text:
After winning re-election in [:1916] with the slogan "He Kept Us Out Of War," Woodrow Wilson quickly found in early 1917 that the drift toward war was becoming more difficult to resist. However, he was at pains to make clear that the United States would only enter the war with its own aims, which would be different from those of the European powers.On January 22, 1917, Wilson addressed the U.S. Senate on the subject:
The present war must first be ended, but we owe it to candor and to a just regard for the opinion of mankind to say that, so far as our participation in guarantees of future peace is concerned, it makes a great deal of difference in what way and upon what terms it is ended. The treaties and agreements which bring it to an end must embody terms which will create a peace that is worth guaranteeing and preserving a peace that will win the approval of mankind, not merely a peace that will serve the several interest and immediate aims of the nations engaged.
While the allies were anxious to have American support, they viewed Wilson`s premises as completely naive and had no intention of following his precepts when they negotiated peace with the Germans after the armistice of November 1918.Representatives of the German government were summoned to Paris and on May 7, 1919, presented with the fruits of the peace negotiations. After examining the more than 200-page document, the Germans were outraged. They believed that they had been lured into an armistice with the promise that the Fourteen Points would serve as the backbone of the peace treaty. What they found instead bore little resemblance to Wilson's even-handed proposals. Thus, the stage was set for two decades of German poverty, hunger, privation and World War II.Peace with Germany, like most complicated issues, required compromise. Despite German anger, the result of the negotiations was much more moderate than the harsh terms of Brest-Litovsk, but still far from the spirit of the Fourteen Points.The treaty contained more than 400 articles, but the major issues can be summarized by the following:
Alsace and Lorraine were returned to France.
German colonies were assigned to victorious nations as "mandates" under the League of Nations.
The Saar Basin was assigned to France for 15 years, then a plebiscite was to be held to determine the area`s allegiance.
Poland was reestablished as an independent nation and granted access to the sea through a strip of land that came to be known as the Polish Corridor.
The amount of German reparations was to be determined by a Reparations Commission.
Germany was forced to accept responsibility for all losses and damages in the conflict in what was termed the "war guilt clause" (Article 231).
Germany was required to disarm, specifically:
The drafting of military personnel as prohibited.
The Rhineland was demilitarized.
The German army was limited in size to 100,000 men.
The German navy and air force were severely reduced.
The German general staff was abolished.
Restrictions were placed on the manufacture and importation of war matériel.
The Covenant of the League of Nations was included as part of the treaty.
Wilson's victories included the creation of a modern Poland, the pledge of support for disarmament, the establishment of colonial trusts and, of course, the creation of the League of Nations. However, in order to obtain these provisions, he acquiesced to the demands of the Allies on reparations, stripping Germany of its colonies and the near total destruction of the German military — all of which contributed to an undercurrent of anger in the defeated nation.Opposition to the treaty came from many different sources. Besides the isolationists who felt the treaty forced to United States to do too much, another view was that it did little to prevent future global conflicts. This point, made in an editorial that appeared in the New Republic on May 24, 1919, expressed this view:
Yet if they connive at this Treaty they will, as liberal and humane American democrats who seek by social experiment and education to render their country more worthy of its still unredeemed national promise, be delivering themselves into the hands of their enemies, the reactionaries and the revolutionists. The future of liberal Americanism depends upon a moral union between democracy and nationalism. Such a union is compromised so long as nationalism remains competitive in policy, exclusive in spirit, and complacently capitalist in organization.
Wilson was well aware of the objectionable features of the treaty, but believed that they could be overcome in the future by actions of the League of Nations.The formal signing of the treaty took place on June 28.
NOTE: The Treaty of Versailles was designed to establish the terms of peace between the Allied Powers and Germany; it was one part of what is generally regarded as the Peace of Paris, which also includes separate treaties with Hungary, Turkey and Austria.
See also Wilson`s Search for Peace and map depicting German territorial losses.
The 5 best beaches in Cancun
How have the valuations of premium examples of legacy technologies behaved historically, around their time of becoming obsolete?
Has an individual ever purchased an entire country?
Invincible Class Aircraft Carrier
1,200 Students participate in the III edition of Arqueocerámica
The Noailles burin, the predecessor of the sewing needle
New York City as "the greatest city in the world" in 1776?
Tupolev Tu-2
Curtis P-6 Hawk - History
USS Long (DD-209) rolling in Alaskan waters
Copyright © ciwanekurd.net | Treaty of Versailles...


Just got pre-approved for a home loan? Congratulations! That's a huge step in life - and it takes you so much closer to living the American Dream!
When you find it, you know it. And then everything happens quite fast: Your agent might help you sign the paperwork, you put in an offer, and then you wait, chewing nails, to hear back whether it was accepted. "Hurry-up and Wait" is probably a good way to describe it.
How far is the nearest fire station?
Don't get bounced by a trampoline (or pool).
Part of a homeowner's association? Check your association's Master policy.
Located in a flood zone? You might need flood insurance.
Your offer was accepted and now the real paperwork begins. Who would have thought that would be so many documents to produce, papers to sign, and questions to answer? When your broker assures you that she can take care of the rest, it's easy to agree.
It's a couple of months after you're moved in that you have the peace of mind to take a closer look at the papers...and discover that your homeowner's insurance rates are high. Very high! To say the least.
Just like property taxes have become commonly rolled into an escrow account and made part of a mortgage payment, so have homeowner's insurance payments. Every lender requires that you have homeowner's insurance in place. But that doesn't mean that your mortgage company has to determine your insurance!
We can help you find the right protection at the best rate for your new home. All you have to do is get us involved early. Follow this short checklist, and with your permission, we can process everything so much faster when the closing date comes. Keep in mind that deadlines are always tight in the mortgage business.
You've always dreamed about a house in the woods. Kind of out there, away from all the hustle and bustle. And you found it, the perfect home, on a perfect 5-acre lot, not too far away...but far enough.
However, as you receive your homeowners insurance statement, you swallow hard. The house isn't that big...actually, it's about the same square footage as your previous house in the suburbs. So why did the rate increase?
The distance of the nearest fire station has an impact on your homeowner's insurance rates. The further away the nearest fire station is located, the higher the rate. This makes sense, in a way: If, heaven forbid, you ever had a fire, every second counts. With no fire department nearby, the risk for a major or total loss of your home is much higher. And insurance companies charge for higher risk.
Just a piece of information to be aware of as you search for your dream home. For detailed rate quotes and more information about potential insurance rates for a house you're looking at, just give us a call. We are here for you!
Now this house is really great! Not only does it come with a big yard and a swing set, but it also comes with a trampoline! Or maybe it's a pool! The kids would love it! Unfortunately, the backyard isn't fenced, but...well...a fence could be added later, plus the row of trees provides some privacy.
This doesn't sound bad at all, does it?
Unfortunately, from an insurance standpoint, it does. There are an estimated 3 million trampolines in US backyards, and injuries caused by trampolines are steadily on the rise, averaging over 240,000 medically treated injuries every year. Over 180,000 involve children aged 14 and younger. Severe spinal cord and head injuries abound.
That's why it is very difficult to find insurance for a trampoline. If the trampoline is located on your property and somebody - invited or not - gets injured while using it, you will be held responsible for the damages. That may mean many years of medical payments, or worse.
A trampoline is considered an "attractive nuisance". Most insurance carriers have recognized the dangerous risk exposure that comes with a trampoline. Some carriers will not write a homeowner's insurance policy for a home with a trampoline. Others may write it only if the trampoline is inside a 6-foot fenced area with a self-locking gate. Swimming pools are also considered "attractive nuisances" and are treated similarly.
Living in a homeowner's association can have its pros and its cons. But fact is that more and more condo, town-home, and other communities are bound together by a Homeowner's Association. For each homeowner, that usually means an annual fee.
What does the homeowner's association have to do with your insurance? Quite a bit, actually: From an insurance standpoint, if anybody got insured on the association's joint property, such as, for example, a walkway or a playground, the association would be held liable. That's why each homeowner's association has a Master policy in place. The question is: What does the Master policy cover?
Some Master policies don't provide high enough protection, so if a loss occurs that exceeds the policy limits, each homeowner in the association will share an equal part in the liability. In that case, you may be required to pay a share of hundreds or even thousands of dollars.
If you don't have the right protection on your homeowner's insurance, that money would need to come out of your pocket. Luckily, your homeowner's insurance can be adjusted to provide you with adequate protection.
We can help you with that (and we can also help in checking your association's Master policy). Give us a call 410-837-2320.
Isn't the sound of water one of the most soothing? Whether it's rainwater drumming on your window, waves crashing on the beach, or a stream babbling away...Not to mention the view! To look out the window and see these sights is priceless.
A house near a river or the beach is a dream for many. But if that dream home is located in a flood zone, your lender may require you to have flood insurance in addition to your standard homeowner's insurance policy.
Get a quote from us. We specialize in all your personal insurance needs and can help you with your flood insurance. Because sometimes, when it rains, it pours.
Kirby Insurance Agency Learn how to protect yourself and your family before it's too late. Read through our buying a home insurance tips. Get your free insurance quote, visit Kirby Insurance Agency to get all the information you need.


text:
<?php
namespace cff\V1\Rest\Banco;

use ZF\Apigility\Admin\Model\DoctrineAdapterResource;

class BancoCollection extends DoctrineAdapterResource
{
}

redpajama_set_name: RedPajamaGithub


Within the exam most of the questions beget been equal to killexams.com free pdf dump, which helped me to storenumerous time and i used to be capable to finish the complete seventy five questions. I moreover took the assist of the reference book. The killexams.com Questions for MB4-219 exam is constantly updated to provide the most accurate and up to date questions. This simply made me feel assured in passing the MB4-219 exam.
have a stare at experts exam bank and dumps to beget superb success.
these days i purchased your certification package deal and studied it thoroughly. ultimate week I handed the MB4-219 and obtained my certification. killexams.com exam simulator was a exotic device to prepare the exam. that superior my self assurance and i easily passed the certification exam! enormously endorsed!!! As I had only one week left for exam MB4-219, I frantically searched for some specific contents and stopped at killexams.com free pdf. It turned into shaped with short query-solutions that had been facile to understand. inside one week, I examine as many questions as viable. within the exam, it changed into smooth for me to control 83% making 50/60 reform solutions in due time. killexams.com become a terrific solution for me. thanks.
do not forget to solemnize these actual test questions for MB4-219 exam.
The short answers made my steerage more handy. I finished seventy five questions out off 80 nicely under the stipulated time and managed eighty%. My aspiration to be an authorized bewitch the exam MB4-219. I were given the killexams.com free pdf guide definitely 2 weeks before the exam. Thank you.
Little beget a stare at for MB4-219 exam, were given first rate success.
Analyzing for the MB4-219 exam has been a difficult going. With so many puzzling topics to cowl, killexams.com prompted the self notion for passing the exam via taking me through seat questions on the state of affairs. It paid off as I should skip the exam with a outstanding pass percent of 80 4%. A number of the questions came twisted, but the answers that matched from killexams.com helped me tag the birthright solutions.
I gave the MB4-219 exercise questions most effective as quickly as in promote than I enrolled for turning into a member of the killexams.com software. I did no longer beget achievement even after giving my adequate of time to my studies. I did no longer realize wherein i lacked in getting fulfillment. But after turning into a member of killexams.com i were given my acknowledge become missing grow to be MB4-219 prep books. It placed any of the matters in the birthright guidelines. Making geared up for MB4-219 with MB4-219 specimen questions is genuinely convincing. MB4-219 Prep Books of different classes that i had did succor me as they had been not enough succesful for clearing the MB4-219 questions. They beget been tough in veracity they did no longer cowl the complete syllabus of MB4-219. But killexams.com designed books are virtually top class.
easy way to pass MB4-219 exam with these free pdf and exam Simulator.
pleasant to hear that actual exam questions present day MB4-219 examination are to be had.
To promote to be a MB4-219 licensed, i was in thrust to pass the MB4-219 exam. I attempted and failed ultimate 2 tries. Accidently, I got the killexams.com material through my cousin. I was very inspired with the free pdf material. I secured 89%. I am so satisfied that I scored above the margin tag with out hassle. The material is rightly formatted as well as enriched with essential requirements. I assume its miles the wonderful desire for the exam.
Great stuff for MB4-219 exam which has really helped me pass. i beget been dreaming about the MB4-219 career for a while, but could never design time to study and actually obtain certified. As much as I was bored with books and guides, I could not design time and just study. These MB4-219 free pdf made exam preparation totally realistic. I even managed to study in my car while driving to work. The convenient format, and yes, the exam simulator is as Good as the website claims it is and the accurate MB4-219 questions beget helped me obtain my dream certification.
I was so much idle and didnt want to labor arduous and always searched short cuts and convenient methods. when i was doing an IT course MB4-219 and it was very tough for me and didnt able to find any guide line then i heard about the site which were very current in the market. I got it and my problems removed in few days when i started it. The sample and exercise questions helped me a lot in my prep of MB4-219 exams and i successfully secured Good marks as well. That was just because of the killexams.
No consume of time on searhching internet! organize exact source of MB4-219 free pdf.
VBA 6.0 opens the door for brand new and more potent line-of-enterprise solutions the exhaust of new points relish multithreaded VBA-based initiatives, developer productiveness add-ins, and back for digital signatures. To design it facile for ISVs to respect VBA 6.0, Microsoft is unveiling its up-to-date VBA net website – http://msdn.microsoft.com/vba/ – the region application providers can gain utility paraphernalia and documentation for without problems integrating VBA 6.0 into their products.
VBA 6.0 extends the achieve of VBA beyond its equitable electricity on the client to plenty of multitier software architectures, as well as to options that combine multiple VBA-enabled products.
assist for COM Add-ins. VBA 6.0 permits back for COM Add-ins, the brand new, consistent add-in architecture supported in Microsoft workplace 2000. The COM Add-in structure allows for developers to create a separate acknowledge that may dash inside multiple VBA-enabled functions.
VBA 6.0 comprises points that enable secure distribution of potent custom-made options and insurance plot against macro viruses. VBA 6.0 additionally gives greater highbrow property insurance policy for builders.
Digital signature assist. VBA 6.0 now gives succor for digital signatures, providing linger users coverage from macro viruses by way of reliably picking the supply of VBA code.
stronger highbrow property protection. VBA now helps 40-bit encryption on project passwords to greater give protection to a developer's intellectual property and to design unavoidable the safety of the answer.
VBA 6.0 allows ISVs to customise the construction atmosphere to optimize it for the needs of their purchasers, combining the merits of a well-recognized know-how with tools aimed toward unavoidable construction needs.
construction ambiance extensibility. The VBA 6.0 IDE can now be tailored and extended by ISVs to supply wizards and productiveness add-ins to assist builders write code and build customized dialogs.
aid for ISV design equipment. ISVs can create personalized, utility- and task-selected design tools that combine directly into the evolution ambiance.
ISVs achieve the VBA 6.0 utility construction paraphernalia (SDK) as the first step towards integrating VBA 6.0 into their applications. The VBA SDK comprises an assessment version of VBA. ISVs that are looking to encompass VBA with their packaged products ought to token a separate license agreement with Microsoft. vendors attracted to evaluating the VBA SDK freed from cost* can order it on-line at the new VBA web website ( http://msdn.microsoft.com/vba/ ).
A member of the visible basic family unit – the language used by over 3.2 million developers global – visible basic for applications is the evolution environment in Microsoft office and is available to 3rd parties through the VBA licensing program. There are at the minute over 80 shipping VBA-enabled purposes from ISVs in dozens of upright and horizontal markets.
centered in 1975, Microsoft (Nasdaq"MSFT") is the international leader in software for personal computer systems. The traffic presents a wide array of items and capabilities for traffic and personal use, each designed with the mission of constructing it less complicated and greater pleasing for americans to bewitch potential of the total power of non-public computing daily.
different product and company names herein may be logos of their respective house owners.
notice to editors: in case you beget an interest in viewing additional info on Microsoft, gratify visit the Microsoft web page at http://www.microsoft.com/presspass/ on Microsoft's corporate counsel pages.
businesses seeking to manage cellular gadgets are the usage of both Microsoft products to obtain the job finished.
corporations fervent to operate cell gadget administration, or deliver-your-personal-device administration, using Microsoft's solutions viable will exigency each device seat 2012 provider Pack 1 Configuration supervisor plus the January 2013 home windows Intune.
That message is emerging this week after Microsoft rolled out these two items, although Microsoft described it a bit bit back in December. On Tuesday, the January 2013 home windows Intune changed into released, together with system core 2012 service Pack 1, which includes Configuration manager (SCCM). windows Intune is a carrier delivered from Microsoft's datacenters that's designed to back IT groups manage PCs and cellular devices. SCCM is a premises-based mostly acknowledge that presents customer gadget and server administration capabilities alongside other profound capabilities for provisioning, upgrading and managing computing environments.
With the brand new product capabilities, it be no longer a windows Intune vs. SCCM 2012 world. fairly, with admire to cell gadget management, Microsoft is recommending using both equipment, exceptionally for greater companies. the brand new SCCM 2012 solution, in keeping with Microsoft's components, will serve as the "single pane of glass" for IT execs.
for instance, windows Intune is attainable under commercial enterprise Agreements (EA) or EA Subscription (EAS) plans, which promote with the rights to exhaust SCCM 2012 and Endpoint insurance plot for $6 per person per thirty days. an extra method, for businesses that already beget licensing for SCCM 2012 and Endpoint insurance plot in region, is to obtain home windows Intune as an add-on, which charges an extra $4 per consumer per month.
those two plans are for gigantic corporations with more than 250 users or instruments. groups with under 250 clients or contraptions can obtain windows Intune during the Microsoft online Subscription program. This "MOSP" alternative costs $eleven per consumer per thirty days, adds software Assurance for one paraphernalia however includes use-rights for four other devices, and additionally offers the organization the rights to design exhaust of SCCM and Endpoint protection, along with the newest windows traffic edition.
In any three instances, the licensing is now provided on a per-consumer groundwork, instead of on a per-device foundation. This per-consumer licensing shift, made by way of Microsoft in December, doubtless will be propitious for companies desirous to succor numerous-gadget scenarios, considering five managed instruments are lined beneath one user license.
"So what they beget performed is we've tried to design the licensing, No. 1, simpler and cheaper because it's per user in region of per machine, however then besides greater bendy in that you're really licensing or procuring what you exigency or need," defined Jason Leznek, director of product advertising of enterprise client management on windows Intune licensing, in a cellphone name on Wednesday.
system middle 2012 contains eight solutions, which will besides be purchased as a complete suite beneath traffic CAL licensing, however the product is additionally offered below Core CAL licensing by which SCCM 2012 is bundled with Endpoint insurance policy, Leznek explained.
"definite add-ons of gadget middle are focused very closely on the desktop or the person device management," he talked about. "in general, it truly is Configuration manager as well as Endpoint insurance plan. these are bought one after the other as a plenary complete product by itself for machine administration, for endpoint administration. it be a allotment of the Core CAL."
Unified device ManagementThe blend of windows Intune with SCCM 2012 is a component of Microsoft's "unified machine administration" idea, wherein SCCM will serve to give a separate view for each cloud and on-premise activities, in line with Leznek.
"There are two main worlds. there's the in-the-cloud and there's the on-premise," he noted. "The on-premise offering is customarily your home windows computing device (desktop or computer), Macs, and many others. which are corporate owned and corporate related, and that i'm going to execute deeper management there as a result of, from an IT point of view, I own the notebook…and that is the understanding what Configuration manager has any the time accomplished. windows Intune is completely within the cloud. As a consumer using it, you beget no infrastructure necessities. or not it's absolutely web based mostly and it be any dealt with on Microsoft's servers."
usually, IT stores would exhaust siloed options to exploit these two worlds, however Microsoft unified the management pane with SCCM 2012 SP1. whereas SP1 makes it viable for SCCM 2012 to exploit home windows to fade and home windows Embedded instruments, as well as Linux, Mac OS X and Unix servers, the broader mobile paraphernalia administration capabilities are present in home windows Intune. The forms of devices that windows Intune can exploit consist of those running home windows 8, home windows RT, windows cellphone 8, Android 2.1 and later versions, and Apple iOS versions 5.0 and 6.0.
SP1 for SCCM 2012 just provides just a few new administration capabilities for some older home windows cell machine scenarios, including management for windows cellular 6.1, windows cellular 6.5 and Nokia Symbian mobile gadgets (enabling discovery, inventory, application distribution, settings administration and faraway wipe), in accordance with Wally Mead, a senior program supervisor on the paraphernalia middle client management group, in a December Microsoft-produced Channel 9 video. Configuration supervisor nevertheless has exchange ActiveSync (EAS) administration capabilities but SP1 adds integration with windows Intune to allow administration through a separate view in SCCM 2012. Mead demonstrated the way to exhaust SCCM 2012 SP1 to create a home windows Intune subscription and how to create an APN (Apple thrust Notification) certificate that's required for managing Apple iOS gadgets.
change ActiveSync RequirementExchange ActiveSync continues to be a requirement for managing some non-home windows instruments below Microsoft's scenario, however the company is relocating away that strategy.
"there's figure of a baseline set of management settings capabilities which are in Configuration manager nowadays, in addition to Intune, and it be referred to as 'change ActiveSync,' and that is the understanding quiet there," Leznek defined. "despite the fact, we're relocating past change ActiveSync for deeper management capabilities, and we're basically using a native administration client, or API set, this is already constructed into the operating gadget. as an example, Apple has a local Apple administration API in iOS. home windows RT has a local management API that's in line with an traffic equitable known as 'OMA-DM' [Open Mobile Alliance-Device Management]. So it be a extra contemporaneous protocol that may assist you to execute deeper manageability beyond EAS. We're placing these investments into Intune. So the deeper cellular gadget management capabilities past just the primary EAS aid is not in Config supervisor, it's in Intune."
Mead pointed out lower back in December that these desperate to manage Android gadgets nevertheless deserve to exhaust EAS to achieve this as a result of Microsoft execute not beget an Android client agent in a position in time for paraphernalia seat 2012 SP1.
"So for the Android ambiance, it be most effective throughout the alternate ActiveSync connector," Mead pointed out. "it be pretty a Good deal the equal facets that I variety of renowned a bit bit previous -- being discovery, a cramped little bit of inventory, a cramped bit of settings management. but we're providing some application distribution capabilities for that as smartly. The rationale this has a cramped little bit of variations in capabilities is they beget not written a shopper agent yet for the Android platform. it be towering on their list to execute however we're just not going to obtain the customer agent equipped for their service Pack 1 unlock, so it is going to be via their alternate ActiveSync connector. For home windows cellphone 8, windows RT and the iOS contraptions, we'll beget over-the-air enrollment. so you, as an conclusion person, supplied you beget got got the capabilities assigned to you from the Config manager administrator, you're going to be in a position to enroll your device into that environment. once you've done that, they are going to be able to obtain inventory on your gadget; they are going to be able to install software focused to you as a person…; you're going to be capable of execute some settings management…; you'll be able to retire a device…, and you will even be in a position to remotely wipe a device."
Self-provider PortalsIn terms of its cell administration method, Microsoft conceives of application distribution to linger users as a cramped bit equivalent to the smartphone model. under this "self-carrier" model, apps can be downloaded with the aid of clients either from a web app shop or from an organization-maintained web portal. Self-service portals for iOS and Android are prebuilt into windows Intune, Leznek defined. Configuration manager has a self-provider portal capability nowadays, but, for cellular contraptions, or not it's customarily handled through Intune.
The portal is usually for offering an region for distributing apps, in preference to gadget administration, Leznek said. There are two techniques for IT execs to ration apps. "Deep linking" is the system wherein an app is published to an app shop, similar to Microsoft's home windows save. The 2nd procedure, known as "sideloading," is the method IT pros would exhaust for publishing company apps. Microsoft is requiring that apps be signed with a certificate when sideloading apps for security reasons.
Google has unique its new games streaming carrier Stadia, a cloud-based mostly video games platform accessed throughout any gadgets.
The carrier will notice users enjoying video games by way of internet streaming on smartphones, pills, desktops, laptops and television by way of a ChromeCast extremely HDMI streamer, with plenary pass-play aid. There is not any hardware required, no committed console.
here is enabled by way of Google's global infrastructure, with information centres in additional than 200 countries and territories around the planet.
The provider will launch later this year. There turned into no point out of how the company mannequin will work, whether users will pay for a subscription or purchase licences for particular person titles, but here is expected to be unveiled later in the 12 months.
As anticipated, the technology builds on the job stream test from October closing year the region assassin's Creed Odyssey was made playable via browsers, with Google CEO Sundar Pichai describing the carrier as "probably the worst-saved stealthy within the trade."
whereas this examine become restrained to 1080p, Google claims Stadia streams may besides be played in up to 4K at launch at 60fps, complete with HDR and surround sound. And there are plans to insert 8K streaming in the future, with the traffic believing its adoption to be inevitable.
Google has partnered with AMD to construct customized GPUs for its facts centres for you to verve video games performed by way of Stadia, claiming the tech has 10.7 teraflops of power -- more than the PS4 seasoned and Xbox One X mixed (which present four.2 and 6.0 teraflops respectively).
as a result of Stadia can besides be accessed by way of any gadget, the addressable audience is already at over two billion, and Google is making it as convenient as viable for these clients to leap in.
The business's Phil Harrison offered the instance of watching a trailer for assassin's Creed Odyssey on YouTube, then being presented with a 'Play on Stadia' button to click on. He claims the online game will launch as quickly as five seconds later, and not using a exigency for downloads, patches or installs.
avid gamers will besides be in a position to switch between devices on the fly, as proven on stage by way of someone relocating from mobilephone to pill to laptop, with Harrison claiming this was the bottom powered computing device Google may discover. every time, the online game picked up precisely where the participant had left off.
Stadia may be suitable with any existing USB controllers and keyboards, but Google is launching its personal committed controller exceptionally for the provider. similar to the patent purposes viewed earlier this month, here's a yardstick gamepad but facets two Stadia-certain capabilities.
One is a trap button that makes it viable for gamers to participate their gameplay by importing it to YouTube. in fact, Google says sessions will involve two simultaneous streams -- one for the linger consumer, and one without retard to YouTube, both at 4K and 60fps.
The other is a Google assistant button that will besides be used to stare for succor while taking allotment in games. the exhaust of Tomb Raider for example, the company showed someone having access to the built-in microphone to query a way to unravel a particular tomb, and the assistant brought up the desirable video sequel from YouTube, showing it in front of the gameplay circulation.
murderer's Creed and Tomb Raider had been the simplest video games proven operating. despite teasers ahead of the convention indicating that everything from pink dead Redemption 2, metal apparatus tenacious V and tremendous Mario dash may well be coming to Stadia, the handiest different validated title changed into Doom eternal, on the way to reportedly dash at 4K and 60fps.
Doom will reportedly dash on a separate GPU at Google's statistics centres, however the traffic is encouraging developers to ascertain the possibilities of video games that dash on assorted GPUs. In a tech demo comparing the change, a game operating on a separate GPU may beget much less subtle outcomes relish water simulation (which is chiefly intensive when it comes to computing energy), but the exhaust of varied GPUs will design this exhibit greater functional.
Rime developer Tequila Works changed into besides on stage discussing the carrier, whereas Q video games' Dylan Cuthbert revealed his studio is constructing an unannounced video game round StateShare, a Stadia role that enables avid gamers to participate video game circumstances from their classes by the exhaust of a link.
Google has besides shaped a brand new internal video games developer studio, catchily titles Stadia games and Entertainmment. The team will construct unique titles that completely blow their own horns the energy of Stadia, and may be led with the aid of the recently hired Jade Raymond.
The team will even be working with accomplice studios to succor them exhaust Stadia's technology to verve their games. an additional new division Stadia companions will besides present counsel to developers the usage of the carrier's tech.
StyleTransferML, as an example, is a computing device discovering tech that can bewitch any image and apply the coloration palate and patterns to a online game atmosphere. in the instance proven, a gray box world become rendered to stare relish Van Gogh's starry nighttime, a sketch pad drawing, or even basic arcade title Pac-Man (youngsters this become generally a witticism because it regarded unplayable).
StreamConnect is a device that helps hyperlink chop up-screen views. Drawing on the vigour of the cloud, Google claims developers will now not exigency to sacrifice visual high-quality or performance when supporting atomize up-display multiplayer and is even opening new probabilities. for instance, a fourth participant can watch the other three on their screen and interact with each and every player's session, placing a beacon on enviornment of pastime.
StateShare, as outlined, allows builders to let players participate the region conditions, their position or elements relish their personality's stock with different people by the exhaust of a link. This may be shared with only one grownup, or with every person via whatever thing relish YouTube. The theory is that clicking that hyperlink would load Stadia and recreate that accurate second or state of affairs for a further player.
The hyperlinks are besides key to spreading Stadia, with Harrison epigram this dramatically improves discoverability for games developers as, "The complete cyber web becomes your store" -- even if hyperlinks are shared by way of the Stadia save, YouTube movies, Gmail, Twitter or some other channel.
Stadia will launch later this year, beginning within the US, UK, Canada and most of Europe. Google promised more guidance to recur any through the summer time -- very nearly actually at E3 in June. For now, developers can head to Stadia.dev to ensue for edifice equipment, while buyers can register their interest and ensue updates at Stadia.com.
Obviously it is arduous assignment to pick solid certification questions/answers assets concerning review, reputation and validity since individuals obtain sham because of picking incorrectly benefit. Killexams.com ensure to serve its customers best to its assets concerning exam dumps update and validity. The vast majority of other's sham report objection customers promote to us for the brain dumps and pass their exams cheerfully and effectively. They never trade off on their review, reputation and character because killexams review, killexams reputation and killexams customer assurance is vital to us. Uniquely they deal with killexams.com review, killexams.com reputation, killexams.com sham report grievance, killexams.com trust, killexams.com validity, killexams.com report and killexams.com scam. In the event that you notice any incorrect report posted by their rivals with the name killexams sham report grievance web, killexams.com sham report, killexams.com scam, killexams.com dissension or something relish this, simply remember there are constantly terrible individuals harming reputation of Good administrations because of their advantages. There are a powerful many fulfilled clients that pass their exams utilizing killexams.com brain dumps, killexams PDF questions, killexams hone questions, killexams exam simulator. Visit Killexams.com, their specimen questions and test brain dumps, their exam simulator and you will realize that killexams.com is the best brain dumps site.
As the main component this is in any aptitude vital here is passing the MB4-219 - Solomon 6.0 Inventory and Order Processing exam. As any which you require is a towering score of Microsoft MB4-219 exam. The only a solitary factor you want to execute is downloading braindumps of MB4-219 exam and memorize. They will not let you downl with their unrestricted guarantee. The professionals in relish way preserve tempo with the maximum best in magnificence exam to offer most of updated materials. Three months free access to beget the potential to them thru the date of purchase. Every candidate can besides undergo the fee of the MB4-219 exam dumps through killexams.com requiring cramped to no attempt. Habitually there is a markdown for every person all.
Inside seeing the bona fide exam material of the brain dumps at killexams.com you could without numerous an enlarge develop your pretense to reputation. For the IT professionals, it is fundamental to modify their capacities as showed through their paintings want. They design it simple for their clients to carry certification exam with the assist of killexams.com confirmed and wholehearted to goodness exam material. For a super destiny in its area, their brain dumps are the satisfactory selection.
A high-quality dumps creating is a basic phase that makes it honest for you to bewitch Microsoft certifications. In any case, MB4-219 braindumps PDF offers agreement for candidates. The IT declaration is a vital tough undertaking if one doesnt ascertain actual route as obvious resource material. Thus, they beget got actual and updated material for the arranging of certification exam.
killexams.com helps a powerful many applicants pass the exams and obtain their certifications. They beget a huge number of effective surveys. Their dumps are solid, reasonable, updated and of truly best character to conquer the troubles of any IT certifications. killexams.com exam dumps are most recent updated in exceedingly outflank way on customary premise and material is discharged intermittently. Most recent killexams.com dumps are accessible in testing focuses with whom they are keeping up their relationship to obtain most recent material.
The killexams.com exam questions for MB4-219 Solomon 6.0 Inventory and Order Processing exam is basically in view of two available arrangements, PDF and exercise software. PDF record conveys any the exam questions, answers which makes your planning less hardworking. While the exercise software are the complimentary ingredient in the exam item. Which serves to self-survey your advance. The assessment apparatus additionally features your feeble regions, where you beget to establish more attempt with the goal that you can enhance every one of your worries.
killexams.com intimate you to must attempt its free demo, you will notice the natural UI and furthermore you will assume that its simple to alter the prep mode. In any case, ensure that, the actual MB4-219 exam has a larger number of questions than the sample exam. On the off chance that, you are placated with its demo then you can buy the actual MB4-219 exam item. killexams.com offers you three months free updates of MB4-219 Solomon 6.0 Inventory and Order Processing exam questions. Their certification team is constantly accessible at back linger who updates the material as and when required.


This tea is grown in the Okabe area, one of the most important areas for cultivation and production of Gyokuro. Its distinctive characteristic is that it is cultivated in the shade. In the days preceding the crop, plantations are covered with sheets that prevent solar rays from penetrating. In this way the plant secretes more minerals and chlorophyll. However, the harvest method is handmade according to tradition. In cup, this Gyokuro is elegant, sweet and vegetable at the same time.
Japan produces only Green Teas. Their characteristic is to be low in caffeine and very rich of vitamin C. The Japanese Teas are digestive and tonic, perfect to be sipped during lunch time or after luch. Ideal Teas to relax and unwind.


text:
Always and Forever, Lara Jean (To All the Boys I've Loved Before #3)
A Vulture Best YA Book of 2017
Lara Jean's letter-writing days aren't over in this surprise follow-up to the New York Times bestselling To All the Boys I've Loved Before and P.S. I Still Love You.
Lara Jean is having the best senior year a girl could ever hope for. She is head over heels in love with her boyfriend, Peter; her dad's finally getting remarried to their next door neighbor, Ms. Rothschild; and Margot's coming home for the summer just in time for the wedding.
But change is looming on the horizon. And while Lara Jean is having fun and keeping busy helping plan her father's wedding, she can't ignore the big life decisions she has to make. Most pressingly, where she wants to go to college and what that means for her relationship with Peter. She watched her sister Margot go through these growing pains. Now Lara Jean's the one who'll be graduating high school and leaving for college and leaving her family—and possibly the boy she loves—behind.
Praise For Always and Forever, Lara Jean (To All the Boys I've Loved Before #3)…
"Sweetly funny."
* "Han's story is timeless. First love, finding one's identity in school and at home, and navigating the delicate transition from child to adult are all universal themes in young adult literature, and Han does a great job exploring them. She has written a sweet, coming-of-age love story that will surely please fans of the first two books who are likely clamoring for this conclusion."
— VOYA, starred review
"Featuring the same endearing sister relationships, mouthwatering descriptions of Lara Jean's cooking, and timeless coming-of-age conflicts, this final volume in the series will continue to delight Han's fans.... Sparkling dialogue, swoon-worthy scenes, and likable characters make this a gem of a selection for most YA collections"
"A must-have conclusion....Fans of Han's best-selling series will turn out in droves to get their hands on this conclusion to Lara Jean's story."
Simon & Schuster Books for Young Readers, 9781481430487, 336pp.
Jenny Han is the #1 New York Times bestselling author of the To All the Boys I've Loved Before series, now Netflix movies. She is also the author of The Summer I Turned Pretty series, Shug, and Clara Lee and the Apple Pie Dream. She is the coauthor of the Burn for Burn trilogy, with Siobhan Vivian. Her books have been published in more than thirty languages. A former librarian, Jenny earned her MFA in creative writing at the New School. She lives in Brooklyn, New York.

redpajama_set_name: RedPajamaCommonCrawl


I used to play a lot of poker when I was in high school, mostly 7 card stud hi/lo. I was never that good, but since I was playing mostly people my own level, I more or less broke even. At least, that's what I think I remember.
I played poker after graduating from college in 84, but again it was 7 card stud hi/lo. And the games were like those in high school, nothing too serious.
About a year ago, I picked up poker once again, playing poker online at Partypoker.com. I lost $50 playing 7 card stud hi/lo, after initially being up $75, and thought that I should permanently retire from the game. Although I really enjoyed playing poker online, I realized that my poker skills were not there, and worried about online cheating.
In an attempt to lure me back, Partypoker has occasionally given me some free money, which I quickly lose. I have been tempted to invest my own money to recoup losses, and perhaps pride, but so far haven't given into temptation. When I was playing online I played Hold'em a few times for real money, and actually won the few games I played. But I'm sure my success was totally due to luck, since I never played the game before in my life. I didn't even the difference between a big blind and a small blind. Pathetic.
This past weekend I was in Vegas (Caesars) with a bunch of college friends, one of which was my friend Seth, who used to be in my high school poker games and in the 90s played quite a bit of Hold'em, and told me that he got to the level where he was winning on average about $500 a week (the guy doesn't lie). He sat down with me at a $4/8 limit game and I experienced my first taste of real live Hold'em. I started with $100 and lost $57 in about two hours. Seth said that I didn't make any stupid mistakes.
The next night I played Hold'em with my friends. We each put in $45 and when the game stopped I was one of three remaining players, although I ended up losing $19. So my cummulative poker losses for Vegas were $76. Fortunately, I won a $20 bet on the Cavliers, getting 5 1/2 points at home. How could I lose?
I really enjoyed my experience playing Hold'em. The small amount of money I lost was well worth it in terms of what I learned. As Bob Dylan says, 'there is no success like failure". There's no way I cannot improve at the game. My current plan is to read Lee Jones' "Winning Low-Limit Hold'em" and play a little online poker (Low-Limit Hold'em) at Titan Poker. Henceforth I shall chronicle my poker experiences and the lessons I learn along the way.


Movie News - More Megamind!
Thanks to Paramount Pictures I have another couple of clips to share with you. I'm really excited for this movie. After watching How to Train Your Dragon it's quite clear that Dreamworks are pulling out the big guns. Hopefully the whole movie is as enjoyable as these clips have been.


Check out these beginner yoga poses for strong arms, plus a free printable. Yoga for beginners - check out these 5 beginner yoga poses for stronger arms. Click through to get a free pdf …... The Sunshine Exchange goes Cruelty Free by The Sunshine Exchange In the past few months, I have made the decision to switch to cruelty free skincare and beauty (to the best of my abilities).
Complete all 5 yoga poses for flat abs, then repeat the workout 2 more times, making sure to switch up the sides of the body. Feel free to stay in any of the stretches for longer than 30 seconds if it feels good on your body or you think you need it.


text:
Gerry Adams waxed: 'Remind me never to do this again'
PJ Hegarty, waxwork sculptor, finds Sinn Féin chief friendly and serious 'peacemaker'
Gerry Adams is the latest subject for wax sculptor PJ Heraghty who has spent four months trying to recreate the Sinn Féin man's likeness. Video: Enda O'Dowd
Peter Murtagh
Sat Jan 9 2016 - 06:00
There's a headless body at the bottom of PJ Hegarty's garden in Leixlip. The head that should be attached to the body is to be found nearby, impaled on a metal rod sticking out of the centre of a wheel, lying flat on the floor of an ivy-clad breeze-block shed.
It is Gerry Adams, though not quite as we have come to know him.
In fact, headless bodies are not what anyone expects to find in the otherwise quiet and unremarkable housing estate that is PJ's home. But all is not what it seems. . .
PJ – Patrick Joseph – is a quietly spoken, white-haired 62-year-old sculptor who has worked for the National Wax Museum in Dublin for more than 30 years, fashioning wax copies of presidents, taoisigh, writers, artists, pop stars, villains, ninja turtles – and now also the president of Sinn Féin, Gerry Adams.
Lisa Jameson, the museum's operations manager, hopes to have the completed wax representation of Adams on public display within weeks. All that remains is for the man himself to supply suitable clothes to dress the mannequin that represents his body, a pair of shoes and spectacles.
"You can't have a naked Gerry Adams," says PJ before being reminded of the infamous Adams tweet about bouncing naked on a trampoline with his pet.
"Oh yes! The dog. Is that a real story?" PJ wonders aloud.
The wax Adams project has been months in the marking.
In early September, after the Sinn Féin leader had agreed to help the museum in its quest to have a copy of him made for its Time Vault display ("the history of Ireland from the Vikings to the peace process," says Jameson), and allow The Irish Times record the process in photographs and on video, Adams took part in the first part of what has been an unusual journey, to say the least.
Numerous shots
He went to the museum in Foster Place where a photographer took numerous shots of his head – front, back, top, both sides – from a variety of angles and with the subject somewhat expressionless. The resultant images show every hair on Adams's head, every blemish on his skin.
Next, Adams was sat on a chair and was wrapped in cling film, neck to feet, rather in the manner of an ancient Egyptian about to be mummified, or a Buddha statue wrapped for shipping overseas.
His clothes suitably protected, a viscous, gray gunge was spread all over Adams's face, only his nostrils being left clear to facilitate breathing. On top of the gunge, the Sinn Féin leader's face was then wrapped in surgeons' plaster gauze to create, when set and dry, a hard bowl shell which the gunge itself sets.
"Remind me never to do this again," said Adams as PJ and a helper lurried on the gunge.
It was alginate, the sculptor explained in an interview this week, a silicone, rubbery substance derived from seaweed, "the same stuff used by dentists", he said, and which would not stick like superglue to Adams's beard.
"I had to use a new material, a silicone material that slips off the hair. It's very good but it was very expensive," said PJ. "Boy, was I worried that it would stick but it worked a treat. You have to be a bit of a chancer in this game."
A couple of hours later, the Sinn Féin leader was off back to politics, leaving in his wake a perfect mask, a mould of his face, retained by PJ. The mould was in effect an imprint of Adams and into it plaster of Paris was later poured to create, when set, a three-dimensional, near- perfect copy of the Sinn Féin leader's face.
Clay bust
This became the model for PJ to fashion a bust of Adams using traditional potter's clay, correcting the distorting effects the silicone mould and gravity had on his subject's skin. He was also armed with a set of detailed measurements he made during a subsequent visit to the Sinn Féin leader's office in Leinster House.
He measured Adams's head – chin to hairline, nose to tragus (the small piece of the ear closest to the cheek and projecting backwards over the concha, the earhole), the width of the ears themselves and the distance from the forehead to the back of the head.
"He's quite a big man, so his head was quite big. His cranium was bigger than normal," the sculptor recalled.
Adams's eyes also struck him as unusual. "They're dark brown with a sort of orange background. I read somewhere that darker brown eyes are a sign of intelligence."
And Adams's hands also surprised. "For a man of his size, his hands seemed artistic or academic. I was expecting labourer's hands."
The clay bust became the base for the final mould – another plaster of Paris mould, this time of the entire head, made in two sections, front and rear. Removed when set from the clay bust and bound back together again with wire, the resultant hollow mould is the receptacle into which molten wax is poured.
That was October and since then PJ has worked, by his own estimation, 40 to 60 hours a week in his garden end studio and workshop, finishing what will soon go on display.
"Sculpting a head is the hardest thing you can do because it's down to millimetres and it's down to good visual information," he says. "It takes so long because, basically, you are putting in hair, one piece at a time, and with a beard, basically you've got two heads of hair, plus the eyebrows."
On his two sittings with Adams – making the original silicone face mould and then taking head measurements – the sculptor found his subject pleasant company.
"I found him quite jovial and calm and friendly and co-operative. That's how I found him. And he turned up for the sitting, so fair play to him."
The headless mannequin at the end of the garden (in a very un-Gerry Adams double- breasted dark blue business suit) leans against a cupboard in PJ's workshop, awaiting the arrival of its subject- approved attire. The room is crammed with clutter: the workbench and shelves are littered with tools, and jars of screws, nails, nuts and bolts; drills and saws; tangled lumps of wire; hessian mesh for plaster moulds; and bags of clay.
A second room, PJ's inner sanctum, has more of the air of the artist's studio about it. "It's my cell," he says.
There's a low-lying work table and several anglepoise lamps. There are numerous former jam jars now bristling with paint brushes, and tubes of oil paint, scrunched up and squeezed from use, their contents smeared on surfaces.
And from low down, impaled on the bar sticking out of the wheel, the head of Gerry Adams – brilliantly executed, the likeness exact and unmistakable, every hair seeming correct in position and colour, the waxen skin toned into life with a delicately applied blush of oil paint, those dark brown eyes looking out (little sign of an orange tinge to the pupils, however).
"I did give him a slight smile," says PJ, "because, although he's a serious man, I found him relaxed and friendly. He is a peacemaker. That's how I see him."
The Sinn Féin leader selected the pose he wished his doppelgänger to adopt in the National Wax Museum. The legs will be a little apart, the left a shade forward of the right, the body leaning back slightly.
The arms will be folded, not tightly and high across the chest, more in a relaxed V across the lower abdomen. And, between them, PJ will place a copy of the Belfast Agreement, the one whose front page was signed by all parties to it, including Adams.
Peter Murtagh is a contributor to The Irish Times
LeixlipLeinsterSinn FeinAdams Un GerryFoster PlaceGerry AdamsLisa JamesonMr AdamsPatrick JosephPeter Murtagh There
First Look: Royal Irish Academy of Music unveils €25m addition to its Georgian Dublin home
Internal report warns of 'unprecedented harm to reputation' of An Bord Pleanála
Dublin ambulance service leaving public at risk, report says
Shortfall of 600 refugee beds in next four weeks predicted


Review of the 60 second binary options trading feature on the SpotOption, 24option and TradoLogic trading platforms.
2014-12-01 · Day Trading 60 Second Binary Options Using Martingale Trading Strategy Trading Binary Options is a very fast-paced binary options, 60 second binary.
© Binary options 60 second trading Binary Option | Binary options 60 second trading Best binary options.


The New York Giants lost their opening game to the Jacksonville Jaguars 15-20. Despite the loss, rookie Saquon Barkley had a very good debut, running 18 times for 106 yards and broke off a 68 yard touchdown run. Already after one game, it seems the Giants will be relying heavily on the rookie running back, which could be favorable for the Giants. Eli Manning went 23 for 37 on the day against a tough Jaguar secondary. He did not throw a touchdown, but he did throw an interception. The Giants will need Odell Beckham Jr. to be more involved this week, especially in the red zone, if they are going to take down a division rival and avoid an 0-2 start.
The Dallas Cowboys only managed 8 points in their opening day game with the Carolina Panthers, which is concern going forward. Quarterback, Dak Prescott did not look very good and with not any elite wide receivers to throw to, it made it very difficult for the Cowboys all game long. Their defense kept them in the game and allowed 16 points to the Panthers, but in the end they could not find enough offensive production to get the job done. Going up against a division rival will be tough, but Dallas will be able to pull this one out if Dak can find time in the pocket to make accurate throws.
[email protected] fan Gregory Hampton was in the hospital when his sons decided to enter him in the contest.
Tonight, he could be crowned the NFL's biggest fan and win 100 years of season tickets.
"Of course I want to play ... I'm also from Kansas City, Missouri. I saw Patrick Mahomes sit for a year and he ended up doing what he did."


Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In this protocol the first part describes the fluorescent labeling by using a precharged tRNA, loaded with a fluorescent amino acid. The second part describes the preparation of a modified aminoacyl-tRNA-synthetase and a suppressor tRNA that are applied to the CHO cell-free system to enable the incorporation of a non-canonical amino acid. The reactive group of the non-canonical amino acid is further coupled to a fluorescent dye. Both methods utilize the amber stop codon suppression technology. The successful fluorescent labeling of the model G protein-coupled receptor adenosine A2A (Adora2a) is analyzed by in-gel-fluorescence, a reporter protein assay, and confocal laser scanning microscopy (CLSM). Moreover, a ligand-dependent conformational change of the fluorescently labeled Adora2a was analyzed by bioluminescence resonance energy transfer (BRET).
G protein-coupled receptors (GPCRs) are involved in vital processes including the regulation of signaling pathways and the subsequent triggering of essential physiological responses. Up to date more than 50% of pharmaceutical drugs are targeting GPCRs and GPCR interaction partners . Therefore, the development of novel production processes of GPCRs for structural and functional characterization remains essential. In a natural environment, membrane proteins are only present in small concentrations . The overexpression of membrane proteins is usually performed in heterologous systems involving a complex vector design. Nevertheless, overexpression obstacles such as low solubility, cytotoxicity, and low stability can occur . In addition, detergents used to solubilize membrane proteins often induce altered ligand binding properties . Cell-free protein synthesis based on translationally active lysates represents an approved alternative for membrane protein synthesis. Eukaryotic cell lysates based on insect, CHO, and human cell lines are predestined for membrane protein synthesis due to the presence of endogenous membrane structures . These microsomes enable an integration of membrane proteins into a biological membrane. In addition, posttranslational modifications such as disulfide bridging, phosphorylation, and partly glycosylation are possible . Furthermore, the CHO cell-free system is characterized by a high productivity of complex proteins .
In the context of protein modification and fluorescent labeling by using genetic code expansion , cell-free systems are of special interest due to their open reaction mode . Exogenously prepared components can be easily and directly added to the cell-free protein synthesis reaction to incorporate a defined non-canonical amino acid site-specifically at the position of a redefined codon within the gene sequence . In a first step, addressing of a defined amber stop codon can be verified by the addition of a precharged tRNA to the cell-free protein synthesis reaction. The tRNAs in this report are aminoacylated to a lysine residue that is coupled to a Bodipy TMR dye (BP). The tRNA recognizes either the amber stop codon or the phenylalanine codon. The lysine residue with the coupled fluorescent dye is subsequently site-specifically (BP-CUA) or statistically (BP-GAA) incorporated. The lack of a re-aminoacylating mechanism of the tRNA leads to the consumption of large amounts of precharged tRNA. Therefore, we describe an alternative system that is inspired by the natural aminoacylation of tRNAs in vivo.
The addition of non-canonical amino acids in vivo that usually do not permeate the cell wall can be realized in cell-free systems due to the missing cell wall. Even high concentrations of non-canonical amino acids can be added to the reaction. Moreover, the suppressor tRNA and the modified aminoacyl-tRNA-synthetase can be applied to the cell-free reaction in defined concentrations enabling an identification of optimal concentrations for highest suppression efficiency. Here, the described experimental setup is based on an evolved aminoacyl tRNA-synthetase (aaRS) derived from E .coli [11, 12] and a modified suppressor tRNA recognizing the UAG amber stop codon . The combination of the aaRS, tRNA, and a phenylalanine-derivate (p-propargyloxy-l-phenylalanine, pPa) with an alkyne group in para-position resulted in an efficient incorporation of the non-canonical amino acid into the model protein adenosine A2A receptor (Adora2a). The human Adora2a is expressed in almost every tissue of the body. Primarily, the membrane protein can be detected in the brain, cells of the immune system, leucocytes, and platelets. In peripheral tissues, the Adora2a is involved in inflammation reactions and affects the control of the cardiovascular system . The interaction of the Adora2a with different neurotransmitters in the brain is of highest interest since the receptor displays an important role in the regulation of dopamine and glutamate release thereby making it a promising therapeutic target for the treatment of depression, insomnia, and Parkinson's disease . Consequently, a better understanding of the detailed interactions between membrane proteins and their specific ligands is mandatory. Information on the structural and functional relationship of a defined GPCR might be gained by a visualization of the target membrane protein in a nature-like lipid environment . A highly preferred method to determine GPCR interaction is based on the spatial proximity of two fluorescent moieties. Interaction partners can be determined by an intermolecular fluorescence resonance energy transfer (FRET) and specific conformation changes by an intramolecular FRET. For the Adora2a, intermolecular as well as intramolecular FRETs were established to analyze the receptors' behavior [16, 17]. In this way, the conformational rearrangement of the helices III and VI induced by agonist binding was elucidated . A coupled fluorescent dye in the region of amino acid 215 will be excited by a C-terminally fused NanoLuciferase (Nluc) resulting in a detectable bioluminescence resonance energy transfer (BRET). A signal change of the BRET value after the addition of a ligand indicates a conformational change of the Adora2a. The main objective of this chapter is to describe a cell-free protein synthesis-based alternative methodology to the standard protein visualization methods typically used in vivo. Our cell-free procedure is based on the site-specific and statistical incorporation of a precharged tRNA and a site-specific incorporation of a non-canonical amino acid into the target protein. The cell-free synthesized protein is subsequently labeled by using a chemoselective reaction. Moreover, we adapted an intramolecular BRET for the Adora2a to show a ligand-dependent conformational change. In addition, we present the preparation of the evolved aminoacyl-tRNA-synthetase and the suppressor tRNA.
Coding sequence for the modified tyrosyl-tRNA-synthetase (eAzFRS, including the mutations Thr37, Ser182, Ala183, and Arg265 [11, 12] and a C-terminal Strep-Tag) from E .coli.
E.coli expression system (RTS 500 E.coli HY Kit, biotechrabbit).
100 mM Isopropyl β-d-1-thiogalactopyranoside (IPTG).
Gravity flow Strep-Tactin® superflow mini-column (0.2 ml).
Strep-Tactin® Purification Buffer Set: 10× Washing Buffer (1 M Tris–Cl, pH 8.0, 1.5 M NaCl, 10 mM EDTA), 10× Elution Buffer (1 M Tris–Cl, pH 8.0, 1.5 M NaCl, 10 mM EDTA, 25 mM Desthiobiotin) and 10× Regeneration Buffer (1 M Tris–Cl, 1.5 M NaCl, 10 mM EDTA, 10 mM HABA (hydroxyl-azophenyl-benzoic acid)).
Zeba™ Spin Desalting Columns (7 K MWCO, 0.5 ml).
Amicon® Ultra Centrifugal Filters (10 K device, 0.5 ml).
Synthetase storage buffer: 50 mM HEPES pH 7.6, 10 mM KOAc, 1 mM MgCl2, 4 mM DTT.
Thermomixer with a microtiter plate adapter and a RTS 500 adapter.
Vector containing the nucleotide sequence of tRNATyrCUA (SupF Gene).
tRNATyrCUA-specific forward primer (5′ CgA gCT CgC CCA CCg gAA TTC 3′) and 2′-OMe reverse primer (5′ Tgg Tgg Tgg ggg AAg gAT TCg 3′).
T7 RNA Polymerase (f.c. 1 U/μl, Agilent).
5× NTP mix containing 18.75 mM ATP, 18.75 mM CTP, 18.75 mM UTP and 7.5 mM GTP.
5× transcription buffer: 400 mM HEPES-KOH, 0.5 mM Spermidine, 50 mM DTE and 75 mM MgCl2.
DNAseI (1 U per μg plasmid DNA).
10× MOPS buffer: 200 mM MOPS, 50 mM NaOAc, 10 mM EDTA, pH 8.0.
MOPS sample buffer: 8% (v/v) formaldehyde, 12 ml formamide, 2.4 ml 10× MOPS buffer, 0.05% (v/v) bromophenol blue to a total volume of 24 ml.
High Performance Liquid Chromatography (HPLC) grade Chloroform.
Coding sequence for the modified adenosine A2A receptor (Uniprot: P29274, amino acids 1-340, with and without amber stop codon at nucleotide sequence coding for P215) with a receptor C-terminally fused Nanoluciferase (Fig. 1). Flanking sequences containing 5′ regulatory sequences (T7 RNA polymerase promotor, Cricket paralysis virus (CRPV) IGR IRES sequence (Genbank accession no. AF218039, nucleotides 6025-6216)) and 3′ regulatory sequences (T7 terminator), cloned with the coding sequence into a plasmid (BioCat or Thermo Fisher Scientific).
CHO lysate prepared as described [19, 20] (see Note 1 ).
10× translation mix: 300 mM HEPES-KOH (pH 7.6), 2250 mM KOAc, 2.5 mM spermidine, 1 mM of each canonical amino acid (Merck) and 39 mM Mg(OAc)2.
5× energy: 100 mM creatine phosphate, 1.5 mM GTP, 1.5 mM CTP, 1.5 mM UTP 8.75 mM ATP and 0.5 mM m7G(ppp)G cap analogue.
100 μM Polyguanylic acid (polyG, IBA).
T7 RNA polymerase (f.c. 1 U/μl).
100 μM Bodipy-TMR-lysine-tRNACUA (BP-CUA, biotechrabbit).
100 μM Bodipy-TMR-lysine-tRNAGAA (BP-GAA, biotechrabbit).
100 mM p-propargyloxy-l-phenylalanine (pPa, Iris Biotech).
5 mM Copper(II) sulfate (CuSO4).
2.5 mM Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, Iris Biotech).
80 mM Sodium ascorbate (NaAsc).
SDS-PAGE Sample buffer: 1× LDS buffer containing 106 mM Tris–HCl, 141 mM Tris base, 2% LDS, 10% glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G, 0.175 mM Phenol Red, pH 8.5 with 50 mM DTT.
Fluorescently labeled protein ladder for SDS-PAGE.
Multimode Microplate Reader Mithras2 LB 943.
μ-Ibidi-Slide (μ-Slide 18 well, flat, Ibidi).
For prokaryotic cell-free synthesis, the eAzFRS gene should be cloned into a vector containing a T7 promotor, ribosomal binding site, and T7 terminator such as pIX3.0, pIVEX2.3d, and pIVEX2.4d vectors or alternatively containing a T5 promotor such as pQE2 vectors as used in this protocol. eAzFRS is synthesized in a cell-free system using an E. coli lysate in a dialysis mode. A typical 1.1 ml reaction is composed of 0.525 ml E.coli lysate, 0.225 ml reaction mix, 0.27 ml amino acids without methionine, 30 μl methionine, 11 μl IPTG for the induction of the protein expression pQE2 vector, 39 μl template containing 110 μg plasmid DNA.
The surrounding feeding mixture contains 7990 μl feeding mix, 110 μl IPTG, 2650 μl amino acids without methionine and 300 μl methionine (see Note 2 ).
Fill the reaction solution into the reaction compartment (marked through the red lid).
Fill the feeding mix into the feeding chamber (marked through the colorless lid).
Insert the prepared chamber into the RTS 500 adapter in a thermomixer. The reaction time is 20 h at 30 °C and a shaking speed of 900 rpm.
For the separation of aggregated proteins from soluble eAzFRS a centrifugation step at 16,000 × g for 10 min at 4 °C is recommended.
Equilibrate two Strep-Tactin columns with 400 μl of 10× washing buffer and add 500 μl of the supernatant of the cell-free reaction to each column.
After the supernatant has completely entered the column, wash each column 5× with 200 μl washing buffer (see Note 3 ).
Elute the protein 6× with 100 μl elution buffer and collect the fractions.
Elution fractions containing the target protein are pooled.
Regenerate the column with 3× 1 ml of 1× regeneration buffer and remove the regeneration buffer 2× with 800 μl of 1× washing buffer. Store the column in 2 ml washing buffer at 4 °C.
The combined elution fractions are applied to Zeba™ Spin Desalting Columns to exchange the elution buffer of the strep-tag purification to a synthetase storage buffer. Therefore, remove the storage solution of the Zeba™ Spin Desalting Column by centrifugation at 1500 × g for 1 min. Add 300 μl of the synthetase storage buffer to the resin bed and centrifuge at 1500 × g for 1 min. Repeat this step 2×. Place the column in a new collection tube and apply 100 μl of the pooled synthetase solution to each column. Centrifuge at 2000 × g for 2 min and collect the synthetase.
The concentration of the synthetase can be performed with Amicon® Ultra Centrifugal Filters. Add up to 500 μl of the synthetase solution to the concentrator and centrifuge at 14,000 × g for 10 min and 4 °C. Collect the concentrated sample and determine the concentration by NanoDrop measurement using the molecular mass (48.6 kDa) and the extinction coefficient (54.3) (see Note 4 ).
The synthetase can be stored at −80 °C after shock freezing in liquid nitrogen.
For specific and homogenous 3′-ends of the suppressor tRNA, an additional PCR step before transcription reaction is included. Therefore, the reverse primer contains a 2′-OMe-group to prevent unspecific nucleotides at the 3′-end of the tRNA that can be added by the T7 polymerase during transcription reaction. Amplify the template by pipetting in a PCR tube final concentrations of 1× Taq Buffer, 0.2 mM dNTP mix, 0.5 μM forward primer, 0.5 μM reverse primer, 2.5 mM MgCl2, 0.01 ng/μl plasmid and 0.04 U/μl Taq DNA polymerase. Fill the reaction with water to a final volume of 250 μl (see Note 5 ).
Thaw the components for in vitro transcription on ice and pipette the reaction at room temperature. Mix 1× transcription buffer, 1× NTP mix, 1 U/μl T7 RNA Polymerase and 8 ng/μl template DNA. Fill the reaction with water to the final volume of 500 μl. Incubate the reaction for 3–6 h at 37 °C and 500 rpm.
Centrifuge the RNA at 12,000 × g for 1 min und use the supernatant for the DNAseI treatment (see Note 6 ) Add 1 U DNAseI per 1 μg DNA. Incubate for 10 min at 37 °C and 500 rpm.
Handle the TRIzol and chloroform reagent with care and use a fume hood. Add a threefold volume of TRIzol to the transcription reaction and mix carefully. Incubate for 5 min at room temperature. Add 200 μl chloroform for 1 ml TRIzol and mix carefully for 15 s by inverting. Incubate for 3 min at room temperature. Centrifuge at 12,000 × g for 15 min at 4 °C. Isolate the aqueous phase (see Note 7 ). Add 500 μl isopropyl for 1 ml TRIzol and mix carefully. Incubate overnight at 4 °C.
Centrifuge at 15,000 × g at least for 1 h at 4 °C and discard the supernatant. Overlay the pellet with 1 ml 75% ethanol for 1 ml TRIzol and incubate for 30 min at −20 °C. Centrifuge at 7500 × g for 10 min and 4 °C. Discard the supernatant and air dry the pellet. Solve the pellet in water. Measure concentration using a NanoDrop and adjust the concentration to 100 μM.
Fold the tRNA by slowly decreasing the temperature from 80 to 25 °C in a PCR cycler. The tRNA can be stored at −80 °C after shock freezing in liquid nitrogen.
Thaw all required components for cell-free protein synthesis on ice (see Note 8 ).
The cell-free protein synthesis is performed in a coupled mode where transcription and translation reaction take place in one vessel. A standard reaction is composed of 40% CHO lysate, 10 μM polyG, 1× translation mix, and 1× energy mix. Add plasmid at a concentration of 40 nM (see Note 9 ). Add 1 U/μl T7 RNA polymerase for the transcription reaction and 14C-leucine (specific radioactivity of 66.67 dpm/pmol) for further analysis of de novo synthesized proteins. Fill the reaction with water to the final volume of 50 μl.
Thaw the Bodipy-TMR-lysine-tRNACUA (BP-CUA) and Bodipy-TMR-lysine-tRNAGAA (BP-GAA) on ice and keep it dark (see Note 10 ).
Pipette 2 μM of the precharged tRNA to the cell-free reaction and incubate the prepared cell-free protein synthesis reaction by 27 °C for 3 h and shaking at 600 rpm (see Note 11 ). Cover the thermomixer with a lid or aluminum foil to prevent the reaction of any light.
Take a 5 μl aliquot of the translation mixture for SDS-PAGE. Centrifuge the translation reaction for 15 min at 16,000 × g and 4 °C. Take a 5 μl of the supernatant and resuspend the pellet (microsomal fraction) in an equal volume PBS in comparison to the volume of the translation reaction. This step is required for the analysis of the localization of the synthesized protein. The expectations of the incorporation of the different Bodipy-tRNAs are described in Table 1 (see Note 12 ).
An example of the performance of the fluorescent labeling of Adora2a with or without incorporated non-canonical amino acid determined by in-gel-fluorescence and autoradiography, and the measurement of the reporter protein are shown in Fig. 2. In-gel-fluorescence and autoradiography (Fig. 2a, c) showed the expected results as described in Table 1. The reporter protein assay (Fig. 2b) is based on the activity of a receptor C-terminally fused Nanoluciferase (Nluc). Nluc activity can only be measured after the translation of the full-length fusion protein. The Adora2a synthesized by using the DNA construct without an amber stop codon (Adora2a + BP-GAA/BP-CUA) resulted in a fusion protein with a detectable Nluc activity. The fusion protein translated from the amber stop codon DNA construct (Adora2a_amb + BP-CUA) showed a fivefold reduced Nluc activity. This result implicates a rather low incorporation efficiency of the precharged tRNA addressing the amber stop codon. The combination of Adora2a_amb with BP-GAA showed no luciferase activity since only the termination product was translated. Protein yields were calculated by scintillation counting and resulted in approximately 20 μg/ml in translation mixture. In the microsomal fraction and the supernatant, approximately 6 μg/ml and 14 μg/ml were detected, respectively. A similar Adora2a distribution was obtained by in-gel-fluorescence, luciferase activity, and autoradiography. Previously reported distributions of membrane proteins are comparable to the here described distribution . A comparable protein yield was calculated for the termination product.
The full-length protein is not labeled with the fluorescent dye and the ncAA is not incorporated.
The full-length protein is fluorescently labeled. The fluorescence signal has a lower intensity in comparison to the statistically labeled Adora2a due to the site-specific incorporation of the ncAA at one defined position. Termination product is not labeled.
The termination product is highly fluorescently labeled due to the statistical incorporation of the ncAA. No full-length product is visible.
Site-specifically label the Adora2a_amb using the precharged tRNA Bodipy-TMR-lysine-tRNACUA as described in Subheading 3.3.1, steps 1–3.
Resuspend the microsomal fraction of the Adora2a_amb in PBS. 5 μl aliquots of resuspended Adora2a_amb were mixed with 5 μl adenosine in PBS with final concentrations of 0 μM, 100 μM, 1000 μM, and 5000 μM adenosine (see Note 13 ).
10 μl samples were applied for the luminescence and fluorescence measurement. In a first step, the luminescence of the Nluc was detected using an OD2 filter. In a second step, the fluorescence of the coupled Bodipy dye, excited by the Nluc emission was detected.
As a control 5 μl of the resuspended microsomal fraction of the full-length Adora2a protein without any fluorescent label is treated with the same concentrations of adenosine (step 2) to determine background fluorescence caused by the broad emission spectrum of the Nluc and possible interactions of adenosine with the Nluc.
Herein, the calculated BRET ratio (Fig. 3) showed a change in the relation of the fluorescence of the Bodipy in comparison to the Nluc luminescence after the addition of different adenosine concentrations. Only a minimal increase of the BRET ratio can be seen after addition of higher adenosine concentrations (above 100 μM) indicating that at lower concentrations all receptors are occupied with adenosine. The result indicates a conformational change of the helix III and the connected third intracellular loop that is expected for the Adora2a.
Additional components are required for the recharging of the suppressor tRNA and a subsequent incorporation of a non-canonical amino acid. Therefore, add the p-propargyloxy-l-phenylalanine, tRNATyrCUA, and eAzFRS in a specific order (Table 2) (see Note 14 ).
Incubate the prepared cell-free reaction at 27 °C for 3 h by gentle shaking at 600 rpm. Keep the reaction in the dark (see Note 15 ).
The membrane protein is translocated and integrated into the microsomal membrane during the cell-free reaction. Therefore, separate the microsomal fraction by centrifugation at 16,000 × g for 15 min at 4 °C. Resuspend the pellet fraction in PBS. Use an equal volume of the cell-free reaction for resuspension.
For the labeling reaction prepare the labeling mix as follows: combine 200 μM CuSO4 with 600 μM THPTA, 5 mM NaAsc, PBS and a final concentration of 3 μM Sulfo-Cy5-azid to a final volume of 5 μl. Add 5 μl of the resuspended microsomal fraction to the labeling mix. Incubate the labeling reaction at room temperature for 1 h. Keep the reaction dark (see Note 16 ).
Centrifuge the labeling reaction for 15 min at 16,000 × g and 4 °C. Discard the supernatant and resuspend the pellet in 10 μl PBS. This step removes excess fluorescent dye to decrease the background signal in the subsequent fluorescent analyses.
The analysis of the synthesis of the full-length protein by using the Adora2a construct with or without an amber stop codon was performed by autoradiography and a reporter protein assay (Fig. 4). The autoradiography shows for both synthesis reactions a similar band pattern (Fig. 4a). Interestingly, the incorporation of pPa led to a comparable band signal as obtained for the full-length protein translated from the DNA construct without an amber stop codon. This result implicates a high incorporation efficiency of the non-canonical amino acid. In addition, no termination product is detected in the autoradiograph. The high incorporation efficiency is the basis for a further coupling reaction to a fluorescent dye. In addition, this result is supported by the Nluc assay (Fig. 4b). The measured luciferase activity of the suppression product reaches up to 80% of the luciferase activity of the full-length product. The protein yield and protein distribution (Fig. 4c) is comparable to the previously described results (Fig. 2, see Subheading 3.3.1, step 4).
Fluorescently labeled GPCRs should be detectable in microsomal structures (Fig. 5). It is recommended to visualize the labeled sample first by in-gel-fluorescence (Fig. 5, left panel). The synthesis reaction in the presence of all orthogonal components led to a specific band at the expected molecular weight. The control reaction without addition of the modified synthetase resulted in no visible band. The success of the microscopic analysis highly correlates to the quality of the in-gel-fluorescence. High background fluorescence during in-gel-analysis often results in unspecific staining of the microsomes. The microscopic analysis clearly shows a difference in the fluorescence intensity of the labeled Adora2a and the unspecific staining of the microsomes (right panel).
After the reaction is completed collect 2 × 3 μl of the translation mixture. Centrifuge the remaining mix at 16,000 × g for 15 min and 4 °C and collect 2 × 3 μl of the supernatant. Resuspend the microsomal fraction in an equal volume of PBS in comparison to the volume of the translation mixture. Collect 2 × 3 μl of the microsomal fraction.
Mix each aliquot with 3 ml TCA and incubate in a water bath at 80 °C for 15 min. Store the aliquots for 30 min on ice or overnight at 4 °C.
The mixture is applied to a vacuum filtration system to separate non-incorporated 14C-leucine from the radioactively labeled protein. Filters with the collected protein are washed twice with TCA and twice with acetone. Dry the filters under the hood.
The filters are transferred into the scintillation vials and overlaid with 3 ml scintillation cocktail. After an incubation time of 1 h with gentle shaking, scintillation vessels are counted in scintillation counter.
For preparation of SDS-PAGE samples take a 5 μl aliquot (Subheading 3.3.1, steps 1–3) or 10 μl site-specifically labeled aliquot (Subheading 3.3.3, steps 1–5) of each prepared sample. Add 45 μl water and 150 μl cold acetone to the 5 or 10 μl aliquots and incubate for 15 min on ice. Keep the fluorescently labeled samples in dark during the whole procedure. Centrifuge the samples at 16,000 × g for 10 min at 4 °C and discard the supernatant.
Dry the pellets for 1 h at 45 °C in a thermo mixer with a shaking speed of 1000 rpm.
Resuspend the dried pellets in 20 μl SDS-PAGE sample buffer and load the samples on a prepared 10% SDS-PAGE gel. Use a ladder with fluorescently labeled bands. Run the gel.
Transfer the gel to the fluorescence imaging system and detect the labeled protein bands. For Bodipy-TMR-lysine use a 532 nm laser and a 580 nm emission filter. Sulfo-Cy5 can be detected with extinction at 633 nm and emission at 670 nm.
Afterwards dry the gel for 60 min at 70 °C using a unigeldryer. The dried gels are exposed on a phosphorscreen for minimal 3 days and read out using a multi-mode imager.
For confocal laser scanning microscopy use 5 μl of the fluorescently labeled protein in the microsomal fraction and dilute the sample in 20 μl PBS. Add the mixture to a μ-Ibidi-Slide.
Fix the slide. Use a plan-apochromat objective with a 60× or 100× magnification. Microsomal structures usually have a diameter of 1–10 μm.
Adjust the beam path to the coupled fluorescent dye. Standard dyes such as Cy5 and FITC usually have a preset configuration. Cy5 is excited at 633 nm and the emission is detected with a long-pass filter above a wavelength of 670 nm.
Adjust the microscope settings (laser intensity, gain master, focus, pinhole) according to the individual sample (see Note 17 ).
Preparation of CHO lysate: The cultivation was carried out in a Biostat B-DCU II bioreactor (Sartorius Stedium Biotech GmbH) at 37 °C with a chemical defined and serum-free media. Cells were harvested at a density of 3.5–5 × 106 cells/ml by centrifugation at 200 × g for 5 min. The cell pellet was resuspended in 40 mM HEPES-KOH, pH 7.5, 100 mM NaOAc and 4 mM DTT. The cell suspension was passed through a 20-gauge needle to mechanically disrupt the cell membrane. Nuclei and cell debris were removed by a centrifugation step at 10,000 × g for 10 min. Raw lysate is applied to equilibrated Sephadex G-25 columns. Elution fractions with the highest measured RNA concentration were pooled and treated with micrococcal S7 nuclease to digest endogenous mRNA. The inactivation of the calcium-dependent nuclease was performed by adding 6.7 mM EGTA to complex the calcium ions. The lysate was further supplemented with creatine kinase (f.c. 100 μg/ml) to ensure the regeneration of ATP out of creatine phosphate. The prepared lysate was shock frozen in liquid nitrogen and stored at −80 °C.
The cell-free reaction in a dialysis mode is performed in a two-chamber device. The reaction chamber (1.1 ml, red lid) and the feeding chamber (11 ml, colorless lid) are separated by a semipermeable membrane with a molecular weight cut-off of 10 kDa. Whereas inhibitory byproducts such as accumulating phosphates are removed, amino acids and energy components are delivered to the reaction chamber.
It is recommended to collect samples of the translation mix, supernatant, purification steps including flow through, washing fractions and elution fractions as well as buffer exchange procedure and concentration. The aliquots can be diluted in SDS-PAGE sample buffer and loaded to the SDS-PAGE in order to monitor the purity of the aminoacyl-tRNA-synthetase during the preparation.
Concentrate the synthetase to a concentration of 5 g/l to ensure a minimal final concentration of 100 μM. If necessary repeat the concentration step.
The PCR product is purified with the QIAquick PCR Purification Kit and the concentration is determined by using a NanoDrop 2000c. For further analysis prepare a 1% (w/v) agarose gel and load 1 μl of the PCR product. The expected band size is 123 bps.
The prepared RNA can be analyzed by gel-electrophoresis run in 1× TBE buffer (Subheading 2.2.1, item 11). Therefore, prepare a 2% (w/v) agarose gel. Mix 2 μl of the RNA with 6 μl MOPS sample buffer (Subheading 2.2.2, item 6) and load the sample to the agarose gel. Use a RNA ladder. The expected band size is around 200 bps.
After centrifugation three phases are present: on top the aqueous phase with approximately 50% of the total volume, containing the RNA; a middle interphase that is nearly invisible and below the red phenol/chloroform phase. Try to isolate only the aqueous phase.
It is important that the components are dissolved completely to ensure the correct concentration. Vortex the components and store them in aliquots to avoid repeated thaw and freeze cycles.
The plasmid concentration can be varied and can be dependent on the chosen vector backbone. It is recommended to apply different plasmid concentrations in the range of 20–100 nM to the cell-free protein synthesis reaction.
The fluorescent dye is susceptible to light. An illumination will decrease the fluorescence intensity of the dye. Use colored tubes or wrap the tube with aluminum foil. In addition, keep in mind that the precharged tRNACUA will address the amber stop codon whereas the precharged tRNAGAA will address statistically phenylalanine codons.
The optimal temperature for cell-free protein synthesis reaction in a CHO lysate is 30 °C. However, a temperature series is recommended to determine the optimal conditions for an individual protein in terms of folding and activity. The Adora2a-Nluc construct showed the highest Nluc activity at 27 °C.
It is recommended to analyze the incorporation efficiency of the non-canonical amino acid. With a low efficiency the subsequent labeling reaction will yield as well in a low amount of labeled protein. Therefore, two different methods can be utilized.
Adding a reporter protein downstream of the amber stop codon. The reporter protein will only be translated if the amber stop codon is addressed by the tRNA and the non-canonical amino acid is transferred to the polypeptide chain. The intensity of the reporter protein signal is directly correlated to the amount of full-length protein and in conclusion to the incorporation efficiency. A comparison to a DNA construct without amber stop codon is possible.
Determine the amount of full-length protein with autoradiography. If the termination product has an adequate amount of incorporated 14C-leucine during the cell-free protein synthesis reaction and a detectable size, an additional band should be visible in the autoradiography. The ratio of full-length product and termination product can be calculated.
It is recommended to evaluate different agonist concentrations because at a defined concentration the binding sites of the cell-free synthesized Adora2a should be completely occupied resulting in a saturation effect in the BRET signal. The saturation effect supports the specific conformational rearrangement after ligand binding.
The non-canonical amino acid is solubilized in 0.5 M NaOH. The alkaline pH of NaOH will shift the pH of the lysate that might result in an inactivation of enzymes involved in protein synthesis. Therefore, dilute the ncAA in translation mix, water and components that are not affected by an alkaline pH.
Reactive groups of ncAA are often instable and susceptible to illumination. If the reactive group is inert a following coupling to a fluorescent dye is not possible. Therefore, keep the ncAA in the dark by using colored or wrapped tubes.
Copper has a toxic effect on proteins due to oxidative damage, but it is necessary as catalyst for the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC). It is recommended to use different copper concentrations for the labeling reaction. For the Adora2a_amb construct we have seen a highest labeling efficiency with 200 μM copper. The lowest copper concentration resulting in a detectable fluorescent band was 50 μM. Keep in mind that the concentration of THPTA has to be adjusted as well (threefold concentration of THPTA to CuSO4). In addition, it is recommended to adjust the incubation time of the labeling reaction. In general, CuAAC is a fast and efficient click reaction. A decrease in incubation time might enhance proteins activity.
The same microscopic settings that are chosen for the fluorescently labeled sample should be applied to the negative control to exclude an unspecific labeling of microsomal structures. Fluorescent dyes are usually highly hydrophobic and tend to stick unspecific to lipid membranes. It is recommended to evaluate the unspecific binding of different dyes to figure out the most suitable dye for certain applications. For our images a pinhole of 1 Airy unit (118 μm), laser intensities of 22% (bright field) and 8% (fluorescence) resulted in the optimal recording of fluorescently labeled Adora2a.
This work is supported by the European Regional Development Fund (EFRE), the German Ministry of Education and Research (BMBF, No. 031B0078A), and the German Research Foundation (DFG Priority Programme 1623).


text:
2010 Pennsylvania elections
Elections in Pennsylvania
Federal government
U.S. President
U.S. Senate
February 1814
December 1801
U.S. House of Representatives
5th sp
1st sp
12th sp
3rd sp
2nd sp
22nd sp
18th sp Jun
18th sp Dec
21st sp
23rd sp
at-large sp
7th & 15th sp
State government
Lieutenant Governor
Attorney General
Auditor General
State Treasurer
State Senate
State House Elections
Other municipal elections
Pennsylvania held various elections on November 2, 2010. These include elections for a Senate seat, a gubernatorial race, and many state legislature races.
1 Federal
1.1 United States Senate
1.2 United States House
1.2.1 Twelfth District special election
1.2.2 General election
2 State
2.1 Governor
2.2 State Senate
2.3 State House of Representatives
2.4 Judicial positions
2.5 Ballot measures
Main article: United States Senate election in Pennsylvania, 2010
Former Republican, now Democratic, Senator Arlen Specter was defeated in a primary election to Joe Sestak, who then faced Republican Pat Toomey. In a narrow race, Pat Toomey was victorious over Sestak.
United States House
Twelfth District special election
Main article: Pennsylvania's 12th congressional district special election, 2010
A special election was held on May 18, 2010 to fill the seat left vacant by the death of Democratic U.S. Representative John Murtha.[1] On March 8, 2010, the Pennsylvania Democratic Party's Executive Committee nominated Mark Critz, Murtha's former district director.[2] On March 11, a convention of Republicans from the 12th district nominated businessman Tim Burns.[3] The Democrats held the seat in the special election, with Critz defeating Burns.[4] Both would face each other again in November's general election, with Critz winning again.[5]
Main article: United States House of Representatives elections in Pennsylvania, 2010
All 19 seats will face an election. Pennsylvania is expected to lose one congressional seat after the 2010 census.
Main article: Pennsylvania gubernatorial election, 2010
A new governor was elected(incumbent Governor Ed Rendell (D) is term limited), Tom Corbett, the Republican, won the general election with 55% of the vote against the Democrat, Dan Onorato, who carried 45% of the final vote.
Main article: Pennsylvania Senate elections, 2010
State House of Representatives
Main article: Pennsylvania House of Representatives elections, 2010
Judicial positions
Pennsylvania holds judicial elections in odd-numbered years.
Pennsylvania judicial elections, 2010 at Judgepedia
At least one statewide ballot question has been proposed for the November 2 ballot:
1. Call for a Constitutional Convention
Pennsylvania 2010 ballot measures at Ballotpedia
^ Catanese, David (February 17, 2010). "Murtha special election set". The Politico. Retrieved February 17, 2010.
^ Becker, Bernie (March 8, 2010). "Dems Choose Nominee for Murtha Seat". The New York Times. Retrieved March 9, 2010.
^ Faher, Mike (March 12, 2010). "GOP chooses Burns for special election in 12th". The Tribune-Democrat. Retrieved March 12, 2010.
^ "Dem Critz holds Murtha's Pa. seat". Politico.com. May 18, 2010. Retrieved July 9, 2010.
^ Associated Press (May 18, 2010). "Critz to face Burns again in November". Tribune-Democrat. Retrieved July 9, 2010.
Voting and Elections at the Pennsylvania Department of State
Candidates for Pennsylvania State Offices at Project Vote Smart
Pennsylvania Polls at Pollster.com
Pennsylvania at Rasmussen Reports
Pennsylvania Congressional Races in 2010 campaign finance data from OpenSecrets.org
Pennsylvania 2010 campaign finance data from Follow the Money
(2009 ←) 2010 United States elections (→ 2011)
Delaware (special)
Massachusetts (special)
New Hampshire
New York
New York (special)
North Carolina
North Dakota
Democratic primary
South Carolina
South Dakota
West Virginia (special)
American Samoa
District of Columbia
New Jersey
New Mexico
Northern Mariana Islands
Rhode Island
U.S. Virgin Islands
West Virginia
Utah (special)
Delaware House
Iowa Senate
Maryland General Assembly
Massachusetts Senate
Montana House
New York Senate
Oregon legislative
Honolulu (special)
Long Beach
New Orleans
Orange County, Florida
San Jose
(Legislative)
This page was last edited on 12 January 2021, at 04:20


I don't know the boy dolls well, so didn't realise that was who he was when I bought him (I just thought he looked interesting). He is wearing a Moxy Teenz jacket and possibly Barbie jeans, so needs to be re-dressed. I'm not sure which version he is as his hair has been messed with a bit, but I don't really like either outfit!
These were produced by ToyBiz in 1996. There were babies and animals.
These are the modern version with rubber clothes.
The cat is in love with bunny and keeps licking it! The inner pocket goes all the way down to it's bottom, and it's so soft. These cost about £12 new and come in all kinds of colours.
What was within this old plastic picnic basket, I wondered? All sorts of goodies! There were two Mr Men jugs with lids, two Country Cousin plates with one cup, and an unknown set with teapot, two cups, four little plates and one bigger plate. The Mr Men and Country Cousin items are marked "Made in Italy by Suci". The other set has no markings at all.
All three series in a nice boxset.
I was looking for something with a strawberry or melon print to make some doll clothes from. I found both!