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Respiratory Syncytial Virus F Envelope Protein Associates with Lipid Rafts without a Requirement for Other Virus Proteins
Elisa H. Fleming, Andrey A. Kolokoltsov, Robert A. Davey, Joan E. Nichols, Norbert J. Roberts, Jr.
Elisa H. Fleming
Andrey A. Kolokoltsov
Robert A. Davey
Joan E. Nichols
Norbert J. Roberts
Like many enveloped viruses, human respiratory syncytial virus (RSV) assembles at and buds from lipid rafts. Translocation of the envelope proteins to these membrane subdomains is essential for production of infectious virus, but the targeting mechanism is poorly understood and it is not known if other virus proteins are required. Here we demonstrate that F protein of RSV intrinsically targets to lipid rafts without a requirement for any other virus protein, including the SH and G envelope proteins. Recombinant virus deficient in SH and G but retaining F protein expression was used to demonstrate that F protein still localized in rafts in both A549 and HEp-2 cells. Expression of a recombinant F gene by use of plasmid vectors demonstrated that F contains its own targeting domain and localized to rafts in the absence of other virus proteins. The domain responsible for translocation was then mapped. Unlike most other virus envelope proteins, F is unusual since the target signal is not contained within the cytoplasmic domain nor did it involve fatty acid modified residues. Furthermore, exchange of the transmembrane domain with that of the vesicular stomatitis virus G protein, a nonraft protein, did not alter F protein raft localization. Taken together, these data suggest that domains present in the extracellular portion of the protein are responsible for lipid raft targeting of the RSV F protein.
An essential step in replication for many viruses is assembly at and budding from the cell membrane, frequently using specialized domains termed lipid rafts. Lipid rafts are liquid-ordered, plasma membrane microdomains that are enriched in sphingolipids and cholesterol (44). At low temperatures, lipid rafts resist extraction with nonionic detergents and can be isolated on density gradients as a low-density fraction. Based on these criteria, lipid rafts are also termed detergent-resistant membranes (DRMs) (5, 6). The saturated hydrocarbons of the lipid raft sphingolipids and phospholipids create a tightly packed subdomain within the plasma membrane. Rafts may allow compartmentalization of the membrane, and raft-localized proteins that demonstrate an energetically favorable association with rafts cluster within these microdomains to create specialized regions for protein-protein interactions (44). This sequestration of membrane proteins is thought to enhance protein-protein interactions needed for diverse cell functions, including receptor ligand-mediated signaling and endocytosis (5, 15, 20).
Many enveloped viruses are thought to exploit membrane microdomains for assembly by providing a means to bring together virus structural proteins on the cell surface at the site of budding. Human immunodeficiency virus (HIV), measles virus, and influenza virus all appear to use rafts or raft-like membrane domains (28, 35, 42). For each virus, at least one of the envelope proteins demonstrates an intrinsic ability to associate with lipid rafts, potentially providing a nucleation event for assembly of new virus (41, 45, 47). Human respiratory syncytial virus (RSV) has also been shown to assemble at plasma membrane domains that are enriched in raft proteins, such as caveolin-1 (Cav-1) and GM1, indicating that RSV structural proteins are also able to target rafts (7, 8).
RSV has three envelope proteins referred to as the small hydrophobic protein (SH), G protein, and F protein. The G protein has been shown to facilitate binding of RSV to the target cell, but the function of SH remains unclear. F protein mediates virus-to-cell and cell-to-cell fusion, creating the characteristic syncytia for which the virus is named (14, 17, 22, 25, 46).
Unlike with other paramyxoviruses, the F protein is sufficient for cell-to-cell transmission since virus deficient in G and SH remains infectious (22, 37, 46). During infection, all three proteins have been shown to localize at rafts (18, 30, 40). F protein specifically has been found to localize in raft-like domains of infected cells and was found to be required for localization of M protein to DRMs (18). However, the mechanism whereby F targets to rafts has not been determined.
Since F protein is an integral factor in the propagation of infectious virus, it is important to determine if F protein can associate with lipid rafts or DRMs without the need for other viral proteins. The purpose of these studies was to evaluate whether F can directly target rafts and, if so, to identify the targeting domain within the protein. By use of both a recombinant virus expressing F in the absence of G and SH and an F protein expressed from a synthetic construct, the results demonstrate that F localizes to lipid rafts independently of other RSV proteins. Furthermore, the results show that the targeting domain is unusual in that it resides in the extracellular domain of the F protein.
Cells and virus.A549 cells (CCL-185; American Type Culture Collection, Manassas, VA) were grown in Ham's F-12 supplemented with 2% sodium bicarbonate, 1% penicillin-streptomycin, and 10% fetal calf serum. HEp-2 cells (CCL-23; ATCC) were maintained in Eagle's minimal essential medium supplemented with 1% penicillin-streptomycin and 10% fetal calf serum. RSV Long strain was maintained as described previously (10). rgRSV-SGF and rgRSV-F viruses were provided by Mark Peeples, Ohio State University, and were described previously (46). Both viruses encode the green fluorescent protein (GFP) at the 3′ end of the genome, and GFP expression was used to monitor growth and evaluate virus titers by fluorescence microscopy. rgRSV-F was derived from the cDNA of rgRSV-SGF by deletion of the G and SH genes. Virus was grown, virus titers were determined, and analysis by reverse transcription-PCR was done as described elsewhere (46). A549 and HEp-2 cells were infected at a multiplicity of infection of 1 and analyzed at 18 h postinfection.
Synthesis of RSV F gene, expression vector construction, and mutagenesis.The native coding sequence of RSV was codon optimized for ectopic expression in human cells as described elsewhere (9). At the same time, potential splice sites were identified using the SpliceView algorithm (SpliceView, http://l25.itba.mi.cnr.it/∼webgene/wwwspliceview.html) and eliminated to produce an optimized coding sequence. Overlapping oligonucleotides (60 nucleotides each), corresponding to the optimized sequence of both sense and cDNA strands and terminal restriction enzyme sites, were purchased from Midland Company, Midland, TX (sequences are available upon request). The synthetic F gene was assembled as two fragments flanked by BamHI and EcoRI and EcoRI and NotI, respectively (see Fig. 2A). PCR-mediated assembly of both segments was performed under the following conditions: 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min for 30 cycles, using all oligonucleotides except for those at the termini. Final products were isolated by a second round of PCR using primers complementary to the 5′ and 3′ ends that included the restriction enzyme sites used for insertion into the expression vector. Conditions for the second PCR were as follows: 90°C for 30 s, 55°C for 30 s, and 72°C for 1 min for 30 cycles. The two fragments were enzymatically digested with their respective restriction enzymes and sequentially cloned into pCDNA3 (Invitrogen, Carlsbad, CA). The resultant inserts were sequenced (UTMB Protein Chemistry Core), and errors were corrected by oligonucleotide-directed mutagenesis using a Quikchange kit (Stratagene, La Jolla, CA). The final pCDNA3 construct encoding full-length F was designated pCDNA3-SynF. All DNA constructs were purified using an Endofree plasmid maxi kit (QIAGEN, Valencia, CA).
F protein mutagenesis.C-terminal truncations of F were generated by PCR using oligonucleotides that incorporated stop codons at the 3′ end of the synthetic F gene. The primers used were 5′ GAGGCGGCCGCTCATTAGCAATACAGCAGCAGGCCGAC 3′ for Δ551-574 (which had amino acids 551 to 574 deleted), 5′ GAGGCGGCCGCTCATTAGCTGCGGGCCTTGCAATACAG 3′ for Δ555-574, and 5′ GAGGCGGCCGCTCATTAATACAGCAGCAGGCCGAC 3′ for Δ550-574. The forward primer was the same for all F mutations and corresponded to the 5′ end of the SynF gene. AccuTaq polymerase (Sigma-Aldrich, St. Louis, MO) was used, and conditions were as follows: 95°C for 30 s, 55°C for 30 s, and 72°C for 2 min for 35 cycles. The resulting fragment was digested with EcoRI and NotI endonucleases and inserted into pCDNA3. All constructs were sequenced and changes confirmed. For single amino acid changes, necessary mutations were incorporated into oligonucleotides and introduced into the expression construct using a Quikchange kit. PCR conditions were as follows: 95°C for 30 s, 55°C for 1 min, and 68°C for 2 min for 12 cycles. The mutations were confirmed by sequencing each construct. For the substitution of the vesicular stomatitis virus G protein (VSV G) transmembrane (TM) domain and cytoplasmic tail, an XbaI restriction endonuclease site was introduced at homologous positions in VSV G and F coding sequences (amino acids 561 and 527, respectively). DNA fragments encoding the RSV F ectodomain and the VSV G transmembrane and cytoplasmic tails were then joined by sequential insertion into the expression vector.
Transient transfection of expression constructs.Transient transfection of plasmid DNA into epithelial cell lines A549 and HEp-2 was accomplished by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, with adjustments. Cells were grown to 90% confluence. Transfections were carried out in OptiMEM (Invitrogen) with a DNA-to-Lipofectamine 2000 reagent ratio of 1:2. Resultant DNA complexes were incubated with cells for 4 h at 37°C prior to replacing media with antibiotic-free medium.
Fluorescence confocal microscopy.Adherent cells were grown on four-well culture slides (BD Falcon, California). All cell culture samples were labeled directly on slides. For indirect labeling of RSV F, the primary antibody was a mouse anti-RSV F antibody (MCA490; Serotec, Raleigh, NC) and the secondary antibody was an anti-mouse immunoglobulin G conjugated to fluorescein isothiocyanate (FITC) (STAR9B; Serotec) or Alexa Fluor 350 (Molecular Probes). For cholera toxin labeling, cells were labeled at 4°C with an Alexa Fluor 594-labeled cholera toxin B subunit (CTxB) (Molecular Probes). Colabeling for RSV F protein and transferrin receptor (TfR) was performed similarly to the described staining method involving CTxB, with replacement of CTxB by Alexa Fluor 594-labeled transferrin (Tfn) (Molecular Probes). Samples were fixed with 2% paraformaldehyde. DAPI (4′,6′-diamidino-2-phenylindole) mounting medium (Molecular Probes) was used to counterstain nuclear DNA where indicated. For confocal microscope analysis, a Zeiss LSM 510 UV Meta laser scanning confocal microscope was used at ×63 magnification. Images were analyzed using Zeiss imaging software.
Colocalization analysis.Confocal microscope images were analyzed for colocalization of RSV proteins, cholera toxin subunit B, or transferrin by use of ImageJ (W. S. Rasband, National Institutes of Health, Bethesda, Md., 1997-2004, http://rsb.info.nih.gov/ij/). Acquired images were analyzed as individual green and red channels corresponding to anti-RSV F protein staining and CTxB or Tfn staining, respectively. Background was corrected using the background subtraction function on ImageJ. Regions of interest were selected based on patching patterns in both red and green channels. Areas that contained staining artifacts, as indicated by intense regions of fluorescence in both channels, were excluded from analysis. Manders overlap coefficient, a measure of overlap of the red and green signals, was generated using the Manders coefficient plug-in (27) which is included in the WCIF version of ImageJ (http://www.uhnresearch.ca/facilities/wcif/imagej/), with regions of interest for analysis set for the green channel. Coefficients for three separate images were averaged, and a paired Student t test value was calculated using Excel (Microsoft, Redmond, WA) to test overlap of F protein staining patterns with that of CTxB or Tfn staining.
Flow cytometry.HEp-2 cells were infected with RSV Long strain at a multiplicity of infection of 1 and harvested after 24 h of infection. Cells transfected with the synthetic RSV F expression construct or vector control were fixed and labeled, as described above for F expression, by use of anti-RSV F primary antibody and FITC-conjugated secondary antibody. Control samples were labeled with the secondary antibody alone. Twenty thousand events were analyzed for each sample on a BD FACSort instrument using CellQuest software.
Separation of DRMs.Transfected or infected cells were harvested by scraping and pelleting. Samples were disrupted by Dounce homogenization in TNE buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA) containing serine and cysteine protease inhibitor cocktail (Roche, Indianapolis, IN) at 4°C. Nuclei were pelleted by spinning for 10 min at 1,000 × g, and Triton X-100 was added to postnuclear supernatants to give a final concentration of 0.25% (vol/vol). After 30 min on ice, homogenates were mixed with 60% Optiprep (Sigma-Aldrich) to give a 40% (vol/vol) solution (1.2 ml total). This was then overlaid with 2.4 ml of a 30% Optiprep solution (diluted with TNE). TNE was overlaid on the top of the gradient, and the tube was spun in an SW60 rotor at 45,000 rpm for 4 h at 4°C. After centrifugation, eight equal fractions were manually collected from the top of the gradient. Protein was precipitated using a methanol-chloroform extraction of 150 μl of each fraction (50), and each fraction was analyzed by Western blotting.
Western blot analysis.Cell samples were lysed in sample loading buffer (50 mM Tris-HCl, pH 6.8, 1% sodium dodecyl sulfate, 10% glycerol, and 0.05% bromophenol blue) supplemented with 2.5% β-mercaptoethanol and protease inhibitor cocktail (Roche) and boiled for 5 min. Proteins were separated on a 4 to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Novex; Invitrogen) and transferred onto a nitrocellulose membrane. After blocking in 5% skim milk in Tris-buffered saline with Tween 20 (0.02 M Tris, 0.5 M NaCl, pH 7.5, 0.05% Tween 20), membranes were probed with a rabbit anti-RSV F1 antibody, raised against residues 255 to 275 and used at 1:500 (provided by Jose Melero, Instituto de Salud Carlos II, Madrid, Spain). Membranes were then probed with a secondary anti-rabbit antibody conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA). Precipitated DRM fractions were also analyzed for TfR (non-DRM resident) (16) and Cav-1 (DRM resident) (24) with antibodies from Santa Cruz Biotechnology. For analysis of the RSV F/VSV G hybrid protein, blots were probed with rabbit antiserum reactive to the cytoplasmic tail domain (CTD) of VSV G (provided by Michael Whitt, University of Alabama—Birmingham).
RSV G and SH are not required for F to localize to lipid rafts.Previous studies have demonstrated that RSV envelope proteins SH, G, and F are able to associate with lipid raft microdomains of the plasma membrane of infected cells (8, 30, 40). Using an RSV recombinant virus (46) deficient in G and SH (rgRSV-F), we were able to demonstrate that the F protein associates directly with lipid rafts and does not require G or SH for this localization. This was established using two independent methods. First, rgRSV-F-infected A549 cells were labeled for GM1 and F protein colocalization. GM1 is a glycosphingolipid that localizes to plasma membrane lipid rafts and is used as a raft marker (38). GM1 can be specifically labeled by binding of fluorescently conjugated CTxB (33). A549 cells grown on culture slides were infected with rgRSV-SGF or rgRSV-F for 18 h to allow F expression and virus budding. Cells were labeled simultaneously at 4°C with a fluorescently labeled CTxB and an antibody specific for RSV F protein. Raft domains in cells are too small to visualize by standard light microscopy and normally give a diffuse membrane staining pattern. To promote visualization of raft-associated F proteins, cells were labeled with a fluorescently conjugated secondary antibody specific for mouse immunoglobulin G, thus cross-linking the membrane-associated F proteins and rafts in which they were embedded. Similar cross-linking methods have been used in other studies to promote visualization of raft domains (13, 16). We confirmed previous reports (8, 30) that the F protein from wild-type virus colocalized with GM1 (Fig. 1A) and was associated with viral filaments. In the current experiments, the F protein from rgRSV-F-infected cells also demonstrated such colocalization and association with the filaments (Fig. 1B).
Colocalization of F protein with a lipid raft marker in the absence of G and SH. Cells were infected with rgRSV-SGF (A) or rgRSV-F (B), viruses engineered to express GFP as a marker of infection. rgRSV-F has the G and SH coding sequences removed and does not express the G and SH proteins. Infected A549 cells grown on culture slides were stained for surface RSV F protein and GM1 at 18 h postinfection. For staining, cells were incubated with a primary antibody specific for RSV F protein and CTxB (binds to GM1) on ice, followed by incubation with secondary antibody conjugated to FITC that binds the primary F antibody for indirect staining of F protein. Fixed cells were then analyzed by confocal microscopy. For panel B, two representative fields are shown (top and bottom rows). Colocalization is shown in the overlay panels and appears pink, and the portions indicated by the boxes are magnified approximately ×5 in the insets. The F protein was not detected in samples of uninfected A549 cells (not shown). (C) RSV F protein expression from both A549 and HEp-2 cells infected with rgRSV-F was analyzed for detergent insolubility as a measure of lipid raft association. Cells were treated with 0.25% Triton X-100 at 4°C and subsequently separated on a density gradient. The lipid raft domains are shown in fractions 1 and 2 as determined by Cav-1 localization. Lanes 6, 7, and 8, are the nonraft or detergent-soluble fractions as determined by TfR localization.
In a second approach to detect F protein raft association, detergent solubility tests were performed with extracts from RSV-infected A549 and HEp-2 cells. Lipid rafts resist extraction with Triton X-100 at low temperatures, and raft-associated proteins, such as Cav-1, remain associated with the low-density complex (6, 24). We tested whether F protein could be detected in DRM-containing fractions from both A549 and HEp-2 cells. After detergent extraction and density gradient fractionation, each fraction was analyzed by Western blotting and probed with both an antibody specific for amino acids 255 to 275 of the F1 subunit and antibodies for Cav-1 and a nonraft marker, TfR. F was consistently found to colocalize with Cav-1 in the DRM fraction (Fig. 1C). Some F protein was also detected in soluble fractions, which has been observed for F expressed during infection by wild-type virus (30) as well as for other well-characterized raft-associated proteins, including virus envelope proteins (12, 52).
Design of a synthetically engineered F gene and characterization of its protein expression.To further investigate the ability of F protein to localize to lipid rafts, we tested the ability of F protein to target rafts in the absence of all other viral proteins. F expression is usually studied using a vaccinia system (3, 17), which usually results in abnormally high expression levels. Plasmid-based F expression vectors were designed to overcome issues of excessive expression altering F localization as well as to eliminate any potential influence of vaccinia proteins on localization. Unfortunately, cDNA constructs of F corresponding to the virus coding sequence were very poorly expressed in mammalian cells. This may have been due to poor codon utilization or RNA secondary structure, as seen for HIV envelope protein genes (11). Gene expression may also be suboptimal since wild-type RSV replicates in the cytoplasm and the genome of the virus could contain cryptic RNA splice sites. To achieve optimal expression of F protein, the RSV F DNA sequence was optimized for high-level expression by eliminating potential splice sites and optimizing codon usage while retaining the native amino acid sequence (9). Oligonucleotide-mediated gene synthesis was used, and a synthetic RSV F gene (encoding SynF) was cloned into pCDNA3. RSV F is made as a precursor termed F0 and is cleaved at two sites to yield F2 and F1 subunits (Fig. 2A). Expression of F was first evaluated by Western blotting for detection of the F1 subunit using an anti-F1 antibody specific for amino acids 255 to 275 (Fig. 2B). F1 protein was detected both in cell lysates of the virus-infected cells and in those transfected with SynF. To determine if F was expressed on the cell surface, SynF-transfected HEp-2 cells were labeled using an antibody specific for RSV F without permeabilization of cells. Flow cytometry evaluation of expression levels on cells demonstrated F protein expression levels that were similar to those attained during RSV infection (Fig. 2C). Subcellular localization of F protein was determined by confocal microscopy for both RSV-infected and SynF-expressing cells, which demonstrated similar staining patterns and levels of expression (Fig. 2D). Optical slices (0.2 μm) of cells also demonstrated labeling on the cell surface (not shown). Similar expression patterns were observed for SynF-transfected A549 cells and 293 fibroblasts (Fig. 3 and data not shown). These data suggested that SynF was made and processed similarly to the F protein produced during virus infection of cells.
Synthetic F construct design and expression. A synthetic F gene was constructed for optimal expression in mammalian cells while preserving the amino acid sequence. (A) The gene was constructed in two sections and is flanked with restriction enzyme sites and contains a cytomegalovirus promoter indicated by the arrow. S-S indicates disulfide bond. (B) The synthetic F gene product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting of HEp-2 cell lysates either infected with wild-type RSV (RSV) or transfected with the vector control (Vec), pCDNA3, or SynF gene. The F0 (uncleaved precursor) and F1 (cleaved) forms of F were detected using an anti-F1 antibody. Cell surface expression was demonstrated in HEp-2 cells by labeling cells on ice without permeabilization with an anti-RSV F antibody and analyzing by both (C) flow cytometry and (D) confocal microscopy. SynF-transfected cells (green line) were compared to RSV-infected cells (black line), and pCDNA3-transfected control cells (solid peak) were labeled with a primary antibody specific for RSV F and analyzed by flow cytometry. For each sample, 20,000 events were acquired. Surface expression of F was also seen in SynF-transfected cells that were labeled on ice with an anti-RSV F antibody (green) and analyzed by confocal microscopy. Cells were also labeled with DAPI (blue) for nuclear labeling. Images shown are optical slices from one field of cells.
RSV F colocalizes with GM1 more than with transferrin receptor in the absence of other viral proteins. Cells were transfected with the SynF gene and labeled on ice with an anti-RSV F antibody (green) and either CTxB or Tfn (red). Samples were analyzed by confocal microscopy, with colocalization shown in yellow in the overlay panel. The portions indicated by the boxes are magnified approximately ×5 in the insets.
Localization of F protein in lipid rafts.To determine if SynF protein colocalized with lipid rafts, transfected cells were stained for F protein and GM1 by use of F-specific antibody and fluorescently labeled CTxB, respectively, by the same methods described for Fig. 1. In these experiments, the F protein was detected at the cell surface and colocalized with GM1 (Fig. 3). In contrast, staining for TfR, a non-raft-associated protein, using fluorescently labeled Tfn showed that SynF was largely excluded from sites that stained for TfR (Fig. 3 and insets). SynF also localized to insoluble fractions after Triton X-100 extraction, as did Cav-1 (see Fig. 5), thus confirming the microscopy analysis.
RSV F palmitoylation, transmembrane domain, and cytoplasmic tail are dispensable for lipid raft association.Lipid rafts are composed of sphingolipids and cholesterol, which allow a more ordered lipid phase than the surrounding membrane (43). The long acyl chains of sphingolipids account for this property (32). One mechanism to promote raft association is posttranslational modification with addition of palmitate or myristic acid groups to cysteine residues (32). HIV and murine leukemia virus are two examples for which palmitoylation of envelope protein TM cysteines are important for raft association (4, 26). RSV F contains one reported palmitoylation modification of Cys550 (Fig. 4A), which is predicted to be located in the TM domain adjacent to the cytoplasmic side of the plasma membrane (2). To determine if this modified residue was important for raft association, we mutated the Cys residue of SynF to serine (C550S) and determined raft association of the modified protein (41). Surprisingly, the F protein lacking this palmitoylation site maintained its ability to colocalize with GM1 (Fig. 4B) and DRMs (Fig. 5).
RSV F protein membrane-proximal palmitoylation, TM domain, and CTD are not required for lipid raft association. (A) SynF sequences for TM domain and CTD mutants are listed. The final construct is a chimeric protein between the VSV G TM domain and CTD and the ectodomain of F protein. *, palmitoylation site. (B) Cells were transfected with the RSV F protein mutants, and colocalization of GM1 and the altered F proteins was analyzed by confocal microscopy. Labeling was done as described in the legend for Fig. 3, and cells were analyzed by confocal microscopy. The portions indicated by the boxes are magnified approximately ×5 in the insets.
DRM association of F protein with mutations to the palmitoylation site, TM domain, and CTD. Detergent solubility of RSV F protein was examined in cells transfected with the various F mutation constructs or with VSV G. Postnuclear supernatants were treated with 0.25% Triton X-100 at 4°C and separated on a density gradient. Subsequent blots of the fractions were probed with either an anti-RSV F antibody or an antibody specific for the CTD of VSV G (anti-VSV G), as specified to the right of the respective blots. The SynF mutant is labeled to the left of the blot. Also shown are representative blots probed with anti-Cav-1 and anti-TfR.
In addition to palmitoylation, subdomains of the envelope proteins can facilitate targeting to rafts. For Newcastle disease virus fusion protein and influenza virus spike glycoproteins, DRM association mapped to the cytoplasmic tail domain (12, 52). We made a series of C-terminal truncations to test if the RSV F CTD contained a raft-targeting motif (Fig. 4A). Each C-terminally truncated F protein retained its ability both to colocalize with GM1 (Fig. 4B) and to associate with DRM fractions (Fig. 5). Furthermore, a mutated F protein lacking both the CTD and the palmitoylated C550 showed such localization.
The retention of lipid raft association in the truncated F proteins indicated that the raft-targeting region must be present in the TM domain or the ectodomain of F protein. For influenza virus, the TM domain of the hemagglutinin protein has previously been demonstrated to be responsible for DRM association (45). VSV G is considered non-raft associated since it is absent from DRM fractions and does not colocalize with GM1 by microscopy (6, 28, 52). To determine if the TM region of F protein was responsible for raft association, the RSV F TM domain and CTD were replaced with the corresponding region of VSV G (Fig. 4A). The chimeric protein was expressed at the cell surface similarly to the SynF protein; furthermore, it retained association with GM1 (Fig. 4B) and was DRM associated (Fig. 5). In contrast, VSV G remained in the soluble portion of the density gradient (Fig. 5).
Transferrin receptor does not enrich in rafts and so should not colocalize strongly with RSV F protein (16). To test this, live A549 cells expressing RSV F and modified F proteins were labeled for F protein expression and for TfR. By confocal microscopy, none of the modified F proteins demonstrated strong colocalization with TfR (Fig. 6). To obtain a measure of colocalization, the images of cells colabeled for RSV F and GM1 or TfR were evaluated by determining Manders overlap coefficients (Table 1) . This calculation is based on the overlap of staining patterns, with values ranging from 0 to 1 indicating no to complete overlap, respectively (27, 53). With CTxB colocalization, each of the modified F proteins gave high Manders coefficient values that were comparable to that of SynF (wild-type protein sequence). In contrast, the colocalization of wild-type or modified F proteins with Tfn was low (Manders coefficient average of 0.502) and was significantly lower (P < 0.01) than that for CTxB staining (Manders coefficient average of 0.830). While not showing a result of 0 (indicating a low-level overlap of signal), this analysis indicated that the wild-type and modified F proteins each were enriched in domains stained with CTxB and were weakly associated with domains stained with Tfn. Furthermore, the modified proteins gave similar Manders coefficients, indicating that raft association must be dictated by the ectodomain of F.
Transferrin receptor localization in cells expressing RSV F proteins with mutations to the TM domain and CTD. A549 cells transfected with RSV F proteins were labeled for F protein (green) and for TfR with Tfn (red) as described in the legend for Fig. 3. Samples were analyzed by confocal microscopy, and a representative image is shown for each transfection group. The portions indicated by the boxes are magnified approximately ×5 in the insets.
Colocalization of RSV F proteins with CTxB and Tfna
The objectives of this study were to determine if RSV F protein was able to target to lipid rafts independently of other viral proteins, including the G and SH envelope proteins, and to determine if palmitoylation, the cytoplasmic tail domain, and the transmembrane domain were involved in F association with rafts.
Incorporation of envelope proteins into the plasma membrane is thought to be a key nucleation step in viral assembly and budding. Several enveloped viruses, including measles virus and HIV, have been shown to incorporate their envelope proteins into specialized microdomains of the plasma membrane termed lipid rafts (4, 28, 35). Rafts may provide a means to localize membrane proteins within the cell membrane to enhance protein-protein interactions. For an enveloped virus, such interactions are important, as envelope proteins must associate and interact with capsid in the cytosol and also cellular components for proper assembly to take place. Human respiratory syncytial virus has three envelope proteins, SH, G, and F, together with matrix and capsid proteins. The matrix protein has previously been demonstrated to be associated with DRMs (18). Similarly, in cells infected with RSV, the envelope proteins were shown to associate with GM1 (8), a marker of rafts (38), and F and G were found in detergent-insoluble fractions (30). However, it was unclear if raft association of the envelope proteins was driven by contact with matrix proteins or by signals present in the envelope proteins themselves.
For measles virus, the F envelope protein associates with rafts independently of not only its H envelope protein but all other viral proteins (47). To address whether RSV F protein also exhibited an intrinsic ability to associate with rafts, we began by using a virus deficient in G and SH. We were able to demonstrate that the F protein was able to colocalize with GM1, perhaps even more distinctly than in rgRSV-SGF-infected cells. F protein fractionated with DRMs of both A549 and HEp-2 cells. This demonstrated that F contains targeting sequences sufficient to bring F to lipid rafts in the absence of other virus envelope proteins.
In addition to the RSV envelope proteins being localized to lipid rafts, the matrix protein, nucleoprotein, and phosophoprotein also migrate with DRM fractions on gradients (30, 31). In order to determine whether F protein required any of these other RSV proteins for raft localization, a synthetic F gene product (SynF) was constructed based on the wild-type gene sequence that eliminated cryptic splice sites and was codon optimized for expression in human cells while retaining the wild-type amino acid sequence. This approach avoided potential problems from expressing F from a vaccinia-driven system, including introduction of vaccinia proteins that may alter cell functions, such as membrane trafficking and localization to the plasma membrane (21, 49).
Expression of SynF in A549 cells did not produce filaments detectable by our methods, as seen with RSV and rgRSV-F infection, suggesting that factors other than the F protein are required for filament formation. To further support this, Oomens et al. observed filament formation in cells infected with a virus with all three of the envelope proteins replaced with a VSV G/RSV F CTD hybrid protein (36). Regardless, the F protein still associated with GM1 and the SynF protein was distributed to DRM fractions. Additionally, F protein was largely excluded from locations that labeled for transferrin receptor, a marker for nonraft regions (16). Together, these observations indicated that signals present in the F protein are sufficient to target this protein to lipid rafts in the absence of all other virus proteins.
Palmitoylation of Cys residues in the TM domain of transmembrane proteins has been shown to be one of the requirements for lipid raft targeting (32). Murine leukemia virus and HIV contain glycoproteins with these acylation modifications on Cys residues that are crucial to DRM association (26, 41). RSV F protein contains one Cys residue located in the TM proximal to the cytoplasmic leaflet of the membrane. In infected cells, this Cys residue is modified by palmitoylation (2). Replacement of Cys550 with a Ser residue or deletion of Cys550 did not prevent association of F protein with rafts, indicating that fatty acid modification on this residue was not crucial for lipid raft association. Similar observations were found with Epstein-Barr virus LMP1, which has one palmitoylated Cys in its TM but does not require this modification for raft association (19). The role of the palmitoylation remains unclear, but it may help to anchor the envelope protein to the membrane or possibly to enhance lipid raft association determined by other signals in the F protein.
It has been demonstrated that the CTD region of both Newcastle disease virus F protein and influenza hemagglutinin contained important signals for determining DRM association (12, 52). To determine if this region was important for RSV F in raft targeting, truncations of F that removed portions or all of the 24-amino-acid CTD of RSV F were made. Unlike with other viruses, CTD truncation did not alter RSV F protein association with lipid rafts nor did the combined absence of Cys550 and the CTD.
The TM of membrane-spanning proteins may contain other acylated amino acids or other properties which make association with the raft regions favorable. The RSV F TM domain and CTD were replaced with those of VSV G, which is known not to associate with DRMs (28). Inclusion of the VSV G TM domain produced membrane localization, and surprisingly, raft association was maintained. All F protein mutations that were generated demonstrated colocalization with GM1 by microscopy. DRM fractionation confirmed localization of the mutated F to the insoluble fractions. There was variation in the distributions of the mutated F proteins among the insoluble, soluble, and intermediate fractions. This variation could be explained by fractionation errors or possibly by differences in strength of association of each modified F protein with the lipid rafts. The latter may reflect weak versus strong association of the different mutated F proteins with the lipid rafts (13). This variation could be evaluated further by careful examination of the effects of various temperatures or detergent concentrations/types on DRM association (29).
The DRM analyses of cells infected with rgRSV-F and those expressing SynF or SynF mutant proteins did not demonstrate cosedimentation exclusively with insoluble fractions, but rather a portion of the F protein was detected in soluble fractions as well. Past studies with virus-infected cells also showed F present in detergent-soluble as well as -insoluble fractions (30), which was attributed to budded RSV remaining associated with the cell membrane at non-lipid raft regions. The mechanism by which the budded virus remained cell associated was not determined. In our studies, it appears that F association with nonraft regions is a function of the F protein itself, since wild-type-virus-infected and rgRSV-F-infected cells show similar distributions of F protein in insoluble fractions. Similarly, F protein expressed independently of the other RSV proteins was found in both soluble and insoluble fractions. Therefore, it is likely that G or SH or other virus proteins are not involved in controlling F raft/nonraft region localization. Independent analysis by microscopy of the raft distribution of F protein supported the DRM results. Again, while the bulk of F was associated with GM1-rich regions of the cell (detected by cholera toxin labeling), some F protein was associated with transferrin receptor (detected with labeled transferrin). This distribution of F protein in both raft and nonraft fractions may indicate two different forms of F protein or may represent the affinity of F for different membrane microdomains. Partitioning to independent regions of the cell membrane may provide a means for the virus to affect the function of the cell in multiple ways. For example, raft-associated HIV Nef protein can downmodulate major histocompatibility complex class I expression to a greater extent than the non-raft-associated form of Nef (1). RSV F may demonstrate a similar phenomenon.
Overall, the data suggest that neither the TM domain nor the CTD contains domains that direct the association of the F protein with lipid rafts. This indicates that the raft-targeting region of F protein is located in the ectodomain of the protein. This is atypical, but other examples where ectodomains are important for raft association have been reported previously. The prion protein PrP associates with rafts through a targeting domain located at its N terminus (48). Epidermal growth factor receptor (EGFR) also contains raft-targeting information in its extracellular region (51). Interestingly, RSV is known to activate EGFR, with a resultant inflammatory response characterized by release of interleukin-8 (34). The mechanism for this activation has not been determined. It is possible that RSV interacts with EGFR in these raft regions to trigger the inflammatory response. RSV infection is also known to alter function of other raft-residing proteins, such as RhoA (30). Raft association through interaction with raft-resident proteins has also been described previously for the c-Cbl-associated protein, which binds to the raft-resident flotillin through a sorbin homology domain (23). RSV F protein may contain a similar domain involved in such an interaction. Future studies will examine the F ectodomain for its ability to target to lipid rafts.
While the CTD of F protein may not be needed for raft association, it likely serves other important roles in virus assembly. In particular, F protein is required for M protein to associate with DRM (18); however, the mechanism of association has not been investigated yet. As with influenza virus, RSV M protein polymerization may cause rafts to congregate through its interaction with F and create large viral assembly centers (39, 52). Along with viral proteins, host raft residents would also be sequestered, and this could have an impact on normal cellular functioning.
Future work will determine the requirements for these components in viral assembly and budding. The F and M proteins are envelope-associated proteins required in the production of infectious virus. Since M requires F to go to rafts but F can locate to rafts independently of M, it is possible that F homing to rafts is an important step in virus assembly and budding. The mutant proteins described in this work provide a means to test this concept and define F and M interaction sites.
We thank Eugene Knutson and Tom Albrecht of the Infectious Disease and Toxicology Optical Imaging Core of UTMB for their excellent assistance with the confocal microscope imaging and analysis. We thank Drew Deniger, Mohammad F. Saeed, and Tonya Colpitts for scientific discussion of the experiments. We thank Jose Melero of the Instituto de Salud Carlos II for the anti-F antibodies used for Western blotting. We thank Michael Whitt of the University of Alabama for both the VSV G cytoplasmic tail domain antibody and the construct expressing the VSV G transmembrane domain and cytoplasmic tail domain (pCAV 711V). We thank Mark Peeples of The Children's Research Institute at Ohio State University at Columbus, Ohio, for the use of his recombinant viruses and advice on their maintenance and for excellent scientific discussion.
E.H.F. was supported by the Robert D. Watkins Graduate Fellowship through the American Society for Microbiology.
Accepted 15 September 2006.
↵*Corresponding author. Mailing address: Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555-0435. Phone: (409) 747-0236. Fax: (409) 772-6527. E-mail: nroberts{at}utmb.edu.
↵▿ Published ahead of print on 27 September 2006.
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Journal of Virology Nov 2006, 80 (24) 12160-12170; DOI: 10.1128/JVI.00643-06
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Vol 6, Supplement 7 (October 2017): Translational Cancer Research /
Combining loco-regional treatments in early hepatocellular carcinoma: new achievements overcoming old limitations
Nelci Antunes de Moura, Fernando Gomes Romeiro
Department of Internal Medicine, Botucatu Medical School, UNESP – Univ Estadual Paulista, Botucatu, Brazil
Correspondence to: Prof. Fernando Gomes Romeiro, PhD. Distrito de Rubião Jr., Zip Code 18 608 917, Botucatu, São Paulo State, Brazil. Email: fgromeiro@fmb.unesp.br.
Provenance: This is an invited Editorial commissioned by Section Editor Dr. Xingshun Qi (Department of Gastroenterology, General Hospital of Shenyang Military Area, Shenyang, China).
Comment on: Vasnani R, Ginsburg M, Ahmed O, et al. Radiofrequency and microwave ablation in combination with transarterial chemoembolization induce equivalent histopathologic coagulation necrosis in hepatocellular carcinoma patients bridged to liver transplantation. Hepatobiliary Surg Nutr 2016;5:225-33.
Submitted Sep 20, 2017. Accepted for publication Sep 27, 2017.
doi: 10.21037/tcr.2017.10.01
Hepatocellular carcinoma (HCC) is the most common primary liver tumor and one of the few malignant diseases that is still increasing around the world. Countless patients are stricken by HCC worldwide, and the tumor incidence is notably linked to the prevalence of other liver diseases such as hepatitis B, hepatitis C, alcohol abuse, non-alcoholic steatohepatitis and any other disease that can cause liver cirrhosis. In the Western countries, it is estimated that more than one million people die due to HCC each year (1). The impact of HCC has led to the development of numerous treatment modalities and algorithms in order to provide better strategies to the patients. However, curative treatments are only proposed for a restricted group of cases that had the tumor diagnosed in the early stage, before the emergence of any type of local or systemic complications (2,3).
Current recommendations regarding curative strategies comprise tumor ablation [such as radiofrequency ablation (RFA)] and surgical procedures (such as liver resection or transplantation) (2,3). As a rule, the increasing number of HCC cases and the shortage of liver donations make resections and ablations a growing necessity. Consequently, the use of these procedures has been encouraging for more and more patients, including those waiting for liver transplantation (LT). Thus, if a given patient with HCC is supposed to spend more than 6 months on the LT list, the current recommendation is to perform a local treatment as a bridge therapy, such as partial resection, transarterial chemoembolization (TACE) or RFA (4). The strategy of using loco-regional procedures for HCC treatment has also been a reason to exceed the parameters stated by the guidelines by offering a curative treatment even when the tumor cannot be included within the limits proposed for a surgical treatment. For instance, many studies have accomplished good results by combining treatment modalities that could never be used together for a given patient, because they are recommended for different tumor stages.
This is the case when TACE, a procedure applied for patients with intermediate tumors, is combined with treatment modalities used for advanced or early tumors. Many articles have been written about the success rates achieved by combined treatments for patients with intermediate and/or advanced tumor stages, because the amount of patients with such conditions has been higher than the number of cases with early stage HCC (5-9). However, the spreading of surveillance programs around the world has been increasing the number of tumors diagnosed in the early stage. As a result, many trials have combined two treatment modalities for patients with small tumors, which are very useful because these tumors have a better prognosis and can be submitted to curative therapies, even when surgical procedures cannot be offered.
In an interesting work, Vasnani et al. compared two modalities of tumor ablation performed after TACE (10). The subjects were submitted to RFA from 2005 to 2009, and microwave ablation (MWA) from 2009 to 2015. One day before the ablation therapy, all the patients were submitted to a drug-eluting bead TACE (DEB-TACE). The results obtained were assessed by multi-phase computed tomography (CT) before and at three-month intervals for the first year, and then at six-month intervals until LT. To assess local response, the authors applied the European Association for the Study of the Liver (EASL) criteria (11). After LT, all the explanted livers were evaluated to calculate the percentage of tumor coagulation. A remarkable finding is that even patients with three or more tumors obtained good results, particularly in the RFA group. Comparing their results with trials using only RFA, the authors showed a striking achievement by combining DEB-TACE with RFA or MWA.
Although the article aimed to compare the results obtained by the two strategies, other findings must be highlighted. First, the authors showed that the ablation techniques performed after DEB-TACE achieved outstanding radiological results, with most subjects attaining complete response. Second, they had excellent histopathological results regardless of the ablation technique. The mean tumor coagulation was around 90% in the explanted livers, showing a strong agreement between radiological and histopathological results. The correlation between the EASL criteria and the tumors examined in the explanted livers was also registered in a similar study performed by Gordic et al. (12), which was somewhat different from the one published by Vasnani et al. For instance, this latter study included some patients with advanced cirrhosis and used MRI instead of CT, because the aim was to compare the performance of various MRI-based response criteria. Even so, the agreement between the EASL criteria and the tumors evaluated in the explanted livers was similar.
Combining loco-regional therapies is a promising strategy to work around the shortage of organ donations, thereby avoiding the tumor growth while LT is not available. It can also be a means for treating tumor recurrence or new lesions after a putatively curative treatment such as resection. At best, it can achieve tumor coagulation of 100%, which can be viewed as a temporary curative procedure. However, the risk of recurrence and/or the occurrence of new lesions must be always kept in mind. Worth mentioning, in all these settings the loco-regional therapies are still limited to patients submitted to strict surveillance programs to diagnose tumors in the early stage. The study performed by Vasnani et al. included only two patients with intermediate-stage HCC (3% of the DEB-TACE/MWA group). Unfortunately, the results of TACE and RFA procedures for intermediate stage HCC are very different (13-17).
In spite of the methods applied, HCC treatment is highly dependent on specific patients' conditions. For instance, patients with HCC graded as B1 or B2 according to the subclassification of the intermediate stage (18) are more likely to achieve better survival rates after RFA, even without TACE, but this outcome is not attained by those with tumors graded as B3 or B4 (13). In the future, other strategies can also be combined to accomplish better results, such as adjuvant therapy. Since RFA and TACE induce transient devascularization followed by neo-angiogenic stimuli (19), some authors have investigated whether using additional drugs for patients submitted to loco-regional procedures would be valuable (19-21). Some studies achieved good results while other seemed disappointing, but this matter still requires a deep analysis after many more clinical trials. For now, the message of Vasnani and collaborators is that treating HCC can attain outstanding successful rates by combining early diagnosis with the best treatment strategy according to each patient's condition.
Funding: The authors would like to acknowledge the financial support received from São Paulo Research Foundation, FAPESP Grant no. 2016/07117-9.
Conflicts of Interest: The authors have no conflicts of interest to declare.
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Cite this article as: de Moura NA, Romeiro FG. Combining loco-regional treatments in early hepatocellular carcinoma: new achievements overcoming old limitations. Transl Cancer Res 2017;6(Suppl 7):S1285-S1287. doi: 10.21037/tcr.2017.10.01
Aims and scope Translational Cancer Research (Transl Cancer Res TCR; Print ISSN: 2218-676X; Online ISSN 2219-6803; http://tcr.amegroups.com/) is an Open Access, peer-reviewed...
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Gland Surgery
Gynecology and Pelvic Medicine
Hepatobiliary Surgery and Nutrition
Journal of Emergency and Critical Care Medicine
Journal of Gastrointestinal Oncology
Journal of Hospital Management and Health Policy
Journal of Laboratory and Precision Medicine
Journal of Medical Artificial Intelligence
Journal of Public Health and Emergency
Journal of Spine Surgery
Journal of Thoracic Disease
Journal of Visualized Surgery
Journal of Xiangya Medicine
Longhua Chinese Medicine
Mesentery and Peritoneum
Microphysiological Systems
Non-coding RNA Investigation
Precision Cancer Medicine
Quantitative Imaging in Medicine and Surgery
Shanghai Chest
Stem Cell Investigation
Therapeutic Radiology and Oncology
Translational Andrology and Urology
Translational Cancer Research
Translational Gastroenterology and Hepatology
Translational Lung Cancer Research
Translational Pediatrics
Video-Assisted Thoracic Surgery
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redpajama_set_name: RedPajamaCommonCrawl


On May 17-19, the famous Obando Fertility Rites or simply Obando Festival will again be the center of attention. Obando?! Where exactly in Bulacan? I am not familiar with its exact location really or how to reach it by commute. So before the main event takes place, I did a little familiarization tour.
I asked some fellow bloggers on how to get there, and they really are of great help.
From Manila, I went first to Victory Mall at Monumento, Caloocan via LRT Line1 (P15). At the back of the mall, there's a terminal of jeep bound to Paco, and these jeeps will pass by the Obando Church. It was like a 20 minute ride from Victory Mall to Obando Church (P15).
However, the church is closed when I got there at around 2:30 PM so I was not able to check the inside.
Across the street is the plaza and beside it is the Colegio de San Pascual Baylon. I can't imagine how many visitors will fill the area on the day of the feast.
Here's my Obando Fiesta (2011) Experience.
btw, you need to know, kapag fiesta, hanggang OEG grocery store lang yung jeep. hindi aabot ng church. well, fmailiar kna naman, lakarin mo nalang.
wait, sa May 17-19, everyday may sayaw? parang sarap nga manuod ha! di rin pala siya ganun kalayo!
opo sir jerik... umaatikabong sayawan ng mga gustong mgka-anak.
when po yung araw ng sayaw? kelan ba yung feast ni san pascual de baylon? what tie po ba nag start yung sayaw?
Anonymous.. nako pasensya na.. haha hindi ko pa din alam yung sched ng upcming fiesta.. bsta from May 17-19 meron sayawan yun..
sama akooooo .. lol .. i wanna witness it .. do you have any idea kung mga anong oras kaya?


text:
The conservation and restoration of Bhutan and the Himalaya's tangible heritage
There are over 2275 monasteries in the Kingdom of Bhutan with over 500,000 artefacts that are in dire need of preservation.
Our monk conservators have, as of 2019, restored over 200 artifacts and over 10 monasteries(in collaboration with Department of Culture, MoHCA) since 2013.
This includes the conservation of 35 Tashigomangs (portable miniature altars unique to Bhutan) and a one-of-a-kind 11 Guru Padmasambhava thangka set amongst others.
Royal Dumtseg Project
The Royal Dumtseg Project is under the patronage of Her Majesty Royal Grandmother Kesang Choeden Wangchuck.
Dumtseg Monastery is the only stupa monastery of its kind in the Kingdom of Bhutan.
It is a national heritage site that is closely associated with the history and local community of Jangsa village, Paro.
Due to its importance, BACCC is dedicated to renovating the site without compromising its historic integrity.
Follow its progress:
Completed November 11th 2019


Large, folding city plan of Paris, by Logerot, 1847, gently restored, cleaned and linen-backed. Excellent condition, 26.5 x 38.75 inches. See TOW098.
This entry was posted in Acquisitions on July 3, 2010 by maps_wp_admin.
A lovely little black and white engraved map of Martha's Vineyard, the Elizabeth Islands, and part of the coast of Cape Cod. With considerable detail of topographical features, roads, towns, villages, and meeting houses. Engraved by P.F. Tardieu for the Paris edition of Letters from an American Farmer in 1787. See MAS461MB.
This entry was posted in Featured Maps on July 3, 2010 by maps_wp_admin.


text:
Ludtke v Kuhn, 1977
In 1977, Melissa Ludtke, a female sports reporter for Sports Illustrated magazine, filed a civil rights action against Major League Baseball Commissioner for the New York Yankees. The action sought to prevent the New York Yankees from enforcing a ban of accredited female sports reporters from entering the team clubhouse in Yankee Stadium.
The Court determined that the policy violated Ludtke's fundamental right to pursue a career under the equal protection and due process clauses guaranteed by the Fourteenth Amendment.
Complaint from Melissa Ludtke and Time, Incorporated v. Bowie Kuhn, Commissioner of Baseball, et al.; Court case to provide equal access to athletes for women sports reporters, 12/29/1977.
National Archives, Records of District Courts of the United States.
This document is being featured in conjunction with the National Archives' National Conversation on Women's Rights and Gender Equality.
The "National Conversation on Rights and Justice" is presented in part by AT&T, Ford Foundation, Seedlings Foundation, Carnegie Corporation of New York, and the National Archives Foundation.
Special thanks to Perkins Coie for their support in this event in New York City.
Emancipation Proclamation & General Order No. 3
Getting to the National Archives in Washington, DC


12 months~6 years. Stx-ld0015. Jwz1/cc5-2. Fyec049. Damping. Quintus. Carrycot pram. ShadesGaojingguan. Green/blue/red/purple. Wholesale car sticker.
Hand push: Dijia. Stx-404. 7kg,5kg,12kg,8kg,9kg,6kg,11kg,10kg. Loading: 6 months - 6 years old. 716848. Selleing cart. Baby buggy cup stroller. 9kg,12kg,5kg,10kg,6kg,8kg. 5 colors choose. Shandong,china. 36x31x18cm.
Cochecito. Wholesale pram basket. Bicycles kids. Organizer buggy. Four-wheel baby stroller folding light portable baby carriage. Wholesale electric car. Place of origin: Crib mobil. Oxford cloth. Bike snow ski. Ksf-031. Luxury baby bassinets. Wholesale 9215 gudi. Baby safety car seat: Baby carrier. Baby cart. Wholesale bodysuit babyMetal material: Ab-338a.
Bq3188. 2007012201236279Al885. Toddlers push car. Small buggyHjp-09. Wholesale dolls umbrella. Insuring new car. No bb. Weight pocket. Basket fabric: Children cart:Model size: Wheel size: Two way comunication. 4 color. With china  3c. 0-3 yeas old. Standardized carts.


LIONEL MESSI was rested for Barcelona's clash with Real Murcia tonight.
Messi remains Barcelona's talisman but the captain was left out of today's Copa del Rey clash.
He has scored 16 goals for Barcelona this season, helping the club through a forward crisis with the injuries to Ousmane Dembele and Luis Suarez.
Messi's form has not been great, however.
He has not scored for the club in any of his last five appearances.
But that was not the reason for him missing Wednesday's 5-0 cup win.
Why was Lionel Messi dropped?
Dropped is maybe a bit harsh in this respect. Messi was rested from Barcelona vs Real Murcia in order to save himself for the Champions League next week.
That was Barcelona's official reasoning when previewing the game.
Barca wrote: "Coming off back-to-back road draws in crucial matches—0-0 at Juventus in the UEFA Champions League and 1-1 at Valencia in La Liga - and playing at home with a comfortable lead over Second Division B side Murcia - Wednesday's match is a perfect opportunity for manager Ernesto Valverde to rest some of his key players.
When will Lionel Messi be back?
The likelihood is is that Messi will return to Barcelona's XI for Saturday's La Liga clash with Celta Vigo at the Nou Camp.


text:
Lista är en halvö i Farsunds kommun i södra Norge belägen mellan Listafjorden i väst och Lyngdalsfjorden, Krossnesundet, Framvaren i öst.

Listahalvön är förbunden med fastlandet genom det 1,3 kilometer breda näset Listeid mellan Framvaren och Eidsfjorden, en arm av Listafjorden. På halvöns sydkust finns en angöringsfyr, Lista fyr, och en väderleksstation. Högsta punkt är Storfjellet, 346 meter över havet. Landskapet påminner om Jæren i ett mindre format. Åtskilliga fynd från stenåldern och yngre arkeologiska perioder har gjorts på Lista, som också omnämns flera gånger i sagalitteraturen.

Källor
Store norske leksikon, snl.no, "Lista. – halvøy i Farsund", läst 2009-06-14 (CC-by-sa, GFDL)

Halvöar i Norge
Farsunds kommun

redpajama_set_name: RedPajamaWikipedia


text:
=====
Usage
=====

To use PyEDGAR in a project::

	import pyedgar

redpajama_set_name: RedPajamaGithub


It's October, which means it's time for new iPads. And Apple's planning to hold an event for just that on October 16th, says Recode, which has a very good track record with these dates. That's just a few days earlier than when Apple held an event last year where it debuted the iPad Air, the iPad Mini with Retina display, and launched its revamped Mac Pro. Expected at this one is an updated version of the iPad Air, a final version of OS X Yosemite, which Apple is in the middle of finishing up, and quite possibly iMacs with Retina displays — something Apple's had trouble keeping a secret.
A separate report last month suggested Apple planned to hold an event on October 21st, something a source told us was not accurate. Before 2012, new iPads were typically released in the spring. Apple's change to the fall meant that the tablet was ready just in time for the busy holiday shopping season, and just after a major new release of iOS with whatever new iPhone had come out.
Apple's last event was in early September where the company debuted the iPhone 6 and 6 Plus, along with a sneak peek of the Apple Watch. That was held at the Flint Center just a few minutes away from its headquarters in Cupertino, where Recode says Apple plans to hold this "more laid-back affair" on the 16th. Apple rented out a much larger venue in San Jose two years ago when first unveiling the first iPad mini and revamped iMac, though kept it low key with an event in downtown San Francisco last year.


In a novel approach to sharing her work, Michelle Richeson, (who's stepping down from her position at Jack Richeson & Co. to devote herself more fully to her painting), has launched an effort to raise money for hospitals and bring a little light into the lives of sick children. Recalling the fear she experienced herself as a young child in a hospital, she's offering giclée prints of North Bay Carousel (pictured here behind the artist) for fund-raising purposes. Check out the full details by clicking here.


Top Lowe's Cedar Lumber Prices deals at … Compare prices and shopping results for Lowe's Cedar Lumber Prices from mySimon.com. mySimon.com has the best deals and .
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Find and save ideas about Lowes lumber prices on Pinterest. . Shop Top Choice 2 x 6 Western Red Cedar Lumber at Lowe's Canada.


An increasing number of companies are moving towards cloud computing and in the present market, it is often difficult to survive without engaging in some form of cloud computing. The cloud has made both small and large companies to access technologies that were ominously expensive and difficult to access. If you have adopted cloud computing, you probably know the importance of hiring IT professionals with the right skills and experience.
Of all these skills, Microsoft Azure is one of the most wanted skill today. With an increasing focus on cloud-related technologies, it only makes sense to hire IT professional who are well versed with Microsoft Azure.
Interview mocha, leading provider of 1000+ online skill tests has Microsoft Azure Test in test library but in case you just want to ask questions yourself here is a list of good application-oriented Microsoft Azure interview questions that you can ask a Windows Azure Developer.
Grill candidates on the below topics, go more in depth. This will provide you a fair view of candidate's knowledge.
Here is the list of Microsoft Azure Interview Questions. Read them, bookmark them, even add your own interview questions in the comments below.
1. How to create a Virtual Machine in Azure?
2. What is Azure Diagnostics? When can we use it?
3. Recommend approaches for controlling access to the service by using the Windows Azure AppFabric Access Control Service.
4. What are blobs, tables, and Queues? Is SQL is the standard way to query blobs, tables, and queues?
5. How to Sync Two SQL Azure Databases?
6. You are designing a strategy for synchronizing an SQL Azure database and multiple remote Microsoft SQL Server 2008 databases. The SQL Azure database contains many tables that have circular foreign key relationships.
7. Recommend an approach to ensuring that all changes in the remote databases synchronize with the SQL Azure database.
9. Explain the troubleshooting procedure to integrate the build system with Azure cloud services for continuous deployments.
10. Explain the troubleshooting procedure to integrate the build system with Azure cloud services for continuous deployments.
11. How can you demonstrate between Azure mobile service and Web API?
12. Explain in detail hybrid and community cloud. When hybrid and community cloud is required?
13. Why should you use Windows Azure Storage as opposed to Local Resources/Storage?
14. How do web role and work role communicate with each other?
15. Explain Windows Azure Cloud Service Lifecycle.
16. Explain Azure Mobile Service. How can we create and integrate new mobile service in the new or existing application?
17. When does a workload on SQL Azure get throttled?
18. What is Text Analytics API in Azure Machine? How does it work?
19. Give some examples of hybrid applications using Windows Azure.
20. While migrating Microsoft SQL Server 2008 database to SQL Azure, what can be done to ensure the database connectivity does not degrade?
A recruiter also faces some recruitment challenges and you must beat them before you hire your next Azure Architect. A Windows Azure Architect should have deep knowledge of the Windows Azure Platform and its technologies. Apart from architecturally designing efficient applications, today's developers need talent that needs to be outside of the development, such as business operations, tools, and integration skills. These questions along with our skill assessment test will help you find the perfect candidate.
For architects, the question needs to be framed in a way that is more related to his personal experience, and which can dig more about him in terms of how well he knows the technology, how he overcame difficult challenges, his interpersonal skills, and ability to lead to stable solutions.
Here's just a quick list of questions, when interviewing candidates for a Windows Azure architect. A seasoned Windows Azure developer would be able to handle the majority of these.
Describe scenarios where they would combine (or separate) tasks into the same (or separate) roles.
Explain how to monitor and scale an app.
Understand the various aspects of upgrading an app.
Know how to install and run 3rd-party tools and alternative (non-.NET) languages.
Architect for cost vs performance vs availability.
Know about storage solutions (SQL, 3rd-party SQL & NoSQL, blobs/tables/queues).
Know the pros and cons of Windows Azure Queues vs. Service Bus queues.
Differences between SQL Server and Windows Azure SQL Database, including federations, security, transient faults, cross-database queries, and transactions.
Understand the capabilities (and limitations) of Content Delivery Network edge-cache.
Technical questions: Ask technical questions that involve a technical discussion of projects the candidate has worked on. Ask the candidate to write code on the whiteboard, or draw solution if needed.
Process thinking questions: Analyze the thought process of the candidate by asking him a technical issue, design question, or problem-solving puzzle. It's not always the end result, but the process the candidate took to get there and the ability to clearly articulate it to others.
In a field like IT, it is important to build a team that possesses the latest skills. Spend a little more when it comes to hiring the right Azure Developer, who will ace your cloud computing strategies.
While spending more time in interviewing, save your time in the initial screening of candidates with the help of Interview Mocha's quality online azure test to hire Windows Azure Developer and weed out the irrelevant candidates with low technical skills.
You can choose from a variety of tests that are well designed to quantify and assess skills of Azure developers and spend time interviewing only the relevant candidates. Evaluate candidate's practical knowledge on Windows Azure concepts with Microsoft Azure Assessment Test.
Our online assessment software is super easy to use. Quality, quick, and fun tests make it a good experience for the candidates as well.
If you have any question on Windows Azure or piece of advice to share, please let us know in the comments below!


2018/12/11 – Morocco's Central Bureau of Judicial Investigation (BCIJ) has dismantled 57 terror cells since its creation in 2015.
Since the beginning of 2018, BCIJ has dismantled eight terror cells. The BCIJ dismantled 9 cells in 2017, 19 in 2016, and 21 in 2015.


No more right hand column.
70% of you ignored the first three results on Google, and never looked at the sponsored results in the right hand column.
So, as of Wednesday February 23, 2016, Google decided to do something about that.
Gone is the right hand column in it's entirety.
Instead of three sponsored links at the top of the page- there are now FOUR.
Then a geographic map- listing Google Local results near you.
Then ten organic results, then three more sponsored search results.
All in the left hand column.
So, this means there are now at least 4 paid results above you- and then, the organic map, then ten results, some of which will almost always be manipulated link farm directories like the Better Business Bureau, Hibu, YP, Angies List, and who knows who else, then, if you are both lucky and good- you.
This means higher bids to be seen for the Search Engine Marketing people to make it into the top 4 positions, and a lot less paid spots on page one.
It also means a lot of white space. The good news, if they are actually searching for your exact business- it will probably show up all by itself on the right.
Time to make sure your Google Local listing is up to date.
Also, since last year, Google will give preference to sites that are both entirely secure (we now offer this as part of our hosting package) and mobile friendly (responsive, or other wise).
Some people speculate that this new Google format is mostly driven by the small screen, guaranteeing that search results, including ads, look the same on mobile as they do on desktop, others, think it's just a way for Google to maximize revenue. Either way, it's a big change, and will take some more time to fully understand how this changes your first page results.


text:
WILLIAMS, James A.
Name: Williams, James A.
Date of death: February 14th, 1945 (Luzon island, Philippines)
Buried on: American Cemetery and Memorial Manila
Major James A. Williams was killed on 14 February 1945 when his Piper L-4 reconnaissance aircraft was hit by antiaircraft fire and crashed. He is buried at Manila American Cemetery and Memorial, Manila/Philippines. Plot: D, Row: 9, Grave: 62.
AIR MEDAL (AM)
Rank: Major
Unit: Headquarters and Headquarters Battery Division Artillery, 40th Infantry Division "Sunburst", U.S. Army
Action: For action performed on 30 January 1945 on the island Luzon, Philippines.
Information source(s): - Author: Division Staff, 40th Infantry Division – The years of World War II, The Battery Press, Nashville, USA, 1995
- Delk, J.D., The Fighting Fortieth – In War and Peace, ETC Publications, Palm Springs, California, USA, 1998


The Pop range offers the ultimate in quick pitch tents. This two person, super-fast pop-up design is perfect for festivals and short stay trips when you just want to pitch and go have a great time. The Pop is light, easy to carry and ideal for first time campers. The Pop features superior fabrics that are fire retardant and waterproof, with an integrated inner tent and plenty of ventilation for a dry, safe and comfortable camping experience. This is the ultimate tent for groups of friends going to many festivals during the summer season.


text:
Lycanthropy: Night of the Full Moon 5
Jacqueline Williamson BBA MPA MS
Ms. Williamson has been captivating the hearts of readers for decades. Her heroes are strong and daring while the antagonists, diabolical!
One legend you don't want to encounter!
New Developments ...
Megan goes with Janice to purchase some additional accessories for her new temporary abode. She seems to be in good spirits and Janice keeps an ever watchful eye on her. By noon, it's time to break for lunch.
"Where would you like to go?" Janice inquires.
"That fantastic steakhouse I've heard advertised." Megan is quick to reply.
"Down on Gillingham Drive? Okay, that's fine. I'll have their Keg Caesar salads." Janice decides, noticing the strange look on Megan's face.
"Is there anything wrong, Megan?"
"I used to really love salads but now a big juicy raw steak seems very appealing to me." Megan confesses.
"A raw steak? Don't you mean medium rare, Megan?" Janice corrects.
"No. I mean rare." Megan is emphatic in her request.
"Okay." Janice replies, heading for The Keg Steakhouse & Bar which is a 20 minute drive. Megan looks out the window, admiring the scenery and then lets down the window to allow the cool breeze to batter her face.
"It's a little chilly for me, Megan. Do you mind letting that window up?" Janice requests feeling somewhat ill at ease.
"It's fine for me but okay." Megan complies.
Moments later, they enter the steakhouse and are seated. The waitress takes their order and is slightly surprised that Megan desires a large Porterhouse Steak barely browned on both sides. She desires the baked potato plain, and very cold water. Janice gets the Caesar Salad with iced tea.
"It's a beautiful place." Janice remarks.
"Yes, it is. I hope the food is as good as advertised." Megan states.
When it arrives, Megan devours the meat with gusto. She leaves the potato and drinks the water. Janice is carefully making mental notes of the entire incident, not missing a single thing.
When they are finished, Janice presents the company credit card to pay for it.
"When will the paintings and other accessories be delivered. I'm most anxious to finish decorating." Megan retorts as Janice leaves a generous tip for the server.
"It should be by this afternoon. I will miss your company at my condo, but I do understand." Janice replies, hiding her inward relief. She enjoys having Janice but there were times when she caught her staring as a predator does when sizing up its prey.
Janice takes Megan back to Albertson Pharmaceutical for her daily workouts in the gym. She sits and observes her while she goes through her routine. First, the weight-lifting, next the strength endurance training and last, the treadmill. Her speed increases each time she mounts it. Later, Megan relaxes in the sauna, giving Janice the opportunity to speak with Dr. Benjamin Riley.
Janice knocks quietly on the door.
"I've finally got a chance to speak with you, Ben." Janice begins slightly breathless.
"Is anything wrong?" Ben offers her a seat.
"Megan is developing quite impressive muscles and her speed on the treadmill is steadily increasing." Janice reports, noticing Ben's raised eyebrows.
"That is interesting." Ben answers while noting it in his tablet.
"Something else." Janice says.
"She downed an enormously large Porterhouse Steak with little effort. It was only browned lightly on both sides." Janice acknowledges and Ben almost drops his tablet.
"It was almost rare?" Ben inquires incredulously.
"That's right." Janice concurs. Ben rises from his seat and walks toward the window.
"It may not be safe for her to stay at the apartment."
"She is so looking forward to being there while she undergoes observation. When will Shirley Crenshaw be here. I hope she will be able to shed more light on this situation." Janice says.
"It should be by the end of this week. She's a doctor now." Ben adds.
"Well, good for her. I knew that she had what it takes to excel. How do you feel about her returning?" Janice tries to gauge Ben's reactions.
"Shirl has never been to Brampton." Ben tries to hedge the subject.
"You know darn well what I mean, Ben." Janice counters.
Will Shirley actually be able to help them or is this a lost cause?
"I really don't know how I feel right now, Jan. I've got to focus on the problem at hand. With the accelerated rate in which Megan is progressing and the advent of a full moon—" Janice immediately interrupts him.
"The full moon! I never thought about that." Janice bemoans.
"I haven't thought of anything else." Ben admits.
"Friday, the same day Shirl is due in Brampton." Ben replies.
"What are you going to tell Ethan?" Janice is very much concerned.
"Be vigilant and keep this gun nearby." Ben produces the same one he used on Jeremy.
"Do you actually think Ethan will be able to kill his own wife?" Janice has strong doubts regarding this.
"I don't really know, Janice. All I can do is to give him the gun. It's up to him what he does with it. I can't be responsible." Is Ben's flippant response.
"Ha, but you are! We all are when we didn't realize how determined Jeremy was. We greatly underestimated him. You did, Ben." Janice puts the blame squarely on him.
Ben winces as if he's been slapped. He looks at Janice, then holds his head down in remorse.
"I guess you're right, Jan." Ben confesses. "I ignored the warning signs. I guess I was a little distraught because he was marrying Shirley Woodard."
"But you didn't want to marry her, Ben." Janice reminds him.
"Yes, I know. I still don't but it's—well, you just won't understand. It's a male thing." Ben rubs his chin, unsure that he does himself.
"Hmm, anyway, Shirley Crenshaw will be here on Friday." Janice repeats his words.
"I need to call Ethan. He needs to be informed." Ben recites in a dismissing manner. Taking the cue, Janice rises to leave his office.
"Tread softly, Ben. Ethan really loves that woman." Janice's parting words as she gently closes the door.
"I'll do the best I can." Ben replies to a closed door. His thoughts are interrupted by the telephone.
"Benjamin Riley here."
"Hello Benjy." A soft voice replies on the other end.
"Dr. Shirley Crenshaw?" Ben responds in complete surprise.
"Right first time. Now give me your version of this caper." Shirley prompts sweetly.
Ben tries to give her the Reader's Digest© version of everything that has happened from the time Jeremy Evans was discovered experimenting on himself to when he bites Megan. It is a 45 minute recital. Then all is quiet on the other end.
"You still there, Shirl?" Ben queries.
"Yes, I'm still here. That's some story, Benjy. You've given me a lot to assimilate." Shirley responds.
"You've dealt with unusual phenomenon before—Experiment 697 and Alpha 1-X." Ben rejoinders.
"Those were animals, and that is where the difference lies. Highly intelligent but still, animals! What you have here—what we have here is a reincarnated legend! A werewolf! Leave it to you, Benjy to excel in creating chaos." Shirley finds this almost laughable.
"It's no joke, Shirl!" Ben insists.
"Yeah, I know, and the full moon is Friday!" Shirley warns.
"What do you think, Shirl?"
"I think I'll see you Friday morning!" With that, Shirley returns the receiver to its cradle. Rising, she walks toward her window in the office. Then she smiles slightly, shaking her head.
"We are in some deep feces!"
To Be Continued ...
Megan feels the transformation. However, it is not the excruciatingly painful experience shown in movies. It is a gradual, methodological and systematical approach. Hands and Feet change. Body hair appears, and then the undeniable face of a Wolf!
© 2019 Jacqueline Williamson BBA MPA MS
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