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The tegument proteins ppUL35 and ppUL82 (pp71) of human cytomegalovirus (HCMV) physically interact and cooperatively activate the major immediate-early transcription. While an HCMV mutant lacking UL82 displayed a multiplicity of infection (MOI)-dependent growth, the biological significance of ppUL35 has not been addressed so far. We generated a mutant virus with a deletion of the UL35 gene. Using an MOI of 0.1, the progeny virus yield of this mutant was reduced by a factor of 1,000; however, when infected at a low MOI (0.01), the gene was essential. Characterization of the replication cycle showed that the mutant virus had two defects: when virus inoculum was standardized by the amount of viral DNA, a reduced immediate-early gene expression was observed, leading to a strongly delayed expression of lytic genes. A second defect was apparent in the virus assembly, as fewer enveloped particles and no dense bodies were present in cells infected with the mutant virus. However, the particles produced by wild-type and mutant viruses did not show significant ultrastructural differences. These results suggest an important role for ppUL35 in immediate-early gene expression and virus assembly.
Human cytomegalovirus (HCMV) is an exclusively human pathogen causing considerable morbidity and mortality in individuals with immature or compromised immune systems (34). HCMV is classified as a herpesvirus belonging to the Betaherpesvirinae subfamily. The genome of HCMV, comprising 235 kbp, is one of the largest genomes of animal viruses, encoding 165 to 190 genes, depending on the method of prediction (12, 30). A substantial part of the coding potential is devoted to virion proteins.
The virion of HCMV is composed of at least 40 proteins and has a tripartite structure, consisting of an envelope, a tegument, and a capsid (16, 43). The tegument is a proteinaceous structure, interspersed between the envelope and the capsid, and is characteristic of herpesviruses. Whereas the envelope and capsid are relatively well defined, characterization of the tegument has lagged behind. Long regarded as an unstructured repository for proteins, the tegument is now thought to have an ordered architecture and multiple functions (27).
At present, about 20 proteins have been localized to the tegument of HCMV, and they have been implicated in virion formation (3, 28), virus transport (41), immunomodulation (8, 10, 17) and transactivation. The first tegument protein which was shown to be involved in the activation of the major immediate-early enhancer-promoter (MIEP) of HCMV was ppUL82 (pp71) (23). Later, another regulatory protein, pUL69, was found in the tegument and was shown to transactivate the MIEP cooperatively with ppUL82 (47, 48). These observations were reproduced and extended, with the HCMV TRS1/IRS1 and UL35 genes added to the list (24, 36). Recently, we were able to show a physical interaction between two of these cooperatively trans-activating proteins, ppUL82 and ppUL35 (38). The UL35 gene encodes two proteins, of which the shorter form, ppUL35A, corresponds to the C-terminal part of full-length ppUL35 (24). Both forms can physically interact with ppUL82, but only full-length ppUL35 cooperates with ppUL82 (24, 38).
The biological significance of several genes encoding tegument proteins has been explored using antisense strategies or virus mutants. These studies showed that two abundant tegument proteins, ppUL32 and ppUL99, are essential for virus growth in cell culture (28, 40). Whereas ppUL32 appeared to play a role in the cytoplasmic accumulation of virions, there was an accumulation of tegumented capsids in the cytoplasm of cells infected with mutant viruses lacking ppUL99. Surprisingly, another abundant protein, ppUL83 (pp65), is not essential for growth in cell culture (39). However, since ppUL83 has immunomodulatory functions (8, 17), it likely plays an important role for the infected organism. The deletion of the gene encoding tegument protein pUL47 led to a 100-fold reduction in viral titers, irrespective of whether infection was done at high (≥1) or low (≤0.01) multiplicities of infection (MOIs) (4).
In contrast, deletions of the two regulatory tegument proteins ppUL82 and ppUL69 led to an MOI-dependent growth defect (19). This means that mutant virus can grow to some extent when infections are performed at high MOIs, but the genes appear to be essential after infection at low MOIs. A common denominator of several of these tegument proteins is that they are implicated in regulatory processes during the immediate-early (IE) phase. A direct effect on IE gene expression has been demonstrated for ppUL82 (6). An MOI-dependent growth phenotype was also observed for mutants of ppUL123 (IE1) (14) and those with mutations in the enhancer region (15) and is therefore consistent with a model in which the level of IE gene expression determines the outcome of infection. A role for virion-associated factors is supported by the observation that growth of HCMV lacking the distal part of the enhancer can be augmented by UV-inactivated HCMV virions (25). Analysis of MCMV with deletion of the enhancer demonstrated that the MOI-dependent growth phenotype is also reflected in a drastically reduced virulence in vivo (15).
Here, we characterized a mutant virus lacking the UL35 gene region. We showed that the deletion of the UL35 gene affects IE gene expression. In addition, we observed an impaired maturation of virus particles leading to reduced numbers of enveloped particles and a lack of dense bodies.
Oligonucleotides.The following oligonucleotides were obtained from MWG (Ebersberg, Germany), Sigma/ARK (Darmstadt, Germany), or Biomers (Ulm, Germany): CMV-IE-Plus, CGTCCTTGACACGATGGAGTC; CMV-IE-Minus, TCGGGGTTCTCGTTGCAATCC; Aktin4, AGCCATGTACGTAGCCATCCAGGCT; Aktin5, GGATGTCAACGTCACACTTCATGATGG; ETH257, TGAACCTCGAGTCTGTCATCACGGTACACAGA; ETH258, GAAGCTTGGCGGCCGCCAACGGGGTTATGGGGGACGTG; ETH417, TGAACGTCGACGACGACCTACGGGACACGCTGATG; ETH418, GAAGCTTGGCGGCCGCCGAACAGACGGTGCCCATCTTG; UL35-ko3, CGACGTCTCATCTTGAGACAGACACGTAGAAAAAGAGGAAAAACCGCTTACAAGGACGACGACGACAAGTAA; UL35-ko5, TTCTCAAGTTCCGCTCTGGTTTTGGTTTCGTTTTCAAAGGGAGCCCCATCCGTCGTGGAATGCCTTCGAATTC; UL35-kon5, CTGGAGAACAATAAAGCG; UL35-kon3, CTGTGTACTTCGCTAGTC; rv35-5, TTCTCAAGTTCCGCTCTGGTTTTGGTTTCGTTTTCAAAGGGAGCCCCATCATGGCCCAAGGATCGCGAGC; rv35-3, CGACGTCTCATCTTGAGACAGACACGTAGAAAAAGAGGAAAAACCGCTTCGTCGTGGAATGCCTTCGAATTC; UL35kon3, CTGTGTACTTCGCTAGTC; UL35kon5, CTGGAGAACAATAAAGCG.
Plasmid constructions.Plasmid pKD46 contains the phage λ recombination genes Redαβγ under the control of the arabinose-inducible ParaB promoter (11). This plasmid also contains the temperature-sensitive origin oriR101 and grows well at 30°C but not at 37°C. Plasmid pCP20 encodes the FLP recombinase and allows growth at 30°C but not at 43°C due to a pSC101-based temperature-sensitive origin (9). Plasmid pSL-FRT contains the kanamycin resistance gene flanked by FRT sites in vector pSL301 (Amersham Biosciences, Freiburg, Germany) (2). Plasmid pBRep-Cre harbors the Cre recombinase expression cassette from plasmid pMC-Cre and the bovine papillomavirus replication origin (18, 21). Plasmid pSL-FRT-UL35 was constructed by excising the UL35 open reading frame as an Asp718I/XhoI fragment from pcDNA3-UL35 (38) followed by insertion into the Asp718I/SalI sites of pSL-FRT.
Fragments corresponding to the sequences adjacent to UL35 were cloned into vector pBlueKan (38), a modified version of pBluescript KS II, where the ampicillin resistance cassette has been replaced by a kanamycin resistance cassette. Plasmid pBlueKan-UL34 was constructed by PCR amplification using primers ETH257 and ETH258 and subsequent cloning via XhoI/NotI. Similarly, UL36 exon 1 was amplified using primers ETH417 and ETH418 and cloned into pBlueKan via SalI and NotI sites.
Engineering BAC mutants.Recombinant bacterial artificial chromosome (BAC) clones were generated by RecET-based recombination using the RecET homologues of phage λ, Redαβγ (44). For deletion of the UL35 open reading frame, we used HCMV AD169-BAC(pHB15), containing the complete AD169 genome (21), and plasmid pKD46 (11) in Escherichia coli strain DH10B. First, the kanamycin gene, flanked by FRT sites, was amplified by PCR with oligonucleotides containing 50 nucleotides of sequence homologous to the region 5′ and 3′ of the UL35 gene (UL35-63/UL35-ko5). The PCR product was then transformed into an E. coli strain containing the HCMV AD169-BAC, where the RecET recombination system catalyzed the exchange of the UL35 open reading frame with the resistance marker. Subsequently, we deleted the kanamycin resistance cassette by FLP-mediated recombination between the two FRT sites flanking the resistance marker. By use of this procedure, the UL35 open reading frame was deleted, thus generating delUL35-BAC. A single FRT site and an additional EcoRI restriction marker remained as marks of the recombination procedure.
In order to generate a revertant virus, we used plasmid pSL-FRT-UL35. A PCR product containing both UL35 and the FRT-flanked kanamycin marker was generated using oligonucleotides containing sequences homologous to the region 5′ and 3′ of the UL35 gene (rv35-3/rv35-5). This PCR product was recombined into the UL35 region of the delUL35-BAC via RecET recombination. Finally, the kanamycin resistance cassette was again deleted by FLP-mediated recombination. Hereby, a revertant BAC, revUL35-BAC, which differs from the wild-type AD169-BAC by a single FRT site and an EcoRI restriction site remaining at the 3′ end of the UL35 open reading frame was generated.
Electrocompetent E. coli DH10B (containing the respective BAC and pKD46) for transformation of the PCR products was prepared as described previously (44). PCR fragments for mutagenesis were prepared using primer pair UL35-ko3/UL35-ko5 and template pSL-FRT for generation of the UL35 deletion virus or primer pair rv35-3-rv35-5 and template pSL-FRT-UL35 for the generation of the revertant BAC. Amplification was done using a touchdown protocol, and PCR products were processed as described previously (44). Briefly, PCR products were purified using a PCR purification kit (QIAGEN, Hilden, Germany), digested with DpnI to inactivate template DNA, and ethanol precipitated. Electrocompetent bacteria were transformed with the processed PCR product, plated onto Luria-Bertani (LB) agar plates containing 25 μg of chloramphenicol/ml and 30 μg of kanamycin/ml, and grown overnight at 37°C. Single colonies were analyzed by colony PCR using primers UL35kon3 and UL35kon5, which bound outside the region involved in the recombination. Colonies containing a correct insertion of the PCR cassette were transformed with plasmid pCP20 to remove the kanamycin resistance marker and grown overnight at 30°C in LB liquid medium containing 25 μg of chloramphenicol/ml and 50 μg of ampicillin/ml. A small aliquot of this culture was then plated on LB agar plates containing 25 μg of chloramphenicol/ml and grown overnight at 43°C to select against plasmid pCP20. Single colonies were analyzed for removal of the kanamycin resistance marker by colony PCR using primers UL35kon3 and UL35kon5.
Southern blotting.Digested BAC DNA was separated on 0.6% agarose gels and transferred to Biodyne B nylon membranes (Pall, Dreieich, Germany). Double-stranded DNA probes were radioactively labeled using the Ready-To-Go kit (Amersham Biosciences) and [α-32P]dCTP. Radioactive hybridizations were performed with a hybridization incubator (Robbins Scientific, Sunnyvale, Calif.) essentially as described previously (46). Briefly, filters were prehybridized for at least 4 h at 42°C in a prehybridization solution containing 50% formamide, 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 50 mM sodium phosphate (pH 6.5), 5× Denhardt's solution, and 1 mg of yeast RNA (Fluka, Taufkirchen, Germany)/ml. Hybridizations were performed for at least 24 h in a hybridization solution containing 50% formamide, 5× SSC, 20 mM sodium phosphate (pH 6.5), 5× Denhardt's solution, and 500 μg of yeast RNA/ml. Thereafter, filters were washed several times with buffer containing 20 mM sodium phosphate-0.1% sodium dodecyl sulfate (SDS) and decreasing concentrations of SSC. For reprobing of the filters, the radioactive probe was removed by two incubations in 0.1% SDS at 95°C for 30 min.
To analyze viral replication, human fibroblasts were seeded in six-well plates at 2.5 × 105 cells/well. One day later, the cells were infected for 4 h with mutant virus (RVdelUL35) at an MOI of 0.1 or with wild-type virus (RVwtAD169) at an MOI of 0.1 or 1. For harvesting, cells were trypsinized and pelleted, and the cell pellet was resuspended in 100 μl of phosphate-buffered saline (PBS). To digest proteins, 3 μl of proteinase K (10 mg/ml) was added, and the samples were incubated for 1 h at 56°C. After inactivation at 95°C (10 min), probes were denatured by the addition of 40 μl of 2 N NaOH and transferred to a nylon membrane by use of a slot blot chamber (Schleicher & Schuell, Keene, N.H.). Viral DNA was detected by hybridization with a radioactively labeled probe for UL36 as described above. For phosphorimager quantitation of the signals, the FLA3000 scanner (Fuji, Tokyo, Japan) and AIDA 2D quantification software (Raytest, Straubenhardt, Germany) were used.
Cell lines and culture.Human foreskin fibroblasts (HFF) were maintained in minimal essential medium (Gibco/BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum, 2 mM l-glutamine (Biochrom AG, Berlin, Germany), 100 U of penicillin and 100 μg of streptomycin (Gibco/BRL), each per ml, and 1× nonessential amino acids (Biochrom AG, Berlin, Germany).
Antibodies.Monoclonal antibodies directed against ppUL69 (69-66), ppUL82 (CMV355), ppUL83 (28-77), gpUL55 (gB) (27-287), and ppUL99 (41-18) have been described previously (7, 33, 45). Monoclonal antibodies E13 (35), CCH2, and Ab-1 directed against HCMV IE proteins, pUL44, and cellular vimentin, respectively, were purchased from Argene (Varilhes, France), DAKO (Hamburg, Germany), and Oncogene Research Products (Boston, Mass.). Rabbit antiserum directed against pUL35 was kindly provided by B. Biegalke (24).
Virus reconstitution, titration, and growth curves.For virus reconstitution, HCMV-BAC DNA was isolated from E. coli by using a Nucleobond AX kit (Macherey & Nagel, Düren, Germany). For electroporation of HCMV AD169-BAC DNA, HFF were trypsinized, washed two times with medium, and resuspended at a density of 107/ml. The transfection mixture, consisting of about 5 μg of BAC DNA, 5 μg of pCMV71 (23), and 0.5 μg of pBRep-Cre (21), was mixed with 400 μl of cell suspension. Electroporation was performed with an Easyject Optima electroporation device (Peqlab, Erlangen, Germany) at 280 V and 1,500 μF. Cells were immediately mixed with fresh medium and transferred to 75-cm2 flasks. After overnight incubation, the cells were washed and supplemented with fresh medium. When most cells showed a cytopathic effect, they were trypsinized, mixed with about 2 × 107 HFF, and seeded in 175-cm2 flasks. Virus supernatant was harvested when a confluent cytopathic effect was apparent. Viral particles were pelleted by ultracentrifugation in a Beckman (Munich, Germany) SW28 rotor at 22,000 rpm for 1 h at 4°C. Pellets were resuspended in a 1:1 mixture of medium-0.2 M sucrose phosphate, and aliquots were stored at −80°C (22). The recombinant viruses (RV) recovered from AD169-BAC, delUL35-BAC, and revUL35-BAC were named RVwtAD169, RVdelUL35, and RVrevAD169, respectively.
For quantitation of DNA in viral particles, a total of 105 PFU was pelleted in an Eppendorf tube by centrifugation at 23,000 × g at 4°C for 2 h. The pellet was resuspended in 50 μl of H2O and 50 μl of lysis buffer B (60 mM Tris-HCl [pH 8.3], 100 mM KCl, 5 mM MgCl2, 0.2 mg of gelatin, 0.9% Tween 20). Proteins were degraded by the addition of 3 μl of proteinase K (10 mg/ml) and incubation at 56°C for 1 h. After inactivation of proteinase K at 95°C for 10 min, insoluble material was pelleted by centrifugation (5 min at 16,000 × g), and the supernatant was used for the determination of the amount of DNA.
Entry of viral DNA into cells.HFF were seeded into six-well plates at a density of 2.5 × 105 cells per well. The next day, the cells were infected at an MOI of 0.1 PFU/cell. At 6 h postinfection (hpi), the cells were trypsinized and transferred to an Eppendorf tube. DNA was isolated essentially as described previously (42), with some modifications. The cell pellet was resuspended in 250 μl of buffer C1 (40 mM Tris-HCl, 20 mM MgCl2, 1.28 M sucrose, 4% Triton X-100) and incubated on ice for 10 min to permeabilize the cells but to leave the nuclei intact. The nuclei were collected by centrifugation at 1,300 × g and 4°C for 15 min, and the pellet was resuspended in 50 μl of buffer C2 (10 mM Tris-HCl [pH 7.5], 2 mM MgCl2, 10% sucrose). After the addition of 50 μl of 2× nuclease buffer {40 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)] [pH 7.0], 7% sucrose, 20 mM NaCl, 2 mM CaCl2, 10 mM β-mercaptoethanol, 0.2 mM PMSF [phenylmethylsulfonyl fluoride]}, the samples were incubated for 30 min at 37°C. After the addition of 6 μl of 0.2 M EDTA and 400 μl of digestion buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 25 mM EDTA, 0.5% SDS, 0.1 mg of proteinase K/ml), the samples were incubated overnight at 50°C. After phenol-chloroform extraction and ethanol precipitation, the DNA pellet was dissolved in 100 μl of H2O. For quantitation, 10-fold dilutions were prepared and used for PCR amplification with primer pair CMV-IE-Plus-CMV-IE-Minus or Aktin4-Aktin5.
Immunoblotting.Cell extracts were separated on 10 or 12% polyacrylamide gels and blotted onto polyvinylidene difluoride membranes (Bio-Rad, Munich, Germany) by semidry electrophoresis using the Transblot SD apparatus (Bio-Rad). To avoid nonspecific antibody binding, filters were blocked for 1 h in 3% skim milk powder-PBS. For detection of specific proteins, filters were incubated with primary antibody at room temperature for 1 h. Rabbit antiserum (anti-UL35) was diluted 1:5,000 in PBS-0.1% Tween 20 containing 1% skim milk powder, whereas hybridoma supernatants (69-66 and 41-18) were used undiluted. After the filters were washed three times for 10 min in PBS-0.1% Tween 20 containing 1% skim milk powder, they were incubated for 1 h with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (DAKO) diluted 1:5,000 in PBS-0.1% Tween 20 containing 1% skim milk powder. Finally, the filters were washed two times for 10 min in PBS-0.1% Tween 20 containing 1% skim milk powder and for 10 min in PBS-0.1% Tween 20. Bound antibodies were detected using the ECL Western blotting detection reagents (Amersham Biosciences) according to the instructions of the manufacturer.
Immunofluorescence.For indirect immunofluorescence analysis, HFF were seeded onto glass coverslips in 24-well plates at 4 × 104 cells per well the day before infection. Cells were infected at an MOI of 0.2. For fixation, the cells were washed three times with PBS, incubated in 4% paraformaldehyde for 10 min, and washed four times with PBS. To permeabilize the cells, the coverslips were treated with PBS-0.2% Triton X-100 for 2 min and finally rinsed in PBS. To block nonspecific binding, the coverslips were first incubated in 10% normal horse serum for 20 min at 37°C. Subsequently, murine monoclonal antibodies directed against ppUL69 (antibody 69-66), ppUL82 (pp71) (antibody CMV355), ppUL83 (pp65) (antibody 28-77), ppUL99 (pp28) (antibody 41-18), or gpUL55 (gB) (antibody 27-287) were added as undiluted cell culture supernatants and incubated at 4°C for 2 h. After being washed three times for 5 min with PBS, the coverslips were incubated at 4°C for 2 h with anti-mouse goat serum labeled with fluorescein isothiocyanate as a secondary antibody (DAKO), which was used at a 1:50 dilution. In some experiments, cell nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole; 1 μg/ml). After two final washing steps in PBS, the coverslips were mounted on slides with Vectashield H-1000 (Vector, Burlingame, Calif.). Images were taken on an Axioscop 2 (Zeiss, Jena, Germany) fluorescence microscope.
Electron microscopy.For electron microscopy, cells were fixed with 2.5% glutaraldehyde and 1% sucrose in 0.1 M cacodylate buffer at pH 7.4. After postfixation in 2% osmium tetroxide, they were block stained in 2% uranyl acetate, dehydrated in a graded propanol series, and embedded in Epon (Fluka). Sections were cut parallel to the substrate to a thickness of 60 nm and contrast stained with uranyl acetate and lead citrate. Images were taken with a model 400 transmission electron microscope (Philips, Eindhoven, Netherlands) at an acceleration voltage of 80 kV.
Generation of a deletion mutant of UL35.To address the in vivo role of UL35, we first deleted the UL35 open reading frame in the viral genome, by using the HCMV AD169 genome as a BAC in E. coli (Fig. 1A). This deletion was achieved by RecET-based marker transfer in which we first exchanged the UL35 open reading frame with a kanamycin resistance gene. In a second step, the kanamycin gene was deleted by FLP-mediated recombination of two FRT sites flanking the resistance marker. The UL35 open reading frame was effectively removed from its start codon to its stop codon, thus deleting the coding sequences for both UL35 isoforms (24). The resulting BAC was named delUL35-BAC. Sequencing showed that an insertion of 80 bp remained as a mark from the recombination procedure. This insertion contained a single FRT site (34 bp) flanked by the two primer binding sites (each 23 bp) that were used for amplification of the kanamycin resistance cassette. An additional EcoRI restriction site was part of one primer binding site. In a similar way, a revertant virus was generated by reinserting the UL35 gene along with the kanamycin resistance marker into delUL35-BAC. Finally, the kanamycin gene was again removed by FLP-mediated recombination. This step resulted in a revertant BAC (revUL35-BAC) with a single FRT site and an EcoRI restriction site remaining at the 3′ end of the UL35 open reading frame. The genomic organization and purity of all BACs were confirmed by PCR (data not shown), EcoRI restriction digestion, and Southern blot analysis, probing for UL35 and regions upstream and downstream of the deletion (Fig. 1B).
Thereafter, the wild-type AD169-BAC, delUL35-BAC, and revertant revUL35-BAC were electroporated into HFF to recover the recombinant viruses RVwtAD169, RVdelUL35, and RVrevUL35, respectively. All viruses grew without the use of complementing cells, indicating that the UL35 gene is not an essential gene. However, recovery of RVdelUL35 took about 3 weeks longer than did the wild-type or revertant virus, indicating a growth defect. Using lysates from infected cells in immunoblotting experiments, we could confirm that RVdelUL35 did not show expression of both UL35 isoforms, ppUL35 and ppUL35A, whereas both proteins could be clearly detected in cells infected with wild-type (RVwtAD169) or revertant (RVrevUL35) viruses (Fig. 2). In contrast, the expression of two structural proteins, ppUL69 and ppUL99, did not show any significant difference in cells infected with the mutant virus RVdelUL35.
As mentioned above, we had noticed a delay in virus replication after reconstitution of the RVdelUL35. In order to investigate this delay in more detail, we compared the growth kinetics of all viruses at different MOIs. At an MOI of 0.1 PFU/cell, RVdelUL35 replicated to titers more that 1,000-fold lower than those of the wild-type or revertant viruses (Fig. 3A). Wild-type and mutant viruses showed similar kinetics and reached a plateau in replication at about 7 days postinfection (dpi). However, at an MOI of 0.01 PFU/cell, the growth defect of the mutant virus RVdelUL35 was much more apparent, as we did not observe a considerable production of infectious virus (Fig. 3B). Since several mutants of HCMV that show MOI-dependent growth have been described, we also compared the yields of wild-type and RVdelUL35 viruses at 10 dpi after infection with different MOIs. Wild-type HCMV (RVwtAD169) showed a gradual rise in viral yield when a higher MOI was used (Fig. 3C). For the mutant RVdelUL35, a comparable rise could only be seen at the higher MOIs of 0.1 and 1, but there was a drastic drop when an MOI of 0.01 was used. Interestingly, the mutant virus did not reach yields equivalent to those of the wild-type virus, even when infections were performed with an MOI of 1 PFU/cell. Thus, the mutant virus shows an MOI-dependent growth.
Uptake of viral DNA is unaffected in RVdelUL35.Since a role for pUL35 in the activation of IE gene expression has been described previously (24, 38), we wanted to investigate the effect of the deletion of UL35 on the IE phase. For this, we first analyzed whether the uptake of regulatory virion proteins was comparable between wild-type and mutant viruses. HFF were infected with the wild-type and the UL35 deletion virus at the same MOI and analyzed for localization of the tegument proteins ppUL82 and ppUL69 at 4 hpi by immunofluorescence. As shown in Fig. 4A, both viruses showed similar uptakes of the tegument proteins ppUL82 and pUL69, indicating a normal entry of viral particles into cells.
Next, we investigated the uptake at the DNA level. For this, we first characterized the relative amount of viral DNA per PFU by serial dilutions of the inoculum followed by a PCR specific for IE genes. For virus preparations that had been normalized for equal infectivities, we consistently observed approximately 10-fold more viral DNA per PFU for the mutant virus (Fig. 4B, upper panel). We then infected HFF with an MOI of 0.1 PFU/cell for both wild-type and mutant viruses. At 6 hpi, cells were harvested and the amounts of viral and cellular DNA were determined by PCR titration (Fig. 4B, lower panels). Consistent with the inoculum results, approximately 10-fold more viral DNA was recovered from cells infected with mutant virus. In contrast, similar levels were detected for the cellular actin gene that was used as a control. Therefore, we conclude that uptake of virus particles was not affected by deletion of UL35, but the viral DNA amount of RVdelUL35 was about 10-fold higher than that of the wild-type virus when cells were infected at the same MOI.
Effect of UL35 deletion on IE gene expression.So far, all experiments were performed using MOIs as determined by a plaque assay. Plaques were counted after infected cells were stained for IE proteins. Thus, an effect of ppUL35 on the initiation of IE gene expression would be obscured by this outcome-based method of inoculum standardization. Our finding that the mutant virus inoculum contained a much larger amount of viral DNA per PFU than did the wild type (Fig. 4B, upper panel) supported the idea that more infectious particles of the mutant virus were needed to start a productive infection. Therefore, to investigate an effect on the IE phase, we decided to standardize the inoculum by determination of the amount of viral DNA in order have a physical measure of infectious particles. We used PCR titration to standardize the inoculum to similar amounts of DNA for both wild-type and mutant viruses (Fig. 5A, 6A, and 7A).
First, we compared the growth levels of wild-type and mutant viruses that had been standardized for equal DNA contents. As can be seen in Fig. 5B, this standardization led to a more drastic difference in virus yields, with only little mutant virus being produced even 10 dpi. When we analyzed viral gene expression, we observed a significant reduction of IE gene expression in cells that were infected with the mutant virus (Fig. 6B). In cells infected with wild-type virus, a strong expression of IE proteins was visible at 24 hpi, while for the mutant virus at the same time only faint bands could be detected. In addition, expression of early and late proteins was hardly detectable. As a result, accumulation of viral DNA was also decreased (Fig. 7B and C, lanes 1 to 8). Therefore, we conclude that deletion of UL35 affects IE gene expression.
Effects of deletion of UL35 on virus assembly.For the investigation of a potential defect during the late phase, we used infection at the same MOI. Under these conditions, there should be no differences in IE gene expression and subsequent expression of other viral genes. To investigate whether this is the case, we characterized viral gene expression after infection of HFF with wild-type and mutant viruses at an MOI of 0.1 PFU/cell. At the indicated time points, cell extracts were prepared and used for immunoblotting to detect viral proteins representative for IE (IE1 and IE2), early (pUL44), and late (ppUL99) phases. As shown in Fig. 6C, both viruses showed similar expression kinetics for all viral proteins.
In addition, we addressed the influence of the deletion of UL35 on the genome replication kinetics of wild-type and mutant viruses. HFF were infected at an MOI of 0.1 and harvested at the indicated time intervals, and viral DNA accumulation was determined by Southern blot analysis. As shown in Fig. 7B and C (lanes 5 to 12), the DNA accumulations of both viruses (RVwtAD169 and RVdelUL35) showed similar kinetics. Thus, the strongly reduced virus yield of RVdelUL35 compared to that of the wild type after infection at equal MOIs is very likely not due to differences in viral DNA accumulation.
This result raised the possibility that the reduced accumulation of infectious virus in the supernatant of cells infected with mutant virus might result from impaired virus assembly. Therefore, we studied the ultrastructural aspects of particle maturation by the use of HFF infected with wild-type (Fig. 8A) or mutant (Fig. 8B) viruses at an MOI of 1. Infection was stopped at 72 hpi, and cells were prepared for electron microscopy. Analysis of the nuclei of infected cells showed that all three types of capsids, A (empty), B (with scaffold), or C (mature, DNA containing), were present. The quantitation of capsids from at least five sections gave similar proportions of all three capsid types in both wild-type and mutant infected cells. A clear difference was the electron-dense accumulations in the nuclei of RVdelUL35-infected cells (Fig. 8C), which appeared to be free of capsids apart from some capsids at their boundaries. Another striking difference was observed after analysis of cytoplasmic sections. In cells infected with mutant virus, almost no dense bodies were found (Fig. 8B). Nevertheless, enveloped particles were present both in cells infected with wild-type or mutant virus. Cross sections of enveloped particles revealed no differences (Fig. 8D and E). When enveloped and nonenveloped particles were counted, the number of enveloped particles appeared to be reduced about 10-fold in cells infected with mutant virus.
As the formation of virions and dense bodies in the cytoplasm requires a correct localization of tegument and envelope proteins (20, 28, 37), we studied the localization of ppUL83 (pp65), ppUL82 (pp71), ppUL99 (pp28), and gpUL55 (gB) by immunofluorescence analysis after infection at the same MOIs. No difference was observed when ppUL99 (pp28) and gpUL55 (gB) were analyzed, as both proteins accumulated at a cytoplasmic location close to the trans-Golgi network (Fig. 9, panels 2e to 2h and 3e to 3h). Staining of ppUL82 and ppUL83 revealed a nuclear localization of both proteins in cells infected with wild-type or mutant virus (Fig. 9, panels 1a to 1d). In cells infected with wild-type virus, however, both proteins translocated to the cytoplasm during the later stages of infection (Fig. 9, panels 2a, 2c, 3a, and 3c). In contrast, in cells infected with mutant virus RVdelUL35, both proteins remained in the nucleus up to 120 hpi (Fig. 9, panels 2b, 2d, 3b, and 3d), the latest time point analyzed. This finding in conjunction with the observed decrease of enveloped particles in cells infected with the mutant virus suggests that late events during assembly are impaired in the mutant virus, thus leading to a reduced release of infectious particles.
The MIEP is a central control unit that processes cellular and viral signals to make a decision between latent or lytic infection of HCMV (26, 31, 32). Among the viral signals, the tegument proteins ppUL82 and ppUL35 have been shown to interact physically and to transactivate the MIEP alone and cooperatively (24, 38). We wanted to analyze the biological role of UL35 proteins by generating a virus mutant with deletion of the UL35 gene. One-step growth curves showed that virus production was decreased 1,000-fold when cells were infected with the mutant virus at an MOI of 0.1. This defect could be reversed when the UL35 gene was reinserted. This observation makes the deletion of UL35 the sole reason for the growth defect.
Our observation of reduced virus production by the UL35 deletion virus is supported by a recent large-scale analysis in which a similar deletion of UL35 was generated (13). In addition, our deletion of UL35 in the genetic background of a clinical isolate showed a comparably reduced virus yield (M. Winkler and K. Schierling, unpublished data). However, in a second large-scale analysis, UL35 was classified as nonessential, with yields reduced less than 10-fold (49). This discrepancy is likely explained by the different targeting strategies, since in the second approach a transposon insertion into the amino-terminal region was created. Since the UL35 gene encodes two coterminal protein species (24), the transposon insertion targeted only the full-length ppUL35, leaving the short-form ppUL35A intact. Therefore, either ppUL35A was able to compensate for ppUL35 or the essential part resides in the C termini of the UL35 proteins. The latter possibility is supported by the observation that the addition of a hemagglutinin tag at the end of the UL35 gene in the context of the viral genome leads to a reduction in viral yield similar to that resulting from the deletion of UL35 (Schierling and Winkler, unpublished data).
It was reported previously that ppUL35 has a role in the transactivation of the MIEP and efficient initiation of IE phase (24, 38). The MOI dependence of the RVdelUL35 virus yield is compatible with this view, since mutations in other genes implicated in IE regulation and gene expression, such as the ppUL82, ppUL69, ppUL123 (IE1) genes, and enhancer deletions showed similar behaviors (6, 15, 19, 25, 29). In addition, when infection was done at an MOI determined by plaque titration with an anti-IE antibody, we observed that the DNA amount in that inoculum of the RVdelUL35 mutant was 10-fold higher than that in the wild-type virus. As the particles appeared to be normal when analyzed by electron microscopy, this observation would mean that a 10-fold-higher number of particles of the mutant virus was required to reach the same infectivity as that of the wild-type virus. Thus, the lack of ppUL35 would be compensated by a higher amount of input viral DNA, by higher levels of other tegument proteins, or by a combination of both. When we standardized our inoculum to equal amounts of input DNA, we observed a delayed IE gene expression and consequently a strongly reduced expression of early and late proteins. This resulted in a significant reduction of viral DNA accumulation and a strongly reduced virus yield. In agreement with an effect on IE gene expression, an MOI-dependent virus yield was observed, rendering UL35 an essential gene when cells were infected at the low MOI of 0.01.
Defects in IE gene expression can be compensated by a high input of viral particles, which has been observed previously in conjunction with an HCMV mutant carrying a deletion in the distal enhancer (25). A possible explanation for the decreased IE gene expression with the mutant RVdelUL35 could be that no ppUL82 was packaged into viral particles, since during late phase there was no translocation of ppUL82 towards the cytoplasm. Due to the low yields of the RVdelUL35 mutant, we were not able to address this point experimentally. When we analyzed the uptake of tegument proteins into cells infected with wild-type or mutant virus, we observed similar numbers of cells staining positive for ppUL82 or ppUL69. Therefore, the defect in IE gene expression should be due mainly to a lack of ppUL35 and a subsequent failure of sufficient cooperative activation of the MIEP.
When infections were carried out using the same MOI for both wild-type and mutant viruses, we observed no difference in viral protein expression. Additionally, the accumulations of viral DNA showed comparable levels and kinetics after infection with wild-type or mutant viruses. As ppUL35 apparently had no influence on early or late gene expression or viral DNA replication, we could use infections standardized to the same MOI to investigate an effect of UL35 during the late phase.
Hereby, we observed an altered virus maturation in cells infected with mutant virus. This alteration was most clearly indicated by the absence of dense bodies. In addition, quantitation of cytoplasmic virus particles showed 10-fold fewer mature enveloped particles when cells were infected with mutant virus. The lack of dense bodies could be explained by the retention of ppUL83 (pp65) in the nucleus, as a virus mutant lacking the UL83 gene also failed to produce dense bodies (39). It is believed that dense bodies accumulate during high-titer passage of HCMV. Therefore, the mutant virus, which produces much lower titers, should be less prone to accumulate dense bodies. However, our reconstituted wild-type virus had the same passage history, and it was the mutant virus for which the higher titers had to be employed for virus stock production. In addition, immunofluorescence staining of cells demonstrated that several viral proteins (ppUL99 and gpUL55) showed normal accumulations and distributions in the cytoplasm of infected cells. It is therefore likely that the failure to produce dense bodies reflects a specific defect in the envelopement of viral particles.
In the nucleus, large accumulations of electron-dense material were observed. At present, the nature of these structures is unclear. Electron density in the nucleus usually corresponds to DNA, indicating that these structures could represent domains of condensed viral genomes. As viral particles can be seen at the margin of this material, a possible reason for this accumulation could be a packaging defect. However, we observed similar ratios of viral capsid types in the nuclei of cells infected with wild-type and mutant viruses, making a packaging problem unlikely. Therefore, these structures require further investigation.
At present, it is unclear how ppUL35 could be responsible for the translocation of ppUL82 and ppUL83 (pp65) from the nucleus to the cytoplasm. A similar phenotype has been described for a mutant of HCMV with a lack of TRS1 (5), and a recently published analysis of this mutant argued that pTRS1 has a role in the encapsidation of viral DNA since the number of C capsids was reduced (1). In our electron microscopic analysis, we did not detect such a difference. In contrast, the lack of dense bodies and the reduced amount of enveloped particles argue for a role of ppUL35 in the cytoplasmic particle assembly. As UL35 proteins interact with ppUL82 but not with ppUL83 (38), their effect on tegument protein localization might be independent of physical interaction. We therefore favor the hypothesis that ppUL35 of ppUL35A might directly or indirectly influence nucleocytoplasmic transport pathways, either enhancing export of tegument proteins from the nucleus or blocking import of tegument proteins into the nucleus during the late phase.
In summary, we could show that ppUL35 plays an important role in both IE gene expression and virus assembly. Further studies will be required to elucidate the underlying mechanisms and to address the question of whether the effects on IE gene expression and virus maturation are direct or indirect.
We thank A. Lüske for technical assistance and H. Hofmann for critical discussion. We also thank U. Koszinowski and M. Wagner for plasmids pSL-FRT, pKD46, pBRep-cre, and HCMV AD169-BAC(pHB15) and T. Stamminger, M. Mach, and B. Biegalke for murine monoclonal antibodies CMV355, 41-18, 28-77, and 27-287 and rabbit antiserum against pUL35.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Wi 1725/2-1), the IZKF Ulm (H4, 01KS9605/2), and the Universität Ulm (P.680) to M. Winkler.
- Received 23 July 2004.
- Accepted 12 November 2004.
- ↵*Corresponding author. Mailing address: Abteilung Virologie, Universitätsklinikum Ulm, Albert Einstein Allee 11, D-89081 Ulm, Germany. Phone: 49 731 5002 3336. Fax: 49 731 5002 3337. E-mail: michael.winkler{at}medizin.uni-ulm.de
- American Society for Microbiology


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Four Weddings star Simon Callow completes line-up
Actor, director and writer Simon Callow completes the line-up for this autumn's festival.
The 'Four Weddings and a Funeral,' 'Room with a View' and 'Shakespeare in Love' star will be with us on Friday 21 October.
Awarded a CBE for services to drama, Simon is one of the UK's finest and best-loved actors, known for his many roles on stage and screen, including Four Weddings and a Funeral – one of the most successful British films of all time.
Not just a great actor, Simon has also proved that he is a gifted writer. In 'One-Man Band', the third volume of his new biography of cinema giant Orson Welles, Callow charts the later years of Welles's life, painting a vivid and often very funny portrait of a troubled genius enduring a cocktail and triumph and decline.
His other books include My Life in Pieces, the autobiographical 'Being an Actor' and his acclaimed Charles Dickens biography.
At the festival Simon will be talking about his life on screen and stage, as well as reading excerpts from his latest book.
He says he still loves being in front of a live audience:
"I've found that people are fantastically warm and keenly interested, each talk is entirely different as people react differently to the – though I say it myself – remarkable story I'm telling. I'm fascinated by my fellow human beings and long to know what makes them what they are."
Councillor Janet Emsley, cabinet member for culture, health and wellbeing at Rochdale Borough Council, said:
"Simon has been treading the boards for many years in the country's top theatres, in addition to his countless film and television roles. As you would expect from such a distinguished actor, this will be much more than a simple reading of excerpts from his book, our audience is in for a treat! He is a superb talent and a perfect final addition to our festival."

subset: commoncrawl


text: DigitalSidecar to Close Ridesharing, Delivery Service on Dec. 31The closure will allow the company to 'work on strategic alternatives and lay the groundwork for the next big thing.'By Brandy Shaul|December 30, 2015 ShareBy Brandy Shaul|December 30, 2015 ShareRidesharing and delivery service Sidecar will close on December 31, according to a blog post from Sidecar co-founder and CEO Sunil Paul.Sidecar was founded in 2011, and allowed users to connect with drivers in their personal vehicles. In February 2015, Sidecar expanded to offer same-day delivery for packages, restaurant orders, flowers and groceries.Paul wrote:Long ago we conceived of the technology that gave rise to the rideshare movement. And nearly four years ago we invented what is now known as "ridesharing" with an app that connected riders with everyday drivers in their personal vehicle. People loved it. It was safe, convenient and affordable, and it quickly caught on …Our vision is to reinvent transportation and we've achieved that with ridesharing and deliveries. It is, however, a bittersweet victory. Shutting down the Sidecar service is a disappointment for our team and our fans. The impact of our work, however, will be felt for generations to come. We changed transportation law, and created a new mode of transportation that has transformed cities and made life easier and better for millions of people.Sidecar's ride-sharing and delivery operations will cease at 2 p.m. PT on December 31. Paul said the closure will allow the company to 'work on strategic alternatives and lay the groundwork for the next big thing.'Readers: Are you sad to see Sidecar shut down its ridesharing and delivery service? Sharehttp://adweek.it/1QZvylU copy Adweek Adweek


text:
Podcast 92: The Shame File
November 26, 2015 by Staff Writer
This week, Kris and JT talk about the A-League heat policy, the shooting down of a Canberra expansion team, preview all the round 8 games, and talk about the big news of the week – the so-called A-League Shame File.
Follow us at @ultimatealeague on Twitter and Instagram, like us on Facebook, or email us at firstname.lastname@example.com
Featuring: Kristian Dwyer and Justin Tickner.
Length: 1 hour, 10 minutes, 59 seconds.


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The Phlebotomy program at Paramount Training Services is a 5 week professional development program and examination preparation for National Certification as a Phlebotomist.
The program starts off with theory, medical terminology, anatomy and physiology. Aspiring phlebotomists learn proper infection control. They receive instruction on how to prepare the blood collection site, how to choose the proper collection tools and how to handle the transportation, processing and management of collected samples. Medical and legal ethics as they relate to phlebotomy services are also taught.
Practicum is incorporated in the class and students practice the blood and specimen collection and handling skills learned. They practice these duties on simulation arms and then students practice drawing blood under supervision of the instructor. Completion of this Certification Training Program in Florida requires passing the practicum portion, which is held on campus, and the course final written exam.
Once the program has been successfully completed, the student can register through the school to take the National Certification Exam, which is held monthly.


text:
The Nexus Player is back again, available for pre-order at the Google Play Store after getting FCC approval. Google had announced the Nexus player officially and put it up for pre-order on the play store last week, but had to remove it as approval from FCC (Federal Communications Commission) was still pending. Nexus Player is a set-top box that allows you to stream music, TV shows and movies, and play Android Games.
In the US, certain electronics devices need to be passed by FCC and it is technically illegal to sell them without it, which is why Google had to stop the pre-orders. Google specifically mentioned at the bottom of the Nexus Player page that "This device has not been approved by the Federal Communications Commission. It is not for sale until approval of the FCC has been obtained." Till the time it was approved, the device was temporarily switched from "Pre-order" status to "Out of Inventory" status.
Luckily, the Nexus Player passed through the FCC over the weekend and now the set-top box is back on Google Play Store for pre-orders at $99. The device will be shipped in 3 to 4 weeks.
The Nexus Player is a small seek Android-based device manufactured for Google by Asus and has the dimensions of 120mm x 120mm x 20mm. It is powered by 1.8GHz quad-core Intel Atom processor and has Imagination PowerVR Series 6 graphics. It has 1GB of RAM, 8GB of internal storage, 802.11ac WiFi and Bluetooth 4.1. The device has a remote control that supports voice search and there is an optional wireless Gamepad costing $39.99 to play Android games on TV.


text:
Contact Information
NEFSC James J. Howard
Marine Sciences Laboratory
74 Magruder Road, Sandy Hook
Highlands, NJ 07732
(732) 872-3000
Sandy Hook Lab: Research Aquarium
Perhaps the most unique aspect of the Howard Laboratory is its state-of-the-art seawater system and the laboratories and aquaria it supports. The wet labs are designed to be exceedingly adaptable to various types of experimental designs and aquaria space needs, are capable of reproducing highly controlled environmental conditions under various water temperature and light regimes, and can provide 24-hour monitoring of experiments through integrated computer and video systems.
High quality sea water is supplied to support research aquaria in 12 laboratories, including a large, two-story 32,000 gallon research aquarium. This research tank is housed in a sound-retardant room in which daily and seasonal changes in light intensity and photoperiod, for any time of the year and for any place in the world, can be simulated. The lighting is controlled by a computer that guides 1,500 flourescent lamps, while video cameras monitor the tank from above and through viewing windows along the sides.


text:
5 reviews
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we have been last night , the food was fantastic and generous portion of food, shisha very nice and lovely fresh orange juice , but the bill was so expensive compared to prices on menu!
we had shish chicken taouk , mixed mezza for 2 and salad chicken plus 3 orange juice and the shisha , our bill should be around £50 but they charged £72!!
placed by layla hassan on the 26th March 2018
Great food and great service!
placed by Claire Baglin on the 25th April 2017
The order was made quickly and the food was nice!
placed by Kameliya Yordanova on the 6th February 2017
We love going to amasi. The chicken shawarma with Lebanese bread is the best!!!!
Definitely recommend it!
placed by Stephanie Jubely on the 18th January 2017
One of my favourite places to order food, well done
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Just a short note to compliment you on the recent Canon Printer that was installed by us.
Now that we got the "hang" of it, really enjoy printing with it. The quality is superb and in my opinion better than the likes of Roland etc.
Do feel free to use as a reference if you want to with new customers


text:
Partner/Chairman, Crispin Porter + Bogusky
Author, Designer & Inventor, Dominic Wilcox
Designer & Consultant,
Artist/Illustrator, Made By Karan
CEO, The One Club For Creativity
Founder, UnitedVisualArtists
Brand & Creative Consultancy, shubhankarray.com
Founder, Yinka Ilori Studio
Founder, Role Models Not Runway Models
Principal Designer & President, NewDealDesign LLC
Creative Director, Dentsu Inc
Illustrator, Kerby Rosanes
Interaction Designer, Pauline Saglio
Graphic & Type Designer,
Digital Artist, Thijs Biersteker
Creative Director/Principal, Foreign Policy Design Group
CEO, Designer & Director, Wearable Experiments Inc
Creative Director, Espadaysantacruz Studio S.L.
CEO & Creative Director, Espadaysantacruz Studio S.L.
Design Director, LAAB
Founder, Kawkab El Rasameen
Founder Director, Cognito
Beatboxer, Emcee and Entrepreneur, Vineeth Vincent


text:
I love wind
I'm a graphic designer, character designer, and Illustrator that lives in New York City.
I grew up in Seoul, South Korea, but now live in south of Brooklyn near the beach.
I love beach life..
I do kite surfing, surfing, biking, hiking, climbing, longboarding.. many activities.
I studied illustration for fashion design at 2003
I was Nail Artist in South Korea for 8 years.
When I've started do graphic design work, It changed my life for better and I love drawing my activities!
I am a very adventurous and brave woman that my life is always challenging…
Looking forward with more challenges…
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Makes a 10-inch cake, serving 10 or more
This is a wonderfully easy cake with flavors for which Piemonte is known. The best hazelnuts in Piemonte are called tonda gentile delle Langhe—the "gentle round one of the Langhe"—and with a bit of chocolate, you have the match made famous in Torino, gianduja. I love this torte for its versatility as well. I serve it simply with powdered sugar or whipped cream, or you can flank it with a scoop of chocolate ice cream or give it a drizzle of warm chocolate sauce. In Piemonte, I've had the torte with zabaglione—and if you turn the page, you'll find a perfect partner in Zabaglione al caffè nero.
- 1 ½ cups hazelnuts, toasted and with skins rubbed off
- 1 ½ cups all-purpose flour, plus some for the cake pan
- 1 teaspoon baking powder
- ¼ teaspoon salt
- 1 cup plus 2 tablespoons sugar
- 6 tablespoons soft butter, plus a bit for the cake pan
- 3 large eggs
- 1 tablespoon extra-virgin olive oil
- ½ tablespoon finely grated orange zest
- 1 cup milk at room temperature
- 4 tablespoons semisweet chocolate, chopped by hand in small pieces
- Garnish: powdered sugar or whipped cream
Chop the hazelnuts in a food processor or mini-chopper to small bits—not to a powder. Set aside. Whisk or sift together the flour, baking powder and salt. Butter and flour the cake pan. Preheat the oven to 350° with a rack in the center.
In the mixer, cream the sugar and butter together until light, smooth and fluffy; scrape the sides of the bowl as needed. Incorporate the eggs, olive oil, and orange zest in several additions, blending each in at slow speed, scraping the bowl, then beating at high speed for a couple of minutes to lighten.
On slow speed, incorporate the dry mix in several additions, alternating with splashes of milk. Scrape the bowl when both are added, and beat briefly on high. Fold in the chopped nuts and chocolate by hand, and blend in well.
Scrape the batter into the cake pan, and smooth the top. Bake until a cake tester comes out clean, about 45 minutes. The top should be lightly browned and just spring back to a light touch.
Cool on a wire rack for 30 minutes or so, remove the side ring of the springform, and let the cake cook completely. Cut in wedges and serve, topped with powdered sugar or whipped cream.
The torte will keep in the refrigerator for a week, well wrapped in plastic, or you can freeze it for longer storage. When serving torte that has been chilled or frozen, toast the cut pieces in the oven (or toaster oven) to bring out the flavors.


text: Please join us! Meet school staff and visit our classrooms. Learn more about how we teach to the needs of each child, our moral focus program, enrichment activities, sports, and so much more. All kindergartners in attendance will receive an invite to a future Chuck E. Cheese's celebration! We look forward to seeing you.


text:
Inground Pool Included !
The Parkside design features 3 bedrooms & separate media room, 2 bathrooms & open plan living & dining areas.
Spacious kitchen with stone bench tops, 1200mm wide front entry door, split system air conditioning throughout & stylish fittings & fixtures.
Turf & landscaping, clothesline & letterbox & so much more.
The in ground pool is complete with exposed agg surrounds, with semi frameless & aluminium pool fencing.


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Yellow Wedding Invitations
This is just a small selection of yellow wedding invitations to give you some ideas – the majority of our wedding invitations on the main page are available with yellow ribbon to match your colour scheme.
Opting for a yellow wedding colour scheme for your big day will have your guests smiling from ear to ear. For a fun and zesty wedding create a rustic wedding theme with a summery burst of yellow.
We stock yellow ribbon in satin and organza.
Order your wedding invitations today.


text:
In our latest installation guide, Saniflo breaks down how to install its Kinemagic Serenity + all-in-one shower cubicle.
The new Kinemagic Serenity + from Saniflo is an all-in-one shower cubicle package, which includes: internal and external panels and door; shower tray; magnetic shower rail; showerhead, valve and all fittings; and a luxury fold-down seat and grab rails.
Ideal as a bath replacement, this walk-in shower fits neatly into the same space as a standard bath, and comes complete with a range of features to make life easy for the user. Follow this simple guide to installation and ensure your customer has a practical and contemporary shower in less than a day.


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Home Health Nurses provide various care and support services to patients at home. When recruiting Home Health Nurses for your agency, modify and use our Boolean search string examples to find resumes and profiles online.
Recruiting Home Health Nurses with Boolean strings
Build search according to your needs:
- -job -jobs -sample -examples, to avoid irrelevant results.
- intitle: or inurl: operator, to include the word "resume" or "CV" in the page title or page url respectively.
- ("home health nurse" OR "home care nurse"), to cover variations of the same job title.
When sourcing EU candidates, please refer to guidance on using social media for recruiting and collecting candidate information as per the General Data Protection Regulation, or GDPR.
Here's an example of a generic search string that you can further modify to find resumes:
(intitle:resume OR inurl:resume) "home health nurse" -job -jobs -sample -examples -template
With this search string, the words "resume" or "CV" have to appear in the page title. And, excluding more terms will reduce false positives.
Examples of Boolean search strings to recruit Home Health Nurses by experience and training
Find Home Health Nurses who:
Are specialized in hospice care
(intitle:resume OR intitle:cv) "hospice nurse" ("at home" OR "home care" OR "patient's house") -job -jobs -sample -examples -"writing service"
Have experience in rehabilitation services
(intitle:resume OR intitle:cv) "home health nurse" rehabilitation -job -jobs -sample -samples -examples
Can care for wounds
(intitle:resume OR intitle:cv) nurse "wound care" ("at home" OR "home care" OR "patient's house") -job -jobs -sample -examples
Are first aid certified
(intitle:resume OR intitle:cv) "home health nurse" ("first aid" OR CPR) -job -jobs -sample -examples
Sample string to search for resumes of Home Health Nurses in various formats
(intitle:resume OR intitle:cv) (filetype:pdf OR filetype:doc OR filetype:txt) "home health nurse" -job -jobs -sample -templates
Examples of Boolean search strings to recruit Home Health Nurses by location
By state or city:
(intitle:resume OR intitle:cv) "home health nurse" (Maine OR ME) -job -jobs -sample -examples -template
(intitle:resume OR intitle:cv) "home health nurse" Philadelphia PA -job -jobs -sample -examples -intitle:indeed
Further reading
Looking for an all-in-one recruiting solution? Workable can improve candidate sourcing, interviewing and applicant tracking for a streamlined hiring process. Sign up for our 15-day free trial today.Get a free trial


text:
Like V.T. my house had reached the point of no return, that is, I wasn't going to be able to sort it without professional help. I was on the verge of seeing if Kim and Aggie were available. However, my Sister found the Aberdeen Clutter Buster website and we contacted Lisa and Denise. They lived up to the ethos as stated on their website, in that they treated me and my possessions with care, dignity and respect and, whilst on site, worked non-stop. I feel that they really earned their money, they have turned my flat back into a home. Lisa & Denise thank you so much.
Hi Denise
Thank you again for helping me declutter my House. Your expertise and work ethic are to be applauded. I need to keep up the good work. Yourself and Lisa have inspired me. I am still working on my husband to go through boxes!
I don't know how high up the list of cluttered up homes I appeared in the eyes of Denise and Lisa. Personally I think I was well ahead of anyone else. I would like the record to show how much I appreciate the hard work done by both ladies, and their consideration of my feelings during a painful couple of days.
It is no easy matter letting go of the past and I couldn't have succeeded without their help. The transformation is nothing short of amazing, but it is the result of hard work not by me. How they managed so much in so short a time I haven't figured out, but if I do the rest of the house will be a doddle.
Aberdeen Clutterbusters have my complete and unreserved endorsement.
David Cameron, Burghead
I called Aberdeen Clutterbusters not knowing what to expect but I shouldn't have worried. Denise and Lisa came to see my flat as we were advised to declutter before it would sell. They cleared and took away all the things we didn't need and restyled the room so the photographer and decorator could come in. It only took two days and the difference was unbelievable. They were so professional in the way they did the job that I have been telling everyone who would listen.
C M, Central Aberdeen
Aberdeen Clutter Busters re-organised our 3 bedrooms and two walk-in cupboards and hallway in our new flat, we were able to settle in really quickly. They did it all in one day, I still don't know how they managed it but they did. Denise and Lisa were really kind to me when I needed help so much so that they are coming back to do my kitchen.
J M, Woodend, Aberdeen
Clutterbusters came in to help when I was beyond the point of helping myself. They were professional but friendly and didn't leave until the final part was finished. All the rubbish was taken away or given to charity - who knew it was possible to have so much stuff you dont need?? I'd highly reccomend Clutterbusters for their completely professional, non judgemental attitude and sheer hard work. Delighted with the outcome, thank you ladies!
V.T. , Aberdeen


text:
Amer Sports (www.amersports.com) is a sporting goods company with internationally recognised brands including Salomon, Wilson, Atomic, Arc'teryx, Suunto, Peak Performance and Precor. The company's technically-advanced sports equipment, footwear and apparel improve performance and increase the enjoyment of sports and outdoor activities. The Group's business is balanced by its broad portfolio of sports and products and a presence in all major markets.
|Senior Analyst Field Channel EMEA GTM
|Annecy / Garching near Munich
As Senior Analyst Field Channel EMEA GTM, you will be supporting EMEA GTM in business analysis concerning field business including ownership of the field sales force target setting process twice a year and being in charge of supporting, implementing and programming certain technologies used by EMEA GTM including Anaplan, Power BI, Qlickview and Excel.
- Defining, implementing and supporting field sales force business analysis (web reporting, Excel, sales KPIs …).
- Developing or adapting Anaplan modules every season for sales force PRP tool.
- Managing distribution of KPI reporting (sales KPIs).
- Supporting, implementing and programming certain technologies in other team members´ projects (Anaplan, Qlickview, Power BI, sales force.com …).
- Training Field Sales Managers, reviewing targets, ensuring guidelines are taken into account.
- Integrating and processing collected sell thru data (from SA/KA, retail or field accounts) into our sell thru database.
- EMEA sales force organization, structure and efficiency KPIs and reporting.
- EMEA premium partner network reporting.
- Being the preferred contact to AS IT regarding technologies used in EMEA sales Ops for business analysis.
- Periodically informing stakeholders on various KPIs and business trends.
- OC campaigns reporting.
- Several years of experience in Business Intelligence area either as a business analyst or in IT supporting business analysts
- Bachelor or Master degree in IT
- Strong Sales background
- Business KPIs understanding (sales, margin, distribution)
- Ability to program technologies like Anaplan, Power BI, ClickView, Microsoft Dynamics
- Understanding of the Target setting process
- Comfortable in manipulation of numbers
- Fluent in English Language
- Proficiency in German and/or French language a plus
- Planning & Organizing
- Analytical skills & Problem Solving
- Listening & solution selling
- Presenting
- Business acumen
- An interesting role in a challenging industry
- Career progression opportunities
- The opportunity for independent and self-reliant work
- A friendly and vibrant working environment in a dynamic and international team
Please apply online.
When making the application please state your salary expectation related to this role.


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site of an ancient Bactrian city that existed from approximately the second century B.C. to the second or third century A.D.; located near the settlement Imeni Nasir Khisrav in Shaartuz Raion, Tadzhik SSR.
Remains of the city walls and towers have been preserved; rectangular in plan, the walls measure about 4 m thick and 6 m high. Excavations conducted between 1950 and 1953 have revealed rectangular chambers adjoining the city wall. Coins, Bactrian pottery, and terra-cotta statuettes have been found.
REFERENCESD'iakonov, M. M. "Arkheologicheskie raboty v nizhnem techenii reki Kafirnigana (Kobadian) (1950–1951 gg)." Materialy i issledovaniia po arkheologii SSSR, no. 37. Moscow-Leningrad, 1953.
Mandel'shtam, A. M., and S. B. Pevzner. "Raboty Kafirniganskogo otriada v 1952–1953." Ibid., no. 66. Moscow-Leningrad, 1958.


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Luxury apartments with simple and colorful theme
Luxury apartments with simple and colorful theme. by using the unique furniture and luxurious, plus moddesain wall and floor which also uses the model full-color. design, designed by Federica Bisazza. These designers use bright colors rich had arranged a celebration of real color in it. Although the major is the dominant white color is a lot of splashes of other bright colors. Some sparks are non-colored furniture and white colors with a few others – painted the walls and ceiling. For example, bright green chairs and orange floor vase made ??of black and white interior of a bedroom more comfortable. The red color tops kitchen make this living space pleasant and happy. One of the more remarkable features of apartments are French windows installed in all rooms. They visually enlarge the space, bringing more light and open views of beautiful.


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The cryptos is slowly moving out of the hype that dominated the market in 2017. Although the people who invested in ICO projects that became successful reaped a fortune, most of the maturity has happened in 2018. The downtrend in the market has forced both investors and cryptocurrency enthusiasts to rethink how they view blockchain projects and particularly cryptocurrencies.
The projects without sound technology have gradually dropped in price and market capitalization giving way to cryptocurrencies that have a future and actually do solve something real world. Such projects are sort after by investors because they have limitless lifespans. In this article we will explore a couple of crypto projects that have the potential become projects that will be reckoned with in the future.
Bitcoin Interest (BCI)
Project Overview
Bitcoin Interest is a cryptocurrency project that was designed to revolutionize the staking process in the crypto industry by decentralizing the entire process. The platform is among the few in the market that reward people for holding tokens. Moreover, the protocol is said to be the first to incorporate ProgPow algorithm. The crypto is a hard fork of Bitcoin Core, although it has recently switched to ProgPow algorithm.
ProgPow algorithm is a version of the Proof-of-Work consensus algorithm that was specifically designed to address the issue of ASIC mining. Mining BCI required equipment such as GPUs, however, the software has been fine tuned for the common GPU hardware as well. The specialized GPU mining protocol ensures that ASIC mining encounters acute inefficiencies leaving the network free of ASIC mining threats.
Bitcoin Interest (BCI) token overview
BCI is the native token on Bitcoin Interest network. The token has utility as an exchange asset on the many crypto exchange platforms around the world. At the moment, BCI is available for exchange on HitBTC (main exchange), Bitfinex, STEX, Trade Satoshi and Extrates.
Bitcoin Interest is currently trading at $1.71 following a 50% surge in the last 24 hours. The crypto is still exploding in fireworks in spite of the stability and consolidation across market. BCI is ranked 152nd with a market cap that stands at $29 million. It has a trading volume of $90 million as per the data on CoinMarketCap.
This coin deserves your attention as an investor. Most digital assets are grappling with ASIC mining but Bitcoin Interest is currently riding above these threats. Besides, users are rewarded for the coins they hold. With BCI you gradually become rich even when you are not buying more coins.
Tokenomy (TEN)
Project Overview
Tokenomy is an exchange platform that supports digital assets only. It has its focus on markets that lack representation such as those in Southeast Asia. The platform is also categorized as a building platform where individuals can develop their tokens and list them for trading on the exchange. Tokenomy is a platform that empowers entrepreneurs by giving them a framework to create their own trusted tokens which they in turn sell to raise capital.
Apart from the ready market in Southeast Asia, the network is working to become an international platform for digital asset exchange. Moreover, future developments will launch the platform on mobile application for both iOS and Android. This will allow users to trade on the go without down times.
Tokenomy (TEN) price overview
The native token, TEN is the center of the Tokenomy exchange platform. It is the lifeline of the network and benefits all the people on the platform including businesses. Users use TEN to buy into other tokens that have been created for exchange or to raise funds on Tokenomy. TEN tokens allow merchants, companies and startups to earn loyalty points when using the platform. In addition, TEN tokens are used to pay for goods and services as the team to push for a cashless economy.
Tokenomy (TEN) is trading at $0.16 following a 10% surge in the last 24 hours. The crypto is also up 12% and 11% against Bitcoin (BTC) and Ethereum (ETH) respectively. According to the data available on CoinMarketCap, TEN is a $33 million asset currently ranked 146th in the market. It has a 24-hour trading volume of $3 million.


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Just how fully can we expect grief to be healed, anyway? Full disclosure, I've had two losses - two babies left my womb and went straight to heaven. The first loss was a still birth at 30 weeks, from a rare illness, and the second was at 8 weeks. The first one was especially traumatic, and one of the books I read about grief mentioned it could take seven years to "recover" or be able to act like life was "normal" again. From talking to other loss mothers, this seemed plausible. To be honest, I didn't know what a "healed heart" looked like, nor did I expect it to happen so quickly for me as it did. First, I want you to know that I don't consider either loss to be "worse" than the other, in the respect that both were babies, both were children that needed to be grieved. But there is something different about being able to hold your newborn in your arms who you'd also never seen take a breath. (What I'm saying is, pain should never be compared, and one life is never worth more than another's. Just that in my experience, the first loss was much harder to recover from.)
But when my sister in law, who was pregnant at the exact same time as I was with Hope, gave birth to a baby girl (we didn't know gender until the birth) - well, I nearly lost my mind. I wanted to die. I wanted somehow to live both in heaven with Hope and to not leave my first baby, Leah (age 2 1/2), behind. No joke - I hurt so bad, I thought I MIGHT die. (What a wondrous gift Leah is to me!)
Well, as with other aspects of my grief, I learned to deal with this development ... or thought I was learning to live with it, by forcing myself to follow everyone else's expectations for steps I could take to heal. Some of them were good, but most of them just made me hurt worse by forcing me to move along much faster than I was actually healing. I thought often, you wouldn't suggest to someone who had lost a limb, to suddenly learn how to live life without it, so why were they expecting me to do "all the things" SO SOON? I had lost part of my heart. I know now that the inside of us is the hardest part to heal. I think that's why Jesus liked to ask the question, which is easier to do, to heal (the physical), or to forgive sins?
God did a lot of comforting during that initial mourning period following Hope's passing. One of my favorite verses to cling to was Isaiah 43:2, which says this:
When you pass through the waters, I will be with you; and when you pass through the rivers, they will not sweep over you. When you walk through the fire, you will not be burned; the flames will not set you ablaze.
Praise Him, He kept me from burning in the fire or drowning until the real help, and complete healing, arrived. However, contrary to what many seem to think, grief does not come in neat little "stages" - instead it came in waves, some bigger than others, that each time felt like they might capsize me. I would be coming along better one day, and suddenly the next day, or even seconds later, a wave would come out of nowhere. During my pregnancy with Gideon, my "double rainbow," I felt especially disappointed in how far I'd come. It was as if I'd suddenly taken 5 steps backward. I will write about his story another day, but for now, I wanted to give a quick glimpse into where I was at when I began my appointments at church for inner healing. I really had "lost hope" in life being anything more than a constant struggle and tug of war between trying to enjoy what I had and feeling a deep sense of loss and brokenness over what I was missing.
Before I end this post, I just want you to know that the God of miracles really does wonderful work on hearts broken with grief. I didn't need to wait 7 years to feel somewhat "whole" again, and neither do you! In a matter of 2-3 months, he COMPLETELY healed my heart and he wants to do the same for you! Sister, if you are in a place where you need this kind of healing, please drop me a comment below and I will be glad to pray for you!


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Walton Arts Center believes it is vital to expose children to the arts. Our Colgate Classroom Series (CCS) provides us with a way to give the children of Northwest Arkansas access to live matinee performances of theater, dance, puppetry and world music. This year, our focused efforts allowed growth in the reach of the program and cultivated new relationships with a Rural School Initiative.
Since 2009, our reach has doubled - with 100 schools attending CCS performances this past year. The number of registered seats grew as well, reaching 34,000 seats, compared to 2011's 30,000. Our goal next year is to register more than 40,000 students for the Colgate Classroom Series.
This growth is due in part to efforts to make participation as affordable as possible for the schools. Two types of partnerships, Smart Partners and 100% Smart Partner Schools, allow us to work with the schools through every aspect of CCS. Both partnerships receive assistance in ticket prices, online performance guides which support classroom instruction by helping educators connect art forms with core curriculum standards, and transportation subsidies from J.B. Hunt Transportation Inc.
Another way we expanded our reach this year was by focusing on rural schools in NWA. Of the 15 schools targeted, one third attended CCS performances, including Gentry, Pea Ridge, Farmington, West Fork and Decatur. Rural schools in the area account for 5500 students, and we are happy to have made great contacts in order to reach these students in years to come. The goal next year is to have participation from at least half of these schools.
The Colgate Classroom Series gives every NWA school and their students access to world-class arts. Through research with the University of Arkansas we're learning more about how attending live performances at Walton Arts Center benefits students. In a recent research project, Junior High School students from Bentonville and Springdale who attended performances at Walton Arts Center as part of elementary school, were surveyed. The results were impressive. Students who attended more performances demonstrated more positive student values, including greater tolerance and empathy and were more likely to read for pleasure compared to students who attended less or not at all. This research begins to quantify some of the many important benefits the arts have for students.
CCS performances are chosen for their ability to enrich class curriculum. To make the process easier for participating schools, we provide a list of the shows offered, a brief synopsis of each and the suggested grade levels that are appropriate for the shows. This past year a few of the select shows were Boats, Grug and Dinosaur Petting Zoo. We are excited to further expand the CCS reach this next year with theater performances like Digging Up Arkansas, dance productions like Company Käfig and world music demonstrations like AnDa Union!
For a complete listing of the upcoming Colgate Classroom Series, and to learn more about the program, visit our website.


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I had the best time wandering around Brooklyn and hanging out at City Hall with these fellow world travelers. Melissa and Jon, congrats again and thanks for having me as your NYC elopement photographer!
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Engraving round or Irregular objects on a CNC
What if I need to engrave something large or round on my desktop CNC?
With a High-Z you have the option of removing the T-slot table so that you can do just that - engrave a bowling ball, a large cabinet, a large odd shaped object, or anything else that doesn't normally fit under the gantry.
You can create your own fixtures, or you can contact us for a custom under frame that can hold your particular items.
If you need to engrave uneven (not flat) surfaces to a consistent depth, we recommend our depth controller.
Need to see some action? go ahead...
Not had enough yet? No problem.
Head on over to the Tube and browse there instead...


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Coming into Tuesday's game against the Michigan Wolverines, the Wisconsin Badgers had not lost a game at home in over a year. But, the Wolverines gave it their best shot, but ended up coming up just short of an upset win.
The first half was a back and forth battle and the Badgers went into the halftime break only up by 5, 26-21.
The second half was just as exciting and the Wolverines were in this game until the final buzzer. Led by Zak Irvin, who scored 20 points and had 6 rebounds, the Wolverines were giving the Badgers a tough time on offense, but just couldn't score enough down the stretch to steal a win on the road.
For the Badgers, they were in need of someone to step up and close the game, but their superstar Ethan Happ couldn't be that guy, even more of a reason for someone else to step up and close the game at home.
The man who would step into that closer role was number 24, Bronson Koenig, who finished the game with 16 points and 4 rebounds, and had a stretch late in the game where he went on his own 10-0 run and gave the Badgers' a 55-49 lead with 3:54 left in the game.
The 10-0 run was made up of back to back lay-ups and back to back three pointers, all four shots had the Wisconsin' fans on their feet. As he says in this ESPN interview, the key for Koenig was staying ready for the moment and taking his shots.
Another player that was big in this game for the Wolverines was Senior guard Derrick Walton Jr.. He finished with 15 points, 5 rebounds, and a pair of assists. He looked to hurt his hip at the very end of the game in a collision with Wisconsin's Nigel Hayes, but we hope it isn't too serious.
Speaking of Nigel Hayes, he had another good game for the Badgers. He would end the game with 13 points, 6 rebounds, and 3 assists, but he did shoot 40 percent from the free-throw line (4-10).
This loss drops the Wolverines to a record of 12-7 overall, 2-4 in Big Ten play, and marks their third loss in the last four games. They shot really well in this game, 43 percent from the field, 48 percent from the 3-point line, and 90 percent from the free-throw line. The difference was that they had 10 turnovers, two more than the Badgers, and had 21 team fouls.
This win for the Badgers extends their record to 15-3 overall, 4-1 in Big Ten, and keeps them at the top of the Big Ten conference. They are undefeated at home, have been for over a year, and have just that one loss in conference play.
Up next for the Wolverines is a home game against the Illinois Fighting Illini, who have the exact same record as the Wolverines in both overall play and conference play (12-7 and 2-4).
The Badgers will take their game on the road and face a much improved Minnesota team (15-4, 3-3). Despite winning tonight's game, they will need to be more efficient on the offensive end and not rely so heavily on Happ for the entire game. They will need to get Koenig and Hayes involved earlier, because this Minnesota team is capable of taking them out on their home court.
Top 5 Plays from the Badgers' here!
-Matthew Handel


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I visited my friend Charlie's shop in Clanton and was taken by this giant piece of Americana, a deluxe wood burning stove that is probably the best and most complete one I've seen in years. He left it sitting on the roller dolly so it can be loaded, as it takes four strong men to lift it.
I already have a cook stove about half this size that I love, or I would be bringing this one home with me. I think the gray color ( spelled grey if you are Old South) would enhance any color scheme.


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Light Phone is the lightest version of your existing smartphone. This secondary phone is intentionally limited to phone calls only. The Light Phone II brings a few essential tools, like messaging and an alarm clock and a navigation home.
Light was founded by Joe Hollier and Kaiwei Tang, an artist and product designer who met inside of a Google incubator in New York City in 2014. They were encouraged to make software apps and taught on a deeper level how and why different apps were getting built and funded. Quickly it became obvious that the last thing the world needed was another app, clearly these products would never align with our actual quality of life.


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Location: All attractions in Zone 1
Get a London Travelcard
Save up to £101 per person compared to buying on the day tickets! The ultimate way to see all of London's unmissable attractions, working out at just over £10 per attraction.
Please note: This pass /e-ticket only allows a maximum of 9 tickets to be purchased in an order.
Why buy this pass?
The Merlin London Big Adventure: 6-in-1 Pass includes entry to:
Please note, children under the age of 16 must be accompanied by a paying adult of 18+ years. Failure to comply, will result in refused entry to the attractions.
This product is sold by VisitBritain and is provided by our supplier, Merlin Entertainments Group.
Madame Tussauds
Get up close and personal with the world's most famous collection of waxworks at the original Madame Tussauds museum. The Madame Tussauds Merlin Pass gives you the opportunity to:
Recommended length of visit: 3-4 hours
The Coca-Cola London Eye
The biggest observation wheel in Europe, a ride on the iconic London Eye gives you incredible views of London – up to 40km on a clear day. The London Eye Merlin Pass lets you:
Length of a ride on the London Eye: approx. 20 minutes
Disabled access: The London Eye is wheelchair accessible. A carer enters free with disabled customers
SEA LIFE London Aquarium
Submerge yourself in a tropical undersea world and meet over 500 species of sea creature at London's spectacular Aquarium, one of the largest in the world. The SEA LIFE London Aquarium Merlin Pass allows you to:
Recommended length of visit: 2-3 hours
Shrek's Adventure! London
Take a magical journey to Far Far Away and meet Shrek and his friends on an interactive adventure. The Shrek's Adventure! London Merlin Pass gives you the chance to:
Length of tour: approx. 80 minutes
London Dungeon
Experience hundreds of years of London's dark and gruesome history at one of the city's most popular and entertaining attractions.
The London Dungeon combines entertaining live actors, impressive special effects, theme park-style rides and unnerving lighting and darkness to guide you through tales of London's history. With the London Dungeon Merlin Pass you can:
London Eye River Cruise
The London Eye River Cruise Experience departs from the pier at the London Eye, great for taking in all the views of the River Thames. See St Paul's Cathedral, the Houses of Parliament, Tower of London, HMS Belfast, Shakespeare's Globe, Tate Modern and the Millennium Bridge. With a London Eye River Cruiser Merlin Pass, you can enjoy live commentary as you cruise the Thames in comfort.
Length of tour: approx. 40 minutes
How to use your voucher
Please ensure that the name on your voucher matches the name on your ID. Failure to do so may result in you being refused entry to the attraction.
When you exchange your Merlin London voucher, you will be given 1 bar coded ticket which will be scanned at each attraction you visit. When you visit the other attractions, please go to:
Please note: E-vouchers and e-tickets purchased are non-cancellable, non-refundable and non-exchangeable
Redemption point (View on map)
Once you've placed your order, you will receive your eTicket via email. This can be downloaded to your mobile phone or printed when visiting the attraction. Please contact us if you haven't received your email confirmation within 2 working days.
eTickets are non-cancellable, non-refundable and non-exchangeable.
Plan your trip to London with our essential guide to the city.
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Travel as much as you'd like on all public transport in London with one ticket. From the Tube to buses and trains, this fuss-free pass has you covered!
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text: The NFU's Poultry Industry Programme (PIP) is calling on anyone planning their next career move to consider opportunities in the poultry sector.If this sounds like you, be sure to join a special hosted seminar at the 2018 Pig & Poultry Fair on 16 May between 11am-2pm.2018 PIP participant Fede Monte is an agricultural consultant for ADAS, who said: "The industry is really calling out for talented individuals and we want to help open the doors for them." David Speller, a successful broiler farmer, will give insight into how his business has embraced cutting edge technology to improve performance, and the hugely varied roles within his company. His seminar will be followed by a light lunch on the NFU stand where you can discuss anything from feed, genetics, breeding, farm management, veterinary, compliance, transport, engineering and more with individuals at different stages of their careers.Register here for free now NFU poultry board chairman Thomas Wornham said: "The poultry sector is an incredibly rewarding, innovative sector that can offer the next generation a fantastic career. That is why the NFU plays a leading role in helping to develop their opportunities through the Poultry Industry Programme."Danny Johnson from PIP sponsors ABN added: "Joining this seminar at the 2018 Fair is a unique opportunity to speak to those in the industry and explore the opportunities and possibilities this broad and exciting sector offers."Sounds pretty awesome from our end - see you there!Meet the current Poultry Industry Programme participantsFind out what else the NFU has to offer visitors to the Pig and Poultry FairRead other news from the NFU poultry team


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Trans*+ing Classrooms: The Pedagogy of Refusal as Mediator for Learning
The Metropolitan Center for Research on Equity and the Transformation of Schools, New York University, Steinhardt, 726 Broadway, 5th Floor, New York, NY 10003, USA
School of Education, University of Colorado Boulder, 1134 CO-93, Boulder, CO 80309, USA
Academic Editors: Maralee Mayberry and Lane Hanson
Soc. Sci. 2016, 5(3), 34; https://doi.org/10.3390/socsci5030034
Received: 30 April 2016 / Revised: 12 July 2016 / Accepted: 18 July 2016 / Published: 28 July 2016
(This article belongs to the Special Issue Transgender Youth: Focusing on the "T" in LGBT Studies)
Gender and sexuality norms, conscribed under cis/heteropatriarchy, have established violent and unstable social and educational climates for the millennial generation of lesbian, gay, bisexual, transgender, intersex, agender/asexual, gender creative, and questioning youth. While strides have been made to make schools more supportive and queer inclusive, schools still struggle to include lesbian, gay, bisexual, transgender*+, intersex, agender/asexual, gender creative, queer and questioning (LGBT*+IAGCQQ)-positive curricula. While extensive studies must be done on behalf of all queer youth, this work specifically focuses on how to support classroom teachers to uptake and apply a pedagogy of refusal that attends to the most vulnerabilized population of queer youth to date, those that are trans*+. A pedagogy of refusal will be explored through an evolving theory of trans*+ness, then demonstrated through a framework for classroom application, followed by recommendations for change. View Full-Text
Keywords: trans*+; sociospatial justice; trans*+theory; trans*space; trans*+pedagogy; preservice teacher education; post trans*+; trans*+/gender creative youth►▼ Show Figures
Figure 1
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited
MDPI and ACS Style
Miller, S. Trans*+ing Classrooms: The Pedagogy of Refusal as Mediator for Learning. Soc. Sci. 2016, 5, 34.
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Miller S. Trans*+ing Classrooms: The Pedagogy of Refusal as Mediator for Learning. Social Sciences. 2016; 5(3):34.Chicago/Turabian Style
Miller, Sj. 2016. "Trans*+ing Classrooms: The Pedagogy of Refusal as Mediator for Learning." Soc. Sci. 5, no. 3: 34.
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When Epic Brewing Company opened their doors in 2010 they offered a unique craft beer that was a little hard to find in other areas in the state. They sold a high alcohol content beer in 220z bottles, and were empowered by new liquor laws that allowed them to sell product directly from the brewery. Their goal then, and still today, was to turn craft brewing on its head. And indeed they have.
#56 – Project Pineapple: Vacation In A Pineapple
While Utah is known for its premier snow, many people don't quite realize is that the majority of the state is a desert. Which means it gets hot. Really hot. And while we find ways to enjoy the warm and sunny days, the need for a cold treat is frequent. And as the saying goes, when you stumble upon a lemonade stand, put shaved ice in a pineapple.
#50: Chedda Burger – Give It Your All, Or Don't Even Try
One week we're celebrating our one year anniversary, the next, we air our 50th episode! When we launched The Utah Foodie our goal was to create a podcast that dove headfirst into the stories of the creators and innovators in Utah's food scene. Every week we've walked away from our interviews full of inspiration and appreciation. This episode, however, truly highlights how important it is to share these emotional and authentic stories.
When Nick Watts founded Chedda Truck, it was more of an online concept than a real company. Working multiple jobs and needing a creative outlet, Nick would stop by the farmers market during his free time to find the perfect ingredients for his homemade grilled cheese creations. His Chedda Truck Twitter account began to grow, and in a moment of generosity, a friend who owned a tattoo parlor offered him an evening to bring his grilled cheese out into the world for some of his customers.
What began as a low-key offer blew up into a frenzy as news of this grilled cheese launch began to spread through popular food blogs and Twitter accounts. Nick originally anticipated a small crowd, but over 250 customers and grilled cheese sandwiches later, it was apparent that this was more than just a casual evening.
And yet, Nick didn't open his food truck for another six months after his initial launch. After soul searching and working a mind numbing job, Nick knew a drastic change was needed. Hence, Chedda Truck became officially official.
What followed his launch was years of lessons learned, generous friends and strangers, and a full-time investment into Chedda Truck and Chedda Burger that was purely driven out of passion and an unwillingness to give up. With a physical location at 26 E 600 S and two food trucks, Nick's ambition and creativity are inspiring, and he offers wisdom and passion that is infectious.
This episode was sponsored by...
90.9FM KRCL -- Community Connection and Music Discovery
Vive Juicery -- A Utah-based cold-pressed juicery
The Chocolate Conspiracy -- A project in pure, raw, honey-sweetened chocolate.
Blue Copper Coffee -- Specialty coffee hand-roasted in Salt Lake City, with a coffee room at 179 W 900 S.
The Yelp Elite Squad, Utah's five-star fan club of all things local.
This episode was hosted by Chase Murdock and produced by Ryan Samanka. For more information or to browse our episode archive, visit theutahfoodie.com or follow us on Facebook, Instagram, or Twitter. See you next week!
#29: Waffle Love – Build a truck and they will come
When Adam Terry founded Waffle Love in 2012, it was more than a labor of love. It was a dire necessity. With a family to support, bills to pay, and no other options, launching their unique and delicious waffle food truck was fundamental.
Three years, six trucks, and two restaurants later, Waffle Love has grown into a thriving business that continually sells delicious, mouth watering food. With an appearance on The Great Food Truck Race on Food Network, locations in Arizona, and more trucks and restaurants arriving within the next few months, you could say Adam's passion and dedication has worked out.
While many people dream of opening their own restaurant or cooking for a living, many tend to shy away from the gamble that comes from such a competitive industry. But with positivity, hard work, passion, and 110% commitment, finding success is more than achievable. It's possible to get bigger than you ever dared to dream.
Join us on The Utah Foodie as we sit down with Adam, hear his outstanding story, and dive into his infectious passion that will inspire you to tackle all your dreams.
For more episodes and information, visit theutahfoodie.com or follow us on Facebook, Instagram, or Twitter.
#27: Rubadue's Saucey Skillet - Food Trucks, Catering, & Hospital Food
Carl Rubadue has spent over 40 years in the food business, so you can say he's been around the block here in Utah and around the food scene. He got his start at the Mariposa restaurant at Deer Valley resort in 1981. He's since worked for a variety of resorts and catering services, cutting his teeth in kitchens all across the Western US.
A few years ago, Carl Rubadue started his own catering service — Rubadue's Saucey Skillet. If it sounds familiar, it's probably because you've seen his food trucks around Salt Lake Valley or Park City. Saucey Skillet's food truck can be found at "Food Truck Thursdays" at the Gallivan Center, the Utah Beer Festival, or the Twilight Concert Series, and a variety of other places around the state.
In addition to his catering and food truck business, Carl offers cooking lessons through Saucey Skillet Academy.
On today's program we talk with Carl Rubadue about the growth Utah is seeing in its food scene, his time as executive chef of the University of Utah hospital, and his latest venture in food -- a kitchen no wheels. Tune in.
Rubadue's Saucey Skillet http://sauceyskillet.com/
For more episodes and information, visit theutahfoodie.com or follow us on Facebook, Instagram, or Twitter.


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