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Vaccines and Antiviral Agents
α-Glucosidase Inhibitors Reduce Dengue Virus Production by Affecting the Initial Steps of Virion Morphogenesis in the Endoplasmic Reticulum
Marie-Pierre Courageot, Marie-Pascale Frenkiel, Claudia Duarte Dos Santos, Vincent Deubel, Philippe Desprès
Marie-Pierre Courageot
Unité des Arbovirus et Virus des Fièvres Hémorragiques, Institut Pasteur, 75724 Paris, France, 1 and
Marie-Pascale Frenkiel
Claudia Duarte Dos Santos
Departamento Bioquimica e Biologia Molecular, Instituto Oswaldo Cruz, 21045-900 Rio de Janeiro, Rio de Janeiro, Brazil 2
Vincent Deubel
Philippe Desprès
DOI: 10.1128/JVI.74.1.564-572.2000
We report that endoplasmic reticulum α-glucosidase inhibitors have antiviral effects on dengue (DEN) virus. We found that glucosidase inhibition strongly affects productive folding pathways of the envelope glycoproteins prM (the intracellular glycosylated precursor of M [membrane protein]) and E (envelope protein): the proper folding of prM bearing unprocessed N-linked oligosaccharide is inefficient, and this causes delayed formation of prME heterodimer. The complexes formed between incompletely folded prM and E appear to be unstable, leading to a nonproductive pathway. Inhibition of α-glucosidase-mediated N-linked oligosaccharide trimming may thus prevent the assembly of DEN virus by affecting the early stages of envelope glycoprotein processing.
The dengue (DEN) virion is composed of three structural proteins designated C (core protein), M (membrane protein), and E (envelope glycoprotein) (6). The genomic plus-stranded RNA molecule is translated as a single polyprotein precursor which is cotranslationally processed by host cell- and virus-encoded proteinases to give three structural proteins, C (capsid protein), prM (the intracellular glycosylated precursor of M), and E, and at least seven nonstructural (NS) proteins, NS1 to NS5 (5,26). The order of these individual proteins in the polyprotein precursor is C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5.
The first steps of DEN virus assembly take place in association with the membranes of the endoplasmic reticulum (ER) (5, 26). The internal signal sequence at the junction of C and prM directs the translocation of prM across the ER (5, 26) (Fig. 1). The virus-encoded NS2B-NS3 protease complex which catalyzes cleavage of the COOH terminus of the C protein on the cytoplasmic side of the ER may promote signal peptidase cleavage at the junction of C and prM (2,19, 29, 34, 35) (Fig. 1). Indeed, the signal peptidase cleavage site at the NH2 terminus of prM in the C-prM precursor may remain in a cryptic conformation unless the cytoplasmic region is cleaved (2, 19, 33). The junction at prM-E is processed by signal peptidases on the luminal side of the ER membrane (5, 26, 29) (Fig. 1). Type I transmembrane glycoproteins prM and E appear inside the lumen of the ER (5,26). The DEN virion is first assembled as an immature particle which contains prM noncovalently associated with E in a heterodimeric complex (5, 26). In the prME heterodimer, prM prevents an irreversible conformational change of E during virus secretion through acidified sorting compartments (15). Proteolysis of prM leads to the formation of homodimeric forms of E in virus particles before their release from the cell (25, 26).
Production of structural proteins of DEN-1 virus. At the top is a schematic representation of the orientation in the ER membranes of C, prM, and E (5, 6, 26). Signal peptides are shown as cylinders, and stop-transfer sequences are shown as rectangular blocks. The proteolytic cleavage sites are indicated by arrows (open arrow, signal peptidase cleavage site; dark arrow, furin cleavage site) (5, 6, 26, 27). The circled CHOs are potential sites for N-glycosylation (9, 17). At the bottom is shown the amino acid sequence of residues 95 to 114 of C of FGA/89, numbered from the N terminus of C (9). Below the sequence, the open arrow indicates the putative cleavage site that reacts with the NS2B/NS3 protease complex (2, 19, 28, 33, 35). Amino acids 101 to 114 are thought to constitute a transmembrane domain (9; P. Desprès, V. Deubel, and F. Pénin, unpublished results). The model is not to scale.
There is evidence that the N-linked oligosaccharide processing events in the ER are important for the secretion of some enveloped viruses (21). Recently, it has been shown that life cycles of enveloped viruses such as human immunodeficiency virus and human hepatitis B virus are greatly altered in cells in which α-glucosidase-mediated N-linked oligosaccharide trimming is inhibited (3, 4, 11, 20, 21). The initial steps of N-linked oligosaccharide processing on the glycoprotein in the ER involve the sequential trimming of the glucose residues on oligosaccharide precursor Glc3Man9GlcNAc2 after it is transferred from the dolichol diphosphate to the growing polypeptide backbone (30). ER α-glucosidases I and II are involved in the first steps in the trimming pathway. α-Glucosidase I removes the terminal α(1,2)-linked glucose from Glc3Man9GlcNAc2, and α-glucosidase II removes the second and possibly the third α(1,3)-linked glucose residues (13).
Castanospermine (CST) and deoxynojirimycin (DNJ) are ER α-glucosidase inhibitors, and both potently inhibit the early stages of glycoprotein processing. In this study, we investigated whether these α-glucosidase inhibitors affect DEN virus production in a mouse neuronal model of DEN virus infection.
The derivation and characterization of FGA/89, a wild-type human isolate of DEN type 1 virus (DEN-1 virus), has previously been described (9). FGA/89 glycoprotein prM has a single N-linked glycosylation site at position Asn68 whereas glycoprotein E has two potential sites, Asn67 and Asn153 (Fig.1) (9). Both N-glycosylation sites of the DEN-1 virus glycoprotein E appear to be utilized during the N-glycosylation process (12, 17). Previous studies showed that the rate of FGA/89 protein synthesis in mouse neuroblastoma Neuro 2a cells reached a maximum 20 h after infection and then decreased by 25 h when apoptotic cell death occurred (7, 8). To study the effect of the α-glucosidase inhibitors on DEN virus release, FGA/89-infected Neuro 2a cells were incubated with CST or DNJ for 6 h after 19 h of infection. Infective particles produced in the presence of various concentrations of CST or DNJ were harvested and titrated by focus immunoassay on the mosquito cell line AP61 (9). Treatment with CST resulted in a dose-dependent reduction in the amount of infectious virus, down to 5% of the control value at 500 μM (Fig.2a). Similarly, the response to DNJ was dose dependent, and 500 μM DNJ reduced the production of infective particles to 20% (Fig. 2a). Thus, the amount of infectious FGA/89 virus released into the supernatants was significantly reduced by treatment with 500 μM CST or DNJ for 6 h.
DEN virus production by glucosidase inhibitor-treated Neuro 2a cells. Neuro 2a cells were infected with strain FGA/89 of DEN-1 virus at a multiplicity of infection of 400 (7). (a) Three sets of infected cell monolayers were incubated with various doses of CST or DNJ for 6 h beginning at 19 h postinfection. Virus yield 25 h postinfection was determined by focus immunoassay titration (9). Virus titer is expressed as the virus titer in CST- or DNJ-treated cells as a percentage of that in mock-treated cells. Data were compared between groups with the Fisher and Yatest test. P < 0.05 was considered significant. n.s., not significant. (b) Viral protein synthesis in glucosidase inhibitor-treated cells. Infected Neuro 2a cells were incubated without (Control) or with 500 μM CST (+CST) or DNJ (+DNJ) for 3 h beginning 19 h postinfection. Proteins from infected cells were labeled for 1 h at 21 h postinfection. DEN virus structural proteins from cell lysate samples were immunoprecipitated with anti-prM MAb 14E9 or 15H5 or with anti-E MAb 8C2, 9D12, or 4D2 (7, 9). Samples were analyzed by SDS-PAGE under nonreducing conditions.
We investigated the mechanisms by which α-glucosidase inhibitors affect the production of infectious FGA/89 virus. First, we examined whether the failure of virus release was linked to the effects of CST and DNJ on Neuro 2a cell viability. More than 95% of Neuro 2a cells incubated for 6 h with 500 μM CST or DNJ still excluded trypan blue. No obvious morphological changes were observed between drug-treated and mock-treated cells, as determined by fluorescent staining of the actin cytoskeleton (data not shown). These observations suggest that neither α-glucosidase inhibitor had a significant effect on the integrity of Neuro 2a cells.
We further investigated whether the overall rate of host protein synthesis was altered in Neuro 2a cells treated with α-glucosidase inhibitor. To measure total protein synthesis, two sets of Neuro 2a cell monolayers treated with 500 μM CST or DNJ for 2 h were labeled for 1 h with Tran35S-label (100 μCi/ml; ICN Pharmaceuticals Inc.) in the presence of the drugs as previously described (7, 9, 10). The cells were lysed with SUM buffer (1% sodium dodecyl sulfate [SDS], 2 M urea, 2% β-mercaptoethanol, 25 mM glucose, 5 mM EDTA, 20 mM Tris-Cl [pH 6.8]), and samples were boiled at 65°C for 15 min. Radioactive proteins were determined by scintillation counting of trichloroacetic acid-precipitated samples (10). The level of overall protein synthesis in CST- or DNJ-treated cells was about 80 or 70% of normal, respectively. Therefore, α-glucosidase inhibitor treatment resulted in a limited shutoff of host protein synthesis.
The lower rate of intracellular protein synthesis observed in the presence of α-glucosidase inhibitor may be sufficient to affect DEN-1 virus replication in Neuro 2a cells. We investigated this possibility by using a radioimmunoprecipitation (RIP) assay. To study the synthesis of viral proteins, FGA/89-infected Neuro 2a cells were treated for 3 h with 500 μM CST or DNJ after 19 h of infection. Infected cells were labeled for 1 h with Tran35S-label (200 μCi/ml) at 21 h postinfection. Cells were lysed with RIP buffer as previously described (7, 9, 10). Samples were analyzed by SDS–15% polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. A panel of prM- and E-specific monoclonal antibodies (MAbs) directed against DEN-1 virus strain FGA/89 was used to study the synthesis of viral proteins in infected cells in the presence or absence of α-glucosidase inhibitor (7, 9). RIP analysis showed that prM (apparent molecular mass of 17 kDa) was immunoprecipitated with the anti-prM MAbs 14E9 and 15H5 (Fig. 2b) (7). The anti-E MAbs 8C2, 9D12, and 4D2 react with the E protein of cells infected with FGA/89 (7, 9). The epitopes bound by MAbs 8C2 and 9D12 are reactive in their linear conformation, whereas MAb 4D2 binds to a conformational epitope on the mature FGA/89 virion (7, 9). RIP analysis showed that MAbs 8C2 and 9D12 immunoprecipitated a small amount of labeled protein E (Fig. 2b). In contrast, a single species of protein E (apparent molecular mass of 63 kDa) was clearly detected in samples immunoprecipitated with the conformation-sensitive MAb 4D2 (Fig. 2b). This suggests that most of the protein E molecules were correctly folded. Levels of production of glycoproteins prM and E were similar in glucosidase inhibitor-treated Neuro 2a cells and untreated cells, suggesting that CST and DNJ did not affect viral protein synthesis (Fig. 2b, compare lanes +CST and +DNJ with Control). However, anti-prM MAbs and the anti-E MAb 4D2 recognized structural proteins in glucosidase inhibitor-containing samples, suggesting that the reactivities of these MAbs were unaffected (Fig.2b). More labeled prM was present in CST- and DNJ-containing samples, and to a lesser extent, more labeled protein E was present in DNJ-containing samples, than in untreated cells (Fig. 2b). This may be due to the accumulation of newly synthesized structural proteins in glucosidase inhibitor-treated cells because the rate of virus release was low.
The correct folding or secretion of some viral glycoproteins is impaired in cells in which α-glucosidase-mediated N-linked oligosaccharide trimming is prevented (21). We first determined the kinetics of the folding and heterodimerization of prM and E in Neuro 2a cells by pulse-chase analysis. FGA/89-infected cells were pulse-labeled with 500 μCi of Tran35S-label per ml for 10 min at 20 h postinfection and chased as previously described (10). The formation of aberrant disulfide bonds in cysteine-containing proteins during cell lysis was prevented by washing cell monolayers in ice-cold phosphate-buffered saline containing 50 mM iodoacetamide, a sulfhydryl-alkylating agent. Cells were lysed with ice-cold 1% Triton X-100 in TNI buffer (10 mM Tris, 150 mM NaCl, 10 mM iodoacetamide [pH 8.0]) containing a set of protease inhibitors (Complete; Roche Molecular Biochemicals). Cell extracts were treated with normal mouse serum and protein A-Sepharose beads and pelleted. The supernatants were incubated with MAbs and protein A-Sepharose. The folding of protein E and its assembly with prM were assessed by using antibodies that recognized correctly folded protein E (MAb 4D2) and the prME heterodimer (MAb 15H5) (7, 9). The complexes were washed five times with ice-cold 0.1% Triton X-100 in TNI buffer at 4°C, and radiolabeled antigens were released by adding Laemmli sample loading buffer and boiling.
Pulse-labeled prM glycoprotein was immunoprecipitated with the anti-prM MAb 15H5 (Fig. 3a, 15H5). The amount of prM increased linearly during the first 40 min of chase and remained constant until 90 min. The time required for half-maximal prM formation (t 1/2) was 15 to 20 min, indicating that this process is slow (Fig. 3b, top). Several reports provide evidence that prM production from flavivirus structural polyprotein is regulated (2, 19, 28, 29, 33, 35). The disulfide-dependent folding of prM was analyzed by SDS-PAGE under nonreducing and reducing conditions. The reduced form of protein prM migrated more slowly than did its nonreduced counterpart, indicating that correctly folded prM included disulfide bonds, consistent with the presence of six cysteine residues (data not shown). RIP analysis with the anti-prM MAb 15H5 showed that pulse-labeled prM and E interacted to form a heterodimer in FGA/89-infected Neuro 2a cells after 20 min of chase (Fig. 3a, 15H5). The molecular mass of the FGA/89 glycoprotein prM as assessed by SDS-PAGE was reduced by approximately 2 kDa by N-glycosidase F (PNGase F; New England BioLabs) digestion, consistent with the presence of one N-linked oligosaccharide at position Asn68(Fig. 4a) (9). However, the molecular mass of FGA/89 glycoprotein E was reduced by approximately 4 kDa by PNGase F digestion, consistent with the presence of two N-linked oligosaccharides (Fig. 4a) (9). Both DEN virus envelope glycoproteins were completely susceptible to endo-β-N-acetylglucosaminidase H (endo H; Roche Molecular Biochemicals) after 30 min of chase, suggesting that prM and E associated in a compartment upstream from the medial Golgi compartment (Fig. 4a). The amount of E that coprecipitated with prM reached a maximum at 90 min of chase and then declined slightly (Fig.3b, top). The t 1/2 of the association between prM and E was 60 min, suggesting that formation of the prME heterodimer is slow (Fig. 3b, bottom). As prM formation is also a slow process (t 1/2 = 20 min), it may be the rate-limiting step for prME heterodimerization.
Kinetics of FGA/89 envelope glycoprotein processing in Neuro 2a cells. Neuro 2a cells infected with DEN-1 virus (FGA/89) or mock-infected cells (No virus) were pulse-labeled for 10 min at 20 h postinfection and chased for various times. (a) Cell lysates were immunoprecipitated with anti-prM (15H5) or anti-E (4D2) MAb. (b) Samples on gels were analyzed with a PhosphorImager (Molecular Dynamics). Quantitation of the prM and E coimmunoprecipitated by MAb 15H5 (top) and the prM+E/prM ratio were used to define the kinetics of association of the prM and E proteins (bottom). Quantitation of the E protein precipitated by MAb 4D2 is shown at the bottom.
Processing of FGA/89 envelope glycoproteins in Neuro 2 cells. (a) Cell lysates chased for 30 min were immunoprecipitated with MAb 15H5. Labeled antigens were eluted from complexes in SDS and β-mercaptoethanol by boiling, and the denatured samples were divided into three aliquots for endo H (H), PNGase F (F), or mock (−) digestion as previously described (36). Glycosylated (g) and nonglycosylated (ng) forms of prM and E are indicated. (b) Pulse-labeled prME complexes chased for 90 min were subjected to sedimentation analysis on sucrose gradients. Samples were immunoprecipitated from gradient fractions with MAb 15H5 and separated by SDS-PAGE. Protein E that coprecipitated with prM was quantitated with a PhosphorImager (black boxes). The sedimentation of detergent-solubilized glycoprotein E homodimer from DEN-1 virions was determined by treating highly purified virus with 1% Triton X-100 in TNI buffer at 20°C for 1 h (1). Denatured virions were run in the parallel sucrose gradient, and protein E homodimer was detected by enzyme-linked immunosorbent assay using an anti-E specific antibody (open boxes). O.D., optical density.
DEN virus prME heterodimers are transported as oligomeric complexes through the cell secretory pathway (1, 5, 6, 15, 19, 26, 27,31). We examined the oligomeric assembly of DEN virus envelope glycoproteins in FGA/89-infected Neuro 2a cell lysates chased for 90 min by velocity gradient centrifugation. Cells were lysed with 1% Triton X-100 in TNI buffer, and samples were directly applied to 5 to 20% (wt/vol) continuous sucrose gradients in TNI buffer containing 0.1% Triton X-100 (1). The gradients were centrifuged for 16 h at 35,000 rpm in an SW41 rotor at 4°C, and the distribution of prME complexes in gradient fractions was determined by immunoprecipitation with the anti-prM MAb, 15H5. Most mature prME complexes sedimented more slowly than DEN virus E homodimer (predicted size, about 130 kDa), suggesting that the assembly of FGA/89 glycoprotein heterodimer complexes was an inefficient process in Neuro 2a cells (Fig. 4b). However, it is possible that multimeric prME complexes were labile in the presence of 1% Triton X-100 (31).
The folding of FGA/89 glycoprotein E was monitored with the anti-E MAb 4D2. The amount of labeled E glycoprotein was low during the first 60 min of chase and then increased rapidly until 90 min (Fig. 3a, 4D2). The t 1/2 of formation of the conformation-dependent 4D2 epitope was more than 60 min, indicating that FGA/89 glycoprotein E folded slowly (Fig. 3b, bottom).
Thus, we assessed the effects of CST and DNJ on the processing of newly synthesized structural proteins in DEN virus-infected cells. FGA/89-infected Neuro 2a cells were pulse-labeled for 10 min and chased for 45 min (t 1/2 for formation of prME heterodimer) or 90 min in the presence of α-glucosidase inhibitor. CST or DNJ, each at 500 μM, was included in the starvation and chase media. In the presence of the α-glucosidase inhibitor CST, the prM and E glycoproteins migrated more slowly in SDS-PAGE than did their counterparts in mock-treated cells (Fig. 5a, compare lanes CST and Control). It is likely that the presence of triglucosylated N-linked oligosaccharides on the N-glycan sites causes changes in protein conformation. Thus, as the samples were analyzed by SDS-PAGE under nonreducing conditions, the different electrophoretic mobilities may result from different conformations stabilized by disulfide bonds. The mobilities of prM and E in DNJ-containing samples were intermediate between those of their counterparts from mock- and CST-treated samples. This suggests that one or two glucose residues remained on core oligosaccharides due to incomplete inhibition of glucose trimming (Fig.5a, compare lanes DNJ with CST and Control).
In CST-treated cells, the amount of the E glycoprotein immunoprecipitated by the conformation-sensitive MAb 4D2 was about 30% of that in controls at 45 min of chase and only 15% at the end of the chase (Fig. 5a, compare lanes CST and Control, 4D2). Thus, the E-related form carrying triglucosylated N-linked oligosaccharides appeared to be misfolded, leading to a nonproductive pathway. This result demonstrates that glucose trimming contributes to the correct folding of FGA/89 glycoprotein E. Conversely, the formation of the conformation-dependent 4D2 epitope was unaffected by the presence of DNJ (Fig. 5a, compare lanes DNJ and Control, 4D2).
Processing of prM and E in FGA/89-infected Neuro 2a cells treated with α-glucosidase inhibitor. Infected cells were pulse-labeled 20 h postinfection and chased in the presence of the α-glucosidase inhibitor (500 μM) or mock treated (Control). (a) Samples were immunoprecipitated with anti-DEN virus antibody. Proteins prM and E are indicated. (b and c) prM and E coimmunoprecipitated by MAb 15H5 are quantified as described in the legend to Fig. 3.
The amount of the E-related form that coprecipitated with prM from CST-containing samples was about 25% of that of the control at 45 min of chase (Fig. 5a, compare lanes CST with Control, 15H5). It appeared, therefore, that CST decreased the formation efficiency of prME heterodimer in FGA/89-infected Neuro 2a cells. The anti-prM MAb 15H5 immunoprecipitated two closely related forms of prM from CST-treated cells with apparent molecular masses of 18 and 19 kDa; both migrated more slowly than prM from mock-treated samples (Fig. 5a, compare lanes CST with Control, 15H5). The intensity of the prM-related 19-kDa band increased slightly during the chase period (Fig. 5b). Conversely, the intensity of the prM-related 18-kDa band increased twofold, and there was a parallel increase in the amount of the E-related form associated with prM in a heterodimeric complex (Fig. 5b). Therefore, the formation of prME heterodimer appeared to require the prM-related 18-kDa form in CST-treated cells. The position within the NH2 terminus of prM at which signal peptidase cleavage occurs may be affected by CST, causing slower conversion of this site from a cryptic to an accessible conformation on the luminal side of the ER membrane. If this is so, the prM-related 19-kDa form may be a prM precursor containing the signal sequence at its NH2 terminus (see below). However, the association between prM and E does not appear to necessitate the trimming of the glucosylated N-linked oligosaccharides, as these proteins can be recovered as a heterodimeric complex in CST-treated cells.
In the presence of DNJ, the amount of prME heterodimer declined during the chase (Fig. 5a, compare 45 and 90 min, 15H5). At 90 min of chase, the amount of complex formed between the prM and E-related forms in DNJ-containing samples was about 50% of that in controls (Fig. 5c). Thus, the presence of partially deglucosylated N-linked oligosaccharides at the N-glycan sites may interfere with the stability of DEN virus envelope glycoprotein complexes which are probably misassociated heterodimers leading to a nonproductive pathway.
Therefore, inhibition of α-glucosidase-mediated N-linked oligosaccharide trimming seemed to impair the correct folding and optimal assembly of FGA/89 envelope glycoproteins in mouse neuroblastoma cells. We investigated the impact of glucose trimming on DEN virus envelope glycoprotein processing by expressing the FGA/89 cDNA coding for prM and E in Neuro 2a cells, using the ecdysone-inducible gene expression system (22). Transient expression in Neuro 2a cells indicated that the prM gene incis greatly increases the production of correctly folded E protein (data not shown), supporting the notion that prM may have a chaperone-like role in the folding of E in the ER (1, 18). The cell clone N2aprM+E was established. It carries the cDNA comprising the FGA/89 prM+E region (Met95 to Ala773) and the genes encoding the modified ecdysone receptor VgEcR and the retinoic X receptor. The 20 amino acids (Met95 to Ala114) at the COOH terminus of protein C are required for translocation of the prM protein (26, 29, 34) (Fig. 1). In the presence of the ecdysone analog ponasterone A, the retinoic X receptor and VgEcR heterodimerize and then transactivate the ecdysone response element-containing promoter. The ecdysone response elements are upstream from a minimal heat shock promoter that drives the expression of the FGA/89 prM and E genes. After 15 h of induction with ponasterone A (5 μM), recombinant FGA/89 envelope glycoproteins were readily detected in N2aprM+E cells (data not shown).
The electrophoretic mobilities of the recombinant prM and E proteins under nonreducing conditions were identical to those of their counterparts from FGA/89-infected Neuro 2a cells (data not shown). Furthermore, treatment with PNGase F indicated that a single N-linked oligosaccharide was present on recombinant prM and that the two N-linked oligosaccharides were present on recombinant E (data not shown). These results suggest that the newly synthesized prM and E proteins in N2aprM+E cells were correctly processed and folded.
The kinetics of recombinant DEN virus envelope glycoprotein processing were examined by pulse-chase experiments. Induced N2aprM+E cells were pulse-labeled for 10 min after 20 h of induction and chased for 180 min. Recombinant prM glycoprotein was clearly detected 15 min after the pulse (Fig. 6a, 15H5), and the amount of prM was constant until the end of the chase (Fig. 6b, top). Signal peptidase cleavage of the prM signal sequence is modulated by the presence upstream of protein C (2, 19, 28, 34). Thet 1/2 of formation of prM was shorter in N2aprM+E cells than that of its counterpart in FGA/89-infected Neuro 2a cells, supporting the notion that the absence of the C protein enhances the efficiency of prM processing.
Kinetics of recombinant DEN virus envelope glycoprotein processing in N2aprM+E cells. N2aprM+E cells were briefly pulse-labeled 20 h after addition of ponasterone A (5 μM) and chased for various times. (a) Cell lysates were immunoprecipitated with anti-DEN virus antibody; (b) samples on gels were quantified as described in the legend to Fig. 3.
The amounts of recombinant E protein that coprecipitated with prM increased slowly during the first 120 min of chase and then remained constant until the end of the chase (Fig. 6a, 15H5). Thet 1/2 of formation of the prME heterodimer was 75 to 90 min (Fig. 6b, bottom). The size distribution of recombinant prME heterodimers in samples chased for 90 min was examined by sedimentation through a sucrose gradient. The faster-sedimenting complexes cosedimented with the FGA/89 prME heterodimer, suggesting that most recombinant DEN virus envelope glycoprotein complexes are single heterodimers (data not shown).
The amount of recombinant E glycoprotein immunoprecipitated with MAb 4D2 increased linearly during the first 90 min of chase and then declined slowly until the end of the chase (Fig. 6a, 4D2). Thet 1/2 for formation of the conformation-dependent 4D2 epitope was less than 60 min (Fig. 6b, bottom), suggesting that the folding of recombinant E glycoprotein was faster than that of its counterpart in FGA/89-infected Neuro 2a cells (t 1/2 > 60 min).
We examined the effects of trimming inhibition on recombinant DEN virus envelope glycoprotein processing. Induced N2aprM+E cells were pulse-labeled for 10 min after 20 h of induction and chased for various times in the presence of the α-glucosidase inhibitor. In the presence of CST, recombinant DEN virus envelope glycoproteins had a slightly greater electrophoretic mobility than their counterparts from mock-treated cells, suggesting that glucose trimming was efficiently inhibited in N2aprM+E cells (Fig. 7a).
Processing of recombinant prM and E in N2aprM+E cells treated with α-glucosidase inhibitor. (a) Mock-treated (−) or CST-treated (+) cells were pulse-labeled and chased as described in the legend to Fig. 5. Cell lysates were immunoprecipitated with anti-DEN virus antibody. (b) Mock-treated (−CST) or CST-treated (+CST) samples chased for 30 min were immunoprecipitated with MAb 15H5. Immunoprecipitated proteins were directly analyzed (Control) or incubated in the presence (PGNase F) or absence (Untreated) of PGNase F as described in the legend to Fig. 4. (c) α-Glucosidase inhibitor-containing samples (CST or DNJ) or mock-treated samples (Mock) were immunoprecipitated with MAb 15H5.
Detailed analysis of CST-containing samples revealed the existence of a polypeptide with a molecular mass of about 80 kDa (p80) which also reacted with the anti-prM MAb 15H5 (Fig. 7a). The samples were subjected to PNGase F digestion, and the molecular mass of the polypeptide was found to have been reduced by about 7 kDa in SDS-PAGE under reducing conditions, consistent with the cleavage of three N-linked oligosaccharides (Fig. 7b, compare lanes Untreated and PGNase F). Therefore, p80 may be the uncleaved precursor of recombinant prM and E in its glycosylated form. There was significantly more putative uncleaved precursor of recombinant DEN virus glycoproteins in CST-treated samples, suggesting that glucosidase inhibition reduced the efficiency of signal peptidase cleavage at the junction between prM and E in N2aprM+E cells (Fig. 7b, compare lanes −CST and +CST). Thus, the maintenance of triglucosylated N-linked oligosaccharides may change the position of the signal peptidase cleavage site at the NH2terminus of the E glycoprotein, by reducing its accessibility on the luminal side of the ER membrane.
In CST-treated N2aprM+E cells, the amount of labeled recombinant E glycoprotein immunoprecipitated with MAb 4D2 increased slowly during the first 45 min of chase (Fig. 7a, compare lanes +CST with −CST, 4D2). The formation of the conformation-dependent 4D2 epitope was reduced to 50% of that in controls after 60 min of chase (data not shown). Therefore, the folding efficiency of recombinant E glycoprotein was diminished by CST as seen in DEN virus-infected cells.
In N2aprM+E cells treated with CST, the anti-prM MAb 15H5 immunoprecipitated two forms of recombinant prM which migrated with apparent molecular masses of 18 and 19 kDa, higher than the molecular mass of prM from mock-treated cells (Fig. 7a, compare lanes +CST with −CST, 15H5). The amount of the prM-related 18-kDa form increased during the first 45 min of chase (Fig. 7a, lanes +CST, 15H5), and this form was the most abundant after 60 min (Fig. 7c, lanes CST). There was a parallel increase in the amount of recombinant E protein that coprecipitated with prM, suggesting that the prM-related 18-kDa form was required for efficient assembly of prME heterodimer as observed in FGA/89-infected Neuro 2a cells (Fig. 7a, lanes +CST, 15H5).
We investigated whether the lower electrophoretic mobility of the prM-related 19-kDa form in CST-treated N2aprM+E cells was due to the maintenance of the putative signal sequence. Cell lysates chased for 30 min were immunoprecipitated with MAb 15H5, and samples were digested with PNGase F. Under the reducing conditions used for this assay, both prM-related forms were observed, suggesting that the changes in electrophoretic mobility were not due to modifications in disulfide bonding (Fig. 7b, lane Control, +CST). After digestion with PNGase F, which cleaves all N-linked oligosaccharides, the two prM-related forms in CST-containing samples migrated as a single band at the same speed as the deglycosylated recombinant prM (Fig. 7b, compare lanes Untreated and PNGase F, +CST). The signal peptidase therefore appeared to have cleaved prM efficiently at the NH2 terminus in CST-treated N2aprM+E cells. The deglycosylated form of prM in CST-containing samples (Fig. 7b, lane PNGase F, +CST) had a slightly lower mobility than its counterpart in inhibitor-free samples (Fig. 7b, lane PNGase F, −CST), indicating a modification unrelated to N-glycosylation. Further investigation is required to determine whether the 18- and 19-kDa polypeptides in CST-treated cells are incompletely folded or misfolded forms of prM bearing unprocessed N-linked oligosaccharides.
In the presence of the α-glucosidase inhibitor DNJ, the yield of prME complexes in induced N2aprM+E cells was about 60% of the control value after 60 min of chase (Fig. 7c, compare lanes DNJ with Control). Thus, DNJ affected the formation of recombinant prME heterodimer as observed in FGA/89-infected Neuro 2a cells.
We found that DEN virus envelope glycoprotein processing in mouse neuroblastoma cells was strongly affected if α-glucosidase-mediated N-linked oligosaccharide trimming was inhibited. Inhibition of glucose trimming has a major effect on the productive folding pathways of DEN virus glycoproteins prM and E, indicating that N-glycosylation processing is required for folding into the correct conformation. The folding efficiency of prM appeared to be reduced by the presence of triglucosylated N-linked oligosaccharide at residue Asn68of prM, causing a delay in formation of the prME heterodimer. Glucosidase inhibition did not prevent the heterodimeric association between prM and E, but the prME complexes appeared to be unstable, reducing the efficiency of folded prME complex assembly. It is likely that these transient heterodimeric complexes are misassociated prME complexes carrying unprocessed N-linked oligosaccharides. It has been suggested that domain II of flavivirus E glycoprotein is involved in prME heterodimerization (25), and the presence of glucosylated N-linked oligosaccharide within this region at residue Asn67 may destabilize the complexes formed between prM and E, leading to a nonproductive assembly pathway. These findings suggest that the formation of properly folded DEN virus envelope glycoprotein complexes requires a lectin chaperone pathway. The molecular chaperones calnexin and calreticulin have lectin-like affinity for monoglucosylated N-linked oligosaccharides and interact with intermediate oligomeric complexes of viral envelope glycoproteins in the ER (14, 16, 23, 24, 32). However, preliminary results suggest that neither calreticulin nor calnexin associates with DEN virus envelope glycoproteins in FGA/89-infected Neuro 2a cells (M. P. Courageot, V. Deubel, and P. Desprès, unpublished results). Glucose trimming of N-linked oligosaccharides may have a direct effect in promoting the correct folding of DEN virus envelope glycoproteins.
This study shows that N-linked oligosaccharide processing may play a crucial role in the early stages of DEN virus morphogenesis. We also found that α-glucosidase inhibitors can be used to prevent the first steps of DEN virus envelope glycoprotein processing and that the inhibition of glucose trimming has antiviral effects.
We thank Mary K. Gentry and Robert Putnak (Walter Reed Army Institute of Research, Washington, D.C.) for providing MAbs.
This investigation was supported by research grants from the CNRS Interdisciplinaire de Recherche Environnement Vie et Société, program 95N82/0134, DGA, program 99 34 031, and CAPES/COFECUB, program 254/98. M.-P. Courageot was supported by scholarship funds received from the Ministère de l'Education Nationale, de la Recherche et de la Technologie.
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Journal of Virology Jan 2000, 74 (1) 564-572; DOI: 10.1128/JVI.74.1.564-572.2000
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Anita Brown
Noel Forrest
Paul Hebert
Christina Hegarty, PT, DPT
Curtis Garbett
Katie Golden
Mac Howard
Renata Louwers
Nick Myers, PhD
Shirley Norris
Alison Petok, MSW, LCSW, MPH
Jennifer Toth
Jim Van Horne
Guy Wheatley
Lucius Wisniewski
Who Gets Bladder Cancer?
What Causes Bladder Cancer?
Anatomy of the Urinary Tract
How Is Bladder Cancer Classified?
Non-Muscle Invasive Bladder Cancer
Muscle-Invasive Bladder Cancer
Metastatic Bladder Cancer
Prognosis & Survival Rates
Differences in Men vs. Women
Who Treats Bladder Cancer?
Future Treatment Options
Participating in Clinical Research
Blood in Urine
Bone Pain
Decreased Appetite & Weight Loss
Fatigue & Weakness
Inability to Urinate
Pain in Lower Back & Abdomen
Swelling in Feet
Urgency of Urination
Diagnosis & Monitoring
Medical History & Physical Exam
TURBT
Examination Under Anesthesia
What Tests Determine Cancer Spread?
Stages & Grades
Early Stages
Muscle-Invasive Stages
Advanced Stage
Bladder Removal (Cystectomy)
Reconstructive Bladder Surgery
Lymph Node Removal
Maintenance Therapy
Bavencio (Avelumab)
Imfinzi (Durvalumab)
Keytruda (Pembrolizumab)
Opdivo (Nivolumab)
Tecentriq (Atezolizumab)
Intravesical Chemotherapy
Systemic Chemotherapy
Laser Ablation Therapy
Coping & Survivorship
Coping with Symptoms & Side Effects
Fatigue & Difficulty Sleeping
Side Effects of Chemotherapy
Poor Appetite
Urinary Symptoms & Daily Life
Living with Urinary Diversion
Ileal Conduit
Indiana Pouch
Neobladder
Fears of Recurrence or a Second Cancer
Financial Impact
Survivorship Plans
Coping with Late-Stage Cancer
Resources for Support
Body Image After Reconstructive Surgery
By Editorial Team · January 8, 2018
What is body image?
Body image is defined as an individual's perception of the appearance or attractiveness of his or her own body. It has been the subject of media, medical and mental health reporting since the term was first used in the 1930's. Poor body image comes from negative thoughts and feelings about your appearance. The concept of body image affects men, women and children. The interpretation of body image varies around the world based on cultural expectations that help craft the concept of what is desirable.
Many who experience debilitating illnesses or injuries, resulting in a change in health or appearance, may find themselves with a changed sense of self. This can impact their perception of how they look to others. This can be especially true for people with bladder or urinary tract cancers that have had surgical urinary diversion. Urinary diversion is a method of redirecting urine produced by the kidneys when the bladder or other parts of the urinary tracts are not fully functional or have been removed. Urinary diversions can be temporary or permanent. Some diversions require a stoma. A stoma is a surgically created opening from an area inside the body to the outside. Urine can drain into a small pouch that fits over the stoma and attaches to the skin with adhesive. This pouch needs to be emptied and cleaned.
Changing body image after urinary diversion
As if body confidence is not difficult enough to maintain, suddenly finding that your body doesn't work the way it used to can be upsetting. Learning how to go to the bathroom is a behavior learned at a very young age. Since you went off to school you were diaper-free and had control of your bathroom habits. A change in this kind of independent control of your bladder or bowels can have an impact on self-image and self-esteem. If you have been diagnosed with bladder or other pelvic cancers, or urological complications, you may have to undergo a urinary diversion. Suddenly urinating is not as simple as just running to the bathroom. Timing and self-care play a big role in staying comfortable and free of infection.
Feelings play a big role in body image. While others may perceive you as exactly the same person as you were before your urinary diversion, your own internal feelings about the surgery can impact your self-esteem. It's completely normal to feel upset about visible changes in your body, and these feelings can affect your different parts of your life. Some people may grieve over their lost body function while they are getting used to the change.1
Experiencing grief
Having your bladder removed is a loss, and grieving loss is a natural process, even for a physical part of your body or body function. Some people report feeling frustrated, saddened and angry about a change in appearance and irritated by having to make lifestyle changes. Some struggle with how they feel they look in public, of having to make changes in the way they dress, or worrying about having the ability to void (eliminate urine) when needed. This can result in reduced self-esteem.
Creating a care plan
What can be done about it? There are professionals who can assist patients in creating a care plan to make the process of getting used to a urinary diversion as smooth as possible. Some people are able to do this on their own. Others benefit from self-help groups, therapists and guidance from nurses who are specially trained in wound and ostomy care.2 There are 4 fundamental steps:1
Assessment: Evaluating how you feel about yourself and the way you think others feel about you
Intervention: Communicating with people who have had a urinary diversion and hearing their thoughts and opinions
Autonomy: Promoting independence, learning how to care for their new method of elimination (often with help from a nurse or other expert)
Reinforce: Reinforcing self-esteem, accepting the change, and building confidence
The goals of each care plan be specific to the areas of concern for each person. For some, goals may include feeling less isolated. For others, it could be finding ways to feel good about sexual activity and resuming normal activities.
It can take some time to get used to the "new normal" after a radical cystectomy and reconstructive surgery. Recovering from a major surgery, re-learning bathroom habits, and adjusting to the physical changes is no small task. Every person is different, and for some, finding undetectable ways to hide a stoma and bag helps in improving body image. For others, embracing a changed body and focusing on the positive helps to boost body image. There is no right way to cope with the many changes that come with surviving bladder cancer, but reaching out for support can help!
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Salloum, M. Self-esteem disturbance in patients with urinary diversions: assessing the void. Ostomy/wound management. Published December 2005. https://www.researchgate.net/publication/7333472_Self-esteem_disturbance_in_patients_with_urinary_diversions_assessing_the_void. Accessed October 23, 2017.
Ewing, G. The nursing preparation of stoma patients for self-care. J Adv Nurs. 1989;14(5):411- Published May 1989. https://www.ncbi.nlm.nih.gov/pubmed/2738237. Accessed October 24, 2017
Am I Going Bonkers?!
by Anita Brown
Deciding Between BCG and a Radical Cystectomy
by jimvhorne
How Do You Process That?
by Paul Hebert
© 2017–19 Health Union, LLC. All rights reserved. This information is not designed to replace a physician's independent judgment about the appropriateness or risks of a procedure for a given patient. Always consult your doctor about your medical conditions. BladderCancer.net does not provide medical advice, diagnosis or treatment. Use of the site is conditional upon your acceptance of our terms of use.

redpajama_set_name: RedPajamaCommonCrawl


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Car Scented Bags to eliminate odours and add a fresh and lively fragrance to your vehicle.
Used for its powerful antioxidant, antimicrobial, sedative, calming and antidepressant properties, lavender essential oil is the most used essential oil in the world today. Perfect for relaxation, insomnia and nervous disorders.
Rose essential oil has been used to treat health conditions and in natural beauty treatments for thousands of years. Used in the treatment of depression, for skin health and libido and hormone health.
Lavender essential oil is the most used essential oil in the world today, perfect for relaxation, insomnia and nervous disorders. Jasmine is a popular natural remedy for improving mood, overcoming stress and balancing hormones. Sandal essential oil is used to promote mental clarity and bring a sense of calmness into your life.
Bring the outdoors inside with these intense fragrant incense cones with this essence of roses. Helps to calm the nerves.
Enjoy the tropical and sweet fragrance of jasmine flowers with these intense fragrant incense cones. White Jasmine is used for purification, confidence, rejuvenation, liberation.


Our traditional homemade version of eggnog combines coconut, spices and of course, rum! No holiday festivity would be complete without this delicious, creamy concoction.
In bowl of blender, add evaporated milk, cream of coconut, coconut milk, sweetened condensed milk, rum (if using), vanilla extract and ground cinnamon. Blend on high until mixture is well combined.Pour coconut mixture into glass bottles or containers. Transfer to refrigerator. Chill until cold. To serve, stir or shake bottle well to combine. Pour coquito into small serving glasses. Garnish with ground cinnamon and cinnamon sticks, if desired.


Honglei Chen submitted Solution 1317852 to Problem 44349. Tick. Tock. Tick. Tock. Tick. Tock. Tick. Tock. Tick. Tock.
Honglei Chen submitted Solution 1314575 to Problem 564. How to subtract?
Honglei Chen submitted Solution 1314571 to Problem 564. How to subtract?
Honglei Chen submitted Solution 1314569 to Problem 564. How to subtract?
Honglei Chen submitted Solution 712915 to Problem 39. Which values occur exactly three times?
The solution is odd for me. Looks like the output starts with [2 3 ...] instead of [1 1 2 3 ...]. I have to use 2 and 3 as initial condition to get a match with the expected result.
Honglei Chen submitted Solution 609967 to Problem 9. Who Has the Most Change?
Honglei Chen submitted Solution 73856 to Problem 129. All capital?


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Aligning AUKUS and the Quad for Australia's defence
To facilitate the development of Australia's defence industrial base, Canberra also needs to engage with Quad partners
By Akash Sahu
The conversation on AUKUS and Australia's defence indicates the need for an advanced industrial base in the country, which can sustain not only the development of nuclear-powered submarines but also align capabilities like hypersonic and Artificial Intelligence (AI)-led military systems. Limitations like the lack of capital and human resources drag down Canberra's ability to develop better weapons.
Navies of India, Japan, Australia, and the US deployed during Malabar quadrilateral naval exercise in the southern sea off Yokosuka in Japan 2022. (Photo: Reuters)
Collaboration with AUKUS partners (the United States and the United Kingdom) is crucial for Canberra to procure, test and commercialise the latest military technologies. At the same time, deeper engagement with Quad partners Japan and India can help ensure a robust supply chain network for Australia's defence industry sector. Both AUKUS and the Quad are driven by shared motives of stabilising regional security in the Indo-Pacific.
The developing geopolitics of the Pacific necessitate credible offensive and defensive capabilities. Mick Ryan from CSIS suggests that Australia should move to short-term 'asymmetric' deliverables that can deter Chinese aggression, instead of its heavy focus on longer-term force structure plans.
Nuclear-powered submarines will take more than a decade to be ready, but escalating tensions demand at least some capability enhancement within 3–5 years. Since the threat is regional, it involves multiple stakeholders. The development of Australia's defence industrial base would be easier and faster through institutional collaboration with like-minded partners in the region.
Though AUKUS and the Quad
are different in their objectives and orientation,
they aim for a stable Indo-Pacific where the rules-based
order prevails. Canberra needs to find ways
of synchronising the two to
deter disruption in its
Pacific neighbourhood
Australia has a strong foundation in research and development. It also exports some defence items, such as the Nulka Active Missile Decoy and Bushmaster Protected Mobility Vehicle. According to the 2020 Force Structure Plan, Canberra will commit more than US$20 billion to develop high-speed weapons and build defence capabilities against ballistic missiles and high-speed weapons.
The Australian government's Defence Innovation Hub allocates grants to businesses developing AI technologies with military applications. Some of these include improving situational awareness using available intelligence and achieving intelligent virtual reality for better simulation, modelling and training of the Australian Defence Force.
Other programs like the Australian Industry Capability program and Local Industry Capability Plans aim to increase the participation of local defence manufacturers in defence infrastructure projects. Agencies such as Austrade have helped domestic tech firms set up research facilities to test advanced technologies and market commercialisation.
Limited capital affects manufacturing for the market and drags down innovation. Even though Australia attracts considerable foreign direct investment, the financing for defence manufacturing remains limited due to reasons including risk, institutional hurdles and lack of trust.
Japan ranked second globally in video game equipment exports in 2020 and has seen strong exports in digital items and semiconductor equipment despite the COVID-19 crisis. In 2021, Japanese foreign direct investment in Asia and Oceania amounted to US$655 billion, with investment in Australia exceeding US$162 billion. Japanese banks have led the growth in project finance in Asia and are apt contenders for financing progress in critical technologies.
The signing of a Reciprocal Access Agreement between Japan and Australia was accompanied by increased bilateral cooperation in the digital and cyberspace sectors. There is potential for work on innovative technologies, AI and autonomous robotic systems that target the grey-zone operations of an adversary.
India has a formidable base in cyber services and digital technologies. A Defence Artificial Intelligence Council and Defence AI Project Agency have been created within the defence establishment to work on AI-enabled defence products. sThe Indian Navy is working on 30 AI-related projects covering areas such as maritime domain awareness and autonomous systems, while the Indian Army has set up an AI centre of excellence in Mhow.
India's defence minister released 75 AI-related products at the 'AI in Defence' symposium in July 2022, which have been developed through partnerships between the armed forces, defence research organisations and private industry. India also declared massive 'Make in India' initiatives for self-reliance in defence manufacturing, which may be vital for critical defence supply chains in Canberra's defence industrial development plans.
Even though the Quad members have unique strengths in AI, their collaboration and investment in AI-related projects are minimal. For Australia, it may be useful to align more with Japan and India for focussed efforts on the military applications of AI technologies.
Washington has robust cooperation and investment across all three countries on AI. But there are reasons to believe that among the AUKUS partners themselves, technology sharing is a sensitive issue and there are comprehensive regulations in place to prevent it. The role of the US Congress in reducing export controls and streamlining information exchange will be critical to allowing greater collaboration.
Loosening export controls can help Canberra to capitalise on the strengths of Japan and India in developing Australia's defence base. The policy discourse around AUKUS in London will be important in engaging the Quad for achieving AUKUS objectives and Britain's own 'Indo-Pacific Tilt'.
The Sydney-based United States Studies Centre recommends an 'AUKUS Visa' to ensure a steady flow of talent to sustain the momentum in defence production under AUKUS. To facilitate the development of Australia's defence industrial base, Canberra also needs to engage with Quad partners.
Though AUKUS and the Quad are different in their objectives and orientation, they aim for a stable Indo-Pacific where the rules-based order prevails. Canberra needs to find ways of synchronising the two to deter disruption in its Pacific neighbourhood.
Akash Sahu is a researcher based in New Delhi who specialises in Indo-Pacific geopolitics and Southeast Asian studies.
Source: East Asia Forum
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UK Conservatives are on life-support
Evidence is mounting that Covid is bad for the brain
Russia and Asia within emerging new international order
Recollection of Mirpur Liberation on January 31, 1972
Gen Z at risk as technology changes work culture again
Doomsday Clock jitters and 'how to fix a broken planet'
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We pride ourselves on creating the highest quality design and installation of anyone in the Henderson, Nevada. No matter what size job is, our quality is always "top of the line".
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Transforming yards into imaginative outdoor living spaces is what Backyard Connection is all about,We approach each project on an individual basis, using space, sound and color to capture the essence of your home.
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For the best lawn care services in the local area, contact us today! We look forward to beautifying your outdoor space.


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Home / North West / Property / £19bn congestion-busting schemes for Greater Manchester unveiled
£19bn congestion-busting schemes for Greater Manchester unveiled
Nick Jackson
PLANS have been unveiled for several major new link roads costing up to nearly £19bn to ease congestion in Greater Manchester.
The proposals are contained in the DfT report, named the Manchester North-West Quadrant Study.
The new "Northern Corridor" proposal is in a report from the Department of Transport involves the M60 motorway, between junctions 8 at Stretford and 18 at Bury and the possibility of a new link road from the M6 junction at Orrell near Wigan to the M61 junction 5, at Westhoughton, near Bolton.
Traffic would then travel south on the M61 to junction 2 where the A666 joins the A580 (East Lancs Road), before joining a new direct route to the M62, connecting at Simister Island junction 18, of the M60.
Also included is the creation of a new orbital ring road outside of the existing M60 motorway, linking the M62 at junction 12, with drivers travelling along the M61 and on to the M62 at junction 18.
A final 'in-corridor package' could involve a programme of improvements to "significantly" enhance the capacity, performance and resilience of the M60 in an area including Bolton, Bury, Salford and Rochdale.
The study proposes that the Northern Corridor package could cost around £5.5bn, with the other In Corridor Package listed at more than £6bn and the Orbital or 'ring road' package predicted to cost as much as £7.3bn.
The DfT says all of the proposals are "speculative" and there are no firm details about what type of roads could be built, motorways, A-roads or dual carriageways.
A spokesman for the DfT said: "We don't have any firm details at this point on dates and costs — the report is just a guide at this point. The costs are speculative and are very unlikely to be that high in reality."
Former mill can be 'jewel in the crown' of...


Member states of the International Maritime Organization (IMO) have committed to developing a ban on heavy fuel oil (HFO) from Arctic shipping, along with an assessment of the impact of such a ban. Namely, a sub-committee (PPR6) was directed to develop a ban on heavy fuel oil use and carriage for use by ships in the Arctic, "on the basis of an assessment of the impacts" and "on an appropriate timescale". The decision was made during the 72nd session of IMO's Marine Environment Protection Committee, which resulted in the adoption of shipping's Green House Gas (GHG) strategy. Under the scheme, the shipping sector is required to reduce its emissions by at least 50 pct by 2050 compared to 2008. The Clean Arctic Alliance, a coalition of 18 non-governmental organisations working to end HFO use as marine fuel in Arctic waters, called for member states to make every effort to adopt and rapidly implement a ban by 2021, as proposed by eight IMO Member States and supported by other countries during the meeting.
The proposal for a ban, along with a proposal to assess the impact of such a ban on Arctic communities from Canada, was supported by Australia, Belgium, Czech Republic, Denmark, Estonia, France, Ireland, Japan, the League of Arab States, Poland, Portugal, Spain, Switzerland, and the UK. The coalition believes the ban is the best way to protect the Arctic communities and ecosystems from the threat of an oil spill, and the impact of black carbon emissions.


text:
Goldberg to headline 2018 WWE Hall of Fame class
July 14, 2020	0	By JohnValbyNation
Confirming what had been expected, WWE announced this morning that Goldberg will be inducted into their Hall of Fame over WrestleMania weekend in New Orleans.
WWE revealed the news via ESPN. "At the end of the day, any business, any venture that you embark upon, you want some type of acknowledgement," Goldberg told ESPN.com. "You always try to make it to a top. There's no reason to do anything unless you want to be the best."
Goldberg returned to WWE television for the first time in nearly 13 years towards the end of 2016. His in-ring return came after he was chosen to be the cover star of that year's video game, and things went better for Goldberg this time around than they did during his initial stint with the company in 2003 and 2004. He squashed Brock Lesnar in his first match back, then their program continued until WrestleMania as Goldberg dropped the Universal Championship to him.
Goldberg cut a post-show promo on the Raw after WrestleMania where he didn't close the door on stepping back into the ring. Before his WWE stints, he was the last true superstar that WCW produced.
Dave Meltzer had confirmed in the most recent edition of the Wrestling Observer Newsletter that Goldberg would be the headliner for this year's Hall of Fame class and wrote that the announcement was expected to be made soon. Goldberg being inducted into the Hall of Fame this year was first reported by freelance journalist Brad Shepard on Twitter.
The 2018 Hall of Fame induction ceremony will take place at the Smoothie King Center on Friday, April 6th.


My entire life I've been told I have an active imagination. Put me in a room with my dolls as a kid and I would've come up with an 8-part mini-series complete with detailed character backgrounds and an epilogue. While the label "imaginative" was given as praise, I've always loathed how I let my imagination get the best of me.
You see, my brain orchestrates these incredibly thorough stories for every aspect of my life. I have crazy ideas about how my prof interprets the phrasing of my emails, or why my friend had to reschedule plans. While I know that the tales I tell myself are ones of complete fiction, they are glued to the walls of my mind, tightly weaved into my perception of the world around me.
I presume that this is a mechanism to make sense of the information my brain has collected. The process of understanding the past and predicting the future is constructed from limited knowledge and I must fill the gaps to gain some semblance of control.
In some ways, I'm grateful for my imagination. As my mind runs through the endless questions and possibilities, it paves way for creativity. I can see different perspectives, feel more self-assured. However, it can also be limiting. I can become stubborn in thought, circulating around the same minute details and jumping to conclusions.
There's a natural inclination to attach ourselves to expectations, especially around things or people we care about. However, centering happiness or self-perception around an outcome will only heighten the fall. This is when the stories we tell ourselves, as riveting and truthful as they may seem, become destructive.
We become so comfortable in the scripts we create that we relate to the action in our heads more than the reality in front of us. Thus, we try to construct our reality to follow what's been playing in our head.
In times of panic, often as a result of anxiety, our reactions can be self-sabotage masked as self-preservation. When there is too much ambiguity, it is easy to feel vulnerable. It's easy to let the chaos in our head leak out in our behaviour, and without sufficient support, the floodgates will open. When the thoughts dry up, we harden even more around our insecurity. We build walls to conceal the breakdown of the interior.
These scenarios are often brought to life by external sources. School, work, relationships – we interpret facts by creating narratives, unaware of how they shape our wellbeing. We can disassociate from those emotions when the scenario is in hindsight; when the story has a clear conclusion. Our impulse to overthink and predict will still continue if we're unable to close that chapter. In a social media age, many of the main characters of our self-told stories orbit our lives, liking photos or sending texts. In the "privacy" of our electronic world, a personal notification still feels fairly intimate. The appraisals of lingering interactions are different than those of a run-in at Metro. We're unable to disassociate value from what we see, and we ultimately misplace our individual importance. Thus, the stories have not ended, and our imagination cannot completely subside.
It's impossible to not tell ourselves these stories. It's frustrating to admit that we don't know everything, that our inferences and analyses are incorrect. However, to realize our limitations may be the only way to achieve peace of mind. Our experience feels collective, but our interpretations are not universal. How we think and act is a reflection of a world we've constructed for ourselves.
Our imaginations are healthy until they aren't. I'm slowly learning to see that magic exists within the uncertainty. So, I will tell you what I've been (trying) to tell myself: there's nothing wrong with daydreams but remember that the stories we tell ourselves are just that – stories.


Levi's 512 Slim Taper Fit jeans. Slim fit jeans with tapered leg. 512 Slim Taper Fit jeans deliver everything you like about the 511 Slim but in a more tapered fit. This pair also has just the right amount of stretch for maximum comfort all day long.


She was speaking at a session on 'Global economy in transition' on the last day of the five-day World Economic Forum Annual Meeting 2019 here.
She said even central banks, banks, insurance companies and almost all industries would need to take climate change into account.
While some industries may be impacted more and some relatively less, all need to do their bit, she added.
"It's very easy to be lost in the headlines ... but what we really want to do ... is not focus on the headlines. So I'm not going to give you the economic outlook," Lagarde said.
"We are going to really focus on risks, opportunities, that are out there ... I'm going to start with one which is clearly on everybody's mind - climate change."
The planet's average surface temperature has risen by 1 degree Celsius since the late 19th century, she said, and most of this warming has occurred in the last 35 years.
World Bank's CEO Kristalina Georgieva said climate change is not only happening, it is happening faster than we thought it would. This is having a huge impact on the economy, she added.
An increase of 2.5 degrees Celsius shaves 15 per cent off the global GDP, she warned.
"(If) it goes up to 3 degrees we lose 25 per cent of the GDP and the cost in terms of suffering is unmeasurable," she added.


Do you have a question on how we can solve your technology solution for your business? Let us know how we can help, call us 1-212-947-2348 Option 0.
Please be sure to let us know when you are having a problem with any of your products or services. We're always here to help. Fill out the service request form or call us 1-212-947-2348 Option 2.
Let our experts build the perfect product for your business, fill out the service request form or call us 1-212-947-2348 Option 0.
Do you have a question on how we can solve your technology solution for your business? Let us know how we can help, call us 1-844-522-5333 option 0.
Please be sure to let us know when you are having a problem with any of your products or services. We're always here to help. Fill out the service request form or call us 1-844-522-5333 option 2.
Let our experts build the perfect product for your business, fill out the service request form or call us 1-844-522-5333 option 1.
Established in 2013, Smart Start Networks, Inc. has rapidly become a leader in the low voltage voice and data cabling industry. A New York based firm with over 50 years of combined industry experience; we offer you technology and security solutions for your peace of mind.


text:
Q: How to prevent 2.6 rounding off to 3 in visual basic? I am working on a program and the only problem i am facing is that a decimal like 2.6 is being rounded off to 3 or any other number like 1.7 is being rounded off to 2.
How can I make it to round 1.6 off to just 1?
Any help will really be appreciated.
Thanks in advance.

A: That feature is called "Truncate".  There's a Math.truncate() function.  From the docs:
Dim decimalNumber As Decimal

decimalNumber = 32.7865d
' Displays 32
Console.WriteLine(Math.Truncate(decimalNumber))

decimalNumber = -32.9012d
' Displays -32
Console.WriteLine(Math.Truncate(decimalNumber))

A: You are looking for Math.Floor Method (Double) as of from .NET Framework 2.0
Examples:
Floor(2.10) = 2
Floor(2.00) = 2
Floor(1.90) = 1
Floor(1.80) = 1
Floor(1.70) = 1
Floor(1.60) = 1
Floor(1.50) = 1
Floor(1.40) = 1
Floor(1.30) = 1
Floor(1.20) = 1
Floor(1.10) = 1
Floor(1.00) = 1

redpajama_set_name: RedPajamaStackExchange


So take time off for an underwater adventure. Let the breathtaking colours of the vast collection of fish and coral take your breath away.
Or imagine standing knee deep in warm waters as you cast your line in the backdrop of a setting sun. Or even a sunset cruise alongside the prancing dolphins as they head back home.
At Dhonveli, the in-house experience is unparalleled. Dhonveli have an array of fabulous options for you to select from. The choice of accommodation varies from garden bungalows to beach and water bungalows. But nevertheless, each option is unique and memorable.
The gastronomical experience of freshly caught sea food awaits you. Apart from that, Dhonveli have a choice of culinary delights guaranteed to tickle your taste buds and leave you feeling sated.
Take time off to visit Dhonveli Chavana spa. Let your mind experience the bliss of undisturbed peace as nimble hands work away the stress of another world. This is what relaxation is all about. Dhonveli sundown entertainment includes disco nights, live music and fun themes. A fascinating Maldivian Cultural show will enthrall you.
Amongst these pearl-like islands is one that is whiter than the rest called �Dhonveli� which means �white sand� in Divehi, the local language. Dhonveli is home to Chaaya Island Dhonveli, one of the world�s best resort hotels, boasting newly refurbished picturesque accommodation, soft pristine beaches, abundant vegetation, a seductive ocean and lagoon, and celebrated surf and dive sites.


text:
The Time Out
Suzanne Black
This article is from 2011
Interactive theatre with a motivational aspect
An interactive piece for 12 players who get to don swimming caps (with headphones inside) and become a water polo team. If this fills you with trepidation, it's meant to. The focus shifts between Ken, the cliché-spouting coach with motivational tidbits about the game ahead, and instructions relayed via the headsets from an unseen performer. The audience are enticed into revealing personal truths, taking part in trust exercises and forming bonds with a bunch of random strangers.
Exploring this 'loaded moment' (the charged minutes before an act), South-East of England-based Non Zero One pushes the bond of trust between audience member and performer. There's not just the trust that nothing humiliating will happen during the performance, but also that there will be a pay-off, a uniting theme for the disparate interactive elements.
The different sections of The Time Out work well together to create a coherent whole, in which the separate stages work up to fostering a certain relationship between members of the audience. It's an interesting exploration of group psychology where the techniques of interactive theatre are employed to good effect.
Forest Café, until 27 Aug, 1pm, free.
The last five minutes of a water polo match, and you get to decide the outcome. With motivational speeches, projections and soundscapes, The Time Out draws relationships out of strangers.
www.forestfringe.co.uk
It's Like He's Knocking (23 Aug 2010)
The magic of coincidence revealed, with free vodka
Sporadical (21 Aug 2009)
Sea shanties and your long-lost auntie
The theatre shows to look out for at the Edinburgh Festival Fringe 2011(9 Jun 2011)
Dance Marathon, Ten Plagues and Tuesdays at Tescos among highlights
Five groups preserving the spirit of the Fringe in 2009(23 Aug 2009)
5 Questions: Debbie Pearson from the Forest Fringe(6 Aug 2009)
A Rubbish Midsummer Night's Dream (27 Aug 2021)
High-energy and kid-friendly Fringe adaptation of Shakespeare's magical comedy is the most fun you'll find in a fitted bedsheet
Tickbox (20 Aug 2021)
An autobiographical Fringe work that tracks how a strong mother helped her daughter to cope with life as an Asian in Glasgow
Patricia Gets Ready (for a date with the man who used to hit her) (18 Aug 2021)
A restrained yet passionate Fringe monologue about domestic abuse that eschews traditional tropes of victimhood
Edinburgh Festival Fringe
Forest Fringe
Non Zero One


text:
Only One Thing Adds Up for Johnson
I wish I had nothing better to do than sit around and do 7 years worth of math. But, apparently that's what a number of old school Petty and Earnhardt fans have been doing since Sunday in an effort to discredit Jimmie Johnson's 7 Cup championships.
No math in the world is going to discredit what Johnson has accomplished. He is, and will be remembered as, one of the greatest, if not the greatest driver this sport has ever seen.
Richard Petty himself said last week that you can't compare eras. Well, you can't compare championship formats either. And, don't start trying to tell me that Johnson would have only won X titles if he'd had to outpoint the competition over 36 races like Petty and Earnhardt did.
Based on what? Your math?
That logic is flawed. Maybe Johnson didn't finish with the most points over 36 races because he didn't have to! The system he won under didn't require it. Who's to say he wouldn't have if he'd needed to?
All you need to know is that three guys have won 7 and no one else has more than 4. Under any format, that's impressive.


text:
Q: negative powers $(x^{-2} = 1/x^2)$ I need clarification for negative power of a number.
I understand $x$ to the power of $2$ is equal to $x\cdot x$
But how $x$ to the power of $-2$ is equal to $\dfrac{1}{x^2}$ ?

A: If you agree that for $x\neq 0$ you have $x^0=1$  then $x^{m-m}=1$ for any positive integer $m$
If you agree that  $x^a\cdot x^b=x^{a+b}$ then you have $x^{m-m}=x^m\cdot x^{-m}=1$
If you agree that $pq=1\Rightarrow q=\frac{1}{p}$ then you have $x^{-m}=\frac{1}{x^m}$

A: This is just a rule of exponents:
$$\frac{1}{x^n}=x^{-n}$$

A: For $n\in\mathbb N$, the equation $$x^{-n} = \frac{1}{x^n}$$ is a matter of definition. Mathematitians decided to extend the notation of $x^n=x\cdot x\cdots x$ (with $n$ repetitions) to negative values of $n$. Since, for positive numbers of $m,n$, you have $$x^{m+n} = x^mx^n,$$ you want to define $x^{-n}$ in such a way that it will still be compatible. This means that, for $m,n\in\mathbb N$, you want $$x^{m-n}$$ to equal $$x^m\cdot x^{-n},$$ and this can only be achieved by setting $x^{-n}$ as $\frac1{x^n}$.


text:
Feb. 3, 2014 10:30 am
This challenge has middle schoolers design hotels with cost-benefit analysis
Trey Smith wanted to find new ways to teach math in the classroom, so he organized the Philadelphia Engineering and Math Challenge as a lab for problem-solving-based approaches to teaching.
By Frida Garza / contributor
About Comcast View Open Positions
Student teams went head to head at the January 2014 Philadelphia Engineering/Math Challenge.
(Photo by Frida Garza)
Roughly 40 high school students are giving up their day to do math problems.
They're seated at round tables in the multipurpose room of Drexel University's James E. Marks Intercultural Center, each with 50 plastic building blocks in front of them, and are waiting for instructions on what to do with them.
This is the second event for the 2014 Philadelphia Engineering and Math Challenge. The students participate in four different projects throughout the day, including this engineering challenge and a task in which they must make a video to solve a math problem.
PEMC is a year-long education initiative started by Boys Latin Charter School teacher Trey Smith, with help from Drexel's Math Forum and the Philadelphia Education Fund's Math + Science Coalition and funding from Philly SEED. It's the first initiative of its kind, according to Smith, who would travel with his students from Boys Latin to out-of-state Quiz Bowl competitions, but didn't see any comparable programs in Philly.
At the front of the room, Smith takes the stage and addresses the kids. He introduces the next challenge: design a hotel that minimizes construction costs in order to get the greatest profit.
The students get a set of expressions, each detailing a specific cost associated with building. They need to come up with their design, build it and then calculate the profit correctly. They are essentially conducting a cost-benefit analysis, but they're middle school and high schoolers—and they aren't allowed to ask for help from the teachers and coaches who roam the room.
The day's event is designed to allow students to think about math differently: rather than learning a skill and doing a bunch of practice problems, the students take a hands-on, problem-solving approach to math. Smith hopes that teachers will also benefit from the challenge, taking the tools they learn back into their classrooms.
There's a Quiz Bowl portion to the event, but it's stuff like the hotel-construction challenge that feels new and different to many of the kids.
Christopher, an 8th grader from Germantown's STEM-focused Hill-Freedman World Academy, says he felt unsure about his math skills before he joined his school's team.
"I don't really like math," he said. "But I wanted to get it better at it for high school. I decided to join the team and it's been a lot of fun."
This is PECM's "pilot year," as Smith calls it. There are two more events this year, on March 19 and June 4. If the rest of their events are successful, Smith hopes to expand the challenges so that any middle schooler and high schooler can join.
Find the PECM winners below.
1st: Academy @ Palumbo
2nd: Boys' Latin
Engineering Solutions Event
2nd: Freire Charter School
Problem of the Week Presentations
1st: Olney High School
2nd: Academy @ Palumbo
Companies: Academy at Palumbo, Hill-Freedman World Academy, Drexel University, Math + Science Coalition
People: Trey Smith
Projects: Philadelphia Engineering/Math Challenge
Access | philly
Science Center's new $4M federal grant will bring STEM education to 300+ kids
Software Development | philly
Code Ninjas, a game dev and programming bootcamp for kids, is coming to Horsham
Educator Melanie Young is behind Math Girl Movement, a curriculum aiming to get girls into STEM
Watch: Students from Science Center's FirstHand program share its impact in a new short film


text:
Genesis Rabba (B'reshit Rabba  בראשית רבה) – najstarszy midrasz, tzn. komentarz haggadyczny, do Księgi Rodzaju. Jego autorstwo jest przypisywane rabinowi Hoszaji, działającemu w III w. n.e. w chrześcijańskiej  Palestynie. Jest częścią większego zbioru Midrasz Rabba.

Genesis Rabba w sposób ciągły, wers po wersie, nieraz słowo po słowie, komentuje pierwszą księgę Pięcioksięgu. Ciągłość komentarza została przerwana jedynie pod koniec księgi. Opuszczone zostały też genealogie i fragmenty nienadające się do wyjaśnień, jak. np. słowa Labana z Rdz 24,35-48.

Tekst zachowany jest w j. hebrajskim, zawiera jednak także fragmenty pisane po aramejsku, z widocznymi wpływami greki i łaciny.

Przypisy

Bibliografia

Linki zewnętrzne
  The Bereshith or Genesis Rabba wybór powiedzeń po angielsku dokonany przez Samuela Rapaporta na stronie sacred-texts.com.
 Midrash Rabbah Genesis, tom I, na stronie "Internet Archive"
 Beresheit Rabba po hebrajsku

Pisma judaizmu

redpajama_set_name: RedPajamaWikipedia


text:
Surgical Masks - Requirements and Test Methods
Face Mask Making Machine - Product Catalog
Raw Material Size List and Requirements for Different Face Masks
National foreword This British Standard is the UK implementation of EN 14683:2014. Itsupersedes BS EN 14683:2005 which is withdrawn. The UK participation in its preparation was entrusted to TechnicalC
National foreword
This British Standard is the UK implementation of EN 14683:2014. It supersedes BS EN 14683:2005 which is withdrawn.
The UK participation in its preparation was entrusted to Technical Committee CH/205/1, Medical textiles.
A list of organizations represented on this committee can be obtained on request to its secretary.
This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.
© The British Standards Institution 2014. Published by BSI Standards Limited 2014
ISBN 978 0 580 78520 7
ICS 11.140
Compliance with a British Standard cannot confer immunity from legal obligations.
This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 April 2014.
Medical face masks - Requirements and test methods
This European Standard was approved by CEN on 4 February 2014.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.
Previous : Raw Material Size List and Requirements for Different Face Masks
Next : No Information


Star Bazaar opens at South Mumbai with a focus on fresh food. Spread over 7500sq.ft area, the supermarket has everything to shop across multiple product categories with modern fixtures and amenities.
Star Bazaar, a unit of Trent Hypermarket Pvt. Ltd has launched its sixth store in the southernmost precinct of Mumbai. Spread over 7500 sq. ft., the store will serve as a convenient shopping destination for the residents of South Mumbai. To enable a seamless shopping environment, the store has been designed with modern fixtures and amenities.
The range offered by the multi format retail chain has been carefully selected to meet the everyday needs of the catchment with an aim to provide a superlative shopping experience with high quality products at great value. It offers customers a wide range of fresh fruits, vegetables, exotic produce, groceries & daily essentials, cheese, bakery products and a lot more. The store has a dedicated section of fresh meat, fish and cold cuts and positioned well to provide a modern shopping environment for customers with a focus on efficient customer services and convenience.
On the store inauguration, Noel Tata, Chairman Trent Ltd, Sanjiv Mehta, CEO and Managing Director, Hindustan Unilever Limited and Jamshed Daboo, Managing Director Trent Hypermarket Limited were present.
Star Bazaar is a joint venture between Tata and Tesco Enterprise a Tata-Tesco enterprise. Currently the grocery retail chain Star operates stores across Mumbai, Pune, Hyderabad Bengaluru and Kolhapur.


text:
Q: PHP - div inside echo function I want to get the article-loop divs to populate inside the echo function in the while loop. I'm trying to create a dynamic pagination that prints the article-loop divs dynamically per page. This is pretty straightforward so for example, when the user changes the page, I want the next few divs to display on that page and so on.
<?php
class Pagination {
public $current_page;
public $per_page;
public $total_count;

public function __construct($page=1, $per_page=20, $total_count=0) {
    $this->current_page = (int)$page;
    $this->per_page = (int)$per_page;
    $this->total_count = (int)$total_count;
}

public function offset() {
    return ($this->current_page - 1) * $this->per_page;
}

public function total_pages() {
    return ceil($this->total_count/$this->per_page);
}

public function previous_page() {
    return $this->current_page - 1;
}

public function next_page() {
    return $this->current_page + 1;
}

public function has_previous_page() {
    return $this->previous_page() >= 1 ? true : false;
}

public function has_next_page() {
    return $this->next_page() <= $this->total_pages() ? true : false;
}
}

$page = !empty($_GET['page']) ? (int)$_GET['page'] : 1;
$per_page = 2;
$total_count = 10;
$pagination = new Pagination($page, $per_page, $total_count);
?>

    <?php
        $i = $pagination->offset() + 1;
        $limit = $pagination->per_page;
        while($i<=$pagination->total_count && $limit>0) {
            echo '**IN HERE POPULATE THE ARTICLE-LOOP DIVS**';
            $i++;
            $limit--;
        }
    ?>

    <?php
        if($pagination->has_previous_page()) {
            echo '
previous_page().'">«
';
        } else {
            echo '
«
';
        }
    ?>
    <?php
        for($i=1; $i<=$pagination->total_pages(); $i++) {
            echo '
'.$i.'
';
        }
    ?>
    <?php
        if($pagination->has_next_page()) {
            echo '
next_page().'">»
';
        } else {
            echo '
»
';
        }
    ?>

A: Instead of
<?php
    $i = $pagination->offset() + 1;
    $limit = $pagination->per_page;
    while($i<=$pagination->total_count && $limit>0) {
        echo '**IN HERE POPULATE THE ARTICLE-LOOP DIVS**';
        $i++;
        $limit--;
    }
?>

you can use something like
<?php
    $i = $pagination->offset() + 1;
    $limit = $pagination->per_page;
    while($i<=$pagination->total_count && $limit>0) {
?>
        **IN HERE POPULATE THE ARTICLE-LOOP DIVS**  // html content

<?php
        $i++;
        $limit--;
    }
?>

You should use second one. Many at times first one doesn't work properly.


The day before the charity event, during an 8-hour session, Beady Eye went into RAK studios to record and mix their version of the song from The Beatles' "Let It Be" album.
Beady Eye have just made the track available for a limited period to download through their website. All proceeds from the sale of the track after VAT, credit card fees and mcps, (62p) will go directly toward the British Red Cross Japan Tsunami Appeal.
To download your copy, go here.
The Japan Disaster Benefit has so far raised over £150,000 for the British Red Cross Japanese Tsunami Appeal. To read more about the Red Cross' work in Japan and to donate, click here.