text:
First look: Voters ready to sacrifice - Politics wrap: Lugar out - Executives blast Obama on capital gains/dividends - Greece nears showdown
( firstname.lastname@example.com; Twitter: @morningmoneyben)
FIRST LOOK: VOTERS READY TO SACRIFICE - From results of new poll of 1,000 registered voters being released later today by the Peter G. Peterson Foundation: "81% ... say that the national debt is a major problem that must be addressed now ... 86% ... agree that reducing the deficit would help the economy ... 87% ... believe that to solve the long-term debt problem, Republicans will have to agree to some tax increases and Democrats will have to agree to some spending cuts ... 53% would be more likely to support a candidate for Congress if they knew that the candidate supported a bipartisan agreement that included both spending cuts and tax increases ... 76% of middle class voters believe that the middle class should contribute to the solution. ...
Story Continued Below
SUPPORT FOR TAX HIKES/BENEFIT CUTS - More from the survey: "66% of voters are willing to pay 1% more of their income in taxes to help reduce the national debt ... 76% ... would be willing to pay 1% more of their income in taxes to help protect Social Security and Medicare for future generations. ... 76% of voters approve of providing smaller but more accurate cost of living adjustments for Social Security benefits each year, including 74% of Democrats and 78% of Republicans ... 66% would be willing to give up 1% of their Social Security benefits so that Social Security is stronger and available for future generations.
PETE PETERSON, in an interview with M.M., said the study shows "the country is ahead of its leadership" on the subject of deficit and debt reduction. More Peterson: "What's encouraging is [voters] know it's a very serious problem and by large margins they know sacrifice is going to be involved and they feel that if everyone is involved in the sacrifice they would be willing to join in. ... It was interesting that such a large proportion knew that it was going to take both revenue increases and spending cuts. There is less ideological or theological rigidity on the part of the public on this ... If you ask the question in the abstract about how people feel about raising taxes that's one thing. But if it's in the context of a grand bargain or a balanced package or whatever euphemism you use, the approval goes up quite a bit. ...
FALSE CHOICE ON STIMULUS VS AUSTERITY - More Peterson: "Picking either austerity or stimulus is an utterly false choice. The fundamental problem is a long term structural problem. If markets thought you were making some progress on that it could easily accommodate some stimulus over the shorter term. You can do both. You just can't keep doing stimulus and pretending we don't have a longer term problem. Because we do."
SUMMIT - The Peter G. Peterson Foundation is hosting its 3rd Annual Fiscal Summit in D.C. next Tuesday, May 15th. Participants will include former President Bill Clinton, House Speaker John Boehner, Treasury Secretary Timothy Geithner, Sen. Rob Portman, Rep. Paul Ryan, Rep. Chris Van Hollen, Rep. Xavier Becerra and former Senator Alan Simpson. Details: http://bit.ly/JpBCAA
POLITICS WRAP: LUGAR OUT - POLITICO's Bill Nichols: "Sen. Dick Lugar, the political gold standard in Indiana for nearly 40 years, suffered a once unthinkable primary defeat Tuesday, marking a seismic shift in his state's political culture and sending yet another warning to longtime Senate incumbents. Lugar's losing battle against Indiana state Treasurer Richard Mourdock, the tea-party-backed victor, was the marquee race on a primary day in which North Carolina voters approved a gay marriage ban ... A Lugar loss marks a sea change in both Indiana and Washington, where Lugar has long played the role of Republican wise man, a sober internationalist who could reach across the aisle to solve difficult problems. In today's political climate, however, Lugar's bipartisan leanings were part of his undoing" http://bit.ly/IBCHT2
BANKER REACTS TO LUGAR - From an industry executive on Lugar: "The retail industry saw one of their lead supporters of Durbin Interchange lose tonight making their next effort (credit card interchange) in serious jeopardy. In fact, 7/11 and other retailers spent heavily in the final days in an effort to assist Lugar. Will be very interesting to see if the retailers support the Democrat Donnelly and alienate Republican Leader McConnell."
TOO BIG TO FAIL HEARING PREP - Here is the staff memo for today's Senate Banking subcommittee hearing on Too Big to Fail institutions, featuring former Fed Chair Paul Volcker. http://bit.ly/ISU717
M.M. BOOK NOOK: "BETTER, STRONGER, FASTER" - Yahoo!'s Dan Gross emails: "'Better, Stronger, Faster: The Myth of American Decline and the Rise of a New Economy' is officially out ... Media blitz now in Bataan death march mode. ... Next 36 hours: Morning Joe, Dylan Ratigan, Kudlow, Marketplace, Fox & Friends, etc." That's impressive! So is the book! Buy it here: http://politi.co/IWAGWs
ICYMI: ZOMBIE BANKS - You should also pick up "Zombie Banks" by Bloomberg's Yalman Onaran: http://amzn.to/L5y995
FACEBOOK E-BOOK - Reuters BreakingViews' "Facebook: A Like Story" "shows why its business model, corporate governance and exuberant valuation shouldn't make it a love story." http://bit.ly/JcnMDI
GOOD WEDNESDAY MORNING - Do or die for the Caps tonight at Verizon at 7:30 p.m. Rock the Red.
** Flood the Hill Week: The nation's leading real estate, financial services, and construction industry trade associations take to Capitol Hill this week to urge Congress to reauthorize and reform the National Flood Insurance Program before it expires on May 31. More than 5.6 million Americans depend on flood insurance. Visit www.floodthehill.com and join us on Twitter at #FloodtheHill **
DRIVING THE DAY - House expected to reauthorize the Ex-Im bank despite some conservative opposition ... Senate Banking "Too Big to Fail" hearing begins at 2:00 p.m. ... Another Senate Banking subcommittee holds a hearing at 10:00 a.m. on reauthorizing the National Flood Insurance Program, which expires at the end of this month. Insurers, realtors and other groups want a reform bill not just an extension ... Data calendar is light following yesterday's strong NFIB report which showed a spike to 94.5, a 14-month high. MBA Mortgage Applications out at 7:00 a.m. expected to rise from last week's 190.7 ... Wholesale trade inventories expected to increase 0.7 percent ... Mitt Romney campaigns in Colorado (swing state) and Oklahoma (non-swing state)
ALSO TODAY: MANUFACTURING REPORT - From a Commerce Dept/ESA report that Secretary Bryson will unveil at a conference at MIT entitled "The Future of Manufacturing in the U.S." From the findings: "[T]he report shows that manufacturing workers earn pay and benefits about 17 percent higher than non-manufacturing workers. During the remarks at MIT before an audience of CEOs, educators, and students, Bryson will also touch on a variety of ways that the U.S. can stay competitive in the manufacturing space, including simplifying and making permanent the R&D tax credit, committing to a renewed focus on STEM education and rewarding companies who bring jobs back to the U.S."
BIG NOT ALWAYS BAD - From blog post by Rob Nichols, President and CEO, Financial Services Forum ahead of the Senate Banking hearing: "Large financial institutions deliver significant and unique value to the U.S. economy and American businesses, investor, and savers. ... On Monday, Berkshire Hathaway CEO Warren Buffett dismissed criticisms over the size of large banks in a CNBC appearance saying, 'Our banks in this country are less concentrated than many other countries... I don't have a problem with the size of banks.' ... Presently, America's largest bank is only the 10th largest in the world, and only three U.S. banks are among the top 20."
BRITISH PM SAYS EURO ZONE NEEDS ONE GOVT - Reuters/London: "A successful euro zone requires a single government if it is to work properly, British Prime Minister David Cameron said in a newspaper interview on Wednesday. 'There's nowhere in the world that has a single currency without having more of a single government,' Cameron told Britain's Daily Mail. 'Making sense of the euro for me would mean that those euro zone countries would have to have much more co-ordinated economic policy, much more co-ordinated debt policy,' he said. Cameron, who opted out of a new European economic pact late last year, advocated Britain's position outside the euro and its ability 'to do things to ourselves, for ourselves, by ourselves.' ... Cameron said, however, that it is in Britain's interest to see a return to growth in the euro zone, which accounts for 40 percent of UK exports." http://reut.rs/LJqbp3
HOT CLICK: JOB CREATION BIGGER UNDER DEM PRESIDENTS - Bloomberg Government compared job growth under Democratic and Republican presidents over the last five decades: "The BGOV Barometer shows that since Democrat John F. Kennedy took office in January 1961, non-government payrolls in the U.S. swelled by almost 42 million jobs under Democrats, compared with 24 million for Republican presidents" http://bloom.bg/LGcI1l
EXECUTIVES BLAST OBAMA ON CAPITAL GAINS/DIVIDENDS - From an open letter to Treasury Secretary Geithner by 18 top executives published in POLITICO: "We are concerned about a specific part of the Obama administration's fiscal year 2013 budget proposal — increasing the tax rate on dividends and capital gains — and the adverse effect it will most likely have on our economy and job creation. Equity capital is the lifeblood of investment and job creation ... We believe the administration's proposal will limit corporations' ability to raise new capital and will undermine economic growth. ... The administration's plan to increase the top tax rate on dividends from 15 percent to 39.6 percent in 2013 will very likely have a seriously disruptive effect on this economic sector, reducing the incentive to pay dividends. ... The threat of looming tax increases on dividends and capital gains could also increase volatility in the stock market this year. Investment advisers are already cautioning clients to consider potential tax law changes for dividends and capital gains while making investment decisions." http://bit.ly/LJd0Vh
MORTGAGE REFI MARKET CLOGGED - WSJ's Nick Timiraos and Ruth Simon on pg. A1: "Clogged mortgage pipelines have created headaches for hundreds of thousands of Americans trying to take advantage of low mortgage rates, which averaged 4.05% for the week ending April 27, according to the Mortgage Bankers Association. Those rates have helped thousands of Americans free up cash or retire debt. On average, borrowers that refinanced during the first quarter of 2012 reduced their first-year interest payments by $2,900 ... Overall, refinancing over the past three years has unlocked savings worth $46 billion in their first year, according to Moody's Analytics. But considering how far mortgage rates have dropped, the refinancing burst has been lackluster by historical standards. ... It now takes the nation's biggest mortgage lenders an average of more than 70 days to complete a refinance, according to Accenture Credit Services, up from 45 days a year ago" http://on.wsj.com/IT4Ngl
GREECE NEARS SHOWDOWN - FT's Kerin Hope in Athens and Alexandra Stevenson in London in the front page splash: "Greece is heading for a clash with international lenders as the radical leftwing party that came second in the weekend's election called for the ripping up of a 'barbarous' austerity programme underpinning its bailout ... Alexis Tsipras, the 38-year-old leader of the Syriza party that surged in popularity in Sunday's poll, outlined a five-point plan to be put to conservative and socialist leaders on Wednesday as he attempts to build a coalition, demanding the reversal of fiscal and structural measures that have enabled Greece to slash its budget deficit. However, in an unusually blunt intervention, Jörg Asmussen, a European Central Bank executive board member, for the first time raised the possibility of a Greek exit from the euro ... Fears of a Greek exit hit financial markets, with stock markets across Europe falling, the US S&P 500 hitting a two-month low ... and investors buying safe US Treasuries, German bunds and UK gilts"" http://on.ft.com/IH8dOj
WHAT'S THE ALTERNATIVE TO AUSTERITY? - NYT's Nathaniel Popper and Jack Ewing on pg. B1: "As voters across Europe call for fewer government cutbacks and more spending on public programs, investors are asking a simple question: How would it be paid for? The option of raising money through bond sales appears to be shrinking as investors worry about the ability of euro zone nations to repay their loans. Attention has shifted to the [ECB], which could use its deep pockets to issue more inexpensive loans to commercial banks, but has shown no inclination to do so ... And Germany remains unwilling to let the rest of the euro zone trade on its good name by issuing debt jointly. ... Investors sent stocks tumbling around the world on Tuesday on fears that an austerity backlash in Greece could lead to its exit from the euro zone, but also on broader worries that other euro zone governments may not find a way to balance the conflicting demands of voters and investors. ... Spooked investors pushed up the borrowing costs for Italy, Spain and France, while the cost of insurance on the debt of those countries also climbed. The situation in Europe represents a conundrum for investors who generally recognize that the cuts that have been made in recent months by countries like Italy, Spain and Greece have caused their economies to shrink. ... But Italy and Spain have already largely lost the confidence of bond investors around the world." http://nyti.ms/IJKrpX
FIRST LOOK: FLOOD INSURANCE TESTIMONY - From Property Casualty Insurers Association of America (PCI) president and CEO David A. Sampson's prepared remarks at the Flood Insurance Program hearing: "PCI and its members believe the NFIP is vitally important to our nation and the economy, and we support your efforts to pass bipartisan legislation that includes a long-term reauthorization and meaningful reforms. I would like to emphasize three key points in my testimony today: first, it is vitally important to avoid another lapse in the National Flood Insurance Program (NFIP), as lapses have real-world negative consequences for consumers and the economy; second, as confirmed by the Government Accountability Office (GAO), the program needs meaningful, structural reforms; and third, privatization of the flood program is not feasible under current conditions."
CLEVELAND ROCKS (WITH HELP FROM GOLDMAN) - Cleveland's News Net 5's Chris Flanagan: "Cleveland-area small businesses are getting a $15 million boost to help them grow and create more jobs in northeast Ohio. The city of Cleveland is teaming up with Cuyahoga County Community College (Tri-C) and Goldman Sachs for the initiative. Cleveland is the 7th city chosen nationally for the program, which was launched two years ago. The eligible businesses will receive the money over the next five years. ... A big part of the program will be business classes at Tri-C for small business owners. Some of the topics will include leadership, hiring the right people and preparing financial statements. Small business owners can apply for classes that will be offered starting this fall or apply for grants or loans" http://bit.ly/KL4JeC
MORE REVELATIONS IN CRAZY CHESAPEAKE STORY - Reuters' Jennifer Ablan: "In the weeks before Chesapeake Energy CEO Aubrey McClendon was stripped of his chairmanship over his personal financial dealings, he arranged an additional $450 million loan from a longtime backer ... That loan, previously undisclosed, was made by investment-management firm EIG Global Energy Partners, which was at the same time helping arrange a major $1.25 billion round of financing for Chesapeake itself. The new loan brings the energy executive's total financing from EIG since 2010 to $1.33 billion and his current balance due to $1.1 billion, this person said. It was secured by McClendon's personal stakes in wells that have yet to be drilled by Chesapeake - and by his own life-insurance policy.... The latest insight into McClendon's personal financial deals comes in the wake of an April 18 Reuters investigation that found McClendon had borrowed heavily against his interests in wells owned by Chesapeake, mostly from EIG." http://reut.rs/JdTRY2
GOOD SIGN ON JOBS - Bloomberg's Shobhana Chandra: "Employers in the U.S. were seeking to fill more jobs in March than at any time in almost four years, showing growing confidence in the U.S. economy. The number of open positions increased by 172,000 to 3.74 million, the most since July 2008, from a revised 3.57 million the prior month that was larger than previously estimated... More vacancies are a sign American companies were planning to expand at the end of the first quarter, undaunted by the jump in fuel costs or concerns that global economic growth will slow. The pickup is a 'positive' development after a report last week showed payrolls in April grew at the slowest pace in six months, said economist Lou Crandall. 'Businesses were becoming a bit more willing to commit to new hires,' Crandall, chief economist at Wrightson ICAP ... said in an interview. 'The progress is incremental at best and the levels are still very low.'" http://bloom.bg/IHbDAA
** Flood the Hill Week: The nation's leading real estate and financial services trade associations take to Capitol Hill this week to urge Congress to reauthorize and reform the National Flood Insurance Program (NFIP) before the program expires on May 31, 2012. Floods are not just a coastal issue. More than 5.6 million Americans across the country depend on the NFIP to protect their homes and businesses. Failing to reauthorize the NFIP could jeopardize nearly 40,000 mortgage closings per month, according to the National Association of REALTORS. Visit www.floodthehill.com to see why the NFIP plays a pivotal role in the U.S. economy. Follow us on Twitter at #FloodtheHill **
** A message from U.S. Bank: U.S. Bank strives to be a partner of choice to make a meaningful impact in communities where we live and work. The U.S. Bank Supplier Diversity program works to identify, develop and build strong relationships with certified small and diverse business enterprises that are qualified to supply U.S. Bank with quality products and services. Over the past five years, U.S. Bank has increased its spending with diverse suppliers by 36 percent. Read more: https://www.usbank.com/en/corporate_responsibility/diversity-and-inclusion.html **


text:
Municipality Of Velsen: My Neighbourhood In Holland
Taking you on a tour through the municipality of Velsen, where I was born. Highlights are Beeckestein mansion and its coastal region.
Today my dad took me on a tour through the municipality of Velsen, after dropping off my sister at Haarlem Station.
First we drove to my dad's cottage and checked out the garden, my dad grows a lot of plants so he needs to checkup regularly.
In the afternoon we drove to Wijk aan Zee which is located above the North-Sea channel.
The coastal location gives a breathtaking view of my suburb.
For snacks we went to the snackbar and ordered some fries with peanutsauce, mayonaise and onions - a Dutch delight also called a 'patatje oorlog'.

subset: commoncrawl


text:
Dell Laser Printer 5100CN Driver
Dell Laser Printer 5100CN Driver
This printer lets you include enough paper drawers with no moment's delay to achieve to a substantial info limit. The default paper drawer stands up to Dell Laser Printer 5100CN Driver linens, including lawful cardstock size, while the fold-down multi-purpose plate bolsters as much as 150 sheets. Merely the multipurpose menu backings imprinting upon thicker media, for example, envelopes and titles. You can heap a discretionary 500-sheet cardstock drawer, estimated on $280, under your printer; $500 gets you one more 1000-sheet drawer, and you'll consolidate both of the with the default plate to acquire a huge aggregate cardstock limit of 2150 linens. An inherent duplexer gives you an opportunity to print twofold sided pages, and your done printouts drop right 250-sheet yield canister about the printer.
The Dell Laser Printer 5100CN Driver must be sensibly practical to perform. Dark toner cartridges, fit for printing as much as 9000 pages much like Dell's page-yield lab tests, cost just $50. Each and every shading cartridge (cyan, fuchsia, and yellow) costs $170, appraised for 8000 pages. When you analyze the $140 expense of supplanting your drum each thirty-five, 000 pages, Dell gauges, you'll pay only around 1 penny to its consumables every very contrasting page.
We additionally timed the Dell Laser Printer 5100CN Driver among the quickest shading lasers within our execution tests. Inside monochrome this design release 21. 1 pages every moment; within shading it designed 6. 1 ppm. We wish it had fared better within our quality tests, regardless. Despite the proven fact that its content publishing wasn't too bad, we did view some barbed and fluffy edges within lettering. Our line-workmanship analyze showed some banding and draining between just about divided lines. The grayscale picture had lots of tight even banding and indicated observable moiré designs on garments. Nonetheless, the grayscale image likewise showed even tonal changes and a lot of subtle element inside the darker territories. On shading photographs, most of us saw great fine detail in shadows, nonetheless speck examples have been excessively obvious within light regions, adding to the photograph's standard non-abrasiveness.
Introducing the printer on our system at the Computer World Test Centre through its built in ethernet port has been clear. When most of us utilized the command board (which incorporates an illuminated, two-line LCD screen) for you to allocate a new IP address and stacked the driver on our server, we had the capacity produce from our Computer without stacking a lot more programming. The printer's likewise has parallel and USB 2. 0 ports. Dissimilar to its little office step-brother, the Dell Laser Printer 3000cn, your Dell Laser Printer 5100CN Driver backings PostScript publishing.
The Web-based business programming is adequate for a little office with number of printers. The printed documentation covers a setup distribution, a fast guide sheet, a manager manual, and a client guide. The recent two have any cover, with the customer aide concentrating upon establishment issues as well as the proprietor manual focusing on printer support (counting information on utilizing the printer having a Mac). The client aide comes just as a Windows Help record about the product CD-ROM.
The Dell Laser Printer 5100CN Driver is quick and must be a temperate decision for a workgroup that devices through paper, however its picture quality could have been something more.
The Supportsdriver.com You can download Dell Laser Printer 5100CN drivers that can be downloaded directly and very easy to download because no redirection to another website, to download the driver on this website can be directly downloaded on the download link I have provided below.
Dell Laser Printer 5100CN Driver
Dell Laser Printer 5100CN Driver
Dell 5100cn drivers for Windows Xp 32 – Bit
Dell 5100cn drivers for Windows Xp 64 – Bit
Dell 5100cn drivers for Windows Vista 32 – Bit
Dell 5100cn drivers for Windows 7 32 – Bit
Dell 5100cn drivers for Windows 7 64 – Bit
Dell 5100cn drivers for Windows 2003 32 – Bit
Dell 5100cn drivers for Windows 2000
Dell 5100cn drivers for Windows NT 4.0


text:
I used to love their arrabiata. And I thought for the longest time they had the best bruschetta in the city (where did they get real tomato tasting tomatoes all year around?).
Has anyone else noticed a change since the new owners? And does anyone know if the owners still have their second restaurant, and if so, where / what it is?


text:
Autopower Industries Offers Roll Bar and Cage for BMW 1-Series Coupe
Autopower Industries is now offering their full lineup of roll bars and cages for the BMW 1 Series coupe 2007- 2013 (E82). Autopower's 4-point race roll bar (PN: 60233) features different options and meets with SCCA and NASA approval. This roll bar is a bolt-in application that allows you to retain your BMW's stock interior. See below for photos, and feel free to contact one of our sales professionals at 1.480.966.3040 with any further questions.
Two-Door Coupe, E82
Race Roll Bar w/ Options (#60233)
Tags: 1 Series, application, Autopower Industries, BMW, Bolt in, Coupe, interior, racing, roll bar, Roll cage, wholesale


text:
|Home | About | Journals | Submit | Contact Us | Français
Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome1,2. The ability of the myristoyl group to facilitate dynamic protein–protein and protein–membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems3. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus4. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen5,6. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.
While seeking to understand how bacterial pathogens regulate the host secretory pathway7, we found that S. flexneri potently inhibited cargo transport through the Golgi apparatus (Supplementary Fig. 1). Further investigation showed that the Golgi was severely fragmented after Shigella infection (Fig. 1a). By contrast, neither Listeria monocytogenes nor Salmonella typhimurium disrupted the Golgi, suggesting that Shigella has evolved a specific, yet poorly understood, mechanism to regulate host organelle function.
Shigella infection requires translocation of more than 20 bacterial 'effector' proteins into host cells through the Mxi-SPA Type III Secretion System (T3SS)8. Deletion of mxiD, a component of the needle complex required for T3SS, eliminated Golgi destruction by Shigella infection (Fig. 1d). To identify the specific effector protein required for this activity, Golgi morphology was assessed after transient transfection of 20 effector genes (Supplementary Fig. 2a, b). Both IpaJ and VirA induced profound Golgi fragmentation (Fig. 1b) and inhibited hormone trafficking through the secretory pathway (Supplementary Fig. 2c). However, the other T3SS effector proteins had no discernible effect on Golgi structure or function (Supplementary Fig. 2a).
Comparison of cells infected with Shigella ΔipaJ and ΔvirA gene deletion strains showed that these mutants induced abnormal Golgi morphologies (Fig. 1c) and variable degrees of Golgi disruption (Fig. 1d), yet neither mutant alone fully attenuated Golgi destruction. By contrast, the Golgi remained intact and functional in cells infected with ΔipaJ/virA double mutant strain (Fig. 1c, d). The Shigella ΔipaJ/virA strain showed normal host cell invasion, intracellular replication and actin-based motility, suggesting Golgi disruption was specifically caused by these effector genes (Supplementary Fig. 3). Next, in vivo bacterial replication was evaluated using an established mouse model of mucosal infection9. The number of recoverable bacteria was sharply reduced (by 100-fold, P < 0.001) by deleting either ipaJ or virA genes compared with wild-type Shigella (Fig. 1e). As expected, bacterial replication was further attenuated in the Shigella ΔmixD mutant lacking all T3SS function (Fig. 1e). We also found reductions in host inflammatory cytokines in mice infected with each Shigella mutant strain (data not shown). Taken together, these data demonstrate that two Shigella effector proteins, IpaJ and VirA, each harbour Golgi inhibitory activity, and are essential and play non-redundant roles for optimal in vivo virulence.
Although VirA was recently shown to inactivate Rab1 GTPase signalling pathways5, extra mechanisms are necessary for Golgi destruction during Shigella infection6. We therefore focused on IpaJ because its molecular mechanism of action is poorly understood10,11. BLAST database searches identified IpaJ homologues in numerous bacterial species, but this sequence-based alignment did not offer any clues to its function (Supplementary Fig. 4). In contrast, structural-based bioinformatics showed that IpaJ is distantly related to the cysteine peptidase C39-like family of domains of unknown function (annotated DUF3335) (Fig. 2a). C39-family members cleave leader peptides from bacteriocins with anti-microbial and quorum-sensing activities12. Although IpaJ is unlikely to function in these capacities, it harbours the catalytic cysteine (C), histidine (H) and aspartate (D) residues required for peptide bond hydrolysis (Fig. 2a). As predicted by this alignment, alanine substitutions at C64, H206 or D218 abolished the ability of IpaJ to disrupt host Golgi morphology in transfection studies (Fig. 2b). In addition, complementation of Shigella ΔipaJ/virA deletion strain with plasmid expression of IpaJ induced Golgi disruption phenotype whereas IpaJ C64A, H206A and D218A had no effect (Supplementary Fig. 5).
Previous studies have shown that IpaJ induces growth arrest phenotype in yeast13. A similar growth inhibitory activity was found with IpaJ, whereas yeast grew normally in the presence of IpaJ C64A, H206A or D218A catalytic mutants (Fig. 2c). Using a multicopy suppressor screen, we identified three distinct genomic loci that, when introduced on high-copy plasmids, suppressed IpaJ activity in yeast. Two loci encoded ARF1p and ARF2p GTPases, and the third loci encoded VPS15p, a phosphatidylinositol kinase required for yeast vacuole fusion14 (Fig. 2d). Although these data suggest that IpaJ may regulate several host signalling pathways, we focused our initial efforts on ARF GTPases because these enzymes are master regulators of cargo trafficking through the Golgi apparatus4. Overexpression of either ARF1p or ARF2p rescued the yeast growth arrest phenotype, thereby defining ARF GTPases as potential substrates of IpaJ (Fig. 2e).
ARF1 functionally couples guanine-nucleotide exchange (GDP for GTP) and membrane binding (through N-myristoylation) to the recruitment of COPI coat-complex to Golgi membranes as a key step in vesicle fission4. Surprisingly, IpaJ had no effect on the guanine-nucleotide cycle or GTPase-dependent interactions of ARF1 in vitro (Supplementary Fig. 6). We therefore searched for IpaJ-induced post-translational modifications on ARF1 by 'top-down' mass spectrometry15,16. ARF1 bearing a carboxy (C)-terminal Strep affinity tag (ARF1-strep) was expressed in HEK293T cells and purified by Strep-Tactin chromatography. We observed an intact monoisotopic molecular mass of 26,895.95 Da and subsequent tandem mass spectrometry (MS/MS) resulted in 30 fragment ions that, together, provided an unambiguous assignment of N-myristoylated ARF1-strep protein (Fig. 3a and Supplementary Fig. 7a). Cellular expression of IpaJ resulted in a 266.35 Da decrease in molecular mass of ARF1-strep, which corresponds closely to the mass of the N-myristoyl group and the amino (N)-terminal glycine (267.22 Da) (Fig. 3b). Indeed, the MS/MS data support the idea that IpaJ cleaved the amide bond between glycine-2 and asparagine-3, liberating the N-myristoylated glycine from the ARF1 GTPase domain (Fig. 3d and Supplementary Fig. 7b). ARF1 cleavage was not observed in IpaJ C64A-treated samples, consistent with the function of IpaJ as cysteine protease (Fig. 3c and Supplementary Fig. 7c).
Next, we devised a protocol to reconstitute the ARF1 cleavage reaction in vitro (Fig. 3e). Because Escherichia coli does not express a system for N-myristoylation, ARF1-His was co-expressed with yeast N-myristoyl transferase (NMTp) in the presence of exogenous azide-conjugated myristic acid. The lipid state of purified ARF1-His was visualized by fluorescently labelling the azido-myristoyl group by click chemistry17 (Fig. 3f, lanes 1 and 2). Importantly, IpaJ cleaved N-myristoylated ARF1 in vitro whereas this reaction was less efficient with IpaJ C64A (Fig. 3f, lane 3 and 4). Similar results were also observed when IpaJ was incubated with ARF1-strep isolated from mammalian cellular lysates following a similar procedure (Fig. 3g).
Having established that IpaJ cleaves ARF1, we revisited our studies in yeast showing that IpaJ may target several host substrates including VPS15p, a lipidated kinase14. Although ARF1 and VPS15p are both N-myristoylated on their glycine-2 residue, they do not share any sequence or functional similarity. These observations suggested that IpaJ might cleave a broader range of substrates than initially suspected. To test this hypothesis, we labelled numerous of N-myristoylated proteins found in HEK293T cells and found that a large proportion of these proteins were demyristoylated in IpaJ-treated samples (Fig. 3h, arrows). The spectrum of N-myristoylated proteins was unaltered by IpaJ C64A mutant, thereby verifying the proteolytic-basis of this reaction. IpaJ had no effect on either palmitoylation or geranylgeranylation, indicating that the protease specifically cleaves proteins modified by the myristoyl group (Fig. 3h).
N-myristoylated proteins share a conserved glycine but their sequences are otherwise highly variable (Fig. 4a). To determine how IpaJ recognizes diverse substrates, we first generated chimaeric enhanced green fluorescent protein (EGFP) fusion proteins that expressed 10 amino-acid leader peptides derived from c-Src, MARCKs, hVPS15 and GRASP65, N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function, respectively (Fig. 4a)18–21. IpaJ cleaved each of these lipid-modified peptides in a reaction requiring its cysteine protease activity (Fig. 4b). The ability of IpaJ to cleave unrelated peptides suggested that the protease might recognize the lipidated glycine-2 common to all of its substrates. We found that soluble myristic acid was unable to inhibit ARF1 proteolysis, suggesting that IpaJ does not simply recognize long hydrocarbon chains (Supplementary Fig. 8). However, protein lipidation was essential for proteolysis because IpaJ was unable to cleave non-myristoylated ARF1 in vitro (Supplementary Fig. 9). When taken together, these data indicate that IpaJ specifically recognizes the conserved glycine-2 of substrates in context of its amide linkage to N-myristic acid.
N-myristoylation is unique among lipid modifications in that it undergoes a dynamic conformational switch in response to upstream signalling events (termed the 'myristoyl switch')3. For example, the myristoyl group is sequestered in GDP-inactive ARF1 and liberated for membrane binding and signalling by GTP exchange (Supplementary Fig. 10)4,22–24. We found that IpaJ was unable to cleave ARF1 T31N (a GDP-bound mutant form), but efficiently cleaved ARF1 Q71L (a mutant locked it the GTP-bound active state) in cells (Fig. 4c). These findings suggest IpaJ recognizes substrates as they cycle between activity-dependent conformational states. Consistent with this interpretation, microinjection of purified IpaJ induced the release of activated ARF1 from Golgi membranes just before organelle fragmentation (Fig. 4d). ARF1 was also displaced from Golgi membranes by wild-type Shigella infection but not by ΔipaJ single mutant (note that the Golgi is fragmented by VirA in this infection) or ΔipaJ/ΔvirA double mutant strains (Fig. 4e). Extra cell-based experiments support the notion that GTP-active ARF1 is a physiological target of IpaJ during Shigella infection (Supplementary Figs 11–13), thereby defining a new mechanism for inhibition of cytokine secretion and immune detection by a bacterial pathogen.
Here we have determined that IpaJ is a cysteine protease that cleaves the N-myristoylated glycine from ARF1 and potentially numerous host cellular substrates. This discovery adds significantly to the small but increasing number of bacterial proteases that cleave lipidated proteins as a mechanism of microbial pathogenesis25,26. Importantly, the ability of IpaJ to eliminate the N-myristoyl modification of unrelated proteins may allow Shigella to exploit signalling pathways in a variety of host cellular contexts27. Although extra studies will be needed to unravel the hierarchy of these events, our discoveries provide a unique framework from which to explore the functional consequences of protein N-myristoylation in human health and disease.
Bacterial effector genes were amplified by PCR from S. flexneri M90T and cloned into pEntr/D, generating a Gateway™ compatible entry clone (Invitrogen) according to the manufacturer's recommendations. Twenty S. flexneri effectors were cloned into pEntr/D vector: IpaA, AAK18443.1; IpaB, AAK18446; IpaH1.4, AAK18594.1; IpaH2.5, AAK18367; IpaH4.5, AAK18395; IpaH7.8, AAK18394.1; IpaH9.8, AAK18544; IpaJ, AAK18440; IpgD, AAK18452; IcsB, AAK18450; OspB, AAL72323.1; OspC1, AAL72322.1; OspC2, AAW64906; OspD1, AAW64782; OspE1, AAW64916; OspE2, AAW64805; OspF, AAW64770; OspG, NP_085391; VirA, AAK18501. For mammalian expression, the effectors were then recombined into modified pcDNA3.1 vector carrying N-terminal eGFP or mCherry in frame with the Gateway cassette (Gateway™ vector conversion system, Invitrogen). For bacterial expression, a 50-amino-acid N-terminal deletion of IpaJ (IpaJΔ50; residues 51–259) was cloned in frame into pGEX-4T1 (GST-tag) (Amersham), yielding intact catalytic domain of IpaJ C-terminally fused to GST-tag. N-terminal deletion of ARF1 (ARF1Δ17; residues 18–181) was subcloned by PCR into pGEX-4T1 vector. Human GGA1 (GAT domain, residues 76–215) was amplified and cloned by PCR in frame into pET28b-MBP-His vector. For complementation of S. flexneri knockout strains, ipaJ was amplified and cloned by PCR into pBad/Myc-His vector to be expressed under arabinose-inducible promoter. mCherry was expressed from the rpsD promoter in pDP151 vector in Shigella strains. For galactose-induced yeast expression, ipaJ was recombined from pEntr/ D into Gateway™-compatible pYes-Dest52 vector (Invitrogen) for expression under galactose-inducible promoter. Yeast arf1 and arf2 (SGD: S000002351, SGD: S000002296) were amplified by PCR from Yep13 genomic clones and cloned into p415 vector for yeast expression from a constitutively active promoter (GAPDH). Human full-length arf1 was cloned in frame into modified pcDNA3.1 vector carrying C-terminal mCherry or eGFP. Human GRASP65 was cloned in frame into peGFP-N2 vector. For expression of Strep-tagged full-length human ARF1 in mammalian cells, the arf1 complementary DNA (cDNA) (Missouri S&T cDNA Resource Center; ARF0100000) was first cloned into pEntr/D entry vector. The gene was then recombined into modified pcDNA4T/O vector carrying Gateway™ cassette and two repeats of C-terminal Strep-tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, IBA GmbH). For expression of leader peptides bearing myristoylation signal sequences derived from c-Src, MARCKs, hVPS15 and GRASP65, cDNA sequences encoding 10 N-terminal amino acids (see Fig. 4a) were cloned into modified pcDNA4T/O vector carrying C-terminal GFP and two repeats of Strep-tag. All site-directed mutations were generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). More primer and plasmid information is available upon request.
For Shigella infections, HeLa cells or HeLa cells stably expressing a GFP tag attached to the N-terminal Golgi retention signal of N-acetylglucosaminyltransferase I31 (NAGFP HeLa) were seeded onto cover-slips in six-well dishes the day before infection. Sodium butyrate at 5 μM was added to NAGFP cells after seeding to stimulate production of Golgi-localized GFP marker31. Shigella strains were inoculated from frozen stocks and grown overnight at 30 °C in brain–heart infusion media (BHI) (Difco™, BD Biosciences). Bacteria were then back-diluted 1:50 and incubated at 37 °C until reaching D600 ~ 0.5–0.6. Bacteria were then washed in 1× phosphate buffer saline (PBS) and incubated at 37 °C for 15 min in 0.003% Congo red. Twenty microlitres of bacterial suspension were then added to each well of a six-well dish of semi-confluent HeLa or NAGFP cells and centrifuged for 10 min at room temperature (1000g) to facilitate bacterial adherence. The plates were then incubated for 90 min at 37 °C, 5% CO2. The media was removed and the wells were washed three times with gentamicin (150 μg ml−1) followed by three washes with sterile PBS. Fresh antibiotic-free DMEM was added to each well after that and cells were incubated for an extra 2–4 h (37 °C, 5% CO2). After incubation, the slides were fixed in 3.7% formaldehyde and prepared for microscopy. Golgi and F-actin were visualized with anti-GM130 (BD Transduction Laboratories) and rhodamine-phalloidin (Molecular Probes) respectively. For infections with S. flexneri knockout strains complimented with IpaJ (wild type and C/H/D mutants), overnight bacterial cultures were diluted 1:50 in BHI containing 2% arabinose and incubated until reaching D600 ~ 0.5–0.6 as described above. Infections were performed in low-glucose media containing 2% arabinose to allow expression of IpaJ from arabinose-inducible promoter. For infections with S. flexneri strains expressing mCherry, ampicillin was added both to BHI and DMEM culture media.
A protocol for Salmonella infection of HeLa cells was adapted from previous studies32. S. typhimurium was grown overnight at 37 °C in a glass flask with shaking then subcultured (1:30) and grown for 3 h. One millilitre was pelleted, suspended in 1× PBS and added dropwise at various concentrations to semi-confluent HeLa cells in low-glucose DMEM+10% FBS. Cells were incubated (37 °C, 5% CO2) for 10 min, washed three times with PBS and incubated for an extra 15 min (37 °C, 5% CO2) in fresh low-glucose DMEM + 10% FBS. Cells were washed again with PBS and incubated in DMEM + 10% FBS + 100 mg ml−1 gentamicin before fixing with 3.7% formaldehyde at various time points. Cellular phenotypes were visualized as described.
L. monocytogenes was grown and prepared for HeLa infection as described33. Various concentrations of bacteria were added to semi-confluent HeLa cells in DMEM + 10% FBS from an overnight culture of Listeria grown in BHI media. Cells were centrifuged (250g, room temperature, 5 min) and incubated (37 °C, 5% CO2) for 10 min, then washed three times with 1× PBS. Fresh media containing 10 mg ml−1 gentamicin were added and cells incubated for an extra 4–6 h before fixation and visualization.
The virA, ipaJ and mxiD genes were individually disrupted using the λ red recombinase-mediated recombination system28. Briefly, a kanamycin resistance cassette flanked with 50 base pairs homologous to the gene of interest (virA, ipaJ or mxiD) was amplified from plasmid DNA (pKD3) (primer sequences are available upon request). PCR products were electroporated into S. flexneri strain M90T carrying the red recombinase plasmid pKD46. Transformants were selected by growth on Luria broth agar plates containing kanamycin (50 μg ml−1) and simultaneously cured of pKD46 by growth at 42 °C overnight. The kanamycin resistance gene was eliminated through the introduction of the pCP20 helper plasmid that contained the FLP recombinase. Subsequent curing of pCP20 was done by growing strains at 42 °C for 5 h. Disruption of the virA and ipaJ genes was confirmed through DNA sequencing of the respective genetic loci. To generate the double strain (Shigella ΔvirA/ΔipaJ), the ipaJ locus was disrupted from a Shigella ΔvirA strain following the protocol described above.
Female C57Bl/6 mice were purchased from the National Cancer Institute and used between 6 and 8 weeks of age. For infection, S. flexneri M90T or mutants were grown and back-diluted from the stationary to early log phase growth (D600 ~ 0.1) in BHI media (BD Biosciences) at 37 °C, washed and diluted in sterile saline, and administered dropwise (106 c.f.u. in 30 μl volume) into the external nares of mice anaesthetized with ketamine/xylazine as described9. At the indicated time points after infection, mice were euthanized, and the recoverable bacterial c.f.u. enumerated by plating serial dilutions of the lung homogenate on BHI plates. Statistical significance was determined by a one-way analysis of variance (P < 0.01) Tukey post hoc test. The amounts of the TNF-a and IL-6 in the serum and lung homogenate were enumerated using commercially available enzyme-linked immunosorbent assay (ELISA) reagents (R&D Systems). All experiments were performed under University of Minnesota or Cincinnati Children's Hospital Institutional Animal Care and Use Committee approved protocols (S.S.W.).
As previously described7,34, HeLa cells (50% confluence) were transfected with 1 μg of 4× FKBP-hGH (Ariad Pharmaceutical; source of material, D. Bernstein) and either 0.5 μg eGFP–IpaJ, eGFP–VirA or pEGFP control plasmid with Fugene6 (Roche). Sixteen hours later, the medium was replaced with medium containing AP21998 (final concentration 2 mM) or vehicle control. AP21998 was incubated with the cells for 2 h before the supernatant was collected. The supernatant was then diluted 100-fold and compared against an hGH standard curve (12.5–400 pg ml−1) for the quantification of hGH released using an hGH enzyme-linked ELISA (Roche). For no drug controls, 100% ethanol (2 μl) was incubated with the cells for 2 h.
HHpred was used to detect known Pfam domains (profile database search used: pfam version 25.0) with distant structural homology relationships to full-length IpaJ (GenInfo Identifier (GI): 12329066) with default parameter settings35. The DUF3335 (C39-like peptidase family) gave a probability score of 90.0. One hundred proteins in the Pfam database contain this domain. PROMALS3D was used to produce a multiple-sequence alignment with the eight IpaJ and 100 DUF3335 family members to identify invariant catalytic residues and confirm conserved secondary and hydrophobicity patterns29.
Recombinant proteins (IpaJΔ50, ARF1ΔN17, GGA176–215) were expressed in BL21–DE3 E. coli strains by induction with 0.4 mM IPTG for 16 h at 18 °C. Pellets were lysed with His buffer (100 mM HEPES, pH 7.5, 300 mM NaCl) or GST buffer (Tris buffer saline (TBS); 50 mM Tris pH 7.5, 150 mM NaCl, 2 mM DTT) supplemented with protease cocktail (Roche). Proteins were purified with nickel agarose (Qiagen) or glutathione Sepharose (Amersham Biosciences) following the manufacturer's instructions. Eluted proteins were buffer exchanged into TBS using concentration centrifugal columns (Millipore); glycerol was added to 15% and the proteins were then stored at −80 °C.
HEK293A, HeLa and NAGFP HeLa (see above) cells were transfected using FuGene6 (Roche) and incubated for 16–18 h. For expression of Strep-tagged proteins, HEK293T cells were transfected using calcium phosphate and incubated for 18–24 h. Equal amounts of DNA were used for co-transfection. Cells were then lysed (lysis buffer: 20 mM Tris HCl pH 7.5, 1.5 mM MgCl2, 350 mM NaCl, 0.5% NP-40, 5% glycerol) and sonicated for 5 s. The lysate was then clarified by centrifugation (15,000g, 10 min) and applied to Strep-Tactin Superflow Plus resin (Qiagen). After incubation (90 min, 4 °C) the column was washed three times (washing buffer: 20 mM Tris HCl pH 7.5, 1.5 mM MgCl2, 150 mM NaCl, 0.2% NP-40, 5% glycerol) and Strep-tagged proteins were eluted with 2.5 mM desthiobiotin (100 mM Tris•HCl, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin, pH 8.0). Microinjections of IpaJΔ50 were performed using a semiautomatic InjectMan NI2 micromanipulator (Eppendorf). Recombinant proteins were diluted in 1× TBS with Cascade Blue (Invitrogen) fluorescent dyes until the final concentration of the protein of 1 mg ml−1 (21 μM). Cellular concentration of microinjected protein was estimated as 1 μM. For IpaJ C64A and ARF1 co-localization studies, HeLa cells were treated either with Brefeldin A (2.5 μg ml−1) for 20 min or with nocodazole (10 μg ml−1) for 1 h before fixation. Immunofluorescence images in all experiments were acquired with Zeiss AxioVert 200 fluorescence microscope. F-actin was visualized by rhodamine- or Alexa Fluor 350-conjugated phalloidin (Molecular Probes), Golgi was detected by GM130 antibodies (BD Biosciences) and secondary anti-mouse IgG antibodies (Thermo Scientific). NAGFP HeLa cells stimulated for production of GFP marker were additionally stained with anti-GFP antibodies (Clontech) and fluorescein-conjugated anti-rabbit IgG secondary antibodies (Thermo Scientific) to enhance the fluorescent signal.
Yeast InvscI strain was transformed with pYes-dest52 (Invitrogen) vector carrying ipaJ gene (wild type or C/H/D catalytic mutant) under the GAL1 inducible promoter. Yeast was streaked onto Yc-U agar media containing glucose or galactose and rafinose as carbon source. Yeast was cultured for at 30 °C for 3–5 days and the plates were visually inspected for growth. For the yeast multicopy suppressor screen, the ipaJ gene was stably integrated into Y7092 yeast strain genome (MATα can1Δ::STE2pr-Sp_his5 his3Δ1leu2Δ0ura3Δ0met15Δ0 lyp1Δ) as previously described36. The resulting strain Y7092-IpaJ (MATα can1Δ::STE2pr-Sp_his5 his3Δ1leu2Δ0ura3Δ0met15Δ0 lyp1Δ trp1Δ::GAL1-IpaJ-URA3) carryied a single copy of the ipaJ gene under control of GAL1 galactose-inducible promoter. Y7092-IpaJ cells were then transformed with the S. cerevisiae genomic library in the Yep13 vector (ATCC) and transformants were selected for survival on Yc-UL media containing 2% galactose and 1% raffinose as carbon sources. Positive clones were isolated and library vectors were sequenced. For the complementation assay, PCR-amplified yeast arf1 and arf2 were expressed in yeast under GAPDH promoter (see above).
Strep-tagged ARF1 protein was co-transfected with IpaJ or control vector. ARF1-strep was purified with a Strep-Tactin Superflow Plus column and eluted with 2.5 mM desthiobiotin buffer (see above). Purified ARF1-strep was used for LC-MS/MS analysis.
LC-MS/MS was generally as previously described30. Optima-grade solvents and acids (Thermo Scientific) were used. Reverse-phase liquid chromatography (RPLC) capillary columns were packed in-house to a length of 15 cm with 5 μm diameter C18 Poroshell-300 resin (Agilent Technologies) in 75 μm inner diameter × 360 μm outer diameter Picofrit columns (New Objective) with 15 μm inner diameter integrated micro-electrospray tips. A capillary column heater (Analytical Sales & Services) was used to maintain the column temperature at 60 °C during analysis. RPLC mobile phase A included 0.025% TFA, 0.3% formic acid and 5% acetonitrile in water. RPLC mobile phase B included 0.025% TFA, 0.3% formic acid and 20% isopropanol in acetonitrile. The elution gradient was 0% B at 0–3 min, 45% B at 3.01 min and 45–60% B from 3.01 to 23 min. Flow was regulated by an 1100-nano-LC (Agilent) at a flow rate of 0.5 μl min−1.
Before analysis, the protein was de-salted using a C4 ZipTip (Millipore) as described previously and suspended in 0.2% formic acid in water. Approximately 1600 fmol of protein was loaded onto the capillary column per injection. Analysis was on an LTQ Orbitrap XLTM (Thermo Scientific) in full MS mode at 60,000 Fourier-transform mass spectrometry resolution, two microscans, maximum ion accumulation time of 500 ms, scan range from 800 to 2000 m/z, source-induced dissociation =25 V, tube lens 100 V, capillary voltage 50 V, capillary temperature 275 °C. MS/MS data were collected with the source-induced dissociation =60 V, tube lens 100 V, capillary voltage 50 V, capillary temperature 375 °C. The [M +6H]6+ charge state of ubiquitin was used to tune the mass spectrometer. The 'Xtract' function in the XcaliburTM (Thermo Scientific) data system with a signal:noise of 3 was used to extract intact protein and fragment masses from the raw spectral data. All fragmentation data presented were the summation of scans across the entire protein elution profile. MS/MS data sets were interrogated with Prosight PC 2.0TM (Thermo Scientific). Analysis of the MS/MS data against the known ARF1 sequences without the correct modification state led to few or no b-ion matches. Replicate analysis was performed on each sample and assigned fragment ions were manually validated in raw data.
E. coli BL-21 cells expressing ARF1-His with or without yeast NMTp were grown overnight at 37 °C in LB media. Cultures were diluted 1:50 and incubated at 37 °C until reaching D600 ~ 0.5–0.6. Myristic acid and azide myristic acid were added at the concentrations 50 μM and 5 μM respectively, and cells were additionally incubated for 30 min. Protein expression was then induced with 0.4 mM IPTG and cells were further incubated at 37 °C for 3 h. We estimated that N-myristoylated ARF1 is bound in a GDP:GTP ratio of 1:1 as assessed by GGA-binding assays. Expression of IpaJ or IpaJ C64A mutant was performed by the same method without adding myristic acid. ARF1- and IpaJ-expressing cells were lysed by sonication for 1 mn separately (lysis buffer: 20 mM Tris HCl pH 7.5, 1.5 mM MgCl2, 350 mM NaCl, 0.5% NP-40, 5% glycerol) and the lysates were mixed together and incubated for 30 min at 37 °C. ARF1 was then purified using nickel agarose (Qiagen). N-myristoylated proteins were labelled with Alex Fluor 647 Alkyne using Click-iT Reaction Buffer Kit (Invitrogen) according to instructions with modifications. Specifically, click labelling was performed on the proteins still bound to the column. The column was then washed and proteins were eluted with 500 mM imidazole/100 mM HEPES containing 1% SDS. The N-myristoylation status was analysed by in-gel fluorescence and the equal protein load was confirmed by Coomassie stain. For cleavage inhibition, free myristic acid or vehicle control (DMSO) was added to bacterial lysate containing ARF1 before adding IpaJ. After cleavage reaction, ARF1 was then labelled and purified as described above. To test the cleavage of non-myristoylated ARF1, a similar experimental setup was used with the following modifications: E. coli BL-21 cells expressing ARF1-His (in the absence of NMTp or exogenous myristic acid) were purified with Ni-NTA agarose, eluted with 500 mM imidazole/100 mM HEPES and processed for mass spectrometry.
For metabolic labelling in mammalian cells, myristic acid-azide (Invitrogen) was added to the cell cultures 6 h after transfection at final concentration 10 μM and incubation was continued overnight. The next day, Strep-tagged proteins were purified using Strep-Tactin SuperFlow Plus columns (see above). N-myristoylated proteins were labelled with Alex Fluor 647 Alkyne as described above. The column was then washed and proteins were eluted with 2.5 mM desthiobiotin elution buffer. Purified proteins were separated on SDS–polyacrylamide gel electrophoresis gel and transferred onto nitrocellulose paper (Biorad) for probing with STREPtactin-HRP (Biorad). Fluorescence was analysed both in SDS-gel and on nitrocellulose paper (collectively 'in-gel fluorescence'). Expression of IpaJ–GFP (wild type or catalytic mutants) was confirmed by probing cell lysates with anti-GFP antibodies (Clontech) and secondary HRP-anti-rabbit IgG antibodies (Invitrogen). Expression of GRASP65– and hVPS15–GFP chimaeric proteins was probed in cell lysate, before protein purification. For in vitro cleavage reaction, ARF1-strep transfected cells labelled with myristic acid-azide were lysed and 3 μg of recombinant IpaJ Δ50 (wild type or C64A catalytic mutant) were added into 20 μl of the lysate. After incubation at 37 °C for 30 min, myristoylated ARF1-strep was labelled and analysed as described above. For analysis of cleavage of multiple proteins, HEK293T cells were cultured in the presence of azide-modified moieties (myristic acid, palmitic acid, geranylgeranyl alcohol) according to the Click-iT protocol. Cell lysates were incubated with 3 μg of IpaJ Δ50 (wild type or C64A) for 30 min at 37 °C. Proteins containing azide-modified moieties were then labelled with Alex Fluor 647 Alkyne and purified according to the Click-iT labelling protocol.
We thank our colleagues at University of Texas Southwestern Medical Center, specifically K. Orth, Y. M. Chook and J. Seeman, for discussions in preparing this manuscript. We are particularly indebted to L. Kinch, N. Grishin, D. Mitchel, X. Guo, D. Trudgian and S. Perelman for their contributions. We acknowledge the services of the University of Texas Southwestern Medical Center proteomics core, supported by a Cancer Prevention and Research Institute of Texas grant RP120613. A.S.S. was supported by the Howard Hughes Medical Institute International Student Research fellowship. B.A.W. was supported by a National Institutes of Health training grant (NIAID; 5T32AI007520) and S.S.W. by the Burroughs Wellcome Fund. This work was supported by grants from the National Institutes of Health (NIAID; RO1AI083359 and NIGMS; R01GM100486), the Welch Foundation (I-1704) and the Burroughs Wellcome Fund to N.M.A.
Supplementary Information is available in the online version of the paper.
Author Contributions N.B. and N.M.A. conceived the general ideas for this work. N.B. and N.M.A. planned, performed and interpreted experiments. T.G.F. and A.S.S. generated bacterial knockout strains and performed GTPase assays, respectively. J.M.E. and S.S.W. performed mouse infection studies. B.A.W. performed the bioinformatics studies on IpaJ. D.A.P. and S.M.P. performed top-down mass spectrometry. N.M.A. and N.B. wrote the manuscript and all authors provided editorial input.
Author Information Reprints and permissions information is available at www.nature.com/reprints.
The authors declare no competing financial interests.
Readers are welcome to comment on the online version of the paper.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
No one has a reputation for cheese and wine quite like the French! This très chic French Wine and Cheese hamper comes with a bottle of our favourite French wine perfectly matched to 4 of our cheesemonger's cheeses, and includes Delicias Spanish quince paste and housemade lavosh.
1 X 750ml Bottle Cotes du Rhone
200g Marcel Petite Comte
250g Epoisses
200g Herve Mons Fourme d'Ambert
200g Delice des Cremiers
240g Delicias Spanish Quince Paste
1 x Packet Milk the Cow Housemade Lavosh


text:
Learn how to protect yourself from Internet scams. Read our online fraud prevention tips
Pets ClassifiedsLocal LA Classified Ads
180 listings match, 11-20 shown
Can't find what you're looking for? Try our
Advanced Search
Champion bloodline!!!CFA reg EXOTIC SHORTHAIR Black and White Male kittens, Would be a great Christmas Gift. Looking for a Grumpy Kitty to Love? Here he is. We are a small Cattery in South Mississippi, not far from New Orleans. They were Born Aug 3, 2017. He is so Cute. Quality kittens and health guarantee. Call or text anytime at 601-916-0372. Also go to our Facebook page and Our Website and check it out!! You will love our babies! Pictures of Kittens and Mom (Snickers) and Dad (ElGatto). Will be vaccinated and dewormed. Our kittens are litter trained. Looking forward to hearing from you. We except Paypal and sometimes you can get interest free with them for 6 months. Breeding rights additional fee of $500. Shipping available or we can meet at a distance agreed upon. Our Parents are PKD Neg. look us up !!! www.grumpykitty.net
Published in The Times-Picayune 11/19. Updated 11/23.
Adorable Yorkie Pups and Pomeranian Pups. Teacup and Toy. CKC, S/W, Delivery Available (601) 517-6291
Published in The Times-Picayune 11/17. Updated 11/23.
Beautiful Yorkie Puppies Registered, Microchipped. S/W. Health Guarantee. $800 251-229-4958
Published in The Times-Picayune 11/17. Updated 11/23.
AKC Reg Boxer Pups beauti ful fawn, 1st shots, (M) only, 6wks $500 each 601-551-3405
Published in The Times-Picayune 11/17. Updated 11/23.
CHIHUAHUA PUPPIES CKC Reg 8 wks. Long Short Hair. 2M 2F , 1st Shots. Ready Nov 16th. $500 985-264-8455
Published in The Times-Picayune 11/15. Updated 11/23.
Rottweiler Pups, AKC, Male Female, previous litter weighed over 90lbs at 7months, call or text 985-514-0998. Dad weighs 150 lbs and Mom weighs 110 lbs. Prices start at $1000.00 up to $1500.00. My website is Von Gueldner Rottweilers.com. The puppies are 9 weeks old and are ready to go. They will have their first 2 vaccinations and multiple wormings.
Published in The Times-Picayune 11/15. Updated 11/23.
Razor Edge Pitbull Puppies For Sale. $200. Call 504-905-5991 985-402-5553
Published in The Times-Picayune 11/15. Updated 11/23.
AKC German Shepard Pups, Excellent Bloodline, Parents on Site. Solid Blk Blk Tan. $800 985-507-7503
Published in The Times-Picayune 11/15. Updated 11/23.
Published in The Times-Picayune 11/12. Updated 11/23.
French Bulldog Pups , AKC, rare colors $2400. 601-297-1330. www.topbulldogs.com
Published in The Times-Picayune 11/12. Updated 11/23.
180 listings match, 11-20 shown
Find A Local Business


text:
Relic Finley Tote Bag - Grey
This sophisticated carry-all will keep you organised to a tee. Equipped with internal and external pocket storage, and adjustable carry handles ready to sling over your shoulder, it's the perfect size for travel or everyday use.
- Relic handbags
- Style: Finley Tote
- Colour: Grey
- Zippered closure
- Double carry handle
- Silver-toned hardware
- Pockets/compartments:
- Main compartment
- 1 x zippered pocket
- 2 x slip pockets
- 1 x external zippered pocket
- 2 x side pockets
- Relic keychain detail
- Smooth finish
- Materials: Synthetic/leather-look finish
- Drop length (approx. cm): 25
- Dimensions (approx. cm): 31 (H) x 35 (W) x 10 (D)
- Please note: Measurements taken from sample and are approximate. Bag width is inclusive of side gusset while empty and may vary slightly dependent on contents. Depth taken from base of bag.
- Country of origin: China
A Bit About The Brand / More Info
Edgy meets sophistication, that's what you'll get with every bag, wallet, purse or accessory from Relic. Try one of these well-crafted pieces and see what we're on about.
Customer Reviews
5 Star
4 Star
3 Star
2 Star
1 Star


text:
Last night at 5:40 p.m, approximately 20 minutes before the Postal Service takes the last set of packages out of the APC machine for the night (i.e. the last shipment of the day), I realized that I didn't have any more "Definitely Not Having A Silent Birth" t-shirts in small. This was highly unfortunate as M (keeping it anon to protect the innocent) had ordered one. I freaked out, because that's what pregnant women who own their own business do. I decided to call M and ask him what he wanted to do. Which is what I did.
Crap. A woman. Possible THE PREGNANT WOMAN. Now I had to be professional.
K: Hi, may I speak with M?
W: He's not here. May I take a message?
K: This is Kristen calling. I'm calling in reference to an online order he made. My number is 867-5309.
Just kidding. I didn't say that was my number. Now that would have been funny. Except if she was pregnant, she wouldn't have thought that was funny. At all. And she would have cried.
He finally called back after I had already made an executive decision and mailed out the package.
K: Did you perhaps order a maternity t-shirt?
M: I did.
K: Is the person I spoke with earlier THE pregnant person?
M: Yes.
K: CRAP!!! OK, I just want you to know that I was VERY worried that she was so DON'T WORRY. I was very coy. I was all "this is in reference to an online order he made" and my husband thought I was a freak.
M: (laughing) Great.
K: No, not GREAT. M, WE HAVE A PROBLEM. WE DON'T HAVE ANY SMALLS IN YOUR SHIRT SO I WAS CALLING TO ASK WHAT TO DO!!!!
In case you were wondering, I was yelling. Derek was laughing, M was laughing and I sounded like I was certifiable.
M: That's great. We'll make it work no problem.
K: I also threw in another shirt in Small–the Baby Brewing shirt I was actually calling to see if you wanted another free shirt instead of that one but then I was all, "I don't know if they are heavy drinkers like we are and would want the Mommy Wants a Cocktail shirt."
M: (still laughing) Actually there is a little of that going on over here.
K: Well, I sent the safe bet.
M: We really could make the medium work. Is there any way to take out the free shirt? I don't want you to have to do that.
K: Nope it's gone you know the Postal Service says it will get to your house in 2 days but are those people on CRACK and I know this from personal experience there is no way that shirt is getting there in two days and what if you wanted to give it for the holidays and it wasn't there on time so I just wanted to get it on its way so the chances of it getting there would be better if I sent it tonight and I didn't hear back from you in time before I sent it and if she just wears it to her doctor's appointments and tells everyone she got the shirts at Baby Brewing then it will be GREAT and I am 8 months pregnant so I am a lunatic.
And then I breathed. Which made my husband VERY happy.
M: Congratulations on being pregnant! We will tell EVERYONE where we got the shirts!!! Talk to you soon.
I love M. He's not my favorite customer. That would be Tara. But I still love M.Share on Facebook


text:
Battery on a Law Enforcement Officer in Florida
Florida vigorously prosecutes people alleged to have acted physically obstructed law enforcement officers and imposes heightened penalties on civilians who batter law enforcement officers while are carrying out their public duties.
Under Florida Statute 784.07(2)(b), the crime of Battery on a Law Enforcement is defined as the touching or striking of a law enforcement officer engaged in the lawful execution of a legal duty.
Penalties for Battery on a Law Enforcement Officer
In Florida, the crime of Battery on a Law Enforcement Officer is a Third Degree Felony and punishable by up to five (5) years in prison, five (5) years of probation, and a $5,000 fine.
Battery on a Law Enforcement Officer is assigned a Level 4 offense severity ranking under Florida's Criminal Punishment Code. A judge may sentence a person convicted of Battery on a Law Enforcement Officer to probation, but may also impose a sentence up to the statutory maximum of five years in prison. [1]
Defenses to Battery on a Law Enforcement Officer
In addition to the pretrial defenses and trial defenses that can be raised in any criminal case, specific defenses to the crime of Battery on a Law Enforcement Officer are:
On The Job
A conviction for Battery on a Law Enforcement Officer requires proof that the officer was "engaged in the performance of a lawful duty when the battery occurred," not just "on the job." [2]
This requirement means that even if a uniformed law enforcement officer is working off duty for a bar, convenience store, as mall security, or for any private employer, they are merely "on the job" for the private employer and are not afforded enhanced protection under the statute if they should be struck while working for the private employer. [3]
However, if while working for a private employer, they become engaged in "activities of an official police nature," such as breaking up a fight, escorting a trespasser of the premises, or arresting someone, a person who then batters the officer is subject to being convicted of Battery on a Law Enforcement Officer.
Self Defense
The use of violence against an officer who is effectuating an arrest is unlawful regardless of whether the arrest is technically illegal.
However, apart from an arrest scenario, a person is authorized to use reasonable force to defend themselves against other unlawful actions committed by law enforcement officers. [4]
Examples of unlawful actions that a person may defend against are:
- Unlawfully entering a private home;
- Unlawfully frisking a person; or
- Unlawfully detaining someone.
Nevertheless, regardless of whether an officer is engaged in unlawful activity, a person who uses force against an officer does so at his own risk; as the State Attorney's Office will usually err on the side of the law enforcement officer when filing charges.
Excessive Force
If an officer uses excessive force when making an arrest, a person is justified in the use of reasonable force to defend himself or herself.
Additionally, the danger of excessive force or police brutality need not have been actual to justify a self-defense claim. In such instances, you are allowed to defend yourself if the appearance of excessive force or police brutality appeared to be imminent.
However, a person can only defend themselves to the extent reasonably believed that such force was necessary. And the determination of whether the resistance with violence was justified is based upon the circumstances at the time.
Unknown Status
While a person is not justified in using force to resist a police officer who is "known, or reasonably appears to be a law enforcement officer." A person accused of Battery on Law Enforcement Officer must have reason to know that the "victim" was actually an officer and not someone impersonating an officer.
Contact Criminal Defense Lawyer Richard Hornsby
If you have been arrested or charged with the crime of Battery on Law Enforcement Officer in Central Florida or the Greater Orlando area, contact Orlando Criminal Defense Lawyer Richard Hornsby today.
The initial consultation is free and I am always available to advise you on the proper course of action that can be taken.


text:
Thursday, September 17, 2009
Nannio's Mitts
In this picture, my daughter looks different to me. Sometimes, I think certain photos seem to flash forward to a glimpse of the future of a child. She looks older than her 5.84 years! I love that expression : )
So, Nannio made a scarf and I made the matching mitts. I didn't like this yarn much, either. It's got too many sparkly bits in it. But the mitts are cute. I'd like to make some more of theses....
Here's them on my second daughter...
And of course, here's what they look like on the puggy : ) Sorry, you know I can't resist!
Yarn: Bulky Italian yarn
Gauge: 6 sts/2"
Needles: size 11
Pattern: Cast on 20 sts and work in the round in k1p1 rubbing for 17 rows. Change to stockinette stitch and work 12 rows in the round. Work back and forth for 6 rows to allow for a thumb opening. Rejoin to work in the round for 5 more rows- bind off. Weave in loose ends.
These work up very fast, as you might imagine. Hopefully they will help Nannio beat the chill this fall.
Posted by Wishful Knitting at 7:51 AM 2 comments:
Wednesday, September 09, 2009
Anastasia's Shawl....
I knit this shawl for my big sister. Although it didn't take long to knit, it has taken me a long time to finish it up. Seems I have several projects that are near completion - over 90% done, yet they remain 10% undone.
I have really changed to a process-type knitter and not a project oriented knitter, but STILL!
The yarn was not my favorite. My knitterly friends seem to think the finished product looks nice, but it's hard for me to see past the yarn. As usual, I am not a fan of novelty yarn.
Yarn: Karabella Gossamer, black
Pattern: Karabella KK230
Yarn used: 3 skeins, 220 yards EACH
Gauge: 14-16 sts/4 inches
Needles: size 10
Mods: Cast on 30 rather than 16 and then I just kept knitting and increasing until it was the length I wanted from neck to elbow. The final measurements are:
neck to elbow-19"
neck down back- 25"
length across at widest point-46"
I knit this with smaller needles than called for, so it has a tighter feel and should be warmer. At the recommended gauge, the stitches seemed too big to me- it had too many "holes." So, I used 3 skeins rather than 2. Even with the extra skein, I had to make the ruffle smaller so I didn't run out of yarn.
It's a very simple pattern and a ruffle is always nice.
Look for this in the mail soon....sis!
Posted by Wishful Knitting at 8:58 AM 3 comments:
Labels: Scarves
Subscribe to: Posts (Atom)


text:
Training survey
Thank you for your interest in our training program. Please complete this short survey to help us determine the most appropriate and relevant training course for you. After the survey, you'll be directed toward the appropriate course application.
Need more information? Learn about our course options here.
Training course placement survey


text:
|09 Jan 2012
Here's what my programs look like now (see below).
However, I also want to delete IE and game folders from my Program and Program x86 folders, but one says I need permission from TrustedInstaller and the other requires permission from SYSTEM.
I'm the only user of this machine and the administrator. How do I get rid of this stuff (and if I need to login as the administrator, please tell me how).
|My System Specs
|09 Jan 2012
Vista, Windows7, Mint Mate, Zorin, Windows 8
IE is an integral part of the system. If you delete it, you will have a lot of trouble in the future because many programs/websites look for it.
Games is another story. I delete those too - but not the folder. Who cares, it takes such a small bit of disk space.
I also deleted Adobe Air. Lots of luck with MSE. It got me into trouble.
|My System Specs
|09 Jan 2012
I run Mozilla, but I can't trash IE? Oh well. What I'm trying to do is remove all distractions on this beast before I rebuild so the games and such need to go. I'd really like to just delete the folders. Once it's all squeaky clean, I'll burn a system image and backup.
|My System Specs
|10 Jan 2012
|My System Specs
|Thread Tools
|Similar help and support threads
Eliminated admin password in boot, now can't get past blue screen of d
Big problem, when i first turned on my computer in the past i got a blue screen and needed to enter a password to get to the page that gave me an option of clicking on admin account or user account. To get rid of the first screen which was a hassle because i'm the only one who uses the computer i...
|General Discussion
How do I get rid of bloatware?
I have a HP Pavillion. and I love it. But.. It is full of bloatware. What is, a real easy way of getting rid of all these programs that I don't want, nor need ? Also, it has a lot of games installed. I don't want them either. I don't want peripheral.. I want tunnel.
Can't Reverse an Anti-"Pinning" Tweak & Eliminated Likely Suspects ...
I'm embarrassed, as I should have taken notes on how to reverse this at whatever point in the past I implemented this tweak. I cannot pin an application to the taskbar. I do not believe it's the IsShortcut problem, and I've tried the LNK restore anyway. I do have the "Pin to Start Menu"...
|General Discussion
HP Bloatware And Beyond
Sorry if this is in the wrong section. I've had this laptop for probably over a year now and its been long overdue for a clean up (never did initially). I'll leave all the excess details out of it as much as possible (I rant about it). I have a desktop, its been in pieces since September when...
What is bloatware
There is often a mention of so called "Bloatware" the comes with OEM installation. But what is bloatware as per your definition - I am sure we will get as many definitions as we get posting. 1. Let me first tell you what i consider as bloatware and what I delete immediately: 1.1. All games...
|General Discussion
Many people voice complaints about the so called Bloatware that is being put on preinstalled systems. I have a more positive attitude towards some of those programs and like to invite you for a discussion. A) The 3 months trials for security programs such as Norton or McAfee are very useful...
|Our Sites
|Site Links
|About Us
|Find Us
Windows 7 Forums is an independent web site and has not been authorized, sponsored, or otherwise approved by Microsoft Corporation. "Windows 7" and related materials are trademarks of Microsoft Corp.
© Designer Media Ltd
All times are GMT -5. The time now is 10:18.


text:
Oakland, Berkeley, And East Bay News, Events, Restaurants, Music, & Arts
Given the size of the store, selection in some genres (jazz excluded) can be limited, but the emphasis is on quality. You're apt to find rare treats at decent prices, and owner Dave Kloski is more than happy to help.
Tags: Record Stores


text:
LAS VEGAS, Jan. 5, 2016 /PRNewswire/ -- At CES® 2016, LG Electronics (LG) officially unveiled its new LG SIGNATURE product lineup, bringing the company's best technology and designs together under a single name.
The vision of the LG SIGNATURE brand extension is to stay pure and sophisticated, true to each product's essence. Initial offerings include a stunning new HDR-enabled OLED 4K TV, a more advanced TWIN Wash™ washing machine, a cutting-edge Door-in-Door™ refrigerator and a futuristic hybrid air purifier, with others to follow.
OLED TV with Maximum Technology for Maximum Immersion
The LG SIGNATURE OLED TV boasts the ultimate in elegant simplicity and takes viewing to the next level with a design that is no thicker than a sheet of glass, featuring LG's OLED panel spanning one-tenth of an inch and a sound system that would be the envy of any audiophile. To achieve this sleek profile that naturally and seamlessly blends into any interior environment, designers streamlined key features and placed the speakers inside the stand.
The LG SIGNATURE OLED TV has received the Ultra HD Premium seal of recognition from the UHD Alliance (UHDA), signifying that it meets the most demanding performance standards for resolution, HDR (high dynamic range), peak luminance, black levels, wide color gamut and immersive audio, among other criteria. With HDR capabilities, Pixel Dimming, 10-bit panel/10-bit processing power, ColorPrime Pro and new webOS 3.0, the LG SIGNATURE OLED TV represents the ultimate in TV technology.
Refrigerator that Unveils the Freshness Within
The LG SIGNATURE refrigerator combines fresh technology with fresh design to help keep food fresh longer. As a nod to its innovative spirit, LG has taken its award-winning Door-in-Door design one step further by adding a KNOCK-ON feature that makes the opaque Door-in-Door compartment transparent with a simple knock. The transparent surface allows consumers to peer inside the refrigerator without having to open the door, offering significant energy savings by limiting cold air loss.
The interior of the refrigerator is composed of stainless steel for more effective cold air retention and longer food storage. Additionally, the new AUTO DOOR feature automatically opens the door when an individual approaches the refrigerator, allowing for quick and easy access even if one's arms are full of groceries. And because the LG SIGNATURE refrigerator is smart-enabled, the door does not automatically open when approached by pets or babies.
Washing Machine Like No Other
The LG SIGNATURE washing machine is an advanced version of the revolutionary LG TWIN Wash, first introduced to the public at CES 2015. The LG SIGNATURE 24-inch front-load washing machine is more convenient and sophisticated than ever and features the unique SideKick™ pedestal washer under the main unit, allowing users to do two separate loads of laundry at once.
The LG SIGNATURE washing machine also comes equipped with LG's proprietary Centum System, which improves the washing machine's durability and energy efficiency with an advanced suspension system that reduces tub vibrations and noise levels during the wash cycle.
The minimalist design of the LG SIGNATURE washing machine is highlighted by the beautiful Quick Circle User Interface, which is touch-enabled and embedded in the glass door at a 17-degree angle that makes it easy to see when standing. The PUSH OPEN washing machine door can be opened even when one's hands are full by simply pushing lightly, the same way LG Door-in-Door refrigerators are opened. And the enamel coating gives the washing machine a sophisticated and sleek look. The U.S. version will feature LG's new Black Stainless Steel design.
Air Purifier that Cleans Air Before Your Very Eyes
LG believes that nothing is more important than clean air. The exquisitely designed LG SIGNATURE air purifier allows the air cleaning process to be a visual experience with its innovative transparent panel. Equipped with an advanced Aqua Cyclone feature, this air purifier harnesses the power of water to filter out harmful chemicals and contaminants while quickly bringing the room to comfortable humidity levels. The unit is equipped with LG's semi-permanent filter system, allowing consumers to remove, wash and reuse the filters for up to 10 years, saving both time and money.
While fine dust particles are typically invisible to the naked eye, consumers can see the air quality of their indoor environment by simply glancing at LG's Smart PM 1.0 Indicator. Positioned around the control display, the indicator uses four different colors of light to signify the concentration of super-fine dust particles.
"Our driving principle is to ensure that the essence of our products is preserved from idea generation to the user experience," said Brian Na, executive vice president and LG's global marketing officer. "LG SIGNATURE is the embodiment of everything that is important to LG, and we hope to convey that philosophy to consumers who appreciate great products from strong brands."
LG SIGNATURE will begin rolling out in key target markets in North America, Europe and Asia soon after CES. CES attendees are encouraged to stop by LG's booth from Jan. 6-9 at the Las Vegas Convention Center, Central Hall #8204 to experience LG SIGNATURE in person. For more information about other LG products at CES, please visit http://ces2016.lgnewsroom.com.
About LG Electronics, Inc.
LG Electronics, Inc. (KSE: 066570.KS) is a global leader and technology innovator in consumer electronics, mobile communications and home appliances, employing 83,000 people working in 119 locations around the world. With 2014 global sales of USD 55.91 billion (KRW 59.04 trillion), LG comprises four business units ― Home Entertainment, Mobile Communications, Home Appliance & Air Solution, and Vehicle Components ― and is one of the world's leading producers of flat panel TVs, mobile devices, air conditioners, washing machines and refrigerators. LG Electronics is a 2015 ENERGY STAR Partner of the Year. For more news and information on LG Electronics, please visit www.LGnewsroom.com.
About LG Electronics Home Entertainment Company
The LG Electronics Home Entertainment Company is a global leader in television, audio video systems, monitors, personal computers, digital signage and commercial displays. The company opened a new era of innovation in the TV market, pioneering the use of OLED technology in home theater displays and implementing an intuitive operating system to allow customers to experience the full benefits of smart TV technology. LG is committed to improving customers' lives with innovative home entertainment products led by its award-winning 4K OLED TVs, ULTRA HD TVs and webOS TVs. For more information, please visit www.LG.com.
About LG Electronics Home Appliance & Air Solution Company
The LG Electronics Home Appliance & Air Solution Company is a global leader in home appliances, air conditioning and air quality systems. The company is creating total solutions for the home with its industry leading core technologies. LG is committed to making life better for consumers around the world by providing thoughtfully designed products, including refrigerators, washing machines, dishwashers, cooking appliances, vacuum cleaners, built-in appliances, air conditioners, air purifiers and dehumidifiers. Collectively, these products deliver enhanced convenience, superb performance, great savings and compelling health benefits. For more information, please visit www.LG.com.
About LG Electronics USA
Named 4K Ultra HD Partner for CES 2016, LG Electronics USA, Inc., based in Englewood Cliffs, N.J., is the North American subsidiary of LG Electronics, Inc., a $56 billion global force and technology leader in consumer electronics, home appliances and mobile communications. LG Electronics, a proud 2015 ENERGY STAR Partner of the Year, sells a range of stylish and innovative home entertainment products, mobile phones, home appliances, commercial displays, air conditioning systems and solar energy solutions in the United States, all under LG's "Life's Good" marketing theme.www.lg.com.
Photo - http://photos.prnewswire.com/prnh/20160104/319171
Photo - http://photos.prnewswire.com/prnh/20160104/319169
Photo - http://photos.prnewswire.com/prnh/20160104/319170
Photo - http://photos.prnewswire.com/prnh/20160104/319168
Photo - http://photos.prnewswire.com/prnh/20160104/319172
Photo - http://photos.prnewswire.com/prnh/20160104/319167
To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/lg-signature-ultra-premium-oled-tv-and-home-appliance-products-unveiled-at-ces-2016-300199136.html
SOURCE LG Electronics USA


text:
Optical Jobs - Zest Optical Recruitment
Zest Optical Recruitment. With a visionary approach to recruitment, we've placed 100s of happy optometrists, dispensing opticians, optical assistants, and associated optical staff, in 100s of fantastic jobs with the UK's best independent opticians and multiples. We are an established optical recruitment consultancy with decades of combined recruitment experience, attracting large numbers of candidates every day for our client's optical positions. This coupled with our high rate of referrals and extensive optical network, puts Zest Optical in a superb position to provide our clients with a fully managed consultancy service and ensures our candidates a huge choice of high quality optical jobs.
If you are seeking a new optical opportunity or an optical business seeking to recruit some fantastic staff, Zest Optical will help you find the perfect match.


text:
Network Switches
Be in control with our huge range of network adaptors. Connect your computer to a specific network through a USB port instantly with these tools. This is also an ideal tool to use if you have a gaming console that needs network access in an area with no wireless capability.


text:
Finally, I'm at a point where I can say I'm happy with something I designed for myself. It's been an ongoing project for about 3.5 years now, with all sorts of renditions. I've finally settled on something I'm proud of – a clean, sans serif typeface that I designed from scratch (meaning, I built each letter myself by drawing it out by hand first and then building it digitally using shapes) and a 'tagline' I find to-the-point and simple in one of my favorite existing fonts. Previous to this one I had settled for the following, which uses the typeface "Neutral:"
Even farther back, this was my first branding and kind of my logo:
I've been researching for a long time now, really trying to learn as much as possible and to get to a professional, well thought-out brand. Stand tuned for more in the coming months!


text:
- Description
DetailsKnight Power Oil is one of the best massage oil formula. It helps to give strength to the tired muscles of the private organ. Providing sexual predominance. Increasing libido, erectile and sexual strength. Regaining lost confidence. Prevents premature ejaculation. Stimulates sexual desire and increases vigor. Retains puberty in old age. Helps to remove impotency. Removes sag during sexual activity.
- Additional Information
Additional Information
Manufacturer IMC Pvt.Ltd.
- Reviews


text:
Fur Chewing (Barbering)
How serious is it? Not serious health wise, but the underlying reason is cause for concern.
What is it? A chinchilla who chews his/her own fur or the fur of another chinchilla.
How can I recognize it? Patchy, uneven fur that looks cropped in areas.
How do chins get it? Could be stress related, genetic, or a symptom of dominance by another chin.
How do you treat it? Treat the underlying cause. If the chin is stressed out, change the environment. If it is nutritionally based, make sure your pet has plenty of hay and other stimulating chew toys of all textures.
How can I avoid this happening?
A while ago, we took in a chinchilla who had excessively chewed his fur. When he came to us, in fact, he had chewed off every bit of fur that he could reach-- in some places, down to his very skin. We constantly commented on how he looked-- like half a chin, like a kangaroo, and so on. We dubbed him Buffalo Bill.
Now, three months later to the day, Buffalo Bill is beginning to show his improvement. He enjoys hanging out near the boy chin in the next cage over. He has stopped chewing his fur and you can see it's growing back in tufts. This pattern of fur growth is completely normal. In just a few more months, he will look like a regular, fluffy chinchilla again. Check out how he's coming along!
This group of chins were surrendered severely fur chewed, emaciated and beat up.


text:
With an Intel® Extreme Masters Certified PC.
With an Intel® Extreme Masters Certified PC.
Check out event highlights and the Intel® Experience Zone powered by the Intel® Core™ i7 processor in the video.
Catch up on the final results and highlights of the biggest eSports event in Australia history ›
IEM Certified PC is assembled from components made by the same companies that supply equipment for official IEM tournaments. Inside you will find a 7th Generation Intel® Core™ Processor, Intel® Solid State Drive and HyperX* memory.
Each PC and Laptop comes with a holographic sticker that ensures its uniqueness.
By purchasing this set you are supporting eSports by donating prize money to the Intel Extreme Masters.
Corruptor Gaming System
PCCG Master 1080 Gaming System
PLE Destroyer Custom Gaming PC
Resistance VR Fury - GTX 1070
By using the #IEMSYDNEY hashtag, you grant us a license to include on intel.com ("Our Pages") your social media display name and photo with your post using #IEMSYDNEY. To remove your post from Our Pages, send an email to firstname.lastname@example.com. You represent and warrant that you have all necessary permissions (including copyright and right of publicity) to grant us license to repost or reblog your post. To learn more about Intel's privacy practices, please visit http://www.intel.com/privacy.
Intel, the Intel logo, the Intel Inside logo and Intel Core are trademarks of Intel Corporation or its subsidiaries in the U.S. and/or other countries.
Other names and brands may be claimed as the property of others.


text:
Skyjacker CR26S Softride Leaf Spring
Skyjacker CR26S Softride Leaf Spring Lift Height 6 in. - w/56 in. Rear Spring - Single
See Full List of Vehicle Applications »
Skyjacker - Softride Leaf Spring P/N CR26S Features:
- Constructed As Progressive Multileaf Packs
- Load Capacity Will Be Equal or Greater Than OE
- Made With American Steel
- Limited Lifetime Warranty
View the Full List of Automotive Applications »
Below is an abbreviated list of applications for the Softride Leaf Spring.
K10 Suburban, K15 Suburban, K15/K1500 Suburban, K1500 Suburban, K20 Pickup, K20 Suburban, K25 Suburban, K25/K2500 Suburban, K2500 Suburban, K30 Pickup, K35 Pickup, K35/K3500 Pickup, K3500 Pickup, V10 Suburban, V1500 Suburban, V20 Pickup, V20 Suburban, V2500 Pickup, V2500 Suburban, V30 Pickup
- 803696121499
- Skyjacker
- New
Shipping Information
- Item Requires Shipping
- 77.0 lbs.
- W14.0000" x H2.5000" x L54.0000"
- Free Shipping!
- Free Shipping!!
Customer Reviews
Total Reviews (0)
Write the First Review!
You must login to post a review.


text:
A simple utility intended for generation of C-Verilog synchronization components of a test system:
- TestBench (a Verilog wrapper around the target module that launches a test scenario and generates a periodical clock pulse);
- VPI-Mediator (a C API for accessing the target module's inputs and outputs);
- C-Interface (C data structures modelling the inputs and outputs as well as functions for synchronizing those structures with the real signals);
- VPI-SysTF (VPI functions being called from TestBench: scenario launcher and clock pulse handler).
The tool is implemented as a back-end for the open-source Verilog simulator Icarus Verilog.
Manager: Alexander Kamkin
Developer: Mikhail Chupilko, Sergey Smolov
Reporter: Николай Пакулин


text:
Profile Stats
- Total Profile Views:
- 233
- Profile Views Last 7 Days:
- 0
- Member Since
- April 2011
Movie Ratings and Reviews
This is one of my favorite films of all time. I had only seen it for the first time a couple of years ago and I had heard so many great things about it. It was much better than I had expected. It was funny, it was clever, and there should be absolutely no reason to hate this film whatsoever. I love Zemeckis's films and I have always been a fan of Christopher Lloyd. And, of course, Michael J. Fox as Marty McFly was definitely the right choice for this film. "Roads? Where we're going, we don't need roads."


text:
Using fans can save your money and add to your comfort and healthiness!
If you have air conditioning, using a fan can help save your money. A single-room fan can cost 0.7 cents per hour, while whole-home AC will cost $1 – 2 per hour, depending on the age & efficiency of the unit. By using a ceiling fan, you can raise the thermostat about 4° with no reduction in comfort!
How Do Fans Work?
Fans create a wind-chill effect that cools your body. Bodies cool down through 1) convection – when heat is carried away via moving air, 2) radiation – when heat radiates from other objects or from you to cooler objects, and 3) perspiration – the body's powerful cooling mechanism, where departing moisture takes heat away with it. Since heat rises, ceiling fans can pull heat up and away. Ceiling fans are also able to switch direction in winter, pushing heat back down.
Increasing the Effectiveness of Your Fan
A ceiling fan works best about 7-9 feet above the floor and no less than 8" below the ceiling. Opening windows upstairs to pull heat from down below, and, of course, cross-ventilation (especially on longer paths directed by fans) are most effective. And, in San Diego, many of us are lucky enough to benefit from the off-shore breezes at night, so a fan is adequate to provide a comfortable sleeping climate.
Benefits of Different Types of Fans
Larger fans can do more than smaller fans, and larger blades can have the same effect at a slower speed. Exhaust fans in the kitchen and bath can remove heat and humidity, as well as indoor air pollutants, which accumulate when homes or buildings are closed up. Another option is a whole-house fan. This system pulls air from open windows and exhausts it through attic and roof, also ventilating the attic! They provide 30-60 air changes per hour and are sized in cubic feet per minute (CFM). Installation, including circuit wiring and vent installation, should be done by professionals. And always remember to check noise ratings when purchasing a fan.
Beat the Heat!
Solar energy is a major source of unwanted heat in most climates – AND appliances, lights, and occupants only add to the temperature! In addition to fans, some preventative ideas include additional attic insulation, reflective roofing, sun-blocking window treatments, and reducing the use of hot water and appliances. And finally, wear cool clothing, such as cotton instead of polyester, in order to stay comfortable.
Become a Fan!
Check back often for new and exciting information: http://hubbellandhubbell.com/blog/


text:
John Barry And ECO - Eternal Echoes (Music CD)
Release Date: 22 October 2001
Artist: John Barry And ECO
£7.99 includes FREE UK Postage (other delivery options available)
Shipping Destinations for this item
UK - Shipping Included
United Kingdom
Europe Zone 1 - £1.29
Austria, Belgium, Denmark, France, Germany, Ireland, Luxembourg, Netherlands, Switzerland
Europe Zone 2 - £1.49
Andorra, Croatia, Czech Republic, Finland, Liechtenstein, Monaco, Norway, Poland, Sweden
Europe Zone 3 - £1.69
Bulgaria, Cyprus, Estonia, Faroe Islands, Gibraltar, Greece, Greenland, Hungary, Iceland, Italy, Latvia, Lithuania, Malta, Montenegro, Portugal, Romania, San Marino, Serbia, Slovakia, Slovenia, Spain, Vatican City
North America - £1.69
Canada, USA
Australia and Far East Asia - £1.69
Australia, Israel, New Zealand, Taiwan
Japan - £1.69
Others - £1.79
Falkland Islands
Sorry, this product is currently unavailable.
Track List
Eternal Echoes
Returning Home
Crazy Dog
Slow Day
Fred & Cyd
Blessed Illusion
Get Over It
First Steps
Release Date: 22 October 2001
Artist: John Barry And ECO
For more information and our Delivery Policy see here.
For more information and our Returns Policy see here.
Catalogue No: 4667652