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Clostridium perfringens causes several diseases in domestic livestock, including necrotic enteritis in chickens, which is of concern to the poultry industry due to its health implications and associated economic cost. The novel pore-forming toxin NetB is a critical virulence factor in the pathogenesis of this disease. In this study, we have examined the regulation of NetB toxin production. In C. perfringens, the quorum sensing-dependent VirSR two-component signal transduction system regulates genes encoding several toxins and extracellular enzymes. Analysis of the sequence upstream of the netB gene revealed the presence of potential DNA binding sites, or VirR boxes, that are recognized by the VirR response regulator. In vitro binding experiments showed that purified VirR was able to recognize and bind to these netB-associated VirR boxes. Furthermore, using a reporter gene assay, the netB VirR boxes were shown to be functional. Mutation of the virR gene in two avian C. perfringens strains was shown to significantly reduce the production of the NetB toxin; culture supernatants derived from these strains were no longer cytotoxic to Leghorn male hepatoma cells. Complementation with the virRS operon restored the toxin phenotypes to wild type. The results also showed that the VirSR two-component system regulates the expression of netB at the level of transcription. We postulate that in the gastrointestinal tract of infected birds, NetB production is upregulated when the population of C. perfringens cells reaches a threshold level that leads to activation of the VirSR system.
Clostridium perfringens is a Gram-positive, anaerobic, spore-forming rod that secretes many toxins and extracellular enzymes. It is widespread in the environment and in the gastrointestinal tracts of both humans and animals. In addition to human diseases, such as gas gangrene and food poisoning (33, 34), C. perfringens is also the causative agent of several enterotoxemic diseases of animals, including necrotic enteritis (40, 41). In chickens, necrotic enteritis presents as either an acute clinical or subclinical disease and has a significant global economic impact; it is estimated to cost the international poultry industry approximately US$2 billion each year (43, 45). The acute form of this disease is believed to involve several predisposing factors (44) that allow C. perfringens type A to multiply to high numbers in the small intestine, and it results in the concomitant elaboration of extracellular toxins that damage the intestines. Tissue disruption ranges from macroscopic lesions on the small intestinal mucosa to extensive necrosis of the mucosal surface in severe cases (45).
Early studies suggested that alpha-toxin was the major virulence factor involved in the pathogenesis of necrotic enteritis (1-3). Although this toxin has been shown to be critical in human gas gangrene (4), recent virulence tests with chickens using defined chromosomal alpha-toxin mutants clearly demonstrated that it was not essential for necrotic enteritis (20). A novel toxin, NetB, was identified and shown to be a critical virulence factor in this disease. C. perfringens netB mutants were unable to induce disease in chickens, which was reversed by complementation with the wild-type netB gene (19). Both native and recombinant forms of NetB were cytotoxic to chicken Leghorn male hepatoma (LMH) cells in vitro but not to several other cell lines. A gas gangrene strain that does not produce NetB but produces alpha-toxin and perfringolysin O (PFO) was not toxic for LMH cells (19).
At present, no information is available regarding how the production of this key toxin is regulated. In the human gas gangrene isolate, strain 13, the production of alpha-toxin and perfringolysin O, the toxins implicated in gas gangrene, is regulated by the VirSR two-component signal transduction system, which comprises the VirS sensor histidine kinase and its cognate response regulator, VirR (9, 22, 38). Since its initial identification, it has become the best-characterized two-component system in the clostridia. It is a global regulatory network, since it not only regulates toxin production but controls the expression of housekeeping genes (7, 18, 29, 39) and genes encoding several regulatory RNA molecules (7, 31, 39).
The VirSR phosphorelay appears to be mediated by quorum sensing (30). In this system, it is proposed that threshold concentrations of a peptide that is synthesized and secreted by the product of the agrBD genes are detected by the VirS sensor histidine kinase. Autophosphorylation of VirS is followed by phosphotransfer to VirR (9). The VirR response regulator then recognizes and binds independently to two imperfect directly repeated sequences, called the VirR boxes, located upstream of the target genes (12). We have previously shown that VirR directly activates transcription of the perfringolysin O structural gene, pfoA, and that the maintenance of the integrity, spatial organization, and helical phasing of the VirR boxes is crucial for optimal levels of perfringolysin O production (10, 12). VirR boxes are located upstream of several other genes in three different strains of C. perfringens, and we have shown that VirR recognizes and binds to these alternative binding sites in vitro and activates reporter gene expression in vivo (10, 26).
In this article, we report the identification of VirR boxes upstream of the netB gene in the necrotic enteritis strain EHE-NE18, which led to the hypothesis that expression of netB, and hence the subsequent production of the toxin, is regulated by VirSR. Using in vitro binding assays and reporter gene expression assays, we show that these VirR boxes are functional. Mutation of the virR genes of EHE-NE18 and a second necrotic enteritis-causing strain, 56, reveals that the VirSR two-component signal transduction system is involved in the regulation of NetB production. Furthermore, quantitative real-time PCR (qRT-PCR) analysis of RNA isolated from EHE-NE18, its isogenic virR mutant, and a complemented derivative demonstrates that netB expression is regulated at the level of transcription. In summary, these results have revealed another toxin gene that is regulated by the VirSR system, thereby adding to the repertoire of genes in the ever-expanding VirSR regulon.
Bacterial strains, plasmids, and growth conditions.The bacterial strains and plasmids used in this study are shown in Table 1. Escherichia coli strains were grown at 37°C in 2× YT agar or broth or SOC broth (36) supplemented with chloramphenicol (30 μg/ml) or erythromycin (150 μg/ml). C. perfringens strains were cultured at 37°C in brain heart infusion broth (Oxoid), Trypticase-peptone-glucose (TPG) broth (35), fluid thioglycolate medium (FTG) (Difco), or nutrient agar supplemented with 30 μg/ml chloramphenicol (NACm30), 7 μg/ml or 50 μg/ml erythromycin (NAEm7 or NAEm50, respectively), or 10 μg/ml rifampin and 10 μg/ml nalidixic acid (NARif10Nal10). For the screening of perfringolysin O (PFO) production, C. perfringens transformants were grown on horse blood agar (HBA) (22). All agar cultures of C. perfringens were incubated in an atmosphere of 10% (vol/vol) H2 and 10% (vol/vol) CO2 in N2.
Molecular techniques.Plasmid DNA from E. coli cells was routinely isolated using an alkaline lysis method (25). When DNA was used for sequencing, an additional polyethylene glycol precipitation step was included, as outlined in the instructions of the Prism Ready Reaction DyeDeoxy terminator cycle sequencing kit (Applied Biosystems). Restriction endonucleases and other enzymes were used as specified by the manufacturer (Roche Diagnostics or New England Biolabs). All oligonucleotide primers are listed in Table 2.
Competent E. coli (17) and C. perfringens (37) cells were prepared and transformed as described previously unless otherwise indicated. C. perfringens genomic DNA was isolated from 5 ml FTG broth cultures as was done previously (28). PCR amplification was carried out as described previously (10). PCR products were either purified directly using the QIAquick PCR purification kit (Qiagen) or extracted from agarose gels using the QIAquick gel extraction kit (Qiagen), according to the manufacturer's instructions.
Cloning of the netB VirR boxes into pJIR2373 was carried out as described previously (10). The resultant construct, pJIR3573, was used to transform the C. perfringens pfoA mutant, JIR4228 (Table 1), to yield JIR12345.
Construction of the vector used in targetron mutagenesis, pJIR3566.As part of another ongoing study, a new targetron vector was constructed for the mutagenesis experiments reported here. The vector, pJIR3566, utilizes a clostridial promoter and contains an ermB retrotransposition-activated marker (ermB-RAM). To construct pJIR3566, the Pgdh promoter region from Clostridium difficile strain 630 was PCR amplified using primers JRP2712 and JRP2713. The resultant 207-bp fragment was then digested with HindIII and SphI and cloned into the targetron vector pJIR750ai (8), generating pJIR3753. The RP4 oriT region was PCR amplified from plasmid pJIR1456 (21), cloned into pCR2.1-TOPO (Invitrogen), digested with SphI and SpeI, and subcloned into the XbaI and SphI sites of pJIR3753, resulting in pJIR3754. The replication region of pMTL540F (15) was then excised using NheI and BstZ17I and subcloned into the FspI and SpeI sites of pJIR3754, leading to the construction of pJIR3464.
To facilitate one-step selection of chromosomal integrants, an ermB retrotransposition-activated marker (ermB-RAM was constructed by a series of stepwise splice overlap extension (SOE) PCRs (16) and cloning reactions that led to the insertion of the td group I intron and its minimal exon sequences (13) in reverse orientation within the ermB coding region, immediately downstream of the ermB start codon (Table 2). The native promoter of the ermB-RAM cassette was subsequently replaced with the strong Clostridium pasteurianum ferredoxin promoter (Pfd) (pJIR3756) to maximize expression of ermB, and the modified cassette was cloned into the MluI site of pJIR3464 to give pJIR3562. Finally, to facilitate blue/white screening of retargeted targetron elements, the 5′ (retargeted) end of the group II intron within the resultant plasmid, pJIR3562, was replaced with the lacZα gene, leading to the construction of the final clostridial targetron plasmid, pJIR3566.
Construction of the virR targetron plasmid.To identify potential targetron insertion sites, the nucleotide sequence of virR was submitted to the Sigma TargeTron design site (http://www.sigma-genosys.com/targetron/ ). Of the predicted sites, insertion into the sense strand at position 546/547 from the ATG start codon in virR was selected for targetron modification. To retarget the group II intron, primer-mediated mutation by PCR was carried out with the IBS, EBS2, EBS1/δ, and EBS universal primers (Table 2) in accordance with the instructions from the TargeTron gene knockout system (Sigma-Aldrich), with the exception that Phusion High-Fidelity DNA polymerase (NEB) and FailSafe buffer E (Epicentre Biotechnologies) were used in place of the JumpStart REDTaq Ready mix. The 350-bp gel-extracted retargeting PCR product was digested with HindIII and BsrGI and ligated into pJIR3566 DNA that had been digested with the same enzymes and alkaline phosphatase treated. The ligation mixture was used to transform E. coli TOP10 cells (Invitrogen), and plasmids from a selection of the resultant transformants were isolated and sequenced. A vector, pJIR3608, that contained the desired altered nucleotides, was chosen for subsequent mutagenesis studies.
Construction of virR mutants using the Targetron mutagenesis system.Approximately 5 μg of pJIR3608 was used to transform C. perfringens strains EHE-NE18 and 56 by electroporation. Transformed cells were inoculated into 20-ml brain heart infusion (BHI) broths and incubated overnight at 37°C. The resultant cultures were subsequently passaged twice, over a 24-h period, in 20 ml of BHI broth. Transformants containing potential intron insertions were selected on NAEm7. Following overnight incubation at 37°C, selected colonies were patched onto NACm30 and NAEm7. Transformants that were resistant to erythromycin but sensitive to chloramphenicol were chosen for further analysis, since they represented derivatives that contained an inserted intron but were cured of pJIR3608. These transformants were also screened on HBA to assess the effect of the mutation on perfringolysin O production.
To show that the intron had inserted into the virR gene, genomic DNA was isolated as described previously (28) and analyzed by PCR with the primer pairs JRP2813/JRP182 or JRP4240/JRP4241. The former primer pair flanks the site of insertion within the virR gene, while the latter primer pair amplifies the ermB-RAM within the intron. To confirm the PCR results, Southern hybridization analysis was carried out. Briefly, genomic DNA of each strain was digested with BglII, separated by electrophoresis, transferred to a nylon membrane (GE Healthcare), and probed with PCR-amplified, digoxigenin (DIG)-labeled virR, ermB, catP, or intron-specific probes (Table 2). Blots were developed as per the manufacturer's instructions.
Perfringolysin O. C. perfringens strains were grown to late log phase before culture supernatants were collected by centrifugation as described previously (4). Perfringolysin O activity was determined by measuring the hemolysis of horse red blood cells in a doubling dilution assay, as described previously (42). The perfringolysin O titer was defined as the reciprocal of the last well that showed complete hemolysis, which was indicated by a significant decrease in absorbance at 570 nm. The PFO titers represent the means obtained from duplicate assays using supernatants derived from three independent cultures of each strain.
Gel mobility shift analysis.The 183-bp DNA target fragments (12) were PCR amplified with oligonucleotides JRP589 and JRP618 using the pfoA reporter constructs as templates. These DNA fragments subsequently were labeled with digoxigenin-11-ddUTP (Roche) as per the manufacturer's instructions. Gel mobility shift assays were performed with purified VirR protein as described previously (10, 12).
Isolation of RNA and qRT-PCR.EHE-NE18 cells were harvested at late logarithmic growth phase. Total RNA was isolated using the TriZol reagent (Invitrogen) as described previously (11). Conversion of 2 μg of RNA to cDNA and quantitative real time PCR (qRT-PCR) analysis were carried out as before (9) using rpoA or netB primers (Table 2). Total RNA or genomic DNA was isolated from at least three biological replicates from each strain and assayed in triplicate. The values obtained were normalized to that of the rpoA gene for each strain, and the results were expressed as a proportion of that for the wild type.
Western blot analysis.Two methods were used to analyze the production of NetB toxin. To determine when NetB toxin was produced, culture supernatants were harvested throughout the growth of EHE-NE18 in TPG broth. Each sample was separated by SDS-PAGE on a NuPAGE Novex 4 to 12% Bis-Tris gel (Invitrogen) in NuPAGE morpholineethanesulfonic acid (MES) SDS running buffer (Invitrogen). Proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (PALL) and probed with rabbit polyclonal rNetB antiserum (19). Blots were developed with the ECL Western blotting kit (Amersham Biosciences) as per the manufacturer's instructions.
To examine the production of NetB by EHE-NE18 and 56 virR mutants, culture supernatants were harvested at late logarithmic growth phase. The total protein concentration of the supernatants was determined using the bicinchoninic acid (BCA) protein assay kit. Approximately 200 μg of protein from each sample was separated by 12% SDS-PAGE. Proteins were then transferred and immobilized onto a nitrocellulose membrane (Millipore). The membrane was probed with polyclonal rabbit NetB antiserum (1:200) (19), followed by diluted (1:2,000) goat anti-rabbit antiserum secondary antibody (Millipore). Bound antibodies were detected using the Western Lightning Plus chemiluminescence reagent (Perkin Elmer) as per the manufacturer's instructions. Chemiluminescence was detected using X-ray film (Fujifilm).
Cell line and cytotoxicity assay.The chicken hepatoma cell line LMH (ATCC CRL-2117) was maintained in Earl's minimum essential medium (EMEM) supplemented with l-glutamine, 10% fetal calf serum (FCS), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), penicillin (100 U/ml), streptomycin (100 μg/ml), and Fungizone (100 μg/ml) (LMH cells also require 0.2% gelatin for adherence to surfaces) and incubated in a humidified environment of 5% CO2 at 37°C.
To test for cytotoxicity, LMH cells were cultured to 70% confluence in 96-well plates (Nunc) grown in EMEM growth medium, with 5% FCS and without phenol red, at 37°C. C. perfringens strains were grown in TPG broth to a turbidity at 600 nm of 0.6. Culture supernatants were obtained by centrifugation at 18,000 × g for 10 min and then dialyzed in phosphate-buffered saline. Volume-normalized, dialyzed culture supernatants were added to the LMH cell medium and incubated for 4 h at 37°C. Lactate dehydrogenase (LDH) release into the supernatant was measured as an indicator of cytolysis using the Cyto-Tox (Promega) kit and expressed as a percentage of cytotoxicity.
VirR activates pfoA reporter gene expression by binding to netB VirR boxes.Sequence analysis of the netB promoter region of EHE-NE18 revealed the presence of potential VirR boxes upstream of the putative −35 box (Fig. 1 A). The sequence and spatial organization of these potential VirR binding sites were similar to those of the VirR boxes located upstream of the pfoA (10), vrr, virU, virT, and ccp genes (31), all of which have been shown to be recognized by VirR (10) (Fig. 1B). Gel mobility shift experiments using the purified VirR protein and DIG-labeled target DNA demonstrated that VirR was able to recognize and bind to the netB VirR boxes in vitro. The two shifted bands, complex I (CI) and complex II (CII), were similar to those observed with the control pfoA VirR box region (Fig. 2 A).
To determine if the netB VirR boxes were functional, pfoA reporter plasmids were constructed as described previously (10) and used to transform the JIR325-derived pfoA mutant, JIR4228 (5). Perfringolysin O activity was used to measure the expression of the pfoA reporter gene. As expected, the results showed that the positive-control strain, which carried the pfoA VirR boxes cloned upstream of the reporter gene, produced levels of perfringolysin O activity that were similar to those of the wild type, while no activity was detected from the negative-control strain harboring the shuttle vector, pJIR751 (Fig. 2B). Background levels of perfringolysin O activity were observed from the strain carrying the pJIR2373 pfoA reporter gene vector, as previously observed (10). In contrast, a wild-type level of perfringolysin O activity was observed when the netB VirR boxes were cloned upstream of the reporter gene (Fig. 2B).
NetB is produced at high cell density.To determine when NetB is produced, Western blot analysis using polyclonal NetB antisera was carried out on culture supernatants that were isolated at various time points during the growth of strain EHE-NE18. The results showed that under these experimental conditions, NetB production begins 4 h after inoculation (logarithmic growth phase), with increasing production at later time points (Fig. 3).
Construction of virR mutants of EHE-NE18 and 56.Previous work showed that VirSR-regulated genes that have correctly spaced and positioned VirR boxes in the upstream promoter region are directly regulated by VirR (10, 12, 26, 31). To determine whether VirR regulated netB expression, the virR genes of two chicken necrotic enteritis-causing isolates, EHE-NE18 and 56, were insertionally inactivated by use of targetron technology (47). To aid in the construction of these mutants, we used a clostridial targetron vector, pJIR3566, which replicates in the clostridia, utilizes a strong clostridial promoter to drive targetron expression, and contains a retrotransposition-activated marker (47) that encodes erythromycin resistance to facilitate one-step selection of chromosomal integrants. Plasmid pJIR3566 also facilitates blue/white selection of retargeted introns due to the inclusion of the lacZα gene fragment between the HindIII and BsrGI restriction sites, which are utilized during the intron "retargeting" procedure.
Of the predicted targetron sites, the insertion site within the region encoding the VirR DNA binding domain (FxRxHrS motif) was selected, since we previously demonstrated that mutation of this motif eliminated VirR function (23). The vector containing the virR targetron, pJIR3608, was introduced into EHE-NE18 and 56 by electroporation. As a result of the presence of the ermB-RAM, transformants containing potential insertions were selected on NAEm7. Verification of targetron insertion into the desired site in the resultant mutants was obtained by PCR and Southern blotting (data not shown).
Mutation of the virR gene in the gas gangrene isolate strain 13 led to elimination of perfringolysin O production (38). Similar results were obtained with the EHE-NE18- and 56-derived virR mutants. When grown on HBA, the mutants showed no hemolytic activity (data not shown). This phenotype was confirmed by quantitative perfringolysin O assays. Compared to the wild-type strains, the virR mutants of EHE-NE18 and 56, JIR12405 and JIR12409, respectively, did not produce any detectable levels of perfringolysin O. Production of this toxin was restored to wild-type levels when the virR mutations were successfully complemented in trans by virR alone or by the wild-type virRS operon (Fig. 4 A). The assay results confirmed that the targetron insertion had successfully inactivated VirR function.
VirR regulates NetB production in necrotic enteritis strains EHE-NE18 and 56.Culture supernatants were assayed to determine the effect of the virR mutations on NetB production. Western blot analysis using polyclonal NetB antisera demonstrated that NetB was being produced by both EHE-NE18 and 56. In contrast, no NetB was produced by the previously isolated EHE-NE18 netB mutant (Table 1) and the strain 13 derivative, JIR325. Very low, almost undetectable levels of NetB were observed with the virR mutants of both strains. The production of the NetB protein was restored when the mutation was complemented in trans with the wild-type virR genes or with the virRS operon (Fig. 4B). These results demonstrated that the production of NetB in these strains was positively regulated by the VirSR system.
To quantitate the level of NetB toxin activity, supernatants were analyzed in an LMH cell cytotoxicity assay, using lactate dehydrogenase (LDH) release from the cells as an indicator of cytolysis (19). The level of cytotoxicity observed with culture supernatants derived from the virR mutants was significantly reduced compared to the wild-type controls. In contrast, effectively wild-type levels of cytotoxicity were observed when supernatants from the complemented strains were tested (Fig. 5 A). These results are consistent with those obtained in the Western blots and confirmed that NetB production was VirSR dependent.
To determine if regulation was mediated at the transcriptional level, the amount of netB transcript in EHE-NE18, its virR mutant, and the virR mutant complemented with the virRS operon was quantitated by use of qRT-PCR. The results showed that netB transcription in the virR mutant was significantly reduced compared to that in the wild type but that expression was restored to wild-type levels upon complementation (Fig. 5B).
In this study, we identified potential VirR boxes upstream of the netB gene in the chicken necrotic enteritis-causing strain, EHE-NE18. Comparison of the VirR boxes found upstream of the pfoA gene with those associated with the netB gene revealed some sequence variation, but despite these differences, VirR was still able to recognize and bind to the netB VirR boxes in vitro and stimulate the expression of the pfoA reporter gene in vivo. Taken together, these results indicated that the VirR boxes upstream of netB were functional and suggested that netB transcription was regulated by the VirSR two-component signal transduction system. This hypothesis was confirmed by the construction of virR mutant derivatives of two necrotic enteritis strains, EHE-NE18 and 56, and their complementation by either virR or virRS in trans. Reduced NetB production was observed in the virR mutants and restored in the complemented derivatives. Further analysis showed that the low level of NetB produced in the virR mutant of EHE-NE18 was consistent with the significantly reduced level of netB transcript in comparison to those of the wild-type and complemented strains. These experiments provide clear evidence that NetB toxin production is regulated at the transcriptional level by the VirSR system.
These findings have significant implications for our understanding of the pathogenesis of avian necrotic enteritis infections. The VirSR system may function in a manner similar to that of the staphylococcal Agr quorum sensing system (27). Recent studies have indicated that in C. perfringens strain 13, the VirSR system is important in sensing an extracellular signal that leads to the transcriptional activation of toxin genes (30). In the staphylococcal Agr system, the extracellular signal is an autoinducing peptide that is a derivative of an AgrD propeptide that is modified by AgrB upon secretion (32). The Agr system represents a quorum sensing system that is responsive to cell density. We have identified similar agrB and agrD genes in the strain EHE-NE18 genome (data not shown), and other workers have proposed that in strain 13, the product of these genes is the autoinducing peptide that activates the VirSR system (30). Furthermore, in this study we have shown that NetB is not produced constitutively; it is produced only when the cells reach late logarithmic phase. Therefore, we postulate that VirSR-dependent NetB toxin production in C. perfringens is responsive to cell density.
Based on the data obtained in studies on strain 13 (30, 46), the results presented here, and our understanding of the pathogenesis of avian necrotic enteritis (45), we suggest that in the normal avian gastrointestinal tract, the low levels of C. perfringens cells are not sufficient to lead to induction of the VirSR system and significant NetB production. However, when the birds are suddenly changed to a high-energy, protein-rich diet, the growth of C. perfringens is stimulated. Therefore, its population density increases and the amount of autoinducing peptide secreted into the lumen of the gastrointestinal tract increases to the point where its concentration is high enough to lead to the induction of NetB toxin production by the induction of VirS, which subsequently activates the VirR response regulator. This mechanism would represent a highly efficient environmental adaption, since cytolytic toxins like NetB would be produced only when the C. perfringens population density was high and there was an increased probability of subsequent nutrient limitation. We propose that as with other C. perfringens toxins, the primary function of NetB is to increase nutrient availability by releasing complex macromolecules from cells of the host, not to cause any disease pathology.
To date, most studies on the VirSR system have been carried out on human gas gangrene or food poisoning strains of C. perfringens. Recently other workers showed that exposure of a C. perfringens type C strain to CaCo2 cells leads to VirSR-dependent upregulation of β-toxin and perfringolysin O production (46), and we have now shown that NetB toxin production in avian isolates of C. perfringens is VirSR regulated. Taken together, these studies demonstrate that the regulation of toxin production by the VirSR system is not limited to human isolates but also extends to strains that cause disease in animals. Despite very significant differences in the diseases that these strains cause and the hosts that are affected, the method by which toxin production is regulated is essentially the same. These findings once more illustrate the global nature of this quorum-sensing VirSR two-component regulatory system and can again be interpreted on the basis that the major reason that C. perfringens strains produce specific hydrolytic and cytolytic toxins is to provide host-derived nutrients under conditions of high cell density.
This research was supported by the Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics with funding from the Australian Research Council and by grants from the Australian National Health and Medical Research Council and the Australian Poultry Cooperative Research Centre.
- Received 7 February 2010.
- Returned for modification 11 March 2010.
- Accepted 3 May 2010.
- Copyright © 2010 American Society for Microbiology


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ARTICLE SYNOPSIS...Charles White Of Tucson Asset Management by Thom Hartle
Charles White's career has ranged broadly from fixed-income portfolio manager to Commodity Trading Advisor. Today, as one of the three principals of Tucson Asset Management, White is doing it all a
AUTHOR:�Thom HartleDATE:�FEB�1998
ARTICLE SYNOPSIS...Gilbert Raff: From Medicine To Money Management by Thom Hartle
Stocks & Commodities readers have suggested that we interview someone who did not start his or her career in the financial community but was successful enough in the markets to change career
AUTHOR:�Thom HartleDATE:�MAY�1994
ARTICLE SYNOPSIS...John Bollinger Of Bollinger Capital Management by Thom Hartle
John Bollinger, C.F.A., C.M.T., former market analyst for CNBC/FNN, president and founder of Bollinger Capital Management, author of the ""Capital Growth Letter"" a market letter for the aver
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ARTICLE SYNOPSIS...Mary Pugh of Pugh Capital Management by Thom Hartle
The year 1994 will be known for the debacle in the derivatives markets. But what are these instruments that seem to have left a number of money managers holding the proverbial bag? To answer this quest
AUTHOR:�Thom HartleDATE:�FEB�1995
ARTICLE SYNOPSIS...The Value Of Valuation
Peter Mauthe Of Core Asset Management
Money management is simply a process of identifying what to own and when. While there are a number
of ways to do so, one unique way is the work of Core Asset Management's Peter Mauthe. STOCKS
AUTHOR:�Thom HartleDATE:�DEC�1994
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Australia, New Zealand create history with first day-night Test
Australia and New Zealand have created history by playing first day-night Test match at the Adelaide Oval Stadium ( New Zealand ). After 138 years, for the first time, the five-day game was played under lights and for this pink leather ball was used.
This match is a real departure from tradition, with the relatively untested pink leather ball replacing the standard-issue red for the first time in a format that dates back to the 1870s. The first day-night test match was the third match of the series between Australia and New Zealand played for 2015 Trans-Tasman Trophy.
Traditionally red ball is used for Test cricket. However, it is not as visible at night as during the day. Hence, pink ball will be used at Adelaide Oval. Kookaburra is the manufacturer of the pink ball. Australia have trialled pink balls in domestic matches and are satisfied with the results.
Test Number 2188 has a five-day limit, six-ball overs and a pink ball. Same 6 hours of play was witnessed as in all Test matches. 90 overs to be bowled per day.This match is being played as per recommendations of the MCC World Cricket Committee to keep the long format test cricket alive.
Idea behind hosting day-night test match
To keep the long format test cricket alive the idea to host day-night Test match par with One Day Internationals (ODIs) was mooted by Cricket Australia chief executive James Sutherland. It was approved by International Cricket Council (ICC), an apex body for regulating cricket in July 2015.
Significance of the match:-
World Series Cricket, established by Australian media baron Kerry Packer in the late 1970s, revolutionised the game when it threatened to go stale, using the one-day format, a white ball and coloured uniforms, and playing under lights for the first time in order stop the dwindling audiences of longest version of game.
With lack of crowds at stadiums for Tests, cricket administrators and players felt day-night matches are the way forward to attract fans back to the grounds the changes in timings of test matches would facilitate audiences to watch live second half of the day's action of test match on Television in prime time or watch it directly from ground after work.
Crticism :-
Critics say it won't be durable enough to last 80 overs because of colour deterioration as it gets scuffed and the white seam will make it difficult for batsmen because of a lack of contrast. There's also the question of whether the ball could swing considerably more during the moister, cooler evening sessions than it does during the day.


text: At first I thought the reason Edward didn't have one was because at the time it made sense. He was a sort of rogue assassin and the fact he didn't have the traditional beaked hood reflected that. But when we played the game every single assassin we met had the same regular hood and I thought maybe it was just the assassins solely in the west-indies that did this. Now the Unity trailer releases and it clearly shows the new protagonist doesn't have a beaked hood either. Of course we know nothing about what he fights for or what his role is in the order, but it's looking to be Ubisoft has just actually ditched the beaked hood but why? Someone that has become so iconic to just do away with doesn't make sense. I hope it's at least explained in the story and not just something they wanted to subtly remove like they did in black flag.


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5 Reasons the Google Pixel Phone Will Flop
The Google Pixel phone Google Unveils New Pixel Phones, Chromecast Ultra, & More... Google has unveiled a host of new hardware, including new Pixel phones, the Chromecast Ultra, the Daydream View, Google Home, and Google Wifi. Read More is finally here. Rumors had been swirling for months, but it wasn't until Google's unveiling event in early-October 2016 that its existence was officially confirmed.
The phone is being billed as an iPhone competitor. The old Nexus line of handsets were cheaper and more accessible to the masses, but the Pixel will go head-to-head with Apple on price, features, and build quality. We'll finally get the showdown we all dreamed about as the two most preeminent tech companies in the world get ready to do battle.
But will be Pixel phone be a success? I think there's a strong argument to suggest it'll be a giant flop. Here's why.
1. Price
What was the best thing about the Nexus range of phones and tablets? A lot of people would say it was the pure "stock Android" experience. A lot more would say it was the price.
The Nexus offered everything that was great about the Android operating system for a fraction of the price of the top-end Samsung models. No bloatware, no manufacturer overlays, and no exploding devices.
That "pureness" won't change with the Pixel, but the amount you'll be paying for it will.
The 32 GB entry model costs $649, and the 128 GB version will set you back $749. The entry model is exactly the same price as the newly-released iPhone 7 and just $20 cheaper than an unlocked Samsung Galaxy S7.
Compare that to the recent Nexus models. The Nexus 5 entered the market for $349 in 2013, and its successor, the 5X, was only $379 when it hit the shelves in 2015.
Whether or not long-time Nexus users will be prepared to shell out almost twice as much for a largely similar experience remains to be seen.
2. Distribution
At the unveiling event, the phone was only available for pre-order in five countries: the U.S., the U.K., Canada, Germany, and Australia. India was added to the list shortly after.
At this stage, there are no official plans to expand that market — and that puts it at a serious disadvantage.
Roll the clock back to 2008 when the first iPhone was released. Apple made it available in more than 80 countries worldwide. Today, there are very few countries where you can't buy an iPhone — they're mainly places such as Cuba and North Korea where the United States has trade embargoes.
The Pixel might sell very well in the areas where it's available, but the global sales figures won't be close to either Apple or Samsung.
3. Rooting
One of the reasons tech enthusiasts love Android so much is because of the customization options available 9 Amazing Android Customizations Worth Rooting For Haven't rooted your device yet? Once you get a look at all this awesome root-only customizations, you might change your mind. Read More .
Want a new launcher What Is the Best Free Android Launcher? Nova launcher, Apex launcher, and dodol launcher are all solid free launchers, but which is the best? We put them to the test. Read More ? No problem. Don't like being forced to use a certain built-in app Replace Samsung Apps With These Better Alternatives Whether you have a Samsung device and want to avoid the built-in apps, or you just want some of those cool Samsung features, these are apps you should check out. Read More ? Get rid of it. Want to overclock your device? Easy.
The catch? To be able to enjoy the vast majority of these customizations, you need to root your device. For those who aren't sure what I mean, rooting gives you to access the phone's entire operating system What Is Rooting? What Are Custom ROMs? Learn Android Lingo Ever had a question about your Android device, but the answer had a bunch of words in it that you didn't understand? Let us break down the confusing Android lingo for you. Read More . You can then install, tweak, and replace anything you want.
Sadly, rooting the Pixel will be harder than ever. This will be a shock to Nexus-lovers. Although the tide was starting to turn, the Nexus devices were historically some of the easiest to root and had some of the largest and most vibrant modding communities as a result.
Since the release of Android Marshmallow Android 6.0 Marshmallow: What It Is and When You'll Get It Android Marshmallow is here -- but why should you care? Read More , Google implemented a new check that would make the phone fail to boot if the /system file was modified. Modders got around the problem by editing the ramdisk, but according to reports, the ramdisk will be in the /system file on the Pixel.
Will the community find a way around the issue? Almost certainly. But the process is becoming less and less appealing to the average user.
4. Daydream
Say hello to Google's stab at virtual reality (VR). Called Daydream, it's one of Google's big selling points, and one they hope will revolutionize the way we use our devices. The software is baked into the Pixel phone, and the supporting headset is on sale for $79.
But do people want it Virtual Reality Still Has 5 Big Problems to Overcome Despite all of the hype surrounding the recent surge in virtual reality products, there are many challenges that still need to be addressed before VR goes mainstream. Read More ?
Sure, the advancements in VR over the last few years have been impressive. Devices from Oculus How the Oculus Rift Works and Why You Need One Why is there so much excitement over the Oculus Rift? Because it's a technological marvel. Here's how it works and why you're going to love it. Read More , HTC, and Sony have changed the way we think about the sector. But is mobile VR going to become a thing? The jury is still out. Maybe Google should've focused its efforts elsewhere.
5. Waterproofing
More and more phones are now becoming water resistant What Makes a Device Waterproof or Water-Resistant? A lot of devices are marketed as waterproof, but that can be severely misleading. Most are actually water-resistant. What's the difference? Here's everything you need to know about it. Read More (the days of pushing people into swimming pools are set to return!), but the Pixel's waterproofing is conspicuous by its absence.
The Pixel's two biggest competitors, the Samsung S7 and iPhone 7, are both water resistant. Apple's offering has an IP67 classification, which means it can be submerged in water to a depth of roughly one meter for up to 30 minutes. The Samsung phone goes one better — it's IP68, meaning it can be put into a depth of 1.5 meters for 30 minutes.
Why did Google omit what would certainly be a popular feature? You tell us!
A Success or Failure? You Decide
Will the Pixel range of phones be a long-term success? Very possibly.
The Pixel 2 — whenever it is released — is potentially the phone you should be waiting for. There is every chance that it will be the device that many onlookers hoped this Pixel would be. It will have more features, the teething problems from the original Pixel will have been ironed out, and Google will have a clearer idea of their long-term vision for the phone.
I've explained five reasons why I think the Pixel phone is doomed to flop, but I am sure many of you won't agree.
Whether you think my points are valid concerns or meaningless worries, I'd love to hear from you. Let me know your thoughts on the Pixel phone in the comments section below.
Related topics: Google, Google Nexus, Google Pixel.
Affiliate Disclosure: By buying the products we recommend, you help keep the site alive. Read more.


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CETNER LISLE, NY – Thunder Mountain Speedway wrapped up its 2019 season by celebrating the accomplishments of the speedway's most talented drivers. Over $30,000 was presented to those in attendance.
Shaun Walker picked up the $5,000 modified championship over runner up Alan Barker. Walker's consistent top five finishes helped him to solidify the grand prize. Alan Barker, Ryan Jordan, Nick Nye and Eric Leet round out the top five.
Dennis Clapperton and Chance Spoonhower battled most of the season for the open sportsman championship. In the end, the two tied for the title and each was presented with a $1,000 payday. Chris Jordan claimed third place followed by Tom Eiklor and Tyler Keener.
Past TMS 602 crate sportsman champion, Tommy Collins picked up the title for 2019. Kenny Hammond brought home a solid second place point finish followed by Dale Welty, Pat Jordan and Rich Talada.
Street Stock driver Earl Zimmer pulled double duty picking up the Thunder Mountain Speedway championship title as well as a championship at The Hill Speedway. Mike Wilbur, Trevor Dudley, Jerry Lobdell Jr., and Gene Sharpsteen round out the top five.
The 600cc modifieds saw returning 2018 champion Chris Jordan defend his title for the back-to-back championship win. He was in a tight point battle with the young Jared Green with just four points separating the two drivers. Third place went to Mark Golden followed by Justin LaDue and James Randall.
Matt Meade had a fantastic year taking home six victories and the championship title. Tyler Banks followed with Erica Bowman, Bryan Pospisil and Bryan Bartos rounding out the top five.
The IMCA Modified champion, Tyler Stoddard, overtook Matt Roberts by a small margin to claim his title. Third place went to Brandon Smith followed by Gary Roberts and Brad Smith.
The 4-cylinder stock point battle was also close between Eric Rowley and Stanley Mathews. In the end, Rowley would take the victory. Third place went to Dane Hedlund followed by Austin Edwards and Dakota Fuller for the top five.
Special awards
Rookie awards went to Eric Leet (Modified), Joe McCabe (Open Sportsman), Zachary Deidrickson (602 Crate), Shane Moshier (4cyl Stock).
Sportsmanship awards went to Matt Meade and Shayne Spoonhower who both gave up their cars for a fellow driver in the points chase.
The Forkey Fabrication & Construction Iron Man award was given to rookie sportsman driver, Bryan Bartos.
The Car Owner of the Year award was presented to Mike Costello, owner of the champion #21 Shaun Walker modified.
Racing legend Mike Colsten received the Dedication to Racing award for his contributions to racing he has made over his long racing career.
It was announced that Raymond Kennerup would take on the head flagger position for 2020. Kennerup has a strong background in racing and is looking forward to making the mountain his home on Saturday nights.
We look forward to rolling into the 2020 season with Hoosier Tire, VP Racing fuel and DIRTcar. Stay tuned for more exciting announcements and the 2020 race schedule.


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On These Shoulders
Thursday Nov. 15, 2012
High Tea with Marcus and Mary
By Ruth Broyde Sharone
The Interfaith Observer
TIO Correspondent Posted on Thursday, November 15, 2012 at 11:00AM
The English landscape rushed by the bus window, lush green hills alternating with roads that twisted and turned through leafy glens.
Excitement welled up in me, the possibility of resolving a mystery: Why is the Rev. Dr. Marcus Braybrooke, a retired Anglican parish priest living near Oxford and the author of more than 40 books on interfaith engagement – the acknowledged 'historian' of the interfaith movement – known to so few, even within the interfaith movement itself? To be sure, the movement has been spontaneous, not planned, so often the left hand has no idea what the right hand is all about. Still, the author of the classic Pilgrimage of Hope: One Hundred Years of Global Interfaith Dialogue (1993) needs to be known!
Today I was going to tea at the home of Marcus and Mary Braybrooke, hoping to shed some light on the mystery.
Dr. Marcus Braybrooke is president of the World Congress of Faiths, which he joined after receiving his Masters degree in 1964, affording opportunities while he was still young to meet religious luminaries from around the world. He served as executive director of the Council of Christians and Jews from 1984-1988, and he has been Patron of the International Interfaith Centre at Oxford. In September 2004, he was awarded a Lambeth Doctorate of Divinity by the Archbishop of Canterbury "in recognition of his contribution to the development of inter-religious co-operation and understanding throughout the world."
In the past half-century, Dr. Braybrooke has attended hundreds of conferences and interfaith gatherings, usually as a presenter.Despite illustrious credentials, though, many in the interfaith community, particularly the newcomers arriving all the time, have not heard of him. In part, Marcus' natural modesty doesn't seek out the limelight. Even at the global conferences, which he still attends month after month, Rev. Braybrooke keeps a low profile. You have to seek him out to find him, and I was about to find him.
My thoughts were interrupted as the bus driver turned to me, indicating we were nearing my destination. Marcus told me on the phone he would pick me up at the Oxford bus station, then drive us to the cottage he shares with his wife of 47 years, Mary, a social worker and a magistrate.
Brahma Kumaris Global Retreat Centre, Oxford -
I spotted him immediately – a bespectacled, ruddy-cheeked Englishman, congenial in every manner. Marcus was wearing a tweed jacket, v-neck sweater, and tie, a picture-perfect Oxbridge intellectual straight out of central casting. He wanted to make a quick stop before heading home to show me the Oxford headquarters for the Brahma Kumaris' community. They recently hosted an "Assisi-like" symposium for world religious leaders. We drove around theirsprawling property while he told me about his long-standing, close relationship with the leaders of the Brahma Kumaris, including their head administrator, Dadi Janki. As I would discover, Marcus is close friends with many world religious leaders.
Highlights from Marcus Braybrooke's Bibliography:
Pilgrimage of Hope: One Hundred Years of Global Interfaith Dialogue (1992)
This is the best history of the interfaith movement available, a 360-page tome surveying a hundred years interreligious relationships in great detail.
Faith and Interfaith in a Global Age (1998)
A much briefer interfaith history is offered here; it continues the Pilgrimage of Hope story through the five years following the 1993 Parliament of the World's Religions, and it reflects on major themes emerging from this new interplay of the world's religions.
1,000 World Prayers (2003)
This collection of prayers from all ages and dozens of different traditions is a testament to the spiritual impulse that runs throughout the human family, a river of wisdom and inspiration.
Peace in Our Hearts, Peace in Our World:
A Practical Interfaith Daily Guide to a Spiritual Way of Life (2012)
A practical guide to a peaceful heart and to helping create a more peaceful world. For each day of the year you'll find a quotation, a meditation, and a suggestion for practical action. It draws on the teaching of many world religions and spiritual traditions while considering the difficulties of the world in which we live. Available only by downloading.
Beacons of the Light: 100 Holy People Who Have Shaped the History of Humanity (2009)
Of all Braybrooke's books, this may be the best to live with. Short but substantive essays profile 100 religious-spiritual leaders who have made a difference in the story of humankind. They range from Abraham to Krishna, Confucius to Hildegard of Bingen, Aurobindo to Black Elk to Desmond Tutu. Taken alone, each profile is full of interesting information about people that are unknown or just names to most of us. Taken together, the scope and wisdom surveyed here is breath-taking.
At the Braybrooke cottage, Marcus's wife Mary came out to greet me and immediately made me feel at home. Photographs of the family were prominent everywhere in the living room, and their charming caramel-colored poodle, appropriately called Toffee, sat quietly in Mary's lap during the interview, as well-versed in interfaith etiquette as the rest of the family.
Together for nearly 50 years, Mary and Marcus have two children and six granddaughters.
She is his interfaith partner, they both emphasized. In fact, Mary was exposed to interfaith prayer services before she met Marcus, as a teenager attending Unitarian interfaith services in her church. "We thought it was the most natural thing to be working together with people of other faiths for justice and peace," she says. "Decades later interfaith services suddenly were considered a novelty," she points out bemusedly. "In the fifties we also studied world religions, but many people today believe that was a much later development."
Attracted to an Interfaith Model
Marcus traces his interfaith initiation to the sixties, when, after graduating from college, he studied in India with Hindu professors and lived with Hindu families. The year was formative for him, he confesses, especially visiting a leprosy clinic for the poor, run by a Hindu doctor with the help of a Christian from Sri Lanka and a local Muslim. "When I saw in India how religions could come together to help the less fortunate, I was profoundly affected by that interfaith model," he recalled. "I also found it extremely refreshing and unexpected that the Hindus in India spoke about direct experience with God, at a time when 'God is dead' was a catch phrase among Western Christian theologians."
As a student, Marcus noted that "the academic student of religions may be content with exterior dialogue." But his mentor, Fr. Murray Rogers, taught that the "exterior dialogue" needed to be accompanied by "interior dialogue" so that listening to people of another faith, observing their spiritual practices and studying their scriptures "should go hand in hand with inner reflection or dialogue with the Lord."
After being exposed for many years to the idea of "respect for the other,'' the main mantra of interfaith engagement, Marcus developed his own theory in an article called Mutual Enrichment in Dialogue and Alliance. In it he claims that respect for the other is not enough. Interfaith engagement at its best, he proposes, leads to "Mutual Enrichment" or "Mutual Irradiation" – an approach reflected in his Peace in Our Hearts, Peace in Our World (2012), an e-book.
Mutual Irradiation. His choice of words and their capitalization point to his belief that something of great import can take place in the interfaith process. Indeed, he seems to suggest that, when interfaith engagement is at its best, cellular changes will occur and the people involved will never be the same again.
"Interior dialogue is not a matter of comparing religions or trying to prove the superiority of one's own faith," Marcus underscores. "Mutual Enrichment both deepens and broadens one's faith." He quotes Christian missionary C. F Andrews, a friend of Gandhi and Rabindranath Tagore, who said that through deep contact with other faiths, "Christ has become not less central but more central and universal: not less divine to me, but more so because more universally human."
"Is religion life-giving or death-bringing?"
In the late seventies Marcus studied for a time in Israel/Palestine and soon afterwards became director of the (British) Council of Christians and Jews. Several of his books reflect the new and growing understanding and friendship between Jews and Christians. In 1997 he co-founded the Three Faiths Forum with Sir Sigmund Sternberg and Sheik Zaki Badawi. In recent years he has given special attention to relations with Islam and counts many Muslims as his friends. "If only we could have responded to the terrorist attacks on the Twin Towers and elsewhere with spiritual rather than military force, how different the last decade would have been," he conjectures.
Marcus took this photograph of three other interfaith pioneers, Paul Knitter, Maha Ghosananda, and Irfan Khan, protesting a massacre in Acteal, Mexico. "The violence of those who acted in the name of Allah was so unlike all that I had learned of Islam, from friendship with Muslims, from reading the Qur'an and above all the inspiration of the Sufis, that I continue to find it sad to see Islam so misrepresented in the media," he notes, and tells a story. "Some years ago a Muslim explained to me that the inner conflict within the world of Islam is between those who say 'God is most great' and those who say 'Islam is most great.' There is a struggle for the soul of Islam. So as a Christian I have felt it right to do what I can to help people discover the true Islam. As has been said, the question today is not so much which is the true religion - but what is true religion? Is religion life-giving or death-bringing?"
Marcus Braybrooke's prose is poetic and lucid as he explores complex subjects with a directness and eloquence that has to be read to be appreciated. In a field where many use quotes for substantiation, Marcus uses them for elucidation and inspiration. He has been profoundly impacted by the people he has met during a lifetime devoted to interfaith activism, and the personal experiences he accumulated, with his wife at his side, have informed his writing as much as his scholarship.
It was time for high tea. I was introduced to Mary's friend, Susan Dixon, at that time educational officer of the Abbey at Dorchester-on-Thames. The four of us sat down to tea and a table piled high with treats: a walnut cake that Mary had baked for the occasion, as well as chocolate cake, scones, English muffins, butter, and homemade jelly. In short, a sweet, high-caloric bacchanal.
It was a typical and colorful English ritual, celebrated for centuries. For me it was the last lap of my treasure hunt. Marcus Braybrooke, an exceedingly humble man of letters, was no longer a mystery to me. An important scholar, chronicler, and a grass roots activist and delightful raconteur all rolled into one, he will undoubtedly continue to enrich the global interfaith community, however many know him by name.
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Thanks for the informative and fun article HIGH TEA WITH MARCUS AND MARY
Those in the SF Bay area might want to know that Marcus followed Matthew Fox as one of the Principal Speakers at the 1992 Pacific Parliament of Religions in Berkeley... which was promoted both by the IARF and by NAIN. Meetings were at First Congregational Church of Berkeley and at the Pacific School of Religion. It was a warm-up to the 1993 celebrations of the Centennial Anniversary of the 1893 Chicago Parliament of the World's Religions.
Any of the over 20 books written or edited by Marcus is worth a read. His book PILGRIMAGE OF HOPE is probably the best single volume taking the reader through the 1893 Parliament and the 100 years of Interfaith celebrated in 1993. I have the copy which Marcus gave us in 1992, and am referring to it for the article I am writing on the IARF for THE INTERFAITH OBSERVER
Dr. Jim Wiggins
Monica Willard
JW Windland
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© TIO 2011. Credit The Interfaith Observer for reproduction of any materials from TIO. If TIO credits additional sources, do so as well.
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Super Turbo (GGPO)
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O. Sagat wins and Roo is the father.
Pick Cammy and spiral arrow to win Battle by the Bay FREE or visit the wiki Super Turbo wiki
CWheezy - Canada West Coast
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boxw3ll - US East Coast (Maine) - Guile P.S. I'm terrible
captain_broseph - US West Coast
PurpledrinksSerhcv - US Midwest (best st player in the universe)
YamiAtomsk - US West Coast
creepr - US West Coast
Mr igloo - US West Coast (Previously worst Blanka now currently worst Guile)


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Phone 01666 511854 or email email@example.com
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Mon, Nov 17, 2014
Le Méridien Dubai Hotel and Conference Centre in Dubai, UAE, has opened Le Royal Club, its new 196-room signature wing.
Built as part of a Dh450 million ($122 million) expansion, the exclusive wing was inaugurated yesterday by Sheikh Ahmed bin Saeed Al Maktoum, President of Dubai's Civil Aviation Authority, CEO and Chairman of Emirates Group and Chairman of Dubai Airports.
Spread across 38 acres of landscaped gardens, the eight-storey wing boasts some of the largest hotel rooms in the city. Measuring a spacious 52 sq m, each room is equipped with every modern comfort and the latest in technology.
In addition, personalised check-in lounges on each floor make guests feel welcome and relaxed.
Developed by wasl Asset Management Group, the annex houses a rooftop pool and an exclusive Club lounge.
It offers Le Royal Club benefits along with signature services such as a 24-hour valet service.
"Le Royal Club is an important new development for both wasl and for the city of Dubai," says Hesham Abdullah Al Qassim, CEO wasl Asset Management Group. "This is the first time our organisation has been involved in such a hospitality project, so it marks a major milestone for the wasl portfolio. It is also a significant development for Dubai's business travel segment, with the luxury annex situated in a prime location close to Dubai International Airport. This convenience, allied to its impeccable standards of appointment and services, means that we are expecting Le Royal Club to enjoy high occupancy rates."
"We are delighted to have opened the doors to our Le Royal Club as part of our expansion to cater to the growing segments of our business. By second quarter of 2015 we will be offering state-of-the-art event and conference facilities to complement the 580 rooms and 18 food and beverage venues of our hotel," says Ranjan Nadarajah, General Manager of the hotel.
Le Méridien, the Paris-born hotel brand currently represented by nearly 100 properties in more than 40 countries, is part of Starwood Hotels & Resorts Worldwide.
Bulgari to open hotel in BeijingTue, Aug 8, 2017
Hong Kong suite gets the party startedSun, Aug 6, 2017
Elements is a winner at luxury awardsThu, Aug 3, 2017
Corinthia London dishes out Arabian treatsWed, Jul 26, 2017
Dubai gets third DoubleTree by HiltonTue, Jul 18, 2017
Nobu opens doors in ShoreditchMon, Jul 3, 2017
Ritz-Carlton enters AstanaSun, Jul 2, 2017
Hilton resort on way for Yas IslandSat, Jul 1, 2017
Address to manage Makkah propertyThu, Jun 29, 2017
InterCon expands in Abu DhabiThu, Jun 22, 2017
Four Seasons Riyadh unveils Ramadan offeringsWed, May 24, 2017
Authentically AegeanSpring 2017
A haven for family escapesWed, Apr 26, 2017
Mandarin welcomes Geneva auction-goersTue, Apr 11, 2017
$600m luxury resort rebranded as Muscat BayThu, Apr 6, 2017


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This year's employee giving campaign is off to an exciting start. Raising Hope at Work now combines the UF Health Shands and UF College of Medicine campaigns, harnessing The Power of Together.
For more than a decade, employees have demonstrated incredible generosity through the Raising Hope at Work campaign. Last year, UF College of Medicine faculty and staff participated for the first time.
Brian L. Hoh, M.D., FACS, FAHA, FAANS, a UF College of Medicine neurosurgeon, was a Team Champion during last year's campaign, a title given to a department's team leader.
"The department of neurosurgery achieved 100 percent faculty participation and contributed nearly $400,000 in support of the UF Health Neuromedicine Hospital," he said.
Neurosurgery faculty members were inspired by the work and achievements of the department's first chairman and internationally renowned neurosurgeon, Albert Rhoton Jr., M.D., who passed away earlier this year.
"Every member of the neurosurgery faculty felt personally indebted to Dr. Rhoton for the legacy that he built here at UF," Hoh said. "It was important that we honor his work by contributing toward naming some part of the UF Health Neuromedicine Hospital in his memory."
Hoh added, "We were also inspired by the 100 percent participation of the UF Health Shands Hospital Neuro ICU Unit 82 and Neurosurgery Unit 11-5 nurses. We are all a team."
Justine Abram, M.S.N., R.N., CNRN, Neurosurgery Unit 11-5 nurse manager, emphasized the importance of investing in her team's future together.
"Our unit is committed to Raising Hope at Work because this is going to be our new home," she said. "We think of ourselves as a work family."
This year, employees can contribute to support the UF Health Heart & Vascular Hospital and UF Health Neuromedicine Hospital, or the George T. Harrell, M.D., Medical Education Building.
Stephan J. Moore, M.H.A., FACHE, CMPE, UF Health Shands Transplant Center administrative director, is a Team Champion for his area, and encourages his staff to participate by discussing the campaign at each meeting. Moore strives to personally thank those who participated in previous campaigns and show his appreciation for their commitment to UF Health.
"We are fortunate to be part of such a dynamic and growing academic medical center where we can truly participate in owning our future together by contributing in whatever way we are able to the campaign," he said.
As we work toward our greater goal of improving health care, we ask for your help. Please join Raising Hope at Work by supporting our latest initiatives in patient care, research and education. Your gift is an investment to create a strong and successful future for UF Health.


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Dear Kadeejah: Can I Keep Living With My Ex?
There's nothing more inconvenient than a breakup. And when you live together, it's not always easy to move on — literally. It's not just the shared experience you're leaving behind, it's the shared utilities, rent, and groceries that come with a price tag that looks so much sexier when it's split between two people. For some people, breaking up doesn't mean saying goodbye to these perks or each other.
I sat down with exes Ashley and John to get the scoop on why they chose to continue to cohabitate post-breakup. I'm a staunch defender of those who remain friends with their exes, but I had to take a pause when I heard about their bold living arrangement. John and Ashley may be doing the impossible, but frankly they're living the dream.
Take a look…


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[bibshow format=apa sort=author order=asc file=custom://data]
Sémagramme is a follow-up to the computational linguistic activities of the former INRIA team Calligramme.
The overall objective of the Sémagramme project is to design and develop new unifying logic-based models, methods, and tools for the semantic analysis of natural language utterances and discourses. This includes the logical modelling of pragmatic phenomena related to discourse dynamics. Typically, these models and methods will be based on standard logical concepts (stemming from formal language theory, mathematical logic, and type theory), which should make them easy to integrate.
The project is organized along three research directions:
Syntax-Semantics Interface
The Sémagramme project intends to focus on the semantics of natural languages (in a wider sense than usual, including some pragmatics). Nevertheless, the semantic construction process is syntactically guided, that is, the constructions of logical representations of meaning are based on the analysis of the syntactic structures. We do not want, however, to commit ourselves to such or such specific theory of syntax. Consequently, our approach should be based on an abstract generic model of the syntax-semantic interface.
Here, an important idea of Montague comes into play, namely, the "homomorphism requirement": semantics must appear as a homomorphic image of syntax. While this idea is almost a truism in the context of mathematical logic, it remains challenged in the context of natural languages. Nevertheless, Montague's idea has been quite fruitful, especially in the field of categorial grammars, where van Benthem showed how syntax and semantics could be connected using the Curry-Howard isomorphism [bibcite key=vanBenthem:1986]. This correspondence is the keystone of the syntax-semantics interface of modern type-logical grammars [bibcite key=moortgat:1997]. It also motivated the definition of our own Abstract Categorial Grammars [bibcite key=degroote:2001].
Technically, an Abstract Categorial Grammar simply consists of a (linear) homomorphism between two higher-order signatures. Extensive studies have shown that this simple model allows several grammatical formalisms to be expressed, providing them with a syntax-semantics interface for free [bibcite key=degroote:2004,degroote:2002,degroote:2003,pogodalla:inria-00107768,kanazawa-salvati:2007,retore:2010:inria-00409557_1].
We intend to carry on with the development of the Abstract Categorial Grammar framework. At the foundational level, we will define and study possible type theoretic extensions of the formalism, in order to increase its expressive power and its flexibility. At the implementation level, we will continue the development of an Abstract Categorial Grammar support system.
As said above, to consider the syntax-semantics interface as the starting point of our investigations allows us not to be committed to some specific syntactic theory. The Montagovian syntax-semantics interface, however, cannot be considered to be universal. In particular, it does not seem to be that well adapted to dependency and model-theoretic grammars. Consequently, in order to be as generic as possible, we intend to explore alternative models of the syntax-semantics interface. In particular, we will explore relational models where several distinct semantic representations can correspond to the same syntactic structure.
Discourse Dynamics
It is well known that the interpretation of a discourse is a dynamic process. Take a sentence occurring in a discourse. On the one hand, it must be interpreted according to its context. On the other hand, its interpretation affects this context, and must therefore result in an updating of the current context. For this reason, discourse interpretation is traditionally considered to belong to pragmatics. The cut between pragmatics and semantics, however, is not that clear.
As we mentioned above, we intend to apply to some aspects of pragmatics (mainly, discourse dynamics) the same methodological tools Montague applied to semantics. The challenge here is to obtain a completely compositional theory of discourse interpretation, by respecting Montague's homomorphism requirement. We think that this is possible by using techniques coming from programming language theory, in particular, continuation semantics [bibcite key=strachey-wadsworth:1974,barker:2002,barker:2004,shan:2004] and the related theories of functional control operators [bibcite key=felleisen-et-al:1987,felleisen-hieb:1992].
We have indeed successfully applied such techniques in order to model the way quantifiers in natural languages may dynamically extend their scope [bibcite key=degroote:2006]. We intend to tackle, in a similar way, other dynamic phenomena (typically, anaphora and referential expressions, presupposition, modal subordination…).
What characterize these different dynamic phenomena is that their interpretations need information to be retrieved from a current context. This raises the question of the modeling of the context itself. At a foundational level, we have to answer questions such as the following. What is the nature of the information to be stored in the context? What are the processes that allow implicit information to be inferred from the context? What are the primitives that allow a context to be updated? How does the structure of the discourse and the discourse relations affect the structure of the context? These questions also raise implementation issues. What are the appropriate datatypes? How can we keep the complexity of the inference algorithms sufficiently low?
Common Basic Resources
Even if our research primarily focuses on semantics and pragmatics, we nevertheless need syntax. More precisely, we need syntactic trees to start with. We consequently need grammars, lexicons, and parsing algorithms to produce such trees. During the last years, we have developed the notion of interaction grammar [bibcite key=guillaume-perrier:2009] and graph rewriting [bibcite key=guillaume:hal-01188694] as models of natural language syntax. This includes the development of grammars for French [bibcite key=perrier:inria-00184108_1,guillaume:hal-01188694], together with morpho-syntactic lexicons. We intend to continue this line of research and development. In particular, we want to increase the coverage of our grammars for French, and provide our parsers with more robust algorithms.
Further primary resources are needed in order to put at work a computational semantic analysis of utterances and discourses. As we want our approach to be as compositional as possible, we must develop lexicons annotated with semantic information. This opens the quite wide research area of lexical semantics.
Finally, when dealing with logical representations of utterance interpretations, the need for inference facilities is ubiquitous. Inference is needed in the course of the interpretation process, but also to exploit the result of the interpretation. Indeed, an advantage of using formal logic for semantic representations is the possibility of using logical inference to derive new information. From a computational point of view, however, logical inference may be highly complex. Consequently, we need to investigate which logical fragments can be used efficiently for natural language oriented inference.


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Bed and Break Fast Home Stays
in and around Valparai
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Karamalai Annai Vellankanni Church
Tour Pakages
Nearest Places of interest
Athirapilly Falls (Kerala)
Athirappilly is popular among tourists. Athirappilly Falls (Punnagai Mannan Falls) is one of the best places to visit in Kerala. Another popular waterfall to visit is the Vazhachal Falls.
Topslip is located above the 800 feet from the sea level on the Anamalai mountain range and in the Anamalai Tiger Reserve.
Parambikulam Tiger Reserve is the most protected ecological piece of Anamalai Mountain sub part of Western Ghats,
Aliyar Dam is a reservoir located 27 KM away from pollachi on the foothills of Valparai, in the Anamalai range of the Western Ghats, Park, aquarium and Monkey Falls are the best spots.
Places of Interest


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There's more than one reason to make financial plans. We have been helping individuals plot their own unique path in life for over 300 years, and we are on hand with the insight and expertise you need to help you set off in the right direction.
Getting to know you
Our Private Banking Service advisers will get to know you and provide you with a consistent point of contact. We will consider your lifestyle and priorities and point out the opportunities and challenges ahead.
We will get to know your banking and borrowing needs so you can decide which options suit you best.
Every day banking and specialist services
We will work with you to understand what elements of the banking service suit your lifestyle and ambitions. We offer the following banking services:
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- Mortgages, loans and overdrafts, subject to status and application.
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Our banking services are complemented by Internet, Mobile and Telephone Banking, 24 hours a day, seven days a week.
Please note that although our lines are always open, not all Telephone Banking services are available 24 hours a day. There may be limits on payments you can make and we may not be able to start processing all payments instructed out of normal banking hours.
Lending and financing products may require security. Lending is subject to our responsible lending criteria. How much we lend, the period and rate available are subject to our assessment of your circumstances.
Specific eligibility criteria and conditions apply for certain types of lending. You must be 18 or over and a UK resident to apply. Lending is subject to status and application. Security may be required. Overdrafts are repayable in full on demand.
Structuring and transferring your wealth
As a Private Banking customer, you will have access to additional products and services depending on your needs. Private Banking can introduce you to specialists that can help you get a clear idea of your current position and get appropriate advice for:
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Find out more about structuring your wealth
Financial Planning
Some of our products and services require financial advice. We provide this service through our partnership with Schroders Personal Wealth. We will introduce you to an adviser to help you make financial planning and investment choices based on your financial goals.
Your Schroders Personal Wealth adviser will explain what advice they can give. They'll also tell you which products and services this covers and any advice or product charges that apply and agree these with you.
Bank of Scotland plc and Lloyds Bank plc are both part of Lloyds Banking Group. More information on Lloyds Banking Group can be found at www.lloydsbankinggroup.com
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Registered Office: 25 Gresham Street, London EC2V 7HN. Registered in England and Wales No. 2065. Authorised by the Prudential Regulation Authority and regulated by the Financial Conduct Authority and the Prudential Regulation Authority under number 119278..


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The process of applying for grants has never been uniform or easy. Different foundations or funding bodies have different processes, rules, guidelines and eligibility requirements. That being said, hopefully the following considerations will help:
- Do your homework
This sounds simple, but in reality so many applicants don't take the small amount of time required to look up the website of the funder to find out what they do and don't support and the process they use in doing so. Rather than getting on the phone straight away, spend some time on website to find out more.
- Ask questions – don't pitch projects
If after reading the website and supporting information you find that you still left with questions, by all means get in touch and ask away. Please don't see this as an opportunity to pitch your project. Funders generally don't have the time to sit there and listen to applicants pitching projects. In fact, many avoid this in order to keep it fair for all applicants.
- Be courteous
This one should be standard as it is simply good manners. Being rude to any staff member will not work in your favour. The 'word' always gets around.
- Manage your existing relationships
There is great value in nurturing relationships with your network of current grant funders, partners and supporters. Get in touch in between reporting times to advise of progress made on your project. Also, don't be afraid to get in touch early when things aren't going to plan. Funders want to see you succeed and will often do what they can and work with you to help get the project back in the right direction.
- Follow the process
When a funding body has a clear grant application process, applicants have a responsibility to obey the procedures in place and not look for ways to 'cut corners' just to try to make sure their project makes it across the line. Having an institution target and accost trustees, directors and review panel members of a funding body is simply bad form – both before and after decisions are made. Rest assured that a well-prepared application for a high quality, well thought out project that meets the objectives of the funding body will always stand on its own two feet.
- Focus your efforts on the right funding bodies
If a funding body stipulates that they don't support something, please don't try to twist an application so that it might be perceived to align with the guidelines. In most cases this will result in a waste of the applicant's time and will also waste the resources of the funding body. It's best to accept that this is simply not what they do and instead focus your efforts on working with the funding bodies that support your type of project.
- Stay in the good books
Some foundations keep track of all 'dud' applications and it may actually hinder an applicant's (and institution's) future efforts should they one day have a project that aligns with the foundation. Likewise, some also 'black list' institutions from which grant applicants have failed to acquit. Be the person everyone wants to work with.
- Request a relevant amount
There is no use applying for a $200,000 project from a funder that generally provides $50,000 grants. Most funders indicate the average and maximum amount of their grants on their website. If this information is unavailable on their website, it can often be found under grants funded in annual reports. Funders find it very frustrating when applicants stretch their budgets to fit within a few dollars of the maximum grant amount. In fact, some avoid advertising maximum amounts as they know applicants will apply for what they think they can receive rather than what is necessary. These are often the same applicants that ask 'how much can I apply for?'. The correct question should be 'how much do you really need to successfully delivery the project?'
- Get assistance from someone with a different background from yours
Once your application is prepared, ask someone with a different background from yours to review your proposal. They will often pick up on key points you may have forgotten or address questions from a different perspective. It's also a good idea to contact your industry engagement office to get some help where appropriate.
- Always acquit
Grant makers understand that grantees are busy – everyone is – but it's important for them to find out about the project's achievements so they can in turn communicate this to their board, donors and key stakeholders. As mentioned before, regular and brief communication outside of reporting times also goes a long way. Acquitting on today's grant may help secure tomorrow's.
Tags: applications, foundations, funders, grants, impact, innovation, medical research, trusts


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House Refurbishment Financing
Home renovations and remodeling had been getting you headache till now? Well, the time isn't to opt for just the conventional methods of arranging for money. There are numerous methods available now to get you that extra cash. But, may be not all the options are made for you to pick. Every money lending option has its own pros and cons, and you need to take a closer look at which ones suit you. Ideally, what money borrowing options you pick depends upon your personal factors, as to how much loans you already have, or how much money could you pay every month.
Take a look at the below things which you absolutely need to know before you pick any option for collecting money for your home remodeling. These steps apply to most of the homeowners despite their specific project-needs; hence ensure that you keep these things in mind before you are sure of what you need from the financing option.
Things to know while borrowing
Estimate the project cost: The very first thing needs to be the estimate of the total cost. You could either do it yourself, or take a professional help to understand as to how much money you would be spending on remodeling. You can consult with your architect and take the price of everything coming under the project, and list down the total money. Add some safe extra amount to this cost, as the final project may go slightly up in total expenditure. Mark the final amount to be what you would need to borrow money for.
List down your personal work-cost: There won't be everything that the architect would do. There would be several things that would be brought by you, and you won't be able to list down an estimated price unless you go to the field and check-out the prices. On that also keep a safer edge. Be lenient in calculating the extra cost, which shouldn't be neither too higher than actual nor too low. Too high estimate would get you into too much deeper mortgage without any need in remodeling, while too fewer estimates would burden you later and might stop the project in between in unforeseen conditions.
Keep credit report clean: If you have not checked your credit report since long, it's preferable to do it now, before you apply for a refinance loan. Ideally, the score of the credit report plays a major role in getting the homeowner clearance fast. This report would ensure that you have paid your scheduled installments in time and not missed any. If you don't have your credit report ready, you may get it prepared and keep it ready.


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This fresh, summery charred corn salad is packed with quinoa, herbs, avocado and feta cheese and tossed in a light white-wine vinegar & lime juice vinaigrette. Perfect for a barbecue side dish!
Corn on the cob. To me, it is summer.
For some reason, corn on the cob makes me nostalgic for those hot summery days when I was growing up. We would play outside for hours, playing basketball, skipping rope, drawing with sidewalk chalk and making hopscotches. Then, we would drag ourselves in, happily tired, and were served up something delicious from the barbecue, a fresh salad, and corn on the cob.
We would slather it in butter. Stick it with those little prongs to keep our kid hands from burning. And go nuts.
You were guaranteed that someone was going to pretend to be a typewriter. Chomp-chomp-chomp-chomp-DING! Start over. That's just how things worked.
This week, I was super excited to see that they had fresh corn on the cob at the grocery store. I fought my way through a rabid mob of people, and came away victorious, with an armload of corn!
I guess we're a bit boring when it comes to corn here, though. Normally we just eat the corn on the cob, and that's that. Although, Ben often cuts the kernels off and eats it with a fork…what a snob! He says the kernels get stuck in his teeth when he eats it right off the cob.
I came across this salad on the Food & Wine website, I thought it sounded like a pretty delicious alternative! But I wanted to put my own spin on it, so I threw in some quinoa, feta cheese, and {duh!} avocado.
If there is one word to describe this charred corn salad it is this: fresh. Fresh parsley, cilantro and mint. Fresh corn. Salty feta. Fluffy quinoa. Tossed in a white-wine vinegar/lime juice/honey vinaigrette that brings even more freshness to the party.
I really try to make sure all of my recipes are super fast and simple. This charred corn salad recipe will take a tiny bit of extra planning, since you need to cook the quinoa and the corn ahead of time and allow them to cool.
I actually grilled the corn and cooked the quinoa the night before shooting these pictures, which made throwing the salad together a task that took probably less than 15 minutes.
This charred corn salad would be perfect for a barbecue! We enjoyed ours with Ben's famous BBQ ribs. It was the perfect, refreshing side to enjoy with the greasy, messy ribs. And being packed with quinoa and corn, this means you can feel a little less guilt about reaching for just one more rib 😉
More summery salads for us!
- this Summer Barley Salad with Grilled Corn & Blueberries is amazing!
- and this Corn & Peach Salad Recipe with Crispy Prosciutto has amazing flavor contrast
- and this Grilled Potato Salad with Onions & Arugula is perfect for a picnic
Charred Corn Salad with Feta, Mint and Quinoa
- 1/2 cup quinoa uncooked
- 3 ears corn
- 1 tablespoon oil
- 1/3 cup feta cheese crumbled
- 1/4 cup parsley chopped
- 1/4 cup cilantro chopped
- 1/4 cup mint leaves chopped
- 1/4 red onion in slivers
- 1 avocado cubed
- 2 tablespoons olive oil
- 2 tablespoons white wine vinegar
- 1 teaspoon lime juice
- 1 teaspoon honey
- salt and pepper to taste
- Cook quinoa according to package directions. Set aside to cool.
- Brush corn with oil. Grill over medium heat, for 20 minutes (or until corn is cooked to your liking), turning every 5 minutes. Set aside to cool. When cooled, cut corn kernels off.
- Assemble quinoa, corn and remaining salad ingredients in a medium bowl.
- Shake together dressing ingredients and toss with salad.
- Inspired by this Food & Wine recipe


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If you require more than one gift box please specify in "Additional Comments".
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Please allow 2-5 days for engraving (if requested).
'Her insight and understanding is profound. She sees both sides of the coin and her ability to rationalise and yet empathise ensures her constant support to those who surround her. She does not judge. She listens. Her warmth has a ripple effect. Her wisdom radiates.'
Thoughtfully designed and lovingly made, the Wisdom Amulet features bespoke line work inspired by profound insight and understanding. Classic in its form and beautiful in its meaning, this illustration has been cast onto a satin disc, and can be worn layered or steadfast on its own. The smooth reverse side of this amulet is engravable.
Shot on location in the heart of Morocco our latest campaign encompasses a two-part collection. She. Part One is a five-piece amulet capsule, which features hand drawn line work illustrations that capture the beauty of the feminine form and embrace the unique qualities found within each of us. She. Part One centers around five key attributes: Valour, Belief, Authenticity, Wisdom and Vitality. Beautiful in their simplicity, each amulet tells a story; with subtlety and strength woven throughout. Curated for you, as well as the women and sisters we all know and love. She. signifies the desire of the heart. She. represents the triumphant action taken throughout one's journey. She. summons the powerful attributes we all have within.
Made with a sterling silver base and plated in 18k gold vermeil. Vermeil is an historical French plating method which uses a palladium barrier layer and heavy 18k gold or rose gold plating.
Personalisation has always been the central focus to Kirstin Ash, so it's with great excitement we are now offering engraving on select pieces from our collections.
Our engraving service allows you to select your desired font, size and text dependant on the piece. From monograming to coordinates, creating a truly bespoke piece is now possible.
Click the ENGRAVE ME button to start your design. Please note that each piece may vary in the number of characters possible due to sizing.
All engraving is complimentary and done in house. Please allow up to 2-3 business days for us to process your order (excluding delivery). All items in your order will be sent together with the engraved designs.
See our delivery estimates per country below:
Please note: Due to the nature of engraving, we cannot accept returns with engraved designs.
ORDER PROCESSINGCurrent Status: Orders made before 1pm are shipped same day. Engraving orders are done in-house and require 1-2 business days to process.
PACKAGING RESTRICTIONSPlease note, our gift box supply is restricted due to COVID-19 impact. Orders may come packaged different than usual. Thanks for your understanding!
30 DAY RETURNSYou may return jewellery that is in original condition within 30 days of the order date. We offer a refund or credit voucher for returns, please note this excludes engraved orders & due to hygiene reasons, earrings cannot be returned.
SHIPPING TIMELINEAll Australian orders are sent with Express Shipping.
Australia Post have announced some delays and have temporarily suspended the next day delivery guarantee. Express Post is still in operation, however, parcels may not be delivered next business day every time. Please refer to Australia Post for delivery updates: https://auspost.com.au/about-us/news-media/important-updates/coronavirus#domestic
For further details on shipping, including international orders please click here: https://au.kirstinash.com/pages/shipping
To lodge a return or for our full returns policy, please visit: https://au.kirstinash.com/pages/returns


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Feather Edge
Slightly different than Graduated Mask.
This is a simple opacity gradient that can be applied to a clip. Use the OnScreen Controls to set the opacity points: the "white" control for full opacity, and the black control for transparent. Parameters published for each point so that this effect can be animated over time via keyframing.
What makes this different than Graduated Mask? Effect masks are available with this template which further enhances its power and effectiveness. Graduated Mask has no access to Effect Masks (Color and Shape).
Add as many as you need — create non-rectangular shapes!
See Video for a demo.
You can stay up to date by following Sight-Creations on Twitter and Facebook.


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2018 Speaker Series Review: The Men Who Would Be Kings with Jesse Donaldson
We're excited to have our 2018 Speaker Series in full swing again! This year's theme, Vancouver Noir, delves into the city's salacious history of crime and corruption from the late 1920s to the early 1970s. To kick things off, our first speaker, Vancouver author, historian and Sins of the