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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?
Aug 21 · 0 comments
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
Aug 21 · 0 comments
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."
Aug 21 · 0 comments
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.
Aug 21 · 0 comments
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).
Aug 21 · 0 comments
Quantum chemistry structures and properties of 134 kilo molecules

AbstractComputational de novo design of new drugs and materials requires rigorous and unbiased exploration of chemical compound space. However, large uncharted territories persist due to its size scaling combinatorially with molecular size. We report computed geometric, energetic, electronic, and thermodynamic properties for 134k stable small organic molecules made up of CHONF. These molecules correspond to the subset of all 133,885 species with up to nine heavy atoms (CONF) out of the GDB 17 chemical universe of 166 billion organic molecules. We report geometries minimal in energy, corresponding harmonic frequencies, dipole moments, polarizabilities, along with energies, enthalpies, and free energies of atomization. All properties were calculated at the B3LYP/6 31G(2df,p) level of quantum chemistry. Furthermore, for the predominant stoichiometry, C7H10O2, there are 6,095 constitutional isomers among the 134k molecules. We report energies, enthalpies, and free energies of atomization at the more accurate G4MP2 level of theory for all of them. As such, this data set provides quantum chemical properties for a relevant, consistent, and comprehensive chemical space of small organic molecules. High throughput screening represents the most straightforward approach towards materials design1. However, it presupposes that all assumptions and approximations inherent to the employed modeling techniques are applicable to the entire chemical compound space, which is the space populated by all stable molecules or materials2. Furthermore, due to the combinatorial scaling of chemical space with molecular size, it is difficult to explore or even navigate. Conclusive insights about the domain of applicability (transferability) are lacking even for the most popular first principle quantum chemistry methods. For example, the reliability and accuracy of density functional theory is known to dramatically depend on chemical composition and atomistic configurations3, highlighting the importance of reliable experimental4 or high level quantum chemistry state of the art results5,6. Unfortunately, the systems reported are typically small, which implies the existence of severe selection bias. One can therefore question how representative they are. Reymond, J. L. Virtual exploration of the small molecule chemical universe below 160 daltons. Angew. Chem. Int. Ed. 44, 1504 1508 (2005). Grimme, S. Mindless DFT benchmarking. J. Chem. Theory Comput. 5, 993 1003 (2009). M., Contreras Garca, J., Wipf, P., Yang, W. Beratan, D. N. Stochastic voyages into uncharted chemical space produce a representative library of all possible drug like compounds. J. Am. Chem. Soc. 19, 7296 7303 (2013). " href=/articles/sdata201422ref10>10.Here, we report molecular structures and properties obtained from quantum chemistry calculations for the first 134k molecules of the chemical universe GDB 17 data base11, covering a molecular property set of unprecedented size and consistency. The data set corresponds to the GDB 9 subset of all neutral molecules with up to nine atoms (CONF), not counting hydrogen. The molecular size distribution of all 134k molecules is shown in Fig. 1. This data set contains small amino acids, such as GLY, ALA, as well as nucleobases cytosine, uracil, and thymine. Also pharmaceutically relevant organic building blocks, such as pyruvic acid, piperazine, or hydroxy urea are included. Among the 134k molecules, there are 621 stoichiometries, among which C7H10O2 dominates with 6,095 constitutional isomers for which atomization energies and radii of gyration also are on display in Fig. 1. number of electron pairs, Nep=Ne/2. Each black box denotes the number of constitutional isomers for one out of the 621 stoichiometries present in the 134k molecules. The two left hand side insets correspond to zoom ins for smaller compounds. global minimum) potential energies of atomization E versus molecular radius of gyration, Rg. Joined projected distributions are shown as well.For all 134k molecules, we have calculated equilibrium geometries, frontier orbital eigenvalues, dipole moments, harmonic frequencies, polarizabilities, and thermochemical energetics corresponding to atomization energies, enthalpies, and entropies at ambient temperature. These properties have been obtained at the B3LYP/6 31G(2df,p) level of theory which forms the basis for the more accurate state of the art Gn methods which are on par with experimental accuracy12. For the 6,095 constitutional isomers of the predominant stoichiometry, C7H10O2, we report the energetics at the significantly more accurate G4MP212 level of theory.This report is structured as follows. We first describe the genesis of the results. Thereafter, we discuss the validation of our DFT results by comparison to (i) G4MP2, (ii) G4, and (iii) CBS QB3 results for 100 molecules, randomly chosen out of the 134k set. This data can serve the development, training and evaluation of inductive statistical data analysis based machine learning (ML) models13. It might also assist the search and discovery of hitherto unknown trends, structure property relationships, and molecular materials design1,14,15.MethodsGeneration of atomic coordinatesStarting with ref. 11, we use all SMILES16 strings for molecules with up to nine heavy atoms. Cations, anions, and molecules containing S, Br, Cl, or I, have been excluded, resulting in 133,885 molecules. 1,705 zwitterions have been kept in the data due to their occurrence in small biomolecules, such as amino acids. Initial Cartesian coordinates for all molecules were generated by parsing the corresponding SMILES strings using Corina (Version 3.491 2013)17. We subsequently carried out geometry relaxations at the PM7 semi empirical level of theory using MOPAC (Version 13.136L 2012)18. In the PM7 calculations, we invoked tight electronic and geometric convergence thresholds, using precise keyword. PM7 equilibrium geometries have subsequently been used as input for B3LYP geometry relaxations using Gaussian 0919. We iteratively refined the electronic and geometry thresholds. For the first iteration, Gaussian 09's default electronic and geometry thresholds have been used for all molecules. For those molecules which failed to reach SCF convergence ultrafine grids have been invoked within a second iteration for evaluating the XC energy contributions. Within a third iteration on the remaining unconverged molecules, we identified those which had relaxed to saddle points, and further tightened the SCF criteria using the keyword scf(maxcycle=200, verytight). All those molecules which still featured imaginary frequencies entered the fourth iteration using keywords, opt(calcfc, maxstep=5, maxcycles=1000). calcfc constructs a Hessian in the first step of the geometry relaxation for eigenvector following. Within the fifth and final iteration, all molecules which still failed to reach convergence, have subsequently been converged using opt(calcall, maxstep=1, maxcycles=1000). calcall constructs a Hessian for all steps through the the geometry relaxation. After all these measures taken, eleven problematic molecules still failed to converge to a minimal geometry. Out of these eleven molecules, six can be converged with low threshold using the opt(loose) keyword. In the remaining five there were two near linear molecules which converged to saddle points with very low imaginary frequencies (0i10cm1 for the lowest mode).In the case of the 6,095 constitutional isomers of C7H10O2, all molecules converged to local minima during the B3LYP geometry relaxation. The XYZ format (originally developed for the XMol program by the Minnesota Supercomputer Center) is a widespread plain text format for encoding Cartesian coordinates of molecules, with no formal specification. It contains a header line specifying the number of atoms na, a comment line, and na lines containing element type and atomic coordinates, one atom per line. We have extended this format as indicated in Table 2. Now, the comment line is used to store all scalar properties, Mulliken charges are added as a fifth column. Harmonic vibrational frequencies, SMILES and InChI are appended as respective additional lines.Table 2: XYZ like file format for molecular structure and properties. Full size tablePropertiesAll molecular geometries copy handbag Hermes were relaxed, and properties calculated, at the DFT/B3LYP/6 31G(2df,p) level of theory. The list of properties of the 134k molecules is summarized in Table 3. For a subset of 6,095 isomers of C7H10O2, energetics (properties 12 16) were additionally calculated at the G4MP2 level of theory. For a validation set of 100 randomly drawn molecules from the 133,885 GDB 9 set, enthalpies of atomization were calculated at the DFT/B3LYP/6 31G(2df,p), G4MP2, G4 and CBS QB3 levels of theory.Table 3: Calculated properties. Full size tableTechnical ValidationValidation of geometry consistencyTo validate the consistency of the relaxed B3LYP geometries, we have used them to generate the corresponding InChI20 strings with Corina and Open Babel (Version 2.3.0 2011)21. InChI corresponds to 'IUPAC International Chemical Identifier'. The resulting strings have been compared to the InChI strings obtained from the initial Cartesian coordinates (generated by Corina using the original GDB 17 SMILES strings). See Fig. 2 for a flow chart of this consistency check. Out of the 134k molecules, 3,054 molecules did not pass this test. This is due knock off Hermes handbag to the fact that SMILES and InChI representations are not unique because transformation of Cartesian coordinates to string based chemical identifiers is prone to implementation specific artifacts. For molecules with same topology, small differences in interatomic distances, bond and dihedral angles can lead to different molecular graphs encoded by the string. To quantify this artifact, the distribution of Coulomb matrix distances13, DIJ [Hartree], using the Manhattan or L1 metric, between Corina generated Cartesian coordinates (see Fig. 2) and B3LYP coordinates is on display in Fig. 3 for all the 3,054 molecules.Figure 2Schematic flow chart used for geometry consistency check.Consider, for example, molecule indexed 58 in the 134k set, which is among the 3,054 molecules for which the consistency check failed. Its original GDB 17 SMILES corresponds to NC(=N)CN, and Cartesian coordinates can be generated using Corina. When feeding back these coordinates to Open Babel to perform the inverse task of reproducing the initial SMILES string, [NH]C(=[NH2])CN is obtained instead. By contrast, performing first a geometry relaxation of the Corina generated Cartesian coordinates using PM7 followed by B3LYP, and only then parsing through Open Babel, recovers the original SMILES string. In this case, DIJ using the Coulomb matrices of Corina and B3LYP geometries is rather small (27 Ha) when compared to distances between geometries of other failed molecules, see distance distribution plot in Fig. 3. By contrast, failed molecules with large DIJ between Corina and B3LYP geometries, see Fig. 3, correspond to molecules for which the B3LYP relaxation induces dramatic distortion with significant alteration and rearrangement of covalent bonding patterns. As a result, parsing back these geometries with Open Babel yields hermes replica bag different SMILES strings. Note that all the 6,095 constitutional isomers of C7H10O2 for which G4MP2 calculations have been performed, passed this geometry consistency check, shown as a flow chart in Fig. 2.Validation of quantum chemistry resultsAll 134k molecules have been modeled using B3LYP/6 31G(2df,p) based DFT. Previously, B3LYP has been validated for several subsets, containing up to a few hundred small molecules. These benchmarks are of limited use since they are not necessarily sufficiently representative for gauging B3LYP's performance in general. In the case of DFT's systematic errors this issue is particularly pertinent22. Experimental data assembled in the NIST database is very sparse by comparison to our 134k organic molecules made up of CHONF atoms. Consequently, we have performed additional benchmark calculations for a imitation Hermes Kelly handbag subset of 100 randomly selected molecules using high level theories G4MP212, G423, and CBS QB324,25.The predictive power of the G4MP2 method is widely considered to be on par with experimental uncertainties. For example, comparison to the G3/05 test set26,27 with 454 experimental energies (including enthalpies of formation, ionization potentials, electron affinities, proton affinities, and hydrogen bond energies) of small molecules yields MAE, and RMSE of 1.0, and 1.5kcal/mol, respectively12. For the same properties and molecules, the slightly more accurate, and considerably more expensive method G412,23 yields errors of MAE=0.8kcal/mol, RMSE=1.2kcal/mol. G4MP2 has been shown to deviate only by 1.4kcal/mol from 261 bond dissociation enthalpies computed with the highly accurate W1w composite procedure28,29 for the BDE261 data set28. Consequently, we believe these calculations to be sufficiently suitable to validate the quality of the B3LYP energetics predictions. Various resulting deviations are summarized in Table 1. For the 100 molecules, the mean absolute error of B3LYP heats of atomization amounts to no more than 5kcal/mol.
Aug 21 · 0 comments
Producer Dean Thullner quits St

The founder/producer behind a popular St. Paul's fundraising gala has resigned after allegedly being told to tone down the show's LGBTQ presence.

Until just a few weeks ago, Dean Thullner was the producer of Brilliant! an event that over three years has raised $2 million for HIV and AIDS, mental health and addiction programs at St. Paul Hospital.

Thullner, who is living with HIV and AIDS and once was a patient at St. Paul Hermes Kelly bag fake said he first heard about the push to remove any mention or representation of the LGTBQ or same sex relationships from the show from an employee of the St. Paul's Hospital Foundation (SPHF) last summer.

Thullner said he was upset, but decided to press on with the show, which relies on the talents of hundreds of Vancouver designers, makeup artists, musicians, hair stylists, models and dancers. In the end the show raised $1.5 million for mental health and addictions (HIV and AIDS had been removed from the equation by SPHF in 2014).

He decided to quit last month after he was told by SPHF representatives that he had to remove LGBT references from Brilliant! and that the show, usually held in the fall, was going to move from the Commodore in downtown Vancouver to a larger venue.

Thullner said he told SPHF's Monica Chan And Renee Fisher during the March meeting: " is ridiculous.' said he was told specifically that they "didn't want any gay on the runway," and that the show had to be tightened up from 90 minutes to 40 minutes. Further, he said, he was informed: " are going to tell you want we think is acceptable. said the issue really is taking the event away from the LGBTQ community, who started it and support it every year.

"Basically it's greed," alleged Thullner, who owns the Volume Studio hair salon in Davie Village. "They want to take this event. They want to hermes replica bag tell us what to do with it."

Thullner said he was told that the push to pull any gay references from the runaway was requested by SPHF CEO and president Dick Vollet, whose wife Evelyn works for the Roman Catholic Archdiocese of Vancouver.

"I want to be clear. I quit. I am so grateful to St. Paul's Hospital and this has nothing to do with the angels that work in St. Paul's Hospital every day dealing with gay issues, straight issues, transgender issues," said Thullner, who sent a letter to the SPHF board of directors outlining his concerns at the beginning of March.

"It's Hermes Kelly bag replica hearsay, I think, right now," said Vollet, who said he was surprised about Thullner's resignation.

"Dean and I talked about a lot of the elements of the show, and frankly he was quite flexible. He actually controlled and terminated a couple of salons that were going to be part of Brilliant! because he knew it wasn't a direction we collectively wanted to go.

quite frankly, my wife has attended Brilliant! every year and she loves it."

Without Thullner at the helm it remains to be seen how many past participants and volunteers will be lining up to return for Brilliant! now slated for a new venue, the Rocky Mountaineer.

"The show must go on, absolutely," said Vollet. "We are working collectively with the community."

Thullner doesn't share that view. epaper, Digital Access, Subscriber Rewards), please input your Print Newspaper subscription phone number and postal code.

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Aug 21 · 0 comments
offs by Truck Service and Repair Providers

The dos and don ts of selecting a truck service and repair shop: The success or failure of a truck business depends on how well truck service and repair is executed. A truck repair can kill a business due to the high costs involved. So analyze well before you spend on truck service and repair. Always do your homework when selecting a repair facility. The first step to get your truck serviced is to do some groundwork by visiting a number of shops and analyzing their advantages and the disadvantages. Take a second opinion from your friends and family for good referrals and personally visit them to get an idea about the place. Find out the shop s warranty or guarantee policy before taking the plunge. Choose one based upon its reputation, quality, and workmanship guarantee and ensure that he provides you with a written warranty on the repair. After you are convinced, take your truck in for maintenance or minor repairs before trusting the shop with major fake hermes leather handbags work. Rule out the "cheapest" price factor, it is not fair. Your truck needs to be serviced by someone with the right skills, service, knowledge, and reputation. For that reason, many insurance companies tie up with repair shops on the basis of an agreement to include the shop on the insurer's "list" in exchange for discounts, use of non original Hermes bag season with the main color coordination parts and other cost replica hermes handbags cutting techniques. Even basics can how much is a hermes bag be helpful. For example, if your truck is disabled and you don't trust the diagnosis from a new mechanic, it's worth the extra hassle to tow it to another shop if need be. If by some chance, you have a problem after getting your truck repaired, take it back to the shop as soon as possible. Give them a chance to fix the mistake. How they handle the situation will help you decide whether to re visit or not. Last but not the least; be aware of your rights. Know the right forum to approach in a worst case scenario. Such unpleasant instances can be avoided it you spend enough time to do research and analysis.
Aug 20 · 0 comments
prep' Veneers in cosmetische tandheelkunde

"No prep"porseleinen veneers zijn een relatief recente concept in cosmetische tandheelkunde. Zoals de naam al aangeeft, deze fineer vereist zeer weinig of geen voorbereiding op alle tanden. Dit elimineert een van de belangrijkste beperkingen van traditionele fineer behandeling waar het proces onomkeerbaar is zodra de tanden zijn opgesteld. Bovendien, tooth voorbereiding een hoge mate van precisie om ervoor te zorgen dat de totale esthetische waarde niet in gevaar in het uiteindelijke resultaat komt.

No prep fineer zijn geavanceerde eigenschappen gemaakt van ultra dunne schelpen van porselein. Deze shells zijn zo dun dat ze vrijwel onzichtbaar zijn wanneer geplaatst op de voorzijde van de tanden. Het is niet nodig om extra ruimte voor deze fineer vanwege hun te verwaarlozen dikte. Deze fineer zijn gebonden met een speciale lijm om ervoor te zorgen dat ze permanent op hun plaats blijven. De geavanceerde kwaliteiten van neen prep fineren kunnen gemakkelijk duren tot 20 jaar of zelfs meer in sommige gevallen.

De procedure wordt eenvoudiger replica hermes handbags outlet met neen prep fineer omdat plaatselijke verdoving in dit geval kan worden vermeden. In het geval van conventionele fineer is plaatselijke verdoving belangrijk op het moment van tand voorbereiding. Sommige patinten kunnen zich tooth gevoeligheid of pijn de fake hermes leather handbags tand voorbereiding procedure how much is a hermes handbag te volgen. Met neen prep fineer krijgen deze problemen volledig gelimineerd.

Enkele van de populaire merken voor neen prep porseleinen veneers omvatten Lumineers, Vivaneers en Durathin. Deze kwaliteiten zijn meestal duurder dan de traditionele kwaliteiten, maar bieden ze de voordelen van gemak, lange levensduur van resultaten en superieure esthetiek in de meeste gevallen. Minimale voorbereiding of neen prep fineer zijn ook ideaal in situaties waar de patint lijdt aan afgestoken of gebarsten tanden. Deze tanden kunnen niet weerstaan de behandeling voorbereiding zodat neen prep alternatief kan worden gebruikt met succes in deze gevallen.

Patinten die lijden aan fake hermes kelly bagd iets onregelmatige, ongelijke of verkeerd uitgelijnd tanden kunnen profiteren van deze fineer. Als er kleine gaten tussen tanden en kiezen of de tanden overvol zijn, zijn neen prep fineer een betere optie. Sommige patinten lijden aan tanden met de leeftijd versleten. Deze voorwaarde kan worden verbeterd met neen prep kwaliteiten omdat het vermijdt enige verdere voorbereiding op tanden die al verslijten.

Verwante artikelen in tandheelkundige zorg

Mensen die genteresseerd zijn in het bovenstaande artikel zijn ook genteresseerd in de aanverwante artikelen hieronder:

Moderne tandheelkundige implantaten bestaan al sinds de jaren 1960, maar heeft alleen hun waarde bewezen in de afgelopen jaren. Voordat ze populair werd, dentale implantaten vroeger ongelovig duur en buiten het bereik van de meerderheid. Destijds was het gemakkelijker en goedkoper om gewoon kunstgebit dan omgaan met de complexiteit van tandheelkundige implantaten.

Zodra uw behandelplan ingevoerd is, zal uw aligners worden gemaakt en uw orthodontist contact met u op zodra ze klaar zijn. U krijgt de eerste set van aligners die u zal moeten dragen voor ongeveer twee weken. De aligners moeten gedragen worden in alle tijden behalve wanneer tandenpoetsen en eten.
Aug 20 · 0 comments
Photo of Kansas dog with a mouth full of Asian ladybugs freaks out pet owners

The graphic image shows a pooch named Bailey (pictured), whose owner says he likes to chase bugs, with between 30 to 40 Asian ladybugs attached to his mouth tissue

'Not true guys chill out. There's only one case ever reported and that's because the dog are loads of them. So guys let's have some proof if you say you treated them. Because this sure looks like the photo from 2015,' one unbelieving user wrote.

But Dr Lindsay Mitchell, who posted the photo, said it was not the first case she has seen, which can cause long term health issues if untreated in dogs.

'This is the second pup I have seen like this today,' the October 17 post read.

'If your pet is drooling or foaming at the mouth look for these ladybugs.

'They cause ulcers on the tongue and mouth and have a very painful bite.'

Asian ladybugs produce a protective secretion which apparently makes them stick, which is why Bailey had a mouthful.

The longer they remain attached to the animal's mouth, the harder they are to remove and can also cause ulcers, according to Dr Mitchell.

Bailey's incident comes amid an infestation of the bugs in Hoisington, with harvest season in Barton County attracting more ladybugs than usual, KAKE reported.

The infestation is also affecting animals that are eating them, including Bailey.

His owner, Frances Jiriks, said one night when he came in to eat, he was lethargic, foaming at the mouth, and did not eat.

After the scary sight, she took him in to see Dr Mitchell and had the ladybugs removed.

Dr Mitchell noted that the replica hermes birkin handbags cases seen in her office are rare and said she shared the photos in hopes of educating pet owners.

'I posted the photo not to freak people out, but to provide pet owners with an action to take in case they find one of their pets drooling excessively or with apparent foaming at the mouth,' Dr Mitchell told the Great Bend Tribune.

After the frightening discovery, she took him in to see Dr Mitchell and had the ladybugs removed

'They could avoid a trip to the veterinarian's office if they check their pet's mouth and if they find the beetles, they can simply remove them with their finger or a tongue depressor.

'They aren't like a tick, so there is no worry that a head or any part of the animal will be left behind to hurt the animal further.'

As for the reason there have been swarms of ladybugs in Barton County recently, one agricultural official told KWCH it is in response to the sugarcane aphid and the replica hermes handbags outlet lady beetles laying more eggs because their food source 'was so abundant.'

'With a lot of aphids, they produced a lot of ladybugs to be able to attack and eat and control the aphids.'

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Aug 20 · 0 comments
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