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The exocyst is an essential protein complex required for targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane. To study the function of the exocyst complex, we performed a structure-based mutational analysis of the Saccharomyces cerevisiae exocyst subunit Sec6p. Two "patches" of highly conserved residues are present on the surface of Sec6p; mutation of either patch does not compromise protein stability. Nevertheless, replacement of SEC6 with the patch mutants results in severe temperature-sensitive growth and secretion defects. At nonpermissive conditions, although trafficking of secretory vesicles to the plasma membrane is unimpaired, none of the exocyst subunits are polarized. This is consistent with data from other exocyst temperature-sensitive mutants, which disrupt the integrity of the complex. Surprisingly, however, these patch mutations result in mislocalized exocyst complexes that remain intact. Our results indicate that assembly and polarization of the exocyst are functionally separable events, and that Sec6p is required to anchor exocyst complexes at sites of secretion.
In eukaryotic cells, membrane-bound vesicles are required for carrying protein and membrane cargo between functionally distinct organelles and to the plasma membrane for exocytosis. The precise spatial and temporal regulation of vesicle fusion is achieved by a number of essential proteins (Wickner and Schekman, 2008 and references therein). Specific SNARE proteins, present on both the vesicle (v-SNARE) and the target (t-SNARE) membranes, are critical for membrane fusion. Tethering proteins, which are proposed to bridge the vesicle and target membranes at a distance, are either dimeric coiled-coil proteins or components of multisubunit protein complexes (Sztul and Lupashin, 2006 ). These proteins interact with the donor and target membranes through specific interactions with small Ras superfamily GTPases and phospholipids (Wu et al., 2008 ). Many tethering proteins bind directly to SNAREs and the regulatory Sec1/Munc18 family proteins and likely play crucial roles in the temporal and spatial regulation of SNARE complex assembly before membrane fusion (Cai et al., 2007 ; Kummel and Heinemann, 2008 ).
The exocyst is a conserved multisubunit protein complex required for tethering and fusion of vesicles at specific sites of polarized secretion on the plasma membrane (TerBush et al., 1996 ; Guo et al., 1999 ; Munson and Novick, 2006 ; Wu et al., 2008 ). This complex is distantly related to tethering factors found in other trafficking pathways, including the GARP (Golgi-associated retrograde protein), COG (conserved oligomeric Golgi), and Dsl1 complexes (Whyte and Munro, 2002 ; Koumandou et al., 2007 ). The exocyst is comprised of eight subunits: Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p. These proteins localize to regions of exocytosis and membrane growth in eukaryotic cells, where their activity is regulated by small Rab, Rho, and Ral GTPases (Wu et al., 2008 ). Temperature-sensitive (ts) yeast exocyst mutants accumulate secretory vesicles at sites of secretion (bud tips and mother-bud necks) at a stage before exocytic SNARE complex assembly. The exocyst also plays critical roles in Drosophila, mammals and plants, not just in exocytosis, but also in endocytic recycling and cytokinesis (Gromley et al., 2005 ; Sommer et al., 2005 ; Oztan et al., 2007 ; Hala et al., 2008 ).
Elucidation of the molecular function of the exocyst requires detailed knowledge of each of the subunits and their assembly into functional complexes. High-resolution structures of four exocyst subunits have been determined: the C-terminal domain of Drosophila melanogaster Sec15 (Wu et al., 2005 ), the nearly full-length yeast Exo70p (Dong et al., 2005 ; Hamburger et al., 2006 ; Moore et al., 2007 ), the C-terminal domain of yeast Exo84p (Dong et al., 2005 ), and the C-terminal domain of yeast Sec6p (Sivaram et al., 2006 ). They fold into remarkably similar elongated helical bundle structures with diverse surface features (Munson and Novick, 2006 ; Sivaram et al., 2006 ). Protein–protein interactions within the exocyst complex and with small GTPases have been identified genetically, by qualitative in vitro binding studies and by yeast two-hybrid analyses (reviewed in Munson and Novick, 2006 ). The exocyst associates with secretory vesicles through the interaction of Sec15p with the Sec4p GTPase (Guo et al., 1999 ), and most of its subunits appear to traffic with secretory vesicles to sites of secretion (Boyd et al., 2004 ). Exocyst function is regulated by interactions of Sec3p and Exo70p with membrane-anchored Rho GTPases and phosphoinositides (Adamo et al., 1999 ; Guo et al., 2001 ; Zhang et al., 2001 , 2008 ; He et al., 2007 ), as well as interactions with the yeast lethal giant larvae (Lgl) homologues Sro7p and Sro77p (Lehman et al., 1999 ; Zhang et al., 2005a ; Grosshans et al., 2006 ; Hattendorf et al., 2007 ). Most of the subunits are essential in yeast, except Sec3p, although sec3Δ cells grow quite slowly (Wiederkehr et al., 2003 ). In addition, the lethality of sec5Δ and exo70Δ can be bypassed by overexpression of either SEC1 or SEC4 (Wiederkehr et al., 2004 ). These structural, biochemical, and genetic studies indicate that each of the subunits plays diverse roles in exocyst function; however, many questions remain about the specific function(s) of the exocyst and its subunits.
Here, we focus on mutational studies of the yeast exocyst subunit Sec6p, which forms a direct link between the exocyst complex and the exocytic plasma membrane SNARE protein Sec9p and has been proposed to regulate SNARE complex assembly (Sivaram et al., 2005 ). Qualitative protein–protein interaction studies also indicated that Sec6p can dimerize in vitro (Sivaram et al., 2005 ), interact with the exocyst subunits Sec10p, Sec8p, and Exo70p (Dong et al., 2005 ; Sivaram et al., 2006 ), and bind Rtn1p, an interaction that is important for cortical ER inheritance (De Craene et al., 2006 ). We identified and mutated two patches of residues on the surface of Sec6p that are highly conserved from yeast to humans and show that these mutants have ts growth and secretory defects in yeast. These phenotypes are due to a loss of polarized localization of the exocyst subunits at nonpermissive conditions. However, unlike other exocyst ts mutants, exocyst complexes are intact at the nonpermissive conditions, indicating that the Sec6p patch residues are necessary to anchor functional, assembled exocyst complexes at sites of secretion. The conservation of these patch residues suggests an anchoring role for Sec6p in all eukaryotes.
Mutations (sec6-49: L418A, Y422A, W433A, Q470A, and Q474A; sec6-54: D607A, T632A, E635R, Y636A, and D639R) were introduced into the SEC6 gene by PCR, and cloned into the NdeI and BamHI sites of pET15b (Novagen, Madison, WI) containing an N-terminal His6 tag. For analysis in yeast, constructs were placed into the BamHI and NotI sites of pRS315 (LEU2 CEN) including the endogenous promoter of SEC6 (0.5 kb of flanking 5′ and 3′ genomic DNA). A C-terminal triple hemagglutinin (HA) tag was added to the pRS315 constructs for use in indirect immunofluorescence and immunoprecipitations. All clones were confirmed by sequencing. The sec6 LEU2 CEN plasmids were introduced as the sole copy of SEC6 in yeast by plasmid shuffling the wild-type SEC6 URA3 CEN plasmid out of MMY204 (Table 1). For green fluorescent protein (GFP) and synthetic lethal analyses, MMY204 was mated with strains containing either a single C-terminally GFP-tagged exocyst subunit (Huh et al., 2003 ) or a single ts mutation (gifts from P. Novick, Yale University; W. Guo, University of Pennsylvania; and J. Jäntti, University of Helsinki). Diploid strains were sporulated and dissected, and the mutant plasmids were introduced into the GFP+ sec haploids as described above. Media, growth conditions, and genetic methods for yeast were described previously (Munson et al., 2000 ). Yeast strains used in this study are listed in Table 1. Invertase secretion was performed as in Adamo et al. (1999) .
His6-Sec6p, His6-Sec6-49, and His6-Sec6-54 were expressed and purified as described for His6-Sec6p (Sivaram et al., 2005 ), with the following change for the His6-Sec6-49 anion-exchange buffers: 10 mM HEPES, pH 7.4, and 1 mM DTT ± 1 M NaCl. Protein concentrations were determined by ninhydrin protein assay (Rosen, 1957 ). Circular dichroism spectroscopy was performed as described (Sivaram et al., 2005 ). Maltose binding protein (MBP) pulldown binding assays were performed as described (Sivaram et al., 2006 ) using a 20 mM HEPES, pH 7, 100 mM NaCl, 1 mM EDTA, 0.1% NP-40, and 1 mM DTT binding buffer. For experiments with MBP-Exo84CT, the soluble C-terminal domain (residues 523-753; Dong et al., 2005 ) was subcloned into pMALc2X (New England Biolabs, Beverly, MA). MBP-Exo84CT was expressed at 20°C for 3 h after induction with 0.1 mM IPTG and purified by amylose resin affinity chromatography.
Strains containing wild-type or mutant Sec6p with C-terminal HA3 tags were grown to log phase in synthetic complete (SC) media at 25°C and either fixed or shifted to nonpermissive conditions (prewarmed YPD at 37°C for 1 h) and then fixed with 37% formaldehyde. Cells were spheroplasted, permeabilized in HS/SDS buffer (0.1 M HEPES, pH 7.4, 1.0 M sorbitol, and 0.5% SDS), and washed twice in HS buffer. Permeabilized cells were placed on slides (Electron Microscopy Sciences, Fort Washington, PA) prepared with 0.1% polylysine. A 1:400 dilution of α-HA-AlexaFluor 488 (Molecular Probes, Eugene, OR) was added to visualize Sec6p-HA3. For Sec4p, a 1:500 dilution of α-Sec4 antibody (P. Brennwald, University of North Carolina) was used, followed by washing and incubation with 1:1000 goat α-mouse-AF488 (Molecular Probes). Differential interference contrast (DIC) and fluorescent images were obtained at room temperature using an Axioskop2 plus epifluorescent microscope (Zeiss, Thornwood, NY) fitted with a 100× Plan-NEOFLUAR (Zeiss 1.30 NA oil immersion) objective lens. Images were collected using a Diagnostic Instruments camera (Sterling Heights, MI; model 2.1.1) and 3rd Party Interface Advanced (ver. 3.5.4 for MacOS) software. Immunofluorescent images were adjusted for total contrast in Adobe Photoshop (San Jose, CA).
Strains containing C-terminal GFP-tagged exocyst subunits and either wild-type or mutant Sec6p were grown to log phase in SC media at 25°C and shifted to nonpermissive conditions for 1 h. Samples were immediately resuspended in PBS containing 10% glycerol, fixed with 37% formaldehyde for 10 min, washed, and resuspended in PBS with 10% glycerol. Cells were viewed as described above. Localization of the GFP-tagged proteins was quantitated in Adobe Photoshop by counting cells with localized or mislocalized exocyst subunits. For each strain and condition, n > 100 cells were counted for two replicates each.
Strains were grown to log phase in SC media at 25°C or shifted to nonpermissive conditions for 1 h. One hundred OD units of cells were pelleted and washed with 10 mM Tris, pH 7.4, and 10 mM NaN3 at either 25 or 37°C. Supernatants were removed and pellets were resuspended in lysis buffer (50 mM HEPES, pH 7.4, 150 mM KCl, 0.5% NP-40, 1 mM DTT, and 1 mM PMSF). Cells were lysed by bead beating, and the lysates cleared by centrifugation and normalized for total protein concentration by Bradford assay (Bio-Rad, Hercules, CA). The supernatants were added to prewashed protein A beads (GE Healthcare Bio-Sciences, Piscataway, NJ) and mixed for 30 min at 4°C. Input samples were collected, the remaining supernatants were placed in fresh tubes, and 1:50 dilutions of HA antibody (Roche) were added. Samples were incubated at 4°C for 1 h. Lysate-antibody mixes were added to fresh, prewashed protein A beads. These mixtures were incubated at 4°C for 30 min, the supernatants were removed, and the beads were washed with lysis buffer. Input and bound fractions were analyzed by 10% SDS-PAGE gels and Western blotting. Exocyst subunits were detected using antisera raised against C-terminal constructs of Exo70p, Exo84p, and Sec10p (Pocono Rabbit Farm and Laboratory, Canadensis, PA) or antibodies against Sec3p, Sec5p, and Sec15p (generous gifts from P. Brennwald, University of North Carolina), followed by HRP-conjugated goat α-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) and ECL (GE Healthcare Bio-Sciences), and visualized using a luminescent image analyzer (Fuji Film, Tokyo, Japan; LAS-3000).
We previously determined the x-ray crystal structure of the C-terminal domain of yeast Sec6p, residues 411-805 (Sivaram et al., 2006 ). This domain is sufficient for interactions with the exocyst subunits Exo70p and Sec10p, but not Sec8p or the t-SNARE Sec9p (Dong et al., 2005 ; Sivaram et al., 2006 ). Structure-based sequence alignments revealed two highly conserved surface "patches," which we hypothesize are important sites for protein–protein interactions in all eukaryotes (Figure 1A; Sivaram et al., 2006 ). These patches are composed of the following amino acids: L418, Y422, W433, Q470, Q474, and V478 in Patch-1, and D607, T632, E635, Y636, and D639 in Patch-2. Patch-2 overlaps a conserved acidic region, as well as being in close proximity to the original sec6-4 ts mutation (L633P; Novick et al., 1980 ; Lamping et al., 2005 ; Sivaram et al., 2006 ). When these patch residues were individually mutagenized to alanine in the context of the full-length Sec6p and substituted for the wild-type in yeast, no apparent effects on growth rates were observed (data not shown). We reasoned that this lack of phenotype could be due to tight protein–protein interactions at these surfaces and that single alanine perturbations would be insufficient to disrupt them. Therefore, we combined mutations in each patch (Figure 1A). All the residues in Patch-1 were mutated to Ala (called sec6-49). For Patch-2, residues 607, 632, and 636 were mutated to Ala and residues 635 and 639 to Arg, causing a reversal of charge at these positions (called sec6-54). These mutations were anticipated to severely disrupt protein–protein interactions in these regions.
Because these are surface mutations, they should not significantly destabilize the α-helical structure of Sec6p. To test this, we cloned and purified the recombinant Sec6p mutant proteins from Escherichia coli and analyzed their secondary structure and stability using circular dichroism. As expected, the patch mutations did not decrease the stability of the proteins. Both of the mutant proteins showed the same characteristic α-helical signal as the wild-type Sec6p (~50% helicity) and also had similar thermal stabilities to wild-type at 37°C (Figure 1B). In contrast, the protein containing the ts mutant allele sec6-4 must be considerably destabilized, because we were unable to produce any soluble recombinant protein in E. coli (data not shown).
To test the function of these mutant proteins as the sole copy of Sec6p in yeast, we performed a plasmid shuffle experiment to replace the wild-type SEC6 with the mutants. The mutants were expressed under control of the endogenous promoter from a low-copy (CEN) plasmid and had equivalent expression levels to the endogeneous Sec6p at both 25 and 37°C (data not shown). Both patch mutants were able to replace the wild-type SEC6 URA3 plasmid when grown on SC plates containing 5-fluoro-orotic acid (5-FOA) at 25°C. However, on SC plates at higher temperatures, both mutants exhibited ts growth defects (Figure 2A). In addition, both sec6-49 and sec-54 showed severe growth inhibition when plated on rich YPD medium, even at temperatures that were permissive for growth on SC medium (Figure 2A). The growth rates of the mutants in SC liquid cultures were similar to wild-type at 25°C and were two- to threefold slower than wild-type at 37°C, and the mutants did not grow in liquid YPD at 37°C (data not shown). The slow growth on rich YPD media may be explained by the higher rate of exocytosis compared with synthetic media, which would exacerbate the secretory defect. Similar results have been observed for other exocyst mutants (Haarer et al., 1996 ; Wiederkehr et al., 2003 ). Consistent with their growth phenotypes, the mutant strains were defective for secretion of the marker protein invertase at the nonpermissive conditions (Figure 2B). Because the recombinant mutant proteins were stable both in vitro and in vivo, we conclude that Sec6p's function, rather than its stability, is compromised by mutation of residues in these conserved patches.
To determine the defect(s) caused by mutations of the conserved patch regions, we examined the phenotypes of the mutant yeast strains in more detail. First, we monitored the localization of the Sec6p mutants at the permissive and the restrictive conditions. The mutants could not be visualized using a GFP tag, because placing a GFP tag at either the N- or C-terminal end resulted in slow growth, even at conditions permissive for growth of the untagged mutant (data not shown). However, when we tagged the mutants at their C-termini with a triple HA epitope tag, these strains grew similarly to the untagged strains (data not shown). Immunofluorescence experiments demonstrated that the patch mutants were properly polarized to tips of the growing bud and mother-bud necks at the permissive conditions (Figure 3). After a shift to the nonpermissive conditions (YPD at 37°C for 1 h), the mutant proteins were completely mislocalized (Figure 3), suggesting that the mutations disrupt the ability of Sec6p to localize to, or remain localized at, sites of secretion. Similarly, the sec6-4 protein was not polarized at the nonpermissive temperature (Lamping et al., 2005 ).
To rule out that mislocalization of the Sec6p mutants was caused by a general disruption of the secretory pathway, we monitored Sec4p localization by immunofluorescence. Sec4p is the Rab family GTPase associated with secretory vesicles; its localization is sensitive to perturbations of the secretory pathway (Ayscough et al., 1997 ; Walch-Solimena et al., 1997 ; Roumanie et al., 2005 ). We found that Sec4p localization was unperturbed in the sec6-49 and sec6-54 mutant cells after 1 h at nonpermissive conditions (Figure 3), as was previously observed for sec6-4 and other exocyst ts mutants (Walch-Solimena et al., 1997 ; Roumanie et al., 2005 ). Therefore, although the Sec6p patch mutant proteins are mislocalized at the nonpermissive conditions, the sec6 mutant cells appear fully competent in localizing secretory vesicles to sites of polarized secretion. Moreover, consistent with the observation that Sec4p trafficking is normal, we observed no obvious morphological defects in the mutant cells (data not shown).
We next tested the localization of the other exocyst subunits at the nonpermissive conditions to investigate whether mislocalization of Sec6p mutants would also affect exocyst complex localization. On the basis of previous results with sec6-4 and other exocyst ts mutants (Finger et al., 1998 ; Roumanie et al., 2005 ; Zhang et al., 2005b ; He et al., 2007 ), we expected to find mislocalization of most of the subunits due to disruption of complexes at the nonpermissive conditions. The exceptions to this would be subunits proposed to localize and/or stabilize the complex at sites of secretion: Sec3p, Exo70p, and Exo84p. To visualize each of the exocyst subunits individually, we constructed strains in which the genomic copy of one of the exocyst subunits was C-terminally GFP tagged (Huh et al., 2003 ), and the only copy of SEC6 was either the wild-type or one of the patch mutants (Table 1). We observed significant mislocalization of all the exocyst subunits at the nonpermissive conditions (75–90%, except Sec3-GFP, which was ~50%; Figure 4, A and B). As a control, we also observed similar mislocalization of each of the exocyst subunits in sec6-4 cells (data not shown), consistent with previous results (Roumanie et al., 2005 ). This suggested two possibilities: 1) the exocyst complex was being disassembled and dispersed from sites of secretion; or 2) interactions with a factor(s) at sites of secretion were destabilized, and hence the subunits became mislocalized.
We reasoned that the conserved patches on Sec6p are important for interactions with other exocyst subunits. If true, mutation of these patches would therefore cause global destabilization and disassembly of the exocyst, resulting in dispersal of the subunits. This phenotype has been previously reported for the sec6-4 ts allele, as well as for ts alleles of several other exocyst subunits (TerBush and Novick, 1995 ). To examine whether the exocyst complex is disassembled at the nonpermissive conditions, we examined the state of the complex by coimmunoprecipitation analyses. For these experiments, we immunoprecipitated the wild-type and mutant Sec6p-HA proteins with α-HA antibodies, and assayed for the presence of the other exocyst subunits by Western blot analyses. Surprisingly, we found that, even after 1 h at nonpermissive conditions, similar amounts of the exocyst subunits were coimmunoprecipitated with wild-type and the Sec6p mutants—indicating that the complexes were still assembled (Figure 5A).
The presence of assembled exocyst complexes at nonpermissive conditions is clearly distinct from similar immunoprecipitation experiments using the sec6-4 ts strain; the complexes in sec6-4 cells were completely disassembled and/or degraded at the nonpermissive temperature (TerBush and Novick, 1995 ). To demonstrate that the exocyst is capable of disassembly under our experimental conditions, we immunoprecipitated wild-type Sec6p-HA in a strain containing the sec5-24 ts allele as the sole copy of SEC5. We used sec5-24 because degradation of the sec6-4 protein precluded the use of the Sec6-4-HA for immunoprecipitations. We found, at nonpermissive conditions, nearly complete loss of the other exocyst subunits bound to Sec6p (Figure 5B). Another possibility is that exocyst complexes are in a dynamic equilibrium and can reassemble after lysis during the course of our immunoprecipitation experiments. To exclude this possibility, we performed a lysate mixing experiment using two strains with differently tagged subunits (Figure 5C). The first strain contained SEC8-GFP and untagged SEC6 (Huh et al., 2003 ); the second had SEC8-MYC3 and SEC6-HA3. The strains were lysed, either kept separate or mixed in a 1:1 ratio, and then the Sec6p-HA was immunoprecipitated with α-HA antibodies. The Myc-tagged Sec8p coimmunoprecipitated with Sec6p-HA (from the same strain), but the GFP-tagged Sec8p did not bind Sec6p-HA (from the mixed lysates), indicating that exocyst complexes do not form during the immunoprecipitation experiments. Therefore, we conclude that exocyst complexes remain intact in the patch mutant strains, even at nonpermissive conditions where mislocalization occurs.
The finding that the exocyst remains assembled at nonpermissive conditions suggests that the conserved patches are not required for Sec6p to interact with other subunits of the exocyst complex. We have previously shown that the C-terminal domain of Sec6p is sufficient for binding both Exo70p and Sec10p (Sivaram et al., 2006 ). In contrast to the full-length Sec6p, however, this domain is not sufficient for binding Sec8p and the t-SNARE Sec9p, nor for dimerization (Sivaram et al., 2005 , 2006 ). Here, we examined the interactions between the full-length Sec6p patch mutants and Exo70p, Sec10p, and Sec8p using qualitative pulldown assays and found that the Sec6p mutants bound with affinities similar to that of wild type (Figure 5D). As a negative control, we found that neither the wild-type nor the mutant Sec6p proteins interacted above background with the C-terminal domain of Exo84p (residues 523-753; Dong et al., 2005 ). Furthermore, as expected from our previous data, gel filtration experiments demonstrated that the mutants were still dimeric and capable of binding Sec9p (data not shown). Together, these binding results corroborate the immunoprecipitation results, and indicate that the binding partner(s) for the conserved patches is not one of the exocyst subunits, nor Sec9p.
To test this possibility further, we examined genetic interactions between the mutants and the other exocyst subunits. We first tested for suppression of the growth phenotypes by overexpression of the other exocyst subunits, as well as SEC4, SEC1, and SEC9, the other plasma membrane t-SNARE, SSO1, and the secretory vesicle v-SNARE, SNC2, from 2μ plasmids. We also tested for suppression by overexpression of an "open" mutant of Sso1p (Munson and Hughson, 2002 ). None of the exocyst subunits or SEC4 or SEC1 suppressed the sec6 mutants (data not shown). These results support the idea that the Sec6p binding partner(s) is not a subunit of the exocyst complex. In contrast, overexpression of SEC9, SSO1, and SNC2 all suppressed the sec6-49 and -54 ts phenotypes (Figure 5E). The likely explanation is that loss of assembled exocyst complexes at sites of secretion leads to a decrease in SNARE complex assembly, which can be rescued by overexpression of the plasma membrane SNAREs. We also expected that the open Sso1p mutant would rescue this defect, because it readily forms SNARE complexes. However, similarly to the synthetic defects observed in combination with other exocyst ts mutants, this mutant failed to suppress the sec6-49 and -54 ts phenotypes (Figure 5E), likely because of a detrimental overaccumulation of SNARE complexes (Munson and Hughson, 2002 ). In contrast, the sec6-4 allele, in which the exocyst complex is completely disassembled (TerBush and Novick, 1995 ), has a more severe block in SNARE complex assembly (Grote et al., 2000 ) that cannot be suppressed by overexpression of SSO1 and SEC9 (Aalto et al., 1993 ). Additionally, unlike the sec6-4 allele, the sec6-49 and -54 mutants were synthetically lethal when crossed with most of the exocyst ts strains (Table 2). The differences in synthetic lethality between our sec6 mutants and sec6-4 (Finger and Novick, 2000 ) may reflect the severity of the ts defects in these cells, especially sec6-54, rather than specific functional differences. However, the combination of destabilization of the exocyst by the sec ts mutants, together with partial mislocalization of the complex by the sec6-49 and -54 mutations at 25°C, could result in more severe defects. Together, the biochemical, phenotypic, and genetic interactions observed for both of the patch mutants indicate that sec6-49 is very similar, albeit weaker, to the sec6-54 allele. In support of this idea, we found that combining the sec6-49 and -54 mutations together resulted in similar ts and YPD growth defects as the individual mutants (data not shown). The patch residues appear to comprise a single binding site for an as yet unknown factor, which is not an exocyst complex subunit or Sec9p.
We have identified a novel class of Sec6p mutants that disrupt the polarized localization of the exocyst complex at nonpermissive conditions, yet do not affect complex assembly. We show that mutation of conserved surface residues on the C-terminal helical bundle domain of Sec6p do not destabilize full-length Sec6p (Figure 1), yet result in severe temperature- and media-sensitive growth defects (Figure 2). At the nonpermissive conditions, vesicle trafficking to sites of secretion appears unimpaired, whereas the Sec6p patch mutants are completely mislocalized (Figure 3). Consequently, all of the other exocyst subunits are also mislocalized (Figure 4). Previous studies of exocyst temperature sensitive mutants showed that this mislocalization is caused by destabilization and disruption of the exocyst complex. However, we show that exocyst complexes remain intact in the sec6 patch mutant strains, even at the nonpermissive conditions (Figure 5).
Unexpectedly, the extensive exocyst mislocalization observed in our sec6-49 and -54 strains at the nonpermissive conditions included Sec3p, which was proposed to be the spatial landmark for exocyst localization (Finger et al., 1998 ). Sec3p-GFP appeared polarized in yeast cells in the absence of ongoing exocytosis and in other exocyst ts mutants (Finger et al., 1998 ; Zhang et al., 2005b ). However, localization of the endogeneous Sec3p by immunofluorescence indicated that the GFP tag may artificially stabilize Sec3p-GFP at sites of secretion (Roumanie et al., 2005 ), although this was recently disputed (Zhang et al., 2008 ). Localization of the endogenous Sec3p in sec6-4 cells (Roumanie et al., 2005 ) was similar to our results with Sec3-GFP in the sec6-4 strain. Several variations between our studies and previous results could account for these discrepancies: the use of plasmid-borne, instead of genomic GFP-tagged Sec3p; the testing of Sec3-GFP in the presence of the endogenous Sec3p; differences in strain backgrounds; and the fact that our construct contains a linker sequence (9 a.a.) between the C-terminus of Sec3p and the GFP (Huh et al., 2003 ). Our data, combined with the observations that Sec3p is not an essential gene and that the other exocyst subunits are polarized in sec3Δ cells at 25°C (Wiederkehr et al., 2003 ; Zhang et al., 2005b ), suggest that Sec3p is not the spatial landmark for the exocyst complex.
Other candidates for targeting the exocyst to sites of secretion are Exo70p and Exo84p (Boyd et al., 2004 ; He et al., 2007 ). However, we found that Exo70p-GFP was mislocalized in the sec6-49 and -54 strains at the nonpermissive conditions (Figure 4). Similarly, although the localization of Exo84-GFP was proposed to be independent of the exocyst subunits (e.g., not mislocalized in previous ts strains; Zhang et al., 2005b ), Exo84-GFP was also mislocalized in the sec6-49 and -54 strains (Figure 4). Therefore, we conclude that the conserved surface patches on Sec6p are required for localization and/or stability of the exocyst complex at sites of secretion and that Sec3p, Exo70p, and Exo84p are not sufficient to localize the complex in these sec6 mutant strains. Nevertheless, the presence of a landmark is not strictly necessary, because actin cables are polarized independently of the exocyst, bringing vesicles to the correct sites of exocytosis (Walch-Solimena et al., 1997 ). Therefore, we propose that exocyst subunits travel to sites of secretion where they assemble together, and that Sec6p is required to anchor the assembled exocyst complexes at these sites. Perhaps Sec6p acts in cooperation with Sec3p, Exo70p, and/or Exo84p for stabilization of the exocyst complexes on the plasma membrane or perhaps their interactions (e.g., with Rho proteins and phosphoinositides) are more regulatory in nature (Roumanie et al., 2005 ; Wu et al., 2008 ). It may be that either Exo70p or Sec3p are sufficient to localize the exocyst, when wild-type Sec6p is present to stabilize the complex at sites of secretion (Hutagalung et al., 2008 ).
The spatial and temporal regulation of exocyst assembly and disassembly is currently unclear. Most of the exocyst subunits appear to traffic to the plasma membrane with secretory vesicles (Boyd et al., 2004 ), although it is unknown whether they traffic as preassembled complexes or they assemble upon vesicle arrival. Assembly upon arrival at sites of secretion, which was suggested by FRAP data (Boyd et al., 2004 ), is compelling, because the interactions between subunits on the vesicle with those on the plasma membrane could tether the membranes together. If so, then the patch residues of wild-type Sec6p would be necessary to anchor the complex at these sites after vesicle arrival. In the case of the patch mutants, Sec4p would need to hydrolyze the bound GTP, perhaps upon exocyst assembly, to release Sec15p and therefore the rest of the exocyst complex. On the other hand, it is possible that the exocyst assembles at sites of vesicle formation and budding; the patch residues of Sec6p would be required for proper trafficking of the complex to sites of secretion. This could only be the case if vesicles are capable of transport in the absence of assembled exocyst complexes, because trafficking of vesicles containing Sec4p is unimpaired in the patch mutant strains (as well as in the sec6-4 and other exocytic sec mutants). This situation would require that Sec4p on the budding vesicle hydrolyze the bound GTP to release the complex. Further mutational and GFP localization studies will be necessary to distinguish between these scenarios.
Regardless of where assembly takes place, the finding that the complex remains assembled despite a block in exocytosis and in the absence of polarized accumulation suggests two possibilities. First, the complex cannot disassemble, because disassembly is triggered by a factor(s) at sites of secretion or by membrane fusion. If the complex is locked into an assembled conformation in the sec6-49 and -54 mutants, then it may be unable to be recycled for subsequent rounds of vesicle tethering and fusion, thus contributing to the ts phenotype. Alternatively, the complex is assembled in these mutants because it normally does not disassemble unless destabilized. In this case, mislocalization of the assembled exocyst complexes at nonpermissive conditions would lower the concentration of active exocyst complexes that are available to function at sites of secretion. This would ultimately result in decreased SNARE complex assembly and membrane fusion.
Conservation of these Sec6p patch residues suggests an important anchoring function for Sec6p in all eukaryotes. Our analyses indicate that the binding partner for the patches on Sec6p is not a subunit of the exocyst complex, nor is it the t-SNARE Sec9p (Figure 5). Therefore, further genetic and biochemical experiments will be necessary to identify the anchoring factor and analyze its role in polarizing Sec6p and the exocyst complex at sites of secretion.
In memory of Jen Songer (1979–2008), who died in a car accident on December 9, 2008. Her legacy is a passion for life and science, which continues to inspire everyone who knew her. We miss her very much. We are grateful to J. Towle, N. Croteau, and B. Farley for technical assistance. We thank P. Novick, P. Brennwald, W. Guo, and J. Jäntti for yeast strains, antibodies, and plasmids, C. R. Matthews and his lab for use of the CD, and N. Rhind for use of the fluorescence microscope and advice. We thank R. Gilmore, C. Carr, N. Rhind, and members of the Munson lab for critical reading of this manuscript and suggestions. This work was supported by National Institutes of Health Grant GM068803 to M.M.
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-09-0968) on December 10, 2008.
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With a market cap of has a large market cap size. Goldcorp Inc. (NYSE: GG) has been on the stock market since its IPO date on the 8/18/1995. Goldcorp Inc. is in the Gold industry and Basic Materials sector. Average volume for Goldcorp Inc., is 9039.81, and so far today it has a volume of 9810300. Performance year to date since the 8/18/1995 is 35.77%.
To help you determine whether Goldcorp Inc. is undervalued the following values will help you decide. P/E is *TBA and forward P/E is 23.47. PEG perhaps more useful shows that Goldcorp Inc. has a value for PEG of *TBA. P/S ratio is 3.47 and the P/B ratio is 1.01. The P/Cash and P/Free cash flow is 36.73 and 99.31 respectively.
At the current price Goldcorp Inc. is trading at, 15.61 (2.43% today), Goldcorp Inc. has a dividend yield of 0.51%, and this is covered by a payout ratio of *TBA. Earnings per share (EPS) is -5.37, and this is looking to grow in the next year to 78.28% after growing -90.50% this past year. EPS growth quarter over quarter is -119.60%, and -36.60% for sales growth quarter over quarter.
The number of shares outstanding is 858.89, and the number of shares float is 848.25. The senior management bring insider ownership to 0.20%, and institutional ownership is at 68.80%. The float short is 1.93%, with the short ratio at a value of 1.81. Management has seen a return on assets of -19.60%, and also a return on investment of -27.70%.
The ability for Goldcorp Inc., to deal with debt, means it current ratio is 1.6, and quick ratio is 1.1. Long term debt/equity is 0.22 and total debt/equity is 0.24. In terms of margins, Goldcorp Inc. has a gross margin of 5.20%, with its operating margin at *TBA, and Goldcorp Inc. has a profit margin of *TBA.
The 52 week high is -23.41%, with 65.63% being its 52 week low. The 20 day simple moving average is -14.76% and the 200 day simple moving average is -0.60%.
Disclaimer: Remember there is a risk to your investment, this is not a recommendation, nor personal advice, never invest more than you are able too loose.

subset: commoncrawl


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Saturday's New York Times carried a fascinating Op-Ed by William Ayers, who wisely waited until after the election to speak out, less anything he say be taken completely out of context by McCain/Palin and Fox News. Here's the column. There two passages are particularly interesting:
DeVore's US Senate announcement: the Liberal Translation
by Dan Chmielewski •
You know, there's plain old English and then there's Republican-speak, a twisted and bending way of saying something you truly believe is true even though reality is warped when words are compared to facts. As a Liberal public service, we've translated State Rep. Chuck DeVore's announcement for his run for Senate against Barbara Boxer. Our…
The Juice Gets 15 years in Jail
by Dan Chmielewski •
OJ Simpson has been sentenced to 15 years in jail; we'll likely serve at least 6 years in the big house. See the story here.
Chuck DeVore's First Ad
by Dan Chmielewski •
Geez Louise; the last election is less than a month ago, and Chuck DeVore has launched his first TV ad in his US senate race against Senator Barbara Boxer. [youtube]WrFJRNRDL0M[/youtube] The ad is classic Chuck. Not a single word on why voters should vote for him and he tries to tie Boxer with the financial…
Good Luck at Red County, Thomas Gordon
by Dan Chmielewski •
In reviewing the local blogs this morning, I noted that Thomas Anthony Gordon is now covering Santa Ana city issues for Red County. It's a smart pick up by Matt Cunningham, and while personally I think Thomas is wrong about everything politically, in my numerous phone conversations with him, Thomas is a reasonable person who…
Prop 8 – The Musical
by Dan Chmielewski •
A three minute musical dedicated to the Proposition you love to hate – Prop 8. See more Jack Black videos at Funny or Die
Suhkee Kang's Mayoral Ceremony set for Tuesday, December 9
by Dan Chmielewski •
Mayor-elect Sukhee Kang and City Councilmembers Larry Agran, Steven Choi, and Beth Krom will be sworn-in on Tuesday, December 9th. A reception in honor of our 2008 Elected Officials will be held at 5:00 PM at the Irvine Civic Center. The swearing-in ceremony will begin at 6:00 PM in the City Council Chamber. It's a…


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Latest News
Stop Climate Denier Scott Pruitt from Running EPA
Scott Pruit Climate Denier EPA Administrator Cook Inlet Alaska
Help Stop Exxon from Running our Foreign Policy
Exxon CEO Rex Tillerson had his first confirmation hearing for the Secretary of State position before the U.S. Senate Foreign Relations Committee on Wednesday, January 11. Now it's time to tell our Senators Lisa Murkowski & Dan Sullivan to oppose this reckless nomination.
What the hell happened?
That's the question I've heard most since the election. We've read a lot of the post-mortems, talked to a lot of Alaskans, and spent many hours thinking, alone and with friends, about our world and where it's going. This election turned our thinking on its head. All our assumptions about data and polling and analysis were wrong. That's a pretty big gut-punch. Now, we've elected a President who believes climate change is a hoax.
Shape the Future of Kachemak Bay - Comments Due Nov. 4
The Alaska Department of Fish & Game (ADFG) is revising the management plan for Kachemak Bay and Fox River Flats Critical Habitat Areas. This process is now in the "scoping phase," which means ADFG is looking for the issues it should address in the management plan revision. Scoping comments are due November 4


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Open Cheese and Mango Sandwiches
- 1 small loaf of bread with seeds, sliced
- Spreadable butter
- Selection of cheeses (sliced gouda, spreadable blue cheese)
- 1 jar of Patak's Sweet Mango Chutney. Want it spicier? Use Patak's Hot Mango Chutney instead.
- Few sprigs of thyme
- Assemble the sandwiches by the spreading the butter on one side.
- Top with one cheese, a little chutney and garnish with a sprig of thyme.


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|Home > Artists > Dan Akio >
Dan Akio
Home: UK
UK artist Dan Akio is a polished singer and songwriter who composes and records in the Pop Rock and Soft Rock genres, with a penchant for beautiful melodic hit records. In the tradition of countless top artists, his songs are equally at home on major alternative radio stations and Top 40, as well as independent and studio film soundtracks. Dan began his career as the lead singer in the Edinburgh rock band Dirty Frank before migrating to London's lively acoustic singer songwriter circuit, refining his craft and evolving into a terrific artist, constantly performing live.
Dan Akio's persistence in performing throughout London attracted the attention of a top notch team of producers and writing partners, Aubrey Nunn and Alex Von Soos. The pair has produced a Number 1 hit single in the UK, Black Coffee by All Saints, and has worked with other A-list performers, including Dido, Seal, Corrine Bailey Rae and Faithless, among others. Dan's influences cover a wide range of top artists from several generations, including Paul Simon, Gram Parsons, The Lemonheads, U2, The Killers, and many more. Dan's path as an artist from here is only going to rise, as he gains the recognition he's worked to achieve. Check out Dan's moving music for your next production.
|Instrumental Tracks (1 Tracks) |
Music Genres For Dan Akio
Themes |
|Tracks for This Artist
DIRECT URL: https://www.audiosparx.com/DanAkio
|ARTIST KEYWORDS: Dan Akio, Singer-songwriter, Soft Rock, Pop Rock, production music library, royalty free sound, royalty-free production music, websites music, production music library, royalty-free song, music for video, royalty free music library, download stock music, world music, royalty free music download, company music, license music, websites music, royalty free music library


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Sept. 3, 2004
Baltimore, Md. - Seniors Jay Joyce and Kevin Nash each scored for Loyola in its men's soccer season opener, but a three-goal day for Princeton lifted the visiting Tigers over the `Hounds on Friday afternoon at Alumnae Field.A very even struggle quickly turned into a one-goal lead for Princeton when a foul inside the box awarded Darren Spicer a penalty kick. The Princeton junior converted and after only 3:26 elapsed in the game, the Tigers had the upper hand.Princeton's second goal, a shot by junior Adrian Melville that hit the crossbar and bounced in, left Loyola in an early two-goal deficit. The Tigers threatened to add more with a pair of corner kicks, but the Greyhounds held firm and would capitalize in the 32nd minute.After John Dalziel's shot sailed wide of the goal, Rade Kokovic sent an arching corner kick to Joyce who was trailing the play from the right side. Joyce beat Princeton keeper Erik White and the `Hounds were back in the game."We've had to come back from a few two-goal deficits and it's never easy," says Joyce. "We really needed something before the half to get us going and that goal did."The Greyhounds went into the half with momentum and came out attacking in the second stanza. After three consecutive corner kicks came up empty, the `Hounds earned the equalizer when Dalziel set himself and shot from outside the 18. Nash re-directed Dalziel's shot with his head and at 56:47 the game was knotted at 2-2."I was very pleased with the way we kept attacking Princeton," says Loyola Head Coach Mark Mettrick. "We really had them on the ropes in the second half."Loyola would only build on its momentum with close calls and tough shots. Sophomore Danny Wheelan had a clear look in the 68th minute and fired a shot off Princeton's post. Joyce kept the heat on with shot that sailed wide minutes later.The Tigers would get the game-winner, however, with Spicer's second goal of the day. The shot came at 80:47, beating Loyola keeper Greg Peters, who finished the game with five saves.The Greyhounds had 10 corner kicks to Princeton's four and both teams ended the contest with 14 shots. The Tigers begin the season 1-0, Loyola, 0-1, returns to action this Sunday when it hosts George Mason at 1 p.m.


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James Blunt - You're Beautiful (Video) MP3
2005 WMG You're Beautiful (Video) Pre-order the 'Apollo Edition' featuring 5 new tracks: http://smarturl.it/ApolloEdition Buy 'Moon Landing' on iTunes: ...
James Blunt - You're Beautiful Lyrics MP3
Please have a listen to a new Estonian song me and my band made: https://www.youtube.com/watch?v=d5id4nphQzo Make sure to leave a like and comment ...
'GAA' Toe-to-Toe: Sharing Your Sexual Past With Your Beau MP3
DL Hughley and Michelle Buteau discuss if it's smart to discuss sexual history with a loved one.
Labrinth - Beneath Your Beautiful ft. Emeli Sandé MP3
Beneath Your Beautiful is available now, click here to download from iTunes http://smarturl.it/BYBDigi?IQid=youtube. The album Electronic Earth is also out now, ...
Beneath Your Beautiful
LabrinthLabrinth You tell all the boys no Makes you feel good, yeah I know you're out of my league But that won't scare me away, oh no You've carried on so long You couldn't stop if you tried it You've built your wall so high That no [...]
Beau Ryan - Where You From? ft. Justice Crew MP3
Music video by Beau Ryan feat. Justice Crew performing Where You From?. (C) 2014 Sony Music Entertainment Australia Pty Ltd. Beau Ryan's fully sick tune ...
Sainte Anthony's Fyre - With Your Beau 1970 MP3
Clap Your Hands (1960) Enhanced Audio - The Beau Marks MP3
The Beau Marks Clap Your Hands Stereo Synch Mix Hands Clapping MP3
A stereo mix with hands clapping of this Quebec's 1960 classic. Un mix stéréo avec les claquements de mains de ce classique du Québec de 1960.
Sainte Anthony's Fire With Your Beau 1970 MP3
Directamente del vinilo original / Straight from the original vinyl.
Madison Pettis does the kiss, marry, unsubscribe tag: Luke, Jai, or Beau Brooks? - IMO Ep. 409 MP3
What's more fun then doing the Kiss, Marry, Unsubscribe Tag? The ladies of IMO do the tag with your favorite youtubers and celebrities! Do you agree with who ...
Beau Monga: Your X Factor NZ winner! MP3
New Zealand, we have an X Factor winner. After 10 weeks of live television, millions of tweets and countless arguments in family living rooms, 21-year-old Beau ...
How to Take The Perfect Selfie MP3
Sorry, my voice sounds sick! I caught a bad flu on the plane :/ Don't forget to subscribe to my channel! It doesn't matter if you don't have a frontal camera lens on ...
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Great 1966 performance of "When It Comes To Your Love" from a exploitation film called Village Of The Giants.
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Beau Mirchoff (Awkward, Desperate Housewives, The Grudge 3) talks about what he does outside of acting and the business, and how to keep stress at bay, ...
David Sabella-Mills sings "Don't Take Your Beau to The Seashore" - By Irving Berlin & E Ray Goetz MP3
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Beau Monga - King and Queen (Original Song - Live) MP3
Original Song "My Queen" by Beatboxer/Singer Beau Monga from New Zealand 2015.
'I Bet' YOUR Beau Isn't Taking You Here For A Date - Trending Topics MP3
Today's Trending Topics: Jay and Bey have a joint album! OR DO THEY? #MYSTERY Kanye + Kim may have a court case. CIARA HAS A NEW BOO? A NEW ...
"In Your Eyes" by Beau LePaige MP3
"In Your Eyes" by Beau LePaige Directed by Liam Dow (C) 2015. Cinematography By Michael Lieberman. Special thanks to Corey Gray for recording this track ...
DIY: Distressed Boyfriend Jeans | LaurDIY MP3
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You'll Be My Ribbon (I'll Be Your Beau) - Original Composition - Blue Sky Mission Club MP3
My special announcement is I now am part of a band called Blue Sky Mission Club :) Ta daa! Here's a song I wrote with band member London McDaniel. Thanks ...
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john wesley - Ride of Your Life (Feat. Beau Warren & Caitlin Hyatt) MP3
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Clap Your Hands par The Beau Marks MP3
L'Écureuil a son voyage,,,,
THE DEVIL LIFTS YOUR ARMS CHALLENGE (With Beau Toone) MP3


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: Proposal:
The "Trade _______ Right ******* NOW" Thread
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Registered User
Join Date: Aug 2009
Location: Detroit
Posts: 10,490
vCash: 500
Originally Posted by
Do people realize how many prospects the Wings have in their pipeline (keep in mind I didn't qualify the abilities of them). Trading away draft picks and prospects is what we SHOULD be doing. We've accumulated so many prospects since the lockout started that we obviously either don't have room for them or are not planning to make room for them.
I'm not claiming they have a ton of value - but being this conservative when we're re-signing Samuelsson, Bertuzzi and probably Cleary this year makes zero sense. So why try to get even more? Unload some for talent that helps us now.
i have absolutely no qualms with trading away a 19th overall pick(our highest pick in two decades that we earned) or prospects for good to elite young players(lets say a subban or a el nino level player) but not a journeyman bottom pairing dman being cast away by a non-playoff team(colorado)
quincey was a reactionary move to acquire a filler with no eye 3 to 5 years down the road.
so yah, if we arent going to be aggressive and "try" and acquire more top end young talent one way or another then its a better idea to just hold onto our picks
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Download the Game Design and Indie Game Marketing Freebook 07/19/17GameDev.net and CRC Press have teamed up to bring a free ebook of content curated from top titles published by CRC Press. The freebook, Practices of Game Design & Indie Game Marketing, includes chapters from The Art of Game Design: A Book of Lenses, A Practical Guide to Indie Game Marketing, and An Architectural Approach to Level Design. The GameDev.net FreeBook is relevant to game designers, developers, and those interested in learning more about the challenges in game development. We know game development can be a tough discipline and business, so we picked several chapters from CRC Press titles that we thought would be of interest to you, the GameDev.net audience, in your journey to design, develop, and market your next game. The free ebook is available through CRC Press by clicking here. The Curated Books The Art of Game Design: A Book of Lenses, Second Edition, by Jesse Schell Presents 100+ sets of questions, or different lenses, for viewing a game's design, encompassing diverse fields such as psychology, architecture, music, film, software engineering, theme park design, mathematics, anthropology, and more. Written by one of the world's top game designers, this book describes the deepest and most fundamental principles of game design, demonstrating how tactics used in board, card, and athletic games also work in video games. It provides practical instruction on creating world-class games that will be played again and again. View it here. A Practical Guide to Indie Game Marketing, by Joel Dreskin Marketing is an essential but too frequently overlooked or minimized component of the release plan for indie games. A Practical Guide to Indie Game Marketing provides you with the tools needed to build visibility and sell your indie games. With special focus on those developers with small budgets and limited staff and resources, this book is packed with tangible recommendations and techniques that you can put to use immediately. As a seasoned professional of the indie game arena, author Joel Dreskin gives you insight into practical, real-world experiences of marketing numerous successful games and also provides stories of the failures. View it here. An Architectural Approach to Level Design This is one of the first books to integrate architectural and spatial design theory with the field of level design. The book presents architectural techniques and theories for level designers to use in their own work. It connects architecture and level design in different ways that address the practical elements of how designers construct space and the experiential elements of how and why humans interact with this space. Throughout the text, readers learn skills for spatial layout, evoking emotion through gamespaces, and creating better levels through architectural theory. View it here. Learn more and download the ebook by clicking here. Did you know? GameDev.net and CRC Press also recently teamed up to bring GDNet+ Members up to a 20% discount on all CRC Press books. Learn more about this and other benefits here.
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About DiegoFloor
DiegoFloor replied to DiegoFloor's topic in Music and Sound FXYeah, I'm working on it. My biggest fear is that once everything is up and running there simply won't be any need to make new layers once we got a few basics working, which defeats the whole purpose of creating the system in the first place. But I'm probably being pessimistic.
DiegoFloor posted a topic in Music and Sound FXHi all I've been looking for a software to help me compose music. I don't want music production software, like most DAWs, nor sheet writing software. I want something focused on composition and there are a few out there, but they are so expensive! So I decided to start working on one. I came up with an idea that seems to fulfill my requisites and is easy enough that I can implement myself. I could really use some feedback though. I mean, it sounds great in my head but I get the feeling my head is not always on par with reality. OK, so it works like this: you get a simple synthesizer to make sound and a simple sequencer. So far so good. Then there's this concept of layers. A layer is something that transforms your sequence of notes in some way, like shifting notes to snap into a scale, or creating notes to form chords, or whatever. These layers operate on the outcome of the previous layer (that's why I call them layers) so you can stack them, modify any layer and all following update. The main gimmick is that these layers are made by the user, so you can come up with your own rules and patterns and try them out quickly. I'm thinking of a visual programming style, where you connect nodes to create functions and define the functionality of the layer. I tried asking for feedback on other communities but I got nothing. I guess people need more than just words, but it's gonna take a while to implement the layer system. If you want to play around with the prototype, you can get it here. But it's just a bare bones sequencer at the moment.
DiegoFloor replied to AGENT47's topic in Game Design and TheoryHah, when you said "keeping the puzzle intact" I immediately agreed with this other person. Remove anything that doesn't need to be there. If the level geometry only plays the role of holding the puzzle elements, then reduce as much as possible. I should emphasize that "doesn't need to be there" doesn't mean you have to make your game black and white with squares, even though that would surely make that puzzle more readable. There are things in a game that contribute to the experience in indirect ways. For example, maybe you need extra geometry in your level because you want it to look like a jungle, and it's important that the player is convinced that is a jungle. In that case, the extra geometry needs to be there, just not because of the puzzle. Does that make sense?
DiegoFloor replied to arnzzz's topic in Game Design and TheoryFirst of all, I should say that without playing there's not much anyone can say. Anyway. There are so many different directions you could take this to make it "more fun". Fun is relative, so I'd start by defining exactly what you mean by that. Maybe you want to make this a brainy game where fun comes from feeling like you overcame a difficult puzzle (so there's some fakery going on, as you can't overwhelm the player). In that case I'd suggest going turn based and working on the rules of the game. But maybe you want something with a little more adrenaline. In that case, maybe you need more gameplay layers to keep the player busier on more than one level. So he's managing events happening on the map level at the same time managing some mini game that happens every time you invade a country or something like that. Maybe there are more levels, "higher" than the map (I hope you know what I mean by that) like an upgrade system.
DiegoFloor replied to d000hg's topic in Game Design and TheoryI'm not aware of anything like that either. In any case, even if there is, it's rare enough that I'd start working on a prototype if I were excited about this idea. And in my opinion it sounds like a great idea! The player overlooks the strategic aspects of battlefields and the AI does the rest. Has an element of luck, and you maximize your odds by making good strategies and/or improving general stats, like gathering intel or simply training units etc. OK, now I wanna play this. Get to work! :P
DiegoFloor posted a topic in 2D and 3D ArtHey all When creating tileable textures there's a very simple trick in photoshop, using the offset tool. If one wants to make an image that serves as a 2d tileset it's not much harder, you make one tileable texture and then create the variations while preserving the borders, after that you can glue all variations in a single image file. I'm working on a 2.5d game tile based. Meaning the world is stored in a 2d array, indicating what's ground, wall, water etc and represented by 3d models. The problem now is to come up with a workflow for creating these 3d tiles! Each tile model has to flow continuously to every other tile, both texture and polygons. I would love to hear some thoughts on the subject and how are people solving that obstacle, and with what softwares.
DiegoFloor replied to DiegoFloor's topic in General and Gameplay ProgrammingI see now why my ideas seem too complicated! I should probably have said that in the first post, but I'm looking for a more general system that could do curved walls and not be restrained to a 2d plane. 2d square dungeons are so popular that this is a natural assumption. (editing the first post now) By the way, thanks for sharing your ideas! It really helps.
DiegoFloor replied to DiegoFloor's topic in General and Gameplay ProgrammingWhen you say pre-made tiles you mean big pre-made tiles, right? like a whole room made of 4 corner pieces etc. My ideas use pre-made tiles as well but in smaller chunks, but I want to avoid having to place them by hand as it's time consuming and not very fun. I might settle for something simpler like placing tiles, if the other alternatives prove to be too complicated for me. I had another idea. Define a floor like a huge plane (or terrain, to have some height variation) and just draw the walls along the floor, click and drag.
DiegoFloor posted a topic in General and Gameplay ProgrammingHi, I hope this is the appropriate forum for this discussion. I'm trying to come up with a dungeon maker system or, more generally, a system for creating rooms and tunnels that is easy and intuitive for the user. edit: The more powerful the creation system the better (obviously) and I'm aiming at something that allows the user the have curved walls and is not restricted to a 2d plane. I'm playing with a few ideas here. The result product of all of them is made with modules, pre made models. The user creates nodes, connects them and they become rooms connected by corridor and hallway. Maybe each node has properties that define what this is going to be in the final product, like room size or something like that. The other idea is by defining the ground. The user draws the ground as splines and the system creates the ground model and adds walls and ceiling. Third idea: The user creates the volumes like small minecraft's cubes and the system does some kind of 'interpolation', using curved walls and such when needed. Any ideas? [img]http://public.gamedev.net//public/style_emoticons/default/smile.png[/img]
DiegoFloor replied to Gnorme's topic in General and Gameplay ProgrammingPython is slow but you can use functions compiled in fortran and C. But I would advice agaisnt it, I regret deeply having used it for my simulations during my masters. It's great for its ease of use, quick prototyping etc. I still use it frequently as an advanced calculator "I wonder what's the sum of all prime numbers up to a million..."
DiegoFloor replied to Dark Star Studios's topic in Game Design and Theory"Cubicle Commando" I'm laughing so much at that one right now...
DiegoFloor replied to Dark Star Studios's topic in Game Design and Theory[quote]you misunderstood how 1337 means leet 7 is not t, 7 is L rotated pi Rad in any direction. and 1 is the t because you can sometimes write 1 with a horizontal line on the bottom and rotated pi Rad it makes a T[/quote] lol You're totally right. Thank you for the explanation. Still looks silly though.
DiegoFloor replied to Dark Star Studios's topic in Game Design and TheoryHow about writing something using that [font=georgia,serif]1337 [/font]way of writing? elite tester = [font=georgia,serif]1337 735732 = 1337 T35T3R = ... [/font] Analyzer = (that's a tough one) [font=georgia,serif][font=arial,helvetica,sans-serif]I don't like it but I'm sharing anyway lol[/font][/font]
[quote]I don't know where you got the definition of Euclidean space[/quote] I got the bad habit of referring to "euclidean geometry" as just "euclidean" during my masters.
You seem to be mixing geometry with topology there. Euclidean means zero gaussian curvature. You can have have a compact space with non trivial connectivity and still be euclidean. If you want to make something that complex (it certainly seems like it) you have to define very precisely where you want to go. Just generally something 'trippy, faceted, non euclidean' isn't enough.


text:
Testers: CentOS 6.5 Emulation and New AppCafe
For those of you on Edge and who wish to test the new Linux emulation and the new Appcafe, Kris has posted the instructions:
CentOS 6.5 emulation has been made the default for the EDGE packages going out this weekend. This replaces the legacy f10 package set. I've tested / confirmed that Flash works properly, haven't tried net-im/skype4 yet, but a package is available for those who want to try.
I've seen a few issues doing the update here with PKG. It seems often the conflict detection won't remove f10 on its own, so I've had to do this:
# pkg update -f
# pkg delete linux_base-f10\*
# pkg install nvidia-driver (Skip this if you don't use nvidia drivers)
# pkg install pcbsd-base
# pkg upgrade
# pc-extractoverlay ports
# reboot
After reboot, you need to recreate the flash plugin:
% flashpluginctl off && flashpluginctl on
Let us know any issues you see with the newer linux emulation layer.
Also, if you run into problems launching / navigating the newer AppCafe, please open bug reports asap. We are working to stabilize that as quickly as possible. Bug reports should be created at bugs.pcbsd.org.
Trackback from your site.
Comments (17)
Cool! Thanks! 🙂
This is great news!
But I had not to use your described steps, since the update/upgrade via
pc-updatemanager pkgupdate
worked without any hassle.
Thanks for your continnued great work!
I have a question
what that means CentOS 6.5 emulation on pcbsd
best regards
It adds the CentOS 6.5 userland so that Linux apps "just work" on PC-BSD.
thank a lot
i have a problem with appcafe: it dosn't running in my pcbsd-10.0.3
best regards
Does this mean we can run nVidia cuda compiled Linux binaries and use the power of GPU?
Good question… Try it and let us know!
The new App Cafe is responsive, but lacked indication that a program is already installed (as the old Cafe did). God Bless and keep up the great work!
Was the new AppCafe just reverted?
I was using the new version until just now, I updated, rebooted and found that the update icon now requests password and the old UI is back.
I still appear to be on the edge packages, did I miss something?
Yup. We decided that there won't be enough time before 10.1 release to polish the new appcafe. Instead, the 10.0.3 version of AppCafe will be the default while the new AppCafe is still available if you type pc-softweb to start it.
Since the update to 10.1 was mentioned. Will we have to do a fresh install or can we just upgrade to 10.1. I guess it is coming up soon but not sure how much after it is released that pcbsd uses it.
Kris is working on an update. Some things changed upstream so there might be special instructions for the update. It is recommended to wait for the official PC-BSD announcement before updating (unless you are just testing on a non-production system).
I am finding that many linux packages in the ports still depend on the fedora core 10 packages, if i wanted tof ix this, is there a sane way to do that without breaking the entire linux compat system?
We've started switching a number of the critical ports to C6, but the bulk of them will have to wait for the porters to catch up. Most of the F10 ports are obsolete and I would imagine we will see a large number of new linux emulation ports hit soon.
Cent OS emulation is great !!
BUT after upgrade as your instruction, Google Japanese, pinyin input method stop working !
Noway to reply east asian emails !
Do you know if the pinyin pkg also got updated? It should not be related to any of the CentOS changes.


text:
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Aundha Nagnath
Aundha Nagnath is considered to not just be one of the twelve auspicious Jyotirlingas in India, but also the very first Jyotirlinga. It is one of Read More
Named after the great Mughal Emperor Aurangzeb, Aurangabad is a popular city in Maharashtra. The word Aurangabad literally means 'Built by the throne'. Aurangabad city is Read More


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Help for the Home (1 of 3) by Marvin D. Patterson
This content is part of a series.Help for the Home (1 of 3)
Series: Fortifying the Family
Marvin Patterson
Deuteronomy 6:1-15
We are going to be looking for the next couple of weeks on "How to fortify the family." I hope this study will help you and your family to be blessed by God, whether you are a single parent, or have ten children, or grandchildren. I hope that you will get all your family and friends here for the next couple of Sunday mornings as we will get God's insights on developing stronger families.
Today we will start in the Book of Deuteronomy chapter six. I've shared with you that we have a boxer puppy around the house, and the whole family has fallen in love with that little fellow! I told Wanda that we needed another one so he would have a buddy! She said "No way hosay! One is enough!" Well, strangely enough, God sent our little boxer puppy a companion. It came in the form of a little black and white kitten. It showed up about two weeks ago, and my boxer and the little kitten have become the best of buddies! They love running around the yard terrorizing each other! It reminds of a true story that I read about this week!
The Pastor's Cat...
Dwight Nelson recently told a true story about the pastor of his church. He had a kitten that climbed up a tree in his yard and then was afraid to come down. The pastor coaxed, offered warm milk, etc.
The kitty would not come down. The tree was not sturdy enough to climb, so the pastor decided that if he tied a rope to his car and drove away so that the tree bent down, he could then reach up and get the kitten.
That's what he did, all the while checking his progress in the car. He then figured if he went just a little bit further, the tree would be bent sufficiently for him to reach the kitten. But as he moved the car a little further forward, the rope broke.
The tree went "boing!" and the kitten instantly sailed through the air-out of sight. The pastor felt terrible. He walked all over the ne ...
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Do I have enough fat on my stomach area to go larger like Amber Rose, Kim K. or Nicki Manji? (Photo)
Doctor Answers 5
Do I have enough fat on my stomach area to go larger like Amber Rose, Kim K. or Nicki Manji? (Photo)
You look like a great candidate for a Brazilian Butt Lift . The common donor sites for this procedure is your posterior waist/flanks, lateral thighs, back, waist, and anterior tummy. A Brazilian butt lift is a safe and effective procedure that combines liposuction of the abdomen hips waist and back and then fat grafting to the gluteal region. This is a safe and effective way to contour the lower body. After a successful Brazilian Butt lift, the fat that remains will stay there for the rest of your life. Like the rest of the tissue in your body this fat will grow and age with you. Choose a board certified plastic surgeon who has done a ton of Brazilian Butt Lifts and has privileges to do this surgery at a local university hospital. This says his credentials have been checked out by fellow Drs. All surgeries carry risk, talk to your plastic surgeon and choose one wisely. Many surgeons, including myself, offer online virtual consultations where you send us your photos and we can estimate the cost. I, like many surgeons, also offer free consultations so that you can be examined and given the most accurate quote.
Learn more: http://spectrum-aesthetics.com/brazilian-butt-lift/
Brazilian Butt Lift/ Fat grafting/Liposuction/ High Definition Procedures/Butt Implants
Thank you for your question.
From your photos, you appear to be an excellent candidate for Vaser Hi definition liposculpture with fat grafting to the buttocks. You should get a great result from liposculpture, giving you a more contoured and hourglass shape with better projection to your buttocks.
Vaser Hi Definition liposuction uses the concepts of negative and positive spaces in relation to the underlying bony and muscle anatomy to define areas such as your six pack and create an athletic looking physique.
The best way to assess and give true advice would be an in-person exam.
Please see a board-certified plastic surgeon that specializes in aesthetic and restorative plastic surgery.
Dr. Schwartz
Board Certified Plastic Surgeon
Learn more: http://beverlyhillsbreastandbodyinstitute.com
Enough Fat For A Butt Like Amber Rose, Kim K., or Nicki Minaj?
You already appear to have a nice baseline volume for your buttocks. As a plastic surgeon who has done many BBLs, I've seen a lot of butts and I would not describe your butt as a "small" butt. It is important to note that for a BBL, it's not just what you add, but what you take away. I've had many patients in whom just liposuctioning the abdomen, flanks, sacrum, and back have created the illusion of a bigger butt WITHOUT adding fat to the buttocks. Treating these areas for you can accentuate the appearance of any fat added to your buttocks and make you look like you have hips. Treating these areas would make your waist look smaller and your waist-to-butt ratio will make you look like you have a bigger butt. Yes, 30-50% of the fat may be absorbed. In the end, you just have to do the best with what you have. You may want to consider adding liposuction of other areas (like the front of your thighs) to get more volume if you do not feel you'll get the results you'll want with abdominal liposuction. Also, remember that how you care for your butt in the recovery period is just as important as the procedure. To avoid flattening your newly injected fat, do not sit, lay, or put any pressure on your buttocks for the first 6-8 weeks after surgery. In my practice, I provide a special sitting roll (NOT a donut roll as that pushes on your butt fat) after surgery so that my patients can sit without putting any pressure whatsoever on the buttocks. Best of luck!
You might also like...
You definitely have enough and plenty fat on your abdomen to add to your buttocks and hips. Also you may want to get rid of the fat on your love handles, for a much tiny waist line.
Best wishes
Do I have enough fat on my stomach area to go larger like Amber Rose, Kim K. or Nicki Manji?
Have you had a procedure done already? If you have never been liposuctioned before, you likely have enough fat in your stomach and love handles to give your buttocks a nice volume increase especially at the lateral aspect as your buttocks have nice projection but is not rounded out on the sides. If you have already had liposuction to your midsection, you likely have scar tissue and the volume of fat that could be collected will not be a fruitful.
These answers are for educational purposes and should not be relied upon as a substitute for medical advice you may receive from your physician. If you have a medical emergency, please call 911. These answers do not constitute or initiate a patient/doctor relationship.


text:
Georgia is a country full of natural beauty which shares the borders with Russia, Turkey, Armenia, and Azerbaijan. It is a small country with an area of 69,700 square kilometers and a population of around 4 million. It is 31 times smaller than Saudi Arabia in size. The capital of the country is called Tbilisi and it is the main attraction for tourists from all around the world. It is situated in the Asia continent. Georgia is very famous for its scenic beauty and historical places. I would encourage all of you to visit this beautiful place. Being GCC and Saudi Iqama (Residence Permit) holders, you are entitled to get visa-free entry to this beautiful country Georgia. However, it is very important to read following guidelines before making your mind.
- In order to get visa-free entry to Georgia, make sure you have a valid passport for at least 6 months. Georgia immigration allows you to stay in the country for 90 to 180 days. If your passport is not valid for that period, you might face difficulties.
- Make sure you are carrying your Iqama (Residence Permit) with you if you want to get visa-free entry to Georgia. The validity of the Iqama (Residence Permit) should not be less than 3 months. Although they don't publicize it on their website, but you might face troubles if your Iqama (Residence Permit) is valid for a time more than you want to stay there.
- Make sure you have booked a return flight from Georgia. You will not be granted a visa-free entry to Georgia based upon your GCC or Saudi Iqama (Residence Permit) if you could not produce a confirmed ticket to return from Georgia after your stay.
- You must provide hotel booking for all the days you are going to spend there. For example, if your flight to return from Georgia is after 10 days, you need to provide them hotel booking of all 10 days. You are unlikely to get visa-free entry to Georgia on your GCC or Saudi Iqama (Residence Permit) if you could not produce hotel booking to immigration authorities.
- Make sure you are taking enough cash with you to meet your expenses in Georgia. I think $100 per day per person is enough in this regard. It is always better to carry USD there as you will get very low exchange rate for Saudi Riyals or other GCC countries' currency. If you don't want to carry that much money, you can show them your credit card along with last month's credit card statement showing your credit limit.
- You can always check if you are required to get a visa to Georgia using this link.
- You can always check if you are required to get a visa to Georgia using this link.
Travelling from KSA to Other Countries
- Procedure to Apply for Schengen Visa from Saudi Arabia
- Procedure to Apply for Malaysian Visit Visa from Saudi Arabia
- Procedure to Apply for Thailand Visit Visa from Saudi Arabia
- Procedure Apply for Turkey visit visa from Saudi Arabia
- Procedure to Apply for the UK (United Kingdom) Visitor Visa from Saudi Arabia


text:
Belle Arti Resort
Via Santa Maria Di Costantinopoli 27, Naples, 80138
Price FromView Rooms
This hotel doesn't have any reviews yet.
About the hotel
Property Location
With a stay at Belle Arti Resort in Naples (Historical Center), you'll be minutes from Second University of Naples and National Archaeological Museum. This bed & breakfast is within close proximity of Galleria Principe di Napoli and Piazza Bellini.
Make yourself at home in one of the 7 air-conditioned rooms featuring minibars.
Your room comes with a pillowtop bed. Complimentary wireless Internet access keeps you connected, and satellite programming is available for your entertainment. Bathrooms have showers and hair dryers.
Make use of convenient amenities, which include complimentary wireless Internet access and tour/ticket assistance. Getting to nearby attractions is a breeze with the area shuttle (surcharge).
Business, Other Amenities
Featured amenities include complimentary high-speed (wired) Internet access, express check-in, and express check-out. For a surcharge, guests may use a roundtrip airport shuttle (available on request) and a ferry terminal shuttle.
Read MoreLess
Facilities Show/hide
- Parking
- Car parking available onsite, Airport Shuttle
- Main
- Laundry/Valet Service
- Business
- Wi-Fi Available In Public Areas
Useful Info Show/hide
- Between 14:00 and 21:00
- By 11:00 hours
- Up to 8 years old
- You must present a photo ID when checking in. Your credit card is charged at the time you book. Bed type and smoking preferences are not guaranteed.Your reservation is prepaid and is guaranteed for late arrival. The total charge includes all room charges and taxes, as well as fees for access and booking. Any incidental charges such as parking, phone calls, and room service will be handled directly between you and the property. Know Before You Go Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation. This property offers transfers from the ferry terminal, airport, and train station (surcharges may apply). Guests must contact the property with arrival details 48 hours prior to arrival, using the contact information on the booking confirmation. Mandatory Fees and Taxes You'll be asked to pay the following charges at the hotel: A tax is imposed by the city: EUR 1.50 per person, per night, up to 10 nights. This tax does not apply to children under 18 years of age. We have included all charges provided to us by the property. However, charges can vary, for example, based on length of stay or the room you book. The LateRooms.com rate for Belle Arti Resort in Naples is the total price of the room and not the 'per person' rate. All taxes are included, with the exception of city tax, which will be charged at 1.00(EUR) per person per night and is payable to the hotel directly.
- Pets are not allowed
Customer Reviews
Our reviews are from genuine LateRooms.com customers. We verify that the customer has stayed before leaving a review.
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text:
|Price incl. VAT
|Price excl. VAT
Indulge yourself in the heights of cloud for the melodious tones that will put facilities Groove Apogee AD / DA. One little box can flourish sculpt each sound to the maximum detail. The music of excellent quality, which is commonplace for these components can put a smile on the face of almost everyone.
APOGEE GROOVE is a box that can change the sound of your music device MAC absolutely perfect and professional symphony tone. Just using a USB cable to connect the accessory to your computer or tablet from Apple. After you turn on the music will sound from the ear speakers or headphones pleasing tones decorated with massive bass, clear highs and booming depths. This component can guarantee quality sound up to 24 bit / 192 kHz. Furthermore, it will expand the range of options when using iTunes, Spotify or Pandora. I will enjoy worked brilliantly colored LED indicator indicates the status and level of musical recordings.
Specifications can be changed without notice. Images are for illustrative purposes only. Retail Price (MSRP) means the manufacturer's/supplier's suggested retail price.
Link to Manufacturer's Website: Enter link to the external review


text:
Fiat Chrysler boss Sergio Marchionne has once again reiterated his lack of belief in electric cars as the future. At least, that's the takeaway from a conversation he had over lunch with CAR Magazine. For example, he was asked whether Fiat Chrysler Automobiles (FCA) could mimic the success Tesla has had in creating a following […]+ Read More
Today Fiat Chrysler CEO Sergio Marchionne said that his company's partnership with Google on autonomous vehicles is just the "first phase" of larger relationship. Fiat Chrysler Automobiles (FCA) is working to put Google's autonomous driving system in 100 Chrysler Pacifica minivans. "This first phase of the operation is very targeted. It's designed to take Google […]+ Read More
Toyota and Mazda are in the final stages of a new agreement to collaborate on eco-oriented technology. More restrictive fuel economy and emissions regulations, along with increased pressure from the competition, led to this partnership, according to the Nikkei Asian Review. "Toyota intends to supply fuel cell and plug-in-hybrid technology … Mazda, which has lagged […]+ Read More
This past Saturday, Fiat Chrysler CEO Sergio Marchionne said that he had a visit with Tesla CEO Elon Musk and Apple's Chief Executive Tim Cook during a recent trip to California. Fiat's CEO said he also reportedly spent 45 minutes in one of Google's self driving cars. Marchionne, the person responsible for bringing Fiat and […]+ Read More
A former executive with the Environmental Protection Agency believes that CAFE regulations won't be lowered during the upcoming midterm review, saying the fuel economy numbers are achievable by the auto industry. Margo Oge, called the "Queen of Cleaner Cars" by some, was the EPA's director of the Office of Transportation and Air Quality when the […]+ Read More
Last January Fiat-Chrysler CEO Sergio Marchionne said FCA has a "massive" program to develop a plug-in hybrid minivan and he's now emphasized FCA wants to be first as the company arguably has before. What many consider one of the first modern conventional "minivans" launched in 1984 as the Dodge Caravan and Plymouth Voyager and these […]+ Read More


text:
Be My Always Necklace
$ 29.00
- Sponsored by MoonMail
- Delicate chain necklace with open circle charm
- Color: Gold
- Your soon-to-be staple piece! This classic, round geometric necklace can be worn daily.
- All KITSCH accessories are PLATED. Remember, anything plated will eventually turn with time and wear. Avoid moisture. Remove at night. Store gold with gold and silver with silver. Do not clean with chemicals. Wipe with a clean, dry cloth to clean.
- Jewelry always fits, so go eat that pizza, girl
- Necklace chain measures 17 inches + 1 inch extender chain. Circle charm measures approximately 0.5 inches in diameter.
- One size


text: Updated from 12:23 p.m. ESTMicrosoft ( MSFT) chief Steve Ballmer repeated his warm words for Yahoo! ( YHOO) at a strategy session Tuesday, helping to float shares again on takeover hopes. Speaking at Microsoft's Strategic Meeting Update presentation, Ballmer said he would like to talk with Yahoo! CEO Carol Bartz to see if the two companies could "somehow get together and find out how to provide more competition" for Google ( GOOG). The comments once again revived the notion that Microsoft still needs Yahoo! to strengthen its Internet weaknesses. The idea helped send Yahoo! shares up 73 cents, or 6%, to $12.70 in pre-market trading Tuesday. This of course is familiar ground for Yahoo investors who watched Microsoft spend the better part of last year pursuing and later withdrawing merger proposals. Microsoft is a distant No. 3 player in Internet search advertising and faces a tough choice of keeping its status quo as Google and Yahoo! take more of the business, or make a costly move to jump in to the market. Yahoo's new chief Bartz is reorganizing the company and expected to make an announcement this week on her new plan. So far, Bartz's comments on the topic of Microsoft have been tepid at best. Bartz has a number of strategic options including Time Warner's ( TWX) AOL franchise if she is deal hunting, as TheStreet.com's Michael Goodman points out. "For advertisers, all of whom will eventually start spending again, AOL's network remains one of the most attractive properties to reach a broad audience," Goodman wrote. In search advertising, Microsoft and Yahoo! could combine to make a formidable foe to Google. And in display ads, where Yahoo! is strong, a hook up with AOL would "make sense," Goodman notes.


text:
Candy Liu
Invisible Target
When the Ronin Gang robs an armored car, three cops -- Chan (Nicholas Tse), Fong (Shawn Yue) and Wai (Jaycee Chan) -- go on the warpath. Each has a personal interest in bringing down Ronin leader Tien (Jacky Wu): he killed Chan's fiancée, humiliated Fong and kidnapped Wai's brother. Outnumbered but determined, the trio launches a deadly war from which only one faction will emerge alive.