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Article| March 03 2003
Infectious Hepatitis C Virus Pseudo-particles Containing Functional E1–E2 Envelope Protein Complexes
Birke Bartosch
Laboratoire de Vectorologie Rétrovirale et Thérapie Génique, Institut National de la Santé et de la Recherche Médicale U412, IFR 128, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France
Jean Dubuisson
Centre National de la Recherche Scientifique-UPR2511, Institut de Biologie de Lille, Institut Pasteur de Lille, 59021 Lille Cedex, France
François-Loïc Cosset
Address correspondence to François-Loïc Cosset, LVRTG, ENS de Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France. Phone: 33-472-72-87-32; Fax: 33-472-72-80-80; E-mail: flcosset@ens-lyon.fr
J Exp Med (2003) 197 (5): 633–642.
https://doi.org/10.1084/jem.20021756
Revision Received:
Birke Bartosch, Jean Dubuisson, François-Loïc Cosset; Infectious Hepatitis C Virus Pseudo-particles Containing Functional E1–E2 Envelope Protein Complexes . J Exp Med 3 March 2003; 197 (5): 633–642. doi: https://doi.org/10.1084/jem.20021756
The study of hepatitis C virus (HCV), a major cause of chronic liver disease, has been hampered by the lack of a cell culture system supporting its replication. Here, we have successfully generated infectious pseudo-particles that were assembled by displaying unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles. The presence of a green fluorescent protein marker gene packaged within these HCV pseudo-particles allowed reliable and fast determination of infectivity mediated by the HCV glycoproteins. Primary hepatocytes as well as hepato-carcinoma cells were found to be the major targets of infection in vitro. High infectivity of the pseudo-particles required both E1 and E2 HCV glycoproteins, and was neutralized by sera from HCV-infected patients and by some anti-E2 monoclonal antibodies. In addition, these pseudo-particles allowed investigation of the role of putative HCV receptors. Although our results tend to confirm their involvement, they provide evidence that neither LDLr nor CD81 is sufficient to mediate HCV cell entry. Altogether, these studies indicate that these pseudo-particles may mimic the early infection steps of parental HCV and will be suitable for the development of much needed new antiviral therapies.
Worldwide, several hundred million people are infected with the hepatitis C virus (HCV;*
Abbreviations used in this paper: GFP, green fluorescent protein; HCV, hepatitis C virus; HCVpp, HCV pseudo-particles; LDLr, low density lipoprotein receptor; MLV, murine leukemia virus; PBMC, peripheral blood mononuclear cell; TU, transducing unit; VLDL, very low density lipoprotein.
reference 1). Progression to chronic disease occurs in the majority of HCV-infected persons. Infection is associated with an increased risk for liver disease and hepato-cellular carcinoma and has become the main indication for liver transplantation. HCV infection also increases the number of complications in HIV-infected people (2). No vaccine is currently available to prevent new infections and the only treatment for chronic hepatitis C is interferon-α therapy, either alone or in combination with the guanosine analogue ribavirin. However, only ∼40% of patients respond to treatment. Clearly, novel therapeutic strategies are urgently required as the health costs for HCV-infected people are predicted to spiral dramatically in the next few decades.
Unfortunately, no efficient and reliable culture system is available to amplify the virus (3), preventing the elaboration of reliable infection assays. Recently, a model for HCV replication, based on the self-replication of engineered minigenomes in cell cultures, has been established (4, 5). Although very useful in the study of HCV genomic replication, this system does not support production of HCV particles (6). HCV virus-like particles have been generated in insect cells or, alternatively, by pseudotyping vesiculovirus or influenza virus particles with modified HCV E1 and E2 glycoproteins, harboring alterations in their transmembrane domains (7–10). However, they were only poorly infectious or not at all, and inconsistencies in the results prevented their use in functional investigations of HCV cell entry (7, 8). Therefore, new approaches are sorely needed to study HCV assembly and infection to design HCV cell entry inhibitors and to study the humoral immune response against HCV. Here, we sought to overcome these hurdles by developing infectious, genetically tagged HCV pseudo-particles harboring unmodified E1 and E2 glycoproteins. High infectivity of these particles allowed the precise investigation of HCV E1 and E2 glycoproteins and their potential receptors in cell entry, HCV host-range, and neutralization by antibodies from HCV patient sera.
HCV E1E2 Expression Constructs.
The phCMV-7a expression vector (see Fig. 1 A) encoding the E1 and E2 glycoproteins from a 1a-type HCV was generated by inserting into a nonpackageable, CMV promoter-driven expression construct (see Fig. 1
B; reference 11) a DNA fragment encoding the last 60 residues of HCV core and all of the E1 and E2 proteins that were isolated from pSKp5E1E2, a construct derived from the pTM1p5E1E2(745) plasmid (12). The phCMV-E1 expression vector (see Fig. 1 A), expressing only HCV E1 glycoprotein, was derived from phCMV-7a by adding a stop codon to the COOH terminus of E1 with primers 5′-actggacgacgcaaagctgc-3′ and 5′-cgcggatcctacgcgtcgacgccggcaaa-3′. The resulting PCR fragment was digested with BamHI and ligated into BamHI-digested phCMV-7a. The phCMV-E2 construct (Fig. 1 A), only expressing an HCV E2 glycoprotein, was obtained by fusing the NH2 terminus of E2 to the COOH terminus of the HCV core using two PCR fragments generated with the two primer pairs 5′-tgcccgcttcagccgaaacccacgtcaccggggga-3′ + 5′-gccagaagtcagatgctcaagg-3′ and 5′-tactctgagtccaaaccg-3′ + 5′-gtgacgtgggtttcggctgaagcgggcacagtcag-3′. The two PCR fragments were fused in a second-round PCR. The resulting DNA fragment was digested with BamHI and ligated into BamHI-cut phCMV-7a. Expression vectors for E1E2 glycoproteins of 1b genotype were constructed by similar strategies.
Packaging and Transfer Vectors Constructs.
The CMV-Gag-Pol murine leukemia virus (MLV) packaging construct, encoding the MLV gag and pol genes, and the MLV-GFP plasmid, encoding an MLV-based transfer vector containing a CMV-GFP internal transcriptional unit, were described previously (see Fig. 1 B; reference 11). The pCMVΔ8.2 (13) HIV-1 Gag-Pol packaging construct contains all HIV-1 genes except env, which encodes the envelope glycoproteins, and nef. The HPPT-EF1α-GFP (14) HIV-1–based transfer vector contains the EF1α internal promoter driving the eGFP marker gene. The gene encoding GFP in this latter vector was replaced by the nlslacZ gene, encoding a nuclear-targeted β-galactosidase, to obtain the HPPT-EF1α-nlslacZ HIV-1–based vector. The phCMV-G (11) and phCMV-RD (15) expression vectors encode the vesicular stomatitis virus (VSV) G protein and the feline endogenous virus RD114 glycoproteins, respectively.
Cells.
The following cells were grown as recommended by the American Type Culture Collection: 293T human embryo kidney cells (CRL-1573); Huh-7 human hepatocellular carcinoma (16); PLC/PRF/5 human hepatoma (CRL-8024); Hep 3B human hepatocellular carcinoma (HB-8064); HepG2 human hepatocellular carcinoma (HB-8065); A431 human epidermoid carcinoma (CRL-1555); Caco-2 human colon adenocarcinoma (HTB-37); HCT 116 human colorectal carcinoma (CCL-247); HOS human osteosarcoma (CRL-1543); HT-1080 human fibrosarcoma (CCL-121); HT-29 human colorectal adenocarcinoma (HTB-38); LoVo human colorectal adenocarcinoma (CCL-229); MCF-7 human breast adenocarcinoma (HTB-22); TE671 human rhabdomyosarcoma (CRL-8805); U118 human glioblastoma (HTB-15); Jurkat human T cell leukemia (TIB-152); CEM human lymphoblastic leukemia (CCL-119); Molt-4 human lymphoblastic leukemia (CRL-1582); Raji Burkitt's lymphoma (CCL-86); CMMT Rhesus monkey mammalian carcinoma (CRL-6299); COS-7 African green monkey fibroblasts kidney (CRL-1651); Vero African green monkey kidney (CCL-81); PG-4 feline astrocyte (CRL-2032); BHK-21 golden hamster kidney (CCL-10); Chinese hamster ovary (CCL-61); BRL 3A rat hepatocytes (CRL-1442); NIH/3T3 mouse fibroblasts (CRL-1658); and QT6 quail fibrosarcoma (CRL-1708).
Cryopreserved primary hepatocytes, isolated from human adult biopsy samples checked for absence of HBV, HCV, and HIV, were purchased from Biopredic International and cultured on collagen I–coated plates according to recommendations of the supplier. Peripheral blood mononuclear cells (PBMCs), derived from healthy blood donors, were isolated, cultivated, and infected as described previously (14, 15).
Production and Detection of Pseudo-particles.
To generate HCV pseudo-particles, 293T cells were transfected with expression vectors encoding the viral components (see Fig. 1 B), i.e., E1E2 glycoproteins, retroviral core proteins, and packaging-competent GFP- or nlslacZ-containing retroviral transfer vectors. In brief, the Gag-Pol packaging construct (8.1 µg), the transfer vector construct (8.1 µg), and the glycoprotein-expressing construct (2.7 µg) DNAs were transfected into 2.5 × 106 293T cells seeded the day before in 10-cm plates using a calcium phosphate transfection protocol (CLONTECH Laboratories, Inc.), as described previously (11). The medium (8 ml/plate) was replaced 16 h after transfection. Supernatants containing the pseudo-particles were harvested 24 h later, filtered through 0.45-µm pore-sized membranes, and used in infection assays. Purified virus samples were obtained by ultracentrifugation of 10-ml viral supernatants through a 1.5-ml 20% sucrose cushion in an SW 41 Beckman rotor (25,000 rpm, 2.5 h, 4°C). Viral pellets were suspended in 50 µl PBS. Immunoblots of producer cell lysates and purified pseudo-particles were performed as described previously (15). Fractionation of the sucrose cushion purified viral pellets was achieved by an overnight equilibrium density centrifugation in a 20–60% sucrose gradient at 35,000 rpm and 4°C in a Beckman SW 41 rotor. Fractions of 0.7 ml were collected, precipitated with TCA, and analyzed by Western blotting.
Infection Assays.
Target cells were seeded in 12-well plates at a density of 8 × 104 cells per well and incubated overnight at 37°C. Unless otherwise indicated, dilutions of viral supernatants containing the pseudo-particles were added to the cells and the plates were incubated for 3 h. The supernatants were removed and the cells were incubated in regular medium for 72 h at 37°C. The infectious titers, expressed as transducing units (TU) per milliliter, were deduced from the transduction efficiencies, determined as the percentage of GFP-positive cells measured by FACS® analysis (15). Transduction efficiency of hepatocytes was determined by counting GFP-positive cells under a UV microscope.
HCV Pseudo-particles Can Be Assembled In Vitro.
HCV proteins are expressed from a single polyprotein precursor and individually released in their respective cell compartments on cleavage by cellular and viral proteases (3, 17). The envelope of HCV is thought to consist of two glycoproteins, E1 and E2, which reside in the ER, where they associate as heterodimers in their prebudding form (18, 19). Therefore, HCV E1E2 glycoproteins were expressed from a polyprotein containing the COOH terminus of the core (Fig. 1 A, C) protein, which serves as signal peptide for E1, and the E1 and E2 glycoproteins (Fig. 1 A; reference 18). This ΔCE1E2 polyprotein was expressed by transient transfection in 293T cells by using an expression construct under control of the CMV immediate-early promoter (Fig. 1 B). As shown by FACS® analysis (see Fig. 2 A) and in situ immunofluorescence (unpublished data) of permeabilized or nonpermeabilized transfected cells, the E1 and E2 glycoproteins were strongly expressed in the cell cytoplasm but could also be detected at the cell surface.
HCV pseudo-particles (HCVpp) were generated by assembling these full-length, unmodified E1 and E2 glycoproteins onto retroviral core proteins derived from MLV. Such pseudo-particles were obtained with E1E2 glycoproteins, derived from HCV genotypes 1a and 1b, that stand among the most prevalent and most resistant to interferon-α therapy (20). Retroviruses were chosen as platforms for assembly of HCVpp because their cores can incorporate a variety of different cellular and viral glycoproteins (15, 21, 22), and because they can easily package and integrate genetic markers into DNA of infected cells (23). HCVpp were produced by transfecting human 293T cells with three expression vectors (Fig. 1 B) encoding a ΔCE1E2 polyprotein, the MLV Gag-Pol core proteins, and a packaging-competent MLV–derived genome encoding the green fluorescent protein (GFP) marker protein. Control pseudo-particles were generated without glycoproteins, with the VSV-G glycoprotein (11), with the feline endogenous virus RD114 glycoprotein (15), and/or with assembly-defective MLV core proteins (MLV-G2A; reference 24). Analysis of immunoblots of transfected cells showed that the structural components of the pseudo-particles were readily detected at the expected molecular masses; i.e., ∼30 kD for E1, ∼60 kD for E2, ∼60 kD for VSV-G (Fig. 2, B and C)
, and ∼70 kD for the RD114 glycoprotein (unpublished data). MLV core proteins were detected as Gag precursors of 65 kD that were partially processed by the MLV protease into mature core components (Fig. 2 B). Viral particles were harvested from the supernatant of transfected cells and purified by ultracentrifugation through high density sucrose cushions, a purification process that removes loosely associated glycoproteins (25, 26). The E1 and E2 glycoproteins were readily detected in the pellets of purified virions generated with the wild-type MLV core particles but not with the viral assembly-deficient MLV-G2A mutant (Fig. 2 B). Comparison of the relative levels of VSV-G or HCV glycoproteins in producer cell lysates versus viral pellets suggested efficient incorporation of E1 and E2 into viral particles. Likewise, E1 and E2 glycoproteins could efficiently assemble on retroviral core proteins derived from HIV-1 (unpublished data), raising the possibility of pathogenic interactions between the two parental viruses in vivo because coinfection of patients with HCV and HIV is prevalent (2). Further evidence that the E1 and E2 glycoproteins were properly incorporated on retroviral core particles was provided by their analysis on density gradients (unpublished data). Immunoblots of viral particles fractionated through an equilibrium density centrifugation in 20–60% sucrose gradients indicated that the same fractions contained the Gag-Pol core proteins and the envelope glycoproteins, for both HCVpp and control pseudo-particles, at a sucrose density that corresponded to that of bona fide retroviral particles. Altogether, these results indicated that transient expression of E1 and E2 in 293T cells leads to specific and efficient incorporation of HCV glycoproteins into pseudo-particles generated with retroviral cores. Although HCV envelope glycoproteins have been shown to be retained in the ER in several cell types (19), our FACS® results of transfected 293T cells show that a small fraction of E1 and E2 reach the cell surface (Fig. 2 A), where MLV budding normally occurs (24), most likely due to saturation/leakiness of the ER retention on overexpression. Therefore, our data suggest that HCVpp may bud from the cell surface and not from the ER lumen.
To further investigate the role of HCV glycoproteins in incorporation on retroviral cores, we designed expression vectors that encoded individually either E1 or E2 glycoproteins (Fig. 1 A). Individual expression or coexpression of E1 and E2 from distinct expression units in trans led to normal levels of synthesis, as compared with expression of both glycoproteins in cis from a single E1E2 polyprotein (Fig. 2 C). Moreover, HCVpp were found to incorporate similar levels of E2 glycoprotein, whether it was expressed alone, or coexpressed with E1 in cis or in trans (Fig. 2 C). Finally, incorporation of E1 expressed alone or coexpressed in trans, with E2, occurred but at reduced levels, consistent with the chaperone activity of the latter protein (19).
HCV Pseudo-particles Infect Hepatic Cells.
We assessed the infectivity of HCVpp on a panel of target cell lines. Cells were incubated for 3 h with dilutions of supernatants containing the HCVpp, washed, and cultured until expression of the GFP marker gene harbored by the virions was measured by FACS® analysis 72 h later. Because HCVpp were generated with replication-defective viral components, this procedure allowed evaluation of the specific infectivity of the pseudo-particles after a one-round infection process. The capsid–protein content of HCVpp and control pseudo-particles generated with VSV-G was identical for virions prepared simultaneously (Fig. 2, B and C), allowing direct comparison of their respective infectivity. However, consistent with previous studies comparing batches of pseudo-particles prepared independently (15), we found important variations in capsid amounts (unpublished data) despite similar infectious titers (Fig. 3
A). Thus, to minimize artifacts due to differences in the quality of HCVpp stocks, infectivity was defined as TU per milliliter rather than TU per nanogram of capsid proteins. Infectious titers of up to 3 × 105 TU/ml (mean, 1.7 × 105 ± 105; n = 6) were reproducibly obtained for the HCVpp on Huh-7 human hepato-carcinoma cells (Fig. 3 A). Upon concentration of the producer cell supernatants by ultracentrifugation, infectious titers of 2 × 106 TU/ml, on average, could be readily obtained (Fig. 3 A). The other target cell types used in the infection assays displayed weaker (PLC/PRF/5, Hep 3B, HepG2, Caco-2, HT1080, HT-29, LoVo, MCF-7, U118, 293T, and Vero) or undetectable (A431, HCT 116, HOS, TE671, Jurkat, Molt-4, CEM, Raji, CMMT, Cos-7, BHK-21, CHO, PG-4, BRL 3A, NIH/3T3, and QT6) levels of infection with the HCVpp (Fig. 3 A), although all these cells were readily infected with control pseudo-particles generated with VSV-G and with titers over 7 × 106 TU/ml (unpublished data). This suggested that all the molecules necessary for HCV entry were sufficiently expressed in the former cell types. Infectious HCVpp could be generated at comparable efficiencies with E1E2 glycoproteins derived from HCV genotypes 1a and 1b (Fig. 3 B, h and i) and/or with retroviral core proteins derived from either HIV-1 or MLV (Fig. 3 B, d and h). Incubation of HCVpp and target cells with AZT, a reverse-transcriptase inhibitor that prevents conversion of the retroviral RNA genome of the HCVpp into integration-competent proviral DNA, inhibited transduction (Fig. 3 B, j). Moreover, long-term expression of GFP could be demonstrated after serial passaging of infected cells for more than one month (unpublished data). These results indicated that infection of the target cells led to integration into the host cell DNA and stable expression of the GFP marker gene transduced by the HCVpp. Additionally, no infection could be obtained with viral particles lacking both E1 and E2 glycoproteins, or lacking core proteins, or, alternatively, when using the MLV-G2A assembly-defective core proteins (Fig. 3 B, a–c). Altogether, these results demonstrate that HCVpp harboring the E1 and E2 glycoproteins and retroviral core proteins were infectious, leading to retroviral-mediated integration of their vector genome. Finally, despite reduced levels of E1 incorporation (Fig. 2 C), HCVpp generated with E1 and E2 glycoproteins expressed in trans, from distinct vectors, were nearly as infectious as those generated with the E1E2 polyprotein construct (Fig. 3 B, g and h). However, HCVpp assembled with either E1 or E2 glycoproteins only were 500-fold less infectious (Fig. 3 B, e and f), demonstrating that both glycoproteins need to be coincorporated on HCVpp to allow efficient virus entry and infection.
Hepatocytes represent the principal site of HCV replication in vivo, yet ex vivo studies have suggested that HCV may also infect lymphoid cells (3, 17). To address whether either cell types could be infected, we transduced human adult hepatocytes from biopsy samples and PBMCs from healthy blood donors. Because vectors derived from MLVs do not efficiently transduce these cells (14, 27), we took advantage of the possibility that HCVpp can be generated with HIV-1–derived vectors (Fig. 3 B) that allow efficient transduction of nondividing target cells (13–15, 27). Relative to infection of Huh-7 cells, these HCVpp could readily infect the primary hepatocytes derived from different donors (Fig. 4, A and B)
, yet the transduction efficiencies were negatively influenced by the quality and cell culture viability of the individual biopsies. In contrast, infection of PBMCs yielded undetectable levels of transduction with the HCVpp, although control pseudo-particles generated with VSV-G could readily infect these primary cells (unpublished data), as shown previously (14, 15). Altogether, these data suggested that HCVpp seem to reproduce the tropism of infection of wild-type HCV and preferentially infect hepatocytes and hepato-carcinoma cells.
HCV Pseudo-particles Are Neutralized by Antibodies and Patient Sera.
We sought to determine whether E1 and E2 and their interactions with cell-surface receptors specifically mediate infectivity of the HCVpp. We used a panel of monoclonal antibodies shown previously to specifically react against HCV-1a glycoproteins (9, 28–31). About half of these antibodies significantly inhibited (i.e., >20% inhibition) the infectivity of HCVpp. The H35 and H48 monoclonal antibodies, which recognize conformational epitopes on E2, reduced the infectivity of HCVpp of genotype 1a by up to 70% (Fig. 5
A). That neutralization was incomplete might be due to the fact that these monoclonal antibodies, which were developed and selected for binding to intracellular E1–E2 complexes, have been shown to be sensitive to post-translational modifications of E2 (30). Indeed, we found that, compared with its intracellular counterpart, E2 associated to HCVpp had undergone sugar modifications (Fig. 2, B and C), most likely as a result of its export through the cell secretory pathway (Fig. 2 A). Interestingly, none of these genotype 1a-specific antibodies could neutralize the HCVpp of genotype 1b (unpublished data) or the VSV-Gpp control pseudo-particles generated with VSV-G (Fig. 5 A). This indicated that infectivity of the HCVpp was specifically mediated by E1 and E2 glycoproteins and suggested that these pseudo-particles represent a valid model to investigate the early steps of HCV infection, i.e., receptor binding, membrane fusion, and envelope uncoating, as well as sero-neutralization.
Next, we investigated whether the infectivity of HCVpp could be neutralized by sera derived from chronically HCV-infected patients. HCVpp were incubated with sera before performing infection assays using Huh-7 as target cells (Fig. 5 B). No or nonsignificant neutralization of control pseudo-particles could be detected with patient sera or with control sera derived from healthy donors. However, most, if not all, of the patient sera could neutralize the infectivity of HCVpp in contrast to sera derived from healthy donors (Fig. 5 B). Neutralization levels varied between the different patient sera from 20 to 90% inhibition of infection. Interestingly, at a 1:50 dilution, sera derived from patients infected with genotype 1b neutralized HCVpp-1a and -1b with similar efficiency, suggesting that cross-neutralization occurs (Fig. 5 B).
LDLr and CD81 Are Not the Major Cell Entry Receptors of HCV Pseudo-particles.
Both the LDL receptor (LDLr) and CD81, a member of the tetraspanin family of receptors, have been proposed as putative HCV receptors (32–34). However, recent studies have questioned the role of these molecules in HCV cell entry, despite their unequivocal capacity to bind HCV particles and/or glycoproteins (35–40). Thus, we sought to investigate the contribution of either cell-surface molecule in the early stages of HCV cell entry by performing receptor competition assays. LDLr binds apolipoprotein B within LDL and apolipoproteins B and E (ApoB and ApoE) within very low density lipoprotein (VLDL) complexes; both complexes have been found associated with HCV particles in the plasma of infected patients (36). Purified LDLs and VLDLs were not found to significantly out-compete infection of HCVpp on Huh-7 LDLr-positive cells (Fig. 6
A). When monoclonal antibodies targeted to the receptor binding sites of ApoB and ApoE were preincubated with the HCVpp and control pseudo-particles, only the anti-ApoE antibodies could inhibit, albeit weakly, the infectivity of the HCVpp of genotypes 1a (Fig. 6 A) and 1b (unpublished data). This is consistent with our observation that HCVpp could not or hardly infect LDLr-positive cell lines such as HepG2, Jurkat, CEM, Molt-4, Raji, and TE671 (Fig. 3 A). Thus, although these results do not exclude a role of LDLr in HCV entry, possibly via association of LDLs/VLDLs with HCV particles (36), they suggest that LDLr is not the major receptor of HCVpp entry. Yet this assertion should be mitigated because in contrast to wild-type HCV (36), our HCV pseudo-particles, which were generated in nonhepatic cells (Fig. 2), may not be specifically associated with LDLs and VLDLs.
Recombinant GST-fusion polypeptides encompassing the large extracellular loop (LEL) of human CD81, which has been shown to bind HCV E2 (29, 34), were then used to investigate the role of hCD81 in HCVpp cell entry. The GST-fusion hCD81-LEL polypeptides, preincubated with the HCVpp, could specifically precipitate E1–E2 complexes (unpublished data), confirming their capacity to bind the E2 glycoprotein (29, 34), and were found to neutralize HCVpp infection on Huh-7 cells, yet with a relatively poor efficiency (Fig. 6 B). Inhibition ranged from 38 to 54% inhibition of infectivity and appeared to be dose-dependent. Consistently, preincubation of Huh-7 cells with JS-81 monoclonal antibody, which blocks binding of recombinant E2 to hCD81 (29), was found to inhibit infectivity of HCVpp in both Huh-7 target cells (Fig. 6 B) and human primary hepatocytes (Fig. 4, A and B). At high antibody concentrations (30 µg/ml), over 90% inhibition of infection could be obtained for HCVpp based on genotypes 1a (Fig. 6 B) as well as 1b (unpublished data). However, several target cells nonpermissive to infection by HCVpp, like TE671, Jurkat, CEM, Molt-4, and Raji cells (Fig. 3 A) were found to express high densities of hCD81 (unpublished data), indicating a lack of correlation between CD81 expression and HCVpp infection. Moreover, expression of hCD81 in nonpermissive NIH/3T3 mouse fibroblasts did not allow infection with the HCVpp (Fig. 6 C). Altogether, these results indicate that although hCD81 binds HCV E2 and might play a role in cell surface attachment of HCV, it is not sufficient by itself to allow HCVpp infection. Additional interactions between the virions and cell-surface components are likely necessary to allow HCVpp entry into cells.
Despite 13 yr of research, no efficient and reliable cell culture system is available to amplify HCV particles, making the study of the virus as well as the design of inhibitors and vaccines very difficult. Here, we have exploited two well-documented properties of retroviruses––the capacity to incorporate foreign glycoproteins (15, 21) and the capacity to integrate and express marker genes from replication-incompetent viral particles (23)––to generate a specific, fast, and reliable in vitro infection assay based on pseudo-particles displaying unmodified E1 and E2 HCV glycoproteins. Thus, we describe for the first time the formation of highly infectious HCV pseudo-particles that may share early cell entry properties with parental HCV.
Our data indicate that the E1 and E2 glycoproteins represent the envelope glycoproteins of wild-type HCV and are both required for infection; each glycoprotein is potentially involved in separate steps of cell entry (9, 17). Production of HCV virus-like particles in insect cells has already been reported (7, 39–41). However, these particles were not secreted and their extraction from intracellular compartments yielded virus-like particle preparations that were contaminated with cellular materials. Moreover, although useful to analyze interactions with the cell surface, they were not infectious and did not permit functional investigation of the putative HCV receptors in cell entry. Pseudotyped VSV or influenza virus particles have also been engineered with chimeric E1 and/or E2 glycoproteins, whose transmembrane domains were modified to allow transport to and assembly at the cell surface (8–10, 42). However, such modifications disturb conformation and functions of the E1–E2 complexes (42), and although VSV pseudo-particles stand among candidate HCV vaccines (43), their use as a tool to investigate HCV assembly and cell entry remains controversial (8). In contrast, we found that no structural modifications of the E1E2 glycoproteins were required for their correct assembly on retroviral and lentiviral cores nor for the generation of high titer infectious HCVpp with functional E1E2 glycoproteins.
Our findings that HCVpp show a preferential tropism for hepatic cells (Figs. 3 and 4), and that they are specifically neutralized by anti-E2 monoclonal antibodies as well as sera of HCV-infected patients (Fig. 5), further support the contention that the E1–E2 complexes assembled on HCVpp mimic those displayed by parental HCV. Therefore, the HCVpp described here may represent a valid model to investigate some of the infectious properties of HCV. Although we found evidence that proposed previously that HCV receptor candidates, LDLr and CD81 (32–34), may be involved in the early steps of infection, by mediating binding of HCV glycoproteins and viral particles (9, 10, 29, 31–40, 42), we show that none of these molecules were sufficient to permit infection (Fig. 6). Indeed, some human cells that express LDLr and/or CD81 were found to be nonpermissive to infection (Fig. 3). Additionally, ectopic expression of human CD81 did not restore infection in nonpermissive murine cells (Fig. 6). One may consider that these LDLr+/CD81+ cells are nonpermissive for HCVpp infection as a result of expression of a factor that prevents infection rather than as a result of lack of expression of a critical HCV receptor component. However, we favor the latter hypothesis because if a negative factor was expressed in these cells, it should restrict infection of both the HCVpp and the control pseudo-particles, for which the only difference lies in the composition of the viral envelope. Indeed, such cells could be readily infected when the HCV-specific receptor/entry phase of infection was bypassed by using alternative glycoproteins such as VSV-G or RD114 Env (Fig. 6 C; unpublished data). These data suggest that, like many other viruses (44, 45), the different steps of HCV entry (i.e., binding, internalization, and cell penetration) are mediated by different molecules. With the HCVpp described here, it should become possible to investigate these early events of HCV infection in detail and to identify novel HCV receptors or coreceptors.
We thank S. Levy for providing the hCD81-LEL purified polypeptides, S. Abrignani for the hCD81-transfected NIH/3T3 cells, M. Andrawiss for MLV-G2A construct, and C. Bain for the HCV patient sera. We thank D. Lavillette for input at the early stage of the paper, P. Kellam, F. Penin, G. Inchauspé, and H. Gilgenkrantz for stimulating discussions, and C. Goujon, D. Olivier, and V. Delauzun for excellent technical assistance.
This work was supported by Agence Nationale pour la Recherche contre le SIDA, the European Community (contracts QLK3 1999–00859 and QLK2-1999-00356), Association pour la Recherche contre le Cancer, Réscov National Hépatite C, Institut Pasteur de Lille, CNRS, and INSERM (Action Thématique Concertée "Hépatite C"). B. Bartosch is supported by a Marie Curie Fellowship from the European Community.
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Maurice, M., E. Verhoeyen, P. Salmon, D. Trono, S.J. Russell, and F.-L. Cosset.
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Sandrin, V., B. Boson, P. Salmon, W. Gay, D. Nègre, R.L. Grand, D. Trono, and F.-L. Cosset.
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. Two point mutations produce infectious retrovirus bearing a green fluorescent protein-SU fusion protein.
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. Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.
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. Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses.
Flint, M., C. Maidens, L.D. Loomis-Price, C. Shotton, J. Dubuisson, P. Monk, A. Higginbottom, S. Levy, and J.A. McKeating.
. Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81.
Flint, M., J. Dubuisson, C. Maidens, R. Harrop, G.R. Guile, P. Borrow, and J.A. McKeating.
. Functional characterization of intracellular and secreted forms of a truncated hepatitis C virus E2 glycoprotein.
Patel, A.H., J. Wood, F. Penin, J. Dubuisson, and J.A. McKeating.
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Agnello, V., G. Abel, M. Elfahal, G.B. Knight, and Q.X. Zhang.
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Monazahian, M., I. Bohme, S. Bonk, A. Koch, C. Scholz, S. Grethe, and R. Thomssen.
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. Characterization of low- and very-low-density hepatitis C virus RNA-containing particles.
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. Binding of hepatitis C virus E2 glycoprotein to CD81 does not correlate with species permissiveness to infection.
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. The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus.
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. Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2.
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. Topologic changes in the transmembrane domains of hepatitis C virus envelope glycoproteins.
Cocquerel, L., J.-C. Meunier, A. Pillez, C. Wychowski, and J. Dubuisson.
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The Rockefeller University Press
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HCV E1E2 and retroviral expression constructs. (A) A cDNA derived from the HCV polyprotein gene was used to express the E1E2 glycoproteins and the COOH terminus of the C protein, which provides the signal peptide for E1 (SP E1). The position of stop codons (asterisk) inserted in the expression constructs to terminate translation of the proteins is shown. The transmembrane domain (TMD) of E1 provides the signal peptide (SP E2) for the E2 glycoprotein (46). NTR, nontranslated region; IRES, internal ribosome entry site. (B) The expression constructs encoding the different components required to assemble infectious pseudo-particles are shown. The shaded boxes represent the viral genes and the marker gene (GFP) transferred to the infected cells. The open boxes show the cis-acting sequences. LTR, long terminal repeat; CMV, cytomegalovirus immediate-early promoter; PBS, primer binding site; Ψ, packaging sequence; PPT, poly-purine track; polyA, polyadenylation site. Vector particles were produced by cotransfection of plasmids harboring the packaging functions, the transfer vector and the viral glycoproteins (GP) into 293T cells. The viral GPs were the HCV E1 and E2 glycoproteins, expressed individually or as a ΔCE1E2 polyprotein (Fig. 1 A), the VSV-G, or the RD114 glycoproteins. The supernatants of transfected cells were collected during transient expression, concentrated by ultracentrifugation, and used for target cell transduction.
Assembly of HCV pseudo-particles. (A) Detection of E2 glycoproteins by flow cytometry. E1E2-transfected 293T cells were or were not permeabilized before staining with A4 or H53 monoclonal antibodies against E1 (28) and E2 (black areas; reference 47). Cell-bound antibodies were incubated with FITC-conjugated antibodies before FACS® analysis. The background staining was provided by staining the cells with the conjugated antibodies only (white areas). (B) Immunoblots of lysates of 293T-transfected cells and of pseudo-particles pelleted through 20% sucrose cushions are shown. Expression of E1 and E2 glycoproteins from HCV-1a genotype and of MLV core proteins was revealed in reducing and denaturing conditions with A4 and H52 monoclonal antibodies against E1 (28) and E2 (29) or with an anticapsid (MLV CA) antiserum. VSV-G expressed in control pseudo-particles was detected with the monoclonal antibody P5D4. The positions of the molecular mass markers (kD) are shown. E2 present within the viral pellets migrated slightly slower than the cell-associated forms due to modifications of the associated glycans by Golgi enzymes (not depicted). The presence of VSV-G in viral pellets generated with MLV-G2A assembly-defective core proteins is due to empty vesicles formed by VSV-G itself (48). (C) Immunoblots of lysates of 293T producer cells and of purified pseudo-particles generated with E1 or E2 expressed alone, from two separate expression units, or coexpressed in trans or in cis, from a ΔCE1E2 polyprotein (E1E2).
Infectivity of HCV pseudo-particles. (A) The results of experiments performed with different target cell types are displayed as TU per milliliter of supernatant (mean ± SD of up to six experiments) for HCVpp of 1a genotype. The infectivity on Huh-7 cells of HCVpp concentrated 100× by ultracentrifugation is shown. Similar results of infectivity and host-range were obtained for HCVpp of 1b genotype (not depicted). The infectivity of control pseudo-particles generated with VSV-G ranged from 7 × 106 to 2 × 107 TU/ml, depending on the target cell type (not depicted). (B) Infectivity of HCV pseudo-particles generated without E1E2 (a), without retroviral core proteins (b), with MLV-G2A assembly defective core proteins (c), with HIV-1 core proteins (d), with E1 or E2 alone (e or f, respectively), with E1 + E2 expressed in trans from two independents vectors (g), with HCV-1a E1E2 expressed from the same vector in cis (h), or with HCV-1b E1E2 (i). HCVpp were treated with 25 µM AZT (3′-azido-3′-deoxythymidine) before and during infection of target cells (j). Infectious titers (TU/ml) were determined on Huh-7 target cells and are displayed as mean ± SD of up to four experiments.
Results of infection on human primary hepatocytes. Infection assays were performed with HCV pseudo-particles generated with core proteins derived from HIV-1 rather than from MLV, which do not permit transduction of nonproliferating target cells (11). (A) Photographs of human adult primary hepatocytes infected by HCVpp of genotype 1a carrying a lacZ marker gene encoding a nuclear-targeted β-galactosidase. The cells were stained 3 d after infection with X-Gal, as described previously (26). Magnification, 150×. The specificity of infection was demonstrated by the absence of transduction when target cells were infected with HIV-1–derived pseudo-particles lacking glycoproteins (pp cores) or by the reduced levels of transduction when target cells were preincubated with 30 µg/ml JS-81 anti-CD81 antibodies before infection (+JS-81). (B) Quantitative analysis of the infectivity of HCVpp on hepatocytes derived from two donors is expressed as a percentage of infectivity determined on Huh-7 cells.
Neutralization of HCV pseudo-particles. (A) Results of neutralization of HCVpp-1a generated with HCV-1a E1E2 and with MLV core proteins preincubated before infection of Huh-7 cells with 20 µg/ml saturating concentrations of monoclonal antibodies against E1 (A4) or E2 (H31, H33, H35, H44, H48, H53, H54, H60, and H61) glycoproteins of genotype 1a. Hmix: neutralization assays with pooled antibodies. Negative control experiments were performed using no antibodies (Control) or using pseudo-particles generated with VSV-G (VSV-Gpp). Neutralization of the infectivity of these control pseudo-particles was achieved by using the VSV-G neutralizing 41A.1 monoclonal antibody. Results are expressed as the percentages of inhibition of the average infectious titers ± SD relative to incubation in the absence of antibodies. (B) Results of neutralization of HCVpp with HCV patient sera. HCVpp of genotypes 1a or 1b were preincubated for 30 min at room temperature with sera from chronically HCV-infected patients diluted 1:50 before infection of Huh-7 target cells. The genotype of HCV diagnosed in these patients is indicated in brackets. Results are expressed as percentages of inhibition of the average infectious titers ± SD relative to incubation with control sera from healthy individuals. Control experiments were performed using pseudo-particles generated with RD114 glycoproteins (RD114pp), rather than with VSV-G, which exhibits sensitivity to human complement (15). Efficient neutralization of the control pseudo-particles (not depicted) was demonstrated with a hyper-immune goat serum raised against the RD114 SU glycoprotein.
Cell entry receptor usage by HCV pseudo-particles. (A) Results of LDL receptor competition assays was performed with 10 µg/ml purified LDLs and VLDLs or using the monoclonal antibodies 5E11, 4G3 (anti-ApoB), or 1D7 (anti-ApoE) preincubated with the pseudo-particles before infection at the indicated concentrations (µg/ml) are shown. Mix: the 5E11, 4G3, and 1B7 antibodies were mixed at the indicated individual concentrations in the neutralization assays. (B) Results of CD81 receptor competition assays are shown. hCD81-LEL GST-fusion polypeptides were preincubated with the pseudo-particles before infection or JS-81 anti-CD81 antibodies were preincubated with the target cells before infection at the indicated concentrations (µg/ml). Results are expressed as percentages of inhibition of the infectious titers obtained on Huh-7 cells relative to titers obtained in the absence of inhibitors. Control experiments were performed using pseudo-particles generated with VSV-G (VSV-Gpp). (C) Comparative results of infection obtained on NIH/3T3, NIH/3T3-hCD81, and Huh-7 cells infected with HCVpp and with VSV-Gpp. Infectious titers (TU/ml) are displayed as mean ± SD of up to three experiments.
Hepatic Endothelial CCL25 Mediates the Recruitment of CCR9 + Gut-homing Lymphocytes to the Liver in Primary Sclerosing Cholangitis
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Tag Archives: David Spade
New Releases for the Week of September 14, 2018
(20th Century Fox) Boyd Holbrook, Trevante Rhodes, Jacob Tremblay, Keegan-Michael Key, Olivia Munn, Sterling K. Brown, Jake Busey, Thomas Jane, Yvonne Strahovsky. Directed by Shane Black
A young boy accidentally triggers the return to earth the greatest hunters the universe has ever seen – further evidence that kids suck. A disgruntled science teacher and a jaded crew of ex-soldiers can prevent the annihilation of the human race at the hands of the predators who just to make matters a bit worse have given themselves an upgrade.
See the trailer, clips, interviews, video featurettes and B-roll video here
For more on the movie this is the website
Release Formats: Standard, D-BOX, Dolby, IMAX, RPX, XD
Now Playing: Wide Release
Rating: R (for strong bloody violence, language throughout and crude sexual references)
A Boy. A Girl. A Dream.
(Goldwyn) Omari Hardwick, Meagan Good, Jay Ellis, Dijon Talton. On the night of the 2016 Presidential election when dreams were busy dying, a jaded L.A. club promoter meets a down-to-earth Midwestern girl. She helps him find the strength to chase his broken dreams, while he gives her the insight to discover her own.
See the trailer here
Release Formats: Standard
Now Playing: Regal Pointe Orlando
Rating: R (for language)
(Lionsgate) Anna Kendrick, Blake Lively, Henry Golding, Rupert Friend. When her best friend suddenly and mysteriously disappears from their small town, a mommy vlogger takes it upon herself to investigate. What she finds is a web of deceit, secrets, betrayal and revenge.
See the trailer and video featurettes here
Rating: R (for sexual content and language throughout, some graphic nude images, drug use and violence)
Unbroken: Path to Redemption
(Pure Flix) Samuel Hunt, Merritt Patterson, Gary Cole, Bob Gunton. The sequel to Unbroken, the 2014 biopic of Louis Zamperlini a former Olympic athlete turned prisoner of war in Japan during World War II, follows Zamperlini during the post-war years. He finds himself a wife but nightmares and other symptoms of PTSD plague him and threaten his marriage until Billy Graham helps him find a path to redemption.
See the trailer here.
Genre: Faith-Based Biographical Drama
Rating: PG-13 (for thematic content and related disturbing images)
(Veritas) Tammi Blanchard, Bruce Dern, David Spade, Dwight Henry. A single mother living hand to mouth inherits her grandfather's farmhouse whose lucrative water rights are coveted by her grandfather's business rival. With his grandson eager to prove himself ready to take over the family business, goons are hired to intimidate the young mother. Things begin to escalate out of control as the grandson fails to take into account how far a mother will go to protect her own daughter.
Now Playing: Picture Show Altamonte Springs
Rating: R (for violence, sexual menace and references, language and drug use)
(Vertical) Amandla Sternberg, Abbie Cornish, Christopher Eccleston, George Mackay. The daughter of a German mother and an African father faces uncertainty during the Nazi rise to power. Finding a sympathetic friend in the Hitler Youth whose father is a high-ranking Nazi official, she is forced to find her own way as things get more and more dire and her future more and more precarious.
Now Playing: Regal Winter Park Village
Rating: PG-13 (for thematic elements, violence/disturbing images, sexuality and language)
(Columbia) Matthew McConaughey, Richie Merritt, Bel Powley, Jennifer Jason Leigh. The true story of a teenage boy from Detroit who became a drug kingpin and a police informant in the 1980s.
See the trailer, clips and video featurettes here
Genre: Biographical Drama
Rating: R (for language throughout, drug content, violence, some sexual references and brief nudity)
ALSO OPENING IN ORLANDO/DAYTONA:
Sailaja Reddy Alludu
Score: A Film Music Documentary
ALSO OPENING IN MIAMI/FT. LAUDERDALE:
God is Brazilian
Kusama: Infinity
The Wild Boys
ALSO OPENING IN TAMPA/ST. PETERSBURG:
ALSO OPENING IN JACKSONVILLE/ST. AUGUSTINE:
SCHEDULED FOR REVIEW:
Posted in Weekly Preview	| Tagged 2018, A Boy A Girl A Dream, A Simple Favor, Abbie Cornish, Amandla Stenberg, Anna Kndrick, Bel Powley, Blake Lively, Bob Gunton, Boyd Holbrook, Bruce Dern, Christopher Eccleston, cinema, Cinema365, David Spade, Dwight Henry, Films, Gary Cole, Henry Golding, Jacob Tremblay, Jake Busey, Jennifer Jason Leigh, Keegan-Michael Key, Matthew McConaughey, Meagan Good, movies, Olivia Munn, Omari Hardwick, Previews, Rupert Friend, September 14, Sterling K. Brown, The Predator, Thomas Jane, Unbroken: Path to Redemption, Warning Shot, Where Hands Touch, White Boy Rick, Yvonne Strahovsky	| Leave a reply
Posted on February 9, 2016 by carlosdev
Drac is back.
(2012) Animated Feature (Columbia) Starring the voices of Adam Sandler, Selena Gomez, Andy Samberg, Kevin James, Steve Buscemi, David Spade, Keegan-Michael Key, Mel Brooks, Asher Blinkoff, Fran Drescher, Molly Shannon, Megan Mullally, Nick Offerman, Dana Carvey, Rob Riggle, Jonny Solomon, Chris Kattan, Jon Lovitz, Nick Swardson, Rose Abdoo. Directed by Genndy Tartakovsky
People and monsters may look different, but inside they really aren't. The things that don't conform are usually the things we fear; if we can't keep everything in its proper compartment, the world becomes a much scarier place.
Dracula (Sandler) is in a happy place. His daughter Mavis (Gomez) is happily married to human Johnny (Samberg) and the two have had a baby named Dennis (Blinkoff). Proud grandpa is sure that the baby is going to be a monster but Mavis isn't so sure that Dennis won't turn out to be human and therefore be raised among humans. This terrifies Drac who doesn't want Mavis to leave for the human world of Santa Cruz (where Johnny's parents live) and lose both his daughter and grandson, so he concocts a plan to force Johnny to "get his fangs" which must happen before his fifth birthday.
So as Mavis is checking out California, Drac and his friends are trying to find ways to force little Dennis to find his inner monster before the dreaded fifth birthday. Drac is desperate as Mavis seems to lean more to going human – so desperate he's even willing to enlist the help of his own father Vlad (Brooks) who doesn't approve of human-monster intermarriages. And when all these monsters and humans get together, all hell is going to break loose.
Director Tartakovsky is proving himself to be one of the ablest directors of animation in the business currently. I can only imagine what he would do with the resources of a Pixar, but nonetheless he generates some of the best stuff out there. This really isn't up to par with the best of his work but it's still better than most of the animated features out there.
Sandler's live action output in the last few years has been mostly lackluster but he seems more at home here. Many of his pals from his live action films are here, including James, Spade, Riggle and Samberg are here mainly reprising roles from the first film. Having Smigel in as a writer helps a lot; the plot is actually a little bit more sophisticated than the first film
The animation is pretty solid, although there is a bit of a cartoon-y feel to it which is just fine. The level of sophistication skews younger here but again, that's five. There's nothing here ostensibly offensive and parents won't be bored watching this with their kiddies. Certainly the writing is better than most, as the movie explores themes of tolerance and acceptance and will give the kids something to think about – maybe their parents too.
Still, this isn't anything that's going to make the animated fan sit up and take notice. It's pleasant and charming and of course anything with Mel Brooks in it is worth investigation, even if the comedy legend is barely in the movie at all. It's a diversion for the older crowd and a video babysitter for the younger. I won't recommend it highly but it's still a recommendation.
WHY RENT THIS: Kids will undoubtedly love this.
WHY RENT SOMETHING ELSE: Not terribly memorable.
FAMILY VALUES: Some mildly scary images and rude humor.
TRIVIAL PURSUIT: Jonathan's parents are played by real-life married couple Offerman and Mullally.
NOTABLE HOME VIDEO EXTRAS: Lots here as with most kid movies. There's a music video from 5th Harmony as well as a look at the development of the drawings from sketch to finished product. The Blu-Ray edition also includes a karaoke track, instructions on how to throw a Hotel Transylvania-themed party, a how-to guide on drawing the various characters and a feature on how the sound effects were made.
BOX OFFICE PERFORMANCE: $468.2M on a $80M production budget.
SITES TO SEE: Netflix (DVD/Blu-Ray Rental only), Amazon, iTunes, Vudu, Google Play, M-Go
COMPARISON SHOPPING: Mad Monster Party
NEXT: Jane Got a Gun
Posted in DVD Review	| Tagged Adam Sandler, Andy Samberg, animated feature, Blob, Chris Kattan, cinema, Cinema365, Columbia Pictures, Dana Carvey, David Spade, Dracula, DVD Reviews, Films, Fran Drescher, Frankenstein's Monster, grandfather, Hotel Transylvania 2, human, Invisible Man, Jon Lovitz, Keegan-Michael Key, Kevin James, Megan Mullally, Mel Brooks, Molly Shannon, Monsters, movies, Nick Offerman, Nick Swardson, Rob Riggle, Santa Cruz, Selena Gomez, Steve Buscemi, Transylvania, Wolfman	| 1 Reply
The Ridiculous 6
The Old West was never this wild.
(2015) Western Comedy (Netflix) Adam Sandler, Luke Wilson, Rob Schneider, Nick Nolte, Jorge Garcia, Terry Crews, Will Forte, Steve Zahn, Harvey Keitel, Jon Lovitz, Whitney Cummings, David Spade, Danny Trejo, Nick Swardson, Blake Shelton, Vanilla Ice, Julia Jones, Saginaw Grant, Lavell Crawford, Steve Buscemi, John Turturro, Chris Kattan, Norm McDonald, Jackie Sandler. Directed by Frank Coraci
With Westerns making a bit of a comeback lately, it's inevitable that there would be movies that poke fun at the genre. With Adam Sandler involved, that means there are a segment of people who will tune in no matter what. Others will stay away in droves.
White Knife (Sandler) is an orphan, taken in by the Apache when his mother was murdered. The Apache chief Screaming Eagle (Grant) teaches the young white boy how to fight, and the ways of the Apache warrior, which turn out to be somewhat more Zen than Caucasian culture gave them credit for. He is engaged to Smoking Fox (Jones), the most beautiful woman in the tribe. When a would-be outlaw (Zahn) tries to make trouble with her, White Knife makes short work of him.
However, there is trouble on the horizon. White Knife's biological father, whom he never knew, shows up at the camp. His name is Frank Stockburn (Nolte) and he wants to make get to know the son he never knew. Just then, his old outlaw gang led by the notorious Cicero (Trejo) shows up and Stockburn hides his stash with the tribe, knowing Cicero will take it. Instead, Cicero takes Frank who tries to lead the gang away from the peaceful Native Americans by saying the stash is buried by an old windmill. White Knife knows that unless Cicero gets the $50,000 that Frank had taken, the old man would be killed.
Having just met his dad, White Knife isn't willing to let him die. He heads out after them, vowing to obtain the money one way or another to rescue dear old dad. However, it turns out Dear Old Dad was very busy. White Knife discovers he has five half-brothers of other mothers – Ramon (Schneider) the Mexican bandito with the amazing diarrhea donkey, Lil' Pete (Lautner) who's the village idiot for more than one village but has a curiously strong neck, Chico (Crews) a saloon pianist who doesn't use his fingers to tickle the ivories but something a little more genitalia-like, Herm (Garcia) the nearly unintelligible moonshiner and Danny (Wilson) who was Abe Lincoln's bodyguard at Ford's Theater who inadvertently showed John Wilkes Booth (Kattan) the road to infamy.
All six of these men have peculiar talents. All six are eager to rescue their father. And all six are incredibly, incredibly ridiculous. The Old West will never be the same once they've hit town.
Sandler as I alluded to earlier seems to affect people in extreme ways; either they are utterly devoted to him, or they hate him with a passion. He seems to inspire no middle ground. I try to be as objective as I can about him but I find that when he tends to be a little more serious I actually appreciate him more; his humor tends to be a little more scatological and quite frankly, a bit more juvenile-appealing than is my own personal taste.
He has assembled an impressive cast, several of whom (Crews, Schneider, MacDonald) are all veterans of SNL or of Sandler films, as well as folks like Nolte – who does a fine job here unsurprisingly – and Lautner, who does a really good job here, surprisingly. The latter hasn't really exhibited much in the way of comedy chops previously, having done mostly action roles in movies that weren't all that good. However, he proves to have some timing and comic presence, neither of which are easy tasks. I found myself liking him here, which isn't my usual reaction to his performances.
Part of the problem here is that a lot of jokes fell flat for me, and it appears to a lot of other critics as well (see scores below). The whole thing about the amazing crapping donkey is humor at the level of five-year-olds and I know Sandler is better than that. Still, one can't argue with success and most of the movies of his that reap box office gold have been the ones that have been, to me, the most childish. I think that says a lot more about the movie-going public than it does about Sandler.
Westerns tend to lend themselves to wonderful vistas and extraordinary cinematography and this movie was no different as veteran Dean Semler gives us some pretty pictures to look at. This is one of Sandler's most cinematic films which makes it a bit ironic that it was released directly to Netflix as part of his four-picture deal with the streaming giant. However, it wasn't for lack of trying; the film was in development at three different theatrical studios until Netflix finally came in and got it made.
There has been some controversy about the portrayal of Native American culture and I don't intend to ignore it. While some outlets got nearly hysterical about it to the point of knee-jerkiness, the fact is that that several Native American extras had some concerns about the jokes made at the expense of their culture and eventually walked off the set when those concerns weren't addressed. The initial reports made it sound like there was an uprising the size of Little Big Horn; in actuality the affair involved four extras, far less than the 150 Native American extras who were employed by the film. Watching the movie, I didn't see anything that was more than culturally insensitive but the movie seemed to be that way to nearly everyone, in particular the white culture itself. Perhaps if the movie had been better written the insults would have seemed less egregious.
REASONS TO GO: A really good cast with Nolte and Lautner actually doing some good work. Lovely cinematography.
REASONS TO STAY: Unnecessarily dumb gags and situations. Attempts at parody miss the mark.
FAMILY VALUES: Some rude humor, mild profanity and sexual innuendo.
TRIVIAL PURSUIT: A group of Native American extras walked off the set due to what they perceived as negative and inaccurate portrayals of their culture; while initially the number of extras involved was reported to be about a dozen, sources close to the film put the number at four actors.
BEYOND THE THEATER: Netflix
CRITICAL MASS: As of 12/18/15: Rotten Tomatoes: 0% positive reviews. Metacritic: 18/100.
COMPARISON SHOPPING: A Million Ways to Die in the West
NEXT: Angels Crest
Posted in New Releases	| Tagged Adam Sandler, Apache, banditos, Blake Shelton, Chris Kattan, cinema, Cinema365, Danny Trejo, David Spade, diarrhea, donkeys, Films, half-brothers, Harvey Keitel, John Turturro, Jon Lovitz, Jorge Garcia, Julia Jones, kidnapping, Lavell Crawford, Luke Wilson, moonshine, movies, Netflix, Nick Nolte, Nick Swardson, Norm McDonald, outlaws, reviews, Rob Schneider, Saginaw Grant, Steve Buscemi, Steve Zahn, stiff neck, Terry Crews, The Ridiculous 6, Vanilla Ice, western comedy, Whitney Cummings, Will Forte	| Leave a reply
(Columbia) Starring the voices of Adam Sandler, Kevin James, Andy Samberg, Selena Gomez, Steve Buscemi, David Spade, Keegan-Michael Key, Mel Brooks. Directed by Genndy Tartakovsky
The Hotel Transylvania, once a refuge where monsters got away from it all, has now opened its doors to humans. After all, proprietor Dracula has a human son-in-law, right? And he also has a half-human half-vampire grandson, and therein lies the problem. His beloved daughter Mavis is becoming infatuated with the human world and is proposing to live in it and her son has shown absolutely no vampire traits whatsoever. Drac reasons that if her son is a vampire, Mavis might stay so that he can learn what it means to be a vampire. As every attempt to make his powers develop fails, Dracula will have to resort to the one thing he didn't want to have to do in a desperate attempt to keep his daughter close at hand – seek the help of his father, Vlad who is none too happy about the invasion of humans into the world of monsters.
See the trailer and a promo here.
For more on the movie this is the website.
Release Formats: Standard (Opens Thursday)
Genre: Animated Feature
Rating: PG (for some scary images, action and rude humor)
The Green Inferno
(Blumhouse Tilt) Lorenza Izzo, Ariel Levy, Aaron Burns, Kirby Bliss Blanton. A group of student activists travel from New York City to the Amazon, hell-bent on saving the rainforest. In the eternal tradition of "no good deed goes unpunished" they soon discover that they are not alone and that presence in the rainforest is hungry. From master horror director Eli Roth.
See the trailer, a featurette and a promo here.
Rating: R (for aberrant violence and torture, grisly disturbing images, brief graphic nudity, sexual content, language and some drug use)
(Warner Brothers) Robert De Niro, Anne Hathaway, Rene Russo, Anders Holm. A 70-year-old widower finds that he just isn't suited for retirement; he decides to get back into the workforce by getting a senior internship at a fashion company. The company's founder and CEO is at first skeptical of what her new intern brings to the table before discovering that he is a far greater resource than she ever thought possible.
See the trailer, clips, interviews and B-roll video here.
Rating: PG-13 (for some suggestive content and brief strong language)
(Bleecker Street) Tobey Maguire, Liev Schreiber, Peter Sarsgaard, Lily Rabe. At one time, Bobby Fisher was a household name in the western world. He was America's chess prodigy, perhaps the only one who was realistically able to compete against the Russians who dominated the game back in the day. However, Fisher had a whole bus full of demons haunting his every move and the higher the pressure was, the more bizarre his behavior became. Fisher walked a tightwire between genius and madness and would eventually fall off, turning from prodigy to legend.
See the trailer, interviews, clips and a featurette here.
Now Playing: AMC Altamonte Mall, AMC Downtown Disney, Regal Waterford Lakes, Regal Winter Park Village
Rating: PG-13 (for brief strong language, some sexual content and historical smoking)
(Roadside Attractions) Jeremy Irvine, Jonny Beauchamp, Jonathan Rhys Meyers, Ron Perlman. I will probably use this in the review (to be published tomorrow) but the Stonewall Riots of 1969 for the LGBT community has a very similar emotional resonance as Selma does for the African-American community. This is a fictionalized version of events with a young naive gay man coming to Christopher Street in New York City, then the center of gay activity basically in the country. He observes directly the violence directed at gays by the police, the institutional repression of gays and the marginalization. Joining a crew of street kids, he searches for his own identity while rejecting the labels put on him by the rest of the world. In the meantime, caught between two different worlds, his frustration and resentment grows until it boils over on one fateful night. An unusual turn of styles for director Roland Emmerich, who is better known for big budget sci-fi extravaganzas.
See the trailer, clips and a featurette here.
Genre: True Life Drama
Rating: R (for sexual content, language throughout, some violence and drug use)
Turbo Kid
(Epic) Munro Chambers, Lawrence Leboeuf, Michael Ironside, Edwin Wright. In an alternative future where the world ended in 1997, the Kid, a comic book-obsessed scavenger trying to survive in the Wasteland, meets up with a beautiful but mysterious young girl. They try to lay low but eventually run afoul of the sadistic self-proclaimed ruler of the Wasteland. Now The Kid will have to become the hero he's always dreamed of, armed only with an ancient weapon and blind faith. Could be a cult classic one day.
See the trailer and an interview here.
Release Formats: Standard (playing midnight on Friday and Saturday nights only)
Genre: Retro Apocalyptic Sci-Fi
Now Playing: Enzian Theater
Posted in Weekly Preview	| Tagged 2015, Adam Sandler, Andy Samberg, Anne Hathaway, cinema, Cinema365, David Spade, Eli Roth, Films, Hotel Transylvania 2, Jeremy Irvine, Jonathan Rhys Meyers, Keegan-Michael Key, Kevin James, Liev Schreiber, Lily Rabe, Mel Brooks, Michael Ironside, movies, Pawn Sacrifice, Peter Sarsgaard, Previews, Rene Russo, Robert De Niro, Ron Perlman, Selena Gomez, September 25, Steve Buscemi, Stonewall, The Green Inferno, The Intern, Tobey Maguire, Turbo Kid	| Leave a reply
Posted on July 23, 2013 by carlosdev
Adam Sandler tries to convince a mob of angry critics not to throw him and his friends over the cliff.
(2013) Alleged Comedy (Columbia) Adam Sandler, Kevin James, Chris Rock, David Spade, Salma Hayek, Maria Bello, Maya Rudolph, Nick Swardson, Steve Buscemi, Colin Quinn, Tim Meadows, Jon Lovitz, Taylor Lautner, Shaquille O'Neal, Alexander Ludwig, Georgia Engel, Peter Dante, Oliver Hudson, Allen Covert, Steve Austin, April Rose. Directed by Dennis Dugan
Growing older is not merely learning to cope with the aches, pains and infirmities that are visited upon our bodies; it is also learning to deal with our own increasing irrelevance as those who are younger are catered to and worshiped while those who were once sitting pretty in the sun are shuffled aside into the shadows. Growing old sucks.
But it doesn't necessarily have to for Lenny Feder (Sandler). A successful career as a Hollywood agent has allowed him to move his family back to Amoskeag Lake where he grew up and where his friends Eric Lamonsoff (James) – a world class mama's boy – Kurt McKenzie (Rock), a sharp-witted man and lady's man gone to seed Marcus Higgins (Spade). All are family men who are feeling a little bit of middle age envy as their responsibilities as husband and parents have forced them to put aside the fun and games of their younger days.
Lenny's restlessness increases as the last day of school coincides with the anniversary of Kurt and Deanne (Rudolph) which Kurt wants to celebrate with a big party at Lenny's house, much to the surprise of Lenny's wife Roxanne (Hayek) who although not consulted is all for an 80s-themed shindig.
But there's a hell of a lot going on. When Lenny and his bro's head on over to the quarry that they swam in as young men, they find it infested with the college kids who are none too happy to find mere townies invading what is their space. Particularly put out is Andy (an uncredited Lautner), leader of a bunch of preppy frat rats who bump chests, give weird intricate handshakes and spout off things like "Yeah, that just happened" and "Boo-yah!" ESPN couldn't pay enough for the free advertising.
Now the Hideous Preppy Frat Villains are out looking to crash Lenny's party and put a beat-down on him and his 2OldCrew. Lenny's high school nemesis, Tommy Cavanaugh (Austin) who terrorized him back in the day, is the boyfriend of Lenny's daughter 's hot dance teacher (Rose) and continues to frighten Lenny even now, a trait which he's passed on to his son.
All of the buddies are basically dealing with mid-life issues from Marcus' attempts to bond with switchblade-wielding son he never knew he had to Lenny's feeling that he's missing out on things because he spends so much time doing the "right" things. Can they resolve them, show the frat kids who's boss and throw one hell of a party?
I have to admit that I didn't mind the first Grown Ups so much. It had some laughs and some real moments in which the talented comics (which also included Rob Schneider who wasn't able to fit this movie into his schedule – and when Rob Schneider can't find time to do your movie, you know there's a problem) bonded and showed some genuine warmth. It looked like a lot of fun to make which made a second film inevitable (as did the massive box office the first film did). Sadly, I'm not looking forward to a third film, not even a little bit.
This is as bad a movie as I've seen this year. There's nothing remotely funny going on here, at least other than in the trailer in which you can see all of the movie's best moments. And as for plot, what you have here is a series gags strung together without rhyme or reason. It's very much throwing whatever you can find against the wall and hoping it sticks and ladies and gents, moose piss sticks to any wall just fine.
This is a real waste of talent and time. I wish it were different but I had more fun glancing at Da Queen and shaking my head than I did watching the goings on onscreen. I can't put it any more plainly – this is the very worst movie you'll see this year.
REASONS TO GO: Because you missed the movie's trailer where all the best moments can be found.
REASONS TO STAY: It's. Not. Funny.
FAMILY VALUES: Quite a bit of crude and sexually suggestive language and some male butt shots.
TRIVIAL PURSUIT: This is Adam Sandler's first sequel.
CRITICAL MASS: As of 7/23/13: Rotten Tomatoes: 7% positive reviews. Metacritic: 19/100; critics hated this and for once I can scarcely blame them.
COMPARISON SHOPPING: I Love You, Beth Cooper
NEXT: Pacific Rim
Posted in New Releases	| Tagged 80s party, Adam Sandler, Amoskeag Lake, Chris Rock, cinema, Colin Quinn, Columbia Pictures, comedy, cops, David Spade, Films, Georgia Engel, Grown Ups 2, home town, J. Geils Band, Jon Lovitz, Kevin James, last day of school, Maria Bello, Maya Rudolph, mid-life crisis, movies, Nick Swardson, quarry, reviews, Salma Hayek, Saturday Night Live, school bus, Shaquille O'Neal, SNL, Steve Austin, Steve Buscemi, Taylor Lautner, Tim Meadows	| Leave a reply
Posted on October 8, 2012 by carlosdev
Count Dracula dispenses some fatherly advice.
(2012) Animated Feature (Columbia) Starring the voices of Adam Sandler, Selena Gomez, Andy Samberg, Kevin James, Steve Buscemi, Fran Drescher, David Spade, Cee-Lo Green, Molly Shannon, Jon Lovitz, Brian James, Luenell, Rob Riggle. Directed by Genndy Tartakovsky
Being the father of a teenage daughter is a special kind of hell. We, as dads, know what the world is capable of and it's natural to be a bit overprotective of our baby girls. Still, it must be way harder to raise a daughter in a world where the majority wants to kill off your entire species.
Count Dracula (Sandler) has more reason than most to fear the humans. While vacationing in Hawaii he met a beautiful vampire named Martha whom he married and had a daughter with. However, angry locals discovered that they had two vampires in their midst and set fire to the house. Dracula was able to rescue his daughter but not his wife. Enraged over the loss of his wife, he swears to protect his daughter from the real monsters and builds a castle in Transylvania that will be forever hidden from human view, a place where his fellow monsters can relax, retreat and be themselves. It's Hotel Transylvania and every boy and ghoul is just dying to get there (couldn't help it).
Operating on a strictly humans forbidden basis, the hotel becomes a success. Drac's daughter Mavis (Gomez) is now 118 and getting restless with her protected lifestyle. She wants to live (which is a bit problematic for the undead), travel, see the world and experience everything. Dracula seems to be all for it at first, but this turns out to be a bit of a ruse.
All of their friends are gathering, from the henpecked Frankenstein (James) and his shrewish wife Eunice (Drescher) to the exhausted werewolf dad Wayne (Buscemi), his also exhausted wife Wanda (Shannon) and their brood of…I don't know, say 150? – werewolf pups. Then there's the coolest mummy ever, Murray (Green). They're all gathering for Mavis' birthday, an annual event.
Into this chaos rolls (or rather walks) Johnny (Samberg), a hiker who could pass for a surfer or a stoner or both. Rather than being terrified (although at first he does have a bit of a panic attack), he becomes fascinated by the monsters and one in particular – Mavis with whom he is instantly smitten.
Dracula is in a quandary. Not only must he keep his daughter safe from this human, he must keep the guests from finding out about him or else their confidence in their safety at the hotel would be compromised. The problem is that Johnny really isn't a bad guy once you get to know him. And Mavis has developed feelings for him as well. What's a dad – and the king of the undead at that – to do?
Tartakovsky, best known for his Cartoon Network classics Star Wars: Clone Wars, Dexter's Laboratory and Powerpuff Girls tackles his first animated feature with an all-star cast and a much more detailed animation than you usually get on the hideously bad CN. It doesn't hurt that he has an all-star cast to work with.
Sandler usually has a tendency to be over-the-top but here he's actually fairly restrained. We get all of the things that are endearing about him and none of the things that are irritating. It's one of his better performances in the past five years. He gives the overprotective Dracula a touch of humanity that many other performances lack. Yes this is a comedy and meant to be about as scary as Mickey's Not-So-Scary Halloween Party at Disney World but that doesn't mean it has to be depth-less. Sandler gives the character a whole lot of reasons for us to identify with him.
Most of the other characters are given less to work with, although Samberg actually acquits himself nicely as the heart-of-gold stoner dude and Lovitz gets to go a little bit over-the-top with his Quasimodo chef's role. Sadly, that's about the extent of it. While there are plenty of in-jokes that adults – particularly those who love classic horror films – are going to chuckle at, there really isn't much in the way of story which we've all seen more than once before.
The universe inhabited here is familiar and fun and makes internal sense. While the ending scene with the rap concert at Mavis' party is unnecessary and simply awful, almost Disney Channel-esque in it's bad rappery (Cee-Lo baby – you're better than this, dawg) most of the rest is merely predictable. There are some fun little gags – like Dracula making a demonic face every time he is annoyed.
This isn't groundbreaking or head-turning in any real way. It's merely pleasant entertainment that will keep the kids satisfied and the parents won't necessarily be squirming in their chairs waiting for the show to end. It will probably end up being a Halloween perennial, showing up on cable and later on broadcast TV every October without fail. In that sense it will become a classic because of repeated viewings but it will be one that while inoffensive isn't necessarily a classic because of exceptional merit.
REASONS TO GO: Nicely drawn universe. Plenty of in-jokes for classic horror fans.
REASONS TO STAY: Story is kind of ho-hum. Rap scene at the end gratuitous and stupid.
FAMILY VALUES: There are a few scary images, some rude humor and a bit of cartoon action.
TRIVIAL PURSUIT: The film was released on World Rabies Day.
CRITICAL MASS: As of 10/8/12: Rotten Tomatoes: 45% positive reviews. Metacritic: 48/100. The movie is getting seriously mixed reviews..
MONSTER LOVERS: Among the famous movie monsters that make an appearance here are Dracula, Frankenstein's monster, the Werewolf, the Invisible Man, the Mummy, the Blob, the Fly and Quasimodo.
NEXT: The Hunt for Red October
Posted in New Releases	| Tagged Adam Sandler, Andy Samberg, animated feature, birthday party, Cee-Lo Green, cinema, Columbia Pictures, David Spade, Films, Fran Drescher, Genndy Tartakovsky, Hotel Transylvania, humans, Jon Lovitz, Kevin James, Molly Shannon, Monsters, movies, persecution, reviews, Rob Riggle, Selena Gomez, spooks, Steve Buscemi, Transylvania, vampires	| Leave a reply
(Columbia/Sony Animation) Starring the voices of Adam Sandler, Selena Gomez, Andy Samberg, Kevin James, Cee-Lo Green, Steve Buscemi, David Spade. Directed by Genndy Tartakovsky
In an effort to insulate his willful daughter Mavis from the world, Count Dracula decides to turn his castle into a hotel for monsters only. There, he and Mavis can hang out with Frankenstein and his bride, the Wolf Man, the Mummy, the Invisible Man and all of their friends. However when a human hiker stumbles into the Castle and Mavis takes a liking to him, all manner of chaos will ensue. However, I don't know how eager I'd be to have Dracula as my father-in-law.
Rating: PG (for some rude humor, action and scary images)
(Tri-Star) Joseph Gordon-Levitt, Bruce Willis, Emily Blunt, Paul Dano. In the future time travel has been discovered but it's illegal; so only criminals use it, to dispose of their "problems." They send the people they want to whack back in time to the present day where an assassin – called a Looper – murders them and disposes of the body. Nice. Neat. But for one Looper, he is put in a very disconcerting situation when his assignment turns out to be his own future self. So would that be homicide or suicide?
See the trailer, an interview and a featurette here.
Rating: R (for strong violence, language, some sexuality/nudity and drug use)
OMG – Oh My God!
(Viacom18) Mithun Chakraborty, Paresh Rawal, Akshay Kumar, Poonam Jhawer. When an antique shopkeeper loses everything to a tornado, his faith in God wavers to the point where he makes it his mission to convince others of the non-existence of God. This leads to mayhem until he gets an unexpected visit from Lord Krishna himself.
(Universal) Anna Kendrick, Brittany Snow, Anna Camp, Rebel Wilson. A shy young girl who has just enrolled in college is roped into the a cappella group, even though she'd much rather listen to music than make it. However she resolves to take the group from their traditional arrangements to mash-ups of modern hits which might lead them to success at competitions. Or unfair comparisons to "Glee."
See the trailer and featurettes here.
Genre: Musical
Rating: PG-13 (for sexual material, language and drug references)
(Goldwyn) Frank Langella, Susan Sarandon, James Marsden, Liv Tyler. The concerned children of a retired thief are worried that he is unable to care for himself living alone. So against the old man's wishes, his son buys him a robot that is programmed to improve his physical and mental health. The old man soon finds his life changing in some ways – and returning back to what it used to be in others.
Rating: PG-13 (for some language)
(Radius) James Purefoy, Max von Sydow, Pete Postlethwaite, Alice Krige. A soldier in the 16th century discovers his actions have damned his soul. He vows to redeem himself and refrain from violence for the remainder of his days, but when a supernatural threat descends upon the land, he discovers that his skills may be the only thing to save his home and people.
See the trailer, featurettes, a clip and a link to stream the full movie from Amazon here.
Rating: R (for violence throughout)
Won't Back Down
(20th Century Fox) Maggie Gyllenhaal, Viola Davis, Oscar Isaac, Holly Hunter. Two mothers, horrified at the state of education in their poverty-level neighborhood, resolve to improve the quality of the education in their area. Met with opposition from the city and education bureaucracy they find themselves forced to take the fight much further than they thought possible.
Rating: PG (for thematic elements and language)
Posted in Weekly Preview	| Tagged Adam Sandler, Akshay Kumar, Alice Krige, Andy Samberg, Anna Kendrick, Brittany Snow, Cee-Lo Green, cinema, David Spade, Emily Blunt, Films, Frank Langella, Holly Hunter, Hotel Transylvania, James Marsden, James Purefoy, Joseph Gordon-Levitt Bruce Willis, Kevin James, Liv Tyler, Looper, Maggie Gyllenhaal, Max von Sydow, movies, OMG - Oh My God!, Oscar Isaac, Paul Dano, Pete Postlethwaite, Pitch Perfect, Previews, Rebel Wilson, Robot & Frank, Selena Gomez, Solomon Kane, Steve Buscemi, Susan Sarandon, Viola Davis, Won't Back Down	| Leave a reply

redpajama_set_name: RedPajamaCommonCrawl


Popovers are, or at least my goal is to make them be, virtually hollow inside, a little custardy, with a delicious crusty/chewy brown exterior. Steam escapes as we break them open and dip into a puddle of maple syrup. A dab of raspberry preserves is another taste treat. Butter is a traditional condiment.
This recipe has less than half the fat, more protein, and triple the fiber of a traditional recipe. And they still pop!
1 Preheat oven - and popover pan - to 450°F.
2 Allow milk, eggs, and flour to come to room temperature. This step is not negotiable.
Note: You can substitute low-fat or whole milk for the fat-free milk. Milk can be microwaved for a few seconds to speed warming. Microwaving eggs is not recommended.
3 Sift salt with flours. Whisk eggs and egg white in a medium-sized bowl. Whisk in milk. Whisk in melted butter. Whisk in flour, stirring thoroughly to break up lumps and create a smooth batter. Let the batter sit at room temperature for at least 30 minutes, preferably one hour. This waiting period is also not negotiable.
Notes: Using all all-purpose flour works best in this recipe. I've substituted white whole wheat flour for 1/2 of that, with good results. When I use more whole wheat flour the results are not as puffy and a little heavier but certainly not inedible.
I have pop-over success without using baking powder. You can use a pinch (1/4 teaspoon) if you find your popovers fail to popover.
Many recipes will instruct to whisk the batter just until small lumps appear. This advice is more suitable when a leavening agent such as baking soda is used; a lumpy batter slows the leavening reaction during mixing, keeping acid and base away from each other until baking is underway. Since popovers rise with steam, this keep-batter-lumpy instruction is moot. In fact, the waiting period I describe as not negotiable encourages formation of gluten which creates a strong framework as the popovers puff. Gluten is formed when water contacts wheat protein, and breaking up lumps puts more moisture in contact with protein.
4 Carefully remove the popover pan from the oven, spray cups with oil (one instance PAM is useful), ladle batter evenly into cups (they'll be about 1/2 full), get pan back into hot oven pronto.
5 Bake for about 20 minutes at 450°F until puffed and golden. Reduce oven temp to 350°F and bake for another 15 minutes. Remove popovers from pan immediately, sliding a knife around edges if they stick. Serve immediately.
Note: If you'll be holding popovers before serving, they can be kept warm in a very low oven. Slit them gently to let steam escape - this will keep them from collapsing, although you'll forfeit the steamy tableside opening. What's life without steamy tableside openings.
this turned out wonderful - i used a 2% milk, and a ramekin, to bake - thought u should know.
Being from England, Yorkshire pudding, aka popovers are traditionally eaten with a lamb or roast beef dinner and have gravy on them, not maple syrup or jam.


Scrumptious campari tomato withstands the big three threats - blight, early blight and fruit cracking.
Count on a crop of sweet, great tasting, 56-85g (2-3oz) globe shaped fruits with a sweet flavour. Best disease resistant variety available.
Sow seeds underglass 0.5cm (1/4 in) deep in pots or trays of good seed sowing compost and lightly cover with a sprinkling of vermiculite or compost. Place containers in a propagator at a temperature of 18–21°C .
Prick out seedlings, once large enough to handle, into 9cm (3 1/2in) pots. Transplant to the greenhouse border soil, or individual large pots, or 3 plants per growbag when 20cm (8in) high. Can also be planted outdoors once all risk of frost has past. Plants require sideshooting and support for the best yields. Water and feed plants regularly with a high potash feed once the first truss has set.
If growing outside pinch out the growing tip after 4-5 trusses of fruit have set.
High in vitamin C and lycopene.


text:
#import "RHActionTabDelegate.h"
#import "RHAppDelegate.h"
#import "ThreeDView.h"

@implementation RHActionTabDelegate

- (void)tabView:(NSTabView *)tabView didSelectTabViewItem:(NSTabViewItem *)tabViewItem {
    if(tabViewItem==app->composerTab || tabViewItem==app->slicerTab)
        view->act = app->stlView;
    else if(tabViewItem==app->printTab)
        view->act = app->printPreview;
    else if(tabViewItem==app->gcodeTab)
        view->act = app->codePreview;
    [app->openGLView redraw];
    [app->openGLView updateButtons];
}

@end

redpajama_set_name: RedPajamaGithub


text:
A Ordem do Mérito das Forças Armadas é uma condecoração criada pelo Decreto nº 91.343, de 18 de junho de 1985, e tem por finalidade premiar os militares do Exército, da Marinha, da Aeronáutica que tenham prestado serviços relevantes as Forças Armadas como um todo, ou a uma Força singular de per si, com reflexos em benefício das demais; os integrantes das Forças Auxiliares que hajam prestado assinalados serviços, às Forças Armadas; os civis nacionais, e os militares e civis estrangeiros que hajam prestado assinalados serviços às Forças Armadas e, as Organizações militares e civis, nacionais ou estrangeiras, que se tenham tornado credoras da homenagem especial das Forças Armadas.

A Ordem do Mérito Forças Armadas foi substituída em 2002 pela Ordem do Mérito da Defesa, sendo todos os seus membros, transferidos para os quadros da nova Ordem. Os agraciados da Ordem do Mérito Forças Armadas passaram a integrar os quadros da Ordem do Mérito da Defesa.

Características
A insígnia é uma Cruz de Malta, com os braços esmaltados em branco. O anverso traz o símbolo das Forças Armadas rodeado por um círculo esmaltado, em azul, onde está gravada a legenda Mérito das Forças Armadas. O reverso traz ao centro as Armas da República rodeadas por círculo onde estão gravados Brasil e EMFA. A fita é de gorgorão chamalotado nas cores verde, branca e azul. Os graus possuem desenhos diversos para homens e mulheres.

Graus
 Grã-cruz
 Grande Oficial
 Comendador
 Oficial
 Cavaleiro

Ligações externas

Condecorações militares brasileiras
Forças Armadas

redpajama_set_name: RedPajamaWikipedia


text:
Wootton Bassett Arts Festival promises a weekend of talent
By Emma Dunn
Wootton Bassett Arts Festival organiser Miranda Shirnia
TALENTED musicians, actors and public speakers will be taking to the stage this weekend for the Wootton Bassett Arts Festival.
More than 250 people are taking part in the event at Royal Wootton Bassett Academy, which starts at 9.30am on Saturday and finishes with a highlights concert on Sunday evening.
Organiser Miranda Shirnia said people who come to see the performances are in for a treat.
"We want people to come along and watch. All the people who have attended are competing against each other," she said.
"It is always lovely to have an audience. Usually the people taking part bring family and friends, but anyone is invited to come. The festival allows people a chance to be assessed by an independent judge who is experienced in that field and can tell them how they are doing and what they need to do to improve. That's invaluable."
Events include speech, drama and classical guitar. It will start with poetry and prose reading on Saturday morning, which will be followed by solo, group and Shakespearean acting.
There will also be classical guitar performances from 1pm.
Mime, poetry and Bible readings will be performed on Sunday from 9.30am, and classical guitarists will also be taking to the stage again.
The festival has become so big that it has to be held on two occasions throughout the year.
The first part of the festival was in June, when musicians and singers were showcased.
The event this weekend concludes with a highlights concert at 6.30pm on Sunday, which will showcase special performances from this weekend's event and the June music festival.
"For me, the best thing is Sunday evening when we have the highlights concert. It is the best of the best," added Mrs Shirnia.
"People who were paricularly outstanding from the June music festival have been invited to come along. We will have people from this weekend too."
Tickets, which are available on the door, cost £2.50 for the day or £4 for both days. The highlights concert is free.
Programmes are available online, on the door or from Brian Taylor Music in High Street.