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Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line.
Methodology and Principal Findings
To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus.
Author Summary
Bunyamwera and serologically related viruses are widely distributed in tropical and sub-tropical regions, and cause febrile illness in man. The viruses possess a trisegmented genome and can evolve by genetic reassortment generating viruses with different pathogenicity, like Ngari virus, a reassortant between Bunyamwera and Batai viruses, which causes haemorrhagic fever in humans. Like other arthropod-transmitted viruses, Bunyamwera virus can replicate efficiently in both mosquito and mammalian cells. Infected mammalian cells are killed by the virus whereas mosquito cells become persistently infected. Understanding the molecular basis for this difference may be crucial in designing new approaches to control bunyavirus disease. The viral non-structural NSs protein is the major virulence factor, which counteracts the innate immune defences of mammalian cells. In contrast, the role of this protein during infection of vector mosquito cells is unknown. We compared the replication of wild type virus and a genetically engineered virus that does not express NSs in various cultured mosquito cell lines and in Aedes aegypti mosquitoes. We showed that some cells did not support mutant virus replication, implying a role for the NSs protein. NSs protein was also important for efficient replication and dissemination in potential vector species.
Citation: Szemiel AM, Failloux A-B, Elliott RM (2012) Role of Bunyamwera Orthobunyavirus NSs Protein in Infection of Mosquito Cells. PLoS Negl Trop Dis 6(9): e1823. https://doi.org/10.1371/journal.pntd.0001823
Editor: Brian Bird, Centers for Disease Control and Prevention, United States of America
Received: May 18, 2012; Accepted: August 6, 2012; Published: September 27, 2012
Copyright: © Szemiel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a Biotechnology and Biological Sciences Research Council Studentship (www.bbsrc.ac.uk), Wellcome Trust Programme Grant 079810 (www.wellcome.ac.uk), and the Pasteur Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family. It was originally isolated from a pool of several Aedes spp. mosquitoes collected in the Semliki Forest in Uganda [1]. Based on detection of antibodies to BUNV in human sera and isolations of BUNV from patients suffering febrile illness, the virus is widely distributed in several regions of sub-Saharan Africa [2]–[4]. BUNV is maintained in nature by a propagative cycle involving blood-feeding mosquitoes and susceptible vertebrate hosts, probably small rodents [5]. BUNV can replicate efficiently in both vertebrate and invertebrate cells in culture but with different outcomes: in mosquito cells no cytopathology is observed and persistent infection is established, whereas in mammalian cells infection is lytic and leads to cell death [6]–[8]. From a practical standpoint, this is shown by the ability of the virus to form clear lytic plaques in cells of vertebrate origin but not in those derived from insects.
Like all bunyaviruses, BUNV is an enveloped virus containing a tri-segmented, single stranded negative-sense RNA genome that encodes four common structural proteins: an RNA-dependent RNA polymerase (L protein) on the large (L) segment, two glycoproteins (Gc and Gn) on the medium (M) segment and the nucleoprotein (N) on the smallest (S) segment. BUNV also codes for two non-structural proteins, NSm on the M segment and NSs on the S segment [9]. The segmented nature of the genome allows for reassortment between closely related orthobunyaviruses to generate viruses that may have altered biological properties, such as Ngari virus, which is associated with human haemorrhagic fever in East Africa, whose genome comprises L and S segments from BUNV and M segment from Batai virus [10]–[12]. BUNV continues to serve as a convenient laboratory model to study the molecular biology of bunyaviruses in general, and an understanding of many aspects of gene function and viral replication have been developed using BUNV, including the establishment of a robust reverse genetics system [13]–[15].
The BUNV NSs protein is a nonessential gene that contributes to viral pathogenesis. It has been shown that in mammalian cells, NSs induces shut-off of host protein synthesis [16], [17], which leads to cell death. It also counteracts the host cell antiviral response and seems to be the main virulence factor [18]–[21], acting at the level of transcription by inhibiting RNA polymerase II–mediated transcription [22]. In mosquito cells neither host cell transcription nor translation are inhibited [17], and although so far no function for the orthobunyavirus NSs protein has been found in mosquito cells [23], it seems the differential behaviour of NSs could be one of the factors responsible for different outcomes of infection in mammalian and mosquito cell lines.
To date, studies on BUNV replication in mosquito cells have been limited to the C6/36 line [24] from Aedes albopictus [6]–[8], [16], [17], [25]–[27]. As differences in the appearance of cytopathic effects, cell death and viral morphogenesis were observed in various Ae. albopictus subclones infected with Sindbis virus [28]–[32], we have compared the replication of BUNV in two additional Ae. albopictus cell clones, C7-10 [33] and U4.4 [34]. All three cell lines were independently obtained from the original Singh cell line derived from Ae. albopictus neonate larvae [35], which has been shown to comprise a heterogeneous population of cell types. More recently it has been reported that these cell lines differ in their RNAi responses: C7-10 [36] and C6/36 [37]–[39] cells have impaired Dicer 2-based RNAi responses, whereas the U4.4 cell line encodes a fully functional Dicer 2 gene [36], [40]. Secondly, we have investigated the ability of BUNV to replicate in Aedes aegypti (Ae) cells, and have compared this to the infection in living Aedes aegypti mosquitoes. Here we report that Ae. albopictus U4.4 and Ae. aegypti Ae cells are refractory to infection with a recombinant BUNV lacking the NSs gene (rBUNdelNSs2; [16], [17]), and that expression of NSs influences the efficiency of viral replication in Ae. aegypti mosquitoes.
Materials and Methods
Cells and viruses
Aedes albopictus C6/36, C7-10 and U4.4 and Aedes aegypti Ae cells were grown at 28°C in Leibovitz 15 medium (Gibco) supplemented with 10% foetal calf serum (FBS) and 10% tryptose phosphate broth. Vero E6 cells were maintained at 37°C in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% FBS. Working stocks of wild-type Bunyamwera virus (wtBUNV), the recombinant NSs deletion virus (rBUNdelNSs2; [17]), rBUNGcGFP [41] and rBUNdelNSs-GcGFP [42] were prepared as described previously [18], [43].
Virus growth curves and titration
Mosquito cells were infected at an MOI of 1 PFU/cell. After 1 h incubation at 28°C, the inoculum was removed and the cells were washed once to remove unattached virus. Supernatants were harvested at various times post-infection and assayed for virus by plaque assay on Vero E6 cells as previously described [13], [44]. In our laboratory, we routinely titrate BUNV on Vero E6 cells as these give the most easily discernible plaques. We have also performed immunostaining assays of viral foci produced in C6/36 cells, and observed that the efficiency of plating compared to Vero E6 cells is in the range 0.5 to 1 (data not shown). This is similar to the plating efficiency for other arboviruses, like Dengue, West Nile and St Louis encephalitis viruses [45], when comparing titration on C6/36 and vertebrate cells.
Analysis of protein by Western blotting
At different times after infection, cell lysates were prepared and equal amounts of sample were separated on 12% SDS-PAGE. Separated proteins were transferred to Hybond-C Extra membranes (Amersham). The membranes were incubated with rabbit anti-BUN N protein antibody (1∶2000 dilution) and mouse anti-tubulin antibody (Sigma; 1∶10000) as a loading control, followed by incubation with anti-rabbit horseradish peroxidase-coupled antibody (Cell Signaling Technology). Immunocomplexes on the membranes were detected by SuperSignal West Pico Substrate (Pierce) according to manufacturer's instructions.
Indirect immunofluorescent staining
Mosquito cells were cultured on glass coverslips and infected with rBUNGceGFP or rBUNdelNSs-GcGFP at MOI of 1 PFU/cell. At various times post infection, cells were fixed with 4% formaldehyde and washed with PBS. The cells were incubated with rabbit anti-BUN N (1∶200) and mouse anti-tubulin (Calbiochem; 1∶100) antibodies, followed by incubation with Texas Red-conjugated anti-rabbit (Cell Signaling Technologies; 1∶200) and CY5-conjugated anti-mouse (Sigma; 1∶400) antibodies. Nuclei were stained with DAPI. Prepared slides were examined with a Zeiss LSM confocal microscope.
Mosquito infections
Laboratory-bred Ae. aegypti (Paea strain) mosquitoes were reared as previously described [46]. Female mosquitoes were selected and exposed to blood-meal containing approx. 108 PFU/ml of wtBUNV or rBUNdelNSs2 as described previously. At various times post infection, mosquitoes were anesthetized and either homogenized whole or the midguts and salivary glands were dissected. Organs and whole mosquitoes were homogenized in 100 µl of Leibowitz 15 medium (Gibco). Twenty-five microliters of each sample were used for titration by plaque assay on Vero E6 cells.
Production of double-stranded RNA and transfection of mosquito cells
dsRNA approx. 1000 bp long was prepared using the Megascript RNAi kit (Ambion). Virus specific dsRNA were prepared from linearized plasmids containing full-length cDNAs of the BUNV genome segments pT7riboBUNS(+) and (−), pT7riboBUNM(+) and (−) and pT7riboBUNL (+) and (−) [13]. Renilla-specific dsRNA were prepared from plasmid phRL-CMV by adding a T7 promoter sequence at each end by PCR as described previously [40]. Purified dsRNA was stored in aliquots at −80°C. A total of 3×105 cells per well were cultured in 24 well plates. 100 ng of Renilla dsRNA or 100 ng of a 1∶1∶1 mixture of S-, M-, and L-segment specific dsRNA was used for transfection. One microliter of Lipofectamine-2000 (Invitrogen) was used per 100 ng and the transfection mixes were prepared in the final volume of 100 µl as per the manufacturer's protocol; this was then applied onto the cells with 400 µl fresh complete L15 medium. After 5 hours incubation at 28°C, 500 µl of fresh medium was added. The cells were infected with wtBUNV or rBUNdelNSs2 24 h post-transfection, supernatants were collected at various times thereafter for assay of released virus by plaque titration on VeroE6 cells.
Growth of BUNV in various Ae. albopictus cell lines
In mosquito cells no shut-off of host cell transcription or translation has been observed. Therefore, we first confirmed that BUNV NSs protein is expressed in these cells. Ae. albopictus C6/36 cells were infected at MOI of 10 pfu/cell and the cells were harvested for protein analysis by Western blotting at different time points (Figure 1). Although BUNV N and NSs proteins are translated from the same mRNA, NSs was produced, though slightly later in the course of infection than the nucleoprotein. This is in line with previous observations [8] based on radiolabelling of infected cells.
Ae. albopictus C6/36 cells were infected at an MOI of 10 PFU/cell and samples were harvested at the indicated time points. Cell lysates were electrophoresed on a 4–12% NuPAGE gel (Invitrogen). After transfer the membrane was cut into three and strips were incubated with anti-BUNV NSs protein (NSs), anti-BUNV N protein (N) or anti-tubulin (T) antibodies as indicated.
We next compared virus replication in Ae. albopictus C6/36, C7-10 and U4.4 cell lines. Cells were infected at an MOI of 1 PFU/cell and the samples were collected at various times post infection. wtBUNV was able to replicate in all three cell lines but with different kinetics (Figure 2A). Growth was most efficient in C6/36 cells, with maximum titres of released virus exceeding 108 pfu/ml, 10-fold and 100-fold higher than in C7-10 or U4.4 cells, respectively. rBUNdelNSs2 grew more slowly than wtBUNV in C6/36 cells and yielded maximum titres about 100-fold less, confirming previous results [16], [17]. In C7-10 cells, the mutant virus showed similar kinetics to wt BUNV. In marked contrast, no increase in titre of rBUNdelNSs2 in culture medium of U4.4 cells was detected. (The detection of some rBUNdelNSs2 virus in supernatant medium (Figure 2A) represents residual virus that remained after removal of the inoculum and replacement with fresh medium; these cells did not adhere firmly enough to the culture vessel to permit extensive washing).
A. Growth curves. C6/36, C7-10 and U4.4 cells were infected at an MOI of 1 PFU/cell. At the indicated time points, supernatants were collected and viral titres were determined by plaque assay. B. Viral N protein expression. Cells were infected at an MOI of 1 and at the indicated time points (hpi), cells were lysed, and proteins separated by 12% SDS-PAGE. The proteins were transferred to a membrane and incubated with anti-BUNV N protein (N) or anti-tubulin (T) antibodies.
These data were supported by Western blot analysis to detect accumulation of the viral N protein (Figure 2B). In C6/36 cells infected with wtBUNV, N could be seen as early as 16 h post-infection (hpi), whereas in cells infected with the NSs deletion, mutant N was not detected until 36 hpi. In the C7-10 cell line, no significant difference between the two viruses was observed in terms of accumulation of N protein, though growth was slower than in C6/36 cells, with N protein first detectable at 48 hpi. In U4.4 cells infected with wtBUNV, N was detectable as early as 42 hpi whereas no N protein could be detected in cells infected with rBUNdelNSs2. These results suggest that BUNV NSs protein is not essential for growth in either C6/36 or C7-10 cell lines, but is required for successful replication in U4.4 cells.
Morphological phases of infection in mosquito cells
To analyse how BUNV spreads in mosquito cells, Ae. albopictus C6/36, C7-10 and U4.4 cells were infected with recombinant BUNV expressing green fluorescent protein [41], either rBUNGc-eGFP or rBUNdelNSs-GcGFP at an MOI of 3 PFU/cell. Three different stages of infection, as proposed by Lopez-Montero et al. (2009), were observed in all the Ae. albopictus cell lines infected with rBUNGc-eGFP. These phases were defined by changes in cell morphology due to virus replication, most notably that infected cells produced projections extending towards neighbouring cells (Figure 3A). During the early phase of infection with the wild type version of the eGFP-expressing BUNV, the levels of viral proteins were relatively low and the cells resembled uninfected cells in shape (Figure 3B). However, as infection progressed and the levels of viral proteins increased, cells transitioned into the acute phase. This phase was characterised by formation of filopodia that were most abundant between the 24 hpi and 48 hpi; over this time period, cells maintained physical contact via the projections. During the acute phase, the levels of eGFP-tagged Gc increased and the glycoprotein was also found in the filopodia (Figure 3B). The late phase of the infection was characterised when the cellular filopodia started to disappear (from 48 hpi), which was also manifested by elevated levels of Gc glycoprotein detected in the cells. Later on in the infectious cycle, the cells returned to their normal form (Figure 3B).
Mosquito cells were infected with rBUN-GcGFP or rBUNdelNSs-GcGFP at an MOI of 3 PFU/cell. The results present cells stained with anti-BUNN/anti-rabbit TexasRed (red signal), and with anti-tubulin/anti-mouse CY5 (light blue signal). The green signal shows autofluorescence of GFP-tagged Gc and the blue signal is DAPI staining of nuclei. A. C6/36 cell infected with rBUN-GcGFP virus during the acute stage of infection. B. C6/36, C7-10 and U4.4 cells were infected with fluorescent viruses. Three morphological stages of infection are presented: early, acute, and late. Only the merged images are shown.
These results were consistent for infection of C6/36 and C7-10 cells. Infection in U4.4 cells was slower, and fewer cells were infected in comparison to C6/36 and C7-10 cells. Also, U4.4 cells produced fewer filopodia (Figure 3B). Comparison of the levels of viral proteins in infected cells revealed that BUNV replication in U4.4 cells was less intense than in C6/36 and C7-10 cell lines, as relatively lower amounts of N and Gc proteins were produced. These observations corresponded with the differences in viral titres observed in the previous experiments (Figure 2), where U4.4 cells produced less infectious particles than the two other cell lines.
The course of infection with the NSs deletion recombinant, rBUNdelNSs-GcGFP, was similar to rBUNGc-eGFP in terms of levels of viral protein expression, but the infection started later in C6/36 and C7-10 cells. Investigation of cell morphology showed that fewer filopodia were produced and they were less pronounced in rBUNdelNSs-GcGFP infected cells, suggesting involvement of NSs in this process. Minimal disruption of the normal cell morphology was observed; those changes that were seen were attributed to cell movement and consecutive attachment to the surface, as similar changes were observed in uninfected cells (Figure 3B). These data suggested that NSs protein contributed to the efficiency of viral replication, but was a non-essential protein.
When U4.4 cells were infected with rBUNdelNSs-GcGFP, a few cells (less than 5%) were observed to harbour replicating virus in that synthesis of tagged Gc protein could be observed by its autofluorescence (Figure 3B). This suggests that infection by rBUNdelNSs-GcGFP is abortive and few, if any, new infectious particles were produced, in line with the titration data shown in Figure 2.
In order to rule out the possibility that changes in morphology were due to alterations of Gc glycoprotein caused by fusion of the GFP sequences, another fluorescent Bunyamwera virus was used, with eGFP fused in-frame into NSm [46]. Infection of the C6/36, C7-10 and U4.4 cells with rBUNM-NSm-EGFP, but not with rBUNdelNSs-NSm-EGFP, resulted in similar marked morphological changes as shown by rBUNGc-eGFP (data not shown).
BUNV establishes persistent infection in Ae. albopictus cell lines
It has been previously documented that the outcome of infection of C6/36 cells with wtBUNV is the establishment of a persistent infection [6]–[8]. To determine whether persistent infections could also be established in C7-10 and U4.4 cells, and if the NSs protein participates in this process, cell monolayers were initially infected with wtBUNV or rBUNdelNSs2 at an MOI of 0.1 PFU per cell. After four days, supernatant fluids were collected for titration of released virus, and the infected cells were passaged (split ratio of 1∶5) and grown until confluent. Thereafter, they were regularly passaged and maintained for about 7 months (25 passages). All lines initially infected with wtBUNV or rBUNdelNSs2 continued to shed infectious virus at each passage, though the titres fluctuated widely (Figure 4A). Similarly, N protein was detected by Western blotting in all passaged cells, though the levels varied. Thus wtBUNV could establish persistent infections in all three cell lines, and rBUNdelNSs2 in C6/36 and C7-10 cells. As expected, since U4.4 cells did not show evidence of productive infection by rBUNdelNSs2, no indication of a persistent infection was obtained. Thus the NSs protein was not essential for establishment of persistent infection in C6/36 or C7-10 cells.
A. Titres of virus released into the supernatant from each passage of cells. Cells were initially infected at MOI of 0.1 PFU/cell, and passaged regularly at a 1∶5 split ratio. The supernatant medium was collected at the time of each passage and the presence of virus measured by plaque assay. B. Detection of viral N protein. An aliquot of cells was collected at each passage, lysed and 10 µg of total protein was analyzed by Western blotting with anti-BUNV N protein antibodies. Numbers at the top of the blots indicate the passage number.
In previous studies of C6/36 cells persistently infected with BUNV, the generation of viruses displaying different plaque phenotypes when titrated in mammalian cells was observed [6], [7]. Similarly, when titrating the two viruses released from all persistently infected cell lines described above in Vero cells, we observed the appearance of large, small and cloudy plaques from different passages (data not shown).
Activation of Dicer 2 in U4.4 cells counteracts wtBUNV replication
As mentioned above C7-10 and C6/36 cells are reported to have defective Dicer 2-based RNAi responses [36]–[39], whereas the U4.4 cell line encodes a fully functional Dicer 2 gene [36], [40]. To determine whether the cells could mount an RNAi response against BUNV infection, we transfected C6/36, C7-10 and U4.4 cells with long virus-specific dsRNA, or Renilla luciferase-specific dsRNA as a control, and then infected the cells with either wtBUNV or rBUNdelNSs2 at a MOI of 5 PFU per cell. Supernatant fluids were collected at various times post-infection and assayed for the presence of infectious virus by plaque formation. Each infection was done in triplicate and the experiment was repeated twice. As shown in Figure 5, no effect on virus growth was seen in C6/36 or C7/10 cells infected with either virus. However, in U4.4 cells transfected with virus-specific dsRNA and then infected with wt BUNV, virus replication was inhibited, and no increase in virus titre was observed. In contrast, transfection of Renilla luciferase-specific dsRNA had no effect on wtBUNV growth in any cell line. These results are consistent with U4.4 cells having a functional dsRNA-mediated interference system. Unfortunately, because rBUNdelNSs2 virus does not replicate productively in U4.4 cells it was not possible to determine whether BUNV NSs protein could be involved in evasion of an RNAi response in mosquito cells.
Cells were transfected with BUNV-specific long dsRNA, Renilla-specific long dsRNA or left untransfected (untreated) as indicated. At 24 h post-transfection, cells were infected with wtBUNV or rBUNdelNSs2 at an MOI of 5 PFU/cell. Supernatants were collected at the indicated times post infection for determination of the presence of infectious virus by plaque assay.
BUNV replicates and establishes persistent infection in the Ae. aegypti Ae cell line
Although Ae. albopictus cell lines are widely used to investigate arbovirus replication, the genome sequence of Ae. albopictus has yet to be determined, thus limiting detailed investigation of molecular details of virus-host interaction. To date three mosquito genome-sequencing projects have been completed, Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus [47]–[49], and hence we examined whether cell lines from other mosquito species would be useful to monitor BUNV replication. In preliminary experiments, no evidence for BUNV growth in the Sua4 cell-line derived from An. gambiae Suakoko strain [50] was obtained (data not shown). However, the Ae cell line [51] derived from Ae. aegypti was shown capable of supporting wtBUNV replication (Figure 6). The virus grew to titres approaching 107 PFU/ml, and accumulation of BUNV N protein from 48 hpi was detected by Western blotting. In contrast, rBUNdelNSs2 appeared unable to replicate in these cells, as N protein was not detected by Western blotting (Figure 6B) and no increase in titre of infectious virus in the supernatant medium was measured. (Again as these cells did not adhere firmly to the culture vessel, extensive washing to remove the virus inoculum was not possible, and only residual infecting virus was detected).
A. Growth curves. Ae cells were infected with wtBUNV or rBUNdelNSs2 at an MOI of 1 PFU/cell. At the indicated times post infection supernatants were collected and assayed for the presence of infectious virus by plaque titration. B. Viral N protein expression. Cell extracts were prepared at the indicated times (h P.I.), separated by 12% SDS-PAGE, and proteins transferred to a membrane. The membrane was incubated with anti-BUNV N protein and tubulin (T) antibodies. C. Establishment of persistent infection. The titres of infectious virus in supernatants collected at each passage of infected cells were determined by plaque assay.
To investigate if BUNV was capable of establishing persistent infection in the Ae cell line, cells were infected with wtBUNV or rBUNdelNSs2 at an MOI of 0.1 PFU per cell. Supernatants were collected and infected cells were then passaged using a fifth of gathered cells as it was done with Ae. albopictus cell lines. Cells were maintained for 7 passages. Analysis for the presence of infectious virus in the supernatants showed the Ae cell line to be persistently infected with wtBUNV (Figure 6C). However the levels of infectious virus production remained low and there was no significant difference between consecutive passages as was seen in Ae. albopictus cell lines. Infection with rBUNdelNSs2 showed a different pattern. Infectious virus particles were detected after the first passage, but no virus was detected in the supernatants for the next two passages. However, from passage 4, low titres of virus were detected (Figure 6C). This pattern was reproducibly observed in two further independent infections of Ae cells with rBUNdelNSs2, and also when another Ae. Aegypti cell line, A20 [51] was infected with rBUNdelNSs2: in all cases no virus could be detected by plaque assay of supernatants from the second and third passages, but virus was detected at subsequent passages at low levels (data not shown).
NSs is required for efficient replication and spread in mosquitoes
Having demonstrated that BUNV could replicate in Ae. aegypti cell cultures, we next studied the infection of Ae. aegypti mosquitoes. It has been reported previously that wtBUNV is capable of replicating in laboratory raised Ae. aegypti mosquitoes and of being transmitted via mosquito saliva to mice [52]. By comparing infection with wtBUNV and rBUNdelNSs2, we investigated the role of NSs in an insect vector. Female Ae. aegypti (Paea strain) mosquitoes were allowed to feed on a blood-meal containing wtBUNV or rBUNdelNSs2 (approx. 108 PFU/ml in the blood meal). After feeding, only engorged mosquitoes were kept for the course of the experiment. Three engorged females were collected from each infection group immediately after feeding to determine the amount of virus ingested. Based on the titration results, it was estimated that each mosquito imbibed two to three microliters of blood (data not shown). These blood-meal volumes were similar to those previously published for various mosquito species, including Ae. aegypti [53]–[56]. The survival rates following the blood-meal were calculated by recording the number of dead mosquitoes daily. Survival rates for each virus were above 98%, which suggests that neither wtBUNV nor rBUNdelNSs2 had any detrimental effects on mosquito viability.
At various times post feeding, 8 to 10 female mosquitoes were collected, homogenised and the levels of infectious virus they contained were titrated by plaque assay. Infection rates were calculated by dividing the number of infected female mosquitoes by the total number of engorged mosquitoes tested at a given day post-feeding (Figure 7). For wtBUNV, >70% of mosquitoes contained infectious virus at all time points. In contrast, at day 1 after feeding, only 20% of mosquitoes fed rBUNdelNSs2 showed evidence of infectious virus. However, by 5 days post-feeding, virus was detected in 60% of mosquitoes, and this rose to 80% by day 9 (Figure 7). Thus the lack of NSs seemed to delay the progress of infection.
Following infection via blood-meal, 8 to 10 engorged mosquitoes were collected at the indicated days after feeding, individually homogenized and the presence of infectious virus determined by plaque assay. Mosquito infection rates were calculated as the percentage of virus positive females over total number of engorged mosquitoes tested.
To confirm that lack of NSs results in delayed BUNV replication, in another experiment, midguts and salivary glands were dissected from engorged mosquitoes and examined for presence of virus. At days 1 to 13 post-feeding, nine mosquitoes, and at days 15 to 21 post-feeding, six mosquitoes, were collected, and pools of three isolated organs were made. Infectious virus could be detected in the midgut by 2 days post-infection for both viruses. By 4 days post-feeding, midgut infection rates (calculated as percentage of virus positive midguts among engorged mosquitoes tested) for wtBUNV reached about 80%, and stayed at high levels for the duration of the experiment (Figure 8A). In comparison infection rates by rBUNdelNSs2 were significantly lower, and more variable. The titres of virus in midguts were also different between the viruses, with wtBUNV titres being about 100-fold higher than those of rBUNdelNSs2 (Figure 8B).
Following infection via blood-meal, 9 engorged mosquitoes were collected at days 1 to 13, and 6 engorged mosquitoes were collected at days 15, 18 and 21. Midguts and salivary glands were dissected, pooled into groups of 3 organs, and infectious virus determined by plaque assay. A. Midgut infection rates. The percentage of virus positive midguts over total number of tested mosquitoes was calculated. B. Average virus titre per mosquito midgut. Error bars show the standard error between the different pools. C. Disseminated infection rates. The percentage of virus positive salivary glands over total number of positive mosquito midguts was calculated. D. Average virus titres per mosquito salivary gland. Error bars show the standard error between the different pools.
In order to be successfully transmitted by a mosquito vector to a vertebrate host, following replication in the midgut the virus must disseminate to secondary tissues like muscles, haemolymph, fat body and eventually the salivary glands. If the virus is found only in the midgut, infection is regarded as non-disseminated. Therefore the transmission potential of BUNV in infected mosquitoes was estimated by calculating disseminated infection rates to salivary glands. Both viruses managed to disseminate to salivary glands successfully, though with different kinetics. wtBUNV was detected in salivary glands by 3 days after feeding whereas the NSs-deletion mutant was not detected in salivary glands until 6 days post-feeding (Figure 7C). The titres of wtBUNV were generally higher in salivary glands than those of rBUNdelNSs2 (Figure 7D). These data suggest that the virus lacking NSs had more difficulty in overcoming the cellular defences in the midgut, but when rBUNdelNSs2 did manage to overcome the midgut escape barrier it was capable of spreading throughout the rest of the body, including to salivary glands.
The BUNV NSs protein has been widely studied in mammalian cells where it is has been shown to be a major virulence determinant. NSs counteracts the host innate immune response mainly by globally inhibiting RNA polymerase II-mediated transcription [16], [18]–[21]. On the other hand, BUNV NSs does not affect cellular transcription in infected mosquito cells [17], and for the related La Crosse orthobunyavirus (LACV), no specific function for its NSs protein was found in mosquito cell lines [23]. However, our results presented above show that the BUNV NSs protein could be a crucial factor for efficient infection in certain cultured mosquito cells and in live mosquitoes.
Comparison of wtBUNV and rBUNdelNSs2 showed that NSs is nonessential for replication and establishment of persistent infection in Ae. albopictus C6/36 and C7-10 cells. By contrast, rBUNdelNSs2 seemed unable to replicate productively in neither the Ae. albopictus U4.4 cell line nor the Ae. aegypti Ae cell line, indicating a requirement for the NSs protein. Unfortunately, attempts to express NSs exogenously in U4.4 cells, and thus enable rBUNdelNSs2 replication, have so far been unsuccessful. Comparison of the levels of released wtBUNV and the recombinant NSs deletion mutant suggests that NSs protein enables high-level virus replication in all cells except Ae. albopictus C7-10, where expression or not of NSs had little effect. It is becoming clearer that, much like with mammalian cell lines, different mosquito lines differ in their response to viral infection and their ability to reproduce accurately events in whole mosquitoes [31], [57], [58]. Our data suggest that of the Ae. albopictus lines, the U4.4 cell line could similarly be a good tissue culture model to study BUNV replication and the role of NSs in infection of mosquito cells.
The three phases of infection, early, acute and late, described by Lopez-Montero and Risco [27] in BUNV-infected C6/36 cells were also identified in the other two Ae. albopictus cell lines. These phases were characterized by changes in the location of viral proteins and changes of the cell morphology throughout each stage. Microscopic observations of the wild type and NSs-deleted viruses showed that lack of NSs reduced the extent to which mosquito cells underwent morphological changes during the acute stage of infection. Possibly these changes are driven by a defense mechanism that allows the cells to cope with severe viral infection, and Lopez-Montero and Risco [27] suggest that the filopodia-like projections could be involved in spreading "protective signals" among the cells. Branch-like projections have also been observed in cells infected with a rodent-transmitted bunyavirus, Sin Nombre hantavirus, and suggested to be sites where progeny particles were released [45], [59]. A release method that does not involve rupturing the cell membrane could explain why virus replication does not kill mosquito cells and persistence is maintained.
To date the only mosquito-borne bunyavirus that was shown to induce RNAi response in mosquito cells is La Crosse orthobunyavirus [23], [37], [39]. We have also detected, by conventional Northern blotting analysis, virus-specific small RNAs (<30 nucleotides) in all Ae. albopictus cell lines infected with wtBUNV (data not shown). Soldan et al. (2005) showed that La Crosse virus replication could be inhibited in C6/36 cells pre-treated with virus-specific small interfering RNAs [60]. Here we showed that an RNAi response could be efficient in inhibiting BUNV infection too. When we transfected cells with long virus-specific dsRNA, BUNV replication was only reduced in U4.4 cells, which have previously been shown to have fully functional Dicer 2 [37], [39], suggesting that the dsRNA was processed efficiently to generate small inhibitory RNAs. Bunyaviruses efficiently avoid dsRNA-based RNAi responses by coating their RNA segments with the nucleoprotein, thereby avoiding the formation of dsRNA species [61]. Our transfection experiments showed that if specific dsRNA species were produced in abundance, mosquito cells could overcome wtBUNV infection. Interestingly, the BUNV NSs deletion mutant was capable of efficient replication only in Dicer 2 incompetent cell lines. There are no studies showing involvement of any bunyavirus NSs protein in overcoming RNAi response in mosquito cells, but the NSs protein of La Crosse virus has been shown to inhibit RNAi antiviral activity in mammalian cells [60]. Further work is required to investigate whether BUNV NSs has an effect on mosquito Dicer 2 activity, or if exogenous expression of NSs in U4.4 cells would render them permissive for rBUNdelNSs2 replication.
The lack of genomic sequence data for the Ae. albopictus mosquito makes it a less attractive model in which to study host-virus interactions. Therefore, we investigated whether cells derived from Ae. aegypti, whose sequence has been determined [48], were permissive for BUNV replication. Our results showed that BUNV growth in Ae cells resembled that in U4.4 cells, and that the NSs protein also proved to be necessary for efficient replication. Thus Ae. aegypti cells could be a useful tool in studying BUNV infection and identification of cellular components that are important for viral replication.
There is only one study of BUNV replication in mosquitoes; Peers [52] reported that BUNV multiplied in the gut, disseminated to salivary glands and was transmitted to suckling mice in Ae. aegypti mosquitoes more efficiently than in Ae. vexans and Ae. canadensis. Our results for wtBUNV showed similar kinetics of viral replication and dissemination in Ae. aegypti to those obtained by Peers. Fewer mosquitoes were infected with the NSs-deletion mutant. In addition, we showed that the NSs protein contributes to high-level virus replication in that the mutant virus lacking NSs grew to lower titres. Similarly, the wild-type virus disseminated to salivary glands more efficiently than rBUNdelNSs2, and the lower levels of rBUNdelNSs2 in salivary glands could affect the transmission potential of the virus. This requires further investigation.
Our experiments showed that BUNV replication in Ae. aegypti mosquitoes resembled replication in Ae. aegypti Ae cells, as well as Ae. albopictus U4.4 cells. These data corroborate previous conclusions that these cell lines are the most appropriate mosquito cell culture models to study arbovirus infection. The NSs protein was required for efficient replication in both mosquito cells with a competent Dicer 2-RNAi system and in adult mosquitoes. In a proportion of mosquitoes, however, the mutant virus could eventually overcome host defences, though remained constrained as evidenced by lower virus titres. As BUNV is a relatively fast growing virus perhaps its reproduction rate is able to counteract the host's inhibitory responses. The NSs protein of Rift Valley fever phlebovirus (also in the family Bunyaviridae), although being quite distinct in size, amino acid sequence and expression strategy from BUNV NSs, plays a similar role in mammalian cells in overcoming innate immune responses via global shut-down of cellular transcription [62]. Two recent papers investigated the role of Rift Valley fever virus NSs in infection of mosquitoes, and neither observed any difference in infection or dissemination rates between wt and NSs-deleted viruses [63], [64]. Interestingly, deletion of another non-structural protein, NSm, from Rift Valley fever virus almost completely abolished its ability to replicate in mosquitoes [64]. These results illustrate the diversity and complexity of virus-host interactions within the Bunyaviridae family.
We thank Dr Denis Brown for providing C7-10 and U4.4 cells, Dr Xavier de Lamballerie for providing Ae cells and Dr Michèle Bouloy for help in facilitating the mosquito infection experiments in the Institut Pasteur.
Author Contributions
Conceived and designed the experiments: AMS A-BF RME. Performed the experiments: AMS A-BF. Analyzed the data: AMS A-BF RME. Contributed reagents/materials/analysis tools: AMS A-BF RME. Wrote the paper: AMS RME.
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text:
Jelly Bean needed a new logo design and created a contest on 99designs.
A winner was selected from 97 designs submitted by 17 freelance designers.
Jelly Bean Photography
I am a mom and a self-taught photographer. Photography has always been my passion. After I had my first little girl, I decided to make my passion and dream a reality. I would call myself a lifestyle photographer.My aim is to bear witness to life's journey. My style of photography is a melting pot of candids, whimsical, vintage and contemporary. I am not your typical studio/portrait photography. I appreciate the art in photography and enjoy capturing those rare, special moments that you want to cherish forever. I shoot on location or at your home. I don't believe in fake smiles. I see the beauty in frowns and lost teeth, in boo-boos and bare feet.
My target audience are mommies who want to capture their children and families' life moments.
I want the logo to incorporate jelly beans.
I am fine with cartoons
The logo should be unique, playful,modern
Please feel free to experiment with colors!
Do not be too fancy
Please no birds, hearts or butterflies...jelly beans and cameras and subjects need to be incorporated into the logo
The files we will require are:
Print file format as well as Web file formats
(my web site requires a png image)
Please do not use any stock images or clip art in the logo
go to my web site at http://www.jellybeanfoto.com to browse my style
Find out how a design contest works or learn more about our logo design services.

subset: commoncrawl


text:
Emerson CF152WW 52" Curva Sky 52" Ceiling Fan in White
- Minimal and contemporary design
- Perfect for tight or confined spaces
- Set of 3 curved appliance white blades
- Reviews
- Description
- Specifications
- Benefits
- Resources
Emerson CF152WW 52" Curva Sky 52" Ceiling Fan in White elegantly cools and ventilates even some of the most stuffy rooms.
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May be subject to restocking fee
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- Model: CF152WW
- Product Type: 52" Ceiling Fan in White
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- Type: Ceiling Fan
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- 3 appliance white blade set included
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- Exterior is finished in appliance white
- Modern design is elegant and universal
- Remote control and built-in downlight included
- Warrantied for life to ensure reliable performance


text:
Former Bulls star Dennis Rodman has been found in contempt of court and ordered to pay $500,000 in back child support to his ex-wife, her attorney said Thursday.
The flamboyant basketball player known for his off-court antics was sentenced to informal probation, his ex-wife's attorney, Mary Ann Noiroux, told City News Service. Orange County Superior Court Commissioner Barry Michaelson warned Rodman could face jail time if he doesn't pay the child support, she said.
Rodman's attorney, Linnea Willis, didn't return an email from The Associated Press and a phone number did not take messages.
Another attorney for Rodman's ex-wife argued in court that Rodman owed his...


text:
1989 Toyota Pickup - Mike's Red ToyPosted in Features on April 1, 2006
The '89 Toyota truck spread across these pages rolled off the assembly line some 16 years ago. It lived a very gentle existence for the first half of its life as commuter and weekend-trip transportation. In 1996, the present owner, Mike Lewis, purchased the lightly used rig and began using it as his daily driver. An added camper was removed from the bed and Mike used the truck for some light-action wheeling running 31-inch tires in the desert near Phoenix.
For a number of years, the truck stayed mostly stock as Mike explored back roads and trails. The IFS served him well with a comfortable street and off-highway ride, getting him through most of what he demanded of it.
A few significant events happened and things started to change. On one trail outing, Mike bent the rear driveshaft on a boulder and was forced to drive the truck some considerable distance with it bent and vibrating badly. He was stuck for nearly a week commuting to work and back with this malady, and that's when something went awry. While driving down the highway, the tranny could take no more of the shaking and split its entire aluminum case in half. At this point, it was clear to Mike that he was going to need more clearance if he wanted to play in the rocks.
Other subsequent events also pushed the truck toward a makeover. As it would happen, twice within a period of several months the Toyota fell off a service lift in a repair garage, damaging the bed each time. It seemed that if the rocks weren't going to chew at the bed, something else would. The transformation began as the crinkled bed was removed to make way for a custom-designed and -fabricated tube bed. Mike's friend, Larry Zager, bent and crafted a good-looking and functional rear bed from 1-3/4-inch round tubing. This area still serves to haul cargo and camping gear but is much less vulnerable to rock rash than the sheetmetal was. The bed is painted to match the factory Metallic Red of the cab and sports aluminum diamond-plate panels. Rear lighting is provided by a big rig taillight panel that was modified as needed, and removable mudflaps help keep the truck legal on the road.
Further tube work was crafted inside and up front. Larry fabricated a front bumper that protects the front end and increases approach angle and clearance. Toyota cabs are well designed and strong, but Mike wanted added safety assurance in the event rubber should point skyward. A full inside cage was added. Tubing passes around and across the top inside of the cab and has been skillfully run forward through the dash area down to the floor.
Next on the upgrade list was the removal of the stock IFS and the swap to a front straight axle. Fellow club members from the Built-To-Grind Rockcrawlers pitched in to complete the transformation over a weekend. Plasma arc and grinding were used to strip a big pile of stock bracketry off the front of the frame and clean up the framerails. On went a custom front drop spring mount and shackle hangers. From there, they hung a set of All-Pro leaf springs with a trussed and armored front axle. Axle upgrades included 5.29 gears and Lock-Right locker, Long Enterprises heat-treated Birfields, Kong's high-steer crossover with 9/16-inch knuckle stud upgrade, and upgraded chrome-moly hub studs. Power is transmitted to the front axle via an All-Pro long-travel driveshaft, while Bilstein 7100 remote-reservoir shocks hang from Zager-fabricated hoops.
10PhotosView Slideshow
Rear suspension currently consists of a basic 4-inch spring and block lift damped with Rancho RS9000 adjustable shocks. While this setup offers reasonable performance, the light weight of the rear of the truck does not allow the suspension to flex significantly. A custom four-link suspension with Fox air shocks is in the works.
Motivation comes from a venerable 22R-E engine backed by a five-speed tranny. A Marlin Crawler 4.7:1 transfer case gearset can offer enough low gearing to keep the 37-inch Goodyear MT/Rs wrapped on Mickey Thompson Classic II wheels moving over rough terrain.
2PhotosView Slideshow
Creature comforts got some attention throughout the build of this rig as well. Inside is a set of 3R Racing seats and APC steering wheel. Lance at Creative Car Stereo filled the cab with tunes via a Pioneer CD system bellowing 600 watts of acoustical energy. Behind the seats, a custom enclosure houses 10-inch Rockford Fosgate subwoofers.
The project continues on as most projects do. The truck is continually evolving to improve its performance and fun factor. Thanks go to friends Mark Mason, Tony Owens, and Mark Owsiany for all their help. Mike also feels a special bond with his truck since some of the upgrade parts he acquired from the parents of his good friend Steve Custer who died in an untimely auto accident. Now Mike feels that maybe Steve's spirit lives on as his "spotter in the sky."


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EWG created Skin Deep so that you could make safer choices about personal care products for you and your family. We count on supporters like you to keep our research going.
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text:
Kihnu [1] is the largest island in the Gulf of Riga but the smallest municipality (together with neighboring islands) in Estonia.
It's located south east from Saaremaa Island and west from the mainland.
For many years, the men of Kihnu have been frequently gone to sea while the women ran the island and became the guardians of the island's cultural heritage which includes handicrafts, dances, games and music. Unlike men, they also wear their national costumes in everyday life. So it's quite normal to see an old woman dressed in traditional clothing driving a motorbike or even a tractor.
UNESCO proclaimed Kihnu's cultural space and traditions as a Masterpiece of the Oral and Intangible Heritage of Humanity on November 7, 2003.
Because of the surrounding sea the climate on the island is soft and warmer than the Estonian average.
Get in
By ferry
Kihnu Island is connected by a frequent ferry service to Munalaid and Pärnu on the mainland. The ferries also carry cars. Bookings can be made via phone or internet [2].
Where to stay
Try to visit Kihnu during traditional celebrations of popular calendar or church holidays like Christmas, Midsummer's day and St. Catherine's Day, where you can witness genuine old traditions.
It's a good location for walking, fishing and bicycling (bikes can be rented on the island).
Get out


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Supporting Documentation
If you answered questions on the FAFSA indicating that you are a dislocated worker, the government might require additional paperwork. You can check on the Student Connection whether you need to submit documentation.
If you must provide information, please submit documentation of involuntary loss of employment for you and/or your spouse (Independent students) or your parent(s) (Dependent students). Documentation may include receipt of unemployment due to being laid off; self-employed but unemployed due to economy or natural disaster; or displaced homemaker (previously provided unpaid services to the family, if no longer supported by husband or wife, is unemployed or underemployed , and is having trouble finding or upgrading employment).
Please refer to our Special Considerations page if your current income is not properly reflected on your FAFSA and you would like to have your situation reviewed by the Financial Aid Office.


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All Miyabi Artisan SG2
Miyabi Artisan SG2 Collection 7-Piece Knife Block Set
Miyabi, a Japanese aesthetic ideal translated as "elegance" or "refinement," is now synonymous with Zwilling J.A. Henckels' line of Japanese cutlery. Following in the tradition of samurai sword makers, Miyabi produces knives that are extraordinarily sharp and exceptionally...


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Boating - Office Locations
You may conduct any registration and titling business in person at the two locations listed below.
Columbia - | Directions | Map |
S.C. Department of Natural Resources
Rembert C. Dennis Building
1000 Assembly Street, Room 104
Columbia, S.C. 29201
Telephone 803-734-3857
Charleston - | Directions | Map |
S.C. Department of Natural Resources
217 Fort Johnson Road
Charleston, S.C. 29422
Telephone 843-953-9301
The following offices offer limited services such as:
- forms
- applications
- renewals
- duplicate registration cards
- duplicate boat and motor decals.
Office hours vary so, please call before coming.
|DNR at the Market
326 Little Brooke Ln
West Columbia, SC 29172
Telephone 803-734-3833
|Florence Office
295 S. Evander Drive
Florence, SC 29506
Phone: 843-661-4766
|Clemson Office
311 Natural Resources Drive
Clemson, SC 29631
Phone: 864-654-1671
|| Directions | Map |
|| Directions | Map |
|| Directions | Map |
Directions to Farmers Market Office
DNR at the Market is located on the grounds of the SC State Farmers Market off of US Hwy 321/Charleston Highway.
Directions to Columbia Office from Florence
Take I-20 West
Merge onto SC-277 South via Exit 73A toward Columbia
Turn Right onto Elmwood Avenue/US-76
Turn Left onto Assembly Street/SC-48 South
When you cross over Gervais Street the Rembert C. Dennis Building where SCDNR is located is on the left hand side at 1000 Assembly Street, Columbia, SC 29201-3117 US
It is located across from the International House of Pancakes (IHOP)
Direction to Columbia Office from Aiken
Take I-20 East
Merge onto US-76 East via Exit 64A toward Columbia
Merge onto Huger Street/US-176 E/US-21 S/US-321 South
Turn Left onto Gervais Street/US-378 E/US-1 North
Turn Right onto SC-48 S/Assembly Street
The Rembert C. Dennis Building where SCDNR is located is on the left hand side at 1000 Assembly Street, Columbia, SC 29201-3117 US
Map to Columbia Office
Directions to Charleston Office
GPS Coordinates are: 32°44'56.7"N, 79°54'16.8"W
Take I-26 East
Take the US-17 South via Exit 221A toward King Street/Savannah
Turn slight right onto US-17/Septima Clark Expressway. Continue to follow US-17 South
Turn slight right onto Spring Street
Turn left onto Lockwood Boulevard
Merge onto SC-30 West/Robert B. Scarborough Bridge/James Island Expressway via the ramp on the left toward James Island/Folly Beach
Take the Harborview Road exit - Exit 2
Turn right onto Harborview Road
Turn left onto Fort Johnson Road
Boating and Title office is the first left after the large SCDNR sign
Map to Charleston Office


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Last Saturday we had a small cookout with two couples from Prince Charming's work. We had a great time grilling out, playing board games, laughing at the kids and having a few cocktails.
After dinner and a rousing game of electronic catch phrase we were all sitting around starting to get a little bored. We began suggesting different things to liven up the party or at least provide some mild entertainment. One of the guys suggested that it would be funny if someone were to ride Alex's skateboard down the swing set slide.
Yes, I know. Bells are starting to ring, aren't they?
Well, Prince Charming being much more a kid at heart than the old man he pretends to be, decided to relive his glory days. And so begins the the tale of why I pulled a week long disappearing act.
He grabbed the skateboard and headed to the top of the slide.
I shook my head. Everyone else laughed in disbelief. The kids got excited. I yelled that I had best not ever see them try this if they wanted to have a good summer. And I shook my head again.
Everything seemed like it was going to go exactly as planned, until he place the skate board on the slide. The theory was that the slide would act as a sort of track for the skate board. The problem was that Alex doesn't have typical skateboard wheels. He has these big monster ones for 'all terrain' type skateboarding.
Prince Charming realized that the whole 'track' theory wasn't going to work out. But at this point he was committed to providing us all with some entertainment and proving that he was a candidate for backyard x-games.
So he placed the skate board on the slide, leaving one set of wheels on the edge and the other inside the sides. He took his position in an impressive surfer style pose. And proceeded to head down. It all went very quickly, but he did manage to do it. Almost. We had not realized that the slide was not on even ground and therefore the very bottom of the slide was crooked. And it was the opposite side of the wheels riding the edge.
As he made an impressive ride down, the skate board hit the crooked bottom and then the ground. At this point the skate board was no longer a skate board. It became a flying board. It hit the ground then took flight right out from under Prince Charming's feet. Prince Charming attempted to save the landing, but it was too late and he fell in a twisted tangle of arms and legs.
Don't feel bad. It's okay to shake your head and laugh. We all were.
He got up and said that his foot hurt, but there was no swelling or bruising and it started to feel a little better as he walked on it. So we all had a really good laugh and continued on with our evening. However as everyone got up to go and we headed into the house after sitting for about 3 hours, Prince Charming stood up and almost fell. His foot was in a lot of pain and he could put no pressure on it. By this time we had the kids in bed and thought maybe it was just from the cold and sitting so we went to bed to see how it would be in the morning.
It wasn't better the next morning and was actually much worse. So we all helped him to the car and headed to our ER. After x-rays and an examination he walked back out in a cast and crutches with orders that he could not return to work for a full week. He has fractured the bones on the top of his foot and also torn ligaments on the side of his foot. It is very swollen and bruised now and will be in the cast for at least 3 weeks then we find out if it's healed or needs further attention.
For the past week I haven't been online because I've had all 3 kids home due to summer vacation and my husband confined to a couch for the majority of the week. Anyone who has had a sick hubby knows that it is a tad difficult. On top of the fact that I'm a person who likes my daily routine and this threw it into total chaos.
He has been bored and annoyed and in pain. I've done everything I can to help make it easier and relieve some of his 'home bound' tension. And he's been wonderfully grateful and even let me get out of the house twice this week to meet up with my girlfriend and a few (much needed) drinks after the kiddies were tucked in for the night. I guess he knew this was the best way to save my sanity.
The one good thing that has come out of this was a wonderful discussion, complete with visual aides, with Alex and Liberty about why you should never take a dare to impress your friends and that health, safety and using your brain are more important than being cool. After seeing what happened to daddy, they both agree.
Of course, the reality is:
Prince Charming's only regret is that he didn't manage to pull the stunt off.
And that my friends, is what prompted my wonderful graphic (Thanks, mom!):


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It's the movie that demonstrated just how erotic pottery making could be. Take clay, two hot lovers, add water and a little Unchained Melody, then swoon.
Ghost, the 1990 Patrick Swayze-Demi Moore romantic thriller that earned more than half a billion at the box office, was probably a natural for the movie-to-musical treatment that supplies Broadway and London with so many new shows. Indeed, Ghost the Musical, which begins a two-week run at the Broward Center for the Performing Arts on Tuesday, did get its theatrical makeover. But the transformation was neither quick nor simple, nor did the musical enjoy the kind of soaring success the movie achieved.
Bruce Joel Rubin is the guy who dreamed up Ghost, winning an Oscar for his screenplay about a potter named Molly, her banker-boyfriend Sam, and their sometimes-dangerous, sometimes-amusing journey through the spirit realm after Sam's shocking murder. That's not really a spoiler: Sam gets shot by a robber early on, and the thing is called Ghost, after all.
Rubin, who spent seven years getting Ghost to the stage, says he was interested in telling a particular kind of story in the movie and the musical.
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"I wanted to write a story that assumed life doesn't start at birth or end at death," Rubin says. "I wanted to tell it from the point of view of a ghost, to explore what it means to be trapped. It's an extraordinary love story. Sam turns away from the light because of Molly, and he has to earn his way out. He has this unfinished business of never having said, 'I love you.' "
Ghost the Musical, which opened in London in the summer of 2011 and on Broadway the following year, is the work of an impressive creative team. Rubin did the book and wrote some of the lyrics, but the score is principally by rock star (and Annie Lennox's former Eurythmics bandmate) Dave Stewart and songwriter-producer Glen Ballard, the Grammy-winning talent whose credits include Alanis Morissette's Jagged Little Pill and co-writing Michael Jackson's Man in the Mirror.
The show was staged by Matthew Warchus, Tony Award winner and the British director of God of Carnage and Matilda the Musical. To create the other-worldly effects that are so much easier to do on film, illusionist Paul Kieve was hired early on.
Kieve, who created tricks for and appeared in 2004's Harry Potter and the Prisoner of Azkaban, had worked with Warchus before and knew what he was aiming to do with Ghost the Musical.
"Matthew wanted a love story, something like a rock concert, a musical that was very cinematic and a magic show," Kieve says. "He wanted the most spectacular things, but they all had to be story-driven."
One of the show's most stunning effects happens when Sam's ghost passes through a seemingly solid door.
"Walking through the door has caused quite a stir in the magic world," he says. "I haven't shared with anyone except the people involved in the show how it's done. In my life, I'm not a secretive person. But magic is all about enchantment. You can't astonish people if they know the details."
As for the music, the movie's signature song — Unchained Melody, written by Alex North and Hy Zaret, performed by the Righteous Brothers — is incorporated in bits and pieces as Sam and Molly's recurring love song. But otherwise, the original music by Stewart and Ballard is a catchy pop-rock score.
Steven Grant Douglas, who plays Sam in the touring company, remembers that when he first heard the musical's cast CD, "it didn't sound like a musical. It sounded like a band's album."
Ballard and Stewart came into the process with Rubin already having written the lyrics to 20 songs. Some of his lyrics survive, but he listened when Warchus made a suggestion.
"Matthew said, 'Your songs are good, but you have two of the best songwriters in the world here. Why don't you let them have a shot?' " Rubin recalls. "A lot of my lyrics and the DNA of my songs are still there. But theirs are better. They understand more about song structure. They were and are masterful."
"Bruce's lyrics were too literal, but they gave us lots of information. We were more elliptical with the poetry of it," says Ballard.
"Bruce did have some great words, but they weren't in a song structure. He wasn't sure how to turn the written word into song lyrics," Stewart says. "But he got very addicted to the process. He got very good at it very quickly."
Ghost the Musical had a longer run in London (just over 500 performances) than it did on Broadway (39 previews and 136 regular performances). Still, touring audiences have been reacting strongly to the show's romantic story and state-of-the-art design, and the cast and creative team offer numerous reasons for the show's ongoing appeal.
"The effects are really thrilling, and the show has something for everyone: magic, suspense, comedy, a love story," says Katie Postotnik, who plays Molly.
Douglas, who's doing his first major show since graduating from college three years ago, feels the messages he's conveying as Sam.
"Tell the people you love that you love them, because you never know when it will be too late," he says. "And appreciate what you have."
Carla Stewart plays fake medium-turned-psychic Oda Mae, the part that won Whoopi Goldberg an Oscar. She thinks audiences are drawn to Ghost the Musical because "people love love. Just to know that love would be so great that someone would come back for you from the spirit world — it's an emotional roller coaster."
Illusionist Kieve and composer Stewart see something classic in Ghost and Ghost the Musical.
"The idea of a ghost being central to a narrative is Shakespearean," Kieve observes.
Stewart concurs.
"It's that perfect Shakespearean drama: love lost, unrequited love and the question of whether there's an afterlife," he says.


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Use the form on the right to contact us.
You can edit the text in this area, and change where the contact form on the right submits to, by entering edit mode using the modes on the bottom right.
423 Lincoln Blvd
Los Angeles, CA, 90291
United States
Tree Birds upholstery Fabric
100% Polyester
58/60" inches wide
$20 per yard
$16 per yard wholesale pricing when ordering 30+ yards
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After missing the previous five games because of a concussion, Pau Gasol is set to play for the Lakers as they face the Miami Heat on Thursday night at Staples Center.
Gasol will come off the bench for the first time since the 2004-05 season and only the seventh time in 822 career NBA games. Earl Clark will make a fourth consecutive start in Gasol's place.
"It kind of makes sense the first game to come off the bench and ease back into it with this game because they weren't really expecting me to play tonight," Gasol said after the Lakers' shoot-around Thursday. "So maybe we would have been better basketball-wise for me to get conditioning drills, conditioning workouts and playing a little more before I get back, but I want to play, I want to be out there."
Coach Mike D'Antoni said Gasol would move back into the starting lineup once his conditioning improves, likely Sunday against Toronto.
Still, there are other factors that contributed to Gasol coming off the bench.
"We've played a certain way for five games and you've got the defending champions coming in and they play smaller," D'Antoni said, "so it's not something you want to force without knowing what the answer is. I would rather start off just looking at the team we've looked at the last two or three games and then have him. He'll be a big addition for us."


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Reservations (off-season): 802-985-8498, m-f, 9am - 5pm EST.
The Inn at Shelburne Farms on the shores of Lake Champlain was originally the 19th-century country home of Dr. William Seward and Lila Vanderbilt Webb. Restored in 1987, the inn today retains its historic character and continues the tradition of warmth, informal elegance and hospitality enjoyed by guests for more than a century. The Inn features:
- 24 guest rooms individually decorated with many original furnishings; 4 guest cottages.
- Public restaurant serving breakfast, dinner, and Sunday brunch.
- Private dining & meeting room
- 1,400 acres of a working landscape to explore.
Each time you stay or dine with us, you support Shelburne Farms' education programs.


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Reviewed by Keaton & Austen (age 7)
I liked it when they showed us a picture of the SCC, and gave information about the SCC car. The book reminds me of being in my car, with my dad driving down the highway. The pictures are real pictures of cool cars. When I am old enough to drive I hope I can get a cool car.
I recommend this book to people who like cars because the book is full of pictures of cool cars. I think that first and second graders would like this book because of the pictures. The illustrator has done many books on different topics, so if you like the cool car pictures, you will probably like other books by him.


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A quarter century after the worst one-day stock crash in history, measures to prevent a repeat are failing to keep investors from losing confidence in the market.
The 23 percent plunge in the Dow Jones Industrial Average on Oct. 19, 1987, came amid signs of a slowing economy, the threat of higher taxes and concern among individuals that trading was rigged for insiders. Today's investors have pulled $440 billion from U.S. equity mutual funds since 2008 and sent trading to the lowest levels in at least four years, retrenching after the worst financial crisis since the Great Depression and the May 2010 stock crash, data compiled by Bloomberg and the Investment Company Institute show.
While Procter & Gamble Co. and McDonald's Corp. are up more than 800 percent since 1987, protections adopted after the crash couldn't stop unharnessed computer trading from erasing almost $900 billion of value in less than 20 minutes on May 6, 2010, based on data compiled by Bloomberg. E.E. "Buzzy" Geduld, 69, who oversaw about 60 equity traders 25 years ago at Herzog, Heine & Geduld Inc. and now runs investment firm Cougar Trading LLC, says crashes happen when investors become convinced they've lost control.
"In 1987 everybody tried to go to the exit at the same time, but the exit door wasn't big enough," Geduld said in a telephone interview. "You had literally a panic. Fast forward to 2012. The volumes we can handle are gigantic, but the exit door hasn't changed in size."
Abandoning Stocks
Individuals are abandoning stocks even after U.S. Federal Reserve Chairman Ben S. Bernanke held interest rates close to zero for a fourth year, valuations for the Dow remain 23 percent below the level at the market peak in October 2007, and exchanges installed safeguards following the so-called flash crash in 2010. U.S. stocks are in the 44th month of a bull market that has restored $9 trillion in share value, data compiled by Bloomberg show. The Standard & Poor's 500 Index has more than doubled since March 2009 and is up 15 percent in 2012.
Average daily volume for U.S. equities was 6 billion shares in the third quarter, the lowest level since at least 2008 and about half the 10.9 billion average in the first three months of 2009. The total has decreased for 12 of the last 13 quarters as investors pulled money from American stock mutual funds for a record fifth year, according to data compiled by Bloomberg and Washington-based ICI.
Copyright 2014 Bloomberg. All rights reserved. This material may not be published, broadcast, rewritten, or redistributed.
Copyright 2014 Bloomberg. All rights reserved. This material may not be published, broadcast, rewritten, or redistributed.