (:
2011-05-31 [Akayume]: Did you get rid of the link that leads to what makes a good review?.
2011-05-31 [SilverFire]: No? It's there in rule five.
2011-05-31 [Akayume]: Dang. X.x I must be more tired than I thought. I totally didn't see that. Sowwy.
2011-06-02 [Nioniel]: Apple iPad (32 GB) Wi-Fi (MB293LL/A) Tablet review
2011-06-02 [Nioniel]: Cyberhome CH-DVD 300 DVD Player review
2011-06-02 [Nioniel]: Nintendo N64 Expansion Pack for Nintendo 64 review
2011-06-02 [Nioniel]: The Simpsons Chess Set review
2011-06-02 [Nioniel]: Burt's Bees Res-Q Ointment review
2011-06-02 [Nioniel]: Buffy Series 2 Spike review
2011-06-02 [Nioniel]: Fear, Itself Anya review
2011-06-02 [Nioniel]: Princess Leia In Boushh Disguise Figure review
2011-06-02 [Nioniel]: Disney Lady and the Tramp 10" Lady Plush review
2011-06-02 [Nioniel]: Harry Potter Hagrid's New Arrival Statue review
2011-06-02 [Nioniel]: Professor Snape Figure review
2011-06-02 [Nioniel]: Neca Harry Potter Hagrid 9" Deluxe Figure review
2011-06-02 [Nioniel]: Order of the Phoenix Series 1 Sirius Black review
2011-06-02 [Nioniel]: Series 1 Dementor 7 Inch Figure review
2011-06-02 [Nioniel]: Series 1 Harry Potter Figure review
2011-06-02 [Nioniel]: Fellowship of the Ring Flaming Ringwraith review
2011-06-03 [Nioniel]: Super Pet Bunny Shake 'n' Chew Toy review
2011-06-05 [Ravenclaw]: X-Men: First Class review
2011-06-08 [Nioniel]: Kiss My Face Shampoo review
2011-06-08 [Nioniel]: Frontline Plus for Cats review
2011-06-08 [Nioniel]: Burt's Bees Lemon Butter Cuticle Creme review
2011-06-08 [Nioniel]: Planar PL1700 17 inch LCD Monitor review
2011-06-08 [Nioniel]: Logitech Illuminated Keyboard Ultra Thin review Logitech Illuminated Keyboard Ultra Thin with Backlighting
2011-06-08 [Nioniel]: Apple iPhone 4 Black (16 GB) Smartphone review Apple iPhone 4 Black (16 GB) Smartphone
2011-06-08 [Nioniel]: Farouk Chi Ceramic Flat Iron Original Model review Farouk Chi Ceramic Flat Iron Original Model
2011-06-08 [Nioniel]: Booda Clean Step Litter Mat for Cats review Booda Clean Step Litter Mat for Cats
2011-06-08 [Nioniel]: Curls Rock Leave-In Moisturizer review Curls Rock Leave-In Moisturizer
2011-06-08 [Nioniel]: Pantene Pro-V Restoratives review Pantene Pro-V Restoratives
2011-06-08 [Nioniel]: Catwalk Tigi Curls Rock Curly Hair Shampoo review Catwalk Tigi Curls Rock Curly Hair Shampoo
2011-06-08 [Nioniel]: XBALM Extreme Tropical Lip Balm SPF 15 review XBALM Extreme Tropical Lip Balm SPF 15
2011-06-08 [Nioniel]: Kiss My Face Peppermint Foot Cream review Kiss My Face Peppermint Foot Cream
2011-06-08 [Nioniel]: Purell Instant Hand Sanitizer review Purell Instant Hand Sanitizer with Moisturizers and Vitamin
2011-06-08 [Nioniel]: Spotbrites Laser Pet Toy and Exerciser review Spotbrites Laser Pet Toy and Exerciser
2011-06-08 [Nioniel]: Dyson DC25 Bagless Upright Cyclonic Vacuum review Dyson DC25 Bagless Upright Cyclonic Vacuum
2011-06-08 [Nioniel]: Oxi Clean Spray-A-Way Instant Stain Remover review Oxi Clean Spray-A-Way Instant Stain Remover
2011-06-08 [Nioniel]: Tetra Silver Betta Bowl Aquarium w/ Light review Tetra Silver Betta Bowl Aquarium w/ Light
2011-06-08 [Nioniel]: Aquachef Automatic Fish Feeders review Aquachef Automatic Fish Feeders
2011-06-08 [Nioniel]: Penn Plax Tank Stand review Penn Plax Tank Stand
2011-06-08 [Nioniel]: Kaytee Gourmet Recipe Rabbit Food 5 LB Bag review Kaytee Gourmet Recipe Rabbit Food 5 LB Bag
2011-06-11 [Nioniel]: Buffy the Vampire Slayer PALz Angel review Buffy the Vampire Slayer PALz Angel
2011-06-11 [Nioniel]: Buffy the Vampire Slayer Palz Buffy review Buffy the Vampire Slayer Palz Buffy
2011-06-12 [Ravenclaw]: X-Men: First Class Review
2011-06-12 [Mortified Penguin]: I kind of wish most of you would put pictures, movie posters, or screenshots of the things you're reviewing.
2011-06-12 [Ravenclaw]: Wouldn't movie posters go against the uploading art rules?
2011-06-12 [Mortified Penguin]: Not posted in a wiki. Anything goes on wikis! Even hardcore porn, with [Hedda]'s face shopped in!
2011-06-12 [Akayume]: T-Shirt Shrug Project (cut out + keep) review
2011-06-13 [Akayume]: Izze Juice review
2011-06-21 [Alexi Ice]: Ravenclaw - Would you mind rating your review please?
Edit - Nevermind, there are two copies of it for some reason
2011-06-21 [Alexi Ice]: <Updated>
2011-06-21 [Alexi Ice]: Black bird manga review
2011-06-24 [Nioniel]: Cat 'n' Mouse Cat Powered Cat Toy review
2011-06-24 [Nioniel]: Friskies Fancy Feast review
2011-06-24 [Nioniel]: Avon Winter Treats Candy Cane Lip Balm review
2011-06-24 [Nioniel]: Body Shop Body Butter Passion Fruit review
2011-06-24 [Nioniel]: Dr. Scholl's For Her Smooth my Sole review
2011-06-24 [Nioniel]: Bath & Body Works Twilight Woods Body Lotio review
2011-06-24 [Nioniel]: Tempur-Pedic Euro (Prima) Bed review
2011-06-24 [Nioniel]: Buffy the Vampire Slayer PALz Spike review
2011-06-26 [Thunder Cid]: E.T: The Extra-Terrestrial review
2011-06-28 [Nioniel]: Jerome Russell's Punky Colour Violet Hair D review
2011-06-28 [Nioniel]: Physicians Formula Mineral Wear review
2011-06-28 [Nioniel]: Avon Slick Tints Glossy Wine Lip Balm review
2011-06-28 [Nioniel]: Napoleon Prestige BIPT450RBI Gas Grill review
2011-06-28 [Nioniel]: ChocoVine review
2011-06-28 [Nioniel]: Hot Spot Itch Relief Shampoo review
2011-06-28 [SilverFire]: I think we need to make a separate review page for 'Contents of Nioniel's house' <_< :P
2011-06-28 [Nioniel]: Lost: The Complete First Season review
2011-06-28 [Nioniel]: :(
2011-06-28 [Alexi Ice]: LOL, agreed, Silver ^^
2011-06-28 [Akayume]: Brow Precision Sharpener review
2011-06-29 [kians mummy]: If there is a badge for reviews, why haven't these pages got the official badge
2011-06-29 [SilverFire]: The request has been put to our banner-maker, but he's a bit busy at the moment. :)
2011-06-29 [kians mummy]: I'll do it, I can do it now if you like
2011-07-02 [Alexi Ice]: Don't forget that all reviews from this page are moved to miscellaneous reviews page! So if you wish to see the wiki's that have been added you should keep that page on watch!
2011-07-06 [Alexi Ice]: Updated!
2011-07-11 [Ms. Steel]: The Scars by William Tennant review
2011-07-13 [Skydancer]: Wall of Gum review A valid discussion on what is art.
2011-07-17 [Avaz]: HBO: Game of Thrones review A TV series based on George R.R. Martin's book
2011-07-25 [kians mummy]: www.myyearbook.com review
2011-07-25 [kians mummy]: Cat hug review Its all about the cats
2011-07-27 [Alexi Ice]: Don't forget to keep your reviews in the correct category! Thank you <3
2011-07-28 [Skydancer]: SCINTILLATION by Xavier Chassaing review
2011-07-30 [toresimonsen]: EPRM Sunrise/Sunset Review. review Many people took great photos in the sunrise/sunset EPRM.
2011-08-01 [Akayume]: Ostia Antica review A great place to better understand the ancient Roman world.
2011-08-13 [AnAtoliAXE]: review
2011-08-13 [Alexi Ice]: Er, once again, [AnAtoliAXE] your review can not be accepted as it contains nothing. Sorry. For further questions please first note the rules at the top of this page.
2011-08-24 [Paul Doyle]: Iron Maiden---"Brave New World" (2000) 1/2 Very solid first 21st Century Iron Maiden studio album, marking the returns of Adrian Smith and Bruce Dickinson. The now six-piece band is older and wiser, venturing bravely into the realm of progressive metal, the start of an ongoing trend.
2011-10-11 [Akayume]: Lemon Sugar Hand Scrub (Tutorial) review
2011-10-17 [Akayume]: Peppered Shrimp Alfredo Recipe review
2011-10-26 [Avaz]: The Well of Ascension by Brandon Sanderson review
2011-11-22 [Ravenclaw]: Batman Arkham City review
2012-01-26 [Akayume]: BeneficialMinerals - Iced Papaya Eyeshadow review
2012-01-26 [Akayume]: BeneficialMinerals - Story Book Eyeshadow review
2012-01-29 [Alexi Ice]: I plan on updating this tomorrow.
2012-02-01 [Akayume]: Lavender French Clay Mask review
2012-05-13 [Ravenclaw]: I have a question concerning movie reviews.
If [All_Most PUNK] is supposed to organize the Movie Reviews page and hasn't been on in 47 days, why the hell is he in charge of anything?
2012-05-13 [kians mummy]: Council members sometimes need time off to, as they have family members and a life outside of Elftown. xD
2012-05-13 [Ravenclaw]: Forty seven days...
My dad broke fourteen bones and punctured a lung in a motorcycle accident and I only missed a couple days. I understand missing a week or two, even a month, but forty seven days is just a bit much.
2012-05-14 [kians mummy]: I have suggested ways around this before, but I'm not going to bitch about it, if I have suggestions, I go to the Council or Guards.
2012-05-14 [Ravenclaw]: I'm not bitching, I'm just curious. I was under the impression that council members understand that they have a responsibility to the council and the populace to preform their duties as expected. It just seems to me that if a person isn't going to be here, their duties should be delegated elsewhere.
2012-05-14 [kians mummy]: Yeah, and I do agree with you fully on that one, I have offered to help on here if they wamt the help, now it's just a waiting game, they should have a page for Emergency staff, I set one up on Elfpack. :)
2012-05-14 [Ravenclaw]: I'm not council or anything but if it just means editing the page and posting the new movie reviews, I wouldn't mind. Heck, it'd only take a couple seconds right?
2012-05-14 [kians mummy]: Yeah, maybe a little longer, as I work for volunteers on Elfpack, I don't really see what the probem is as I know how to run official pages, but thats up to the Council to decide. :)
2012-05-14 [Ravenclaw]: Maybe I should mention it there... I don't wanna cause anything though...
2012-05-14 [kians mummy]: You won't. :)
2012-05-16 [iippo]: You might do better addressing the Reviews Group directly.
2012-05-16 [Ravenclaw]: thanks.
2012-05-21 [All_Most PUNK]: Hello. I won't explain myself. If I wasn't online for some time, it's my problem why I didn't. But since I do agree that I'm not reliable nowadays, I just let the Council know that I'd prefer to not be in charge of anything.
2012-05-24 [Alexi Ice]: Same here, Punk. We don't get paid to do this job.
2012-05-24 [kians mummy]: So what if you don't get paid, you still do voluntry work for members of the site and without people like you, Elftown would be dead, but the crew have kept it going really well and I can not spot one thing wrong with it, everyone on the crew are brilliant at there job and deserve a special Elftown medal, which I am thinking of creating.
To see how much the crew are dedicated to keep Elftowners entertained, as well as protective and to make them feel safe is such a privalage, and all workers deserve better treatment off normal members as without you, there wouldn't be an Elftown.
2012-05-24 [Mortified Penguin]: Wait, you think the crew deserves their own special medal? Like a badge or something? Only meant for crew members? That distinguishes them from other members? Ridiculousness
And you go on to suggest they be treated different from normal members? Like what? Give them privileges that others don't have? Completely insane. That's never going to happen.
2012-05-25 [kians mummy]: No, not a badge, an award, which is different to a badge, I thought you had at least one brain sell left, but I guess its all the reman your eating.
2012-05-25 [Paul Doyle]: Why do I hear big smooching sounds from Sammie all of a sudden? It's kind of loud in here now.
2012-05-25 [iippo]: Eh, would you guys stop the chatter, please? Or at least write some reviews once and a while.
2012-05-25 [Avaz]: Yeah, this isn't Bob's Diner or now, after all. Sheesh.
2012-05-25 [Mortified Penguin]: >:o
2012-05-28 [Alexi Ice]: I'm retired, actually.
2012-07-09 [elvina0981]: Make Your Business Alive with Ecommerce review To expand the business
2012-07-09 [Paul Doyle]: This link (which is in [elvina0981]'s review) is to a page that is deeply red-flagged by WOT (Web of Trust) . . . beware! http://www.myw
2012-07-17 [SilverFire]: Those all seemed to be "reviews" of the same website, and read far more like adverts than reviews. We do not accept adverts as Reviews. Since they were all for the same website, I deleted most of the comments as spam. I'll leave the comment above, but it won't be added to Reviews.
2012-11-14 [Akayume]: Into the Universe with Stephen Hawking review TV with three episodes, featuring information and theories o
2013-11-19 [iippo]: Updated.
2013-11-19 [Mortified Penguin]: Would audiobook reviews go under Book reviews?
2014-01-15 [pegasus1000]: Yep
2014-02-23 [pegasus1000]: The Dark Tower I: The Gunslinger (Revised) review
2014-11-11 [Sideways]: Middle Earth: Shadow of Mordor review
2015-07-16 [Gran Mitico]: review
2015-07-16 [Gran Mitico]: review
Number of comments: 216
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text:
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text:
We're moving!
Sadly we are leaving our cute little shop on Hayle harbourside and moving to pastures new. In the next couple of weeks we will be packing up everything and moving to a new larger and much bigger shop. Its exciting but also terrifying and a little sad to. We have truly loved our time in Penpol Terrace. While we work, we have been watching the boats go up and down in the harbour as the tide ebbs and flows. Its been wonderful. But things do have to change and for a number of reasons……. We want to expand the shop and to do that we need larger premises. We want to stock more variety of things and to collect more crafts people to sell with us, for that we need space. One the most common things we hear in the shop is, "It looked bigger in the photos", being able to have more than 6 customers in at any one time will be amazing! But also I haven't settled in West Cornwall. I miss my family and friends and I miss having a garden. So when the opportunity came up for us to take on a shop further up in Cornwall, it made sense to move, home and shop closer to those we miss. So the shop has been sold and will soon have new owners. Our house has also sold and we have started looking where to move to. We are not sure where we are going to live, but it's exciting exploring new areas as possible new homes. So we will be quiet for a bit, while we get organised, so please look out on facebook and Instagram for updates and as soon as we officially get the new shop keys, we will tell you where we are. Janice


text: The Habitat Grove, in the center of Moerenuma Park, Japan, was the chosen location for Hidemi Nishida's installation. He installed atmospheric curtains between the trees, in an area 50 by 50 meters in size, adding gradation and depth to the landscape. Blue and white are natural in a snowy environment, actually the color of snow and ice it's self. These are the colors you'll see if you we're in a snowcave, eventhough snow itself is actually colorless: it's white merely because it reflects all the light that reaches it. Adding these colors to the already snowy landscape make it more chilling.


text:
Stair balusters manufacturers Private3 недели назад - Multimedia - Buturlinovka - 63 view
Location: Buturlinovka
Price: -- руб
Raising INOX is private enterprise engaged in the special field of foreign trade. With modern office facilities and efforts of our experienced professionals in foreign trade and management, we have built up very close business relationships with more than 100 reliable domestic suppliers and more than 100 customers from about 50 countries and regions around the world.
Our company mainly deals in import and export of following products:
Metal products: Stainleses stee handrail system, balustrades, railing systems, glass clamps, framless glass balustrade and sliding door system.
Our Product
Railing System, Handrail System, Balustrades
Product Application
Construction and Decoration
Our Certificate
CEstair balusters manufacturers


text:
Jeremy Piven has strongly denied groping a background actress on the set of Entourage in 2009.
The actor, who has already been hit with allegations of sexual harassment and assault in the scandal sweeping Hollywood, has been accused of groping Anastasia Taneie when she was a 23-year-old extra on his TV show Entourage in April 2009.
She claims they got talking during a filming break on location at a Los Angeles theatre and they went off together when the actor suggested he and his manager wanted to talk to her about something. But when they were alone in a dark hallway, he allegedly grabbed her, pushed her up against a wall and groped her breasts and genitals. She alleged that he only stopped when an assistant director walked by.
"I was scared at the time nobody was going to believe me. I didn't want to make a scene. I just wanted to go home," she told BuzzFeed News. "It was the most horrible thing I've ever experienced."
Piven has denied the claim through his lawyers, saying, "Let me be absolutely clear, this simply did not happen. I would never force myself on a woman. I cannot speak as to why a person would create a story like this."
He is also threatening to pursue legal action and has demanded his accusers take a polygraph test, like he did on 13 November in a bid to prove his innocence.
He was asked questions about allegations by Ariane Bellamar, who claimed he touched her inappropriately on the set of Entourage and at a Playboy Mansion party, advertising executive Tiffany Bacon Scourby, who accused him of exposing himself and ejaculating over her in his New York hotel room in 2003, and Amy Meador, who claims Piven forced himself on her after he came round her then-home in Los Angeles in 1995.
He denied all the claims during the session. According to The Blast, the lie detector examiner said Piven "showed no reactions indicative of deception" and therefore passed the test.


text:
batter down(redirected from battering down)
batter down
To remove or destroy a particular structure. A noun or pronoun can be used between "batter" and "down." Which wall will the construction crew batter down?
batter something down
to smash or break down something, such as a wall, door, or any defensive structure. Do they have to batter anything down as part of the construction project? They battered down the wall as a first step in enlarging the house.