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Etk (also called Bmx) is a member of the Btk tyrosine kinase family and is expressed in a variety of hematopoietic, epithelial, and endothelial cells. We have explored biological functions, regulators, and effectors of Etk. Coexpression of v-Src and Etk led to a transphosphorylation on tyrosine 566 of Etk and subsequent autophosphorylation. These events correlated with a substantial increase in the kinase activity of Etk. STAT3, which was previously shown to be activated by Etk, associated with Etk in vivo. To investigate whether Etk could mediate v-Src-induced activation of STAT3 and cell transformation, we overexpressed a dominant-negative mutant of Etk in an immortalized, untransformed rat liver epithelial cell line, WB, which contains endogenous Etk. Dominant-negative inactivation of Etk not only blocked v-Src-induced tyrosine phosphorylation and activation of STAT3 but also caused a great reduction in the transforming activity of v-Src. In NIH3T3 cells, although Etk did not itself induce transformation, it effectively enhanced the transforming ability of a partially active c-Src mutant (c-Src378G). Furthermore, Etk activated STAT3-mediated gene expression in synergy with this Src mutant. Our findings thus indicate that Etk is a critical mediator of Src-induced cell transformation and STAT3 activation. The role of STAT3 in Etk-mediated transformation was also examined. Expression of Etk in a human hepatoma cell line Hep3B resulted in a significant increase in its transforming ability, and this effect was abrogated by dominant-negative inhibition of STAT3. These data strongly suggest that Etk links Src to STAT3 activation. Furthermore, Src-Etk-STAT3 is an important pathway in cellular transformation.
Etk (also called Bmx) is a member of the Btk nonreceptor tyrosine kinase family (48, 51, 57). This family of kinases is characterized by a modular structure, including an N-terminal pleckstrin homology (PH) domain, a Tec homology domain, Src homology 2 (SH2) and SH3 domains, and a C-terminal kinase domain. Many members of the Btk family are predominantly expressed in cells of hematopoietic origin. Btk is expressed in B cells and myeloid cell lineages and is essential for B-cell development and signaling. Loss-of-function mutations in Btk result in human X-linked agammaglobulinemia and murine X-linked B-cell immunodeficiency (50, 53). Itk is expressed in T cells and activated by various T-cell receptors and by FcɛRII stimulation in mast cells (2, 25, 30). Mice lacking Itk have defects in T-cell development and signaling (34). Tec, expressed in various hematopoietic cells and liver, is involved in the intracellular signaling of T-cell receptors and many cytokines, such as c-Kit, granulocyte colony-stimulating factor, interleukin-3 (IL-3), IL-6, and erythropoietin (37, 39, 42, 44, 58). In contrast to these family members, Etk is expressed in a variety of tissues and cell types including hematopoietic, epithelial, and endothelial cells as well as several prostate cancer cell lines and tissues (19, 48, 57, 65). Little is known about the biological function of Etk and the signaling pathways in which Etk is involved. So far, Etk is only known to be required for IL-6-induced differentiation of prostate cancer cells (48) and Gα12/13-induced activation of serum response factor in fibroblasts (40). No downstream effectors for Etk have been clearly defined. Although a recent report indicated the activation of STAT3 by overexpressed Etk in Cos cells (52), whether this involves a direct or an indirect mechanism is not clear. Furthermore, current knowledge on the role of Btk family kinases in signal transduction is largely limited to cells of hematopoietic origin. It would be important to elucidate the signaling mechanism of Etk in other cell types.
Btk family kinases are subject to several modes of regulation. They are activated by Src family kinases (16, 27, 49), phosphatidylinositol 3-kinase (PI 3-kinase) (3, 33, 48), and the α subunits of G proteins, such as Gα12/13 and Gαq (4, 28, 40). Src family kinases have been shown to directly bind and phosphorylate Btk or Itk on a tyrosine residue in the activation loop of the kinase domain. This phosphorylation leads to autophosphorylation with eventual full activation of Btk or Itk (27, 47, 49). The full activation of Btk and Itk is also dependent on the interaction between their PH domains and phosphatidylinositol 3′-polyphosphates, products of the PI 3-kinase (3, 33). A previous study showed that Etk, being analogous to other Btk kinases, is a downstream effector of PI 3-kinase (48). Etk is somewhat atypical in that it lacks a proline-rich region present in the Tec homology domain of all other Btk kinases (48). This region was suggested to mediate an interaction with the SH3 domain of certain Src family kinases (41). Thus, whether Etk is a substrate for Src has yet to be experimentally demonstrated.
v-Src is a potent oncogene which mediates cellular transformation by engaging several signaling pathways. In addition to the well-established Shc-Grb2-Ras-Raf-Erk connection, recent reports indicated that STAT3 is also activated by v-Src (10, 69), which is independent of the Ras signaling pathway (35). Importantly, STAT3 activation is required for v-Src-mediated transformation of NIH3T3 cells (7, 60). Consistent with this view is the increasing body of evidence indicating the roles of STAT3 in cell growth, antiapoptosis, and malignant transformation. For instances, constitutive activation of STAT3 has been detected in cells transformed with various oncoproteins and tumor viruses (10, 36, 43, 69, 71) as well as in many human cancer cell lines and tissues (21, 24, 26, 63, 70). Introducing a constitutively dimerizeable STAT3 into immortalized fibroblasts causes cellular transformation and tumor formation (8). Furthermore, STAT3-deficient mouse embryos implant but fail to grow, suggesting a role in cell proliferation and/or survival (56). In addition, STAT3 is required for the cytokine receptor gp130-mediated G1-to-S phase progression of a pro-B-cell line by regulating several cyclins and cyclin-dependent kinase inhibitors (23) and it mediates cytokine-induced survival of the cell by upregulating Bcl-2 (22). In cell lines derived from multiple myelomas, constitutively activated STAT3 is essential to protect against apoptosis (10).
Not only v-Src is able to activate STAT3; c-Src also plays a role in IL-3- (12) and epidermal growth factor- (46) induced STAT3 activation. A key question, then, is how v-Src or activated c-Src mediates the activation of STAT3. One report showed that v-Src forms a complex with STAT3, suggesting a direct role (10). Other studies, however, imply a more indirect mechanism involving other tyrosine kinases, such as JAK (9). It is likely that different mechanisms are utilized by different cell types.
As part of our effort to understand the mechanisms underlying cellular transformation by Src-like kinases, we investigated the role of Etk in this process. Here, we present evidence which indicates that v-Src is able to transform rat liver epithelial cells and that it does so by activating STAT3 via Etk tyrosine kinase. We show that Etk is a downstream effector of Src; upon activation by Src, it associates with and phosphorylates STAT3. This pathway is crucial for transformation, as dominant-negative mutants of Etk or STAT3 interfere with the transformation process. Furthermore, coexpression of Etk and a weakly transformed Src mutant, c-Src378G, synergistically induces transformation and STAT3 activation. The present work not only illuminates the Etk pathway in several nonhematopoietic cell types but also provides one molecular link between Src and STAT3.
Plasmids.pcDNA3T7Etk and pcDNA3T7EtkKQ are expression plasmids for T7-tagged wild type and kinase-defective Etk, respectively (48). pRK5MEtk and pRK5MEtkKQ, expression plasmids for myc-tagged Etk and EtkKQ, respectively, were constructed by PCR and cloned into pRK5M (20). Deletion mutants of Etk were generated by cloning PCR fragments corresponding to each deletion to pCDNA3T7. To construct pBabevSrc, a retrovirus-based expression plasmid for v-Src, the coding region of v-Src was PCR amplified from pMvSrc (a gift from H.-F. Yang-Yen) and cloned to pBabepuro3. The same PCR fragment was cloned to pRK5 or pBabepuro3 to generate pRKvSrc or pBabevSrc, respectively. pRK5vSrcKD, a kinase-defective v-Src (v-SrcKD) mutant carrying a K-to-R mutation at position 297, was generated by in vitro mutagenesis with the QuickChange site-directed mutagenesis kit (Strategene). To construct pRKSrc527F, a retroviral vector for a constitutively active c-Src, the coding region of c-Src was excised from pUSESrc (Upstate Biotechnology) and cloned to pBluescript. The resulting plasmid was used as a template for in vitro mutagenesis to create pBSSrc527F. pBabecSrc378G, a retroviral vector for a weakly active c-Src, was also constructed by in vitro mutagenesis. The mutagenized Src was then subcloned to pRK5. pCAGGSSTAT3D, an expression plasmid for a dominant-negative STAT3 (45), was kindly provided by T. Hirano. pMc/CMVSTAT3, an expression plasmid for wild-type STAT3, was from X.-Y. Fu.
Antibodies.Polyclonal antibody to Etk was described previously (48). The anti-phosphotyrosine antibody (RC20) was purchased from Santa Cruz. Antibody specific to STAT3 phosphorylated at position 705 was from New England Biolabs. Anti-T7 antibody was from Novagen. Antibodies to JAK2, v-Src, c-Src, STAT1, and STAT3 were from Upstate Biotechnology.
Cell culture and transfection.NIH3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. WB-F344 rat liver epithelial cells (59) were cultured as described previously (29). Human hepatoma cell line Hep3B was cultured as described previously (14). Transfection of NIH3T3 and Hep3B cells was performed using Lipofectamine reagent (Life Technologies) according to the manufacturer's instructions. For transient transfection, cells were harvested 48 h after transfection. For selecting stable clones, G418 (700 μg/ml) was added to culture medium 48 h after transfection.
Construction of recombinant retrovirus and infection.Production of recombinant retrovirus and infection of host cells were carried out following procedures essentially as described by Kitamura et al. (32). Briefly, package cell line Phoenix-Eco was transfected by calcium phosphate method. The supernatant containing viral particles was harvested 48 h after transfection. For infection of WB cells, 2 × 105 cells were seeded onto a 60-mm plate the night prior to infection and incubated with 2 ml of viral stock in the presence of polybrene (6 μg/ml for WB and 4 μg/ml for NIH3T3). The medium was changed to fresh culture medium 18 to 24 h after the beginning of the infection. Two days after infection, cells were selected in culture medium containing 600 μg of G418 per ml or 2 μg of puromycin per ml.
Fusion protein construction and purification.GlutathioneS-transferase (GST)-Etk-K(K445Q) was constructed by inserting a PCR fragment corresponding to the kinase domain of Etk (Etk-K) containing a mutation at position 445 into pGEX-4T vector. Mutation of Y566 was introduced by site-directed mutagenesis, and the corresponding PCR fragments were cloned to pGEX-4T to make GST-Etk-K(Y566F) and GST-Etk-K(K445Q, Y566F). Expression of the fusion proteins was induced by 0.1 mM isopropyl-β-d-thiogalactopyranoside for 3 h, and the fusion proteins present in the inclusion bodies were solubilized by denaturation with 6 M urea. The fusion proteins were allowed to refold by dialysis to remove urea and then purified by incubating with glutathione-Sepharose beads. The beads were washed five times with wash buffer containing 10 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1 mM EDTA, and 0.1% NP-40, and the fusion proteins were eluted by elution buffer containing 50 mM Tris-HCl (pH 8.0) and 5 mM reduced glutathione.
Immunoprecipitations and in vitro kinase assays.Cells were lysed in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% NP-40, 10 mM NaF, 1 mM NaVO4, 1 mM sodium pyrophosphate, 2 μM aprotinin, 2 μM leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The T7-tagged Etk, JAK2, or Src was precipitated from cell lysates containing equivalent amounts of proteins with anti-T7, anti-JAK2, or anti-c-Src antibody, respectively, followed by protein A-Sepharose conjugated with rabbit anti-mouse antibody as described previously (15). The immunocomplex recovered was washed three times with lysis buffer, boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and subjected to SDS-PAGE. Alternatively, the immunocomplex was washed twice with lysis buffer and twice with kinase buffer containing 20 mM HEPES (pH 7.4), 1 mM dithiothreitol, 10 mM MnCl2, 10 mM MgCl2. The kinase reaction was carried out in kinase buffer supplemented with 7.5 μg of acid-denatured enolase or 2 μg of each GST-Etk-K fusion protein and 10 μCi of [γ-32P]ATP at 30°C for 30 min. The kinase reaction was terminated by addition of an equal volume of 2× SDS-PAGE sample buffer. The samples were boiled for 5 min prior to electrophoretic separation by SDS-PAGE.
EMSA.Electrophoretic mobility shift assay (EMSA) was performed according to the procedures described previously (31). Briefly, nuclear extracts containing equal amount of proteins were incubated with 32P-labeled hSIE probe (62) in the presence or absence of cold hSIE or a nonspecific oligonucleotide. The samples were electrophoresed on a 5% polyacrylamide gel in 0.5× Tris-borate-EDTA buffer. For supershift, nuclear extracts were incubated with anti-STAT3 or anti-STAT1 for 20 min before the addition of labeled probe.
Luciferase assay.NIH3T3 and its derivatives were transfected with pGASLuc and pRKβ-gal. One day after transfection, cells were serum starved overnight and then harvested. Luciferase and β-galactosidase activities were quantitated by the Luciferase Assay System and the β-galactosidase Enzyme Assay System (Promega), respectively. The luciferase activity was normalized to β-galactosidase activity to account for transfection efficiency.
Soft agar colony formation assay.WB, NIH3T3, or Hep3B cells stably expressing various proteins were trypsinized, diluted in 0.3% of top agar and spread onto 60-mm plates containing 0.5% of bottom agar. For cell lines WB and NIH3T3 and their derivatives, 5 × 104 cells were seeded in each plate. For Hep3B and its derivatives, 104 cells were seeded. Colonies formed after 1 (for WB) or 4 (for Hep3B and NIH3T3) weeks were visualized by staining with 0.05% p-iodonitrotetrazolium violet dye.
Etk is phosphorylated and activated by v-Src.Src family kinases have been shown to activate Btk family kinases, including Btk, Itk, Tec, and Txk (16, 27, 38, 49). This, however, has not been tested for Etk. To explore the upstream regulators of Etk, we investigated whether Etk could be activated by oncogenic v-Src. Coexpression of v-Src and Etk in 293 cells led to a substantial increase in the in vivo tyrosine phosphorylation of wild-type Etk (Fig.1B, lane 3). Furthermore, v-Src stimulated the kinase activity of Etk, as judged by autophosphorylation of Etk and phosphorylation of exogenous substrates such as enolase in an in vitro kinase assay (Fig. 1A, lane 3). This increase in tyrosine phosphorylation and kinase activity was not observed upon coexpression of a v-SrcKD with Etk (Fig. 1, lane 4), indicating that these events are dependent on the kinase activity of v-Src. Coexpression of v-Src with a kinase-defective Etk (EtkKQ) did not result in an in vitro phosphorylation on enolase or Etk (Fig. 1A, lane 6). Therefore, the in vitro phosphorylation seen with wild-type Etk (Fig. 1A, lane 3) was carried out predominantly, if not exclusively, by Etk, rather than by v-Src or other contaminating kinases in the immunoprecipitates. v-Src also increased tyrosine phosphorylation for the kinase-defective Etk, although the amount was less prominent than that observed for wild-type Etk (Fig. 1B, lanes 3 and 6). This suggests that the increase in tyrosine phosphorylation of wild-type Etk upon coexpression of v-Src is due to the combination of phosphorylation of Etk by v-Src and Etk autophosphorylation.
To further investigate the mechanism of Etk activation by v-Src, we determined the Src phosphorylation site on Etk. Previous studies showed that Src family kinase phosphorylates Btk at Y551 (47, 49). Mutation of an analogous tyrosine residue (Y566) on Etk almost completely abolished the Src-induced tyrosine phosphorylation of Etk in cotransfected 293 cells (Fig. 2A). Phosphorylation of Y566 by Src was also tested in the in vitro kinase assay using chimeric proteins consisting of Etk-K fused with GST as the substrate. To distinguish the event of transphosphorylation of Etk by Src from its autophosphorylation, several GST-Etk-K mutants, in which K445 and/or Y566 were replaced, were compared as the substrates of Src. Because the lack of antibody can effectively immunoprecipitate v-Src, a constitutively active Src (c-Src 527F) was utilized in the assay. As shown in Fig. 2B, c-Src 527F purified by immunoprecipitation from transfected 293 cells effectively phosphorylated GST-Etk-K(K445Q). Phosphorylation of GST-Etk-K(Y566F) was much less efficient, whereas the GST-Etk-K(K445Q, Y566F) double mutant was not phosphorylated. Altogether, these results strongly suggest that Etk is activated by Src via a mechanism similar to that of other Btk kinases, i.e., by direct phosphorylation by Src on Y566, followed by Etk autophosphorylation.
Etk associates with STAT3 in vivo.Having identified Src as an upstream regulator of Etk, we wished to test whether STAT3 is its immediate downstream effector. To investigate whether Etk acts directly on STAT3, we examined their association in vivo. Lysates from 293 cells cotransfected with a T7-tagged Etk and STAT3 were immunoprecipitated with anti-T7 antibody, and coprecipitation of STAT3 was examined by Western blotting. This assay demonstrated the formation of a stable complex between Etk and STAT3 (Fig. 3B, lane 3). Furthermore, association of Etk with endogenous STAT3 was also observed (Fig. 3B, lane 2), indicating that this association occurs under physiological conditions. The associated STAT3 is likely to be phosphorylated by Etk directly, as is evident by the existence of a 90-kDa phosphorylated band corresponding to the size of STAT3 in the anti-Etk immunoprecipitates subjected to an in vitro kinase assay (Fig. 1A). To map the domain in Etk required for this association, we constructed a panel of Etk deletion mutants lacking each of the potential protein-protein interaction domains (Fig. 3A). Coimmunoprecipitation analysis showed that the PH domain deletion mutant could no longer associate with STAT3, suggesting that this domain is required for interacting with STAT3 (Fig. 3B).
Etk mediates v-Src-induced activation of STAT3.STAT3 was shown to be activated in v-Src transformed cells and required for v-Src-mediated transformation (7, 60). The identification of v-Src as an upstream activator and STAT3 as a downstream effector of Etk prompted us to investigate whether Etk is involved in v-Src-induced activation of STAT3 and whether Etk is required for v-Src-mediated transformation. To test these possibilities, a rat liver epithelial cell line, WB, was chosen for the following reasons. First, WB cells are nontumorigenic but can be transformed by many oncogenes including v-Src (17). Secondly, this cell line expresses a relatively high level of Etk as determined by reverse transcription-PCR (data not shown) and Western blotting (Fig. 4A upper panel, lane 1). We therefore tested whether the endogenous Etk could be inhibited by overexpression of the EtkKQ. WB cells were infected with recombinant retrovirus carrying EtkKQ, and four stable lines expressing EtkKQ were obtained (Fig. 4A). A high expressor, EtkKQ-10, and a mixture of all four clones (EtkKQ-M) were chosen for further analysis. In vitro kinase assays showed that expression of EtkKQ significantly decreased the kinase activity of endogenous Etk (Fig. 4A), indicating that EtkKQ functions as a dominant-negative mutant. The parental WB cells and stable clones expressing EtkKQ were infected by recombinant retrovirus containing v-Src and the puromycin resistance gene. After selection with puromycin, three pools of drug-resistant cells (WB v-Src, WB v-Src EtkKQ-10, and WB vSrc EtkKQ-M) expressing comparable amounts of v-Src were generated (Fig.4B).
To assess the activity of STAT3 in WB and its derivatives, nuclear extracts were prepared and used for EMSA with the32P-labeled hSIE probe that binds STAT1 and STAT3 with high affinity. Similar to what was found in fibroblasts, v-Src expression in WB cells markedly increased the DNA binding activity of STAT3 (Fig. 4C, lanes 7 to 10). Importantly, this v-Src-induced activation of STAT3 was inhibited in cells stably expressing EtkKQ (Fig. 4C, lanes 12 and 13). To further characterize the role of Etk in activation of STAT3 by v-Src, lysates from these cells were used for Western blot analysis with an antibody specific to STAT3 phosphorylated at tyrosine 705, an event required for its dimerization and nuclear translocation (54, 66). As shown in Fig. 4D, dominant-negative Etk blocked v-Src-induced tyrosine phosphorylation of STAT3. The expression levels of STAT3 were comparable in all cell lines (Fig. 4D). Noted that the dominant-negative Etk did not generally affect v-Src-induced tyrosine phosphorylation on many cellular proteins (Fig. 4E), indicating that Etk is likely to transduce v-Src signals to only few targets, such as STAT3. We conclude from these observations that Etk links v-Src to the activation of STAT3.
Increased tyrosine phosphorylation on JAK2 in v-Src-transformed WB cells.Previous study showed that JAK1 and JAK2 are strongly and moderately activated in v-Src-transformed fibroblasts, respectively, suggesting that these kinases are involved in mediating STAT3 activation by v-Src (9). To test whether JAKs are activated in v-Src transformed WB cells and whether inhibition of Etk affects JAK activation, we examined the tyrosine phosphorylation of JAKs in WB and its derivatives. Western blot analysis showed WB contains a much higher level of JAK2 than JAK1 (data not shown). Consistent with a previous report (9), JAK2 phosphorylation was indeed higher in v-Src transformed cells (Fig. 5). This increase of the phosphorylation, however, is only moderate, since a large amount (∼1 mg) of protein from cell extracts was required to generate the results shown in Fig. 5. The mechanism of JAK2 activation by v-Src remains obscure. It is, however, clear that this activation is not mediated by Etk, since expression of EtkKQ did not affect this phosphorylation of JAK2 (Fig. 5). These data suggest that JAK and Etk act in parallel in mediating v-Src-induced STAT3 activation. Nevertheless, the contribution of JAK2 to v-Src-induced STAT3 activation is minor, since dominant-negative Etk almost completely abolished the STAT3 phosphorylation and activity (Fig. 4C and D). Therefore, we conclude that, in this cell type, Etk is the primary mediator to transduce v-Src signal to STAT3.
Etk is required for transformation of WB epithelial cells by v-Src.To investigate the role of Etk in v-Src-induced cell transformation, we performed soft agar colony formation assays on WB and its derivatives. Consistent with previous studies (17), WB cells expressing v-Src efficiently formed colonies in soft agar. However, the soft agar colony forming ability of v-Src was markedly diminished by dominant-negative inhibition of Etk (Fig.6). Furthermore, the high expressor EtkKQ-10 exhibited a higher degree of inhibition than EtkKQ-M, indicating a dose-dependent effect. These findings demonstrate a significant role of Etk in v-Src-induced transformation of epithelial cells.
Etk and Src synergistically activate STAT3 and transform NIH3T3 cells.Having demonstrated an inhibition by dominant-negative Etk in v-Src-induced transformation and STAT3 activation, we next investigated whether overexpression of Etk could enhance these effects of Src. The already high endogenous level of Etk impeded our effort to overexpress Etk in this cell line. We thus performed studies in NIH3T3 cells, which contain a much lower level of Etk (data not shown). Etk and/or a partially activated Src mutant (c-Src378G) were introduced into NIH3T3 cells by retrovirus-mediated gene transfer. Pools of infected cells were assayed for their transforming activities. As shown in Fig.7B, cells overexpressing Etk did not grow in soft agar, and cells expressing Src378G were only weakly transformed. However, coexpression of Src378G with Etk resulted in a significant increase in both the number and size of the colonies formed in soft agar. This enhancement in transforming activity could not be attributed to the expression level of the c-Src378G, since the two pools of infected cells contain a similar level of this Src mutant (Fig. 7A). Our result is consistent with previous study in which Btk requires the partially active Src to affect its transforming activity (1).
To further demonstrate an interaction between Src and Etk signaling, we determined the activity of STAT3 in NIH3T3 cells stably expressing Etk and/or Src378G. These cells were transiently transfected with the pGASLuc, a STAT3-driven luciferase reporter plasmid (7). Src378G or Etk alone was capable of driving a moderate increase in the transcription of reporter gene, whereas coexpression of the two genes led to a synergistic activation of STAT3 (Fig. 7C). Taken together, these findings verifies the notion that Src and Etk act together in mediating STAT3 activation and cellular transformation.
STAT3 is a critical component of Etk signaling leading to transformation.The results derived from previous sections provide strong evidence that Etk mediates both STAT3 activation and cellular transformation induced by v-Src. The remaining task was then to demonstrate that the role of Etk in cellular transformation is due, at least in part, to its signaling to STAT3. Our strategy was to investigate the effect of STAT3 inactivation on the Etk-mediated transformation. For this purpose, we needed a cell line that could be readily transformed by Etk overexpression. Human hepatoma cell line Hep3B was a good candidate (see below). Hep3B cells were transfected with expression vectors for wild-type Etk or EtkKQ, and several stable clones were generated. Figure 8A shows the expression levels of Etk or EtkKQ in these clones. The stable clones expressing Etk or EtkKQ were respectively pooled and subjected to soft agar assays. While parental Hep3B cells did not efficiently form colonies in soft agar when plated at a low density (104 cells/plate), overexpression of Etk increased its transforming ability. This enhancement in transformation was not observed with the EtkKQ (Fig. 8B). To assess the role of STAT3 in this effect, we transfected Hep3BEtk cells with a dominant-negative mutant of STAT3, STAT3D, carrying substitutions of A for E at positions 434 and 435 (45). Several stable cell lines were generated and pooled for soft agar assay. A pool of Hep3B stable clones expressing STAT3D generated previously (13) was also included in the assay. As shown in Fig. 9B, overexpression of STAT3D abrogated the effect of Etk on the transforming ability of Hep3B cells. This difference is unlikely due to the difference in expression levels of transfected genes, since the two Etk- and the two STAT3D-expressing lines contain similar amounts of Etk and STAT3D, respectively (Fig. 9A). Thus, STAT3 is involved in Etk signaling leading to transformation. Taken together, the above data show that the Src-Etk-STAT3 pathway plays an important role in the transformation of rat, mouse, and human cells.
The role of Etk in Src-induced activation of STAT3.In this report, we demonstrate v-Src to be an upstream activator and STAT3 to be an immediate downstream effector of Etk. Furthermore, we provide evidence indicating the involvement of Etk in Src-induced activation of STAT3. Several lines of evidence are presented to support this notion. First, we found that expression of v-Src resulted in an activation of Etk and that expression of Etk led to STAT3 activation. Secondly, Src and Etk acted synergistically in the activation of STAT3, suggesting an interaction between Src and Etk signaling. Thirdly, Etk directly complexed with and tyrosine phosphorylated STAT3. Finally and most importantly, dominant-negative inhibition of Etk activity by a kinase-defective mutant was able to inhibit STAT3 activation by v-Src. Thus, these results firmly establish a cascade link of v-Src-Etk-STAT3. Previous study revealed that v-Src associates in a complex with STAT3 (10), suggesting a direct phosphorylation and activation of STAT3 by v-Src. Another study found a constitutive activation of JAK family kinase in Src-transformed cells (9), implicating a role of JAK in v-Src-induced activation of STAT3. Yet, another report showed that it is the Src-like activity, not JAK activity, which mediates IL-3-induced activation of STAT3 (12). However, these mechanisms of STAT3 activation by v-Src need not be mutually exclusive and may vary according to cell types. In this report, we describe a new mechanism of STAT3 activation by v-Src, through the action of Etk. We show here that in epithelial cells, such as WB cells, containing a relatively high level of Etk, v-Src-induced STAT3 activation is primarily mediated through Etk. Although in this cell line JAK2 is modestly activated by Src, our finding that dominant-negative Etk nearly completely abolished v-Src-induced STAT3 activation indicates that other kinases, such as JAK2, play only minor role in linking Src signaling to STAT3.
Most interestingly, a recent study has demonstrated a requirement of STAT3 serine phosphorylation in Src transformation (61). The Ras- and Rac-1-mediated p38 and JNK pathways are activated by Src and responsible for this serine phosphorylation. Our results, which revealed the involvement of Etk in Src signaling leading to STAT3 tyrosine phosphorylation, complement well with this finding and suggest multiple signaling pathways induced by Src converged on the activation of STAT3.
We demonstrate that coexpression of v-Src and Etk led to an increased phosphorylation and activation of Etk. Furthermore, tyrosine phosphorylation in the EtkKQ expressed with v-Src was significantly reduced compared to that in the wild-type Etk expressed with v-Src. In addition, Src directly phosphorylated Etk at Y566, a position equivalent to the Src kinase phosphorylation site on Btk. These findings strongly suggest that Etk is activated by Src in a fashion similar to that in which other Btk family kinases are activated (27, 47, 49). Src is likely to phosphorylate Etk on Y566, which leads to its autophosphorylation and eventually complete activation. In other Btk family kinases, the proline-rich region is implicated in the association with Src family kinases (41). Etk, however, does not contain a typical proline-rich region. The association of Src and Etk could not be reproducibly detected by coimmunoprecipitation analysis (data not shown), which might be due to a transient nature of this interaction. Alternatively, additional components might be required to stabilize this association.
Our observation that Etk associates with STAT3 in vivo suggests that STAT3 is directly tyrosine phosphorylated and activated by Etk. In support of this notion, STAT3 coimmunoprecipitated with Etk was phosphorylated by Etk in an in vitro kinase assay. The interaction of Etk and STAT3 is presumably mediated through the PH domain of Etk, since deletion of this domain abrogated interaction. Nevertheless, it cannot be excluded that such a deletion affects Etk's membrane localization or protein conformation, thus resulting in the disruption of its interaction with STAT3. Interestingly, the PH domain of Etk contains a YPFQ motif which is also found in several other Btk family kinases. Similar sequences (YXXQ) have been shown to function as STAT3 docking sites in many cytokine and growth factor receptors, such as the IL-10 receptor (64), the granulocyte colony-stimulating factor receptor (17), gp130 (55, 68), c-Met (6), and v-Eyk (5). Additional studies will be required to determine precise residues in Etk required for STAT3 binding.
Role of Etk in transformation.This report reveal that Etk is involved not only in v-Src-induced STAT3 activation but also in v-Src-induced cell transformation. The ability of Etk in mediating v-Src-induced transformation is due, at least in part, to its induction of STAT3 activity, since dominant-negative inhibition of STAT3 blocked the enhancement of transformation of Hep3B cells by Etk. Therefore, the v-Src-Etk-STAT3 pathway is important for cell transformation (Fig.10). Emerging evidence suggests that STAT3 plays a critical role in cellular transformation by several oncogenic receptor and nonreceptor tyrosine kinases, including v-Src (7, 60), v-Eyk (5), Ros, and insulin-like growth factor receptor (72). Furthermore, recent studies demonstrated that a constitutively activated STAT3 is capable of inducing cell transformation and tumor formation, indicating the function of STAT3 as an oncogene (8). Although the mechanism by which STAT3 contributes to oncogenesis is not fully understood, its involvement in cell transformation is consistent with several lines of evidence for its roles in proliferation (22, 23, 56, 72) and antiapoptosis (10, 22). In addition, previous studies revealed that the function of STAT3 seems to be more important in anchorage-independent growth than in monolayer growth, implying that STAT3 signaling can fulfill certain adhesion-triggered signaling (72).
Although Etk is clearly required for transforming WB cells by v-Src, it is insufficient to trigger transformation by itself when introduced into untransformed cells such as NIH3T3 cells. The requirement of a partially activated Src as the cooperating oncogene for Etk to efficiently transform NIH3T3 cells resembles the case of Btk (1). Given that malignant transformation of mammalian cells is a complex process involving multiple changes, additional molecules and signaling pathways are likely to cooperate with Etk in transformation. In this regard, we found that Etk is capable of enhancing the transforming efficiency of Hep3B. This hepatoma cell line is marginally transformed and presumably have other cooperating pathways activated already. Experiments are underway to investigate the cooperating pathway to achieve a full understanding of Etk-mediated transformation process.
The present report, taken in the light of a recent finding that Etk is critically involved in an antiapoptotic pathway that protects prostate cancer cells from radiation and thapsigargin (67), suggests a possible role for Etk in the transformation and progression of human malignancies. Although the activity of Etk in various tumors has not been determined, Etk is widely expressed in many epithelial tumors, including hepatoma and prostate cancers (data not shown). Additional studies are needed to further investigate the contribution of Etk in human epithelial malignancies.
Yuh-Tyng Tsai and Yi-Hsien Su contributed equally to this work.
We thank T. Hirano, H.-F. Yang-Yen, X.-Y. Fu, and J. E. Darnell for plasmid constructs, G. P. Nolan for phoenix package cells and instructions on retrovirus-mediated gene transfer, and Rachel L.-C. Chuang for excellent technical assistance.
This work was supported by NSC Frontier Grant 88-2312-B-002-050 to R.-H.C., by intramural funds from National Health Research Institute to Y.-S.J. and H.-m.S., and by NIH grant CA39207 to H.-J.K.
- Received 23 June 1999.
- Returned for modification 11 August 1999.
- Accepted 13 December 1999.
- Copyright © 2000 American Society for Microbiology


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Product Description
4 Dozen Red Roses Basket, Personalized Get Well Soon Card and 10 kg Mix Fresh Fruit Basket for your friends and family in Pakistan.
Send good quality picture with minimum size 500kb at email@example.com with order number.
Order 2 Days in Advance
Delivery Available in all Major Cities

subset: commoncrawl


text:
Pimento Cheese Salad Dressing Mix
Click image to zoom
Click image to zoom
Pimento Cheese Salad Dressing Mix
Two kinds of cheese, pimento peppers, and all natural spices are blended to create a truly marvelous dressing mix. Add buttermilk and mayo for a salad dressing with all the flavor of the South's favorite cheese spread. Great on sandwiches, burgers, and veggies, too!
Net Wt 2 oz
Item# DR-PCDM


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Liquid Chalk (200ml) - The Professionals Choice
Specially designed chalk that dries in seconds to increase grip and reduce hand sweating.
Ideal for weightlifting
Squeeze a small ball of Liquid Chalk onto the palms of your hands. Spread chalk to create an even layer. Allow to dry fully before using equipment. Do not use on broken skin. Test on a small area of skin, if detrimental reaction occurs, cease use immediately and wash off with water.
Alcohol, Magnesium Carbonate, Thickener, Fragrance
- Returns
- Shipping
- Questions?
Shipping is free for orders totalling over £20. For orders totalling less than £20 this will cost £4.99. Depending on the item being order shipping can take 3-7 working days. Shipping codes can be provided to track shipment. First Physique use different courier companies depending on the item ordered. For further information on our shipping policy click here.
At First Physique we are fitness nuts and whilst we may not have all the answers we will find out!
If you have a question on our gym accessories / equipment of even something you are after and cannot find just click here and send us your query now and we will do all we can to help!
Or email us at email@example.com or call us on 0208 144 5084


text: Patricia Freebody has been a USTA/Midwest Section volunteer for nearly 30 years. She is currently the USTA Managing Director for Player and Community Development overseeing the competitive player and recreational player departments. Freebody served as executive director of the USTA/Midwest Section from 1993-1996. In addition, she served as president of the USTA/Midwest Section from 1987-1989, served on numerous USTA committees, and was elected the first woman president of the Chicago District Tennis Association. Freebody received the USTA/Midwest Section Mel Bergman Award in 1989 and is also a recipient of the USTA/Midwest Section Stanley Malless Award. At the USTA level Freebody received the USTA Hall of Fame Merit Award. In addition to her involvement with the USTA and USTA/Midwest Section, Freebody was president of World TeamTennis from 1989-1992 and general manager of Mid-Town Tennis Club in Chicago from 1980-1988.


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I did it!
I jumped in feet first and presented my FIRST Flipgrid FlipHunt! Annnnnnd it was pretty darn AMAZING!!!
So...what is a FlipHunt, you ask? Only the most amazingly awesome way to get your students engaged in learning! A FlipHunt is a video based scavenger hunt! It is run completely through FlipGrid and your students self guide themselves through it with very little facilitation from you!
My reflection:
First, let me say, I am VERY surprised at how easy it was to set it up! Once I came up with the actual questions and created the #FlipHunt document, the only thing left to do was set up the Flipgrid and the topic and set the participants free to hunt! They LOVED it! The energy in the room was so amazing! There was dancing and smiles and a WHOLE lot of laughing! I would DEFINITELY do this again and can't wait to continue to share it's ease of use and engagement with my colleagues and our students!
Setting it up:
So, how do you set it up?
✔ First, have a clear vision for what you want your students to accomplish as a result of completing the FlipHunt.
✔ Put together your actual FlipHunt! You can either put it together yourself from scratch, or you can search the Discovery Library and find one there! This is what I did for the professional development session that I led with educators last weekend! Basically, you are putting together a document with scavenger hunt type questions for your students to discover. They sky is the limit with this. It could be an internet search for information. A getting to know you activity. An activity that has them outside looking for different things in real life. You get the picture!
The FlipHunt that I used is below and it's adapted from one that I found in the Discovery Library! It's hyperlinked with viewing rights! Feel free to make a copy of it and alter it to meet your needs! I made mine in Google Drawings but feel free to use Google Docs or whatever floats your boat!
Here it is in action! That's organized chaos that you hear! Check out the young lady in the back of the room getting ready to do an arabesque! Oh yeah....she did that!
The sound of an AMAZING #fliphunt at Southern Summit last weekend! @Flipgrid @BMcClelland24 @edtechteam pic.twitter.com/4IODvVFYU6— Natasha Rachell,EdS (@apsitnatasha) March 2, 2019
✔ Set up a Grid and your topic! Now that you have your FlipHunt all ready to go, set up a grid with the instructions for the FlipHunt and a link to the actual FlipHunt so they can access it! I actually displayed the FlipHunt on the board in the front of the classroom as well so they could see it instead of having to toggle back and forth between Flipgrid and the FlipHunt instructions.
This particular FlipHunt received 91 videos in less than 15 minutes!!! And that was with educators! Imagine if you did this in your class!
Here are a couple of ideas to get you started!
Math Idea:
What if you are a math teacher and your FlipHunt made students look for Geometry shapes in nature? Each of the questions on your Hunt could be one of the shapes (ie. quadrilateral, parallelogram, octagon, etc). As students found those, they would record a video and explain why that shape is the shape that it is!
Science Idea:
Have students create videos about the weather every day and talk about the different weather patterns that they see. If it were raining one day, they could talk about the water cycle. What if it were snowing? What effect does humidity have? These are all great conversation starters for your students!
English/Language Arts:
Have students create virtual reading logs over the summer and spring break! Students can record their feedback on the books that they've read. Perhaps the FlipHunt has different genres of reading material that you want them to read over the breaks! Once they've read that genre, they record their video and move on to the next!
Social Studies:
Let's say your kids are studying the candidates for an upcoming Presidential election. Your FlipHunt could include questions/prompts about their platforms. As students located/discovered the information about each candidate, they would record their video on the grid and discuss the candidates outlook on certain topics versus their own!
As always, if you need more information about Flipgrid in general, there is a phenomenal resource out there from Sean Fahey and Karly Moura! It's hyperlinked below so be sure to check it out!
Also, here is another resource all about FlipHunts! https://kerszi.wordpress.com/2018/07/26/whats-a-fliphunt/
Have you tried a FlipHunt with your students or staff before? How was it received? Will you try it now?


text: The booth is almost ready. Tomorrow morning we will hang garments. Thank you to the Knitters' Fair committee. They opened the doors earlier than normal for us today. In past years I would be getting home now. Then dinner and a bit of work before bed. I've had dinner and can get a full night's sleep.


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20 January 2020
A by-election countback for the Lake Nagambie Ward was held this morning at the Euroa Community Conference Centre
As a result of this, Robert (Bob) Gardner is a Councillor elect and an official swearing-in ceremony will take place on Tuesday 28 January 2020 at 10am at the Nagambie Visitor Information Centre.
Members of the public are welcome to attend.
The seat was made vacant following the resignation of following the resignation of Cr Debra Bower (formerly Swan).
A postal by-election will be held this March for Honeysuckle Creek Ward within Strathbogie Shire Council.
This follows the resignation of Cr Kate Stothers.
People are reminded that they must be correctly enrolled by 4pm Friday 24 January to be eligible to vote.
There are special enrolment categories for owners, occupiers, company nominees and people who are not Australian citizens but pay rates for an address in Honeysuckle Creek Ward.
Check your enrolment at: https://www.vec.vic.gov.au/Enrolment/
Pictured below at the by-election count are Cr John Mason, Robert (Bob) Gardner, Cr Malcolm Little, Cr Amanda McClaren (Mayor) and Phillippa Heskett (Victorian Electoral Commission).
Strathbogie Shire Council welcomes feedback from its residents and will endeavour to respond to your request as soon as possible.
If you would like to keep in contact with Council and stay up to date with the latest Council news, please register your details here.


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About 134 results.
NOTICE INVITING TENDER BIHAR STATE ROAD TRANSPORT CORPORATION (BSRTC) Parivahan Bhavan (Sultan Palace) Birchand patel Path, Patna.
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Please contact DIC Corporation for further information.
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RFHIC US Corporation RFHIC US Corporation was founded to address the need for cost ...
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Please contact DIC Corporation for further information.
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... 07 Yuzaburo Mogi Kikkoman Corporation www.
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xWDM – which is best? What's next? © 2011 JDS Uniphase Corporation | JDSU CONFIDENTIAL AND PROPRIETARY INFORMATION 2 A quick review of ...
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This Gorgeous Fort Kochi Boutique Stay Costs Only INR 600 A Night
Great For
What Makes It Awesome
A lovely home with rooms to let, Niyati Boutique Stay looks equally inviting whether you are an intrepid backpacker or a couple on vacation. Walk into a hipster space with exposed brick walls, vaulted ceilings, polished wooden floors and teak wood furniture. And to top it all off (quite literally) they have a beautiful rooftop terrace with a million dollar view. A great spot to catch the breathtaking sunrise and sunset.
Located in Fort Kochi, Niyati gives you an opportunity to soak in a bit of culture along with the sun, by visiting the many cafes and galleries that are close by. Or spend a day at the beach. Spacious, comfy, flooded with natural lighting and tidy, the rooms are kept neat with minimal furniture and plenty of space for you to set up your things. Same goes for their bathrooms which are kept spic and span, with an enclosed shower space and stocked with plenty of complimentary toiletries for your use. Some rooms also have desks or study areas, if you're looking to catch up on some work or just spend the day reading or writing.
With Budget Double Rooms and Economy Rooms, that don't come with air-conditioning (for those on a tighter budget), to Deluxe Double Rooms with a garden view and Deluxe Suites if you're looking for a more luxe set up. And yes, they have free Wi-Fi. Room tariffs start as low as INR 600, so book your room right away. They've received rave reviews for its two-person stays (and you know what that means), meaning it's a certified spot for the perfect bae-cation. With beaches nearby and many galleries and restaurants to check out, there's plenty to do in Fort Kochi with that someone special. What more? Explore it all by biking around town as you can rent bicycles from the lodge for an additional charge.


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Oscillating sprinklers are the ideal solution for watering lawns. The quiet operation and gentle watering provides soaking moisture to large areas up to 3,400 sq. ft. with 18 precision drilled brass spray nozzles. The spray is soft enough for newly seeded areas. The built-in watering gauge helps you water just the right amount. Built to last, this sprinkler is designed to add life to your lawn for seasons to come.
3400 sq. ft. Oscillating Sprinkler with Brass Nozzles
• Waters up to 3,400 sq. ft.
• Built-in watering gauge helps monitor the amount of water used
• 18 Precision nozzles ensure even watering
• Adjustable spray range lets you water just where you need it
• Backed by a Limited 7-Year Warranty. We stand behind our products.


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Meet Dominik, Daizy & Meka
*PLEASE NOTE: Only families living in Kansas are being considered at this time.*
Meka, Dominik, and Daizy are bonded siblings who love spending time together playing outside, playing sports or building things.
The oldest, Meka, likes to cheer, dance and participate in track. Her favorite class is science and she would like to be a teacher when she grows up. She is respectful to others and enjoys caring for younger children.
The middle child, Dominik, is described as being "thoughtful, helping, and having a great imagination." He is very good at math and science, and he says he wants to be an archeologist when he grows up. Dominik loves to build things and likes to "find solutions."
The youngest, Daizy, loves to play with dolls and board games. She likes art and science, and when she grows up she wants to be an artist. Many describe Daizy as "helpful, creative and caring." All three children love to laugh!
A family for these three siblings will certainly never be bored and should not be shy about facing any parenting challenge. Each child has unique needs, and it would be ideal if they had a family that was aware of and knowledgeable about available community resources to ensure each child's needs were met.
Are You Interested?
To make an inquiry about this child, please fill out our information request form. Your information will be shared with The Department of Children's Services for review as a potential adoptive parent.


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WINGS STRIVES TO CREATE NOT JUST AN EVENT BUT AN EXPERIENCE FOR YOU AND YOUR GUESTS.Contact Us
Delivering a positive attendee encounter is essential to an event's success, and achieving this goal begins early in our planning process. By managing registration and transportation, and coordinating busy schedules, we aim to eliminate any stress your guests may typically encounter. The Wings team will work closely with you to ensure that you and your guests enjoy a polished, personalized event.
The attendee experience is more than just food and décor. Regardless of the type of event, Wings provides innovative ways to captivate guests. From the start, our team will work within your budget to brainstorm ideas to customize your event in meaningful ways. Our team's precision and attentiveness will ensure that your event flows smoothly and efficiently.
With an event planned by Wings, you will be offering an exceptional attendee experience sure to make a lasting impression on your guests. Our skilled, knowledgeable, and innovative team of event engineers will give your guests the attention they deserve—delivering a maximum impact to all who participate.
Reach out to our team today to get started planning your next event!


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Photo by Kelly Sikkema on Unsplash
Coronavirus 2019 (COVID-19)
As coronavirus disease 2019 (COVID-19) epidemic continues to spread throughout the world, threatening a pandemic status, according to the Centre for Disease Control and Prevention (CDC), to-date there is still no vaccine, specific antiviral treatment, or cure.
On their website, the CDC states that the best way to prevent illness is to avoid being exposed to this virus and advises that people with COVID-19 should receive supportive care to help relieve symptoms and for severe cases, treatment should include care to support vital organ functions.
What is worth noting and sharing is that on the website is a reminder that the CDC always recommends the following everyday preventive actions to help prevent the spread of any respiratory disease:
- Avoid close contact with people who are sick.
- Avoid touching your eyes, nose, and mouth.
- Stay home when you are sick.
- Cover your cough or sneeze with a tissue, then throw the tissue in the trash.
- Clean and disinfect frequently touched objects and surfaces using a regular household cleaning spray or wipe.
- Follow CDC's recommendations for using a facemask.
- CDC does not recommend that people who are well wear a facemask to protect themselves from respiratory diseases, including COVID-19.
- Facemasks should be used by people who show symptoms of COVID-19 to help prevent the spread of the disease to others. The use of facemasks is also crucial for health workers and people who are taking care of someone in close settings, i.e., at home or in a health care facility.
- Wash your hands often with soap and water for at least 20 seconds, especially after going to the bathroom; before eating; and after blowing your nose, coughing, or sneezing.
- If soap and water are not readily available, use an alcohol-based hand sanitizer with at least 60% alcohol.
- Always wash hands with soap and water if hands are visibly dirty.
According to the Center For Disease Control and Prevention (CDC) these are simple measures you can take to help keep yourself and others healthy.
Source: https://www.cdc.gov/coronavirus/2019-ncov/about/prevention-treatment.html
God Is Our Ultimate Protection
As Believers, when it comes to dealing with coronavirus 2019 (COVID-19), it is important for us to use wisdom and follow advised precautions and preventative measures prescribed by governing authorities as best as possible.
However, as we physically do all we can to protect ourselves and our loved ones from coronavirus 2019 (COVID-19), it is even more important for us to know, recognize and understand that our ultimate protection is from God, and as such it is crucial that we fundamentally trust Him and stand on His Word above all.
Psalm 91 is a scripture that celebrates the protection of the Lord; it is a song of confidence and trust meant to build up the faith of those who trust in the Lord for His divine protection.
The Hebrew text has no title or inscription ascribing the Psalm to any particular author or specific event, making it timeless and applicable to every Believer who chooses to so position themselves in God.
Psalm 91 – NKJV
Safety of Abiding in the Presence of God
91 He who dwells in the secret place of the Most High
Shall abide under the shadow of the Almighty.
2 I will say of the Lord, "He is my refuge and my fortress;
My God, in Him I will trust."
3 Surely He shall deliver you from the snare of the [a]fowler
And from the perilous pestilence.
4 He shall cover you with His feathers,
And under His wings you shall take refuge;
His truth shall be your shield and buckler.
5 You shall not be afraid of the terror by night,
Nor of the arrow that flies by day,
6 Nor of the pestilence that walks in darkness,
Nor of the destruction that lays waste at noonday.
7 A thousand may fall at your side,
And ten thousand at your right hand;
But it shall not come near you.
8 Only with your eyes shall you look,
And see the reward of the wicked.
9 Because you have made the Lord, who is my refuge,
Even the Most High, your dwelling place,
10 No evil shall befall you,
Nor shall any plague come near your dwelling;
11 For He shall give His angels charge over you,
To keep you in all your ways.
12 In their hands they shall bear you up,
Lest you dash your foot against a stone.
13 You shall tread upon the lion and the cobra,
The young lion and the serpent you shall trample underfoot.
14 "Because he has set his love upon Me, therefore I will deliver him;
I will set him on high, because he has known My name.
15 He shall call upon Me, and I will answer him;
I will be with him in trouble;
I will deliver him and honor him.
16 With long life I will satisfy him,
And show him My salvation."
A Personal Prayer/Declaration of Psalm 91
Believing in our heart, and confessing and declaring God's Word and promises with our mouth over our life and circumstances in a personal way is a powerful scriptural and spiritual weapon.
When we do this, we are not denying the facts of our situation, but recognizing them and confronting them with the Truth and Power of the Word of God and declaring our faith and trust in Him and what He says more than in them and what they say.
In view of the coronavirus 2019 (COVID-2019), the following are personalized prayer points based on Psalm 91 that you can pray and declare by faith..
- Because I dwell in the secret place of the Most High, I abide under the shadow of the Almighty.
- I say of the Lord, "He is my refuge and my fortress, my God, in Him I trust."
- Surely he shall deliver me from the snare of the fowler and from the perilous pestilence.
- He covers me with his feathers, and under his wings I take refuge; his truth is my shield and buckler.
- I am not afraid of the terror by night, nor of the arrow that flies by day,
- Nor of the pestilence that walks in darkness, nor the plague that destroys at midday.
- A thousand may fall at my side, and ten thousand at my right hand, but it will not come near me.
- Only with my eyes will I look and see the reward of the wicked.
- Because I have made the Lord, who is my refuge, even the Most high my dwelling place;
- No evil shall befall me nor shall any plague come near my dwelling.
- For He gives His angels charge over me to keep me in all my ways.
- They bear me up in their hands lest I dash my foot against a stone.
- I will tread upon the lion and the cobra, the young lion and the serpent I shall trample underfoot.
- Because I set my love upon Him, therefore He will deliver me; He will set me on high because I have known His Name.
- I will call upon the Lord and He will answer me; He will be with me in trouble and deliver me and honor me.
- With long life He will satisfy me and show me His salvation.
When we decree and declare the Word in this manner, we must not interpret this to mean that God will protect us from all problems, for God has not promised to spare us from all difficulty; in fact in John 16:33, Jesus himself said that in this world we will have tribulation, but to be of good cheer for He has overcome the world.
Instead, by decreeing and declaring the Word in this manner from a place of faith, we activate its authority and can be confident that the Lord God Almighty is with us, has the power to cover, preserve and protect us and will be right by our side to help us through whatever danger we may encounter.
A Song Of Faith, Protection And Comfort
"Trust in the Lord with all your heart;
do not depend on your own understanding.
Seek his will in all you do,
and he will show you which path to take."
Proverbs 3:5,6 – NLT
A Final Word
If under the Old Covenant, the blood of lambs was able to cover and protect the lives of the Children of Israel from the destructive plague that passed through Egypt, under the New Covenant, HOW MUCH MORE IS THE BLOOD OF THE LAMB ABLE TO COVER AND PROTECT OUR LIVES AS CHILDREN OF GOD in this threatening pandemic of coronavirus 2019 (COVID-2019),
So in addition to all other measures, be sure to cover yourself and your household in the healing, cleansing, delivering, protecting, powerful Blood of Jesus Christ of Nazareth.
The world may be blindsided, reeling and perturbed by the ominous presence of the coronavirus 2019 (COVID-2019), but God isn't.
Trust in, rest in, hope in, and be guided by Him.
Be safe! Be strengthened! Be encouraged! Be blessed!
Copyright © 2020 by Kim Sweeting, Author of "Unwalled Words" Blog, All rights reserved.
Picture courtesy of "monfocus" – http://www.pixabay.com
Video "He That Dwelleth In The Secret Place" by David W. Morris & Hosanna Music courtesy of jmigueba


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(1820-1891) Graduated #6 in the West Point class of 1840. Rising to be one of the Union's most renowned military leaders, Sherman saw action at 1st Bull Run, Shiloh, Chickasaw Bluffs, Arkansas Post, Vicksburg, Missionary Ridge, Atlanta, the infamous March to the Sea, and the 1865 Carolina's campaign. He received the surrender of Confederate General Joseph E. Johnston's army at Greensboro, N.C., on April 26, 1865. Sherman continued in the Regular Army after the war and became a Lieutenant General on July 25, 1866, and Full General, on March 4, 1869.Signature With Rank, Place & Date: 4 3/4 x 2 7/8, in ink, W.T. Sherman, General, Washington, D.C., Dec. 15, 1887. Very nice and an extremely desirable autograph.
War Between the States
PO Box 267Lady Lake, FL 32158US
Contact Us!
Updated: Monday, July 13 2020
©1995-2020 TIAS.com. All rights reserved.


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Hybrids have become quite the thing, haven't they? In the wake of the diesel scandal of a few years ago, and with electric cars still only now becoming attractive to some motorists, hybrids are filling the gap rather nicely. It helps that, in the past few years, the hybrid tech on offer has really matured, delivering genuinely impressive real-world performance and economy, which wasn't always the case before.
Even given that maturity, Honda has decided to do things a little differently with its new CR-V Hybrid. The Japanese giant has committed to offering electric or part-electric versions of all of its cars on sale in Europe by 2025, and the CR-V Hybrid is the first one of these on sale. In fact, it's the first Honda hybrid you can buy in Ireland since the (rather underrated) Insight and CR-Z went out of production in 2014.
How is this one different, though? Well, with a 'normal' hybrid (it's very hard not to write Toyota hybrid there, as the Toyota Hybrid Synergy Drive has become so ubiquitous in the part-battery world), you get a petrol engine (usually with four cylinders, occasionally with six) running on the fuel-saving Atkinson combustion cycle. This is like conventional four-stroke 'Otto' combustion (intake, compression, ignition, exhaust), but it leaves the exhaust valves open on the compression cycle for a fraction of a second longer than normal. That's good for efficiency, but bad for power output.
So, to cover up the holes in an Atkinson engine's power delivery, you get an electric motor. The motor can spin up in super-fast time, adding torque to the engine's performance, and all of that can be driven through the gearbox (usually either a Continuously Variable Transmission, CVT, or a dual-clutch automatic) to the wheels.
Honda has binned the gearbox.
Well, actually not entirely. Technically, the CR-V Hybrid's intelligent Multi Mode Drive technology (i-MMD) does have a gearbox, but it's gear in the singular sense. Just the one gear, a fixed ratio that transmits power to the wheels from the engine and electric motor combo. Honda claims that this single-ratio system is smaller, easier to package and lighter than a CVT or a dual-clutch setup.
So, the CR-V follows hybrid conventionality up to a point. There's a petrol engine - a 2.0-litre i-VTEC unit with 145hp - and an electric motor with peak power of 184hp. Combine the two and you get a system maximum of 181hp (they don't both produce maximum power at the same time, you see), and that's good enough for a 0-100km/h time of 8.8 seconds.
The CR-V can be driven in three separate modes - Hybrid, EV Drive or Engine Drive. In EV mode, the compact 1kWh lithium-ion battery can drive the CR-V all by itself for up to around two kilometres. Not much, but then that's not the point. The point is that, most of the time, you're going to be driving in Hybrid mode. When you are, most of the driving power is actually coming from the electric motor, and the 2.0-litre petrol engine is reduced to the status of a high-end generator, feeding electric current to the battery and to the electric motor.
Only in Engine Drive Mode does a small clutch close up and allow the engine to power the wheels directly, with the electric motor effectively acting as a reduction gear so that you're not having to scream along at maximum rpm. In fact, Honda says that: "The fixed-gear format of Honda's i-MMD system has been specifically tuned for the European market to provide a linear feel through acceleration, with optimised calibration between throttle input and powertrain response, to ensure the engine sound matches the driver's expectations. In addition, the first application of Active Sound Control to CR-V also enriches the driver's experience delivering the natural sound of acceleration relative to the engine speed." That's probably a bit of a dig at rival Toyota, which still gets criticised for the way it's CVT-equipped hybrids allow their petrol engines to rev high, loud and long when accelerating.
Honda says that it's worked hard to make sure you never know what mode you're actually in, without checking the dashboard display, and that at a constant 65km/h cruise, the CR-V will be under pure electric power for around half the time. Up the speed to 100km/h and it'll be more like one-third of the time, but even so Honda claims that the CR-V Hybrid's entire system has been carefully tuned to give the best possible fuel economy on long motorway runs - historically a hybrid weak point.
The upshot of all of this is impressive efficiency. Under the updated NEDC Correlated fuel economy and emissions test, Honda quotes average fuel consumption of 5.3 litres per 100km (53mpg) and CO2 emissions of 120g/km for the front-wheel-drive CR-V Hybrid, or 5.4 litres per 100km (51.4mpg) and 126g/km of CO2 for the four-wheel-drive variant.
As you drive, you can just leave the system to its own devices or, if you prefer, you can have a go at fiddling with the settings. There's a system that allows you to choose the level of engine braking, too, which you can adjust using paddled behind the steering wheel. You can select EV mode if there's enough charge in the battery, and there's even a Sport mode that sharpens up the steering and the accelerator response a little.


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Santissimo Sudario di Nostro Signore Gesù Cristo is a 17th regional church at Via del Sudario 47 in the rione Sant'Eustachio. It is one of two small churches close together in this short street off the north-west corner of Largo di Torre Argentina, the other being San Giuliano dei Fiamminghi at number 40. Picture of the church at Wikimedia Commons here.
The dedication is to the Shroud of Turin.
This is the church in Rome for expatriates from Piedmont.
Possible predecessorEdit
The French expatriate community in Rome obtained permission in the 14th century to erect a little chapel in honour of King St Louis IX, after his canonization in 1297. (He had died horribly of dysentery during the futile Eighth Crusade in 1270.) The site of this was not that of the present church of San Luigi dei Francesi, but well to the south on the old Via Papalis very near the present Sant'Andrea della Valle. One tradition is that it was on the site of the present church of Santissimo Sudario.
The confraternity that founded the church was made up of expatriates from the Duchy of Savoy, a group which was first recorded in 1537 and which was formally erected as the Arciconfraternita della Santissima Sindone in 1597 by Pope Clement VIII. Back then, the political unit of the duchy included territory on both side of the Alps, with the name coming from what is now French Savoy and the economic base being in what is now Piedmont. Hence, the confraternity was dedicated to the famous relic of the Shroud of Turin.
The present church was built by the confraternity in 1605, the architect being Carlo di Castellamonte who was himself from Turin. The patron was the duke of Savoy at the time, Charles Emmanuel I.
17th centuryEdit
Since Savoy was then an independent state, the church had national status and the confraternity could attract useful funding. In 1660 it began a campaign of amplification and embellishment of their church, overseen by Carlo Rainaldi. This involved a remodelling of the interior and a new façade, but the confraternity seems not to have been satisfied and so Rainaldi added the present façade by 1690 -he died in the following year. He also designed a new high altar, but the interior restoration was supervised by Pier Francesco Garoli. This was finished by 1687, to designs by Antonio Gherardi. The chief craftsman was Pietro Mentinovese.
18th century Edit
In 1720 the duchy became the Kingdom of Sardinia, and Duke Victor Amadeus II the first king. This was a rather complicated process, brought about through Savoy being on the winning side in the War of the Spanish Succession (the island of Sardinia had been a Spanish possession). In the Treaty of Utrecht 1713 that ended the war, the duke was rewarded with Sicily but was forced to exchange that island for Sardinia through diplomatic pressure seven years later.
The reason why Sardinia gave the title to the kingdom instead of Savoy (the power base) was that the latter was still counted as part of the Holy Roman Empire and so the emperor had to give permission to turn it into a kingdom. It was easier not to ask. However, the capital was Turin not Cagliari.
In 1750 the interior of the church was restored, but on the occupation of Rome by the French in 1798 the confraternity was suppressed and the church shut down.
19th centuryEdit
In 1801, King Charles Emmanuel IV had the church re-consecrated while living at Rome as the private guest of the Colonna family. He had been forced to relinquish his mainland possessions to the French in 1798. However, he decided to abdicate altogether in the following year and after that the church was desecrated definitively and turned into a magazine (gunpowder store).
In 1805, after Piedmont was annexed to the French Empire, the church was briefly put under the administration of the other French churches in Rome. In 1809, after the downfall of Napoleon, it was returned to its re-founded confraternity but its status as the national church of the Kingdom of Sardinia was only restored in 1837 with the king as patron.
In 1861, after the Second Italian War of Independence, the Kingdom of Sardinia became the Kingdom of Italy under King Victor Emmanuel II. This was because it had acquired control of the entire peninsula, except for the Veneto under the Austrians and Latium with Rome which was still ruled by Pope Pius IX under a French guarantee.
There was a restoration of the church from 1869 to 1871, ordered by the government of the Kingdom and supervised by Giacomo Monaldi.
Palatine ChapelEdit
The restoration was finished in the year after the Kingdom of Italy finally conquered Rome. The pope refused to accept the reality of the situation, and declared himself the Prisoner in the Vatican (although he was not coerced by the Italian government, so the title was tendentious). The government requisitioned the formal Papal residence of the Quirinal Palace as the new royal palace, and in response the pope put the palace chapel -the Cappella Paolina- under interdict. This meant that no Mass could be celebrated there.
However the church of Santissimo Sudario was already under the patronage of the king, and so became the royal Cappella Palatina or palace chapel in Rome. It was, de facto, the church of the royal court until the Lateran pacts of 1929 established relations between the papacy and the Italian government.
In 1946, the Republic of Italy was founded and the monarchy abolished. The property of the Italian crown passed to the Italian presidency, and so did the church. Because the 1929 concordat was not revised, the church remained a cappella palatina but only functioned as a regional church for the Piedmontese in Rome.
The concordat between Italy and the Holy See was finally revised in 1984. One revision was to abolish the category of cappella palatina, and the presidency relinquished responsibility for the church.
The church is now the responsibility of the Military Ordinariate in Italy, although as to why is not clear. Recently it has become a centre for Exposition of the Blessed Sacrament.
Layout and fabricEditThe church is enclosed by domestic buildings on all sides except the street. It has a simple rectangular plan, with two external side chapels.
It is difficult to view the façade properly owing to the narrowness of the street. It has two storeys, rendered in a cream colour.The first storey has three zones, the central one slightly brought forward. The two side zones do not front the church, but ancillary accommodation which the doorways here access. The central doorway, the one into the church, has a raised segmental pediment but no other decoration. It is flanked by a pair of double rectangular Corinthian pilasters. The two side zones each have a smaller door, above which in turn is a Baruque transom window with a floating gable cornice, and then a rectangular window. The outer corners of the side zones are occupied by another pair of pilasters. These six pilasters support a wide entablature dividing the storeys, but unusually a pair of horizontally rectangular windows is cut into this above the pair of main windows in the side zones. The ends of the entablature bear the two halves of a broken and well-separated segmental pediment.
The second storey, narrower than the first (squeezed by domestic buildings on either side) has a double pair of Ionic pilasters either side of a very large rectangular window and supporting a pediment missing its cornice above the window and with its central angle set back. The window has a cornice on posts supported by corbels. The pediment contains the arms of the House of Savoy in relief, tilted so as to be visible from the street.
Layout and fabricEdit
The simple rectangular interior has five bays, the central third bay being much deeper than the others. Two side chapels lead off this third bay, and the last two bays are occupied by the sanctuary. There is a very shallow sixth bay occupied by the altar aedicule.
The interior decoration of nave and sanctuary is of one design -there is no triumphal arch. The bays are separated by Composite pilasters revetted in red Sicilian jasper, with the walls in between in what looks like green marble with yellow strip inlays. The pilasters support an entablature which runs round the interior and has a green frieze.
The second and fourth bays of the nave each have a pair of side doors with door-cases in a brown-veined marble, above which are panels in a purplish-red stone.
The semi-circular barrel-vaulted ceiling covers both nave and sanctuary. The deep central nave bay has a fresco depicting The Apotheosis of the Saints of the House of Savoy, which echoes the main altarpiece in the sanctuary (see below). As well as BB Louise, Amadeus and Margaret, the work depicts Bl Boniface of Savoy and Duke Humbert III who was never beatified despite attempts.
The two flanking bays on each side are combined in the vault to have octagonal frescoes each with an allegorical virtue (they look like Charity and Religion). The panels to the sides of the main fresco have more allegories (Prudence, Justice, Temperance and Fortitude) with the Savoyard banner (white cross on a red background). The fresco panels are bordered by wide ribs with fern-leaf scrolls in god on red.
These frescoes are by Cesare Maccari 1873. The octagonal fresco panels do not have flanking frescoes, but instead a pair of windows is inserted on each side in round-headed lunettes.
There is a wooden floating organ gallery over the entrance.
The far wall of the sanctuary is taken up by the altar aedicule in a way which integrates it into the overall design. The interior entablature is made to step back twice on each side, the angles of the steps pointing outwards slightly. These angles are supported by four Ionic columns in red marble, hence forming the aedicule. The capitals of these have incurved volutes and little winged putto's heads. Above the entablature is a white stucco relief of God the Father by Antonio Raggi, which fronts its own little aedicule with a segmental pediment and pink pilasters. Angels and putti are in attendance.
Between the entablature and sculpture is displayed a copy of the Holy Shroud, an object of devotion, which was commissioned by "Maria Francesca di Savoia". According to the Ordinariate, it was donated when the confraternity received its charter at the end of the 16th century. This is all very confused, and it seems that the copy was actually commissioned by the Venerable Maria Apollonia di Savoia in 1650. She gave it to Cardinal Giovanni Battista Pallotta, who passed it on to Pope Urban VIII who in turn gave it to the church.
The altarpiece is a Deposition by Antonio Gherardi, with Savoyard saints shown in attendance on the dead Christ. The moment is shown when the Shroud allegedly received the miraculous image of Christ. The saints and beati concerned are (right to left) St Maximus of Turin, Bl Margaret of Savoy, St Maurice, Bl Louise of Savoy and Bl Amadeus IX of Savoy.
The two side wall frescoes show scenes from the lives of SS Francis de Sales and Anselm -both born in areas once ruled by the Duchy of Savoy, although on either side of the Alps. The actual scenes are St Anselm with Pope Urban II at the Council of Bari, and St Francis of Sales with Bl John Juvenal Ancina of Savoy.
Side chapelsEdit
The side chapels are arched niches, flanked by pairs of semi-columns in the same style as the pilasters.
The left hand side chapel is dedicated to St Francis de Sales, with an altarpiece showing his apotheosis by Carlo Cesi. This is just hung on the wall without any aedicule or elaborate frame. The saint was bishop of Geneva, but could never visit the Calvinist-ruled city and so exercised his ministry in Savoy.
The right hand side chapel is dedicated to one of the ruling dukes who was beatified, Amadeus IX of Savoy. The altarpiece showing him having a vision of Our Lady is by Giovanni Domenico Cerrini, and is in an elaborate Baroque frame with an ogee cornice.
The vault in this chapel has an attractive little fresco of putti in heaven, within a stucco flower garland. It is easily overlooked.
According to the Ordinariate (2017), the church is open daily EXCEPT Wednesdays:
10:00 to 12:00, 16:00 to 19:00 (after Mass).
The feast of the Holy Shroud is on 4 May.
The liturgical activity of the church was recently focused on Adoration of the Blessed Sacrament. The Ordinariate gave the following information in 2017:
Exposition takes place after the church is open at 10:00, and continues until 12:00 when the Angelus is recited and the church closes for lunch. Meanwhile, at 11:00 the Rosary is recited.
In the afternoon, Exposition occurs again after opening at 16:00, and continues until 18:00. At 17:30, the Chaplet of Divine Mercy is recited, followed by Vespers.
Mass is celebrated at 18:30, after which the church closes.
The prayer intentions of the Adoration are for peace, the public prayer intentions of the Church and for familiies.
External linksEdit
"DE Alvariis" gallery on Flickr


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Wealth Transfer Lie Exposed: Bill Winston - Part 1
If you believe these lies...Please Read: 1 Timothy 6:6-8, Philippians 4:11-13, Hebrews 13:5, Luke 12:15, 2 Peter 2:1-4, Ephesians 5:3.....
Kenneth Copeland clone Bill "Big Bucks" Winston, has been preaching this prosperity "end times" wealth transfer lie for decades, along with Creflo Dollar, Benny Hinn, Kenneth Hagin Jr. & Sr., and many others. It is amazing how people will believe these lies even though their financial situation worsens and the economic situation of most of their family worsens. This series highlights the WEALTH GAP among the population in the U.S. as well as the CORRUPTION in the FINANCIAL/BANKING Sector. There is a direct correlation between the corruption & the gap in wealth. There is also a correlation between the WEALTH GAP and the "prosperity gospel". You won't hear Winston deal with the REAL issues, you will only hear him preach COVETOUSNESS and GREED!!!! This type of preaching is in direct opposition to everything that The Lord Jesus Christ stands for and against the very essence of The Word of God itself!!! The finanical situation for most people in America will continue to deteriorate...due to injustice in the Financial system, high taxes, aging workforce, technological advancements, emerging markets like China/India, increase in obesity & unhealthy lifestyles, and outright defiance to Our LORD!!! God will judge these false teachers!!!!!
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Changes in circumstances happen every day to people from all walks of life, and often moving homes or offices is the result of these changes. Regardless of where you need to go and how much time you have to move, we, as the experts in Canberra to Melbourne removals will make it happen.
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It's cheaper as the weekend surcharges may apply in weekend. A weekday move's main advantage and the reason many people choose to move during the week is the cost benefits.
It is important to realize that Canberra movers have less availability on the weekends, so weekend surcharges may apply.
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Dawson Moving & Storage is a proud member of AFRA https://afra.com.au/find-a-removalist-2/dawson-moving-storage-victoria/ as this shows our high quality and dedication to providing the best possible service to our customers.
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The cost of your moving varies depending on the amount of furniture being moved and the services required. As a guide a basic 1 bed room apartment ranges from $800.00 – $1800.00. A standard 3 bedroom home ranges from $3500.00 – $5000.00.
Yes we have storage facilities available in almost all locations, including Canberra.
Moving home is considered by insurance companies as one of the highest risk times for damage to occur to furniture. Whilst the ratio of damage is quite low and Dawson Moving & Storage take specific steps to protect and transport furniture safely, there are some things that are out of our control and the risk of damage occurring is there.
We always recommend to our customers they strongly consider taking out insurance. We have multiple policies available.
Yes we can move plants to some locations interstate, some states (W.A. & Tasmania) do have strict quarantine laws prohibiting the transportation of plant material. We would be happy to provide you with a list of restrictions.
In some locations we engage other AFRA accredited Removal Companies that we have partnered with to perform services for our customers.
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