été Out NowThe Complete Phonogram, The Wicked + the Divine 28, Doctor Aphra 6 and Mercury Heat 12 By gillen.cream.org Published On :: Wed, 12 Apr 2017 16:56:16 +0000 Cripes. What a big week. For more details, I direct you to my newsletter which is best for this kind of thing, but in Brief… The Complete Phonogram. 504 pages comprising all three volumes (the first coloured for the first time), all the eighty-odd previously uncollected B-sides and all in an oversized hardback format. If […] Full Article Comics
été I've Written And Deleted So Many Posts By www.eschatonblog.com Published On :: Sun, 10 Nov 2024 14:29:00 +0000 Might be best to shut up for a bit. Full Article
été F&S weten niet of ze volgend jaar weer meedoen (NRC, do, 15-08-24) By www.foksuk.nl Published On :: Thu, 15 Aug 2024 00:00:00 +0200 Full Article
été Lisa Simpson Replaces Bart in Detention in ‘The Simpsons’ Latest Episode By www.cracked.com Published On :: Mon, 11 Nov 2024 13:10:00 -0800 By JM McNab Published: November 11th, 2024 Full Article
été Graphic novels, RPGs and Advice For Young Writers: Interview with Brian McLachlan (COMPLETE THE QUEST: THE POISONOUS LIBRARY) By www.inkygirl.com Published On :: Mon, 12 Jul 2021 18:11:29 +0000 Brian McLachlan is a cartoonist who's worked for Nickelodeon, The Nib and the New Yorker. He writes the monthly comic Spruce Street Squad for Owl Magazine. His book, Draw Out The Story: 10 Secrets to Creating Your Own Comics is an ILA-nonfiction award winner. Each Tuesday he hosts a group of artists in a PG-13 game of Dungeons & Dragons where they live draw what happens in game on Twitch, called Magical Marker ( MM ). You can find Brian at his website, on Twitter, and Instagram. Synopsis of COMPLETE THE QUEST: THE POISONOUS LIBRARY (Imprint/Macmillan): Save your kingdom in this interactive role-playing adventure, which reads like a graphic novel but also plays like a game! It’s perfect for fans of all ages who are interested in the exciting world of fantasy gaming. Q. How did you come up with the idea for Complete The Quest? Complete the Quest in a brand new mix of role playing game (RPG) and comic. It started as an experiment to see what would happen if I took a Dungeons & Dragons or Chose Your Own Adventure multi-path story and made it into a comic. How would the gameplay change by putting it into a visual space? For me, it was a literal game changer, turning the CYOA anxiety of multiple choices, into a laid-out, laid-back invitation to check all paths for your favourite way forward. It also encourages people to come up with their own, better solution if they want. While I always give the reader a clear path forward, I invite them to forge their own. It's a great introduction to co-operative storytelling. Q. What was your writing/illustration process? I draw a scene much faster than I can write about it. A picture's worth a thousand words and all that. So I draw my scripts in rough, laying out out the word balloons, figuring out how much space I need, how the page turns feel, etc. This was especially important for this comic gamebook which also filled with maps for the reader to navigate. We needed to see if the pathing was clear. Creating the tutorial was the most time intensive part, trying to make the most elegant, clear way to ease the reader into how the game part functions. I think we nailed it. I hear from parents who don't play RPGs that they were worried about the reading a gamebook, but they quickly "got it". Q. What advice do you have for young writers and illustrators? Advice is hard to give because everyone's different. Some people need to hear "finish what you start" and some need to hear "stop working on that thing, it's good enough". So my advice is to train your inner advice-giver and follow it. What is it about the way you work that you've been putting off improving, but could do right now? Because you know what type of story or art you're making, and if you're trying to make an apple, the best advice in making a banana isn't going to help. And the best advice in making an apple right-handed isn't going to help you if you're left-handed. Not all advice is for you, it's a skill to figure out what part is for you to take right now, later, or never. ---- Also see my other Inkygirl interviews with book creators and advice for young writers and illustrators from book creators. Full Article
été This Must-Have IKEA Item Completes Your Kid’s Harry Potter Costume By ikeahackers.net Published On :: Wed, 23 Oct 2024 10:00:00 +0000 I couldn’t believe my eyes as I wandered through IKEA’s soft toy section. There, perched among the bins was Hedwig from Harry Potter. It was then I realized I was staring at IKEA’s new SKOGSDUVA snowy owl hand puppet. The resemblance to Harry’s faithful feathered companion was uncanny. While it might not deliver Hogwarts acceptance letters, it certainly brought a touch of magic to my shopping trip. With its fluffy white feathers and expressive eyes, this soft, cuddly version of […] The post This Must-Have IKEA Item Completes Your Kid’s Harry Potter Costume appeared first on IKEA Hackers. Full Article Products Featured Latest on IG Skogsduva
été Bunday: Here Comes Peter Rabbit By cheezburger.com Published On :: Mon, 08 Oct 2012 13:00:00 -0700 This widdle rabbit is obviously a big fan of Beatrix Potter's books about Peter Rabbit. -Sally Squeeps Find more squeedorable moving pictures over at Animal Gifs! Full Article bunny gifs hop mouth rabbit toy categoryimage
été Sleek school building is made with low-carbon concrete By inhabitat.com Published On :: Fri, 23 Jun 2023 19:30:00 +0000 The ways buildings are constructed has to change. It has become something else instead, something more sustainable, something newer. And it doesn't get much more modern than buildings that can actually move and adapt at will.[...] Full Article Green Building toronto school post-format-gallery Schools
été Leonid meteor shower 2024: How to spot 'shooting stars' and 'fireballs' over the US this week By www.livescience.com Published On :: Tue, 12 Nov 2024 17:23:09 +0000 The Leonid meteor shower peaks in North America overnight from Nov. 17 to 18, with fast-moving fireballs possible in US skies. Full Article Space
été VEILDrive Attack Exploits Microsoft Services to Evade Detection and Distribute Malware By thehackernews.com Published On :: Wed, 06 Nov 2024 23:22:00 +0530 An ongoing threat campaign dubbed VEILDrive has been observed taking advantage of legitimate services from Microsoft, including Teams, SharePoint, Quick Assist, and OneDrive, as part of its modus operandi. "Leveraging Microsoft SaaS services — including Teams, SharePoint, Quick Assist, and OneDrive — the attacker exploited the trusted infrastructures of previously compromised organizations to Full Article
été The Giant Fecal Art Object Appeared in St. Petersburg By englishrussia.com Published On :: Mon, 17 Jan 2022 14:53:47 +0000 The post The Giant Fecal Art Object Appeared in St. Petersburg appeared first on English Russia. Full Article Funny Photos crazy
été A Tiny Place for Rent in Saint Petersburg By englishrussia.com Published On :: Tue, 08 Feb 2022 08:27:46 +0000 The post A Tiny Place for Rent in Saint Petersburg appeared first on English Russia. Full Article Funny Photos saint-petersburg
été Rare Natural Phenomenon Observed in Saint Petersburg By englishrussia.com Published On :: Sat, 02 Apr 2022 05:47:05 +0000 The post Rare Natural Phenomenon Observed in Saint Petersburg appeared first on English Russia. Full Article Photos Russian Nature saint-petersburg
été Unusual Residents of Saint-Petersburg By englishrussia.com Published On :: Thu, 14 Apr 2022 11:02:02 +0000 The post Unusual Residents of Saint-Petersburg appeared first on English Russia. Full Article Photos funny saing-petersburg
été BBC Radio 2 In Concert complete show By craigjparker.blogspot.com Published On :: Tue, 05 Nov 2024 04:54:00 +0000 Full Article
été Veterans Day observance to be held at the Princeton University Chapel By www.princeton.edu Published On :: Fri, 08 Nov 2024 10:59:36 -0500 The 9 a.m. service on Monday, Nov. 11, will also be livestreamed. Full Article
été Alban Forcione, ‘unequalled interpreter of Don Quixote’ and ‘infinitely generous mentor,’ dies at 82 By www.princeton.edu Published On :: Mon, 18 Oct 2021 14:53:00 -0400 Alban Forcione, an internationally renowned scholar of 17th-century literature of “Golden Age” Spain, the Walter S. Carpenter Jr., Professor of Language, Literature and Civilization of Spain, Emeritus, and Princeton alumnus, died Sept. 14 at age 82. Full Article
été A Conversation on Middle East Regional Security with Peter Berkowitz By www.princeton.edu Published On :: Wed, 13 Nov 2024 16:45:00 -0500 Peter Berkowitz, Director of Policy Planning at the Department of State from 2019 to 2021 will discuss Middle East regional security. Full Article
été A Marine Corps veteran expresses concerns for the military in a 2nd Trump presidency By www.npr.org Published On :: Mon, 11 Nov 2024 12:07:44 -0500 Essayist Phil Klay says Trump tried to use the military to push his partisan agenda before, and may further erode norms around the military as he looks for those willing to "go with his whims." Full Article
été A 'dama de gelo': quem é Susan Wiles, estrategista de Trump que será 1ª mulher chefe de gabinete de um presidente dos EUA By www.bbc.com Published On :: Sat, 09 Nov 2024 14:15:35 GMT "No início da minha carreira, coisas como boas maneiras importavam e havia um nível esperado de decoro", disse Susan ao site Politico, descrevendo o partido Republicano como significativamente diferente do de várias décadas atrás. Full Article
été Air Force veteran Anna Luna runs Charlie Crist By shark-tank.com Published On :: Tue, 18 Feb 2020 05:08:13 +0000 Air Force veteran and former Turning Point USA spokesperson Anna Paulina Luna has just announced she is running for the U.S. Congress against Democratic Congressman Charlie Crist in Florida’s 13th congressional district. The post Air Force veteran Anna Luna runs Charlie Crist appeared first on Shark Tank. Full Article Featured immigration islam
été Gov. Ivey’s treatment of Bama veterans is a scandal By quinhillyer.com Published On :: Tue, 12 Nov 2024 02:55:07 +0000 (Nov. 9, to kick off Veterans Day weekend) MOBILE, Alabama — On this Veterans Day, Alabama is roiled by a doozy of a veterans-related controversy. Republican Gov. Kay Ivey last month invoked […] The post Gov. Ivey’s treatment of Bama veterans is a scandal appeared first on Quin Hillyer. Full Article Email Featured Gulf Coast Politics National Politics
été Mega meteorite tore up seabed and boiled Earth's oceans By www.bbc.com Published On :: Mon, 21 Oct 2024 23:02:27 GMT It was 200 times bigger than the one that wiped out the dinosaurs nearly three billion years later. Full Article
été Skies to clear for Taurid meteor shower peak on Monday By www.bbc.com Published On :: Mon, 11 Nov 2024 12:05:38 GMT The Northern Taurids are at their peak on 11-12 November - but will the clouds part to let us see them? Full Article
été Why luxury cheese is being targeted by black market criminals By www.bbc.com Published On :: Sun, 10 Nov 2024 08:58:48 GMT How cheese has become so valuable that it's being stolen to order Full Article
été Moscow targeted as Ukraine and Russia trade huge drone attacks By www.bbc.com Published On :: Mon, 11 Nov 2024 05:14:06 GMT Both countries say they shot down dozens of drones over their respective territories. Full Article
été Biden’s Nitwit Dept. of Defense Uses Photo of Chinese Jet in Veterans Day Message By lidblog.com Published On :: Wed, 13 Nov 2024 02:27:52 +0000 Joe Biden’s stupid, corrupt regime can’t even get a simple Veterans Day message right. As the nation came together to celebrate those who served out country, Biden’s Department of Defense pushed out a social media message telling Americans “Veterans Day, honoring all who served.” While, that is a perfectly fine message, the DoD added a […] The post Biden’s Nitwit Dept. of Defense Uses Photo of Chinese Jet in Veterans Day Message appeared first on The Lid. Full Article China Military
été Veterinarians share 10 things you should never do when taking your dog on a walk By www.businessinsider.com Published On :: Tue, 12 Nov 2024 18:10:23 +0000 Some things you should never do on a dog walk include approaching unfamiliar pets, using an extendable leash, and not picking up waste, experts said. Full Article Health freelancer evergreen-story lifestyle pets veterinarian pet-owners vets dogs freelancer-le
été Top Ducati executive explains how the Army helped him succeed and shares 2 traits that make veterans great hires By www.businessinsider.com Published On :: Tue, 12 Nov 2024 20:04:26 +0000 Ducati North America CEO Jason Chinnock enlisted in the US Army out of high school and served with the Third Armored Division in Desert Storm. Full Article Transportation Careers ducati veterans
été 10 Ways to Build Customer Loyalty and Increase Retention By blog.avangate.com Published On :: Mon, 04 Feb 2019 00:00:00 +0000 Selling your services and wares online can be challenging, as today’s digital shoppers are smart and demanding. You need a customer-focused approach right from the start. The customer is the most significant pillar of modern businesses, so you need to build a genuine relationship with them if you want your online store to survive. Read […] Full Article
été futuretext.ai newsletter By www.opengardensblog.futuretext.com Published On :: Sat, 14 Apr 2018 11:07:24 +0000 Full Article BIG DATA FEATURED POSTS LATEST POSTS
été Amazon readies new AI chip to compete with Nvidia By readwrite.com Published On :: Tue, 12 Nov 2024 12:56:28 +0000 Nvidia might have some serious competition in the AI chip space coming. Amazon is now seeking to bring its own… Continue reading Amazon readies new AI chip to compete with Nvidia The post Amazon readies new AI chip to compete with Nvidia appeared first on ReadWrite. Full Article Big Tech Technology Amazon ChatGPT Nvidia
été INTERNal Stupidity: Pentagon Interns Post Pic of Chinese Plane on Veteran’s Day By conservativefiringline.com Published On :: Tue, 12 Nov 2024 23:15:42 +0000 The following article, INTERNal Stupidity: Pentagon Interns Post Pic of Chinese Plane on Veteran’s Day, was first published on Conservative Firing Line. A knock-off Chinese plane similar to the US F-35 was inadvertently posted by the F-35 JPO (Joint Program Office) on Veteran’s Day. Somebody needs to help this administration figure out some basics …oh wait, a new administration is coming in January. Good. The JPO Strike Force account had no clue what they did until an … Continue reading INTERNal Stupidity: Pentagon Interns Post Pic of Chinese Plane on Veteran’s Day ... Full Article Opinion Chinese plane F-35 Fighter jet Joint Program Office Joint Strike Force
été TRUMP TRIUMPHS: Victory Speech As He Completes The YUGE Comeback By clashdaily.com Published On :: Wed, 06 Nov 2024 13:12:14 +0000 Trump asked the question: are you better now than when I was in office? America's answer could not have been more clear. The post TRUMP TRIUMPHS: Victory Speech As He Completes The YUGE Comeback appeared first on Clash Daily. Full Article Videos
été VETERAN’S DAY: You’re Gonna Love What One Patriotic Coffee Company Did For The Marines! By clashdaily.com Published On :: Mon, 11 Nov 2024 10:30:41 +0000 Did you know that the Marines just celebrated their 249th birthday? This is one helluva tribute. The post VETERAN’S DAY: You’re Gonna Love What One Patriotic Coffee Company Did For The Marines! appeared first on Clash Daily. Full Article Videos
été A.F. Branco Cartoon- – The Puppeteer By comicallyincorrect.com Published On :: Mon, 21 Oct 2024 12:00:06 +0000 A.F. Branco Cartoon – Free Market Capitalism is great, but when big corporations merge with the Government, it begins to.. Full Article Political Cartoons Democrats Rinos
été Footage of Jill Biden and Kamala Harris Sitting Side-by-Side at Veterans Day Event Goes Viral By www.westernjournal.com Published On :: Mon, 11 Nov 2024 23:03:57 +0000 There appeared to be a definite frost between first lady Jill Biden and Vice President Kamala Harris during a Veterans Day event at Arlington National Cemetery on Monday. A close-up […] The post Footage of Jill Biden and Kamala Harris Sitting Side-by-Side at Veterans Day Event Goes Viral appeared first on The Western Journal. Full Article Commentary 2024 election Jill Biden Joe Biden Kamala Harris Veterans Day
été CNN Host Reportedly Leaving Network After Short-Lived Stay: His Career Change Is Completely Absurd By www.westernjournal.com Published On :: Tue, 12 Nov 2024 14:42:13 +0000 Three years ago, Chris Wallace walked away from Fox News over concerns that those at the “Fair and Balanced” network were beginning to, as he put it, “question the truth.” […] The post CNN Host Reportedly Leaving Network After Short-Lived Stay: His Career Change Is Completely Absurd appeared first on The Western Journal. Full Article Commentary Chris Wallace CNN Establishment media Fox News Social media
été Newsroom: TikTok surpassed YouTube as the third-most-popular influencer platform among US marketers in 2021 By www.emarketer.com Published On :: Mon, 24 Jan 2022 04:01:00 GMT TikTok will have more US users than Snapchat and Pinterest in 2022   January 24, 2022 (New York, NY) – YouTube is the “OG†of influencer marketing platforms, and it’s […] Full Article
été Belt and Road Is Not a (Completely) Closed Shop By www.chathamhouse.org Published On :: Thu, 22 Mar 2018 09:47:39 +0000 Belt and Road Is Not a (Completely) Closed Shop Expert comment sysadmin 22 March 2018 Though the infrastructure initiative will continue to be led in many places by Chinese investment, where there are benefits of scale, effectiveness and risk management, outside participation will be welcome. — Chinese Foreign Minister Wang Yi at the Belt and Road Forum in Yanqi Lake in May 2017. Photo: Getty Images. First announced in 2013, President Xi Jinping’s Belt and Road Initiative promises, at a minimum, to improve infrastructure and connectivity between China and the rest of Eurasia. Any bold plan to finance infrastructure on a large-scale across so many low-income economies deserves a sympathetic ear and a positive reception. But many wonder how large the role can be for non-Chinese players in what is clearly an initiative of the Chinese government. So far, Chinese state and policy banks account for the overwhelming majority of the financing – and this money then flows to Chinese enterprises, mainly state-owned. One study found that 89% of the work went to Chinese contractors on China-funded projects. Yet, recently in Paris, Chinese Foreign Minister Wang Yi said the Belt and Road was a ‘sunshine initiative’ that was open and for all to benefit from. He declared that there were to be ‘no backroom deals. There is openness and transparency.’ The Belt and Road Initiative will ‘respect international rules’ and ‘will run according to market rules’.There are some notable instances of Belt and Road projects changing to conform more to international rules as they develop. For example, work on the Belgrade–Budapest high speed rail link originally ran into problems. Contracts were allegedly awarded to Chinese companies without following the EU-mandated competitive procurement processes. Now, the most recent round of contracts is to be awarded by tender.Such a switch perhaps does not herald the unrestricted ‘market rules’ that Wang speaks of, but it does highlight that China is willing to turn Belt and Road towards openness in certain instances. These will be cases where it clearly brings benefits for China – but also where private sector players can be convinced of good commercial returns. These benefits are most likely to lie in three areas: scale and access, effectiveness and risk management.Mutual benefitsFirstly, scale and access. Belt and Road can achieve greater scale if additional financing comes in alongside the current Chinese state and policy bank lending. This can come both by working together with multilateral institutions and with private sector institutions. This will in turn require defining projects or structuring financing with attractive risk-return trade-offs. In some cases, this will be feasible – in others not. Getting bigger also necessitates initiating meaningful Belt and Road activity in more countries. As the Belgrade–Budapest rail experience has shown, approaches vary in terms of competitive tendering requirements and consideration of non-Chinese bidders. An approach that works in, say, Tajikistan, may not be effective in Poland. This is a pragmatic recognition of context that is quite separate from debates on what the ‘right’ approach to these standards should be. Both these elements suggest that a more open Belt and Road will be a larger and more far-reaching one.Secondly, effectiveness. Despite extensive experience building infrastructure within China, many Chinese companies are much less familiar operating outside of China. Western, Japanese and Korean companies can bring their own experience to the table and help the success of these projects. By doing so, they also put competitive pressure on Chinese companies to improve and upgrade, while providing opportunities to learn. This in turn creates a stronger, more productive Chinese economy.Beyond physical hardware, the ‘software’ is also critical to success. Again foreign companies have much to contribute. Areas such as maintenance, training, legal and accounting services are all in demand. Indeed, many such British and other companies are already actively engaged in working on projects with Chinese companies to help in these areas.Finally, risk management. Beyond the usual project management risk of large projects, Belt and Road brings additional challenges. Many countries have weak governance, internal divisions and security issues, all within distinctively different cultures and traditions. Chinese companies with little experience of local conditions will struggle. Going it alone may provide what is an illusion of control, but exposure to social and political dynamics can rebound on China in unexpected ways.Cooperating with others who have a history of in-country experience is a way to manage these risks. Examples would include British and Chinese cooperation both at company and governmental level in engaging in particular African countries, in partnership with the relevant country government.Mixed systemMechanisms that encourage competitive choices and restrain corruption are positive, but mechanisms that slow decision-making to a crawl also prevent countries from getting benefits of infrastructure projects. Conversely, continuing along the current path of Chinese-led investment does have some clear attractions in certain settings, at least to those directly involved. It combines the ability of Chinese policy banks to provide large-scale funding in even high-risk environments with the relevant experience and production capacity of Chinese state-owned businesses. It allows for government-to-government deals, pragmatic negotiations and all-encompassing accords, at times out of the public eye. In many cases, it is hard to make a commercial case for the investments.But in cases where there is mutual benefit, engagement and will, there will be a role for international partnerships. Full Article
été Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay Development Using a Fit-for-Purpose Approach By www.mcponline.org Published On :: 2014-03-01 Steven A. CarrMar 1, 2014; 13:907-917Technological Innovation and Resources Full Article
été Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution By www.mcponline.org Published On :: 2007-12-01 Hasmik KeshishianDec 1, 2007; 6:2212-2229Research Full Article
été Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics By www.mcponline.org Published On :: 2012-11-01 Amelia C. PetersonNov 1, 2012; 11:1475-1488Technological Innovation and Resources Full Article
été Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis By www.mcponline.org Published On :: 2012-06-01 Ludovic C. GilletJun 1, 2012; 11:O111.016717-O111.016717Research Full Article
été Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap By www.mcponline.org Published On :: 2005-12-01 Jesper V. OlsenDec 1, 2005; 4:2010-2021Technology Full Article
été Safeguarding Europe: how to defeat and deter Russia By www.chathamhouse.org Published On :: Wed, 23 Oct 2024 14:27:13 +0000 Safeguarding Europe: how to defeat and deter Russia 10 December 2024 — 10:00AM TO 7:30PM Anonymous (not verified) 23 October 2024 Chatham House Threat assessment and practical solutions. Nearly three years into Russia’s full-scale war against Ukraine, the security picture for Europe remains uncertain. Questions of European states’ own resilience and ability to counter Russia and support Ukraine have not been adequately answered by key European powers. So how can European states act proactively to support not only Ukraine but also their own security?Key questions:How resilient is Europe to the challenge posed by Russia?What more can be done to defend EU candidate states?What will be the impact of the US election results?What deters Russia? What have we learnt from Russia’s war on Ukraine?Are the current approaches sufficient?This one-day, in-person conference at Chatham House will bring together experts and policymakers from across the UK, EU and US, as well as drawing from the expertise of Russian analysts in exile. Alongside analysis of Russia’s intentions towards Europe and European strategy, there will be opportunity for in-depth conversation with experts on the geopolitical and defence tools available to European states. Safeguarding Europe agenda (PDF, 0.16MB) This conference will be hosted in-person, with the recording of the keynote address made available on the website following the event.Participants will be selected based on expertise. Please wait for confirmation before attending.Corporate organisations who currently do not support the Russia-Eurasia Programme or Ukraine Forum will be subject to a fee. Your registration will be reviewed by a member of our team before it is approved.The institute occupies a position of respect and trust, and is committed to fostering inclusive dialogue at all events. Event attendees are expected to uphold this by adhering to our code of conduct. Full Article
été Legal Determinants of Health By f1.media.brightcove.com Published On :: Mon, 28 Oct 2019 00:00:00 +0000 Full Article
été A simplified method for the preparation of detergent-free lipid rafts By www.jlr.org Published On :: 2005-05-01 Jennifer L. MacdonaldMay 1, 2005; 46:1061-1067Methods Full Article
été Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells By www.jlr.org Published On :: 1978-11-01 W GambleNov 1, 1978; 19:1068-1070Articles Full Article
été Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations [Molecular Biophysics] By www.jbc.org Published On :: 2020-12-18T00:06:18-08:00 Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization. Full Article