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Engagement of FcεRI causes its phosphorylation by Lyn kinase. Two alternatively spliced variants, Lyn A and B, are expressed in mast cells, and both isoforms interact with FcεRI. Unlike Lyn A, Lyn B lacks a 21-aa region in the N-terminal unique domain. In this study, we investigated the role of Lyn A and B isoforms in mast cell signaling and responses. Lyn B was found to be a poor inducer of mast cell degranulation and was less potent in both inositol 1,4,5-triphosphate production and calcium responses. Expression of Lyn B alone showed reduced phosphorylation of both phospholipase Cγ-1 and -2 and decreased interaction of phospholipase Cγ-1 with the phosphorylated linker for activation of T cells. Lyn B also showed increased binding of tyrosine-phosphorylated proteins, which included the negative regulatory lipid phosphatase SHIP-1. In contrast, both Lyn A and B caused similar total cellular tyrosine phosphorylation and FcεRI phosphorylation and neither Lyn A nor Lyn B alone could completely restore mast cell degranulation or dampen the excessive cytokine production seen in the absence of Lyn. However, expression of both isoforms showed complementation and normalized responses. These findings demonstrate that Lyn B differs from Lyn A in its association with SHIP-1 and in the regulation of calcium responses. However, complementation of both isoforms is required in mast cell activation.
Mast cells are important innate immune cells that can amplify the adaptive immune response (1). They are also known as the central effector cell in IgE-mediated allergic and inflammatory disorders. In an allergic reaction, mast cell activation is initiated through the recognition of an Ag by Ag-specific IgE bound to the α subunit of FcεRI, which is expressed on the cell surface. The Src family protein tyrosine kinase Lyn provides the key recognition signal that inteprets receptor engagement into intracelluar events by transphosphorylating the FcεRI β and γ subunits (2). Efficient phosphorylation of the FcεRI requires specialized regions of the cell membrane that are enriched in cholesterol and sphingolipids (commonly termed lipid rafts) as both Lyn and FcεRI can be concentrated in these domains upon receptor engagement (3). Phosphorylation occurs within the cytoplasmic tails of the β and γ subunits in a domain that encodes the ITAM, which is characterized by a YXXL-X7-YXXL amino acid sequence (4). Once phosphorylated, phospho-ITAMs constitute a novel docking site for the binding and subsequent activation of Src homology 2 (SH2)-domain containing molecules, such as Syk, a tyrosine kinase that is crucial for mast cell activation (5). The activation of Syk results in the phosphorylation of multiple substrates, among which the membrane-localized linker for activation of T cells (LAT) coordinates the assembly of a molecular complex that includes proteins like phospholipase C (PLC)γ. PLCγ catalyzes the hydrolysis of phosphatidylinositol-4,5-biphosphate to inositol 1,4,5-triphosphate (IP3) and diacylglycerol. IP3 binds to its receptors on the endoplasmic reticulum, promoting calcium release from the intracellular stores, which, upon emptying, trigger calcium influx from the extracellular environment via store-operated calcium channels like Orai1/CRACM (6, 7). Diacylglycerol binds the C1 domain of a number of proteins (like protein kinase C) promoting their membrane localization and activity. Both the calcium influx and protein kinase C activation are essential for the release of preformed granule-stored allergic mediators and the de novo synthesis of cytokines and eicosanoids from mast cells (8, 9).
As the key initiating kinase, the therapeutic targeting of Lyn is of interest, because intervening at this step should presumably abrogate mast cell activation. However, recent studies suggest that Lyn has both positive and negative regulatory roles (10–12) and, in the context of a particular genetic background, Lyn deficiency could result in either reduced or increased mast cell degranulation and anaphylactic responses (13, 14). In mast cells as well as in other cell types (15), Lyn kinase exists as two isoforms, Lyn A and Lyn B of 56 and 53 kDa, respectively. These isoforms are generated by alternative splicing and differ by a 21-aa insert found in the N-terminal unique domain of Lyn A (Fig. 1A) (16). Prior studies have shown that both Lyn A and Lyn B coimmunoprecipitate with FcεRI (17). In addition, both isoforms can be found in lipid rafts (18). In mast cells derived from a mouse model of Smith-Lemli-Opitz Syndrome (an inborn error of cholesterol metabolism leading to loss of cholesterol from lipid rafts) both isoforms of Lyn are lost from lipid rafts, and these mast cells showed a hyperresponsive phenotype (19). Although distinguishing the individual roles of Lyn A and Lyn B (or whether one isoform has a more dominant negative function) is of considerable interest, this was not feasible by means of silencing RNA or genetic deletion strategies, as both isoforms arise from a single gene and differ in a relatively short stretch of nucleotide sequence.
In this study, we investigate the roles of the two Lyn isoforms by expressing each individually or together in mast cells derived from lyn−/− mice. Our results show unique properties as well as redundant roles for the Lyn A and B isoforms. Lyn B was found to dominantly associate with the negative regulatory lipid phosphatase SHIP-1 and was less effective in eliciting calcium responses and mast cell degranulation. However, Lyn B was equivalent to Lyn A in total cellular tyrosine phosphorylation and FcεRI phosphorylation. Lyn A showed stronger calcium responses and degranulation than Lyn B and was better in promoting the interaction of PLCγ with phospho-LAT. Importantly, expression of each individual isoform did not fully restore calcium fluxes or degranulation or controlled hypercytokine production, but expression of both isoforms fully normalized these responses. The findings demonstrate differences in the role of these two Lyn kinase isoforms in mast cell signaling and responses. However, they also demonstrate a requirement for both Lyn A and Lyn B in generating normal mast cell responses.
Materials and Methods
Mice used in this study were wild-type (WT) and lyn−/− (129/Sv, or 129/Sv × C57BL/6 [N8]), which were bred in-house. Animals were maintained and used according to National Institutes of Health guidelines and a National Institute of Arthritis and Musculoskeletal and Skin Diseases-approved animal study proposal.
Mouse anti-DNP mAb was purified from culture supernatants of the H1-DNP-ε-26.82 hybridoma (20). Mouse mAb to FcεRIβ was previously described (21
Cell cultures
Total bone marrow from the femurs of lyn−/− and WT 6-wk-old female mice was extracted and used to obtain cultures of bone marrow-derived mast cells (BMMCs). Cells were cultured for 4 wk in the presence of IL-3 and stem cell factor (PeproTech, Rocky Hill, NJ). Cultures were checked periodically for mast cell differentiation and purity by FcεRI and c-Kit expression. Cells were used when cultures achieved ≥95% of the population expressing both markers.
Lentiviral expression constructs
Lentiviral gene transduction
Supernatants containing virus were harvested after a 48 h transfection of 293FT cells with Lyn A or B constructs (Cell Biolabs, San Diego, CA) as previously described (22). Viral particles were concentrated 25× by centrifugation for 2 h at 20,000 × g. BMMCs (10 × 106
Ag stimulation, immunoprecipitation, and Western blotting
For Ag stimulation, BMMCs were sensitized at 37°C for 3 h with 1 μg/ml IgE in RPMI 1640 without cytokines. Cells were washed and stimulated at 37°C with 100 ng/ml Ag (DNP-BSA) or with the indicated concentration in Tyrode's buffer (135 mM NaCl, 5 mM KCl, 20 mM HEPES, 5.6 mM glucose, 1 mM MgCl2, 1.8 mM CaCl2, 0.05% BSA [pH 7.4]). Stimulation time varied with the type of experiment as indicated. Cell lysates were prepared by incubation of 20 × 106 cells in 1 ml BSS buffer containing 0.5% Triton X-100 and 20 mM octyl β-d
Measurement of IP3 production and calcium fluxes
Intracellular IP3 was extracted with 20% cold perchloric acid from IgE-sensitized cells (10 × 106/sample) challenged with 25 ng/ml Ag. The acidified samples were centrifuged at 2000 × g for 15 min at 4°C, and the supernatants were neutralized with a 1:1 (v/v) mixture of 1,1,2-trichlorotrifluoroethane:trinoctylamine. The concentration of IP3 in each sample was measured with a competitive binding assay (IP3-[3H] Biotrak Assay System, GE Healthcare).
To measure calcium mobilization, two methods were used. In some experiments, cells were sensitized overnight and loaded with 3 μM fura 2-AM for 20 min, washed, and aliquoted in 96-well plates (9 × 104 cells/well). Cells were then challenged with Ag (25–50 ng/ml), and changes in intracellular calcium were monitored with a microplate fluorescence reader (Opti-Fluor; BMG Labtech, Cary, NC) as previously described (23). In some experiments, single-cell calcium imaging was performed using a Zeiss LSM510 META confocal microscope (Carl Zeiss MicroImaging, Thornwood, NY). The procedure was essentially described (24). Briefly, 5 × 106
β-Hexosaminidase release assays
Mast cell degranulation was measured by the release of the enzyme β-hexosaminidase as described elsewhere (25). Briefly, cells were sensitized for 2 h with 1 μg/ml anti-DNP IgE in medium without growth cytokines. Cells were stimulated for 30 min with varying concentrations of Ag (ranging from 0.1 to 1000 ng/ml) in Tyrode's buffer followed by supernatant collection. β-Hexosaminidase released into supernatants was measured by incubation with substrate and quantified colorimetrically as described by Blank and Rivera (25).
Cytokine production
For cytokine secretion analysis, 1 × 106 BMMCs were incubated with anti-DNP IgE as described above and stimulated with 100 ng/ml DNP for 4 h. Supernatants were collected, and the amounts of IL-6, IL-13, and TNF-α were measured by Millipore Cytokine Multiplex Panel (Millipore, Billerica, MA).
Statistical analysis
Statistical significance of observed differences (p value) was assessed by two-tailed Student t test using the Prism software (GraphPad, San Diego, CA).
Expression of the individual isoforms, Lyn A or Lyn B, does not alter Lyn-dependent phosphorylation of FcεRI or Syk activation
Lyn A and Lyn B isoforms (Fig. 1A) were expressed in BMMCs derived from lyn−/− mice. Stable expression was achieved after 2 wk of blasticidin selection, as decribed in Materials and Methods, with the protein levels being comparable to those found in WT mast cells (Fig. 1B). Expression of Lyn A or Lyn B alone caused no significant change in mast cell differentiation and FcεRI expression (Fig. 1C). Given the extensive regulatory cross talk among Src protein tyrosine kinases (26), we verified that each individual isoform was active when expressed individually. The kinase activity, after immunoprecipitation of each isoform from cells expressing them individually, was determined in an in vitro kinase assay and compared with the activity in WT cells. As shown in Fig. 1D, each individual isoform was active only in the presence of ATP. Ag stimulation caused only a minimal increase in Lyn activity for both isoforms as measured in vitro, consistent with previously published reports (13), suggesting that large fractions of both Lyn A and B can be readily activated by the presence of ATP in the absence of FcεRI engagement. No kinase activity was found in the immunoprecipitates from Lyn-null mast cells whether transfected to express LacZ or not (Fig. 1D). Thus, expression of the individual Lyn isoforms did not appear to alter their intrinsic kinase activity. To assess the relative activation of Lyn A and B in cells, we measured the phosphorylation of the activation loop Y396 (as recognized by an Ab to Y416 in human Lyn) versus the inhibitory Y507 prior to and after FcεRI stimulation. Fig. 1E shows that, when normalized for the amount of protein expressed, the FcεRI-dependent phosphorylation of Lyn B at Y396 is more prominent than that of Lyn A. Thus, the ratio of Y396/Y507 increased after FcεRI stimulation, whereas the fraction of Lyn A phosphorylated at Y396 showed an initial decrease and a subsequent return to baseline levels. This suggested that Lyn B may be more active in mast cells, but the fraction of Lyn A and Lyn B that is involved in FcεRI-stimulated mast cell responses is unknown, and this would likely determine their relative activities in vivo.
As shown in the control LacZ-transduced lyn−/− BMMCs (Fig. 2A), and also as previously reported (10), the absence of Lyn kinase caused a marked reduction in the total cellular tyrosine phosphorylation in resting and FcεRI-stimulated mast cells. This demonstrates that the dectable tyrosine phosphorylation seen in mast cells is highly dependent on Lyn activity. Therefore, it was of interest to determine if the profile of tyrosine phosphorylated proteins might differ considerably in cells individually expressing Lyn A or Lyn B. As shown in Fig. 2A, the total cellular tyrosine phosphorylation did not differ markedly between the two isoforms, and the gross pattern of phosphorylation observed was similar (albeit not identical) to that of WT mast cells. Because Lyn is the kinase responsible for ITAM phosphorylation of β- and γ-chains of FcεRI, we evaluated the relative contribution of the Lyn isoforms to FcεRI tyrosine phosphorylation following the stimulation of this receptor. As shown in Fig. 2B, both Lyn A and Lyn B were equally effective in the phosphorylation of the FcεRI β- and γ-chains, albeit at levels that appeared to be slightly less than that of WT mast cells. Quantitation of the phosphorylation of the FcεRI β- and γ-chains for all experiments showed that at 3 min poststimulation, the β-chain in WT mast cells had a significantly higher phosphorylation (30%) than in Lyn A- or Lyn B-expressing cells. However, although a trend for increased phosphorylation of the FcεRI γ-chain at 3 min was observed, no significant difference was found for the phosphorylation of the FcεRI γ-chain, and the kinetics of FcεRI phosphorylation were unaltered upon expression of either isoform when compared with WT cells. Thus, Lyn A and Lyn B were equally efficient in the phosphorylation of FcεRI and showed similar ability in inducing total cellular tyrosine phosphorylation.
Given that FcεRI phosphorylation is essential for the activation of Syk kinase (5) and that phosphorylation of mouse Syk on Y346 (which is recognized by an Ab to human Syk Y352, the equivalent site on human Syk) requires Lyn kinase (13), we investigated the effect of Lyn A and Lyn B on phosphorylation of Syk. As shown in Fig. 2C, phosphorylation of Syk at 3 min poststimulation occurred efficiently in cells expressing Lyn A or Lyn B and appeared to be comparable to that seen in WT cells. Varying doses of Ag did not reveal any difference in Syk Y346 phosphorylation between Lyn A- and Lyn B-expressing cells (data not shown). Thus, it is clear that the extent of Syk phosphorylation at Y346 did not differ significantly between Lyn A and Lyn B (Fig. 2C), arguing that both isoforms can promote Lyn-dependent phosphorylation of this site. This was verified, because cells expressing LacZ or Lyn-null cells showed minimal phosphorylation of Y346, consistent with our previous results (13). Thus, both Lyn A and Lyn B are capable of causing Syk phosphorylation at the Y346 site in a manner similar to that of WT cells, consistent with the ability of each isoform to similarly elicit FcεRI phosphorylation.
Lyn A and Lyn B differ in promoting mast cell degranulation but not cytokine production
We assayed cells expressing the individual Lyn isoforms for their ability to promote FcεRI-mediated degranulation, as measured by release of granule-localized enzyme β-hexosaminidase. Our past studies (11, 13) demonstrated that Lyn-null mast cells derived from 129/Sv mice or from early crosses to C57BL/6 mice showed hyperresponsive degranulation. In contrast, mast cells from Lyn-null C57BL/6 mice or mice backcrossed extensively (N8) into this background showed hyporesponsive degranulation when compared with WT mice. This difference was due, in part, to the relative levels of Fyn expression, as C57BL/6 mice showed lower levels of Fyn kinase, and hyperdegranulation was restored by overexpression of this kinase (13).
At the onset of these studies, we decided to use lyn−/− BMMCs derived from mice backcrossed extensively (N8) into a C57BL/6 background. These cells showed a marked loss of FcεRI-induced degranulation (Fig. 3A), thus allowing us to determine if Lyn A or Lyn B or both isoforms were needed to promote degranulation. Cells expressing Lyn A or Lyn B showed reduced degranulation response when compared with WT cells (Fig. 3A), albeit the impairment observed in cells expressing Lyn B was more marked. Although Lyn A-expressing cells also showed a reduction in their degranulation response, in some experiments, these cells approached the degranulation response of WT cells. Moreover, Lyn A-expressing cells consistently showed a degranulation response that was considerably higher than Lyn B-expressing cells. The impaired degranulation of Lyn B-expressing cells was not due to a deficiency in granule content or to a developmental defect in the granule fusion apparatus because PMA and calcium ionophore (A23187) elicited a similar and potent degranulation from all cells, regardless of whether they expressed the individual Lyn isoforms or none at all (Fig. 3A).
Previous work (11) demonstrated that the loss of Lyn kinase led to increased cytokine responses in mast cells and that this phenotype was independent of the genetic background of the mice from which the cells were derived (13). Thus, we explored if the different isoforms of Lyn had differing roles in suppressing cytokine responses. In these experiments, we used BMMCs derived from lyn−/− 129/Sv mice because of their potent cytokine responses relative to BMMCs from lyn−/− C57BL/6 mice. We reasoned that a more potent cytokine response might allow us to more easily ascertain the relative contributions of Lyn A and Lyn B to this response. As shown in Fig. 3B, both Lyn A and Lyn B showed a similar loss in the ability to suppress the hyperproduction of IL-6, IL-13, or TNF, similar to Lyn-null mast cells but markedly differing from WT cells. Neither isoform alone was able to completely restore the negative control of cytokine production to the levels seen in WT mast cells. However, cells expressing Lyn A showed a trend (albeit not significant) toward dampening of the hypercytokine response than those expressing Lyn B. Thus, the findings show that Lyn A more effectively promotes mast cell degranulation and is seemingly better in dampening cytokine responses.
Lyn B is less effective than Lyn A in evoking Ca2+ responses and optimal IP3 production following FcεRI engagement
To explore the underlying mechanism for the observed difference between Lyn A and Lyn B in inducing mast cell degranulation, we investigated whether the two isoforms were similarly able to induce intracellular Ca2+ mobilization upon FcεRI stimulation. A rise in the intracellular Ca2+ concentration that is coupled to the influx of Ca2+ from the extracellular medium is essential for the degranulation response (9). As shown in Fig. 4A, both Lyn A and Lyn B showed altered Ca2+ fluxes when compared with WT cells. For both isoforms, the onset and extent of the calcium response was impaired. Lyn B-expressing cells showed the most marked delay in Ca2+ fluxes relative to Lyn A-expressing or WT cells. Nonetheless, in many experiments, the Ca2+ flux of Lyn A-expressing cells was also delayed when compared with WT cells, and the extent of the response appeared to be diminished for both Lyn A- and Lyn B-expressing cells. Relative to the Ca2+ flux of LacZ or Lyn-null mast cells, both Lyn A and B elicited a significant rise in intracellular Ca2+ concentration, and all the analyzed transductants showed a marked increase in intracellular Ca2+ concentrations when stimulated by PMA and the calcium ionophore A23187 (Fig. 4A).
Because the production of IP3 is key for the mobilization of Ca2+ from intracellular stores, which subsequently leads to extracellular Ca2+ influx, the role of Lyn A and Lyn B in IP3 production was also explored. These experiments showed that both Lyn A or Lyn B alone were unable to generate IP3 at levels comparable to that of WT cells, following FcεRI stimulation (Fig. 4B). For Lyn B, there was a marked impairment in the onset and extent of IP3 production relative to WT cells. In contrast, it was found that WT and Lyn A-expressing cells showed a similar early rise in IP3 production, although the extent of the response appeared to be diminished in Lyn A-expressing cells. Thus, the findings demonstrate that Lyn A and Lyn B differ in their ability to induce IP3 production and Ca2+ fluxes, consistent with their observed ability to elicit mast cell degranulation.
Lyn A and Lyn B differ in their ability to associate with phosphoproteins and LAT
The difference of Lyn A and Lyn B in promoting IP3 production and Ca2+ fluxes suggested that these isoforms might differ in protein-protein or protein-lipid interactions required for these events. To explore such possibilities, we first assessed whether the two isoforms could coimmunoprecipate similar profiles of tyrosine-phosphorylated proteins. Fig. 5A shows that GST-fusion proteins of Lyn A and Lyn B differed in their binding to tyrosine-phosphorylated proteins from FcεRI-stimulated cell lysates. Relative to Lyn A, Lyn B showed enhanced binding to tyrosine-phosphorylated proteins, including a prominent band with the molecular mass of LAT. This was further demonstrated by Far Western. Cells were stimulated with pervanadate (to maximize the abundance of phosphorylated proteins), and the total cellular proteins were transferred to nitrocellulose and probed with GST-fusion proteins encoding Lyn A and Lyn B without their catalytic domains. As shown in Fig. 5B, GST-Lyn B bound more effectively than Lyn A to tyrosine-phosphorylated proteins in cell lysates from both nonstimulated and stimulated cells. Collectively, the results show that Lyn B forms stronger or more abundant interactions with tyrosine-phosphorylated proteins than Lyn A. To further explore whether these differences might be seen in cells, we immunoprecipitated Lyn A or Lyn B or both from cells expressing them individually or from WT cells, respectively. Fig. 5C shows that a dominant band identified as LAT was found to coimmunoprecipiate with Lyn B and not Lyn A (albeit weak interactions with Lyn A could be seen in some experiments). LAT was also found to coimmunoprecipitate with Lyn kinase from WT cells and was present in resting cells, although at reduced levels when all experiments were analyzed.
Lyn B forms a stable complex with SHIP-1 and decreases the interaction of PLCγ with phosphorylated LAT
Among the many proteins described to interact with Lyn kinase, the lipid phosphatase SHIP-1 is key in controlling the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) following FcεRI stimulation (27). SHIP-1 has been shown to interact with PLCγ1 and modulate its activation (28). Lyn is also required for the phosphorylation of the Tec family kinase BTK (29), which was shown to be required for full phosphorylation of PLCγ and increased activity (29, 30). Because both BTK and PLCγ require PIP3 for their membrane localization and activation, we explored if the Lyn isoforms showed differences in coimmunoprecipitation of SHIP-1, which might explain differences in promoting calcium responses. Fig. 6A shows that Lyn B binds SHIP-1 more efficiently than Lyn A following FcεRI stimulation of mast cells expressing the respective kinases. Tyrosine phosphorylation of SHIP-1 could be seen in the Lyn B coimmunoprecipitates, whereas it was difficult to detect in Lyn A coimmunoprecipitates (data not shown). This interaction was confirmed in pulldown assays in which a GST-Lyn B fusion protein showed the increased presence of SHIP-1 relative to a GST-Lyn A fusion protein (Fig. 6B). Moreover, a GST-Lyn SH2 domain construct showed similar efficiency in its interaction with SHIP-1 (data not shown), demonstrating that this domain is key in promoting this interaction. Because of these findings and the observation that Lyn B associates more prominently with LAT than Lyn A, we explored if LAT was required for the interaction of Lyn B with SHIP-1. Fig. 6B shows that the absence of LAT had only a minor effect on Lyn B–SHIP-1 interactions. A more marked effect was seen for Lyn A, particularly after FcεRI stimulation. Moreover, a phosphoprotein of the molecular mass of NTAL/LAB/LAT2 (the Ab recognizing Y191 of LAT also recognizes an equivalent site on LAT2) was also more prominently associated with Lyn B. Regardless, the association of Lyn B with SHIP-1 was not significantly altered by the absence of LAT, showing that this interaction was not LAT-dependent.
Because Lyn B was less effective than Lyn A in inducing Ca2+ fluxes, we further explored if Lyn B-expressing cells might be less efficient in causing PLCγ phosphorylation, which would be consistent with the marked reduction in IP3 production (Fig. 4B). As shown in Fig. 6C, tyrosine phosphorylation of PLCγ1 and PLCγ2 was markedly diminished (albeit not completely absent) in Lyn B-expressing cells when compared with Lyn A or WT cells. In general, cells expressing Lyn A were also slightly impaired in PLCγ1 phosphorylation, although this was not apparent in all experiments. Strikingly, the amount of phosphorylated LAT coimmunoprecipitating with PLCγ1 was reduced in cells expressing Lyn B relative to WT cells (Fig. 6C). Lyn A-expressing cells also showed less binding of phospho-LAT to PLCγ, but this interaction was less affected relative to Lyn B-expressing cells. Thus, the findings demonstrate that Lyn B-expressing cells have an impaired phosphorylation of PLCγ1 and 2 and a reduced association with phosphorylated LAT relative to Lyn A or WT cells. Because Lyn kinase has been reported to phosphorylate the protein tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1), we explored the possibility of differential control of this phosphatase by Lyn A and Lyn B, which might explain the differences in the level of LAT and PLCγ phosphorylation. Coimmunoprecipitation experiments revealed no significant association of SHP-1 with Lyn A or B, under conditions where SHIP-1 was detected, and no difference in the phosphorylation of Y536 on SHP-1 (the site phosphorylated by Lyn) was observed (data not shown).
This demonstrates that the mechanism underlying the ability of the two Lyn isoforms to evoke a Ca2+ response lies in their relative capacity to activate PLCγ and IP3 production, and this appears to be independent of a role in SHP-1 phosphorylation.
Complementation of Lyn A and Lyn B activity is required for normal mast cell activation and responses
The presence of both Lyn A and Lyn B isoforms in mast cells, and their association with FcεRI, suggests that both isoforms are likely to complement for maximal FcεRI-induced mast cell activation. As noted in the above experiments, expression of Lyn A or Lyn B alone did not fully restore many of the cellular responses to those seen in WT cells. In these experiments, we set out to investigate whether expression of both Lyn isoforms in lyn−/− BMMCs complemented to normalize some of the responses, such as calcium mobilization, degranulation, and cytokine production, to the levels observed in WT cells. As shown in Fig. 7A, left panel, the ectopic expression of both Lyn A and Lyn B together was similar to the levels of these two isoforms in WT cells, and FcεRI stimulation did not alter this expression. Analysis of the effect on Ca2+ responses when both isoforms are restored showed that the isoforms complemented in restoring this response to WT levels (Fig. 7A, right panel). Interestingly, in most experiments, the rise in intracellular Ca2+ was still slightly delayed in cells expressing both Lyn A and Lyn B, suggesting that the rate may be determined by factors independent of Lyn or that minor differences in the expression levels of each isoform may affect the rate of this response. As shown in Fig. 7B, the degranulation of cells reconstituted with both Lyn A and Lyn B isoforms was now similar to that of WT cells, and consistently, the response was greater than that of cells expressing Lyn A or Lyn B alone. Thus, the presence of both isoforms caused a full degranulation response in an apparently additive manner (Fig. 7B). Finally, analysis of whether expression of both Lyn A and B would restore control (dampen) on cytokine responses revealed a marked inhibition of IL-6 production (which was similar to the levels of IL-6 production in WT cells) when both isoforms were expressed (Fig. 7C). Collectively, the findings demonstrate that, although Lyn A and Lyn B differ in the efficacy of PLCγ activation and Ca2+ fluxes following FcεRI stimulation, both isoforms complement to achieve a full Ca2+ response, normal degranulation, and to dampen cytokine responses.
The presence of two Lyn kinase isoforms in hematopoietic cells where this kinase is expressed has long been recognized (16). However, how these two isoforms function and whether both isoforms are required for normal cellular responses has long remained a mystery. In the current study, we explored this unknown by reconstituting Lyn A and Lyn B isoforms individually or together in Lyn-null mast cells. This investigation was not amenable to RNA silencing strategies or gene deletion because the two Lyn isoforms are alternatively spliced products from a single gene and differ only in a 21-aa N-terminal insert found in Lyn A and not in Lyn B (16). Moreover, the requirements for the alternative splicing that generates the Lyn B isoform are not understood, hampering a genetic knock-in approach. How the 21-aa difference in Lyn A and B can cause the marked changes in functional responses analyzed in this study is not yet completely clear. However, some clues may be derived from their relative ability to coimmunoprecipitate tyrosine-phosphorylated proteins. The 21-aa insert contains a tyrosine (Y32) residue (Fig. 1) that could potentially be phosphorylated. As identified from Prosite database analysis, the amino acid sequence surrounding Y32 has some structural homology with the Y43 site on enolase, which is a protein readily phosphorylated by tyrosine kinases (31). We explored this possibility and found no evidence for phosphorylation of Y32 nor did a Y32F mutant of Lyn A show altered Ca2+ responses or degranulation (data not shown). In contrast, the marked difference of Lyn A and B isoforms in interacting with phosphoproteins suggests that the 21-aa insert functions to control Lyn A interactions presumably by creating a conformation that limits access to its protein interaction domains (SH2 and/or SH3). How this may occur is undetermined; however, given that GST fusion proteins of Lyn A and Lyn B (which are not tyrosine phosphorylated) maintained their differing ability to bind phosphoproteins, one can conclude that this behavior is not likely to be regulated by tyrosine phosphorylation of the 21-aa insert but most likely results from differences in protein folding/conformation.
Unlike Lyn A, Lyn B poorly induced FcεRI-mediated Ca2+ mobilization and mast cell degranulation. The underlying mechanism for the effect on Ca2+ mobilization and mast cell degranulation was the inability of the Lyn B isoform to effectively promote the phosphorylation of PLCγ and the production of IP3, crucial steps for these responses (9). Phosphorylated LAT1 was coimmunoprecipitated with PLCγ from Lyn A-expressing cells in greater amounts than from cells expressing Lyn B. This suggests that Lyn B fails to promote and/or impairs this interaction. This finding is consistent with the previous observation that loss of LAT1, or mutation of the PLCγ binding site on LAT1, causes loss of PLCγ phosphorylation, impaired Ca2+ responses, and defective mast cell degranulation (32, 33).
The ability of Lyn B, and not Lyn A, to efficiently associate with SHIP-1 may also be the key factor in the impaired PLCγ activity and reduced Ca2+ response. It has been shown that SHIP-1 coimmunoprecipitates with Lyn kinase from monocytes (34) and from mast cells (35). Moreover, SHIP-1 association with FcγRIIB is a key step for the inhibitory properties of this receptor on FcεRI-mediated Ca2+ responses, following coligation of these receptors (36). Recently, the negative regulatory role for SHIP-1 has been extended to its association with LAT1 in both mast cells as well as in T cells. In T cells, LAT1 organizes an inhibitory complex that includes the adapter Dok-2 and SHIP-1, which serves to dampen the extent of early activation upon TCR engagement (37). In mast cells, LAT1 can also exert an inhibitory role under certain circumstances. Analysis of LAT1 mutants showed the recruitment of SHIP-1 into an FcεRI-induced signaling complex (38). SHIP-1 was found to be recruited to LAT1 in a cooperative manner, in which binding of Grb-2 to tyrosine Y195 potentiates the binding of SHIP-1 to tyrosine Y235. However, our findings suggest that LAT1 is not required for the association of Lyn B with SHIP-1. Deficiency in LAT1 had minimal effect on this interaction, suggesting that the interaction of SHIP-1 with Lyn B is not mediated through this adapter protein. Instead, Lyn B may bring SHIP-1 to LAT1, where it might exert its control by destabilizing PLCγ interaction with the plasma membrane (and thus with LAT1), possibly by reducing the levels of PIP3, a lipid required for PLCγ translocation to the plasma membrane. It should be noted that other modes of inhibition by SHIP-1 have been described. Dok-1, an adapter that binds SHIP-1, can associate with and negatively regulate FcεRI signaling without the involvement of inhibitory receptors (39, 40). In mast cells, Dok-1 was found to associate with the FcεRI β-chain (40), and its phosphorylation was demonstrated to be Lyn-dependent (41). Thus, it is possible that Lyn B interactions with SHIP-1 might be mediated through the adapter Dok-1. Nonetheless, our analysis of Lyn B immunoprecipitates for the presence of Dok-1 did not reveal detectable levels of this adapter protein (data not shown). Yet, mutation of the noncanonical tyrosine residue that lies in the middle of the FcεRI β-chain caused increased cytokine production in mast cells and led to a modest loss of SHIP-1 phosphorylation, suggesting that this tyrosine residue might interact with this negative regulator (42).
In this study, we also investigated whether the previously described negative regulatory role of Lyn kinase on cytokine production (11, 13) could be ascribed to Lyn A or Lyn B. Although Lyn A consistently showed a moderate dampening of cytokine responses, when compared with Lyn B-expressing cells, this effect was incomplete. That Lyn A showed a greater dampening effect than Lyn B was unexpected, given that the association of SHIP-1 is more prominent with Lyn B and thus one might expect that Lyn B would have a greater dampening effect. Our findings show that expression of both Lyn isoforms is required to restore the control on cytokine production. It is unlikely that the suppression of cytokine responses is related to dampening of Ca2+ mobilization, because expression of both Lyn A and Lyn B increased Ca2+ fluxes following FcεRI stimulation. A plausible explanation could derive from our previous finding that mutation of the FcεRI β-chain ITAM tyrosines, which causes the loss of Lyn association with this receptor, induced increased IκBα degradation and enhanced nuclear NF-κB activity following FcεRI stimulation (42). It is well established that in mast cells, TNF and IL-6 production are highly dependent on NF-κB activity (43, 44). Thus, both Lyn A and Lyn B may be required to prevent the excessive degradation of IκBα and thus dampen nuclear NF-κB activity. However, a test of this hypoth-esis showed the inverse, as FcεRI-stimulated cells expressing the individual Lyn A or Lyn B isoforms were found to have higher amounts of IκBα protein remaining when compared with cells expressing both isoforms, which had a similar content of IκBα protein to stimulated WT cells (data not shown). Thus, it is unlikely that increased NF-κB activity could account for increased cytokine response of Lyn A- or Lyn B-expressing cells, and how both Lyn kinase isoforms complement to dampen cytokine production remains to be determined.
We now appreciate that the 21-aa N-terminal insert found in Lyn A, and not in Lyn B, determines the efficacy of each isoform in activating PLCγ and generating IP3, and thus in promoting Ca2+ fluxes. Additionally, key differences were seen in the ability of each isoform to associate with phosphoproteins like LAT and SHIP-1, suggesting that the 21-aa insert governs protein interactions. Most importantly, our findings demonstrate that the Lyn A and Lyn B isoforms have distinct roles with regards to mast cell signal transduction. Nonetheless, the two isoforms complement in inducing both positive and negative regulation of mast cell effector functions. These findings provide an explanation for the presence of both Lyn A and Lyn B isoforms in mast cells and likely for other cell types as well.
We thank the Laboratory Animal Care and Use Section, the Light Imaging Section, and the Flow Cytometry Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, for invaluable support.
Disclosures The authors have no financial conflicts of interest.
This work was supported by the intramural program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health.
Abbreviations used in this paper:
- bone marrow-derived mast cell
- inositol 1,4,5-triphosphate
- linker for activation of T cells
- phosphatidylinositol 3,4,5-trisphosphate
- phospholipase C
- Src homology 2
- Src homology region 2 domain-containing phosphatase 1
- whole cell lysate
- wild-type.
- Received December 18, 2009.
- Accepted February 22, 2010.
- Copyright © 2010 by The American Association of Immunologists, Inc.


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Renewal, Tenure, and Promotion CommitteesCollective Agreement[+]Please consult the Faculty Association Collective Agreement, provided here by Human Resources.Senate Bylaws[+]Official documents via the University Secretariat.
ESSENTIALS: A-Z List of Campus Services | Acronyms | Campus Map | Emergencies | FAQ | Find a Person | Important Dates | Important Forms | Policies, Procedures & Bylaws | Workshops & Campus Events
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conveyor belt capacity limestone
DESIGN OF MATERIAL HANDLING EQUIPMENT BELT CONVEYOR SYSTEM FOR CRUSHED BIOMASS WOOD USING V MERGE CONVEYING SYSTEM Since the limestone to be handled is abrasive, heavy, with specific gravity between From Equation (2.4), the overall capacity of the belt conveyor consisting of 3 equal roll idler is 4 tones/hr.Live Chat
Design of a Material Handling Equipment Belt Conveyor
This paper discusses the design calculations and considerations of belt conveyor system for limestone using 3 rolls idlers, in terms of size, length, capacity and speed, roller diameter, power andLive Chat
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Nov 15, 20180183;32;Portable conveyors and conveyor belt systems help transport construction materials from Belt conveyor for stone,coal,cementBelt conveyor Belt conveyor for stone quarry In stone quarry, itLive Chat
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Conveyor Belt For Limestone, Wholesale Various High Quality Conveyor Belt For Limestone Products from Global Conveyor Belt For Industrial Rubber Belt Conveyor for ggregates . Africa Large Capacity PVC and Rubber Conveyor Belt .Live Chat
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A conveyor belt is the carrying medium of a belt conveyor system (often shortened to belt conveyor). A belt conveyor system is one of many types of conveyor systems . A belt conveyor system consists of two or more pulleys (sometimes referred to as drums), with an endless loop of carrying mediumthe conveyor beltthat rotates about them.Live Chat
Limestone Conveying System KWS Manufacturing
Limestone Conveying System. Download PDF Brochure. Plant Name and Location. Duluth Minerals, LLC Raw limestone is delivered by barge to the facility for processing. It is off loaded, stored, crushed, ground and dried. Conveyor Capacity Multiple Capacities Ranging from 2353 toLive Chat
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Former British Ambassador to Syria: UK complicit in Trump's Syria Oil Grab
With the UK Parliament already on alert, how long will it be before Congress wakes up to this scandal-in-the-making?
With the UK Parliament already on alert, how long will it be before Congress wakes up to this scandal-in-the-making?
If the U.S. military, or Israel or Saudi Arabia, had a viable plan to attack Iran without triggering a wider war, they would have done so long ago.
Medea Benjamin
Nicolas Davies
On Saturday, September 14th, two oil refineries and other oil infrastructure in Saudi Arabia were
Medea Benjamin is the co-founder of CODEPINK and author of Kingdom of the Unjust: Behind the US-Saudi Connection. Her new book is Inside Iran: The Real History and Politics of the Islamic Republic.
The "Iranian leadership has to make a decision that they want their people to eat." – US Secretary of State Mike Pompeo, November 7, 2018
Who is responsible for the recent attacks on the fuel tankers in the Gulf of Oman? Washington blames Iran, and has offered what it calls proof—a grainy video allegedly showing what are said to be Iranians removing what is said to be a mine from what is said to be the hull of a stricken tanker. But the video proves nothing more than
Stephen Gowans is a Canadian-based political activist and foreign policy analyst. He is the author of Israel, A Beachhead in the Middle East (Spring 2019); Patriots, Traitors and Empires; and Washington's Long War on Syria. Visit his personal blog at gowans.blog.
While Russia often serves as a useful "boogeyman" for promoting militaristic policies, the odd moments when those same policies actually benefit Russia and avoid strong opposition from U.S. politicians and media provide a rare glimpse into the real motivations behind Cold War 2.0.
In early May, MSNBC news host Rachel Maddow -- known as one of the top promoters of the new Cold War and Russiagate in American media -- emphatically endorsed regime change in Venezuela after she claimed that President Donald Trump's hawkishness towards the South
Whitney Webb is a MintPress News journalist based in Chile. She has contributed to several independent media outlets including Global Research, EcoWatch, the Ron Paul Institute and 21st Century Wire, among others. She has made several radio and television appearances and is the 2019 winner of the Serena Shim Award for Uncompromised Integrity in Journalism.
Think about who gets rich off of the Venezuela regime-change agenda. It's the same people that said we had to invade Iraq in order to prevent nuclear apocalypse. It's the same people who said the world would stop turning on its axis if we didn't carpet bomb Libya and Syria.
Transcript —This video was produced as part of a MintPress News and Grayzone collaboration — Of all the reasons to plot an elaborate and risky coup, there's one reason that always stands out: profit. Money makes the world go around and in far more
Kei Pritsker is a journalist and activist located in Washington DC. Kei focuses on international politics and economics. He previously worked as a producer at RT America.
What makes the Libyan, Syrian and Venezuelan scenarios so similar? Why was the West so eager to viciously attack, and then destroy these three, at first glance, very different countries? The answer is simple, although it is not often uttered in the West; at least not publicly.
Yes, the latest research confirms that Venezuela is so rich in natural resources, that it could single-handedly satisfy all global demand for oil, for over 30 years. And it has much more than oil to offer, in its Orinoco basin and in other areas of the country. But it is not all 'about oil'; actually, far from it. Those who believe that what
Andre Vltchek is a philosopher, novelist, filmmaker and investigative journalist. He has covered wars and conflicts in dozens of countries. Three of his latest books are Revolutionary Optimism, Western Nihilism, a revolutionary novel "Aurora" and a bestselling work of political non-fiction: "Exposing Lies Of The Empire". View his other books here. Watch Rwanda Gambit, his groundbreaking documentary about Rwanda and DRCongo and his film/dialogue with Noam Chomsky "On Western Terrorism". Vltchek presently resides in East Asia and the Middle East, and continues to work around the world. He can be reached through his website and his Twitter.
Israel is now pushing for the U.S. to recognize its sovereignty over the Golan for fear of losing it completely, even though such recognition could result in an all-out war between Israel and Syria, as well as with Syrian allies such as Iran and Lebanon's Hezbollah.
WASHINGTON -- A new bill recently introduced in the Senate, along with its companion bill in the House, would result in the United States government recognizing Israel's sovereignty over Syria's Golan Heights, which has been occupied by Israel since 1967. The
Whitney Webb is a MintPress News journalist based in Chile. She has contributed to several independent media outlets including Global Research, EcoWatch, the Ron Paul Institute and 21st Century Wire, among others. She has made several radio and television appearances and is the 2019 winner of the Serena Shim Award for Uncompromised Integrity in Journalism.


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GL4 Festival in partnership with Strike A Light present…
Doodle Dance
Saturday 19 October | 11:30am & 2pm
The Redwell Centre, Matson
Tickets: £10/£7
The Doodle Dance Show is a playful and innovative show for families that combines drawing and dance. Created by award-winning performers Anna Williams (Russell Maliphant, La Ribot) and Tom Roden (New Art Club) the show invites children and their families to grab a pen and join in the action, as the whole audience comes together to turn a giant piece of paper into a delightfully anarchic world populated by crazy birds, dancing pirates, strange underwater creatures and maybe more…
'Having fun as a family and giving confidence to my 4-year-old!'
Originally produced and premiered by Greenwich Dance in 2015 he show has since been touring the UK, selling out and generating a fantastic response.
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Recommended Age: 4+


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Financial, ethical and other problems roil Oyster Bay town
" . . . There is something rotten in the town of Oyster Bay," U.S. District Judge Leonard Wexler said last week in delaying sentencing for a former town official convicted of income tax evasion.
Ya think?
Because, yes, his honor is on to something — and not just in the case of former town planning director Frederick Ippolito, who in January pleaded guilty to one count of tax evasion.
Where, oh where, oh where do we begin?
How about the town's special treatment of restaurateur Harendra Singh, who was indicted last year on 13 felony counts — including bribing an Oyster Bay official to obtain millions of dollars in town-backed loan guarantees?
Singh has pleaded not guilty. And the town has labeled some of the guarantees invalid because they were not approved by the town board, because town officials don't remember them, because they were not properly archived — and because they are illegal under state law, which bars municipalities from guaranteeing private debt.
But tell that to Singh's lenders, who, as Newsday reported in May, were demanding that the town — and by extension taxpayers — pay up more than $14.5 million in Singh's defaulted loans.
And it looks as if Singh's singular treatment didn't stop there.
In 2013, according to a story earlier this month by Newsday's Ted Phillips, Oyster Bay fast-tracked a payment to Singh for superstorm-damaged professional kitchen equipment that, mistakenly, had been destroyed by town workers.
The town agreed to pay the higher cost of replacing the equipment as if it were new, rather than, as is the more common practice, paying a lower sum based on the condition of the equipment at the time it was destroyed.
Even at that, however, the $155,868 Oyster Bay ended up paying to satisfy Singh's claim — after what town officials called extensive negotiations — ended up being higher than the $130,558 replacement cost quoted by a vendor.
The town's justification for the deal? Officials said they sped up the payout so that Singh, who was behind on payments to Oyster Bay for running concessions at the town golf course and Tobay Beach, could pay what he owed.
Following that logic, Oyster Bay used public money to pay a private contractor, so the private contractor could give that money right back.
Meanwhile, the town took a beating on Wall Street, and got slapped for being stingy with public information.
In April, the bond rating for Oyster Bay, one of the richest towns in the United States, was dropped to junk status by a Wall Street bond rating agency. That's lower than when angry residents in Nassau, one of the richest counties in the nation, voted in the county's first Democratic legislative majority in 70 years — after the county's bond rating fell to one level above junk during an economic boom.
And earlier this week, State Supreme Court Judge Leonard Steinman in Mineola ruled that Oyster Bay must turn over to Newsday documents in seven Freedom of Information Law requests. "It is in the general public's significant interest that Newsday be permitted to conduct the news investigation it is attempting to pursue by examining public documents (or, at least, documents that should have been and now will be public)," Steinman wrote.
Wexler, in delaying Ippolito's sentencing last week until he can get more information related to the case, said, "I have never in my 33 years, as long as I've been a judge, gone beyond the maximum. But there is something rotten in the town of Oyster Bay and this court wants to know what's going on."
The judge said he was "totally confused and wants answers," and, he said, "I'm sure the people of Oyster Bay want answers, too."
Looks like Wexler struck gold with his observations.
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August 1, 2011 -- Partner Anthony Taylor was named one of the top-rated attorneys in Orange County by OC Metro Magazine, August 2011. This annual list of the region's top attorneys is compiled by AVVO, which uses a mathematical model including criteria such as years of experience, education, achievement, and industry recognition.
Mr. Taylor specializes in litigation, and serves as the City Attorney for Suisun City. Mr. Taylor graduated with a B.S. from University of Southern California, summa cum laude and class valedictorian of the School of Public Administration. He received his J.D. from USC Law School.
Mr. Taylor was also named a "Rising Star" for six consecutive years from 2006 to 2011 by Super Lawyers Magazine.


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GoodNotes for Windows
goodnotes desktop version in order to edit files both on tablets and on desktop laptops and sync in between devices. many people in my medical school would appreciate and heavily use this app if it had these options.
45 comments
Andrew commented
need to focus on macOS version first.
toni toni commented
please I need this for my surface pro please!!!! =)
Ian.Xu commented
Very useful! I have been waiting this for a long time
Why still on iOS only? GoodNotes on the coming Surface Neo - wouldn't that be awesome!?
Fabian Kuhn commented
Totally agreed!
Goodnotes could completely replace Evernote if there was a windows app version, where I could edit and take notes.
Shrutika commented
please do this soon i got a 2 in 1 for you app but now i see its not for windows
John commented
Now that the backup file no longer supports PDF's we need a way to at least view documents on a Windows PC.
Sopon Leelathanapipat commented
Do it now!!!!!!! Pleaseeeeeeeeeee
Hdj commented
Pls add win 10 Support!!
Poderia ter uma versão para PC ou que desse para abrir os arquivos no computador.
Paul TIng commented
100% agree, All features and all. Windows has a good pen support as well
diasi commented
I'd love to have desktop linux version of goodnotes. ready to pay for it.
oliver commented
add +1:
in addition to the non-feature parity already mentioned before, one thing missing in the current 'stump'-app is:
- image object rotation
- templates (has been mentioned, kind of...)
Kat commented
Need a desktop version, or access. Being able to edit documents on both the desktop and tablet is what i have been looking for for years. Need to be able to access any document from ipad. but still be able to edit it on pc at some stage to.
Indeed a webapp version would solve a lot of every user's sync issues.
Linnea commented
Is this coming soon? I love ur app but have change to Evernote. Can't wait my whole life my favourite app to evolve. Really love all the work you've done so far <3 Let us know you amazing developers and doers :)
Please. Need a Windows version. Cannot really use as main note app until this happens.
I really would appreciate a desktop version for macbook and the possibility to handle with hyperlinks, please
I wish I could get a refund for the useless desktop version. No sync between iOS and Macbook? I suppose the web builders with know-how to do this are not in you group. Lovely app for iOS. Thank you for that much. And I hope you get full font capabilities for iOS.
siobhan commented
There is a mac client already but there are problems with it:
- It does not have the same functionality as the iOS version. You can easily copy & move pages around on OSX (impossible on iOS), you can import templates easily on iOS (impossible on Mac), . Once templates are imported on iOS, they are not visible on Mac (but if they are used in a notebook, they are visible - this makes no sense), there are no help instructions online for Mac, so I have spent hours trawling the web to see if it is possible/not possible on Mac, iOS has the ability to recognise shapes (drawing a box, snaps to a box shape), Mac does not have this. etc.etc.
- For those having difficulty syncing, I sync my iPhone, iPad & MacBook Pro via iCloud and it does work but it is SLOW & there seems to be no way to 'force' it to sync.


text:
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- Mar 25, 2019 4:10 am GMT
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Dear friends,
I would like to share with you the link to GlobalData's press release "Political Uncertainties Hover on Ukraine's Gas Investment Drive, Says GlobalData," which was distributed on March 20, 2019.
Thank you.
Kind regards,
GlobalData. "Political Uncertainties Hover on Ukraine's Gas Investment Drive, Says GlobalData," in GlobalData, March 20, 2019.
Ukraine is organizing a series of licensing rounds for 2019 with the goal of increasing its gas production, but the political risks linked to the...
Thank Alessandro for the Post!
Energy Central contributors share their experience and insights for the benefit of other Members (like you). Please show them your appreciation by leaving a comment, 'liking' this post, or following this Member.


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REOPro - Real Estate Default Professionals
America's Largest Social Network for Default Real Estate Professionals
Dear Fellow REO agent!
I need your help, and many others might be in the same boat. A month ago Single Source send out an email stating that files were being transferred to Nationstar. But Not to worry they were keeping the listing agents:0)
Well, two weeks later I still have the properties, paying the bills, etc. Now no seller/Lender!
I called the REO department of Nationstar first day, I had…
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The UK's international trade secretary Liam Fox will spend two days in Washington to discuss the details of a post-Brexit trade deal with his US counterpart Robert Lighthizer.
Removing commercial barriers with the US could generate an additional £40bn ($31bn) in trade with the UK by 2030, Fox said on Sunday, but admitted that discussions would be "difficult".
"Our exit from the European Union offers an unprecedented opportunity to reshape our independent trading ambitions and build on the already strong trading relationship with our single largest trading partner — the US," he said in a statement.
Fox will tell lawmakers in Washington on Tuesday that 700,000 US jobs are supported by trade with Britain.
The Department for International Trade added that discussions were expected to focus on "providing certainty, continuity and increasing confidence for UK and US businesses as the UK leaves the EU".
Under EU rules, member states are not allowed to negotiate free-trade agreements so the talks in Washington are only preliminary.
Adam Marshall, director general of the British Chambers of Commerce (BCC) said that it will not be easy for the UK to secure a good deal with the US.
US negotiators have more experience, he told BBC's Today Programme:
We're just getting back into the game of doing this sort of thing after 40 years of doing it via the EU. So I think early on in the process, it would be concerning if the UK were to go up against the US on a complex and difficult negotiation.
Labour Party leader Jeremy Corbyn remains critical of the government's decision to seek a trade deal with the US, particularly because president Donald Trump intends to withdraw from the Paris climate agreement.
After his two-day trip in Washington, Fox will then travel to Mexico and Texas.
Free download worth over $5000Download our 2019 Technology, Media and Telecoms Predictions Report
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I'm trying to pass an extra float to my vertex shader because I need to divide my UV by some value I calculate in order to do projective correct texturing.
The batcher I am using is a modified version of the nez batcher. However after modifying the struct nothing renders, and no errors are thrown.
This is my struct after modifying.
[StructLayout(LayoutKind.Sequential, Pack = 1)]
struct VertexPositionColorTexture4 : IVertexType
//public const int realStride = 96;
// public const int realStride = 0;
public const int realStride = 112;
VertexDeclaration IVertexType.VertexDeclaration { get { throw new NotImplementedException(); } }
public Vector3 position0;
public Color color0;
public Vector2 textureCoordinate0;
public float q0;
public Vector3 position1;
public Color color1;
public Vector2 textureCoordinate1;
public float q1;
public Vector3 position2;
public Color color2;
public Vector2 textureCoordinate2;
public float q2;
public Vector3 position3;
public Color color3;
public Vector2 textureCoordinate3;
public float q3;
I don't actually do anything currently with the extra value so I don't think the issue is in the shader. Is there anywhere else I need to modify in order to do this?
Why mod the vertex structure when you can just pass a variable to the shader, or do you need to make different changes to the UV per vertex?
That does not look like a full vertex definition either....
Yeah it has a different value per vertex. It's to correct the UV when the quad is not a parallelogram.
I worked it out in the end. When creating the vertex buffer I was still telling it to expect a float2. Had to create a new structure with float3s and pass that into the constructor.
vertexBuffer = new DynamicVertexBuffer(graphicsDevice, typeof(P3.VertexPositionColorTexture), MAXVERTICES, BufferUsage.WriteOnly);
Ahh right, bet it was getting confused when Vertex Patching


text:
Monticello Park
About Monticello Park
Seconds from Fort Worth's Cultural District and minutes from Downtown. Low monthly HOA dues pay for a great community pool. Many different floorplans available.
In-Town Living Specialists
Stiles Mund GroupRE/MAX Trinity
Call/Text: 817-835-6150
Interested in this building?Contact Stiles Mund Today!
Monticello Park Pl, Fort Worth, TX 76107
- Year Built1984
- Floors2
- Neighborhood West Fort Worth
- Building Type Townhomes
- Ownership Condo
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Do you happen to have the contact information, number and/or email, to the President of HOA?


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2. Nebuchadnezzar's Dream
And in the second year of the reign of Nebuchadnezzar Nebuchadnezzar dreamed dreams, wherewith his spirit was troubled, and his sleep brake from him. 2Then the king commanded to call the magicians, and the astrologers, and the sorcerers, and the Chaldeans, for to shew the king his dreams. So they came and stood before the king. 3And the king said unto them, I have dreamed a dream, and my spirit was troubled to know the dream. 4Then spake the Chaldeans to the king in Syriack, O king, live for ever: tell thy servants the dream, and we will shew the interpretation. 5The king answered and said to the Chaldeans, The thing is gone from me: if ye will not make known unto me the dream, with the interpretation thereof, ye shall be cut in pieces, and your houses shall be made a dunghill. 6But if ye shew the dream, and the interpretation thereof, ye shall receive of me gifts and rewards and great honour: therefore shew me the dream, and the interpretation thereof. 7They answered again and said, Let the king tell his servants the dream, and we will shew the interpretation of it. 8The king answered and said, I know of certainty that ye would gain the time, because ye see the thing is gone from me. 9But if ye will not make known unto me the dream, there is but one decree for you: for ye have prepared lying and corrupt words to speak before me, till the time be changed: therefore tell me the dream, and I shall know that ye can shew me the interpretation thereof.
10The Chaldeans answered before the king, and said, There is not a man upon the earth that can shew the king's matter: therefore there is no king, lord, nor ruler, that asked such things at any magician, or astrologer, or Chaldean. 11And it is a rare thing that the king requireth, and there is none other that can shew it before the king, except the gods, whose dwelling is not with flesh. 12For this cause the king was angry and very furious, and commanded to destroy all the wise men of Babylon. 13And the decree went forth that the wise men should be slain; and they sought Daniel and his fellows to be slain.
14Then Daniel answered with counsel and wisdom to Arioch the captain of the king's guard, which was gone forth to slay the wise men of Babylon: 15He answered and said to Arioch the king's captain, Why is the decree so hasty from the king? Then Arioch made the thing known to Daniel. 16Then Daniel went in, and desired of the king that he would give him time, and that he would shew the king the interpretation. 17Then Daniel went to his house, and made the thing known to Hananiah, Mishael, and Azariah, his companions: 18That they would desire mercies of the God of heaven concerning this secret; that Daniel and his fellows should not perish with the rest of the wise men of Babylon.
19Then was the secret revealed unto Daniel in a night vision. Then Daniel blessed the God of heaven. 20Daniel answered and said, Blessed be the name of God for ever and ever: for wisdom and might are his: 21And he changeth the times and the seasons: he removeth kings, and setteth up kings: he giveth wisdom unto the wise, and knowledge to them that know understanding: 22He revealeth the deep and secret things: he knoweth what is in the darkness, and the light dwelleth with him. 23I thank thee, and praise thee, O thou God of my fathers, who hast given me wisdom and might, and hast made known unto me now what we desired of thee: for thou hast now made known unto us the king's matter.
24Therefore Daniel went in unto Arioch, whom the king had ordained to destroy the wise men of Babylon: he went and said thus unto him; Destroy not the wise men of Babylon: bring me in before the king, and I will shew unto the king the interpretation. 25Then Arioch brought in Daniel before the king in haste and said thus unto him, I have found a man of the captives of Judah, that will make known unto the king the interpretation. 26The king answered and said to Daniel, whose name was Belteshazzar, Art thou able to make known unto me the dream which I have seen, and the interpretation thereof? 27Daniel answered in the presence of the king, and said, The secret which the king hath demanded cannot the wise men, the astrologers, the magicians, the soothsayers, shew unto the king; 28But there is a God in heaven that revealeth secrets, and maketh known to the king Nebuchadnezzar what shall be in the latter days. Thy dream, and the visions of thy head upon thy bed, are these; 29As for thee, O king, thy thoughts came into thy mind upon thy bed, what should come to pass hereafter: and he that revealeth secrets maketh known to thee what shall come to pass. 30But as for me, this secret is not revealed to me for any wisdom that I have more than any living, but for their sakes that shall make known the interpretation to the king, and that thou mightest know the thoughts of thy heart.
31Thou, O king, sawest, and behold a great image. This great image, whose brightness was excellent, stood before thee; and the form thereof was terrible. 32This image's head was of fine gold, his breast and his arms of silver, his belly and his thighs of brass, 33His legs of iron, his feet part of iron and part of clay. 34Thou sawest till that a stone was cut out without hands, which smote the image upon his feet that were of iron and clay, and brake them to pieces. 35Then was the iron, the clay, the brass, the silver, and the gold, broken to pieces together, and became like the chaff of the summer threshingfloors; and the wind carried them away, that no place was found for them: and the stone that smote the image became a great mountain, and filled the whole earth.
36This is the dream; and we will tell the interpretation thereof before the king. 37Thou, O king, art a king of kings: for the God of heaven hath given thee a kingdom, power, and strength, and glory. 38And wheresoever the children of men dwell, the beasts of the field and the fowls of the heaven hath he given into thine hand, and hath made thee ruler over them all. Thou art this head of gold. 39And after thee shall arise another kingdom inferior to thee, and another third kingdom of brass, which shall bear rule over all the earth. 40And the fourth kingdom shall be strong as iron: forasmuch as iron breaketh in pieces and subdueth all things: and as iron that breaketh all these, shall it break in pieces and bruise. 41And whereas thou sawest the feet and toes, part of potters' clay, and part of iron, the kingdom shall be divided; but there shall be in it of the strength of the iron, forasmuch as thou sawest the iron mixed with miry clay. 42And as the toes of the feet were part of iron, and part of clay, so the kingdom shall be partly strong, and partly broken. 43And whereas thou sawest iron mixed with miry clay, they shall mingle themselves with the seed of men: but they shall not cleave one to another, even as iron is not mixed with clay. 44And in the days of these kings shall the God of heaven set up a kingdom, which shall never be destroyed: and the kingdom shall not be left to other people, but it shall break in pieces and consume all these kingdoms, and it shall stand for ever. 45Forasmuch as thou sawest that the stone was cut out of the mountain without hands, and that it brake in pieces the iron, the brass, the clay, the silver, and the gold; the great God hath made known to the king what shall come to pass hereafter: and the dream is certain, and the interpretation thereof sure.
46Then the king Nebuchadnezzar fell upon his face, and worshipped Daniel, and commanded that they should offer an oblation and sweet odours unto him. 47The king answered unto Daniel, and said, Of a truth it is, that your God is a God of gods, and a Lord of kings, and a revealer of secrets, seeing thou couldst reveal this secret. 48Then the king made Daniel a great man, and gave him many great gifts, and made him ruler over the whole province of Babylon, and chief of the governors over all the wise men of Babylon. 49Then Daniel requested of the king, and he set Shadrach, Meshach, and Abed-nego, over the affairs of the province of Babylon: but Daniel sat in the gate of the king.
We observe with what object and with what confidence Daniel demanded an extension of time. His object was to implore God's grace. Confidence was also added, since he perceived a double punishment awaiting him, if he disappointed the king; if he had returned the next day without reply, the king would not have been content with an easy death, but would have raged with cruelty against Daniel, in consequence of his deception. Without the slightest doubt, Daniel expected what he obtained — namely, that the king's dream would be revealed to him. He therefore urges his companions to implore unitedly mercy from God. Daniel had already obtained the singular gift of being an interpreter of dreams, and as. we, have seen, he alone was a Prophet. of God. God was accustomed to manifest his intentions to his Prophets by dreams or visions, (Numbers 12:6,) and Daniel had obtained both. Since Misael, Hananiah, and Azariah were united with him in prayer, we gather that they were not induced by ambition, to desire anything for themselves; for if they had been rivals of Daniel, they could not have prayed in concord with him. They did not pray about their own private concerns, but only for the interpretation of the dream being made known to Daniel. We observe, too, how sincerely they agree in their prayers, how all pride and ambition is laid aside, and without any desire for their own advantage. Besides, it is worthy of notice why they are said to have desired mercy from God Although they, do not hem come into God's presence as criminals, yet they hoped their request would be graciously granted, and hence the word "mercy" is used. Whenever we fly to God to bring assistance to our necessities, our eyes and all our senses ought always to be turned towards his nlerey, for his more good will reconciles him to us. When it is said, at. the close of the verse, — they should not perish with the rest of the wise men of Babylon, some explain this, as if they had been anxious about the life of the Magi, and wished to snatch them also from death. But although they wished all persons to be safe, clearly enough they here separate themselves from the Magi and Chaldeans; their conduct was far different. It now follows —


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Ability To Compare Custom Time Periods
score 7Open for Voting
It would be handy to easily compare 2 time periods against each other.
e.g. Pick Last week Monday & this week Monday and see how the graphs compare hour by hour but with the same scale. Currently each day would have it's own scale so they're hard to compare


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, combines the wit and levity of Ratchet and Clank with the grim tone of Resistance. It's a combination that shouldn't work, but the characters make it work. Fuse's cast of co-op characters each brings an interesting charm to mask their dark and mysterious pasts. It's not as cliche as it seems. In some cases, you might get uncomfortable.You can learn the who's-who of Fuse's anti-terror group Overstrike 9 in IGN's handy profile series right here.
see deal
Fuse - Xbox 360$11.99on Amazon
Dalton Brooks
Naya Deveraux
Jacob Kimble
Isabelle Sinclair
Mitch Dyer is an Associate Editor at IGN. He's also quite Canadian. Read his ramblings on Twitter and follow him on IGN.
If you buy something through this post, IGN may get a share of the sale. For more, learn more.


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Course description
It is strongly recommended that you complete the Introduction to perfectionism in dance course prior to enrolling this course.
The second part in the Dancing to perfection series. Explore how perfectionism is learnt and reinforced in the dance environment.
What caused you to become a perfectionist and what keeps your perfectionism going? Make clear distinctions between the three dimensions of perfectionism and consider how perfectionism is amplified through dance training.
2hrs time valued on demand CPD. Includes three months access.
Course aims
- Develop an understanding of how perfectionistic behaviour develops.
- Recognise the factors that can reduce or exacerbate perfectionistic tendencies.
- Understand how perfectionism is reinforced through rules, assumptions, and standards
- Make clear distinctions between the three dimensions of perfectionism.
Course outcomes
- Define the learning and temperament factors in the development of perfectionism.
- Elucidate perfectionism that is orientated towards self or others or is socially prescribed.
- Recognise how perfectionism develops over time.
- Consider how perfectionism functions in your own practice.
As a psychologist specialising in the performing arts I work with you on your journey to become the performer, educator or director you want to be. I develop bespoke and evidence based solutions for the challenges you face.
The learning in Dance Knowledge works with the same principles I employ in face to face psychological services.
Practice Fostering mindful practice to establish workable techniques and habits.
Performance Achieving personal goals and acclaimed levels of performance.
Artistry Harnessing and understanding creativity and talent.
Resilience Building strength and flexibility through mental and emotional toughness.
For further information contact


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Pamela ThompkinsJun 20
Hi, BEJ. I enjoy your work ethic and appreciate all the hard work to deliver useful and practical content. However, I would like to see more detailed information (if applicable, at the time of a video shoot) about the process to gain employement as a teacher. I am a US Army Veteran who was stationed in Seoul, Korea many years ago. I was impressed with Ms. Frank (Felicia) and her experience as a teacher working in Seoul. I have a Bachealor's Degree in the Arts of Divinity (course catalog focus in leadership, mentoring, coaching and missions). I am currently doing my masters grauduate studies online at Liberty University. Upon graduation, I will have a Masters of Arts in Interdisciplinary Studies. Any video interview you can do on the process of becoming a teacher abroad is welcomed. Likewise, if there is a specific recognized certification requirement that I need to have is great to know. Blessings, Ms. Thompkins


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LIVE CASE STUDY - Bring The Fresh vs. Extreme Niche Empires vs. Google Sniper 2
I admit it. I have been absorbed by the huge number of moneymaking methods and tactics out there, and have been pulled in 1000 different directions. What's worse, without even knowing if any of them was good.
But some time ago I decided to put an end to this taking MASSIVE ACTION and I started seeing some results.
As my next action-taking step and, at the same time, as a way to give back to this community by sharing my results with you LIVE, I'll run an experiment.
Here's what I've decided to do:
I'll build three sites, following three different methods, put their results side by side, and see which one performs better.
I've chosen three well known methods, which in the end follow a very similar approach: choose a niche and one or a few related products, select some targetted keywords, and build a site around them to make money selling as an affiliate (there's an additional monetization method in one of them, that I'll mention later in the case study).
These methods are:
- Bring the Fresh by Kelly Felix and Mike Long
- Extreme Niche Empires by Sean Donahoe
- Google Sniper 2 by George Brown.
As I build my sites following these methods, I'll be tracking and posting the following magnitudes:
- Time spent working on each one
- Money spent working on each one (I already have hosting, so I won't compute this cost to any of them).
- Position in Google for their main keyword, and maybe some secondary keywords.
- Number of visits to the site
- Earnings
No, I won't reveal the niche or keywords as part of the case study
Apart of that, I'll also be reporting my progress, detailing any roadblocks I may find, and how I overcome any issues.
Of course, I may not be able to give too much detail sometimes, as it's not my intention to reveal the methods themselves here, I have to protect their creators' IP. I hope you'll understand
I'd like to make it as impartial as possible, so that the results are somewhat comparable, so here are the conditions that will rule my experiment / case study:
- I'll use the same niche for the three of them, so that they target the same audience. For instance (I'll use this niche as an example here, but it won't be the real one), they may all target "hard-core gamers".
- I'll promote physical products in all cases, so that no difference arises based on the likelyhood of buying / refunding digital vs. physical goods. For instance, I may promote gaming accessories like headsets, keyboards, mouses, etc.
- I'll also try to use the same marketplace, likely Amazon, so that differences in the checkout process don't affect results.
- So that sites don't compete against each other in google, they'll target different products and keywords. However, I'll try to choose products that serve similar functions and share simlar prices, for the same reason as above.
- If the methods allow it, I'll choose keywords that have similar search volume as reported by the Google External Keyword Tool, and similar easiness to rank or competition levels as reported by Market Samurai / Traffic Travis.
- I'll try to follow all the methods to the letter; if for some reason I had to deviate from them, I'd explain where and why.
- As I have no preference for any of the methods, I'll speak of them and build their sites following an alphabetical order, as I did in the post subject. This does not mean Bring the Fresh is "number one" except for the fact that it starts with a B and the others don't
- If I decide to outsource some task for one site, I'll outsource the same or similar tasks for the others, so that the cost and time spent on each are somewhat comparable.
IMPORTANT DISCLAIMER: I know the results won't be a scientific proof of any kind. So don't take this case study as the end-all be-all of how each of these methods perform by themselves or as compared to the other two.
Who knows, maybe one works like charm for the niche I choose and Amazon products, but another one would have beat it promoting a digital product from Clickbank and the third would have trashed the others had I chosen a different keyword for its site.
Also, my results may not be comparable to yours if you followed any of them, as we all have different technical, marketing and sales backgrounds.
You know, the only way you'll find to BE SURE how good any method works FOR YOU it's by testing it yourself.
But I hope the experiment yields some interesting results that we can all learn from. At least, I hope I'll achieve some results with all of them just because I'LL TAKE ACTION, and we can at least learn from that.
Next stop: niche / keyword research.
As you see I have a daunting task ahead. Wish me luck!
PS: let me know what you think of this case study, and if you think there's something I may be missing, or something else you'd like me to share with you.
PS2: if you like this experiment, please click the thanks button below!
- {{ DiscussionBoard.errors[3941721].message }}


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Research & Consultation Officer
- Recruiter
- Greater London Authority (GLA)
- Location
- Central London
- Salary
- £40,291 per annum
- Posted
- 09 Oct 2019
- Closing date
- 27 Oct 2019
- Ref
- Industry
- Government & politics, Local government, Policy
- Job level
- Experienced (non manager)
- Hours
- Full time
- Contract
- Contract
- Listing type
- Job vacancy
What does London's future look like? How do we create communities in which Londoners want to live, work and play? What will make London the best big city in the world?
These are just some of the questions we're working to answer at City Hall. But we can't do this alone – a dialogue with Londoners is essential, and that's where this job comes in.
Do you have relevant experience of delivering mixed-method social research? Are you passionate about using research to understand how people think, and using this to tackle big policy challenges? This is your chance to do that in an organisation with influence and high national profile.
You'll work in City Hall's Opinion Research team, working across policy teams, to bring new insight from Londoners to develop policies that meet Londoners needs. We are experts at integrating research, data analysis and consultation into meaningful policy conversations, to meet the challenges of population growth, economic and environmental change. We use a range of tools: from traditional forms of consultation and social research, to innovative digital engagement.
An excellent communicator, committed to building partnerships, you will be well-versed in delivering, commissioning and designing qualitative research projects, working with research agencies and policy officers to deliver high quality insight on citizens' opinions, perceptions and behaviours. You will bring a creative approach to research design, using both innovative and conventional methodologies.
An understanding of quantitative research would also be an advantage, but we will consider candidates who are happy to develop and work in this area.
This role is fixed contract until January 2021.
The GLA's research team would like to invite anyone interested in this role to an open evening on Thursday 17 October 2019 at London's City Hall to provide you with a chance to ask any questions you have about this opportunity, and hear a bit more about the role. If you are interested in attending this open evening, Please click here to register.
We are committed to being an inclusive employer and we are happy to consider flexible working arrangements. We would welcome applications from candidates who are seeking part time work as this role is open to job share.
In addition to a good salary package, we offer an attractive range of benefits including 30 days' annual leave, interest free season ticket loan, interest free bicycle loan, childcare voucher scheme and a career average pension scheme.
If you have a question about the role or the recruitment process then please contact a member of the recruitment team via email quoting reference GLA676.
To apply, please click on the apply button to visit our website.
Application closing date: Sunday 27 October 2019 at 23:59 GMT.
London's diversity is its biggest asset and we strive to ensure our workforce reflects London's diversity at all levels. We welcome applications from everyone regardless of age, gender, ethnicity, sexual orientation, faith or disability.


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Bulawayo — President Mnangagwa yesterday declared 2018 as the year for all Zimbabweans, saying his administration will work tirelessly to create a peaceful and stable political and socio economic environment that will ensure the nation's prosperity.
In his New Year Message that was also aired on ZBCTV, President Mnangagwa said his administration will thrive on governing, guided by honesty, transparency, accountability and discipline.
"Fellow Zimbabweans whether at home or abroad, I would like to wish you and your families a very prosperous 2018. I also wish you good health, joy, peace and happiness," he said in his maiden in-studio recording at Montrose Studios in Bulawayo.
"The New Year will undoubtedly have immense opportunities and its full share of challenges. But if we remain united as one people, nothing is insurmountable. Let us resolve to collectively work hard for the betterment of our country. The President implored Zimbabweans to thrive more than ever before, to be united in all endeavours, citing Psalms 133 Verse 1 which reads; "For it is indeed good and pleasant when God's people live together in unity and harmony."
"Government will continue to work tirelessly to create a peaceful and stable political and socio economic environment which will enable us to be a prosperous nation and to realise our full potential. 2018 should be the year of all Zimbabweans, 2018 should be the year of progress. 2018 should also be the year of credible, free and fair elections." President Mnangagwa committed that he will continue to be a listening and responsive leader.
"I urge you fellow Zimbabweans to engage with Government, its institutions and agencies for more transparent, just, accountable and responsible governance. Indeed, no one person or institution has a monopoly of ideas," he said. "Let us equally commit to honesty, transparency, accountability and discipline to ensure accelerated national development and progress. Collectively, we can build a new, democratic and prosperous Zimbabwe."
Since his inauguration on November 24, President Mnangagwa has hit the ground running introducing a cocktail of interventions towards the stimulation of the economy with the aim of returning the country to its lost glory. He has also said in a number of platforms that the country should not focus on politicking but shift focus towards the economy and productivity.


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Meet Me In The Cloud
Professional Videoconferencing at a Fraction of the Cost
Imagine internal and B2B videoconferencing, with no CapEx investment, no bulky hardware to manage, and no new video endpoints to purchase.
Cloud-based Conferencing:
Makes it easier than ever for your employees, partners, customers, and suppliers to join a conference anytime, anywhere.
- Provides customers with high-quality, easy-to-use, videoconferencing functionality, without the problems associated with traditional professional videoconference systems
- Requires zero capital expenditure for video bridging hardware. The conferencing service is run in the cloud.
- Expands your endpoint options. You are no longer constrained by scarce, overbooked video room systems. Participants connect on virtually any device, anywhere.
Benefits of Cloud-based Conferencing
TelePresence in the Cloud
Enterprise-class video solution without the infrastructure costs
Quick and Easy Implementation
Within minutes, enable conference room video, personal video, and set up virtual meeting rooms
Easy to Deploy Video
No server configuration or complex installations. Simple one-click activation and you are ready to make video calls
Simple to Scale
Expands or reduces easily with adding or removing subscriptions
Flexible Deployment Options
Automatically connect from any location
Cost Control
Monthly or annual billing for active subscribers
Redundancy and security built-in
Talk to anyone on any network on any device
Plug and Play
Activation is simple and you have your own, unique calling address
Unlimited Video Calling
Call as much as you'd like to users both inside and outside of your company
Screen Sharing
Show presentations and other documents
Virtual Meeting Rooms
- Secure, open virtual rooms for multiple callers
- Unique address with password protection when needed
- 3rd party integration with applications such as Lync
- Unlimited usage available
- Similar to cell phone usage and billing but with business video
- Start using video immediately
- Unlimited video calling immediately – simple activation, no complex configurations
- Dedicated capacity without your own dedicated network
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- Only pay for active users – Everyone can have access at a low cost