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The mevalonate pathway is essential in eukaryotes and responsible for a diversity of fundamental synthetic activities. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme in the pathway and is targeted by the ubiquitous statin drugs to treat hypercholesterolemia. Independent reports have indicated the cidal effects of statins against the flatworm parasite, S. mansoni, and the possibility that SmHMGR is a useful drug target to develop new statin-based anti-schistosome therapies. For six commercially available statins, we demonstrate concentration- and time-dependent killing of immature (somule) and adult S. mansoni in vitro at sub-micromolar and micromolar concentrations, respectively. Cidal activity trends with statin lipophilicity whereby simvastatin and pravastatin are the most and least active, respectively. Worm death is preventable by excess mevalonate, the product of HMGR. Statin activity against somules was quantified both manually and automatically using a new, machine learning-based automated algorithm with congruent results. In addition, to chemical targeting, RNA interference (RNAi) of HMGR also kills somules in vitro and, again, lethality is blocked by excess mevalonate. Further, RNAi of HMGR of somules in vitro subsequently limits parasite survival in a mouse model of infection by up to 80%. Parasite death, either via statins or specific RNAi of HMGR, is associated with activation of apoptotic caspase activity. Together, our genetic and chemical data confirm that S. mansoni HMGR is an essential gene and the relevant target of statin drugs. We discuss our findings in context of a potential drug development program and the desired product profile for a new schistosomiasis drug.
Citation: Rojo-Arreola L, Long T, Asarnow D, Suzuki BM, Singh R, Caffrey CR (2014) Chemical and Genetic Validation of the Statin Drug Target to Treat the Helminth Disease, Schistosomiasis. PLoS ONE 9(1): e87594. https://doi.org/10.1371/journal.pone.0087594
Editor: Jennifer Keiser, Swiss Tropical and Public Health Institute, Switzerland
Received: October 18, 2013; Accepted: December 21, 2013; Published: January 29, 2014
Copyright: © 2014 Rojo-Arreola 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: LR-A was supported by a UC MEXUS-CONACYT Postdoctoral Research Fellowship. The research reported was supported by the National Institute of Allergy And Infectious Diseases of the National Institutes of Health under award numbers 1R01AI089896 (BMS, CRC, DA, and RS) and R21AI107390 (CRC), and by the National Science Foundation through grant IIS 0644418 (DA and RS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 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.
The mevalonate pathway is essential in eukaryotes , . Branches arising from the central pathway are responsible for a diversity of fundamental synthetic activities, e.g., sterols, dolichol and ubiquinone, and for protein prenylation and glycosylation –. The enzyme responsible for the rate-limiting catalytic step in the pathway, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), is a trans-membrane glycoprotein in the endoplasmic reticulum that is responsible for converting HMG-CoA to mevalonate . Specific inhibitors of this enzyme, termed statins, were first identified in the early 1970's from Penicillium and Aspergillus fungi . Their ability to competitively inhibit cholesterol synthesis led to the development of the now ubiquitous anti-hypercholesterolemia drugs to control cardiovascular disease , . Statins have been ‘blockbuster’ drugs for the major pharmaceutical companies with sales in excess of $12 billion dollars per year between 2005 and 2008 .
In addition to hypercholesterolemia, other potential therapeutic uses have emerged for statins, including the treatment of inflammatory  and immune disorders , and as anti-cancer drugs , . Statins have also been explored as possible treatments for parasitic diseases, including Human African Trypanosomiasis , Chagas' disease , leishmaniasis  and malaria –.
HMGR has been described and characterized in the flatworm parasite, Schistosoma mansoni , , which is one of a number of schistosome species that cause the globally prevalent disease, schistosomiasis , . Whereas in mammals, HMGR homeostasis is regulated both transcriptionally and post-transcriptionally by cholesterol levels and conformational changes induced by phosphorylation state –, the regulation of biosynthesis and activity of the S. mansoni HMGR (SmHMGR) are unknown. The presence of sterols and non-sterol lipids in the diet of the host does not affect parasite enzyme activity and there is no evidence of a reductase phosphatase-kinase system, therefore, it is presumed that the mechanism differs from that described in mammals . Also, in schistosomes, the branch responsible for de novo synthesis of cholesterol is absent – a feature that is shared among worms , , .
The HMGR statin inhibitor, lovastatin, severely impacted egg-production ,  and survival of S. mansoni in a mouse model of infection . Parasite death could be prevented in the presence of excess mevalonate, the specific product of HMGR . Subsequently, our own observations confirmed the lethality of statins to S. mansoni in vitro during a phenotypic (whole organism) screening campaign employing drugs approved for use in humans . These data support the continued investigation of SmHMGR as a possible drug target, especially considering that just one drug, praziquantel (PZQ), is available to treat and control the disease , . Also, most of the erstwhile blockbuster statins are now off-patent relaxing intellectual property constraints for further chemical development.
To follow up on these observations and in the further consideration of SmHMGR as a possible drug target, we measured the in vitro bioactivities of six commercially available statins against both the somule (post-infective larvae) and adult stages of S. mansoni. For somules, we also developed an automated screening strategy to measure statin bioactivity. In addition to the chemical approach using statins, we also employed the reverse genetics tool of RNA interference (RNAi) to understand the contribution of SmHMGR to somule survival both in vitro and in an ‘in vitro-to-in vivo’ method, whereby parasites are first subjected to RNAi in vitro before transfer into a mouse model of infection. Finally, we attempt to identify by which mechanism(s) interference with SmHMGR function leads to parasite death. The results are discussed with respect to the current target product profile for schistosomiasis, not least the goal of achieving single oral dosing, which is the case with the current drug, PZQ.
Materials and Methods
Maintenance and handling of small mammals were carried out in accordance with a protocol (AN086607-03) approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California San Francisco.
Schistosoma mansoni life cycle and parasite maintenance in vitro
The acquisition, preparation and in vitro maintenance of newly transformed schistosomula (somules; derived from infective stage cercariae) and 42-day-old adult S. mansoni (from small mammals) have been described by us previously , .
Chemical screens of somules and adult S. mansoni with commercial statins
Six commercially available statins, namely, atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin (available from Sigma-Aldrich, Tocris or LC Laboratories) were dissolved as 20 mM stock solutions in DMSO. For in vitro tests with somules and adults, serial dilutions were set up as indicated in the results section. Final concentrations of DMSO were never greater than 0.5%. Somules were distributed in 96-well flat-bottomed plates at a density of 300 worms in 200 µL culture medium. Culture medium was Basch medium 169  (custom prepared at the UCSF Cell Culture Facility) containing 5% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin solution. Plates were kept at 37°C and 5% CO2. For screens with adult S. mansoni, five pairs were maintained in 2 mL of the above medium in 24-well culture plates. To assess whether mevalonate, the product of HMGR, could rescue any deleterious phenotype induced by statins, the compound was added at the final concentrations indicated in the results section from a stock solution of 3.87 M (±)-mevalonolactone (Sigma-Aldrich) dissolved in water.
Parasite phenotypic responses to statins were recorded daily out to four and seven days for somules and adults, respectively. For both developmental stages, the process of manually adjudicating the manifold phenotypic responses possible by this parasite has been described  and essentially involves recording changes in movement, shape, translucence, surface integrity and, in the case of adults, the ability of the parasite to adhere to the culture dish surface. With somules, those statins that were bioactive consistently induced a darkening and rounding of the parasite and impaired motility in a process we termed ‘degeneration.’ Each day representative photographic images were taken and the number of degenerate parasites calculated as a percentage of the total number of worms in the given image. Images from each treatment were captured using a Zeiss Axiovert 40 C inverted microscope (10x objective) and a Zeiss AxioCam MRc digital camera controlled by AxioVision 40 (version 126.96.36.199) software.
For adults, we converted the types and number of phenotypic responses recorded manually into a ‘severity score’ ranging from 0 (no effect) to 4 (severely compromised). Each response was awarded a score of 1, except where damage to the parasite's tegument (surface) was evident, in which case the maximum score of 4 was awarded on the assumption that such damage is lethal to adult parasites, including in the mammalian host .
Automated high-throughput approach to screening somules
The automatic approach for identifying and counting ‘degenerate’ and ‘normal’ somules consisted of three main stages. First, an image processing algorithm , developed specifically for segmentation of somules in bright-field photographs, was used to identify individual somules and subsequently extract a quantitative, feature-space representation of each parasite. This segmentation method is generic in that it can be used with any bright-field imaging device and does not rely on any a priori model of parasite shape. Consequently, it is highly suited to screening complex macroparasites due to their natural irregularity of shape and size as well as the unpredictability of phenotypic effects caused by drugs on the parasites. Furthermore, the segmentation method is highly accurate: in evaluations reported , it was found to identify 95.9% of individual schistosomula and to detect the location of their boundaries, i.e., the outer edge of the tegument, with an average accuracy of 1.3 pixels on a data set of 6,960 hand-traced schistosomula imaged under varying conditions. Owing to the accuracy of the method, a predominant majority of touching parasites were separated during segmentation. Consequently, there was no need to discard such parasites using a predefined area threshold as was done in .
Following segmentation, in the second stage, each parasite was represented by a vector of descriptive measurements capturing appearance, shape and texture. These features, expanded on those used previously –, included the statistical moments of image intensities, invariant image moments  and two multi-scale texture measures based on either gray-level co-occurrence matrices  or wavelet decomposition .
In the final stage, vector space representation of each somule was taken as input to a machine learning algorithm which performed the classification of parasites paralleling the role played by the human expert in the manual assay. Specifically, each feature vector was used to classify the corresponding parasite as “normal” or “degenerate” using supervised machine learning with support vector machines (SVM). The SVM is a linear method which finds a hyperplane within the feature space that separates the two classes with the maximum margin, or distance between the decision surface and the closest points , . In the training step of the SVM, a feature space hyperplane suitable for dividing degenerate and normal parasites was constructed using a set of 3,907 segmented parasites in 118 photographs (see above). These somules were manually labeled as either normal or degenerate using a custom, web-based user interface. To ensure consistent manual decision making, for each micrograph where the parasites had to be labeled, the corresponding control micrograph was displayed. The trained SVM was then applied to a separate data set composed of 23,867 individual somules taken from 438 micrographs.
We found it is necessary to model the variation between different populations of somules, captured by individual control experiments conducted simultaneously with particular drug response experiments. This was done using a two-step classification procedure in which initial SVM classification results were used to compile ‘normal’ control parasites, which were used to determine a control medoid for each group of experiments. Final class assignments were obtained from a second classification tier in which the control mediods were used as a baseline for their particular drug-response experiments.
Synthesis of double-stranded (ds)RNA and treatment of somules with dsRNA in vitro
PCR primers were designed to respectively amplify N-terminal and C-terminal segments (positions 302 to 1349 and 1854 to 2626, respectively) of the open reading frame (ORF) of S. mansoni HMGR (GenBank M27294) using adult mixed-sex complementary (c)DNA as template. After sequence confirmation of the amplified DNA, dsRNA templates for the N-terminal and C-terminal segments were obtained by PCR amplification with the same primers now flanked with a T7 promoter sequence . These amplicons were employed as templates for reverse transcription using the T7 RiboMAX express RNAi system (Promega). DsRNA integrity was assessed by 1% agarose gel electrophoresis and its concentration measured in a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies). DsRNA to Discosoma sp. mCherry fluorescent protein, a protein not present in S. mansoni, was also prepared for use as a control.
Incubations of somules with dsRNA were set up in duplicate in 24-well culture plates at a density of 400 worms in 1 mL culture medium at 37°C and 5% CO2. Fifteen µg/mL of each of the dsRNAs to the N-terminal and C-terminal sections of the SmHMGR ORF or 30 µg/mL mCherry dsRNA were employed and medium and dsRNA were exchanged every three days. Incubations were continued for either 7 days for quantitative (q)RT-PCR analysis or 25 days to monitor for gross phenotypic responses of the parasite. For qRT-PCR, quantification of S. mansoni cystatin (AY334553.1) and cathepsin B1.1 (AJ506157) mRNA was also performed to identify possible RNAi off-targeting by the SmHMGR dsRNA. To assess whether mevalonate could prevent the appearance phenotypic responses induced by RNAi of HMGR, the compound was added each time the medium was exchanged at the final concentrations indicated in the results section. Parasite phenotypic responses to RNAi were monitored every day and when apparent we employed the same photographic imaging and manual counting strategy used to measure statin insult as described above.
Treatment of somules with dsRNA in vitro and assessment of survival after transfer into mice
Incubations of somules with dsRNA were set up as six replicates in 24-well culture plates at a density of 500 worms in 1 mL Incomplete Basch medium 169 (i.e., omitting the FBS). DsRNA was added twice daily for 2.5 days each time at a concentration of 60 µg/mL (30 µg/mL of each of the dsRNAs targeting the N-terminal and C-terminal HMGR ORF sections). Parasites were then washed twice in the same medium and the somules from one of the replicate incubations assessed for transcript suppression by qRT-PCR. The parasites from each of the remaining five replicate incubations were injected sub-cutaneously using 25 gauge needles into 4–6 week-old Swiss Webster mice. Thirty-four days post-infection, the mice were euthanized and the portal system perfused: worms were counted and sexed .
Isolation of RNA and cDNA, and qRT-PCR
Procedures for isolation of parasite RNA, reverse transcription to cDNA, primer design and amplification efficiency, and qPCR analysis have been detailed previously . Briefly, parasite material was washed three times in PBS, transferred to Trizol reagent (Life Technologies), flash frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated using the Trizol method up until phase separation in chloroform. The RNA in the aqueous phase was further purified using the RNeasy Mini Kit (Qiagen) according to the supplier instructions. The concentration of RNA was determined using a Nanodrop ND-1000 Spectrophotometer. Single-stranded cDNA was synthesized from total RNA using SuperScript III reverse transcriptase (Invitrogen) and an oligo d(T)18 reverse primer according to the manufacturer's protocol. The resulting cDNA was stored in water at −20°C.
Primers for gene expression analysis were designed with the Primer 3 software (http://frodo.wi.mit.edu ) using the parameters described previously . A BLAST analysis was run to confirm the specificity of each primer. Primers were purchased from Elim Biopharm at the 200 µM scale without further purification. The PCR amplicon generated by each primer pair was visually inspected by 1% agarose gel electrophoresis to ensure the presence and correct size of a single PCR product. The amplification efficiency of each primer pair was evaluated as described ,  and only those primers generating a single dissociation peak were employed. Table S1 lists the primers used for dsRNA synthesis and qPCR analysis.
QPCR was carried out as described  using the LightCycler 480 SYBR Green I Master mix (Roche Diagnostics) in an MX 3005P Real-Time PCR cycler (Stratagene). S. mansoni cytochrome C oxidase I (GenBank accession number, AF216698) served as the sample-normalizing gene transcript , . Controls for genomic DNA contamination (minus reverse transcriptase) and reagent purity (water control) were included for each sample. Duplicate reactions were carried out in 96-well plates for each cDNA sample in a final volume of 20 µL (Stratagene). Using the MxPro qPCR Software version 4.01 (Stratagene), the 2−ΔΔCT method  was employed to compare transcript levels following dsRNA treatments. Transcript levels were expressed as a percentage relative to those after exposure to mCherry dsRNA.
Measuring caspase activities after incubation with statins or HMGR dsRNA
Somules were exposed to statins or dsRNA as described above and then assessed for caspase activity as described . Parasites were washed once in PBS and mixed with 100 µL lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 and 1% deoxycholic acid containing 1X Halt protease inhibitor cocktail (Pierce)). After centrifugation at 1000 g for 10 min to remove debris, 100 µL of caspase reagent containing the peptide substrate, benzyloxycarbonyl (Z)-DEVD-rhodamine R110, were added to the supernatant and the reaction was agitated in the dark at room temperature for 4 h. Fluorescence (excitation/emission: 496/520 nm) was measured in 96-well black plates (Corning Inc.) using a FLEXstation (Molecular Devices).
Adult worms exposed to statins were washed in PBS. Two worms were then ground up using a disposable plastic pestle in a 1.5 mL tube containing 100 µL lysis buffer and 1X Halt protease inhibitor cocktail. After centrifugation, 100 µL of caspase reagent were added to the supernatant and the reaction agitated in the dark at room temperature for 4 h. Fluorescence was measured as described above.
Statins kill schistosomes in a concentration- and time-dependent manner
For six commercially available statins, dose-response curves, based on the appearance of somule degeneration, were prepared both manually and by an automated machine-learning approach (Figure 1). ED50 values were broad. Thus, for the automated method at Day 4, values ranged from 0.05 and 0.06 µM for simvastatin and lovastatin, respectively, to >10 µM for pravastatin. The process of degeneration was consistent among the statins whereby the normally translucent and flexible parasite (Figure 1 panel A) became darkened and ovoid with decreased movement (Figure 1, panel B). Death was invariably the consequence of continued incubation. The dose-response data calculated using the automated method for the six statin compounds correlated significantly with those generated manually: Table 1 lists the Pearson product-moment correlation coefficients obtained between the two sets of curves, as well as the significance (p-value) of the correlation determined by Student's t-test.
Newly transformed somules were incubated out to four days in the presence of the concentrations of statins indicated. The percentage of degenerate worms was measured both manually (dashed curves) and using a new automated algorithm (solid curves) as described in the text. Points represent means ± S.D. across two independent experiments each in duplicate. ED50 values are inserted above the curves. Mevalonate (100 µM) prevented worm-kill by 1 µM simvastatin and is indicated by the single square point. Image panels display somules cultured for four days; (A) DMSO control, (B) 1 µM simvastatin (indicating the rounded and darkened (degenerate) response) and (C) 1 µM simvastatin plus 100 µM mevalonate. Bar in Panel A = 100 µm.
Degeneration of somules was also time-dependent. For example, with simvastatin, degeneracy appeared by Day 2 of the incubation at the greatest concentration of 10 µM and was essentially complete (i.e., dead parasites) by Day 4 at both 10 and 1 µM (Figure S1). Parasite degeneration by simvastatin (Figure 1 panel C and lower right panel) or lovastatin (not shown) was preventable by incubation with excess (100 µM) mevalonate, the product of HMGR. Thus, SmHMGR is the relevant statin target. Rescue by mevalonate was concentration-dependent with a minimum of 100–200 µM being required for maximal protection of the parasite (Figure S2).
Likewise for adult S. mansoni, simvastatin was the most potent drug whereas pravastatin was without effect (Figure 2). Thus, by Day 2 in the presence of 20 µM simvastatin, worms had become slower and darker, and did not adhere to the culture well floor (severity score of 3): the other statins were without apparent effect. By Day 4, simvastatin at 20 µM had shrunk and immobilized the worms, and the parasite tegument had been disrupted (a maximum severity score of 4). By Day 6, the worms were dead. From Day 4 through Day 6, the other statins caused some darkening and slowing of the worms, although, with the exception of lovastatin a severity score of 2 was never exceeded. Incubation of worms for three days with 2 mM mevalonate prior to the addition of 20 µM simvastatin and lovastatin prevented worm degeneration (Figure 2).
Adult worms were incubated out to six days in the presence of the concentrations of statins indicated. A severity score was devised based on previously employed phenotype descriptors that record changes in movement, shape, translucence, surface integrity, and the ability of the parasite to adhere to the floor of the culture plate well . Each descriptor was assigned a score of 1, except where damage to the parasite's surface was evident, in which case the maximum score of 4 was awarded. For each concentration tested, each bar (from left to right) represents simvastatin, lovastatin, atorvastatin, fluvastatin, rosuvastatin and pravastatin. At the bottom, co-incubation with mevalonate (2 mM) for three days prior to the addition of 20 µM simvastatin or lovastatin prevented worm degeneration. Representative data from two independent experiments are shown.
RNAi of HMGR is specific to the target
The above data suggest that HMGR is the relevant molecular target for the cidal activity of statins. To confirm this, we applied transient RNAi as a reverse genetics strategy to target HMGR transcription. In advance, however, we formally demonstrate that HMGR is expressed in somules and other developmental stages (Figure S3). Incubating somules for seven days in the presence of 30 µg/mL HMGR dsRNA (added every third day) decreased HMGR transcript levels by 50% (Figure 3). RNAi of HMGR was apparently specific as no off-target modulation of S. mansoni cystatin or cathepsin B1.1 transcript levels was noted, findings that are consistent with our previous data using transient RNAi in somules .
Newly transformed somules were incubated with HMGR- or mCherry-dsRNA for seven days as described in the text. RNAi was measured by qRT-PCR and data expressed relative to data for mCherry dsRNA controls. To assess for potential off-targeting by HMGR-dsRNA, the expression of a S. mansoni cysteine protease inhibitor, cystatin (Cys: AY334553.1), and the gut-associated cysteine protease, cathepsin B1.1 (AJ506157), was also measured. Bars represent means ± S.D. across two independent experiments each in duplicate. Using Student's t-test, gene suppression in the HMGR-dsRNA treated sample is significantly different from the mCherry controls (p<0.01).
RNAi of HMGR in vitro kills somules, an outcome that can be prevented by mevalonate
No phenotype was evident by the seventh day of incubation with HMGR dsRNA when somules were harvested for qRT-PCR analysis. Therefore, we extended the incubation time in the hope that an obvious phenotype might appear and in the knowledge that HMGR transcripts were only decreased by 50%. We continued to add 30 µg/mL HMGR dsRNA and exchange the culture medium every three days. By day 16, a small but non-significant (relative to mCherry controls) number of the parasites incubated with HMGR dsRNA had become rounded, darkened and less motile (Figure 4). The progress of degeneration continued through the end of the experiment on day 25 by which time 62% of the parasites had degenerated relative to the 21% background recorded for parasites in the presence of mCherry dsRNA. Importantly, RNAi-induced degeneration could be prevented by including mevalonate (500 µM) in the incubation, thus identifying HMGR as the relevant target.
Newly transformed somules were incubated for the times indicated in the presence of mCherry-dsRNA (control) or HMGR-dsRNA with and without the presence of 500 µM mevalonate, as described in the text. Bars represent means ± S.D. across two independent experiments each in duplicate. The number of degenerate worms in the HMGR-dsRNA-treated samples taken on Day 25 is significantly different from both the mCherry controls and HMG-dsRNA-treated samples that had also been incubated in the presence of mevalonate (Student's t-test; p<0.01). Image panels display somules on Day 25 incubated with (A) mCherry-dsRNA (control), (B) HMG-dsRNA and (C) HMG-dsRNA plus 500 µM mevalonate. Arrowheads highlight a dead (left) and degenerate worm (right). Bar in panel A = 100 µm.
RNAi of HMGR in vitro limits subsequent parasite survival in vivo
We asked whether somules exposed in vitro to HMGR dsRNA would thrive when transferred into mice, which are typically used as an animal model of schistosome infection. Somules were incubated in the presence of mCherry or HMGR dsRNA that had been added twice daily at 60 µg/ml for 2.5 days and then injected sub-cutaneously into mice. Mice were sacrificed 34 days later and the hepatic portal system perfused to recover parasites. Irrespective of the dsRNA employed, all parasites recovered appeared normal in size, shape, motility and translucence (not shown). However, relative to mCherry dsRNA controls, the number of worms exposed to HMGR dsRNA was decreased by 81 and 80% for males and females, respectively (Figure 5).
Newly transformed somules were incubated for 2.5 days in the presence of mCherry dsRNA or HMGR dsRNA as described in the text. Parasites were then injected subcutaneously into Swiss Webster mice (5 mice per dsRNA treatment). After 34 days, mice were euthanized and the portal system perfused to recover parasites for counting. Bars represent means ± S.D. across two independent experiments. Using Student's t-test, worm recovery after exposure to HMGR-dsRNA was significantly different from mCherry controls (p<0.01).
Chemical and reverse genetic targeting of HMGR induces apoptosis in somules
To understand the mechanism associated with the lethality of statins to S. mansoni somules and adults, we assayed for the activation of caspases as an indicator of apoptosis, which is a common outcome upon inhibition of HMGR in cancer cells (see Discussion). Somules incubated for two days in the presence of 10 µM simvastatin showed a small but non-significant increase in caspase activity compared to DMSO controls (Figure 6A). By Day 4, however, the activity was five-fold that of control. For both time points, mevalonate at 100 µM prevented the activation of caspase activity and the non-cidal statin, pravastatin (10 µM) was without effect.
A. Newly transformed somules were incubated in the presence of either 10 µM simvastatin (±100 µM mevalonate) or pravastatin for the times indicated. Caspase activity was then measured using the fluorescent substrate Z-DEVD-R110 as described in the text and is expressed relative to DMSO controls. Bars represent means ± S.D. across two independent experiments. Significance (p<0.05) was measured using Student's t-test and is indicated with an asterisk. B. As for A, but employing adult worms. Bars represent means ± S.D. across two independent experiments. Significance (p<0.05) was measured using Student's t-test and is indicated with an asterisk.
Adult parasites that had been incubated for two days with 10 µM simvastatin showed a six-fold increase in caspase activity compared to DMSO controls (Figure 6B). By Day 4, activity was a non-significant eight-fold that of control. For both time points, the non-cidal statin, pravastatin (10 µM), did not significantly activate caspase activity over DMSO controls.
We also assayed for activation of caspases in somules exposed to HMGR RNAi. Somules were incubated in the presence of HMGR or mCherry dsRNA as described above. On day 25, caspase activity was measured in supernatants of lysed worms. Compared to mCherry-exposed controls, RNAi of HMGR induced a four-fold increase in caspase activity: mevalonate at 500 µM prevented caspase activation (Figure 7).
Somules were incubated with dsRNA to HMGR (±500 µM mevalonate) or mCherry for 25 days. Caspase activity was then measured using the fluorescent substrate Z-DEVD-R110 as described in the text. Data are expressed as the fold increase in caspase activity relative to somules exposed to mCherry dsRNA. Bars represent means ± S.D. across two independent experiments. Significance (p<0.05) was measured using Student's t-test and is indicated with an asterisk.
Here, we demonstrate that statin drugs, which are now ubiquitously employed to treat hypercholesterolemia, kill S. mansoni somules and adults in vitro. Statins target human HMGR, the rate-limiting step in the mevalonate pathway that is responsible for biosynthesis of a number of key products required for cell growth and survival, including cholesterol, farnesyl pyrophosphate and geranyl pyrophosphate , –. We show that the SmHMGR ortholog is the relevant target for the schistosomicidal activity of statins as worm-kill is prevented by excess mevalonate, the product of HMGR. RNAi of HMGR kills the parasite and, again, lethality is prevented by providing excess mevalonate. Finally, we demonstrate that HMGR is crucial to the survival of somules upon their transfer to mice post-RNAi of HMGR in vitro. Parasite death, either via statins or specific RNAi of HMGR, is associated with apoptosis. Together, our chemical and reverse genetics data demonstrate that S. mansoni HMGR is an essential gene and the relevant target of statin drugs which may form the basis for a new anti-schistosomal drug therapy.
In terms of the methodologies employed to quantify the cidal activity of statins against somules, the data generated manually were cross-validated with a novel automated approach that involves biological imaging and machine learning using support vector machines. The approach, novel in its application to drug screening for schistosomiasis, can monitor phenotypic changes in multiple parasites simultaneously and provides for determination of complete dose- and time-response curves, including ED50 values. The description of the automated machine learning-based approach along with experiments on a larger dataset of statin and non-statin drugs will be the subject of a separate report.
Our decision to investigate statins and SmHMGR as a possible therapeutic axis for schistosomiasis was triggered by our previous phenotypic (whole-organism) screen of a small molecule collection that included drugs approved for use with humans . Specifically, statins emerged as one of several drug classes that consistently damaged and killed somules after seven days at 1 µM. Our subsequent search of the literature for precedent regarding statins, HMGR and schistosomes identified in vivo  and in silico  studies that further suggested their relevance. Finally, of a number of reports by Bennett and colleagues , , perhaps the most pertinent to this discussion was the demonstration of near cure of adult and juvenile S. mansoni infections in mice that had been provided ad libitum with lovastatin (a.k.a. mevinolin) mixed into mouse chow . Thus, our present data are consistent with and expand upon those from other sources, and encourage the continued exploration of statins and the SmHMGR target to develop a new chemotherapy for schistosomiasis (see below).
We determined that statins and/or RNAi of HMGR induce apoptotic caspase activity in schistosomes as measured with peptidyl (DEVD) caspase substrates. Caspases-3 and −7 (ACU88129 and ACU88130, respectively) are expressed in S. mansoni  and the existence of an apoptosis pathway in S. mansoni  and S. japonicum has been described . Apoptosis is a typical outcome for many cancer cell lines exposed to statins in vitro whereby blockading of what is often a dysregulated mevalonate pathway  leads to a myriad of downstream effects. Indeed, the abundant pre-clinical evidence for statins as potential anti-cancer agents has been translated to over 100 clinical trials currently registered at the ClinicalTrials.gov website (operated by the United States National Institutes of Health; queried on October 10th 2013 with the terms ‘statin’ AND ‘cancer’) to test statins either as a monotherapy or in combination with other agents. Among the defects caused by statins is the defective prenylation of small GTPases (e.g., Ras, Rho and Rac) that are necessary for correct cell division , , . In schistosomes, small GTPases belonging to the Ras superfamily that regulate development are farnesylated. Specifically, RhoA, also known as Rho1, which is geranyl-geranylated, may contribute to regulating the dynamics of the actin cytoskeleton –.
Interestingly, of the statins known to induce apoptosis in cancer cells in culture, pravastatin stands out as being generally the least effective . Pravastatin is the most hydrophilic of the statins in use and is not readily taken up by cancer cells unlike the other more lipophilic drugs. In like fashion, we noted pravastatin's inability to kill both adults and somules, and induce caspases. Indeed, the statin ED50 values against somules in vitro trended with lipophilicity whereby the lipophilic statins (simvastatin>lovastatin ≈ atorvastatin ≈ fluvastatin> rosuvastatin) were generally more active than the hydrophilic pravastatin . Likewise for adult worms, simvastatin and pravastatin were consistently the most and least active of the drugs tested, respectively.
As judged by manual query of the S. mansoni genome (GeneDB, Version 4.0), a complete mevalonate pathway is encoded by the parasite (Figure 8). In addition to SmHMGR, there are a number of other enzymes downstream in the pathway that could be interrogated for development of an anti-parasite therapy based on clinical precedent in oncology (farnesyl transferase –) and osteoporosis (farnesyl diphosphate synthase , , ). Indeed, these interrogation points are also under evaluation for treatment of other parasitic diseases. Thus, bisphosphonate inhibitors of farnesyl diphosphate synthase have been investigated as anti-trypanosomal agents ,  and farnesyl transferase inhibitors inhibit the growth of Plasmodium falciparum, Leishmania mayor, Trypanosoma cruzi, Giardia lamblia and Entamoeba histolytica , .
The enzymes are numbered in bold typeface and their respective products are boxed; synthetic inhibitors are indicated in red. GenBank entries for each enzyme are: 1- CCD58635; 2- CAZ30720; 3- CCD79354; 4- CCD76423; 5- CCD60126; 6- CCD78373; 7- CCD81269; 8- α-subunit- XP_002580188; β-subunit- CCD82339; 9- α-subunit- CCD77887; β-subunit- CCD79051. FTI = farnesyl transferase inhibitor; GGTI = geranylgeranyl transferase inhibitor.
In light of the specific requirement for short course (preferably single dose) oral therapy of helminth diseases (incl. schistosomiasis) of humans , , , the current data for statins suggests that we are still short of an acceptable efficacy profile. First, using lovastatin, Chen et al.  could only demonstrate clearance of S. mansoni from the mouse infection model with an extended dosing regimen of 14 – 17 days and using high concentrations of lovastatin (estimated at 640 mg/kg/day provide ad libitum in mouse chow). By contrast, a single, oral, 500 mg/kg dose of the current anti-schistosomal drug, PZQ, kills >90% of adult worms in mice . Second, in the present in vitro work, whereas > 60% of somules had degenerated by the fourth day of incubation in the presence of 1 µM of most statins, adult parasites were more resilient, i.e., only simvastatin at 20 µM caused severe damage on Day 4, whereas the other five statins had a relatively minor impact. This concentration is well above the approximately 0.1 µM maximal plasma levels of statins used to treat hypercholesterolemia based on a once daily chronic dosing regimen . Encouragingly, however, no dose limiting toxicity has been noted in humans up to plasma levels of 12.3 µM lovastatin when administered every six hours over four days . In fact, maximum plasma levels were achieved within 24 h  suggesting that, under a relatively acute dosing regimen, plasma levels of statins that are safe yet parasiticidal may be achievable. Moving beyond the commercially available statins, there is also likely considerable scope for improving selectivity and inhibition kinetics toward the schistosome HMGR in a formal drug development program employing a statin library and de novo chemical synthesis. In support of a program to optimize a ‘schistostatin’ is the approximately 50% primary sequence difference in the catalytic domains of the human (SQ1L_A) and SmHMGR (AAA29896.1) orthologs that presumably contributed to the demonstration of differences in inhibition kinetics between the enzymes using a small series of statin analogs .
A relevant consideration for developing statins as anti-schistosomals is their cidal activity against immature parasites, both in vitro (see above) and in a mouse infection model , which stands in contrast to PZQ's well-known poorer efficacy against the younger developmental stages , . Not least, the possibility of a combination therapy with PZQ is worth investigating in one effort to preserve the clinical utility of the latter drug given the current momentum to expand its deployment as a monotherapy and the associated increased risk of resistance , . Finally, given the evidence surrounding HMGR and statins in schistosomes and the conservation of HMGR and the mevalonate pathway among helminths, statins may have potential as general anthelmintics; either as the preferred short-course therapy, or, for example with filariasis, for which longer drug administration regimens are under consideration , .
Time-dependent killing of S. mansoni somules by simvastatin in vitro. Newly transformed somules were cultured out to four days in the presence of simvastatin at the concentrations indicated. Time point data represent means ± S.D. across two independent experiments each in duplicate.
The prevention of statin-induced somule death by simvastatin using mevalonate is concentration-dependent. Newly transformed somules were cultured for four days in the presence of simvastatin at 1 µM and the concentrations of mevalonate indicated. Data are displayed as means ± S.D. across two independent experiments each in duplicate.
Sequences of primers employed for dsRNA synthesis and qRT-PCR.S. mansoni developmental stages. Data are displayed as means ± S.D. across two independent experiments each in duplicate. Transcript expression levels are normalized to that of cytochrome C oxidase I (GenBank accession number, AF216698) ,  and the data are displayed according to . NTS = newly transformed somules.
Conceived and designed the experiments: LRA TL DA BMS RS CRC. Performed the experiments: LRA TL DA BMS RS CRC. Analyzed the data: LRA TL DA BMS RS CRC. Contributed reagents/materials/analysis tools: LRA TL DA RS CRC. Wrote the paper: LRA TL DA RS CRC.
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domain: biology


text:
Date: Fri, 19 Feb 1999 00:12:32 -0600 (CST)
From: email@example.com (Rich Winkel)
Subject: Privatization in Bosnia
/** labr.global: 245.0 **/
** Topic: Bosnia Problems With Privatization **
** Written 10:59 PM Feb 17, 1999 by labornews in cdp:labr.global **
/* ————— “ECONOMY-BOSNIA: Problems of Privati” ————— */
SARAJEVO, Feb 14 (IPS)—Delayed and heavily criticised plans to privatize Bosnia's economy have come under renewed fire, this time from representatives of two of the country's religious communities.
In the three years since the end of the Bosnian war ambitious privatization proposals have been faulted for lack of transparency and incompetent drafting of relevant legislation.
There also was a widespread belief that politicians and well-placed civil servants were scheming to buy-up assets at bargain-basement prices.
While the pace of privatization has been glacial in the Serb Republic—one of the two entities into which Bosnia-Herzegovina was divided by the 1995 Dayton Peace Agreement—the parliament of the Muslim-Croat Federation, the other entity, recently adopted a privatization law which opened the way for the sale of public housing.
The process was complicated by the fact that legal tenancy in many cases has been disputed following the forced migration of large numbers of people during the three and-a-half year civil war.
Vouchers to buy privatized state assets started being issued this week, after considerable delay and privatization of the banking sector is scheduled to begin next month.
The process was stepped up at the end of January when Carlos Westendorp, the UN representative, charged with supervising the implementation of the Dayton Peace Agreement, inaugurated a Privatization Monitoring Commission, designed to resolve political deadlock and examine social issues raised by privatization.
This week Westendorp received separate open letters from leaders of Bosnia's Jewish and Muslim communities criticising the latest proposals to sell off state-owned property.
The religious leaders said that property confiscated by the pre- World War II Kingdom of Yugoslavia, as well as property stolen by the wartime Nazi puppet regime and property nationalised by the former communist regime, should not be put up for sale until provision was made to compensate the original owners.
At present, the government has proposed paying compensation, when it is deemed necessary, with vouchers, a form of scrip which has not impressed potential beneficiaries.
In the first post-Communist elections, held in 1990 “all political parties promised restitution,” said Jewish Community President Jakob Finci, “Everything which was taken under socialism was to be turned back over to the original owners.
“Now, after eight years, nothing has been given back, not communal property, not private property.”
Finci stressed that before the Holocaust in the 1940s, Jews in Sarajevo were the owners of “a respectable amount of property.”
“Nationalising things is like making scrambled eggs,” he said “ Restitution is like putting the eggs back together again; you can’t do it easily, but some mistakes can be avoided. You can provide cash instead of certificates and I’m against restitution in valueless paper.”
Similar arguments were used by Mustafa Ceric, head of Bosnia's Muslim clergy, who wrote to Westendorp arguing that “the process of taking property from the Islamic community dates from 1918.”
He said “more than 90 percent of the property of the Islamic community has been taken and the value of confiscated property is put at several billions of Deutsche Marks.” And he underlined the the confiscation of residential and institutional buildings owned by Islamic charitable foundations.
“If restoration of the property is not possible the law provides for compensation through vouchers, and it's well known that vouchers don’t represent fair compensation for property taken,” Ceric said in his letter. “We suggest that religious communities and property holders without any preconditions are given back their residential and commercial buildings.”
Ceric conceded that land which had been developed since it was expropriated could not be returned, but should therefore be the subject of compensation payments.
In the Islamic tradition, maintaining the property of charitable foundations is a religious obligation.
“A ‘vakuf’ (religious foundation) means a permanent charitable deed, lasting in the other world,” explained Professor Resid Hafizovic of Sarajevo University's Islamic Studies Faculty. “That type of property cannot be alienated or given to someone.
“If the Islamic community is the keeper of an old building, for example, their obligation is to maintain the building. If they have to sell it for some reason then they have to buy another building to be used for the same purpose.”
The obligation to maintain charitable foundations—and consequently to seek the restoration of property owned by such foundations—affects a considerable amount of property. In Sarajevo alone around 300 choice residential locations now in state hands and scheduled to be sold off are believed to have formerly belonged to the Islamic community.
The issue of religious property further complicates a Byzantine bureaucratic and political situation.
“The parties now in government promised fast privatization after they won the elections, but that hasn’t happened,” observed newspaper commentator Ibrahim Polimac. “Not more than 5 percent of apartments have been bought by citizens and people are waiting for certificates that are now a year late.”
Among the many practical obstacles to selling off property is the absence of reliable data on ownership. “Statistical information
is much worse than in other countries,” said Polimac, noting that Serb, Croat and Muslim-controlled local authorities have resisted the countrywide collation of information.
“You have three sources, everyone is giving his own source, plus agencies like the World Bank have their own statistics. So there is no exact data, just estimates.”

domain: sociology


Photo by Juan Ocampo
Here’s the official photo of the last diesel bus in Metro Los Angeles’ fleet being towed into retirement this morning.
As we posted earlier in more detail, Metro is now the first major transit agency in the world that has a fleet comprised entirely of clean-burning buses. That includes 2,221 buses that run on compressed natural gas, six gas-electric hybrids and one bus that is fully electric.
And what happens to the above bus? It’s being scrapped and holes are being punctured in its engine block so that it will never again run or burp out those very unlovely diesel fumes.
Categories: Policy & Funding


text:
After two years with no increase in Social Security benefits, senior citizens should see a cost-of-living adjustment in January, a nonprofit group says.
The exact size of the COLA is yet to be determined, but it should be somewhere between 3.5 and 3.7 percent, according to a new report from the American Institute for Economic Research (AIER), a nonprofit research group based in Barrington, Mass.
COLA is a measure that ensures Social Security benefits keep pace with inflation.
Why is that such a big deal? Before 2010, COLA had gone up every year since 1975, the first year that COLA increases went automatic. The size of the COLA automatically adjusts itself based on the annual percentage change in consumer prices. When consumer prices go up, COLA goes up.
But by the start of 2010, in the aftermath of the Great Recession, consumer prices over the 12 previous months had dropped. And before COLA could increase again, consumer prices had to be higher than they were at their peak. That didn’t happen for 2011.
“It’s important to remember that the typical older American today lives on an income of roughly $20,000, and Social Security keeps nearly a third of older Americans out of poverty,” said Tiffany Lundquist, spokeswoman for the AARP, a Washington-based advocacy group for seniors, in a statement. “After two years with no COLA and increasing costs for food, utilities and health care, every dollar of the modest average benefit of $14,000 is critical.”
When the Consumer Price Index goes down, COLA remains flat, because benefits cannot be reduced, by law.
But just because consumer prices and cost of living went down as a whole, that doesn’t necessarily mean that they went down for retirees and seniors in that period.
“The COLA index is based on average price of goods and services as consumed by workers, not by retired people,” says Polina Vlasenko, an AIER research fellow. “Retirees tend to spend more on health care and goods and services, and those prices increase faster than the national average. So COLA may not fully compensate for what that they spend their money on. The index isn’t ideal for retired persons, but it is what it is.”
“The exact size of the cost-of-living adjustment should be known on Wednesday or soon thereafter, when the Bureau of Labor Statistics releases the Consumer Price Index for Urban Wage Earners and Clerical Workers (CPI-W) for September 2011,” the AIER said in a statement.
The cost of living adjustment will trigger other changes, including an increase to the maximum individual earnings subject to Social Security taxes. Currently, an annual income of $106,800 is the maximum, but that number is likely to go up with the COLA increase.
The AIER also expects the increase to affect Supplemental Security Income benefits, which provide aid to the blind, disabled, and those 65 years of age or older with little or no income.
Schuyler Velasco is a correspondent for the Christian Science Monitor.

domain: news


text:
Find a Doctor
Michael Mason, DO, received his medical degree from the Philadelphia College of Osteopathic Medicine. He did his internship and orthopedic surgical residency at Community General Hospital in Harrisburg, Pennsylvania and he continued his subspecialty fellowship training at Massachusetts General Hospital and Brigham and Women’s Hospital, affiliates of Harvard Medical School. Dr. Mason has designed and been involved in the development of multiple artificial joints, and has more than 20 years of experience in joint replacement and reconstructive surgery, most recently at New England Baptist Hospital in Boston.
Dr. Mason is a board-certified orthopedic surgeon with Newport Orthopedics.

domain: medical


text:
CaPao, a mission-based brand created by Mondelēz International's SnackFutures innovation and venture hub, announced the launch of its new Quinoa Squares, which is also one of the first products on the market to carry the Upcycled Food Association's (UFA) Upcycled Certified™ mark on its packaging.
The new addition to the CaPao portfolio combines upcycled cacaofruit with oats and puffed quinoa into portable snack squares that are zesty and sweet, crispy yet chewy, and come in three flavors: Coconut Oat, Ginger Berry and Almond Orange.
In fact, according to the UFA, 60% of people want to buy more upcycled food products, and that's because 95% of us want to do our part to reduce food waste. Additionally, more than 50% of consumers had increased intent to buy Upcycled Certified foods when the mark was on packaging (Source: Mattson), already ranking better than other product certifications on the market. The Mark puts the power in the consumer's hands to reduce global food loss.

domain: food


text:
If many alternatives exist, engineers weigh Each and every design decision centered on their advantage and opt for the solution that ideal matches the requirements. The important and exceptional endeavor in the engineer is always to identify, realize, and interpret the constraints with a design and style so that you can generate a successful final result.
In this project, learners find out how strong state lighting engineering can Enhance the life of individuals inside the establishing planet, generating their evenings extra productive and safer. Inside the laboratory, The scholars design and style a conveyable, solar-charged, LED lamp that can provide two several hours of reading gentle.
The usefulness of such procedures is supported by modern analysis evidencing BRM techniques influencing project accomplishment from the strategic standpoint throughout distinctive nations around the world and industries.
Congratulations to product Miranda Kerr, who'll be a second time mom quickly, expecting her initial child with husband Evan Spiegel, In line with a report from People today.com.
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Be brief. Although some entries inside your lab notebook may well have to have in-depth notes, a lot of of your entries is going to be quick and concise. Comprehensive sentences will not be needed! Each and why not check here every scientist develops her own form of recordkeeping. What is significant is that you document enough information and facts so you totally fully grasp the notes you have made and so which the notes contains all important or vital facts.
All abroad enhancement and aid NGOs make sizeable utilization of engineers to use solutions in catastrophe and progress situations. Quite a few charitable businesses purpose to implement engineering directly for The great of mankind:
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Whether or not you might be striving to learn if engineering is a great vocation option for you, or in order to receive a taste for all times at KU, there is a KU College of Engineering celebration or camp for you.
A pc simulation of superior velocity air move all-around a Space Shuttle orbiter all through re-entry. Options into the move demand modelling with the combined outcomes of fluid circulation and the warmth equations.

domain: design


Element83 is behind plenty of amazing kinetic magnetic sculptures.”Elevation” is also worth a look. It is capable of levitating up to 6mm spheres or cubed magnets. This bismuth kinetic sculpture features dimagnetic levitation. Simply put the base on a surface, slip the magnet between the crystals, and adjust the top magnet to adjust the levitation.
Element83 Bismuth Magnetic LEVITATING desk toy
More gadgets like this: here
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- The deputy governor was caught with someones's wife
- MP Kaluma was among those who felt humiliated
- Kaluma claimed the lovers' right to privacy was breached
On a cool Tuesday, April 24, afternoon an embarrassing video of two adults who seemed to just have been from exchanging bodily fluids surfaced online.
And within no time, the identity of the sweaty fellow was revealed.
More shocking than not, the man was Kirinyaga Deputy Governor, Peter Ndabiri wa Njagi and someone's wife.
Send 'NEWS' to 40227 to receive all the important breaking news as it happens
Homa Bay Township MP Peter Opondo Kaluma was among those who felt dismayed and humiliated.
According to the lawmaker, recording videos of two lovers making out is immorally unacceptable and goes against the right to privacy.
"Right to privacy is guaranteed by the Constitution. This idea of recording people n** and posting them is immoral and unacceptable! It hurts innocent children and shuts their sense of security for life.
"How do you record and publish the nudity of your own wife in such a situation of total helplessness and weakness! No one is infallible," he posted.
It was not immediately established whether the two were ensnared but judging from the viral video, the deputy governor was reaping from where he did not sow.
In a rare kind of scene, the Kirinyaga deputy governor and the woman were busted by her hubby who had been accompanied by a group of men.
Together, they took the woman and Njagi on a torturous journey as they questioned them amidst caning.
According to what TUKO.co.ke gathered from the questioning session, Njagi regretted his move and asked for forgiveness claiming he did not know the woman was married.
Do you have a hot story or scandal you would like us to publish, please reach us through firstname.lastname@example.org or WhatsApp: 0732482690 and Telegram: Tuko news.
Willy Paul Interview for Tuko Buzz: Willy Paul Reveals it All | Tuko TV


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The sister of the man accused of the deadly knife rampage in Sydney has spoken outside the family’s home, saying she is sorry a young woman died in Tuesday’s attacks.
“She was younger than me. She could have been going out within a few hours, to do shopping with her mum, eat dinner with her boyfriend, go speak to her girlfriends … and now she can’t do that, can she?” she said.
“All the words that I can say isn’t ever gonna bring her back, is it? I want to say, I’m really, really sorry. No one should have … she was defenceless.”
— Nine News Sydney (@9NewsSyd) August 14, 2019
Mert Ney, from Marayong, in Sydney’s west, is expected to be charged with murder and other serious offences when he is released from hospital on Wednesday afternoon.
The body of a woman was found at a Clarence Street apartment on Tuesday afternoon, shortly after Mr Ney was sensationally halted by “heroic’ bystanders on his rampage through Sydney’s CBD.
She has since been identified as Michaela Dunn, 24.
The ABC has been told that Ms Dunn, a sex worker, had been sharing the apartment with another woman, also understood to be a sex worker.
Police sources said the flatmate came back to the apartment yesterday afternoon because she had not heard from Ms Dunn, and found her dead with fatal cuts to her neck.
Joan Westenburg, a friend of Ms Dunn, has paid tribute on Twitter, while lamenting violence against women at the hands of males.
’’I’ve known this girl since she was just a kid in high school at 14. Her sister and I were together for 6 years, and Mikki was like my baby sister. I cannot describe how sad and how broken I am at this moment. I loved this kid. She was incredible,” she tweeted.
‘‘I am angry that male violence has taken another victim. Selfishly I am so angry that the victim is a young woman I deeply respected and cared about. Mikki was a true delight to know. The violence in this country cannot continue. It cannot.
‘‘I will not stand for her to be attacked as a sex worker. Sex work is work. She was working. She deserved to be safe while she worked. Her job does not make her a lurid story. She was a person and she is now gone. And she is gone because male violence has taken her from us. I have gone so far beyond angry today. I have gone so far beyond sad.
“Australia has a brutal and deadly problem with violence against women. And i miss her.’’
Australian Associated Press understands the stabbing victim is Linda Bo, 41.
Mr Ney will also face other charges – including serious assault – after a stabbing and chase through the CBD.
Police say they have not yet ruled out terrorism as motive for the knife attacks in the city shortly after lunchtime on Tuesday.
On Wednesday morning, investigators trawled through Mr Ney’s electronic devices, social media accounts and the Marayong family home as they tried to determine why the 20-year-old sought to kill and injure innocent people on Tuesday afternoon.
Mr Ney remained under police guard in hospital. He has not yet been interviewed by police about the incident that brought the city’s streets to a standstill.
He was detained by bystanders who gave chase after he was seen wandering the streets and jumping on a car, brandishing a bloodied knife. He was quickly pinned to the ground under a milk crate and a cafe chair until police could arrive.
NSW Police Commissioner Mick Fuller said the rampage had not been classed as a terrorist incident and that the “lone actor” had no known links to terrorist organisations.
Mr Ney was, however, carrying a USB stick with information about recent mass casualties in New Zealand and the US.
While he had a history of mental health issues and an “unremarkable” criminal history, his motive remains unclear.
“Having some footage saved on a USB drive is not a leap far enough for me to say that this is a terrorist incident – but obviously it’s extremely concerning and it is the starting point of a long-term investigation,” Mr Fuller said.
“What he has on social media, what we may well find in his bedroom, will hopefully give us, and shine more light on, why this crime happened.”
Mr Ney’s sister said she did not believe he subscribed to extremist ideology.
“All this stuff about extremism, white supremacy, stuff like that, that’s news to me … it was using religion as an excuse,” she said.
On Wednesday, detectives searched Mr Ney’s family home in Marayong and a property in Blacktown, where it was believed he had been staying.
Mr Ney’s family had reported him missing and had concerns for his welfare following a domestic violence incident.
“That is not unusual in terms of what we see day-in-day-out in some houses across NSW [but] that is not a common theme for someone to then take the next step of coming onto the streets of Sydney with a knife and killing people and threatening to kill people,” Mr Fuller said.
Mr Ney has had surgery at Royal Prince Alfred Hospital for a cut to his knee. Police hope he will leave hospital on Wednesday afternoon.
“If we can link him to terrorist activity, then the offences that follow would see him locked behind bars for a very, very long time,” Mr Fuller said.
The commissioner again praised the people who helped restrain the 20-year-old, labelling them “true heroes”.
“They saved lives,” he said.
Commenting on the stabbing victim, who remains in a stable condition in Sydney’s St Vincent’s Hospital, Mr Fuller said it was a “miracle” she was alive.
Police Minister David Elliott said he will ensure police consider any exposure Mr Ney had to government services in recent months as part of their investigation.
“We want to make sure that we learn any lessons that need to be learnt from this particular occasion,” he said.
Police have also appealed for any CCTV, dashcam, and mobile phone vision of Tuesday’s stabbing rampage.
The 41-year-old woman is in a stable condition in hospital.


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One of the greatest actors to have ever existed. He has performed exceptionally well in several movies including: Koi Mil Gaya, Krrish, Kaho Na Pyaar Hain, Dhoom 2, and several others.
Hrithik often comes second to many actors including Shahrukh Khan
, Saif Ali Khan
, and Abhishek Bachchan
; but some feel as though he has revolutionized the entire world of Bollywood.
He has dared to do films that no one would have ever attempted in Bollywood.

domain: celebrity


Pierce County prosecutors on Wednesday charged a 32-year-old man with first-degree murder in the Sept. 7 fatal shooting of Michael Ward Jr. outside a Tacoma party.
Vernon Lewis Curry Jr. was arrested Tuesday and arraigned Wednesday in Pierce County Superior Court. A not guilty plea was entered on his behalf. Court Commissioner Meagan Foley ordered him jailed in lieu of $2 million bail.
Police believe Curry was the masked man who walked up to Ward’s car in the 3800 block of South Yakima Avenue and opened fire on him.
Officers later found a ski mask near the scene that they say had Curry’s DNA on it, court records show. Curry’s DNA is on file with the state after a previous felony conviction.
Curry told officers he had nothing to do with Ward’s killing, the records show. He admitted owning the ski mask but told officers he had no idea how it got to the shooting scene.


The RERC on AAC partnered with four student engineering Learning Factory teams at Penn State University in the fall of 2016. The student teams met with David McNaughton on a weekly basis, and developed engineering solutions to communication related challenges. All students were undergraduates in biomedical engineering, computer science, computer engineering, and mechanical engineering. All projects were presented at the Penn State Learning Factory showcase, and will be submitted to the RESNA Student Design Competition for 2017.
Using Head-Tracking and Eye Movement to Control a Cursor in a GUI: The Penn State student team of Ikenna Okafor, Nathan Scribano, David Sun-chu, Hui Xu, Susmita Sanyal and Darshan Shah worked with Tom Jakobs and Ethan Williams of Invotek to address the challenge of integrating information from head- and eye-tracking technology to control a cursor using the Intel RealSense camera. The goal was to develop and evaluate multimodal input strategies to assist individuals with minimal movement ( e.g., persons with ALS, spinal cord injury) in using a computer. The students developed a working prototype solution that will contribute to RERC on AAC project D1: Developing multimodal technologies to improve access
AAC Home Wifi Alert Device: The Penn State student team of Philip Mancuso, Lucas Gramig, Moyo Williams, Tim Lagnese, Ryan Nunamaker, and the Belgium Campus (South Africa) student team of Stacey Nunez, Shane Tshabalala, and Sithembile Ngxongo worked with Anthony Arnold (a person who uses AAC) and Jessica Gormley (Speech Language Pathologist) to develop an inexpensive and durable personal alert system that could be used by persons with complex communication needs to send text messages to others during emergencies. The students developed a working prototype solution that will be further developed in upcoming Learning Factory projects.
Electrolarynx-Holder to Support Single Switch Activation: The Penn State student team of Stephen Monaco, Ian Robbins, and Dhananjay Bhaskov partnered with Dr Richard Hurtig (Voxello) to develop an Electrolarynx holder that could be used by individuals who are temporarily unable to hold the electrolarynx in position for themselves, and who need single switch activation for operation. The students developed a working prototype solution that will be shared with manufacturers of electrolarynx related medical equipment.
Single Switch Technology for Teaching Adolescents to Scan: The Penn State student team of Eric Kilmer, James Udischas, Katelyn McGee, and Meghan Reigel developed an online tool to assist adolescents with severe disabilities to learning how to scan using a single switch. The online tool supports uploading of personally relevant images, and scanning speed and the type of scanning array used can be individualized. As part of their project, the team visited the headquarters for Tobii-Dynvavox in Pittsburgh, PA. The students developed a working prototype solution that will be further developed in upcoming Learning Factory projects, and shared with manufacturers.


This is a very simple Tutorial to make a Sock Owl. I’m using the leggings sock… the one that goes from your ankle up to your knees. See photos below from left to right. Measure 5″ from the rubber and cut. We only need this part, the rest can be kept for other toys ;-). Turn the sock around and sew a straight line on the rubber part. Ones done, go to the other side (where we cut just now), and fold the opening into 3 section like the photos. The section that I am holding in the photo is about 1 cm and it’s the tail of the owl. Then continue to sew the other section (the legs sections should be longer that the tail). Turn the socks around. Here is that body of the owl. Now, use some felt to cut the eyes and nose. Sew the felt on the owl as shown in photos below. Then (refer bottom panel of photos), cut 2 pieces of felt and 2 pieces of socks in another colour (you can also use fabric). Use simple stitches to sew the pink socks on the felt, then sew them on the sides of the owl.
Related: Sock animals


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SIMILAR BOOKS BY CATEGORY
LINK FROM YOUR SITE
100 pages (Summer 2007); 910KB downloadWOWIO Books
; ISBN: WOWIO-00242
The Second Part of King Henry the Fourth continues the storyline of Part 1 with no break in time or action. Prince Hal continues to be a concern to his ailing father, the King; and Falstaff looks forward to the day of Hal's accession to the throne. Shakespeare presents Prince Hal's preparation for the burden of the crown by following dual storylines: he alternates between the world of the court, a world in which Hal seems unsuited to be king; and the world of the tavern, a world in which Falstaff reigns. The two old friends meet in the final scene, and Hal must choose between his old life and a life in which he is responsible to himself and England. It is in this final scene that Hal truly becomes Henry V.
This edition is based on the 1914 Oxford edition, which ranks among the most authoritative of the 20th century.

domain: entertainment


text:
Every day, Tim Hortons team members serve millions of Canadians. As Tim Hortons faces a new era in the quick service restaurant industry, the company is launching a new campaign to help find more than 5,000 individuals across Canada to join the organization's restaurant team. The campaign includes in-restaurant, digital, and print advertising, as well as regional hiring initiatives including job fairs and restaurant hiring events.
The official kick-off to the campaign took place Tuesday in Calgary, where Tim Hortons owners are looking to hire 280 individuals. As part of the launch, Tim Hortons surprised a local neighborhood by transforming a residential home overnight into “The Tims Next Door.” Throughout the morning, neighbors enjoyed a cup of coffee and got to know their local team members, who had the opportunity to give back to their local guests.
"At Tim Hortons, the success of our business relies heavily on our front-line team members, whose energy and dedication serve as the foundation for the exceptional service our guests have come to expect from our restaurants," says Steve Wuthmann, executive vice-president of human resources. "Our restaurant owners are always looking to offer talented, reliable individuals fulfilling employment opportunities that allow them to feel challenged, satisfied and valued in their roles. I encourage Canadians to visit their local Tim Hortons, where owners and managers are on-hand to accept applications and conduct on-the-spot interviews."
According to a survey by Restaurants Canada, 65 percent of restaurant operators in Western Canada said labor shortages were having a negative impact on their business. With a workforce of approximately 100,000 team members in more than 3,630 restaurants across Canada, through its franchise owners, Tim Hortons is the country's largest quick service employer. Tim Hortons is continuously enhancing its efforts to attract employees whose skills and positive energy will serve them well in the fast-paced restaurant environment. This week, Tim Hortons revised its team member dress code policy so that employees can now have visible, non-offensive tattoos.


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Pan Natural Cosmetic Acnicare Lotion
Olchaen for healing skin problems acne with natural extracts.
To enhance the peeling off of Comedone adds radiance to the skin healthy.
Olchaen heal the skin. Additional copies of the fall of Comedone helps care acne problems from it. And dirt clogged facial. With continued exposure will help reduce the accumulation of acne Comedone make the future down. With extracts from natural Mangosteen Extract, along with NANO TT-M innovative of the Pan Cosmetic value of natural herbs. With the combination of nano-particles of Tea Tree Oil and perfectly Emblica Extract helps maintain acne problems arising from it. And dirt clogged. The skin look healthy. Also adds radiance to the skin healthy.
How to use:
paint an New Slovenia Healthcare Corporation after the cleanser around your face 2 times a day morning - cold without washing out.
Size: 20 ml
Contact Name: Michael Wendt
Company Name: Asienservice
Street Address: Leo Residence Room 1001
39M1 Road 304, Tamboon Ladtakiern
Kabinburi / Prachinburi 25110
Member name: asienservice
Member Since: 06 April 2009
Total Leads: 5341 asienservice Import Export Business Leads
Business focus: Distributor, Cosmetic 100% Nature Biologically, Demeter, Food, Washing Cleaning, Chemicals, Steel, Paper, Rice, Fresh Fruits, Sugar
Verify: Safe Import Export Tips
Product Category: Cosmetics

domain: health


text:
Winning images show ‘two sides of law enforcement coin’
Talented photographers have shared a diverse vision of law enforcement from across the world as part of the 11th annual Europol photo competition.
Europol received entries depicting police officers, rescue teams, customs and border guards during moments of their working life – from difficult operations and training exercises, to touching moments during everyday community work.
The agency received entries from as far afield as Australia and Colombia, showing two sides of the law enforcement coin: “dramatic and critical, as well as human and warm”.
Entries were invited across three categories – cybercrime, diversity and inclusion, and making Europe safer – although no winners were chosen for cybercrime. Instead two prizes were given for images depicting officers making Europe safer.
Winners of the 2019 competition were announced on Thursday (October 31).
The diversity and inclusion award was given to Giovanni D’ Amico from Italy for the above warm shot taken in Turin, showing a Carabinieri officer taking the time to get to know a member of the public.
Making Europe safer category
The first award in this category shows a French National Police explosives dog with his handler securing a site. He is pictured here by David Mendiboure.
The second award in this category goes to Peter Monteny from the Netherlands. In this picture, Dutch police officers are keeping an eye on the crowd at Utrecht Central Station – making people, including children, feel safe.
The three winners won a trip for two to the Netherlands, including a visit to Europol. Their pictures will feature in its 2020 calendar and other publications.
The competition is open to law enforcement photographers from the European Union and from countries with Europol cooperation agreements and aims to raise awareness of the work carried out by the law enforcement network across Europe.