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Duck Dynasty Phil Robertson caused quite a controversy last week when GQ published an interview with the reality show co star. After Robertson targeted gays, blacks, Muslims, and "idolaters," among others, the show network, A suspended the celebrity, causing a right wing backlash, led in part by former half term Alaska Gov. Sarah Palin (R).

This week, however, the failed vice presidential candidate made an interesting admission.

Turns out Sarah Palin, who fake hermes leather handbags has been one of Duck Dynasty biggest defenders, hasn actually read the GQ interview that includes cast member Phil Robertson controversial comments on homosexuality and other subjects.

When pressed by Fox News host Greta Van Susteren whether the language Robertson used when talking about his opposition to homosexuality was graphic and offensive, Palin admitted she didn know what Robertson had said.

Specifically, Palin conceded,"I haven read the article. I don know exactly how he said it.

Look, it pointless to get caught up in every minor Palin dust replica hermes bag up, and as a rule, I tend to ignore the various Palin inspired stories of the day.

But there something wonderfully emblematic of this week gem. Sarah Palin took it upon herself to lead the Outrage Brigade, condemning A for taking offense to Robertson bigotry, and using her platform to position herself as a leading national defender of the reality show personality.

Palin did not, however, think it was worth taking a few minutes to do the one obvious thing: read the remarks she was defending.

Incidentally, in the same Fox News interview, Palin added that Robertson "was quoting the Gospel, so people who are so insulted and offended Hermes bag season with the main color coordination by what he said evidently are offended by what he was quoting in the Gospel."

Here the follow up question: if Palin didn read the interview, and has no idea what Robertson specifically said, how does she know he was "quoting the Gospel"?

Indeed, has she ever read the Bible to know whether or not the Good Book was being "quoted" properly? If Palin doesn know what Scripture says, and doesn know what Robertson said, then why is she replica birkin handbags so eager to stoke the fires of this odd culture war skirmish?
zroessgs viesoess Yesterday, 22:58
Ontario man wanted in stepson's death arrested in Walmart replica hermes handbags parking lot

Tuesday 2,000 km from St replica birkin handbags Catharines.

He credited the media and widespread public awareness for the bust.

because of those efforts that Kuijer is in custody today, and the community is safer than it was before his arrest yesterday, he said at a press conference.

Cops say it was a member of the public who had heard of the shocking murder that gave them the break they needed. The citizen recognized Kuijer grey, 2009 van.

Six provincial police officers responded to the tip and arrested Kuijer without incident, McGuire said.

While Kuijer described as a loving father by Nathan mother Whitney had been wanted Hermes bag season with the main color coordination for second degree murder on the Canada wide warrant that all changed.

Now, McGuire said he will be charged with first degree murder.

Kuijer is expected at a bail hearing scheduled for Thursday.

On Friday, Nathan Dumas family plan to say goodbye to the little boy at a funeral service in nearby Thorold.

Detectives remain tight lipped on motive and how Nathan died, although sources told the Sun there were marks around the boy neck.

McGuire added that Nathan mother and the RBC victim were at news of Kuijer arrest.

The bank employee remains in hospital but is expected to make a full recovery, he said.

was truly a kind, loving boy with a big heart, full of compassion for everyone and everything. He was always quick to help anyone who may have been in need, an obituary for the boy in the St. Catharines Standard says.

the polite young gentleman he was, he never missed fake hermes leather handbags an opportunity to open a door, give a gift or come to the defence of a good friend. Nathan truly felt the joy of giving, never looking for anything in return. There are no words to express the unbearable heartbreak we feel in his loss. With files from The Canadian Press
zroessgs viesoess Yesterday, 22:57
Olympic protesters get their game on

Protest sign: Anti poverty activists believe that the Olympic Games have accelerated gentrification and homelessness in the Downtown Eastside. Shaw is a veteran of earlier anti globalization protests sparked my interest and Seattle cemented it but he not out of central casting. At 59, he older than most anti replica birkin handbags Olympic activists. Just like we delayed the torch relay. The relay has been delayed across the country and that is a powerful organizing tool. 22 at Victory Square, in the heart of the Olympic city. More than 200 anti 2010 protesters, some carrying black flags and burning torches, gathered for what had all the hallmarks of a dress rehearsal for the street protests that could erupt during the Winter Games just over two weeks away.

The crowd, mostly young, some wearing bandanas over their faces, had come to march against the "police repression" of anti Olympic activism. A portable audio system jacked up the energy level with Hermes bag season with the main color coordination the opening chords of the Rolling Stones' Street Fighting Man. "Everywhere I hear the sound of marching, charging feet, boy," howled Mick Jagger.

A few protesters with megaphones screamed slogans, often laced with obscenity, at startled motorists: "No 2010!" and "Did You Vote For Gordon Campbell?"

The small but angry far left demo recalled the much larger anti globalization demonstrations of a decade ago: the APEC protest and the Riot at the Hyatt in Vancouver, the epic Battle in Seattle against the World Trade Organization and the violent clash between demonstrators at the Summit of the Americas in Quebec City.

Many alumni of those so called anti globalization convergences were in the crowd, along with younger activists seeking to emulate their radical forebears.

Now 35, Westergard Thorpe is a key figure in the militant Olympic Resistance Network (ORN), perhaps the most radical of the anti Olympic groups behind the "Take Back Our City" march from the Vancouver Art Gallery to replica hermes handbags BC Place Stadium, set for the afternoon of Feb. 12, when tens of thousands of people will be arriving at the stadium for the Olympic Games opening ceremonies.

"I hope it does go down like APEC," said Westergard Thorpe, who was pepper sprayed, strip searched and how much is a hermes bag arrested by police at that protest.

"I hope it does look like when Jean Chretien used to come to the Hyatt and we used to have thousands of people out in the street."

Westergard Thorpe added that the Feb. 12 marchers should be able to proceed toward BC Place as far as any non ticket member of the public can get.

Whether any protesters would try to breach the security fences, she said: "That really depends on what type of police repression we face during the day. You know if we have a day where we're being beaten back by the police when you're simply trying to march, people's tempers can get high."

A 'mega industry event'

As to whether she's hoping a disruption of traffic heading to BC Place might affect the opening ceremonies, set to be watched by millions of people around the world, Westergard Thorpe said: "Absolutely, I would love to disrupt the opening ceremonies."
zroessgs viesoess Yesterday, 22:52
Randi Zuckerberg debuts apparel line at Westfield Valley Fair

Former Facebok exec Randi Zuckerberg introduced a fashion and accessories line, known as 265, at a party celebrating the new luxury wing at the Westfield Valley Fair Mall in San Jose on Nov. 1, saying that "it's through high tech that my partners cartier love necklace yellow gold fake and I can make fashion."

But it's high tech that is making luxury fashion possible at the mall, with the continuing growth of Silicon Valley responsible for creating enough wealthy customers to afford the wares at luxury shops such as Prada, Ferragamo, Burberry, Louis Vuitton, Tiffany, fake cartier love pendant Cartier and the first Northern California locations of Miu Miu and David Yurman, just a few of the 18 high end purveyors among fake cartier love earrings the mall's 250 stores.

One store proprietor said the mall had surpassed sales per square foot of South Coast Plaza in Orange County, among the state's most profitable malls per square foot ( The Grove in Los Angeles or Fashion Valley Mall in San Diego are the top ranked, depending on published reports.)

Caran Sealey, the mall's marketing director, declined to release sales figures but said the aim has been to make Westfield Valley Fair "a destination" in the region.

The event was called "Fashion DuJour," sponsored in part by Du Jour magazine. Several hundred guests sipped Champagne and nibbled on caviar while wandering the mall looking at Emmy nominated costume showcase apparel from shows such as "Mad Men" and "Downton Abbey" on mannequins.

A live design competition pitted students from the Fashion Institute of Design and Merchandising against one another, giving them an hour or so to drape material on a dress form and create full length gowns worthy of a walk on the red carpet. A panel of judges awarded Angela Avanesyan the winner of a $2,500 cash donation to her studies, as well as a merchandising vitrine in the mall for one year to show off her creations to the mall's 16 million annual visitors.

Zuckerberg, whose book "Dot Complicated" about our tangled, wired lives, hits the stores Nov. 5, has also partnered with the South cartier ring necklace imitation Bay's Angelina Haole, a fashion designer for Franco Uomo, and Hasti Kashfia, a Bay Area based celebrity stylist, to create a fashion line called 265. The name comes from the birth months of each of the founders February, May and June.

It features T shirts, tote bags, cell phone covers and bracelets. The apparel is designed with tech life in mind, with programming inspired 1's and 0's as a clothing motif ("If you know binary code, you'll get it," Zuckerberg said."
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  • fuadiskws Yesterday, 22:12
    The famous images of photojournalist Marc Riboud

    Anti Vietnam protest Flower Child World renowned French photographer Marc Riboud, who died on Aug. 20, 2016 at age 93, was most recognized for two very famous photographs: the girl with the flower facing National Guardsmen poised with bayonets during an anti Vietnam march on the Pentagon; and the whimsical image of a painter balancing precariously on the Eiffel Tower necklace cartier copy in Paris. Over the years, Riboud globetrotting and keen eye produced a wealth of imagery from China to Iran to West Africa. war in Vietnam.

    In an interview with The Guardian years later, Kasmir said she didn consider herself fearless: was before Kent State, so who would ever think that they would kill me?

    of them made eye contact. They stonewalled me. But the photographer later told me he noticed them shaking. I think they were afraid they were going to be told to fire at us. Credit: Marc Riboud/Magnum Photos

    Beijing street life A street in Beijing as seen from inside an antique dealer shop in China, 1965.

    Riboud first worked as an engineer before turning to photography and moving to Paris. The legendary street photographer Henri Cartier Bresson, and co founder of the collective, Magnum Photos, was a mentor for Riboud in the City of Light.

    Riboud was one of the first Western photographers allowed into China after the country Cultural Revolution in 1957.

    Much of his work over the years focused on daily life. In places like China and India, that meant often capturing people impacted by major cultural and industrial transformation in the and

    Credit: Marc Riboud/Magnum Photos

    India People dress after bathing in the Ganges in Benares, India, 1956.

    In 1955, Riboud used an old Land Rover to travel from Paris to Calcutta with stops in Istanbul and Afghanistan. He spent a year photographing and traveling through India before eventually making his way to China and Russia.

    His 2012 book, Riboud Into the Orient, was born out of his three year sojourn.

    Credit: Marc Riboud/Magnum Photos

    Great Wall The Great Wall, China, 1971.

    Riboud made repeated trips to China over a 40 year period. Many of his images were published in the book, Riboud in China: Forty Years of Photography in 1996.

    Credit: Marc Riboud/Magnum Photos

    Eiffel Tower Painter Zazou, the Eiffel Tower painter, in Paris, 1953.

    This photo (Riboud first published one) debuted in Life magazine. The Eiffel Tower image prompted an invite from his mentor and war photographer Robert Capa to join Magnum Photos. Riboud remained a fake love ring cartier member replica cartier diamond pendant necklace of Magnum until 1979.

    Credit: Marc Riboud/Magnum Photos

    China A statue of Chinese leader Mao Tse Tung stands before billowing smoke in the industrial center of Wuhan, China, 1971.

    Credit: Marc Riboud/Magnum Photos

    Afghanistan A tribal munitions factory near Kohat Pass on Afghanistan lawless border with Pakistan, 1956.

    Credit: Marc Riboud/Magnum Photos

    China Steel factory engineer in his office in Anshan, China, 1957.

    Credit: Marc Riboud/Magnum Photos

    Marc cartier pendant necklace replica Riboud (1923 2016) Marc Riboud was born on June 24, 1923 in Saint Genis Laval, France. He died at age 93 on August, 30, 2016 from Alzheimer

    During his 60 year career, Riboud work was published around the world, including in Life, National Geographic and Paris Match; exhibited in numerous museums; and appeared in several books.
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  • fuadiskws Yesterday, 22:07
    Real Madrid tops AP Global Football 10 after 20th win

    LONDON Real Madrid replaced Barcelona at the top of the Associated Press Global Football 10 on Tuesday after extending its record run of wins to 20 in all competitions with two four goal victories in a week.

    Madrid followed its 4 0 demolition of Ludogorets in the Champions League with a 4 1 thrashing of Almeria on Friday to take the record tally of consecutive wins for a Spanish club to 20.

    "Whether it's the Champions League or La Liga, Carlo Ancelotti's men how much is a hermes handbag just keep getting the business done," voter Marco Monteverde of News Corp. Australia said.

    Real had two members of its squad in the top 10 players this week, with Cristiano Ronaldo third after fake hermes kelly bagd scoring three times across the two games, and Gareth Bale sixth.

    Arjen Robben is the leading player, after his double for Bayern Munich propelled it to a 4 0 rout of Augsburg to surge nine points clear at the summit of the Bundesliga.

    Pep Guardiola's team is runner up to Madrid in the club vote, with Manchester United third after its 3 0 victory over Liverpool at Old Trafford on Sunday.

    Goalkeeper David de Gea, who made a number of outstanding saves for United, enters this week's poll in second.

    While United easily dislodged Liverpool's fragile defence through the attacking prowess of Robin van Persie, who polls ninth this week after scoring United's third, its fierce rival was repeatedly denied by De Gea.

    "Goalkeepers rarely get the credit they deserve. Even when they make saves, it is the strikers that get the glory," Mike McGrath of The Sun in England said. "But David De Gea rightly took the plaudits for his performance against Liverpool. His saves were the difference between the two teams despite the 3 0 scoreline."

    Sam Tighe of Bleacher Report in England was in agreement, suggesting that De Gea's performance meant the score did not reflect the game.

    "De Gea resembled fake hermes leather handbags a brick wall on Sunday as Manchester United beat Liverpool 3 0. It wasn't a 3 0 game far from it in fact as Raheem Sterling missed three one vs. one chances due to this Spaniard's brilliance," Tighe said.

    Manchester City and Samir Nasri are fourth in the club and player votes respectively.

    The Premier League champion secured its place in the final 16 of the Champions League with a 2 0 win at Roma in the Stadio Olimpico, where Nasri scored a stunning opener for Manuel Pellegrini's side and then laid on his team's second goal.

    Chelsea's Eden Hazard is fifth, after opening the hermes replica scoring in his team's 2 0 win against Hull at Stamford Bridge. The result maintained Chelsea's three point lead over City at the top of the English standings.

    Elsewhere, Olivier Giroud continued his comeback from a broken leg by spearheading Arsenal's attack with two goals in a 4 1 win at home to Newcastle. His performance earned him seventh place in the player vote.

    Arsenal polls fifth this week with the result, which followed another 4 1 win at Galatasaray in the Champions League.

    In France, Monaco beat league leader Marseille 1 0 to poll seventh.

    Paris Saint Germain, a point behind Marcelo Bielsa's team, could not take advantage of Marseille's defeat as it slipped to a similar 1 0 loss to Guingamp, which enters the team poll in ninth.

    Villarreal is eighth, after beating Spanish champion Atletico Madrid 1 0 thanks to an 84th minute goal from Luciano Vietto, who is eighth in the player rankings.

    Basel's qualification for the knockout stages of the Champions League sees it enter the team poll in 10th place following a 1 1 draw against Liverpool at Anfield. Fabian Frei, who scored for Basel, is 10th in the player vote.
    zroessgs viesoess Yesterday, 20:53
    Plasma membrane restricted RhoGEF activity is sufficient for RhoA

    AbstractThe small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.IntroductionRho GTPases belong to the Ras superfamily of small G proteins and are involved in a variety of cellular processes, such as the dynamic regulation of the actin cytoskeleton and cell morphology, cell cycle progression, and gene transcription1,2. It is well known that dysregulation of Rho GTPase function plays a key role in tumor formation, invasion and metastasis3,4. Accumulating evidence points towards Rho GTPases and their effectors and regulators as possible therapeutic targets. Better understanding of the spatiotemporal regulation of Rho GTPase signaling could increase therapeutic success and help in the design of novel therapeutic intervention strategies5,6.Like most typical G proteins, Rho GTPases function as molecular switches by cycling between an inactive GDP bound state and an active GTP bound state7. Three classes of accessory proteins that control the molecular switch kinetics and the location of Rho GTPases in cells have been identified8,9. Rho guanine exchange factors (Rho GEFs) stimulate the exchange of GDP for GTP, resulting in Rho GTPase activation. In contrast, Rho GTPase activating proteins (Rho GAPs) accelerate the hydrolysis of bound GTP to GDP, which abrogates Rho GTPase signaling. Inactive, GDP bound Rho GTPases are sequestered in the cytoplasm by Rho guanine nucleotide dissociation inhibitors (Rho GDIs). The signaling output of Rho GTPases is dictated by spatiotemporal control of GEF and GAP activity and the subcellular location of the Rho GTPase itself.There are 22 Rho GTPases identified in humans, of which RhoA, Rac1 and Cdc42 have been studied in most detail10. RhoA has been linked to the regulation of fake hermes kelly bagd cytoskeletal dynamics, cell migration and cell adhesion2. RhoA is localized to the cytosol in mammalian cells and has been reported to translocate to the plasma membrane upon activation11. However, the precise subcellular site and kinetics of RhoA activation by its GEFs is still under investigation.P63RhoGEF (encoded by the gene ARHGEF25) is a RhoA specific guanine exchange factor12,13, member of the Dbl superfamily of Rho GEFs. Members of this superfamily are characterized by one or more Dbl homology (DH) domains, which are almost always accompanied by a C terminal Pleckstrin Homology (PH) domain14. The DH domain interacts directly with the Rho GTPase and is responsible for the catalytic activity that accelerates the exchange of GDP for GTP on the Rho GTPase7. Indeed, the catalytic DH domain of p63RhoGEF was shown to be necessary and sufficient for its downstream signaling function15, as is the case for many other GEFs.The role of the PH domain is less clearly defined. It has been hypothesized to assist in plasma membrane localization, facilitate Rho GTPase activation, mediate target specificity, function as scaffold for signaling proteins and/or phospholipids, or autoinhibit the catalytic DH domain7. Interestingly, the PH domain of p63RhoGEF has been shown to exhibit an inhibitory function by preventing the DH domain from accessing RhoA16,17. By using biochemical, structural and in vitro approaches it has been shown that activation of the heterotrimeric G protein Gq allosterically activates the GEF activity of p63RhoGEF by binding to the PH domain, which structurally relieves the DH domain from its auto inhibited state16,18.Based on the fact that plasma membrane localization of p63RhoGEF is important for its effective interaction with Gq19,20, we set out to investigate the requirement of plasma membrane localization of p63RhoGEF for the activation of RhoA and subsequent downstream signaling. We have used live cell fluorescent imaging techniques and a novel optimized high contrast FRET based RhoA biosensor to determine the kinetic parameters of RhoA activation by p63RhoGEF via stimulation of endogenous Gq mediated GPCRs in single living cells. Furthermore, we have employed a rapamycin dependent heterodimerization system to create a plasma membrane recruitable RhoGEF, enabling direct spatiotemporal control over the subcellular localization of Rho GEF activity in mammalian cells. This system is combined with several read outs based on RhoA activity, actin polymerization and cellular morphology to arrive at the conclusion that RhoGEF activity at the plasma membrane is sufficient for the activation of RhoA, resulting in actin polymerization.ResultsAn improved FRET based biosensor for RhoA reveals rapid activation of p63RhoGEF after stimulation of an endogenous Gq coupled GPCRThe kinetics of the guanine exchange reaction of p63RhoGEF on RhoGTPases have so far only been characterized in vitro, using purified proteins16. To investigate the spatial and temporal aspects of RhoA nucleotide binding state in individual living cells, a Dimerization Optimized Reporter for Activation (DORA) RhoA biosensor was employed. The single chain FRET based sensor is based on a previously reported RhoA biosensor21. When RhoA GDP is converted to RhoA GTP a PKN1 moiety binds RhoA GTP, resulting in a high FRET state, which is detected as an increase in sensitized emission over CFP ratio (Fig. 1a). To examine the FRET contrast between the 'on' and 'off' state of the biosensor, the emission spectra of a non binding biosensor (RhoAsensor nb), containing a mutation in PKN1 (L59Q) preventing RhoA binding, and a biosensor containing a constitutively GTP loaded RhoA mutant (Q63L) (RhoAsensor ca) were measured in HeLa cells. The average single cell spectra show clearly the CFP emission around 475nm and the (sensitized) YFP emission around 530nm (Fig. 1b). The RhoAsensor ca shows a marked decrease in CFP emission and an increase in YFP emission relative to the RhoAsensor nb, demonstrating substantial FRET contrast that allows differentiation between the two states. FLIM measurements of the RhoAsensor ca and RhoAsensor nb showed that the donor fluorescence lifetime of the constitutively active mutant is reduced compared to that of the non binding version (median values change from 2.9 to 2.4 ns), demonstrating that the RhoA GTP state is accompanied by an increase in FRET efficiency (Fig. 1c).Figure 1: Rapid and reversible GPCR mediated GTP loading of RhoA by p63RhoGEF, measured by a novel high contrast RhoA FRET biosensor.(a) A cartoon of the DORA RhoA biosensor consisting of full length RhoA (shown in light blue) fused to CFP, connected via a linker to YFP fused to the Rho binding domain of PKN1 (shown in lila) (structures are based on pdb entries 1CXZ, 1MYW and 3ZTF). (c) (left) Donor intensity images (top) and phase lifetime images (bottom) of the RhoAsensor ca (left) and the RhoAsensor nb (right) with a false color coded lifetime according to the scale depicted in the combined lifetime histograms of the same experiment (middle). (right) Accumulated FLIM data for RhoAsensor ca and RhoAsensor nb, showing the median phase lifetime from multiple cells (at least 8 acquisitions, n=28 and n=24, respectively). Box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. of p63RhoGEF, containing the plasma membrane targeting sequence, show minimal changes in YFP/CFP ratio (n=29). (e) Time lapse FRET ratio imaging of HeLa cells transfected with the DORA RhoA biosensor and RFP p63RhoGEF (n=71) show fast reversible increase in YFP/CFP ratio, indicating rapid GTP loading of RhoA upon GPCR stimulation. (f) Average ratio images at three time intervals of a single cell from the experiment shown in (e). (g) Pre incubation with the Gq inhibitor QIC (2M) abolishes the DORA RhoA biosensor response by GPCR stimulation in RFP p63RhoGEF transfected cells (n=30). HeLa cells were stimulated with Histamine (100M) at t=40s and the response was antagonized by the addition of Pyrilamine (10M) at t=160s. Average curves consist of data from at least 3 independent experiments, conducted on different days. Width of the individual images in (f) corresponds to 65m.To examine whether p63RhoGEF is capable of activating RhoA in living cells, we performed live cell measurements of HeLa cells transfected with the DORA RhoA biosensor and co transfected with either full length p63RhoGEF or a control construct containing only the first 29 amino acids of p63RhoGEF (p63RhoGEF1 29) (Supplemental Fig. S1). p63RhoGEF1 29 contains only the plasma membrane targeting sequence of p63RhoGEF, and is catalytically inactive. Activation of p63RhoGEF by endogenously expressed histamine 1 receptors (H1R) was achieved by stimulating cells with histamine at the indicated time points, and the response was antagonized by addition of the H1R specific antagonist mepyramine.Control experiments on cells co transfected with p63RhoGEF1 29 showed only a minimal change in FRET ratio upon addition of histamine and mepyramine (Fig. 1d). This small observed response could be attributed to the GTP loading of RhoA by endogenously expressed, Gq mediated GEFs, like Trio22.A rapid increase in YFP/CFP ratio (30 60%) was observed in the cells transfected with p63RhoGEF with t=21.50.7s after stimulation with histamine, reflecting an increase in RhoA GTP levels (Fig. 1e). After addition of mepyramine, the response decreased and the YFP/CFP ratio returned to baseline levels. Single cell ratio images from different time points are shown in Fig. 1f. These images suggest that the RhoA biosensor is predominantly activated at the cell periphery.To verify that the observed effects were mediated by Gq and not by other G proteins, we used the Gq specific inhibitor UBO QIC, a small molecule inhibitor similar to YM 25489023,24. In cells transfected with p63RhoGEF and pre incubated with 2M UBO QIC, no RhoA biosensor response to either histamine or mepyramine was observed, confirming that Gq mediates the response to histamine (Fig. 1g).During the course of our study, Pertz and co workers published an optimized RhoA sensor based on teal fluorescent protein (TFP) and Venus25, referred to as RhoA2G. A comparison in cells revealed that the DORA RhoA sensor shows increased brightness relative to RhoA2G and a better average dynamic range in relevant live cell imaging experiments (for details see Supplemental Note).Thus, p63RhoGEF shows rapid and reversible GEF activity towards an optimized high contrast RhoA FRET based sensor upon stimulation of H1R mediated Gq signaling in HeLa cells.GEF activity of p63RhoGEF at the plasma membrane is sufficient to induce actin polymerizationP63RhoGEF is located at the plasma membrane by palmitoylation of its cysteine residues near the N terminus19,20. To examine whether the plasma membrane location is important for its function, we investigated the effect of multiple p63RhoGEF deletion constructs on actin polymerization.Since it is well documented that removal of the autoinhibitory PH domain results in increased GEF activity16,18,19, we generated two truncated variants of p63RhoGEF, indicated as plasma membrane DH domain (pmDH) and cytosolic DH domain (cDH), both lacking the PH domain (for details see Supplemental Fig. S1). The cDH construct also does not contain the N terminal plasma membrane targeting sequence and resides in the cytoplasm, while the pmDH construct is localized like full length p63RhoGEF, at the plasma membrane. We transfected HeLa cells with either full length p63RhoGEF or one of its truncated variants. After 24hours the cells were fixed and stained with an F actin marker in order to analyze the effect of the different p63RhoGEF constructs on actin polymerization. We noticed that pmDH expression strongly induced actin polymerization, whereas only minimal effects on how much is a hermes handbag actin were observed in the full length p63RhoGEF or cDH conditions (Fig. 2a). This effect was analyzed quantitatively by comparing the fluorescence intensities of the phalloidin staining between the different conditions, which showed a significant difference in fluorescence intensity between transfected and control cells in the pmDH condition (pFig. 2b). From this we can deduce that p63RhoGEF guanine exchange factor activity at the plasma membrane results in actin polymerization, while p63RhoGEF guanine exchange factor activity in the cytoplasm does not.Figure 2: GEF activity at the plasma membrane increases actin polymerization.(a) HeLa cells transiently transfected with YFP p63RhoGEF, YFP cDH or YFP pmDH constructs were stained for F actin after 24hours with TRITC phalloidin and DAPI. The panels show from left to right YFP fluorescence, indicating the transfected cells, F actin staining and the overlay of YFP, actin and DAPI (b) Quantification of F actin in HeLa cells by determining the fluorescent intensity of the TRITC phalloidin staining in transfected cells and normalization to the intensity of untransfected control cells in the same fake hermes leather handbags experiment. YFP p63RhoGEF n=18 (control n=143), YFP cDH n=11 (control n=46), YFP pmDH n=32 (control n=134). Statistical significance per condition was determined by performing a two tailed student T test. Width of the individual images in (a) is 236m.GEF activity of p63RhoGEF towards RhoA is enhanced at the plasma membraneActin polymerization and the often used SRF activity assays26 are a relatively indirect measurement for the activation of RhoA, which might be under influence of considerable crosstalk and signal amplification or desensitization. Therefore, we compared the response of full length p63RhoGEF and the cDH and pmDH truncated variants on the DORA RhoA biosensor to evaluate the influence of subcellular localization on GEF activity towards RhoA in a more direct manner.We transfected HeLa cells with the DORA RhoA biosensor and either one of the p63RhoGEF constructs or a control vector that expressed only a red fluorescent protein (RFP), and measured the YFP/CFP FRET ratio to assess the basal state of RhoA activation in each condition. The minimal and maximal ratios were estimated from the inactive RhoAsensor nb probe and the active RhoAsensor ca probe, respectively. The experimental values of the wild type RhoA biosensor are expected to fall within this range of YFP/CFP ratios.The YFP/CFP ratios of the RhoA biosensor in the condition with wild type p63RhoGEF did not differ significantly from the control condition with cells containing a vector expressing only an RFP (Fig. 3a). In contrast, we found that cells expressing pmDH and cDH had significantly higher YFP/CFP ratios (pFig. 3a). We repeated this experiment in HEK293 cells, which confirmed that cells expressing the pmDH or cDH have a significant higher YFP/CFP ratio than cells expressing p63RhoGEF (Supplemental Fig. S2). This is in agreement with earlier reports of the auto inhibitory function of the PH Hermes Kelly bag replica domain as tested in vitro using purified proteins16.Figure 3: Expression of the differentially localized DH domains of p63RhoGEF, pmDH and cDH, increases Rho GTP levels with opposite spatial distributions.(a) Boxplot showing the basal YFP/CFP ratio of the DORA RhoA biosensor in HeLa cells. Cells transfected with the constitutive active (ca, n=21) or non binding (nb, n=26) RhoA biosensor were co transfected with an empty vector containing just RFP to keep expression levels equal between the different experimental conditions. Wild type (wt) RhoA biosensor was transfected with an empty vector containing just RFP (control, n=29), RFP p63RhoGEF (n=36), RFP pmDH (n=27) or RFP cDH (n=22). (b) Representative ratio images of the pmDH (top panel) and cDH (bottom panel) conditions from the experiment depicted in (a), showing the gradient of RhoA GTP loading state in HeLa cells. (c) Quantification of the spatial distribution of RhoA GTP loading state between the cell cortex (CX) and the cell body (CB) in the pmDH and cDH conditions of the experiment shown in panel (a). The difference value "ratio (CX CB)" plotted on the y axis is a measure of spatial inhomogeneity. A value of zero indicates no spatial differences, while a positive value indicates increased RhoA GTP in the cortex relative to the cell body and a negative value denotes decreased RhoA GTP in the cortex relative to the cell body (for detailed methods see Supplemental Fig. S3). For boxplots in (a,c); center lines represent the median values; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; data points are plotted as open circles. Statistical significance between conditions was determined by performing a two tailed Mann Whitney test. P values are shown in plot for the RhoA wt biosensor conditions with significant different median values. Width of the individual images in (b) is 120m.The finding that the cDH and pmDH expressing cells show similar FRET ratios is surprising, since only cells transfected with pmDH showed increased actin polymerization (Fig. 2a,b). Therefore, we decided to examine the location of RhoA GTP loading activity. Careful examination revealed different gradients of RhoA activity between cells in the pmDH and cDH condition. HeLa cells expressing the pmDH construct show enriched activity around the cell cortex (Fig. 3b), while cells expressing the cDH domain showed an inverted spatial distribution with higher activity in the cell body (Fig. 3b). In order to quantify this effect we subtracted the FRET ratio of a 5 pixel wide region corresponding to the cell cortex from the FRET ratio in the cell body (see Supplemental Fig. S3 for methodological details). We found a significant difference (pFig. 3c).These results provide evidence for the importance of the plasma membrane localization of RhoGEF activity for its signaling efficiency towards its downstream effectors. The difference between the cDH and pmDH condition in the actin polymerization assay in connection with the spatial difference in the DORA RhoA biosensor read out provides evidence for the plasma membrane as platform for RhoA mediated actin polymerization.Recruitment of the DH domain of p63RhoGEF to the plasma membrane is sufficient for sustained activation of RhoATo enable the direct comparison between the effects conferred by cDH and pmDH, we used a rapamycin dependent recruitment strategy27. This strategy allows us to first measure the effects of cytosolic GEF activity and subsequently the effects of plasma membrane located GEF activity, within the same cell.RFP cDH or RFP constructs were fused to an FKBP12 domain, which, upon addition of rapamycin, will be recruited to Lck FRB ECFP (W66A). Lck FRB ECFP (W66A) is a plasma membrane localized FRB construct fused to a non fluorescent ECFP variant (Fig. 4a).
    zroessgs viesoess Yesterday, 20:48

    Hyper Hippo instructions the team behind Experience Capitalist - plus RuneScape developers Jagex have announced a effort that sees the particular Buy Runescape 3 Gold pair begin work with RuneScape: Idle Ventures. Billed as a “miniature RPG experience”, Not doing anything Adventures will permit players enjoy a RuneScape-themed game “in just a couple of moments each day”.

    Role on onto our list of very best RPGs on COMPUTER.

    Due for a spring and coil release on Heavy steam, Idle Adventures might find elements of Hyper Hippo’s popular AdVenture Capitalist sewn into the acquainted RuneScape world aid most notably its pick-up-and-play style.

    Incedently, RuneScape: Idle Adventures signifies the first time Jagex did with an external designer to make a RuneScape online game.

    “In RuneScape: Idle Adventures players take on the role of a magnificent fresh RuneScape hero who also sets out to nurture the planet through noble attempts, including nothing lower than fishing and increasing cabbage, ” deciphers the game’s report. “Players can look ahead to the joys of RuneScape questing and creature fighting seen by way of a very different pair of eyes, working with beloved characters and even mortal enemies likewise. ”

    While not at all opposed to the idea of producing games catered to the people with less time particular hands, I’m uncertain how this design will translate to a RPG. Part of the handle role-playing games, so far as I see it, is the fact you’re expected to devote hours at a time sampling into their worlds together with characters and plotlines.

    Then again, AdVenture Capitalist has shifted above 10 million replicates to date, so what will i know? It’ll end up being interesting to see just how this one pans out in any event.

    “The emerging idle style is starting to produce a real impact, having novel gameplay activities propelling idle online games into the Steam and mobile top chart, ” says Phil Mansell, VP regarding RuneScape. “We consider there are lots more impressive experiences to come, specially when blending narrative as well as RPG aspects by using idle gameplay. ”

    RuneScape: Idle Travels is due to arrive in Steam this originate.

    jiaruli Yesterday, 20:15
    Let's Get Comfortable PHOTOS

    You guys, things are getting real. Our Season 16 Bachelor, Ben Flajnik, has met the parents and is ready to "take things to the next level" with three different "incredible women." All in one week. Thank God he rinses off in all those hottubs, because Sir Dates A Lot has got three different ladies allll over him after Episode 9's overnight dates. Yuck.

    But enough about germs let's talk fashion. Unfortunately, there are only three would be Mrs. Flajniks left. fortunately, these three have been some of my fashion faves throughout the entire season: Nicki, Lindzi and Courtney.

    The gang is in Switzerland in the picturesque town of Interlaken. Fun fact: I have actually visited Interlaken, spending time there with my family when I was just a kid. Who knew that over a decade later, I'd be writing up a reality TV recap about a town where I took a family vacation in 1996? Not I.

    Moving on. These are the overnights, people, so consider the ante upped. Nicki gets the first date, joining Ben for a helicopter ride (haven't these people ever heard of dinner and a movie?). For her big day, Nicki chooses a flattering but rather plain outfit: dark pants, leather boots, a coordinating cobalt sweater and wool coat and a white scarf to top it off. Of course she also had her signature "extras": painted nails and big earrings. Surprisingly, her hair is straight and parted on the side sleek and sophisticated.

    The coat was cute (in a place like Switzerland, it's the outerwear that matters) but I wasn't blown away by the matchy matchy look. In fact, I much preferred Ben's bulky jacket. given that this is a man who occasionally straightens his hair and knock off cartier gold bracelet wears dainty bow ties, a rugged look was a much needed change.

    For the evening portion of the date, Nicki sticks with pants but changes tops: a lowcut white tank, a black jacket/sweater thing and a colorful scarf. The neckwear pulls it together, but she clearly stays cozy and warm. Given that they weren't in Belize or some other warm, exotic location, she did as best she could and looked lovely.

    Also, I would be remiss to note that she also looked great in her turquoise bikini back at the Fantasy Suite. (At least, that's what my male "Bachelor" commenters/hecklers informed me.)

    Next up: Lindzi. The horse lover is subjected to yet another death defying, fear of heights inducing date, which this time includes rappelling down a ravine. Awesome we hope she brought her Depends.

    What she didn't bring was athletic gear. Is it just me, or did it look a little strange to see Lindzi hooked into her rock climbing gear in tight jeans, a light cardigan and leather boots? On the ground, the outfit was just blah; hanging in air, it looked seriously out of place.

    Luckily Lindzi stepped it up in the evening, baring her legs (they are one of her best assets, after all and Ben's a leg man) in a short, dusty purple minidress. She kept things classy with a high neckline and delicate sheer sleeves, adding a hint of glitter with a sparkly necklace. Overall, Lindzi hit the nail on the head.

    Also, it must be noted: BEN IS WEARING ANOTHER BOW TIE. Who does he think he is, Brad Goreski? Ben, when knock off yellow gold cartier love bracelet it comes to quirky fashion signatures, your hair is your "thing." And no one is supposed to have more than one "thing." Give up the twee ties.

    Last but not least, what you've all been waiting for. Courtney! Loathe as I am to say it, Courtney had my favorite look this week. The city chic model arrived for her Swiss date wearing an adorable black coat that flounced out at the waist, giving an otherwise sleek, simple look a flirtatious touch. She kept the rest simple: black pants, black over the knee boots and a black scarf with a colorful rose print. Her outfit was pulled together but actually interesting, as opposed to her two competitors' date looks. Well done, sneaky Courtney.

    The head to toe black proved to be a trend: for the night portion of her date, she wore a stretchy black, off the shoulder dress/tunic with black tights and boots. This didn't work as well and was a bit amateur for someone who's usually so competent in getting dressed. Oh well.

    [Sidenote: Kacie B. returns, which is probably by which I mean definitely a bad idea. But at least she chose a great pair of jeans for a film segment that imitation bangle cartier focused at least 50% of the time on her lower half.]

    Then, for the Rose Ceremony, Courtney slipped into a black, long sleeve lace minidress with black tights. This is a big deal. We almost never see "Bachelor" contestants wearing tights, either because they shoot in warm (and sometimes unbearably hot) locales and/or the ladies are trying to show off their bodies. Neither of those facts applied to Courtney tonight: the ladies were still safely ensconced in their Swiss chalet and Courtney, as opposed to the others women, has never been into showing off her legs. (Her chest, however, she'll show to basically anyone.)

    And while the black tights might not have been the best pick with that dress (it was, after all, a lot of black), it was at least interesting. Lindzi chose a deep navy halter dress with a severely unflattering mid calf, asymmetrical hemline. Nicki, bless her heart, wore another unflattering dress (although not as bad as last week's disaster): a white, one strap cocktail dress that stretched across her chest replica bracelet cartier and emphasized the tops of her arms. Courtney, that's not a good look.

    Also, her hair was sort of frizzy. But I'll let it slide I mean, who hasn't had a few bad hair days?

    Ultimately the hair and dress mattered very little. soon, Nicki had more pressing issues. She sobbed upon her exit, leaving Lindzi and Courtney to battle for Ben's half assed, sorta unenthusiastic love. One bonus: I think I spotted a Cartier tank watch on the Bachelor's wrist as he was holding Nicki's hands and saying good bye. That's pretty sweet.

    But not as sweet as next week's episode, when all the women return for "The Women Tell All"! This is guaranteed to be a fashion bonanza, as the spurned contestants return to show off how good they look since getting dumped. For many it will be their last chance to milk "The Bachelor" TV fame. so they're bound to step up their style game.
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  • fuadiskws Yesterday, 19:55
    Let's bang our stumps

    The clock is rapidly counting down to the July 1 weekend, and celebration of Canada sesquicentennial, or 150th birthday as a nation. There are just 10 weeks to go until Sesquifest, being promoted as a Canada 150 London Signature event, featuring five full days of programming and entertainment. events tied to Canada Day are just part of a year full of activities and events all in celebration of the occasion.

    It seems only fitting that Londoners should go all out for these celebrations and make cartier clou bracelet imitation up for the understated launch of this country of ours.

    In October of 1866, the London Times reported (the other London) on the mayor of Oxford annual dinner and the speech of the Right Hon. E. Caldwell, MP.

    Caldwell spoke of efforts to construct a new community by uniting the various provinces of British North America in one confederation. He was quick to point out that this new country would be of greatness to compare with their powerful neighbour; there will be no rivalry and there will be no jealousy but will constitute no mean community among the nations of the world. that might qualify as being damned with faint praise, George Etienne Cartier, a key Father of Confederation, offered this description of our new country: our confederation there cartier love bracelet fake will be Catholics and fake cartier diamond necklace Protestants, English, French, Irish and Scotch, and each by its efforts and success will add to the prosperity of the Dominion. . . . We are different races. Not to quarrel, but to work together for the common welfare. the bar was still not being raised too high.

    Here in London, the city new mayor at the time, Frank Smith, made no plans to recognize the new national holiday marking the country founding.

    The London Free Press found fault with this decision, noting the day long be remembered as the day on which a new Dominion will be added to the world, and it is meet that all should celebrate the day in a befitting manner. . . . It is to be regretted, however, that no fitting celebration will be made here, but done can be undone. Or vice versa, and consequently it of very little use to harp upon this matter. hundred and fifty years later, London Mayor Matt Brown was inviting Londoners to celebrate this year in Canadian history by celebrating not just on the Canada Day weekend but at a multitude of events planned throughout the year to mark the occasion.

    150 years earlier, Amelia Ryerse Harris of Eldon House in London did observe in her diary for the day that it was Dominion Day, the first day of Confederation. She noted there had been a military review in London that day, but that people have celebrated the day by going on excursion as it is a public holiday. wrote that her family chose to take the train to Port Stanley and then carriages to Port Talbot to celebrate the holiday.

    Back to the present for this celebration of the sesquicentennial, planners are replica yellow gold cartier love bracelet urging that Londoners do exactly the opposite and stay home and join some of the many planned celebrations to mark the occasion over the four day holiday weekend.

    Orlo Miller, in his This Was London history, noted that there was no holiday on July 1st, 1867 for the students at W. G. Montcrieff private school in London.

    Miller wrote that the teacher was determined that they would remember the day, and up and down the room on his wooden leg, repeating over and over, remember this day. I want you to remember it the first Dominion Day in Canada. Will you remember it? wrote that not a single boy or girl in that room seems ever to have forgotten Montcrieff and Canada first Dominion Day.
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