text:
SAN FRANCISCO, Oct 20- Microsoft Corp will provide free cloud-computing and research applications to qualified medical researchers working on the Ebola virus, the software company's chief executive said on Monday. Azure is the name of Microsoft's cloud-computing platform, essentially a group of datacenters that allow users to access large amounts of...» Read More
SAN DIEGO-- Southern California home prices posted a sixth straight annual increase in September as buying shifted to more expensive houses along the coast and the supply of heavily discounted foreclosed properties dwindled, a research firm reported Friday.
NEW YORK-- Soda makers, restaurateurs and other businesses sued Friday to try to block the city's unprecedented move to restrict sales of super-sized, sugary drinks, an effort the city called a coup for public health but the industry views as unfair and undemocratic.
Wells Fargo said Friday that its third-quarter net income and revenue rose as the bank increased consumer lending and collected more fees. In a call with analysts, Wells Fargo Chairman, President and CEO John Stumpf explained why interest income might not be such a grave threat.
NEW YORK-- Analysts expect that Mattel Inc.' s third-quarter net income and revenue to rise as shoppers snap up its Monster High and Barbie dolls, among other toys, ahead of the busy holiday season.
Major indexes closed little changed on Wall Street Friday. Banks fell after Wells Fargo missed analysts' revenue estimates. Falling stocks outnumbered rising ones nearly two-to-one on the New York Stock Exchange.
CHANGE INEVITABLE: With the outcome of next month's presidential election increasingly hazy, here's a shred of clarity that investors can cling to: The tax rates they pay on investment income like stock profits and dividends are almost certain to change.
NEW YORK-- Shares of China's Ninetowns Internet Technology Group Co. jumped on Friday after the company said it had received a preliminary, non-binding proposal from a group including the chief executive to take the company private.
WASHINGTON-- With the outcome of next month's presidential election increasingly hazy, here's a shred of clarity that investors can cling to: The tax rates they pay on investment income like stock profits and dividends are almost certain to change.
AG DRAIN: Wheat prices fell 3.3 percent as weekend storms are expected to deliver much-needed moisture to fields in part of the growing region. SLUGGISH OIL: Oil prices fell after the International Energy Agency predicted a slower pace of growth in demand over the next five years.
Wheat for December delivery fell 29.25 cents to finish at $8.5675 per bushel. The weekend rains are expected to help winter wheat crops that have been plagued by dry weather in western Kansas, western Nebraska and eastern Colorado.
NEW YORK-- Shares of XenoPort Inc. fell Friday after a Piper Jaffray analyst downgraded the stock, saying the company's experimental multiple sclerosis drug is years away from marketing approval or a development partnership.
BETHESDA, Md.-- DiamondRock Hospitality's fiscal third-quarter results improved as room rates climbed, but the company lowered its 2012 adjusted funds from operations forecast and issued fourth-quarter guidance below Wall Street's view.
PARIS-- The Paris prosecutor's office says it has opened a preliminary investigation into scarf and bag maker Hermes' claim that luxury giant LVMH Moet Hennessy Louis Vuitton engaged in insider trading and share manipulation when it took a stake in its rival.
NEW YORK-- Shares of discount store chain Dollar Tree fell on Friday after an analyst downgraded the stock on weakening sales trends. THE SPARK: Jefferies analyst Daniel Binder said in a note to investors that revenue in stores open at least one year, a key retail metric, have been decelerating at Dollar Tree.
TOP- LINE GAINS: Wells Fargo posted record earnings in the third quarter. Wells' net income in the quarter ended Sept. 30 rose 23 percent, to $4.72 billion from $3.84 billion in the same period last year. New mortgage loan originations rose to $139 billion from $89 billion in last year's third quarter.
NEW YORK-- A Citigroup analyst on Friday downgraded the shares of Seagate Technology PLC and Western Digital Corp., two leading makers of hard drives, to "Sell" from "Buy" because he sees weakness not just in the PC sector, but in corporate spending on servers and storage arrays, which also use hard drives.
WASHINGTON-- The federal budget deficit has topped $1 trillion for a fourth straight year. The Treasury Department said Friday that the deficit for the 2012 budget year totaled $1.1 trillion.

domain: finance


One topic that generates comments from time to time at our shoots consistently is the importance of reliability over accuracy. A lot of shooters want a truly accurate firearm. Yet, with accuracy often comes the penalty of reliability. In order to achieve a high level of accuracy and precision, these guns tend to have tighter tolerances, which means they malfunction more often than their less pricey counterparts.
In USPSA (United States Practical Shooting Association) type matches, reliability trumps accuracy. Scoring is a combination of speed and accuracy. Yet, when the A-zone, the highest scoring region on a cardboard target, is about 8 inches in diameter we can get by with a lower level of accuracy.
Malfunctions of any type cost you precious time. A close examination of the hit factor, which is simply the total stage points divided by time, shows that the both time and accuracy matter.:
stage_points / time
10 points/10 sec = 1 hit factor
10 points/9 sec = 1.11 hit factor
9 points/10 sec = 0.9 hit factor
So, you can see that missing the mark or taking too long costs you in hit factor.
Most modern handguns are capable of hitting the A-Zone at a standard engagement distance of 25 feet consistently, even with lousy bullets. Next time you head out to the range and practice on a plate rack, consider this. The minimum safe engagement distance for steel is 25 feet. The diameter of a plate is 8-inches, about the same width as the A-Zone on a USPSA cardboard target. So, if you are able to engage the plates quickly then you will be able to engage the A-Zones quickly.
How to Find a Reliable Firearm
The Internet abounds with information on the hundreds of thousands of rounds fired through many different types of guns as well as how many times they malfunctioned. Because of the ready prevalence of information on this subject, I suggest a search with your favorite search engine.
That being said, I prefer the late model guns, such as Glocks or Springfield XDs, They run super-reliable and with very little hiccups, unlike some of the more expensive and finicky firearms, like the 1911s-style handguns. You can literally drop a Glock magazine into the desert dirt and reuse it without having to break out the magazine brush and dismantle the magazine, a frequent sight with some of the more expensive and higher-tolerance firearms. In fact, I remember only having to clean my Glock magazines once a year. And this is coming from a guy who used to shoot at least 3 matches a month for a year straight!
Malfunctions, Oh My!
You should still learn how to clear malfunctions, if even from the comfort of your home, using inert ammunition and against a good backstop! They can and will happen. You will want to learn how to quickly clear a malfunction and move on. I have frequently seen many shooters become quite flustered whenever a malfunction occurs. Subsequently, they lose track of what they were doing on a stage, re-engaging a target previous shot, walking past a target without engaging it, or giving up outright!
But think about this. What do you think will happen when you make it a habit to give up at the first sign of difficulty at a match? That’s right. Give up clearing a malfunction on the range and you will give up in a real gunfight. A habit is a habit is a habit. I frequently encourage people to clear the malfunction because if you develop this habit of giving up during a drill or just a stage, you will give up in real life. Drill these habits into you. They will also save your life in a real gunfight, which I hope you will never find yourself in.
Choose Reliability Over Accuracy!
So, the next time a burgeoning newbie just getting into the exciting world USPSA asks you what type of handgun they should purchase, suggest recommending reliability over accuracy! Again, the Internet contains a multitude of information on this subject. Your local gun store, especially one with in-house gunsmithing service, will also be knowledgeable in this field.


text:
Or – “From Out Of The Asylum…”
Bluewater Productions gave us the initial Insane Jane miniseries back in 2008, and I have to say I was pleasantly surprised by the book. All at once fun, vaguely silly, and truly chilling, the Insane Jane mini introduced to a young girl named Jane who wanted to escape her humdrum life by becoming a superhero. The familiar premise went places that no one could have predicted, and ended with Jane having an entirely new life, albeit not the one she might have chosen…
Insane Jane -Avenging Star #1
Writer: Darren G. Davis & Zach Hunchar
Pencils: GMB Chomichuk
Cover Artist: GMB Chomichuk
Published by Bluewater Productions
Previously, on Insane Jane: Jane was a mousy young thing who wanted nothing more than to be amazing, spectacular, invincible and other adjectives that Stan Lee was fond of. A chance encounter with a convenience store robbery changed her life forever, filling her with zeal to save the innocent and become… The Avenging Star! With her homemade costume, Jane patrolled the streets fighting evil (or whatever passed for evil.) Her first interactions with a villain called the Shadowy Man sent Jane into a spiral of tragedy as bad things began happening all around her, leaving her life forever changed. After her initial foray into superheroics, Jane (whose last name I’ve searched for and can’t find in my copies of the original miniseries) has been left with scars physical and emotional, orphaned (through means that you honestly HAVE to see in order to believe) and under professional psychiatric care in an asylum. That is where this story begins…
My first impression of this book is how DIFFERENT the art is from the first mini. In her initial outings, Jane was drawn in a style that is reminiscing of Scott Kurtz work on ‘Captain Amazing,’ a soft-edged cartoony art style that evoked a sense of fun and wacky adventures (and really set off the climactic horrors of that first series nicely.) Jane herself was always presented in full color, while the people around her and her day-to-day grind were all black and white, a device that worked very well. This issue’s art is a whole new ballgame, an impressionistic, almost photo-collage that reminds me favorably of the work of Dave McKean and Bill Sienkiewicz. Jane starts the issue in her padded room, hallucinating her wonderful battles as The Avenging Star, before remembering that it’s all hallucination. “I need to make a change,” thinks our heroine. “I don’t want to end up like SOME people here… They’re crazy!” Heh… Given that she says this with her face still scarred by her own actions, there’s some black humor in that statement.
Indeed, there’s black humor throughout the issue, introducing (possibly) a new inmate named Grant who seems to share her love of costumed characters. Insane Jane has a really impressive stream-of-consciousness freakout halfway through the book that ends with her deciding that the nurse who gives Jane her daily meds is the source of all her troubles… and Nurse Gayman MUST BE STOPPED. Jane and Grant become partners in crime, and the issue ends with them making a run for the border in a beautifully illustrated sequence. To be honest, the whole issue is visually stunning, changing the black-and-white to color motif of the first Insane Jane miniseries and swapping it for a new dichotomy: the backgrounds and bit players take on the pea-green cast of the institutional paint used at the asylum, while Jane gets a lively pastel palette, and her hallucinations are drawn in a harsh (but appropriate) blood-red color. The issue also goes places that I never expected, places that the usual “hero feigning madness” stories could NEVER have attempted, allowing us to see something new and different that characters firmly established in a regular superhero reality simply wouldn’t be able to do.
My major concern with this issue is the VAST gulf in tone and art-style from the previous Insane Jane series, a change that could be off-putting to anyone who loved the cartoon adventures of the girl in the homemade cape, but even that doesn’t undermine how neat the visuals are. Setting this “superhero” character in an entirely mundane, real-world setting gives her a depth that could be fun to watch the creators play with, and I like the joy that she takes in her hallucinated hero adventures. It is, admittedly, a dark tale (though not yet as dark as the climax of the first miniseries) and as the first issue of four, it may not accurately portray what the whole mini is really going to be about, but it does give you enough to get you interested in who this girl is, what’s up with her backstory (a bit of business that is touched on multiple times, but never explicitly spoiled in the issue) and what kind of mischief she might be able to get into. The test of success with any title (but especially with many independents) is “Does this story remind me too much of anything else?” Aside from some naming similiarities (Crazy Jane of the Doom Patrol is similarly themed, but a completely different take, while Painkiller Jane… um… exists. I’ve never read any of her stories, but I have it on good authority that her conceptualization is different) this book isn’t a take on somethin we’ve seen before or a love-letter to another body of work. There’s still some (probably intentional) ambiguity regarding Grant and his motives, but overall, it’s an engaging book that I went through more than once just to goggle at the art. Insane Jane – The Avenging Star #1 is interesting, visually stimulating, and creepy-in-a-good-way, earning 4 out of 5 stars overall. Bluewater Productions just got this one on my pull list…
(Insane Jane – Avenging Star #1 will be available in August 2010, and can be ordered out of the current June 2010 issue of Preview.)
Faithful Spoilerite Question Of The Day: Why do so many comic fans seemingly buy their books based on publisher? All Marvel titles aren’t alike, all Image titles aren’t alike, and in this day and age there’s little to no distribution gap to force you to pick one publisher over the other. How does one justify picking something up just because of the DC swirly or the big red R of Radical Publishing?

domain: artistic


text: In Payback! Eric Esor, a former law enforcement agent, forensic specialist, and investigator, following serious on duty injuries, is forced to change careers. Eric learns that his sixteen year old adopted daughter, Briana, was raped by a person who prefers chubby girls. Brianas protection from the boogeyman is to be thin. She turns to an unhealthy peer group, an eating disorder, and experimenting with illegal methamphetamines. Briana is not forthcoming with the police or her parents about being raped, leaving Eric to use his investigative and forensic skills. For more than two years, Eric conducts a lonely hunt for his daughters rapist. His skills pay off but his efforts taint evidence so that it is useless in traditional jurisprudence. Eric Esor now must decide whether or not to become the antithesis of himself. Could he be the polar opposite of the good citizen, son, husband, father, provider, brother, socially active, and well-educated teacher? Eric must decide whether to be investigator, prosecutor, judge, jury, and perhaps executioner. In other words, should Eric Esor seek Payback? If so, how, when and where will he accomplish this? If he does, he will have to leave absolutely no trace behind in order to return to his normal life. The question to be answered in Payback! is, Is it a time to kill?

domain: law


text:
Greens Lake Protection and Marsh Restoration - Phase II
Ducks Unlimited, Inc.
DWH Project Funding
Known Leveraged Funding
National Fish and Wildlife Foundation (NFWF)
NFWF Gulf Environmental Benefit Fund GEBF
Engineering and/or Construction
Complete construction of 3.3 miles of shoreline protection breakwaters along a highly-eroded section of the Gulf Intercoastal Waterway (GIWW) adjacent to the Greens Lake Marsh Complex.
Texas General Land Office

domain: geography


Statement of Wm. McC. Martin, Jr., Chairman, Board of Governors of the Federal Reserve System, before the House Committee on Banking and Currency, on H. R. 4413 March 12, 1963 I appear today in response to your invitation to present the views of the Board of Governors of the Federal Reserve System with respect to H. R. 4413, which would repeal the silver purchase laws and provide for replacement of silver certificates with Federal Reserve notes. The Board believes that it is unnecessary to utilize sil¬ ver as part of the United States monetary system, other than as a material for coinage. There is no need, therefore, to retain the silver purchase provisions that would be repealed by H. R. 4413. As a practical matter, these provisions are inoperative today, because of the rise in the market price of silver. The Board favors the proposed amendment in section 3 of the bill, which would authorize issuance of Federal Reserve notes in $1 denomination in addition to the denominations of $5, $10, $20, $50, $100, $500, $1,000, $5,000, and $10,000 now authorized. Since the bill as introduced would authorize the Federal Reserve System to issue notes in all present denominations of currency except for $2 bills, we recommend broadening the bill to cover $2 notes as well. Although the Board is not in a position to comment on the technicalities of the bill's tax provisions, we perceive no objec¬ tion in principle to repealing the tax on transfers of silver bullion. -2- If this bill is enacted, it is important that the resulting shift from silver certificates to Federal Reserve notes take place gradually. Roughly $2 billion in silver certificates are outstanding. A complete shift of this amount to Federal Reserve notes would reduce the Treasury's free gold stock by $500 million, because of the 25 per cent gold certificate reserve requirement on Federal Reserve notes. I am pleased, therefore, to note Secretary Dillon's statement yesterday that the increase in the required gold certificate reserve resulting from the retirement of silver certificates and their subsequent replace¬ ment with Federal Reserve notes should not exceed $35 million a year. Although some concern has been expressed that removing the silver "backing" from part of our currency might lower its value, I would not agree. The fact is that the stability or instability of prices in our economy does not depend on the amount of silver in the Treasury, The relatively small part of our total money supply repre¬ sented by silver certificates does not derive its value from the silver the Treasury must hold as ''backing" for the certificates. Throughout the history of the silver purchase laws that this bill would repeal, the dollar has been worth more than the silver in it. This is still true today, even after the recent steady rise in the market price of silver. So it would seem that public acceptance of silver certificates must rest on their appraisal of factors apart from the silver "backing". This is further demonstrated by the fact that the public accepts Federal Reserve notes as readily as silver certificates. About $30 billion of Federal Reserve notes are in circulation--fifteen times the amount of silver certificates circulating. -3- It is possible, of course, that the market price of silver could rise above its monetary value if the Treasury's supply of free silver should ever be exhausted. H. R. 4413 is designed in part to avoid the problems that could arise in that eventuality. If this bill is not enacted, the Treasury must continue to issue silver certificates to meet the public's need for $1 bills. Under those circumstances, if the market price of silver went high enough to encourage the public to turn in silver certificates for silver dollars, to be melted down for metal, the Treasury would be faced with the impossible task of trying to meet the public's need for $1 bills by issuing a certificate that would be exchangeable for dollar coins con¬ taining more than a dollar's worth of silver. Consequently, silver certif¬ icates would soon be returned from circulation. This would not only add significantly to the operating costs of the mints and the Federal Reserve Banks, but would also thwart, rather than serve, the public's need for a stable medium of exchange. Unlike gold, the Treasury's stock of silver cannot be used to maintain the role of the dollar as a key international currency, because silver is not a readily acceptable means of settling our accounts with other countries. Offhand, one might suppose it would bolster the value of our currency to keep a valuable commodity such as silver in the stockpile of Government assets. But without this bill, the Treasury sooner or later will be forced to buy more silver for silver certificates, in competition with other buyers who seek it for other uses. It can hardly be supposed that the Government will find itself in a sounder financial position for having -4- been forced to acquire an asset by simultaneously adding an equal amount to its debt, as would occur under these circumstances. There is no point now in renewing the historic controversy over the desirability of the silver purchase program as a means of assuring a favorable price to producers of silver. Today, because our economy has many other uses for this metal, its market price is well above that guaranteed by the silver purchase laws. The time seems ripe to take this step toward a free market in silver.


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VPNArea is a Bulgarian VPN provider hosted in Switzerland. The company was launched in 2012 owned by Offshore Security LTD and they have been regularly updating their software. It provides worldwide servers and delivers secure network at impressive speeds on users’ devices for various purposes. Any of the servers guarantee complete anonymity when surfing the web & or using any geo-restricted services. The VPN utilizes blowfish/AES encryption, all major protocols as well as other advanced technology to keep your online activities private and safe.
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7-day Refund Guarantee
VPNArea does offer a 7-day refund. If you are not satisfied with the service, you can request a refund by sending as email. There are no sneaky “can’t have used more than x amount of bandwidth”-type exclusions. The provider claims on its website that they have never refused a refund request. They have confidence in their product.
VPNArea is one of the best VPN services when it comes to features and affordability. It offers top of the line servers to guarantee you the best experience. You are able to browse freely without limit from anywhere. The bank-grade encryption keeps your online data and connections complete safe. No one can access your information without your authorization. It offers friendly price which starts from $2.99 USD per month. One single plan comes with highest encryption, unlimited bandwidth, dedicated IP option, anti DNS leak, great online protection, a seven day money back guarantee, multiple platform software, instant activation and much more.
Once you finish payment, the website immediately displays a clear page with username, registered email address, and a link to the Members Area where you can log in. Within seconds an excellent welcome email arrived with extra details including links to the main clients and advice on where you can go for support. Once you install and set up VPN, it works for you immediately.
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You can get access to the fast VPN network for great experience. VPNArea develops infrastructure servers in 70 countries with blazing fast 1 Gbps+ speeds. VPN servers are needed for different purposes. Some servers are optimized for streaming to bypass Internet censorship and other restrictions so that you can access your favorite content at home and from abroad. Some servers are specially developed for games and P2P sharing to achieve private and fast connections. The provider also offers special servers, including Double VPN and Stunnel to every user’s need.
High Encryption & Advanced Protocols
To ensure your connection and device get unbreakable encryption, VPNArea utilizes AES-256 cipher with 4096-bit RSA keys and SHA-256 algorithm. This is the strongest encryption configuration available to the public. Though the hackers are good at attacking users on the Internet and ISPs are able to monitor record Internet users’ activities, they can’t decode your content. All they see is encrypted sessions.
VPNArea supports OpenVPN, L2TP and PPTP. OpenVPN is the latest and most secure one. It should be your first choice. If your device does not support this option, you can download the third party OpenVPN software from app stores. The software is free, safe and very easy to install. If you only want fast download speed, PPTP is good. This provider keeps updating their technology to supports forward secrecy. You will always get the best protection.
Unless you use a VPN service, all your traffic flies through the cyber space naked and vulnerable. Users who know little about cyber attack and Identify theft may feel safe browsing on mobile device in a coffee shop or bar. From your ISP to public WiFi snoopers and hackers, there is always someone after your data. The hackers on the unsecured network can spy on everything you look at or type in.
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You simply need to click “Connect” on VPNArea client, and the restrictions just fall like dominoes. You will no longer be a victim to restrictions imposed by web sites, streaming platforms, ISP, company/government firewalls. Enjoy all of your favorite websites safely with VPNArea.
BBC iPlayer & Netflix
BBC iPlayer and Netflix are typical services that they both have geo-restriction and block VPN access. BBC iPlayer is normally restricted from those outside the United Kingdom while it has many fans all over the world. Netflix is a very popular steaming service while you can’t access it outside US. That is very upset as many users have to move to another country for work. Users in other country also want to watch different videos on this great platform.
To access your favorite show, you need VPNArea which is one of the rare VPNs that are still able to unblock BBC iPlayer and Netflix abroad. It is an important feature needed to be considered when selecting a VPN. Its dedicated IP, smart servers and advanced protocol all work together to give you excellent streaming experience on BBC, Netflix and Hulu etc.
Privacy & Incognito Surfing
VPNArea provides new IP addresses from different countries and regions to keep your information private on the Internet. As your IP address is essentially your fingerprint on the Internet which reveals your location and other personal information, this VPN masks and make it virtually impossible for a hacker to target you. The advertiser can track you and deliver annoying ads when you surfing the Internet. The blocking ad feature of this VPN stops your ISP from seeing what addresses you visit with provided DNS servers.The ISP, employer, university or government will not know who you are and what you are doing on the Internet.
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P2P/Torrents/Gaming Optimized & Unlimited Bandwidth
VPNArea is friendly with P2P, torrents and gaming by providing unlimited bandwidth and optimized servers. Some VPNs do support P2P and torrents as they are illegal in some countries, but this VPN helps you do torrent fast and securely. With special streaming servers and double VPN servers, the ISPs or other parties will not know who use P2P/torrent. Your connection goes through 2 of its servers which is double protection. It is necessary to remind that servers in Austria, Australia, France, Japan, Singapore, Sweden, United Kingdom, The Isle of Man and Iceland do not work with P2P/torrent. The rest servers are fine with them. You can download anything you want as it allows unlimited bandwidth.
As some games require certain servers and good connections, you may not be able to play the hit games. You can choose the suitable server provided by VPNArea to make you seem to be from a location where the games have best performance no matter where you are located. You not only can play the game, but also be able to enjoy the fast and stable experience without noticing the buffering.
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For advanced users, there are powerful settings available. Just change the settings according to network and purpose for the best performance. This VPN also works on Chromebook, DD-WRT internet routers, Merlin Firmware and Tomato. As the provider allows a maximum of six simultaneous connections, you can protect all of your devices or share your account with family and friends.
You can contact VPNArea support staff via email, Skype, and live chat. By the way, the live chat option doesn’t operate 24/7
VPNArea company hosts its emails and servers in Switzerland which is the perfect location for privacy. It is well-known that it is a country which highly respects users’ privacy useful laws for privacy protection. They are not subject to European data retention laws. The government cannot force them to provide information, even with a subpoena. Besides, VPNArea does not log any of your traffic, let alone providing your log information to the third parties.
|VPN Brands||Servers||Country||# of Connections||Price(USD)||Official Website|
|3000+ servers in 94 countries.||British Virgin Islands||5||$6.67/Month
|5570+ servers in 60+ countries.||Panama||6||$3.49/Month
|200+ servers in 70 countries.||Bulgaria||6||$4.18/Month
|Servers over 34 countries & Still work in China.||Czechia||5||$4.88/Month
After reading this VPNArea review you understand it is a reliable VPN service that you can trust. Its security features on offer are excellent. It uses a 245-bit AES encryption algorithm along with OpenVPN to encrypt your online sessions. Even the government can’t decode your connections. The large number of servers assists you to access any restricted content without leaving trace. No more government ban or streaming limit. The state of the art protection & equipment of this VPN ensures that you’ll always have the best online experience wherever you are connecting from. VPNArea does not keep any logs of your activities. The VPN app also protects users from DNS leaks and the addition of a Kill Switch makes sure the users remain secure even when offline. The dedicated IP address and auto-IP changer also help you keep anonymous. That means your privacy is completely safe. In addition, the VPN company does try to make up for it with a seven-day money back guarantee and the customer service is also decent enough. They are reliable. Currently, its 36-month plan is 70 % off. It only costs $2.99 USD per month. You can receive the amazing online protection, excellent network speeds, ultimate online freedom and much more by spending less money. Save 70% on VPNArea membership now.
Published by & last updated on February 27, 2020 12:43 am


text:
For the sake of full transparency, let me begin with saying that I have never played a farming simulator before and Stardew Valley probably doesn’t count. However, I tackled Farming Simulator 19 with an open mind and had some big dreams. I wanted to create a place called “Tegrity Farms” and name my character Wessel “Randy” Minnie, and I wanted to face the hardships that farmers face, from predators hunting their livestock to battling against the elements.
After roughly 20 hours with Farming Simulator 19, it was clear that my dreams wouldn’t come true. Not meeting some of my high hopes, however, doesn’t necessarily mean that Farming Simulator 19 is a failure. In fact, there were times that I did enjoy my time on the farm, as the game does deliver a detailed and realistic experience.
The game taught me the intricacies of farming throughout the experience as I struggled to maintain my crops, worked the fields with a variety of vehicles and got my hands dirty. However, I also struggled due to poorly implemented gameplay mechanics and some bugs that made the experience a frustrating one.
Farming Simulator 19 gives you some short tutorials on how to, for example, cultivate the land, plant seeds and cut down trees. Then, when you pick the career mode, it gives you a short guided tour, which lasts around 20 minutes. This is where one of my biggest problems with the game occurred. If you aren’t a farming enthusiast, to begin with, these tutorials won’t be enough, not by a long shot. It is almost as if the game assumes you have played multiple Farming Simulators before. After finishing the guided tour on the easiest difficulty, I didn’t know what to do next. There was no indication what I should be doing, or how 95% of the game mechanics work.
For example, I only knew how to grow, harvest and sell wheat. However, there are a dozen types of plants I could grow, I could even grow trees to forage wood to sell. Then, there are also several livestock which wasn’t even mentioned in the guide, including horses (which are apparently new to the franchise) as well as a tonne of heavy machinery that I could buy and use, but I didn’t know how, and figuring it all out became a massive chore.
Here’s one example of what you need to do just to harvest some plants. First, I needed to cultivate the land to prepare it for sowing. This was done by getting into a tractor, then attaching two pieces of equipment to it. Then, I had to lower the weight piece so that I could actually cultivate the land by driving over it. Next up, I had to connect a sowing machine, connect the seed pallet and then turn on the machine, all while not forgetting to lower it. Then, there is a waiting period for the crops to grow. You can speed this process up by tuning how quick the in-game time passes. After the crops grew, I had to use a combine-harvester, and untold the front piece of the machine, then turn it on and drive slowly over the crops to harvest it. I then had to drop my harvest in a tipper and even had to manually move the pipe from the harvester to do so. Lastly, I drove the tipper to the silo to store my grain.
Farming Simulator 19 sure has a lot of detail in every process and combined with over 300 vehicles and even user created content via mods, it could be a farming enthusiasts dream. However, the game’s UI is very poorly designed and after the tutorial I had no idea what most of the tabs meant. The game didn’t teach me that I could borrow money, how to build new structures like farmhouses, or even how to get a dog.
Hours of trial and error ensued as I desperately tried to figure things out and finally managed to build a place for some chickens, which I picked up in town. Yes, there’s a town where you can purchase a bunch of stuff from, but the town is pretty dead. You only see vehicles driving about, but things like the restaurant and all shops have no NPCs in them. That’s where a big issue with Farming Simulator 19 comes in. The game tries to be so realistic and in the aspect of working the lands, it certainly is. However, in other aspects, it fails at realism, which is the whole point of a simulator, isn’t it?
For example, when picking up wood, you don’t see arms or anything like that, you just see the object floating in the air. You can’t open any doors of your farmhouse (there is one permanently open, even at night!) and there are no hardships to really overcome. I would have loved nothing more than to have events take place where I needed to protect my animals from predators or to hunker down as a tornado approached, or just to fight the fight against drought. However, nothing happened and I just farmed away with no real goal in sight. Just because a game is a simulator, doesn’t mean it can’t be fun and where Farming Simulator 19 fails the most is in providing any kind of fun in the whole experience.
As I mentioned before, the UI is a mess and this can be seen from the map portion to buying land and everything in between. Even fast-travelling to a shop was a chore to figure out, but at least the day-to-day work was relaxing at times. You see, I had to do it all myself and you might be thinking, why not just hire some workers? The game does give you the option to do so, but every time I hired one by entering a vehicle, starting the processes of, for example, sowing seed, and pressing the hire worker button, something went wrong.
The worker appeared to be doing his job and then just stopped midway through, with an unknown error occurring. Then, there’s also texture pop-ins even inside a building and some graphical glitches with flickering to push through, as well as a few framerate drops on the PS4 Pro.
The game also boasts a 6-player multiplayer experience where you can work together. I thought that would really be the best way to play the game as it felt very lonely on my farm. However, the screenshot below showcases my entire multiplayer experience after 3 days of constantly trying to connect.
It’s a real shame that the multiplayer didn’t work and I did see that some servers had 2/6 players on them, but I simply couldn’t connect so I can’t speak to the quality of the multiplayer and what you can do at all.
Farming Simulator 19 can be relaxing at times and I did like the great music featured in the game. It is an ultra-realistic game in the day-to-day aspects, but if you want to make a realistic game, you should go all out. There was nothing exciting about the gameplay and I really wanted to love it and have a relaxing time farming. However, the lack of a decent tutorial or guided mode made everything feel like one big chore.
I’m not quite sure who Farming Simulator 19 is aimed at. Real farmers would probably not want to spend their off time playing this game, not with all the issues. It could be for the farming enthusiast with a lot of time on their hands and a capable gaming device, but then again it feels so inaccessible if you haven’t played previous games in the series.
The only real positives about the game is its music and the attention to detail put into day-to-day farming work, and I simply don’t know if that is enough.
This review was based on a code sent to us by Focus Home Interactive
Available On: PS4, Xbox One & PC | Reviewed On: PS4 Pro | Release Date: 20 November 2018 | Price: R779

domain: hobby


Morgan Housel of The Motley Fool writes a good piece on how difficult it can be buy and hold the very best performing stocks over time. As Housel points out, the best performing individual stock from 1995-2015 was Monster Beverage — up 105,000% over that 20 year period. That return would have turned $10,000 into a cool $10 million.
But, as Housel points out, Monster has been an incredibly volatile stock, so volatile that most investors would not have been able to stay invested in the shares. He writes, Monster “traded below its previous all-time high on 94% of days during that period. On average, its stock was 26% below its high of the previous two years. It suffered four separate drops of 50% or more. It lost more than two-thirds of its value twice, and more than three-quarters once.”
And the pattern is not just with Monster, as Housel highlights. He looked at the top ten performing stocks over the past two decades and all of them “spent a majority of the time well below their previous high mark. They all had multiple declines of 50% or more. A few had multiple 70% drops.”
What can investors learn from this? Housel outlines three takeaways:
1) “Stories change, and nothing is perfect” – it’s easy to look at the best performing stocks today and think the story behind a firm’s success make total sense, but as Housel explains, what looks like a no-brainer, “slam-dunk” story today may not have looked that way when one studies a company’s actual history and the bumps in the road that all firms take.
2) “Investors underestimate how common and severe volatility is, especially among individual stocks” – investors need to understand that individual stocks can, and will, vary widely. If you can’t handle and accept that, you should be diversifying away that stock specific risk.
3) “Accepting this, even at the whole-market level, is the name of the game.” Housel concludes with a quote about Charlie Munger. Munger, when asked in 2009 about Berkshire Hathaway’s 50% decline, said he wasn’t at all concerned about the stock’s drop. Mr. Munger said “you can argue that if you’re not willing to react with equanimity to a market price decline of 50% two or three times a century you’re not fit to be a common shareholder, and you deserve the mediocre result you’re going to get compared to the people who can be more philosophical about these market fluctuations.”


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Baku, August 14, AZERTAC
In his interview with CNN Turk TV channel, President of Azerbaijan Ilham Aliyev highlighted the reasons why the second Karabakh war ended in victory.
“Of course, many factors have played a role here. First of all, of course, the determination of our people, their uncompromising position and our policy. I have been president for almost 18 years, and for all these years I have been saying that we must and we will liberate our lands at any cost. We simply had to do it at the right time, and we did it. We couldn’t afford to make a mistake because this is historical responsibility, and therefore our timely steps have led to such an outcome. The uncompromising spirit of the people is, of course, one of the primary factors. Because we had not forgotten Karabakh. Even the kids who have never seen Karabakh and were born in tent camps lived with a dream and desire to return to their homeland. It was thousands of young people like that who were ready to die,” the head of state said.
“The strengthening of our army, the weapons, equipment and ammunition are also among the key factors. In recent years, weapons based on the most modern technology have been purchased and the professionalism of our army has further increased. Because weapons and technology solve many problems, of course. But it is soldiers who liberate the land and raise the flag there. If they do not go to war with enthusiasm, no weapon can help you. These factors led us to Victory. Our military strategy and the acquisition of weapons served one goal – to liberate these lands with minimal losses, and this is exactly what happened. No-one could believe that our losses were less than 3,000 people. Everyone's life is precious, but the fact that only 2,900 people were killed in this large-scale operation shows, of course, that we tried to protect the lives of every soldier until the last moment,” President Ilham Aliyev noted.
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domain: politics


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This Cornwall painting is a piece of original art painted with acrylic on canvas by Cornish artist John Dyer.
Come and take a stroll through the beautiful city of Truro on a cold winter's day with this delightful painting by John Dyer.
The snow is falling gently all around, draping the buildings and streets in a blanket of white. A couple enjoys a peaceful walk with their dog, while seagulls glide effortlessly through the darkening sky. The lights in the windows create a warm and inviting scene.
This charming painting is sure to add a touch of magic to any room. It would make the perfect gift for someone special or a lovely addition to your own home.
This piece of Cornish art, 'Flurry of Snow, Truro Cathedral' by John Dyer, and other original paintings of Cornwall that are for sale on our online Cornwall art gallery, can be purchased securely online. We provide free worldwide delivery to your door.
||Framed Original Painting
||Cornish Artist John Dyer (born 1968)
|Signed by the Artist
||Signed by John Dyer
||12 x 10 inches
||Acrylic on canvas
||17 x 15 inches
||John Dyer: In the 1950s simple neutral frames became popular for St Ives artists. John chooses an off-white so the lightest tones appear in the painting. The moulding is a variation of 'Salvator Rosa' from Italy. Hand-finished in almond white paint.
|Ready to hang
||Strung with picture cord and ready to hang
Artist Information Links
Read about artist John Dyer
Artist Chronology I Paintings in Public Collections I View John Dyer's Latest Paintings


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MPs will receive a pay rise of 3.1% from April this year, the Independent Parliamentary Standards Authority (IPSA) has said.
The increase, announced on Thursday, will raise MPs’ basic pay from £79,468 to £81,932 and will be effective from 1 April.
It follows a decision confirmed in 2018 by IPSA to adjust the pay of MPs at the same rate as public sector earnings, which are published by the Office of National Statistics.
The announcement marks the first time MPs’ salaries have exceeded £80,000 and is an increase of £16,194 compared to the £65,738 salary in April 2010.
Many MPs who hold other duties receive an additional salary, including chairs of select committees and ministers. Select committee chairs will now be given a further £16,422, up from £15,928 in April 2019.
IPSA has no role in setting ministerial salaries. Cabinet Office figures show in 2019 the Prime Minister was entitled to a second salary of £79,286, while senior ministers including the Chancellor and Secretaries of State were entitled to up to £71,090. The Attorney General is entitled to one of the highest salaries in government, at £98,921 in April 2019.
We’re a thorn in the side of the establishment, but we can’t do it without your help
Your fight is our fight. But as many of you will know, speaking truth to power has never been easy, especially for a small, independent media outlet such as the Canary. We have weathered many attempts to silence our vital opposition to an increasingly fascist government and right-wing mainstream media. Now more than ever, we need your support.
We don’t have fancy offices, and our entire staff works remotely. Almost all of our income is spent on paying the people who make the Canary’s content. So your contribution directly supports our team and enables us to continue to do what we do: disrupt power, and amplify people.
But we can’t do this without you. So please, if you appreciate our work, can you help us continue the fight?

domain: news


The trucking company Knight Transportation has revised downward its financial expectations for the third and fourth quarters of 2013.
Based on preliminary results, Knight now expects earnings per diluted share will range from:
- $0.18 to $0.20 for its third quarter ended September 30, compared with its prior estimates of $0.22 to $0.24.
- $0.20 to $0.23 for its fourth quarter ending December 31, compared with its prior estimates of $0.22 to $0.25.
A number of factors negatively impacted the third quarter of 2013, it said in a release, which has resulted in expected earnings below original estimates. “We experienced lower miles per tractor year over year, despite having 3.6% less trucks in the third quarter of 2013 than in the third quarter of 2012, revenue per mile improvement fell short of our targeted level, and increased driver recruiting, training, and payroll costs,”
Soft freight demand, particularly in the first half of the quarter, challenges related to recruiting qualified driving associates, and the new industry-wide regulations governing hours of service that went into effect in July 2013 were among the factors affecting the quarter, said Knight
''In recent weeks, we have seen improvement in driver recruiting, which reduced the number of unseated tractors, and have begun to see pickup in some previously soft freight markets,” said Kevin Knight, chairman and CEO. “We believe that our industry-leading operating efficiency positions Knight for continued success, and enables us to capitalize on growth opportunities during these challenging times in our industry.''
Knight did not provide any update about its pursuit of Arkansas-based trucking company USA Truck, which has been rebuffed by the company.


EC-OG Launch Crowdfunding Investment Campaign
Aberdeen-headquartered subsea energy storage and energy generation specialists have launched a crowdfunding campaign to kick-start the energy transition and support ambitious growth plans.
EC-OG, the creators of the Subsea Power Hub technology, are seeking support from the public to bring the business’ scale-up plans to reality as they set out to decarbonise Oil & Gas operations in the North Sea.
A clean and renewable energy source, the Subsea Power Hub is designed, programmed and built by the in-house team of electrical, firmware, software and mechanical engineers. The EC-OG team have also developed proprietary lithium-ion battery technology, which forms the basis of the Subsea Power Hub’s Intelligent Energy Management System.
The target for the crowdfunding campaign is £150,000, with minimum investment of £50 to the Invest platform on the EC-OG webpage https://ecog.sharein.com/invest
The Subsea Power Hub device combines the core technologies of subsea energy storage and energy generation to deliver autonomous electrical power to remote subsea locations without a power cable link to surface or shore. Clean energy is produced directly at the point of demand, signifying a reduction in CO2 output when compared to traditional power delivery systems. The Subsea Power Hub was successfully tested at the Shapinsay Sound test site at the European Marine Energy Centre (EMEC) in 2017.
The technology can be applied to a number of applications, including Subsea Automation and the Subsea Internet of Things (SSIoT). The firm recently demonstrated the Subsea Power Hub’s capabilities for powering subsea production equipment using battery power alone.
EC-OG has already experienced significant growth since being established in 2013. The firm has grown from 7 founding team members to 25 employees today, with the support of £1.5 million public sector funding and £2.5 million private sector funding. The crowdfunding platform launch signifies the company’s shift to a fully commercial organisation.
EC-OG co-founder Richard Knox said, ‘We’re asking the public to get behind EC-OG, our team and be part of accelerating the energy transition. The Subsea Power Hub will play a crucial part in decarbonising oil & gas operations and will importantly bring energy storage technology to the subsea environment. We want to share this journey with investors and allow them to be part of our development and the commercial opportunities that lie ahead for us.’
Your capital is at risk and investments are not covered by the Financial Services Compensation Scheme (FSCS). Please read the full risk warning on https://ecog.sharein.com/risk before deciding to invest.


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It’s not just a trip to the dog park. It’s a pet parent’s support system. We provide your pet with exhilarating exercise and loads of affection. We provide you with peace of mind.
We take pride in limiting our pack size to 10 or fewer dogs. This allows our staff to stay in a positive frame of mind and keep your pets safe and happy.
We supply daily photos of every dog park outing showcasing your pet’s adventure with his friends. We keep in close contact about your dog’s health and behavior, and we’re always fine tuning his social skills for you. Our pack leaders are professional, accountable, and appreciate your dog as a companion and equal.
Premier Pet Parents get $2 off every outing!
Premier Pet Parents are clients on a recurring schedule with monthly billing and a valid card on file for payment. That’s up to $40 off per month! What a treat!
This unique service is available Monday through Friday afternoons. A fantastic heart pumping time for social dogs who need to run, or just love the company of other dogs! Your pooch will enjoy a minimum of 60 minutes of off-leash time at Marymoor and a ride to and from the park with his friends.
|Dog Park Day (one dog) …………………………$26*
*Regularly $28, but $26 for Premier Pet Parents!
|Dog Park Day (two dogs) ………………………… $42*
*Regularly $44, but $42 for Premier Pet Parents!
Q: What do the dogs do at the park?
A: They run, chase each other, play, sniff the scents of wild animals who passed through, swim, eat treats, and explore for an hour.
Q: How do you keep track of all the dogs? Is it total chaos?
A: Absolutely not! 40 acre Marymoor dog park on a weekday afternoon is very low key. The park is pretty empty, and most dogs in attendance are being handled by professionals. Dogs in our groups are taught to come when called and are actively supervised by our permitted professional dog walkers who have animal behavior training and lots of experience (and they carry GOOD treats!). We keep the pack moving throughout the park which keeps them together as a group with their leader. Quite different from the chaotic sunny Saturdays at the dog park, when every wound up dog in the Seattle Metropolitan area heads to the same place, at the same time, with amateur handlers. Those sunny Saturdays make us squirm too.
Q: Who is in my dog’s group?
A: A consistent little family of friendly dogs who know each other and know their handler! The great thing about Le Pouf is that we have multiple packs, so if your dog isn’t getting on well with his pack, he can switch to a different little family to find the right fit.
Q: Will my dog get sick from going to the Dog Park?
A: Healthy, immunized dogs derive far more benefit than risk from dog parks. We’ve been guiding packs through Marymoor since 2005, and we have not had greater incidence of illness among dogs in our Dog Park packs when compared to dogs who go on leashed walks. The key here is common sense: if your dog loves to eat poop or rocks, is immuno-compromised, or has not been fully immunized, we do not recommend that they attend a dog park or even walk on a well-used trail.

domain: pet


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Tag Archives: nature
Not far from the hustle and bustle of Hanoi, you can discover spectacular landscapes with karst terrain, canals, caves and rice field stretching along the Ngo Dong river. Make sure you explore this natural site by boat to appreciate breathtaking … Continue reading
No description needed. Amazing and almost empty beaches, warm sea, little bars with Hanoi beer, greenery, slow life, chill days.. Thanks for stopping by!
“Where are you going for your holidays this year?” “To Vietnam.” “Oh, amazing! Who are you going with?” “No one.” “Are you going by yourself?” “Yes.” “Hmm, but how do you do that?” “Well, most probably I get on the … Continue reading
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During the search for haloarchaeal viruses, we isolated and characterized a new pleomorphic lipid-containing virus, Haloarcula hispanica pleomorphic virus 1 (HHPV-1), that infects the halophilic archaeon Haloarcula hispanica. The virus contains a circular double-stranded DNA genome of 8,082 bp in size. The organization of the genome shows remarkable synteny and amino acid sequence similarity to the genome and predicted proteins of the halovirus HRPV-1, a pleomorphic single-stranded DNA virus that infects a halophilic archaeon Halorubrum sp. Analysis of the two halovirus sequences, as well as the entire nucleotide sequence of the 10.8-kb pHK2-plasmid and a 12.6-kb chromosomal region in Haloferax volcanii, allows us to suggest a new group of closely related viruses with genomes of either single-stranded or double-stranded DNA. Currently, closely related viruses are considered to have the same genome type. Our observation clearly contradicts this categorization and indicates that we should reconsider the way we classify viruses. Our results also provide a new example of related viruses where the viral structural proteins have not diverged as much as the proteins associated with genome replication. This result further strengthens the proposal for higher-order classification to be based on virion architecture rather than on genome type or replication mechanism.
Metagenomic studies have increased the amount of information on the nucleotide sequence space in our environment. It has also increased our awareness of the viral abundance and diversity not recognized before (16, 24, 26). Along with this new information, we have learned to acknowledge the significance of viruses in the evolution and behavior of other organisms (55). To reveal the dynamics and molecular interactions in the interplay between a particular virus and its host, however, isolation of single viruses and their hosts is needed. Even though a number of viruses pathogenic to humans, domestic animals, and plants, as well as some bacteriophages, have been studied in great detail, much of the diversity of the archaeal viruses has remained unknown. By the year 2007 only 44 archaeal viruses had been described (2). That embraces less than 1% of all reported viruses. Although the diversity among these few isolated archaeal viruses is considerable, a head-and-tail morphology is prevalent among isolated viruses infecting euryarchaeal cells. In contrast, viruses of Crenarchaeota are diverse and often unusual with no viruses having a head-tail morphology (53).
Archaeal haloviruses infect euryarchaeal hosts living in environments up to saturated salt. This makes them an interesting group of viruses that reside in a very restricted habitat. In samples taken from high salt environments, the Dead Sea and Spanish solar salterns, viral morphotypes most often observed were spindle-shaped, head-and-tail or tailless icosahedral particles (25, 31, 47). Isolated haloviruses, however, do not seem to reflect the proportions of different morphotypes found in the nature as nearly all of the isolates possess a head-and-tail morphology (2). Molecular level studies on only two spindle-shaped (10, 11) and one tailless icosahedral particle have been carried out (37, 51). Virus-like particles of other morphologies have also been observed in high-salt environments (47), but only one additional morphotype has been described in detail (50). This recently isolated lipid containing halovirus, HRPV-1, is the first archaeal virus containing a single-stranded DNA (ssDNA) genome (50). It infects Halorubrum sp. and has a pleomorphic appearance with glycosylated spike structures protruding from its external membrane (49, 50).
The evolution of prokaryotic viral genome sequences is very fast (18), and the assessment of viral relationships using homology of the genome sequences applies only to closely related viruses (17, 19). Current higher-order classification of viruses is based on the host organism, the nature of the genome (RNA/DNA, single stranded versus double stranded) and the virion morphology. Recently, a higher-order clustering of virus families has been proposed based on common principles of virion architectures as well as on the fold of the major capsid protein (1, 6, 12, 13, 42). Consequently, major capsid proteins most probably belong to the vertically inherited viral “self” (4), whereas proteins involved in replication of the viral genome can be swapped by horizontal exchange (21, 63). The proposal is based on observations that structurally related viruses have been found to infect organisms that reside in all three domains of life.
We have isolated a new pleomorphic virus infecting Haloarcula hispanica (Har. hispanica pleomorphic virus 1 [HHPV-1]). Here, we determine the molecular constituents of HHPV-1 and its genetic relatedness to other archaeal viruses and putative proviruses. Sequence homology and gene order (synteny) shows distinct genomic regions shared between four genetic elements separating replication, virus assembly, and integration functions. Surprisingly, in spite of the close relatedness of HRPV-1 and HHPV-1, the genome types of these two viruses differ (ssDNA and dsDNA, respectively).
Modified growth medium (MGM) containing 18% (top-layer), 20% (solid), or 23% (broth) (wt/vol) artificial salt water (SW) was used (52). To prepare top-layer and solid media, 4 and 14 g of Bacto agar (Difco Laboratories) were added per liter, respectively. When samples were drawn for thin-section electron microscopy, cultivation was performed in MGM, in which 1 M Tris-HCl was replaced with 1 M 4-morpholineethanesulfonic acid (MES; pH 6.7). Halophilic archaea and bacteria used in the present study are listed in Table S1 in the supplemental material. All strains were grown aerobically at 37°C.
Virus was isolated from a water sample acquired from a solar saltern in Margherita di Savoia, Italy. Isolation was performed by enrichment culture method in which 2 ml of the water sample was incubated with 10 ml of MGM overnight at 37°C with aeration. Enrichment culture was centrifuged (Heraeus Biofuge, 16,060 × g, 5 min, 22°C) to remove cells and debris. The water samples were then plated on 12 indicator strains (Table S1 in the supplemental material), available beforehand, using 18% MGM. After incubation at 37°C for 3 days a plaque that had appeared on a lawn of Har. hispanica was picked and purified by two consecutive single plaque purification steps.
Haloarchaeal strains, indicated in Table S1 in the supplemental material, were tested for virus susceptibility. Dilutions of virus lysate were plated with different strains using 18% MGM. After incubation for 4 days, the plates were checked for plaques.
Virus stock was prepared from top-layer agar plates, incubated to show semiconfluent amount of plaques by HHPV-1. Soft agar from each plate was collected into 2 ml of 23% MGM and pooled. Culture was incubated at 37°C for 1.5 h with aeration. Cells and debris were removed by centrifugation (Sorvall SLA1500, 9,700 × g, 20 min, 4°C), and the titer (PFU/ml) was determined by plaque assay. Infectivity of HHPV-1 in a stock was sustained for at least 1 month when stored at 4°C.
Viruses were precipitated from the supernatant with 10% (wt/vol) polyethylene glycol (PEG) 6000 MW, collected by centrifugation (Sorvall SLA1500, 15,200 × g, 40 min, 4°C), and resuspended in 18% SW. Titer was determined by plaque assay. Purification of virus preparation was performed by rate zonal centrifugation in a linear 5 to 20% (wt/vol) sucrose gradient in 18% SW (Sorvall AH629, 103,600 × g, 4 h, 20°C). After centrifugation, the light scattering virus zone was collected and designated as “1×-purified” virus. The A260 was measured, and the titer was determined by plaque assay. To produce “2×-purified virus,” 1×-purified virus was layered either on the top of a CsCl solution (0.3 g/ml in 18% SW) or on a linear 10 to 45% (wt/vol) sucrose gradient in 18% SW and centrifuged to equilibrium (Sorvall AH629, 79,300 × g, 16 h, 20°C). The light-scattering virus zone was either collected, or CsCl tubes were fractionated into 2-ml fractions which were analyzed by measuring the A260 and density, as well as determining the titer by plaque assay. Samples were also taken for protein gel and lipid analyses. The collected virus zone was diluted (in volume ratio of 2:1:3 of virus, 18% SW, and 18% SW without NaCl), virus was collected by differential centrifugation (Sorvall T647.5, 177,500 × g, 3 h, 20°C), and the pellet resuspended in 18% SW. For this 2×-purified virus material, the A260 was measured, the titer was determined by plaque assay, and the protein concentration was measured by the Coomassie blue method (15) with bovine serum albumin as a standard. The specific infectivity for the virus was 4.4 × 1012 PFU/mg of protein.
A total of 30 ml of exponential-phase Har. hispanica culture (1.5 × 109 CFU/ml) was infected with HHPV-1 stock (multiplicity of infection [MOI] of 15). After an adequate adsorption period of 30 min (5 min at 22°C with no aeration, followed by 25 min at 37°C with aeration ), the cells were washed with MGM broth to remove the unbound viruses and resuspended into 30 ml of MGM broth. The culture was incubated at 37°C with aeration. The A550 was measured in hourly intervals of the infected and similarly treated uninfected culture. Samples were drawn concurrently with the absorbance measurements, and the number of free viruses and infective centers was determined by plaque assay. The number of viable cells (CFU/ml) was monitored in both infected and uninfected cultures. After incubation for 12 days, different-sized colonies (small, medium, and large) were picked from viable count plates of the infected culture and inoculated into MGM broth. After incubation at 37°C for 8 days with aeration the titer of the culture supernatants was determined by plaque assay.
To determine when virus production begins, a culture was prepared as described above except that an MOI of 1 was used. Samples were drawn at half-hour intervals from 1 h postinfection (hpi) onward for determination of the number of free viruses by plaque assay.
For negative stain transmission electron microscopy, viruses were allowed to adsorb to grids for 1 min and were then stained with 1% (wt/vol) potassium phosphotungstate (pH 6.5) for ~10 s, uranyl acetate (pH 4.5) for ~20 s, 3.32% ammonium molybdate (pH 7.4) for ~15 s, or 1% ammonium molybdate (pH 7.0) for ~15 s.
For thin-section electron microscopy cultures were prepared as described for the HHPV-1 growth curve, except that MOI 40 was used. Samples were drawn 5 or 25 min postinfection or 1, 3, 5, 7, or 23 hpi from the infected culture and 5 hpi from the uninfected culture. Thin-section electron microscopy was carried out as previously described (7).
All micrographs were taken with a JEOL 1200EX electron microscope operating at 60 or 80 kV (Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki).
Chloroform sensitivity was tested by mixing chloroform and virus stock in a proportion of 1:20, followed by incubation at 4°C overnight before titer determination by plaque assay.
The protein composition was analyzed by using a modified Tricine-SDS-PAGE system (devoid of spacer gel ) with 14% acrylamide concentration (wt/vol). The N-terminal sequences of the two structural proteins were determined in the Protein Chemistry Core Laboratory, Institute of Biotechnology, University of Helsinki, as described previously (8).
Lipids from cell suspensions and 2×-purified virus preparations were extracted according to the method of Folch et al. (29) essentially as described previously (8) and stored in chloroform-methanol (9:1 [vol/vol]) at −20°C until analyzed. Total lipid extracts were subjected to thin-layer chromatography (TLC) on silica gel 60 plates (Merck), and phospholipids were separated by using chloroform-methanol-90% acetic acid (65:4:35 [vol/vol/vol]) as the solvent (8, 23). After visualizing the lipids by iodine vapor, the plates were sprayed with molybdate (27) and 0.1% orcinol-sulfuric acid reagents (60) to detect phospholipids and glycolipids, respectively. The phosphorus content of lipid spots was determined as described by Bartlett and Lewis (9). The lipids in each spot of the TLC were identified by performing a mass-spectrometric analysis and comparing their m/z values, as well as product and precursor ion profiles, to those of known lipids from halophilic archaea (8).
The genome was purified from the 2×-purified viral material by dilution of the virus 1:5 in 50 mM Tris-HCl (pH 7.2). SDS was added to a final concentration of 1.5%, EDTA was added to 1 mM, and proteinase K was added to 0.8 mg ml−1, and the preparation was incubated at 56°C for 20 min. Proteins were removed by phenol extraction and ether. Nucleic acids were precipitated with 0.3 M sodium chloride and 2 volumes of ethanol at −20°C overnight. Approximately 1.5 to 2 μg of the genome was used for each digest of DNase I (1 U of RQ1 RNase-Free DNase; Promega), RNase A (4 μg; Promega), or mung bean nuclease (0.5 U). As controls, the single-stranded and double-stranded (RFII [RF for replicative form]) viral DNAs of X174 (New England Biolabs) were used.
To determine the preliminary genomic sequence of HHPV-1, a six-base-tagged (barcoded) library was done for Genome Sequencer FLX pyrosequencing (Roche/454 Life Science). A total of 3,000 sequences were obtained with a mean read length of 206 bp. The reads were assembled by using the gsAssembler, and the obtained assembly was verified with PCR products that covered the whole genome. The final sequence data were obtained by sequencing these PCR products. The primers used for PCR and sequencing are listed in Table S6 in the supplemental material. All sequencing was carried out by using BigDye version 3.1 and analyzed on an ABI 3130 (Applied Biosystems). Sequencing reactions were purified with CleanSeq (Agencourt) using a Beckman NXp robot (Beckman Coulter). Sequences were assembled and edited using GAP4 (61). The pHK2 sequence was determined by primer walking using pMDS1 (33) as the template. The primers used for sequence determination are listed in the Table S7 in the supplemental material. The preliminarily annotated sequence of Haloferax volcanii (Hfx. volcanii) genome was obtained from the HaloLex website (48).
Genome sequence was analyzed for the open reading frames (ORFs) by using the programs Artemis (56), GenMark (14), and pDRAW32 (AcaClone software). The basic properties of the genes and encoded proteins were studied by using the programs of the EMBOSS program package (54) at the IT Center for Science, Ltd. (www.csc.fi), in Finland and the Expasy proteomics server (30). Transmembrane helices in the polypeptides were predicted by using TMHMM server version 2.0 (41) and coiled coils were predicted using the Coils-Server (45). The global pairwise identity between two proteins was determined by using LALIGN (a global alignment method without end-gap penalty  at ch.EMBnet.org) using the default values and the BLOSUM50 matrix. Homologous proteins in the databases were searched by using the BLASTP search engine (3) at the National Center for Biotechnology Information (NCBI).
The nucleotide sequences determined in the present study were deposited in GenBank with the following accession numbers: HHPV-1 genome, GU321093; and pHK2 sequence, GU321094.
HHPV-1 was isolated from a solar saltern in Margherita di Savoia, Italy. HHPV-1 produced plaques on the lawn of a Har. hispanica host but was unable to produce plaques on any other strains tested (see Table S1 in the supplemental material).
Halovirus HHPV-1 production and purification methods were established (see Materials and Methods). The virion has densities of 1.27 and 1.23 g/ml in CsCl and sucrose, respectively. Recovery and specific infectivity (PFU/A260) were monitored during the purification process (see Table S2 in the supplemental material). Virions were extremely sensitive to chloroform (data not shown). Chloroform sensitivity and low buoyant density indicated the presence of lipids as virion constituents. Gradient analysis (Fig. (Fig.1)1) confirmed this as A260, virus titer, proteins, and lipids peaked in the same fractions. Negative-stain transmission electron microscopy (TEM) of HHPV-1 particles purified by equilibrium centrifugation (2×-purified virus) showed pleomorphic particles (Fig. (Fig.1D1D).
HHPV-1 did not lyse the host Har. hispanica cells (Fig. (Fig.2A).2A). Increase in turbidity was similar for the infected and uninfected cultures until 7 hpi, after which the infected culture showed a slightly retarded growth rate. When the number of CFU was monitored, the infected culture resulted in reduced colony formation after 10 hpi compared to the uninfected one (Fig. (Fig.2B2B).
Virus release started 3 to 3.5 hpi, reaching its maximum, ~8.6 × 1011 PFU/ml in the medium, around 25 hpi (Fig. (Fig.2A).2A). The number of infective centers (ICs) started to increase after 3 hpi, indicating that not all of the cells had been infected before the onset of the release of the first viral progeny. Increases of ICs were observed up to ~10 hpi. It should be noted that the amount of ICs could not be detected accurately because viruses seemed to be released continuously from the cells and the detected numbers of ICs were slightly higher than the CFU. Virus production seemed to influence the size of colonies because in the early stages of the life cycle (~1 hpi) colonies formed were mostly large, and cells taken from such colonies did not produce virus. Smaller colonies dominant in the late stages, on the other hand, produced virus.
The life cycle of HHPV-1 was also studied by taking samples for thin-section electron microscopy at different time points during the life cycle. The amount of extracellular viruses was observed to increase in the course of time, and most of the viruses were detected in clusters outside the cells (Fig. (Fig.2C).2C). However, we were not able to detect intracellular viruses or viruses in the process of budding.
Analysis of the lipid extracts by TLC revealed that highly purified HHPV-1 virions contain phospholipids but no significant amounts of glycolipids (data not shown). The four phospholipid classes detected were cardiolipins (CL), phosphatidylglycerols (PG), phosphatidylglycerophosphate methyl esters (PGP-Me), and phosphatidylglycerosulfates (PGS). Comparison of the quantity of the three main classes of phospholipids (i.e., PG, PGP-Me, and PGS) of Har. hispanica, HHPV-1, and the icosahedral lipid-containing halovirus SH1 (8) shows that the composition of HHPV-1 is quite similar to that of the host, except that the PG is slightly enriched in the virion (Fig. (Fig.3),3), whereas the lipid composition of SH1 deviates significantly from that of the host.
Analysis of the virion in Tricine-SDS-PAGE revealed two major protein species with apparent molecular masses of approximately 60 and 12 kDa (Fig. (Fig.4A).4A). The N-terminal sequence analysis of these two proteins gave sequences of AYSNNARGLV for the 12-kDa and VVPVVLPGVA for the 60-kDa protein. These two peptide sequences were identified as the protein products of the ORFs 3 and 4, respectively (see below). The 12-kDa protein is designated VP3 (VP for viral protein), and the 60-kDa protein is designated VP4. The protein sequence analysis also showed that VP4 contains a 50-amino-acid signal sequence that is processed.
Enzymatic digestion of the HHPV-1 genome with DNase I and mung bean nuclease showed that the genome is a dsDNA molecule (see Fig. S1 in the supplemental material). The preliminary sequence of the genome was determined by pyrosequencing, and the whole genome sequence was further confirmed by sequencing overlapping PCR-products that covered the whole genome. The ability to generate overlapping PCR-products revealed the circular nature of the genome. The HHPV-1 genome is 8,082 bp in size, and the GC percentage is 55.8%.
The HHPV-1 genome has eight putative ORFs (Fig. (Fig.4A).4A). The properties of the ORFs and the encoded polypeptides are shown in Table S3 in the supplemental material. ORFs 3 and 4 encoding the two major structural proteins (Fig. (Fig.4A)4A) were designated genes 3 and 4, respectively. The genome organization and protein homology showed that HHPV-1 is closely related to the recently isolated and characterized pleomorphic ssDNA virus HRPV-1 that infects Halorubrum sp. (50). The structural protein patterns are also similar (Fig. (Fig.4A).4A). HHPV-1 ORF2, and five consecutive translated ORFs show significant identity to the translated ORFs and identified genes of HRPV-1 (Fig. (Fig.4).4). The highest identity (55%) was found between the HHPV-1 gene 3 encoding the small major structural protein VP3 (HHPV-1 VP3) and the VP3 protein of HRPV-1. Although both proteins are putative integral membrane proteins, they differ in the number of predicted transmembrane regions (two in HHPV-1 VP3 and four in HRPV-1 VP3). VP4, the other major structural protein of HHPV-1, is similar in size but shows relatively low identity to the HRPV-1 VP4-protein. Both of these two proteins, however, contain a signal peptide that is processed and a predicted C-terminal transmembrane domain that is preceded by a predicted coiled-coil region.
The remaining putative gene products of HHPV-1 that show homology with the translated ORFs of HRPV-1 are >20% identical (Fig. (Fig.4B).4B). The highest identity among these (47%) was found between HHPV-1 ORF7 and the HRPV-1 ORF8. The latter has been shown to encode the minor structural protein VP8 that contains predicted ATPase motifs (50). A search using HHPV-1 ORF7 for conserved domains at the NCBI recognized domains belonging to the P-loop NTPase superfamily proteins (cl09099, Expect [E] value, 0.22). The polypeptide of the predicted ORF1 did not show significant homology to the HRPV-1 ORF1 (9% identity). A BLASTP search of the database at NCBI, however, gives best matches to the putative Rep protein of Halomicrobium mukohataei (GenBank accession no. EEJ07478.1, E-value 9e-38) and to ORF1 of plasmid pHK2 (GenBank accession no. AAB09607.1, E-value 2e-25, see below). Since ORF1 of HRPV-1 is also predicted to be a replication initiation protein (50), the two putative ORF1 polypeptides most probably serve the same function as replication initiation proteins.
Preliminary BLAST searches using the HHPV-1 ORFs gave hits to the ORFs of the plasmid pHK2 isolated from haloarchaeal species Haloferax sp. (34). Since similar observation was made for the HRPV-1 polypeptides (50), we completed the sequence of the pHK2-plasmid using its derivative pMDS1 (33). The previously published sequence of the 4.1-kb fragment of pHK2 (GenBank accession no. L29110.1) (34) was found to diverge from the new sequence in 12 positions. The biggest change was the addition of a G after the nucleotide (nt) 3885 in the previously determined sequence. This change removes the stop codon for ORF4 at nt 3935 and the ORF continues to ORF5. In addition, in the beginning of the previous 4.1-kb region, differences in sequence result in 94% identity between the ORF1 polypeptides predicted to encode replication initiation proteins (data not shown). The sequence obtained from the rest of the pHK2 plasmid showed homologous ORFs in the same order as in both HHPV-1 and HRPV-1 (Fig. (Fig.4B;4B; see also Tables S4 and S5 in the supplemental material). The last annotated ORFs, ORF8 of HHPV-1 and ORF9 of HRPV-1, do not show homology to each other or to ORF10 of pHK2 (Fig. (Fig.4B).4B). Although the HHPV-1 VP4 shows only 19% identity to the VP4 of HRPV-1, the identity between the pHK2 ORF4 encoded polypeptide and the HRPV-1 VP4 is remarkably higher, i.e., 36%. Also, HRPV-1 VP3 shows higher identity to pHK2 ORF3 (58%) than to HHPV-1 VP3 (Fig. (Fig.4B;4B; see Table S6 in the supplemental material). The 10,795-bp pHK2 plasmid has four putative ORFs after the ORF encoding the putative ATPase (HHPV-1 ORF7, HRPV-1 VP8, and pHK2 ORF9). The encoded polypeptides of these ORFs do not show identity to either HHPV-1 or HRPV-1. ORF11, however, encodes a putative polypeptide which shows homology to H repressorlike proteins and ORF13 encodes a putative integrase (see the supplemental material). These features strongly suggest that the pHK2 plasmid actually is a temperate virus related to both HHPV-1 and HRPV-1.
A search through preliminarily annotated Hfx. volcanii genome using pHK2 ORFs as queries resulted in identification of a 12.6-kb gene region containing ORFs highly identical and in the same order as the ORFs in the pHK2 plasmid (Fig. (Fig.4B;4B; see also the supplemental material). The region fulfills the typical features of a provirus integrated into the genome. There are ~450 nt between tRNAArg and the ORF1 encoding a putative replication initiation protein. ORFs 2 to 9 are 96 to 98% identical to the corresponding ORFs in pHK2; the predicted ATPase encoding ORF9 is the last ORF showing high identity. After that there is no detectable similarity except for ORFs encoding the putative H repressor-like protein (HVO_1423) and a putative integrase (HVO_1422) that are in similar positions between pHK2 and the chromosomal region of Hfx. volcanii in the end of these genetic elements (Fig. (Fig.4B).4B). Between the preliminarily annotated ORFs HVO1423 and HVO_1422 there is a short ORF that has not been annotated in the preliminary annotation of the Hfx. volcanii genome. The predicted polypeptide, however, shows high homology to the putative ORF12 polypeptide of pHK2.
Apart from this chromosomal region in Hfx. volcanii there are several regions encoding hypothetical proteins in the genomes of halophilic archaea that show homology to the viral genomic region starting from VP4 encoding gene and ending after the putative ORF encoding an ATPase. As reported previously (50), the same region also shows homology to the similar region in another halophilic virus, His2 (10).
During a search for new haloviruses, a pleomorphic halovirus was discovered from a solar saltern on the eastern cost of Italy. This Har. hispanica pleomorphic virus 1, HHPV-1, has a pleomorphic appearance similar to the recently reported HRPV-1 virus (Fig. (Fig.1D)1D) (50). HHPV-1 production and purification methods were set up to gain highly pure viral material. HHPV-1 virion contains four distinct phospholipid classes and two major structural proteins with predicted transmembrane regions (Fig. (Fig.4A).4A). The other described pleomorphic halovirus HRPV-1 has also been shown to contain lipids and two major structural proteins (50). The almost identical phospholipid compositions of HHPV-1 and Har. hispanica suggest that lipids during the assembly of HHPV-1 are acquired from the host almost without selection. Interestingly, the composition of phospholipids of SH1, an icosahedral virus that also infects the Har. hispanica host, is significantly different from that of HHPV-1 (Fig. (Fig.3).3). This observation indicates that the lipids of SH1 are selectively incorporated into the virion most likely due to the geometrical constraints within the icosahedral protein coat and/or specific lipid-protein interactions (8, 37). Thus, the assembly pathway of HHPV-1 must differ considerably from that of SH1 and related viruses.
HHPV-1 exits the host without causing cell lysis (Fig. (Fig.2).2). Exit without lysis has been observed for several viruses of Crenarchaeota (58, 64) and has also been observed for the haloviruses HRPV-1, His1, and His2 (10, 50). Some of these viruses seem to exit in an as-yet-undescribed way. Interestingly, the two viruses homologous to HHPV-1, also exit the host without cell disruption (10, 50). Such a mode of exit suggests budding.
Genome synteny reveals a close relationship between HHPV-1 and the HRPV-1. In addition, the entire 10.8-kb genome of pHK2 completed in the present study as well as the genomic 12.6-kb region of Hfx. volcanii, show considerable homology to HHPV-1 and HRPV-1 viruses (Fig. (Fig.4B).4B). pHK2 has earlier been reported to be a plasmid (32, 34). Although the viral nature of pHK2 has not been confirmed, the highly homologous genomic region of Hfx. volcanii shows the typical features of a virus integrated into tRNAArg. In addition, both pHK2 and the Hfx. volcanii chromosomal region contain a putative integrase (46), as well as genes encoding H repressorlike proteins. We predict that these two genetic elements can be enclosed into pleomorphic viral particles and consequently will be designated as proviruses in the discussion that follows.
Comparison of the four genomes enables us to detect the most conserved putative and identified proteins of the proposed new group of pleomorphic viruses. Of the two major structural proteins, VP3 and VP4, VP3 shows the highest identity. VP4, on the other hand shows quite high diversity. The VP4 of HRPV-1 was predicted to function as the receptor binding and fusion protein (50). Viral receptor-binding proteins are often the most variable ones among the viral structural proteins (17, 44, 57). Since all of these viruses infect different hosts, variability of VP4 is in accordance with its function as a receptor interacting protein. Apart from these two proteins, ORF5, ORF6, and ORF7 of HHPV-1 show relatively high identity in all four genomes. The region containing identical and almost identical predicted proteins in the pHK2 and Hfx. volcanii elements starts from ORF2 and ends after ORF9. In most of the pairwise alignments (see the supplemental material), the predicted and identified proteins of HRPV-1 are more closely related to corresponding ORF-encoded proteins of either pHK2 or Hfx. volcanii than to predicted and identified proteins of HHPV-1. HRPV-1 genome also contains ORF5, which shows homology to the corresponding ORFs in pHK2 and Hfx. volcanii proviruses. This suggests that HRPV-1 is a closer relative to pHK2 and Hfx. volcanii proviruses than to HHPV-1.
Keeping in mind the high identity of the central region of the pHK2 and Hfx. volcanii provirus genomes, it is surprising that ORF1 and the region starting from pHK2 ORF10 and Hfx. volcanii HVO_1425 varies considerably. HRPV-1 and HHPV-1 genomes do not contain the region starting from the pHK2 ORF10 and including the H repressor and the integrase. This shows that an integrase is not required for the life cycle of these pleomorphic viruses even though under certain conditions an integrase may confer an advantage. Similar observation have also been made for other types of viruses such as the crenarchaeal virus SSV1 (22) and an icosahedral phage P23-77 that infects Thermus thermophilus (38).
The ORF1s of the four genomes shows low identity in the pairwise alignments (the highest being 20% between HRPV-1 and Hfx. volcanii ORF1). They all are, however, predicted to function as replication initiation proteins. HHPV-1 and pHK2 ORF1 polypeptides contain the conserved domain DUF1424 (E value of < 0.01). This domain can be found in the family of haloarchaeal proteins predicted to be involved in replication. ORF1-encoded polypeptides of HRPV-1, pHK2, and Hfx. volcanii provirus also contain the three conserved motifs suggested to be essential for initiation proteins of rolling-circle replication (36). The HHPV-1 ORF1 contains domains 2 and 3 of these conserved motifs. Since at least HHPV-1, HRPV-1 and pHK2 genomes are circular, it is highly likely that also the ORF1 encodes a rolling-circle replication initiation protein. Replication and packaging of a circular ssDNA genome of bacteriophage X174 has been described in detail (40, 63). Replication of the circular ssDNA genome of HRPV-1 and circular dsDNA genome of HHPV-1 may occur in a similar fashion. Packaging of different types of genomes just occurs in different stages of the replication process.
During our studies of new halophilic viruses we have found a new group of pleomorphic viruses including HHPV-1 and HRPV-1 and two putative proviral elements. Based on these results, two major observations can be made that both have consequences as to how viruses are classified. First, we observed that the genome of HHPV-1 is a circular dsDNA molecule, whereas the genome of HRPV-1 is a circular ssDNA molecule (50). In terms of current viral classification, this is unusual, since according to the nucleotide and deduced amino acid sequence data these viruses are closely related and should be grouped together despite the different genome types. This shows that in the era of genome sequencing, higher-order classification according to the genome type may not be justified.
Second, although associated with different hosts, these four viruses show conserved regions, including the two major structural proteins determined for HHPV-1 and HRPV-1 (50). This conserved region containing ORFs 2 to 9 in pHK2 most probably constitutes the core proteins for virion assembly. In contrast, the putative replication initiation proteins show lower identity. In addition, unlike the core proteins, multiple alignment of the ORF1 polypeptides arranges the relationships between the viruses and the proviral elements in a different order than the alignment using the core proteins. Thus, comparisons of the four genomes suggest that the viral world is indeed “… a dynamic network of relationships in which genes have diverse, variously intertwined histories …” (39) obtained both vertically and horizontally. The comparisons of the core proteins of this new pleomorphic virus group show, however, that the structural components are more conserved and most probably obtained by vertical inheritance in order to keep the viral self intact and functional for continuation of the viral lineage (1, 4-6, 12, 13, 42). Similar observations have been made for tailed phages using even more abundant sequence information (20, 21, 28, 62). As a consequence, the mode of the genome replication and the nature of the genome (RNA/DNA, ss or ds) may not be adequate determinants for classifying distant, but related viruses to higher-order taxons. The determinants for the virion architecture instead may better serve this function.
We thank Paula Collin-Olkkonen, Sari Korhonen, Päivi Laamanen, Kirsi Lipponen, Soile Storman, Eeva-Marja Turkki, and Suvi Varjonen for excellent technical assistance. Nisse Kalkkinen and Gunilla Rönnholm are acknowledged for the protein N-terminal sequencing.
This study was supported by Academy of Finland Centre of Excellence Program in Virus Research grant 1129684 (2006-2011) and Academy of Finland grant 1210253 (to D.H.B.).
Published ahead of print on 20 January 2010.
‡Supplemental material for this article may be found at http://jvi.asm.org/.

domain: biology


text:
Madison Oberg is the young beautiful bride of the National Football League quarterback, Carson Wentz who made history as the second overall selection ever during the 2016 NFL Draft. She has become famous as well as making the headlines of many news outlets following her marriage to the American football superstar.
Their marriage was much publicized as a result of the manner it unfolded, having that the celebrated footballer popped the question to his beau in February 2018, out of the excitement of seeing his team claim their first ever Super Bowl Championship along with earning a Championship ring.
Although Carson didn’t play in the Super Bowl LII, having that he was sidelined as a result of his knee injury, the famous footballer wanted to hold tight to the beautiful memory for the rest of his life and that’s why he got engaged immediately after becoming a Super Bowl Champion. See more facts about Carson Wentz’s wife below.
Madison Oberg Biography
The gorgeous wife of the American football star is the third child of Brian Oberg and Lisa Broughton. She is a native of Lexington, Kentucky, United States and was born on 21st June 1997. Madison Oberg first attended Ozark Christian University before she moved to the Missouri Southern State University.
There is no available information concerning the career of Carson Wentz’s beautiful bride as she is yet to be present on social media. However, it has been observed that she was once involved in a charity work that took her to Haiti as a fellow of the Mobilization Team.
Madison Oberg is blessed to be among the few that have settled down with their best friend after a few months of dating. She reportedly met her footballer beau during the time she was on a humanitarian duty in Haiti, having that Carson Wentz was also in the same country at that given time.
Oberg and Wentz reportedly kicked off their relationship in 2017 but decided to keep it private, away from the media. They later made the love affair public in December 2017, when the American footballer underwent knee surgery and uploaded a picture of him and Madison Oberg from his hospital bed on his Instagram account.
Wentz got down on his knees to propose to his girlfriend a few days after his team clinched the Championship. He went on to express his joy of having his best friend say yes to his marriage proposal on his Twitter handle on 6th of February 2018, as he is very active on social media.
A few months later, the couple solemnized their union in a small wedding ceremony held at Bucks County in July 2018. Wentz took to his Instagram on 16th July to share the breathtaking pictures of their wedding and also cited how lucky he is to have an amazing wife in the person of Madison Oberg.
Oberg’s husband, Carson Wentz has become a big name in the American National Football League as a result of his performances on the field of play which has earned him many titles and awards including National Football League Offensive Rookie for the month of September 2016 and Bert Bell Award. The NFL star was picked by the Philadelphia Eagles in April 2016 NFL Draft and he broke the record in the National Football League as the first quarterback to have been selected by the NFL club during the first round of an NFL draft since Donovan McNabb in the year 1999. Carson Wentz has remained with the Eagles till date.
Facts About Carson Wentz’s Wife
1. Madison Oberg has worked as an intern at a charity organisation known as Mission of Hope: Haiti, which spawned her travel to Haiti for humanitarian work.
2. Following her study at Ozark Christian University, the elegant wife of Carson Wentz previously gained employment at Ozark Admission situated at Joplin, Missouri, United States.
3. Oberg’s husband is five years older than her as he was born on 30th December 1992 while Madison Oberg born on 21st June 1997.
4. Her husband has a charity foundation that goes by the name of AO1 Foundation which he launched in 2017. And his foundation has went on to collaborate with a non-profit organisation, Mission of Hope: Haiti to construct a sports complex in the country.
5. The gorgeous bride of Carson Wentz is the youngest child of her parents. She has two older sisters named Lauren Oberg who was born in 1991 and Alexis Oberg born in 1995.

domain: celebrity