What types of matboards are available?
We offer four types of matboards with our framed art: (1) whitecore, (2) premium whitecore, (3) rag, and (4) 8 ply.
What are the differences between the types of matboards?
Whitecore matboards are non-conservation matboards, which provide the aesthetic and design benefits of a bright-white bevel. Whitecore matboards are composed of decorative facing papers, stay-white core and backing, and a surface, core and backing paper buffered with calcium carbonate to alkaline reserve of 3-5%. Whitecore matboards are recommended for decorative framing and general presentations, open end reproductions, and posters and decorative art.
Premium whitecore matboards have the same specifications and uses as the whitecore matboards. However, premium whitecore matboards offer more design options and a much wider selection of surface colors to choose from.
Rag matboards are made from conservation grade surface papers for optimum bleed and fade resistance. Rag matboards are made with 100% cotton fibers that are naturally acid-free and lignin-free. Cotton is naturally free of chemicals and pollutants that can degrade or harm artwork. Cotton is an annually renewable resource that reduces the amount of trees used to make mounting and matting boards. In addition, cotton fiber is processed without harsh chemicals or additives, making it superior to bleached, chemically processed woodpulp that is used to produce other conservation boards. Rag matboards are recommended for fine art prints, limited editions, valuable documents and photographs, and heirloom needle art.
Lastly, 8 ply matboards are also made from 100% cotton, conservation grade papers. 8 ply matboards are acid-free, lignin-free, extra-rigid, and have a solid color throughout. 8 ply matboards provide optimum archival protection for fine works of art on paper. 8 ply matboards are recommended for valuable original works of art, rare and antique documents, and items of historical importance.
What types of frames are available?
We offer five basic types of frames: (1) solid wood, (2) composite wood, (3) polystyrene, (4) metal, and (5) float. Frames are a major price component when designing art pieces. Changing the type and profile of your frame can have a major impact on the price of the piece.
Generally speaking, solid wood frames are the most expensive. Composite wood frames offer a nice less expensive alternative to solid wood. Our composite wood frames are perfectly engineered with a very smooth, fine-textured surface. It looks like wood, cuts like wood, joins like wood, and readily accepts nails and hangers.
Polystyrene frames are made from polystyrene, which is a versatile plastic that can be rigid or foamed. In its solid form, polystyrene is one of the denser plastics and is used for a wide range of moulded frames. Polystyrene frames are durable, lightweight, and cost less than the same look in wood or composite wood.
Our metal frames are made from aluminum and are typically thinner than the other frame types. The largest commonly used size is approximately 1 3/8". However, metal frames do offer a clean look and exceptional durability.
Lastly, float frames give a unique appearance to the artwork because the artwork looks as if it is floating inside the frame.
Clear acrylic is a general-purpose glazing for indoor framing.
Ideal for large pieces where the weight of glass would be excessive or some shatter resistance is needed.
This acrylic product has a matte-like finish that scatters light to reduce unwanted glare.
Ideal for uses where unwanted reflection may impact the viewing of the art and some shatter resistance is desired.
Use on any framing project with up to two mats without significant resolution loss.
Clear glass works well in a controlled lighting environment.
Ideal where high visibility and clarity are desired.
Non-glare glass has a matte-like finish that scatters light to minimize unwanted glare.
Ideal for uses where unwanted glare and reflection may impact the viewing of the art.
The surface of non-glare glass has micro etches that may reduce clarity in addition to glare.
This premier glass product provides the lowest possible reflection rating available with UV protection.
Virtually invisible glazing that will enhance colors, brightness, and contrast levels of all types of artwork.
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Xenia junior Samari Curtis talks about how it felt to score 50 points for the Buccaneers in a loss at Tippecanoe on Jan. 5.
Xenia guard Samari Curtis announced Tuesday he is re-opening his recruitment.
Curtis, a four-star combo guard who has led the GWOC in scoring the past two seasons, committed to Xavier in February.
The Musketeers changed coaches in April when Chris Mack left for Louisville.
Curtis told 247Sports in late April he was looking forward to playing for new Xavier coach Travis Steele, an assistant who was promoted to replace Mack.
The 6-foot-4 scorer averaged 30.4 points per game last season and has moved up in the 247Sports Composite rankings over the past month.
He is the No. 100 prospect overall and No. 12 among combo guards in the class of 2019.
Before committing, Curtis had offers from Akron, Toledo, Kent State and Cincinnati along with XU.
He's the No. 4 prospect in the state and No. 1 among area players, followed by fellow guards Andre Gordon of Sidney and Amari Davis of Trotwood-Madison.
Neither of them have committed yet.
The trio is playing together for the C2K Elite AAU team this spring along with Trotwood-Madison center Justin Stephens and Springfield teammates RaHeim Moss and Sam Towns.


Out-Of-Service or OOS means the operation has no standing to operate due to bad safety performance or a failure to pay a fine. Continued operation is not permitted.
Under § 392.9b Prohibited transportation. (a) USDOT Registration required. A commercial motor vehicle providing transportation in interstate commerce must not be operated without a USDOT Registration and an active USDOT Number.
A deactivated USDOT number, for those in interstate commerce, can result in roadside citations and fines, but not (at this time), being put Out Of Service (OOS).
At a later DOT audit or investigation, your company can be fined $1,000 per day, up to $10,000.
Tip: Check your status at the DOT's SAFER Safety and Fitness Electronic Records (SAFER) System webpage: www.safersys.org (click on Snapshot and put in your DOT number). Update the registration information (at the same link), if your DOT Number is Inactive or deactivated, by clicking on: FMCSA Registration & Updates).
You may call the DOT toll-free for help at 1-800-832-5660.


text:
Q: failed with Received heartbeat from member with the same member ID as ourself:0 Trying to set a replica set configuration for 1 primary , 1 slave and 1 arbitrator.
I have setup this in the /etc/mongodb.conf
replication:
  replSetName: ProductionReplicaSet

but accidentally ran rs.initiate()  on the three servers. now when I'm running rs.add("mongo02....") i'm getting :
{
    "ok" : 0,
    "errmsg" : "Quorum check failed because not enough voting nodes responded; required 2 but only the following 1 voting nodes responded: chef-production2-mongo01:27017; the following nodes did not respond affirmatively: mongo02...:27017 failed with Received heartbeat from member with the same member ID as ourself: 0",
    "code" : 74
}

I read here that rs.initiate() can be reversed but the instructions are not clear.
tried on mongo02:
config = rs.config() //config with _id :0
config["members"][0]["_id"] = 1
rs.reconfig(config)
{
    "ok" : 0,
    "errmsg" : "New and old configurations both have members with host of chef-production2-mongo02:27017 but in the new configuration the _id field is 1 and in the old configuration it is 0 for replica set ProductionReplicaSet",
    "code" : 103
}

any help? i'm open to other solutions.

A: I ran in to this as well.   Turns out it can be caused by two things.
1.  If the node you are adding already has its own dbData.

2.  If you ran rs.initiate() on both nodes.   It is only supposed to be run on the primary.

To resolve this you will need to check /etc/mongod.conf for the "dbPath" attribute and move or remove all the files in that folder.

redpajama_set_name: RedPajamaStackExchange


We're looking for an individual to help spearhead our charge into the world of online marketing. Due to the increased demand from our clients you will have the opportunity to work with a selection of organisations across various markets, each bringing a unique challenge. This role will be yours to grow and develop and will more than likley lead to a full time position within the Full Phat Design studio as we grow with you. This role would ideally suit someone with a few years experience.
Our studio is based in Mansfield, Nottinghamshire, you would be required to work from the studio along with occasional visits to meet clients. This job is initially part time at 21 hours a week but with the view of increasing the number of hours as the client base grows.
Please send your application to work@fullphatdesign.co.uk and include the following….


The next blog posting will be Monday July 24, 2017 as I am working on an upcoming Manual Digital Operating Systems Using Artificial Intelligence, Deep Learning and Bots for the Small and Medium Size Business …. Have a great weekend!!


text:
Destineer Brings Classic Arcade Games to Wii
Thursday, February 10th, 2011 | Permalink
[This unedited press release is made available courtesy of Gamasutra and its partnership with notable game PR-related resource GamesPress.]
MINNEAPOLIS, MN – July 30, 2010 – Experience the fantasy adventures that became a cultural phenomenon! Game publisher Destineer today announces a partnership with Digital Leisure Inc. to develop Dragon's Lair Trilogy, bringing three timeless arcade adventures together on one disc for Wii TM : Dragon's Lair, Dragon's Lair 2: Time Warp, and Space Ace. For the first time ever on a console platform, you can experience the stunning visuals and engaging storylines from all three of these classic arcade games created by legendary American animator Don Bluth. Lead on brave adventurer, your quest awaits!
An instant hit in its 1983 debut, Dragon's Lair is one of the most beloved arcade games of all time. As valiant knight Dirk the Daring, immerse yourself in a fantasy adventure on a quest to rescue Princess Daphne from the clutches of an evil dragon. You control the actions of the daring adventurer, finding your way through the castle of a dark wizard, who has enchanted it with treacherous monsters and obstacles.
Straight off the success of Dragon's Lair, Don Bluth and his creative team began working on a galactic adventure known as Space Ace! In Space Ace you play the heroic Ace who must stop the evil Commander Borf from taking over the Earth! Only two people have the courage and strength to stop Borf and save Earth: the beautiful Kimberly and Ace. But as they approach Borf's stronghold, Ace is hit by the Infanto Ray, changing him into a child, and Kimberly is kidnapped by the evil madman! The fate of Earth is in your hands!
In the sequel to the smash-hit, Dragon's Lair 2: Time Warp allows you to once again play the heroic Dirk the Daring, who must stop the Evil Wizard Mordroc as he has kidnapped Princess Daphne and taken her through time! But you must hurry, for once the Casket of Doom has opened, Mordroc will place the Death Ring upon Daphne's finger in marriage and she will be lost forever… in the Time Warp!
Completely arcade authentic
Original arcade gameplay modes
Remastered animation
Both standard and widescreen editions
All-new move guides for beginners
Watch Mode – View all of the stunning animation without playing!
Dragon's Lair Trilogy will be available this fall for a suggested retail price of $29.99 for Wii.
Click here for information on Used arcade games.
Click here for information on Used amusement park equipment.
Click here for information on Used amusement park rides.
Click here for information on Used laser tag equipment.
Tags: used amusement center equipment, used amusement equipment, used amusement fec equipment, Used Amusement Park Equipment, Used Amusement Park Rides, used arcade games, used laser tag equipment
Silver Strike Bowling
Silver Strike Bowling is the most popular video bowling game in on-premise entertainment. The realistic ten-pin trackball bowler can be found in over twelve thousand bars and restaurants in the U.S., Canada, South Africa, New Zealand, Australia and the U.K. Its lifelike competitive experience and straightforward trackball control has cultivated an army of nearly 5 million Silver Strike bowlers since the game was first released in the summer of 2004.
Up to eight players can play SSB at once, while enjoying three different virtual bowling modes of play. "Vegas Bowling", the most popular play mode, adds a new level of competition by awarding playing cards for every mark a bowler earns. Additionally, it's SSB's realistic physics and 3-D graphics that form the foundation of its success, while the game's competitive, yet casual, competition has continued to drive players to Silver Strike's lanes.
In 2006, Incredible Technologies released the newest version of the game, Silver Strike 2007 Bowler's Club. SSBC is connected online via ITNet® allowing bowler's to track their stats, compete in contests, be a part of local leagues and, most importantly, enter weekly sweepstakes for huge prizes! Whether it's cash, cameras or cars, the Silver Strike Sweepstakes is giving away big prizes to avid bowlers every week!
Posted in Arcade Games General News, New arcade games general news, New arcade games news | No Comments »
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redpajama_set_name: RedPajamaCommonCrawl


text:
Become an OV TOGETHER Member
The Old Vic has been influencing the landscape of British and world theatre for over 200 years. From John Gielgud, Laurence Olivier and Judi Dench to Kristin Scott Thomas, Sheila Atim and John Boyega, The Old Vic has always attracted the best creative talent. Bernard Shaw reviewed plays here. Olivier said it had 'the most powerful actor/audience relationship in the world' and it was the first theatre to perform the complete works of Shakespeare as a series.
Under the leadership of Matthew Warchus, The Old Vic continues to entertain, educate and inspire. Through an exciting and varied programme of work that reflects the theatre's rich history of great acting, dance, music, vaudeville and spectacle, The Old Vic is nurturing a new love of theatre while continuing to appeal to the more experienced theatregoer.
Over the past ten years, the American Associates of The Old Vic (AAOV) have been instrumental in supporting the work of this extraordinary institution. And because the AAOV is a 501(c)(3) non-profit organization, American taxpayers, wherever they live, are able to enjoy the benefits of membership in London, New York, and on tour. AAOV plays an important part in forging transatlantic partnerships and associations that enable American work to be seen in London and British work in New York, a transatlantic exchange of creative talent would not be possible without the help of the American Associates.
JOIN ONLINE DOWNLOAD THE MEMBERSHIP FORM
For more information contact American Associates of The Old Vic c/o Eisner Amper, 733 Third Avenue, New York, NY 10017
Email aaov@oldvictheatre.com.
The American Associates of The Old Vic Theatre, lnc., Tax lD# 51-0389805 is a 501 (c) (3) non-profit organization incorporated in the US as a publicly supported organization. Donations to it are tax-deductible to the full extent allowed by law.
200 Years of The Old Vic


I prefer it over the tech backplate becuase the tank sits closer to my make, making it feel lighter by reducing the lever arm.
It is also much less expensive. D rings can be attached anywhere on the straps, just as with the DIR plate.


I'm really enjoying the Rekindled beta so far. It sends me email collections of passages I've highlighted in Kindle books. There are many books that I remember loving but recall very little from. But I'm also not the type of person who goes back and re-reads books. Rekindled is a nice in-between, since it surfaces specific passages that can be enjoyed on their own or prompt a return to a specific section of a book.


The Laundress Buckets are back!
Store all your detergents in a stylish way!
or to make a unique gift for this Christmas!
Just choose the large white or the extralarge black one and fill it with your favorite products!


In this book "Positive Thinking Quotes: 101 Inspirational, Affirmation and Successful Quotes in Creative Images" you will find what you are looking for.
Whether you have just fought with a friend or a loved one, have to make an important decision in your life, are afraid and worried that things aren't going your way or are just in need a pick-me-up because life has got you down, you'll find a quote that calls to you.
In this book "Positive Thinking Quotes: 101 Inspirational, Affirmation and Successful Quotes in Creative Images" you will find what you are looking for.
Whether you have just fought with a friend or a loved one, have to make an important decision in your life, are afraid and worried that things aren't going your way or are just in need a pick-me-up because life has got you down, you'll find a quote that calls to you.
The compilation is there for your comfort, for your perusal, and for your inspiration. There are 101 quotes that can make any day better and turn your negative thoughts into positive ones.
If you are a writer and that certain inspirational quote is escaping you, browse our pages and you're sure to find one that fits your needs. If you're a student and are writing an essay for class and just can't seem to find the motivation, take a minute.
Open our book, find some inspiration. But most of all, if you are a person who's had a rough day, a lousy week or just want to do some soul-searching, open our book and in between its pages will be quotes that bring you new thoughts and new outlooks on your one very special life.


BELGRADE, 05.02.2109. – The Western Balkan's Regional Platform for Advocating Media Freedom and Journalists' Safety, which represents more than 8000 members, strongly condemns death threats colleagues from N1 received following Milenko Jovanov, vice-president of the ruling Serbian Progressive Party, suggested that N1 was not objective and had recognised Kosovo's independence.
On 4 February N1TV announced its Belgrade office received a series of insults on social networks that branded its journalists as "traitors" and "anti-Serbs". A letter containing serious threats to their lives, those of their families and a message that N1 building will be blown up was also received. It stated: "Do you have families? You're killing our children and grandchildren, and are unaware that if the destruction happens, your building will be the first to be blown up, and you may lose your dearest. That's why we, the veterans, warn you not to play with anyone's destiny because your fate is in our hands." The letter was signed by the "Belgrade Veterans of 1999 war", but the president of this association denied its involvement and called for the police investigation.
In the last three years IJAS recorded more than 80 pressures. Those include public labelling of journalists as "traitors", "foreign mercenaries" and similar by the state officials.
The Regional Platform supports IJAS in its efforts to protect journalists and freedom of speech and call on relevant authorities to investigate the case urgently, bring perpetrators to justice and sanction them adequately.
Additionally, we appeal to the highest officials of Serbia, Aleksandar Vucic, the President and Ana Brnabic, the Prime Minister to condemn these horrific threats and influence state officials to refrain from public labelling of journalists.


At Autobot MC we pride ourselves on the quality of our work. Our team are highly qualified experts using the best products and parts available on the market. We offer diagnostic and MOT work as well as repairs, clutches, cam belts, brakes and exhausts at very competitive prices.
We also do class 4 light 5l and 7 MOT's on site with our state of the art equipment.


This probably The Cheapest natural body oil available, in case you don't want to use virgin olive oil from supermarket:).
Many reviewers compare it to Baby Oil or even Bio Oil, but I would never use either, because they both contain mineral oil that is comedogenic, clogs pores, and doesn't sink in, but only forms a film on the skin.
Palmers, however, is mostly natural (although not organic or virgin) soja oil (only very little of cocoa is in it!), and much better for your skin and environment than any mineral-oil based lotion/cream or oil.
So yes, this Palmers oil has been my every-day body moisturiser on-and off for many years. I actually dry my skin after shower, and apply a small amount on warm skin, put some loose clothes on until it sinks in fully (about 20 min to apply make up etc). It so cheap, very fast relief for dry skin, has a lovely sweet smell that doesn't clash with parfume, if you don't slather a lot of it on, and is just far easier/faster to apply than any lotion.
A very good product that can be easily mixed with a few drops of bergamot or other essential oil to mask the sweet chocolate/vanilla scent it has. As I recently had the bottle leaking in the bag, I poured it into a an emty spray bottle instead, much safer.
I put this in my bath and slather it on before a shower, and then use a body scrub. My skin stays baby smooth all year round with this routine. It's great stuff but does smell a bit cloying if you use it on its own as a moisturiser.
Tip: When my bank balance is in the red, I mix this oil and a handful of sugar for a budget sugar scrub. Has the same effect as commercial ones for a fraction of the price!
UPDATE: Works even better if you apply it to soaking wet skin straight from the bath, pat dry and then apply the lotion. Heaven!
I agree with the other reviewers: Decant this into a spray bottle or you'll be leaving oil slicks in the bathroom and wiping your hands constantly! But-- packaging issues aside-- I love this oil in the cooler months. I mostly use it on my legs, which seem to get the most dry. The scent is very pleasant and sweet, a bit vanilla-ish like most Palmer's products. It does dissipate quickly and doesn't clash with perfumes. The oil is a mixture of plant oils and synthetic emulsifiers, so it feels very light and absorbs quickly. I think the trick is to use sparingly!
I have an absolute addiction to Palmer's products right now! I have the pump lotion, the jar and now have the oil. The Body oil is fantastic, pretty, light and moisturising. I use it in my bath and I apply it in the morning before I dress. I love the scent but can see how some would find it cloying.
UPDATE: I've stopped using this on my body in the morning as I've rediscovered a proudly South African product: Bio-Oil and now only use Palmers oil in the bath as a bath oil.
My HG of body oils. Love the smell of this stuff, quickly absorbing, leaves my skin very soft and with a goregous glow. Definite re-buy again and again.
I'm really happy with Palmer's Cocoa Butter Body Oil. I actually use it as a makeup remover, before I wash with Palmer's Cocoa Butter soap. It removes all of my makeup (Bare Minerals) without a struggle, and my complexion has never looked this good.
I feel a little conflicted over whether I would purchase this again. There are several good things about this product. I haven't had any problem with the bottle so applying it after I take a shower while I am still wet has worked fine for me. I do find the chocolate scent to be very strong. I am not fond of food scents for body products. I have added a ton of orange essential oil (as in a couple of inches) and finally have this product smelling much more like chocolate-orange than just chocolate (which I find much more pleasant than just straight chocolate). If you like the scent of strong chocolate I think you will like this scent. As it is, this scent really lingers on me - I can still smell it on my skin hours after my shower - which is very unusual for me. I think this does an okay job of moisturizing my skin. I normally make my own body oils with a mixture of shea oil, almond oil and jojoba oil. I bought this bottle when I was short on my own supply of oils (which I have to order over the internet) and I was curious to see if I enjoyed the chocolate experience. I mostly likely will stick with my own body oil mixture since it is very inexpensive in the long run and does a great job of moisturizing my skin with natural skin products and no preservatives.
i LOVE THIS OIL!! Has a chocolate scent that works well layering Vanilla Abricot, Serendipity or most other vanilla scents. I love this just for the smell alone.
Argh. Would like this product if the smell didn't make me feel so yucky. This chocolate fragrance just doesn't appeal to me. Went out to buy Neutrogena sesame oil that I used to use years ago.


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Windows Media Player: Tweaking Skinned Windows Media Player with Actual Window Manager
Tweaking Skinned Windows Media Player
Windows Media Player is the most popular digital media player and media library application developed by Microsoft that is used for playing audio, video and viewing images on personal computers running the Microsoft Windows operating system.
Together with the standard Windows Media Player User Interface (UI) today there exists a great number of modern skins. Skin is a non-standard design of UI aimed to make the use of Windows Media Player more enjoyable. Using skins you can easily customize the look of Windows Media Player to match your mood. Skins are quick and easy to download, and they automatically install directly into your player.
The basic difference between standard UI and skin-mode UI is that while skinned Windows Media Player window usually has no standard Windows title bar, therefore it's impossible to tweak skinned Windows Media Player with additional title buttons as it was described in this article. Fortunately, it's still possible to tweak Windows Media Player using automatic triggers available in Actual Window Manager.
Actual Window Manager is an innovative desktop organization application. It has more than 40 handy tools to help you in daily routine window manipulations. In this article we would like to touch upon just automatic features available in Actual Window Manager that will make your skinned Windows Media Player more pleasurable and convenient to use.
- Want to listen music and keep Windows Media Player in background while you work?
Use the Roll Up action to shrink Windows Media Player in such a way that only a small part of its window remains visible. You can teach Actual Window Manager to roll up Windows Media Player automatically when it becomes inactive (and unroll it when it activates back) or unroll while it's being mouse-hovered. Use the Stay Always-on-Top add-in to never lose rolled Windows Media Player fr om sight and have an easy access to it.
- Do you like to have Windows Media Player semi-transparent?
Use the Make Transparent action to toggle the transparency effect for Windows Media Player in Windows� 2000/XP/2003/Vista, ranging fr om 0% (opaque) to 100% (invisible). With the help of Actual Window Manager you will be able to apply the transparency effect to different states of Windows Media Player: at startup, while moving, while mouse-hovering, while inactive and while resizing. This will help you by immediately showing what windows are open now and wh ere they are.
the transparency effect cannot be applied to some skins. Actual Window Manager uses the transparency effect available in standard drawing mechanisms Windows provides for general windows, whereas skinned Windows Media Player uses other specific mechanisms (usually DirectX functions). Therefore such windows are not 'general' in their nature, and that's why the standard transparency effect is incompatible with such windows and produces unexpected results when being applied.
- Need to hide Windows Media Player when working with other windows to free the desktop space?
If you like to listen to music when working with other applications then the Minimize Alternatively action will help you to hide Windows Media Player to a nifty icon placed either to the system tray or anywhere on your desktop. Now straight upon composing a playlist and switching focus to other applications, inactive Windows Media Player will be minimized to the tray. This will make your own taskbar less cluttered and more handy! Click this icon - and Windows Media Player is restored and ready to use!
Use the Move to Monitor function to place Windows Media Player to the desired monitor if you have several monitors connected to your computer. In other words, Windows Media Player will be placed to the desired monitor at its startup. No need to drag it manually!
- Want to remove Windows Media Player from the taskbar?
The Remove Taskbar Entry action will further extend your ability to control Windows Media Player and remove it from the taskbar last and all the time if you wish. Now you will forget about taskbar clutter and will be able to free the precious taskbar space for more urgent applications.
- The music started to stumble and you don't know how to eliminate this handicap?
The most likely reason of this problem is that Windows Media Player lacks for central processor resources. This problem can be solved by increasing the priority level of Windows Media Player upon its startup. Also, you can lower the priority level of Windows Media Player when it is minimized or inactive to provide the maximum system productivity.
- Want to change Windows Media Player window's size and position?
The Resize action lets you change the size of Windows Media Player, which might be useful when watching video to get better view or for extending a playlist. You will get access to the wide range of predefined sizes or specify custom window dimensions yourself as circumstances require. You can maximize Windows Media Player at its startup for watching video or minimize it if you have just a few songs in your playlist.
The Align action provides the automatic horizontal and vertical alignment of Windows Media Player in nine predefined alignment modes. Now you will be able to position Windows Media Player at its startup to the most convenient place on the desktop!
Actual Window Manager makes your work with Windows Media Player and other applications extremely simple and convenient. It will help you to save your time and efforts. It has more than 40 productivity tools which you can use to control every window on your screen.
Interested? Try it out, Actual Window Manager is easy-to-use and very handy!
See the following links that will be your guides in being acquainted with Actual Window Manager:
Download the fully functional 60-days trial version of Actual Window Manager right now. It is completely free!
Actual Window Manager Home - the official page wh ere you can download or order the risk-free version and read what other users say about Actual Window Manager.
Features available in Actual Window Manager - here you can find the full spectrum of what and when Actual Window Manager is able do with windows.
Here you can find the information on how to create custom settings just for the skinned Windows Media Player.
Check out our articles. There you will find many other ways of applying the new title buttons and other features to make your day-to-day work easier, faster and more convenient.
Dual Monitor Desktop Background Management with Actual Multiple Monitors in Windows 8
How to set the slideshow from Internet sources as the dual monitor desktop background in Windows 8? Read the following article.
How to keep system dialogs Always-on-Top
This article describes how you can stay on top any system dialogs such as 'Add/Remove Programs', 'System Properties', 'Time/Date Properties' and doesn't allow other windows to hide it partially or completely.
Improved Windows 8/8.1 Dual Monitor Taskbar
Multiple monitor support in Windows 8.1 contains only several new features, including a limited multi-monitor taskbar. This is not enough to make work with several displays convenient. That is why Actual Multiple Monitors provides a large set of different tools improving multiple-monitors environment. This is a best alternative to the native Windows 8.1 multiple monitor support.
Minimize to tray: Minimize to the system tray any program you want!
How to minimize any program to the tray!
Improve Yahoo Messenger with Actual Title Buttons
Enhance your work with Yahoo Messenger with the help of Actual Title Buttons. Now you can add a whole bag full of new nifty buttons that will make Yahoo Messenger more convenient, stylish and pleasurable to work with.
Optimize Microsoft Word by means of Actual Window Manager!
Actual Window Manager presents a great variety of functions for working in Microsoft Word. By using them, you will be able to decrease the time you usually waste on switching between windows of different documents, keep your desktop in order and work much quicker.
Organizing Standalone Kiosk Access to Intranet with Actual Window Manager
Actual Window Manager will help you overcome the complications associated with the Intranet kiosk access.
Easy Way to Minimize Windows to the System Tray or to the Desktop
Actual Window Manager provides an additional title button to minimize any window to the system tray in one click.


It is the leaves of the basil plant that are used in cooking, and these leaves can range from green to reddish to purple in color, depending on the variety. There are more than 60 different varieties of basil, each with its own distinct flavor. These flavors include hints of lemon, thyme, jasmine, clove, cinnamon, and anise.
For best flavor, the leaves should be picked before the plant begins to flower. As an added benefit to growing your own, basil is said to repel flies, mosquitoes and cockroaches.
Although basil is an annual, it is often pinched back to keep it from flowering and extending its growth period.
Many home cooks keep basil growing year-round in pots indoors in a sunny window to have fresh basil at their fingertips. If you do not have the luxury of a basil plant, you can still find fresh basil leaves in most local grocery stores in the produce department.
Look for fresh, vibrant green leaves with no dark spots or signs of decay. Fresh basil leaves should be layered in damp paper towels inside a plastic bag and refrigerated up to 4 days.
For basil with stalks attached, place in a glass of water and cover with a plastic bag secured to the glass. Store in the refrigerator, changing water daily, and use within a week. Do not wash the leaves until you are ready to use them.
Another option is to put whole or chopped fresh leaves in an ice cube tray and cover with water or broth before freezing.
Once frozen, pop the cubes out into an airtight bag. Use the cubes in soups, stews or sauces.
Basil is readily available in dried form, but it cannot compare in flavor to fresh basil. Dried basil should be stored in a cool, dark place away from heat and light. Dried herbs begin to lose their potency within 6 months, even under the best conditions.
Bear in mind that dried basil can easily have been sitting on your grocer's shelf for months by the time you buy it. So unless you use copious amounts and go through it quickly, it is best to buy small amounts so you can use it up faster. When dried, basil loses much of its intensity.


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'The Shooter' On the Hill
By David Granger Published: Feb 13, 2013
Read the full story on "The Shooter" by Phil Bronstein >>
Yesterday, as Washington, D.C., got ready for the State of the Union, the man at the center of our story "The Shooter" was on Capitol Hill. We had arranged meetings with nine of the most influential members of the Senate and House to discuss the larger issues of the story — the difficulty soldiers, and especially members of elite Special Operations Forces, have transitioning to civilian life and the inadequacy of the assistance provided to them by our government. Our little traveling party, which included Phil Bronstein, who wrote our story and is executive chairman of the Center for Investigative Reporting, met for breakfast at about 9:15 and ended up hurrying out of the Capitol building just as it was being locked down in preparation for the President.
The Shooter is not as large as I expected. He's one of those guys who is sneaky big. He doesn't stand out. In a suit, you can't see his tattoos and you don't realize how solid he is until you stand next to him.
In every meeting, he hit on the same themes. In the years since 9/11, the toll on members of Tier One Special Operations Forces has become even more brutal than in previous eras. As the way we wage war has transitioned from boots on the ground to tactical, highly targeted strikes, those who do the striking are under unprecedented stresses. Without drama, he described his life as a member of SEAL Team 6: Three hundred days a year away from his family. Four-month deployments during which they performed missions — sometimes multiple missions — virtually every night. When not deployed, constant training — parachute jumps, underwater exercises, skills development, conditioning. The toll on the body, and the psyche, is unending, which makes the requirement of twenty years of service to qualify for pension and retirement benefits an unrealistic expectation in the modern era of war.
In one meeting, the Shooter gave an example of one of his own long-term injuries that stuck with me: He has arthritis in his neck that is the result of wearing heavy night-vision goggles every night for 120 nights in a row, repeated over multiple deployments. He's in his mid-30s.
He spoke of how difficult it was to leave the service. His fellow Team 6 members saw it as something of a betrayal, as quitting the mission. The command structure discourages it to keep force levels up. As a result, even though he gave notice of his intentions a year before his actual departure and signed up for another deployment (to Afghanistan, in winter) after the bin Laden mission, assistance in transitioning from military to civilian life was brief and abrupt. Health benefits for his family ended at midnight of his last day; no pension; no security provisions for him or his family.
It was this last point that seemed to have stayed with him on a personal level. The Shooter is self-sufficient, as you'd imagine. He took pains to tell the legislators at each of the gatherings that he was not in D.C. to ask for help for himself — "It's not about me," he said in each meeting — but for colleagues and friends who are still in and concerned about the approaching end of their service. After six months with minimal income (as the story reports), he's been doing a form of consulting that is making him a living.
But, in several meetings, he spoke passionately about the lack of action to provide for the safety of his family. On returning from the bin Laden mission, he asked to have his house fortified by the construction department on base, but was rebuffed. He also talked about more recent measures he and his wife have taken in anticipation of the release of the Esquire story.
During each meeting, the Shooter and the members of the group accompanying him presented the legislators with a three-part proposal for easing the transition out of the military for elite forces that would require no legislation.
• A tiered pension plan that would begin after five years of duty and vary depending on base pay and length of service.
• Enhanced transition services that must be provided on an "opt-out" basis to overcome the fact that many vets are unaware that such services exist or how to apply for them and to include 18 months of comprehensive health insurance available immediately for vets and their families.
• Departure pay based on length and type of service.
In every meeting I attended, the senators, representatives, and their staffs were receptive — in many cases eager to help. They are all extremely attuned to veterans' concerns, as nearly all of them have heard from constituents that the Veterans Administration system is a challenge to navigate and that high percentages of veterans never take advantage of the benefits offered. The Shooter mentioned in most meetings that it was only in recent weeks that he was made aware of the medical benefits he is eligible to apply for through the VA. And in most meetings, the staff members who attended included former military and/or active-duty service members who were on staff or interning in the office.
But it was illuminating to hear what the roadblocks to change will be. While most legislators were entirely attuned to the unique and uniquely punishing demands put on special operators like SEALs, creating a special category of benefits, even if it applies to the elite Special Operations Forces of all branches of the military, will be difficult.
There was discussion that while more effective transitional assistance is essential, it can sometimes be seen by the command structure as providing an incentive for soldiers, especially the elite, to leave the military and thereby deplete force levels. The Shooter's mentor, a former SEAL who attended all the briefings, spoke about both the peer pressure on elite soldiers to continue their mission and also the pressures on the command structure to retain its best talent.
In each legislative office, the story had been avidly read and certain details in particular — such as the discovery of raw opium in bin Laden's compound — elicited comment. In general, there were far fewer questions about the mission than we anticipated, though several legislators were curious about what the Shooter thought of Zero Dark Thirty. As in the article, he was generally complimentary of the movie, with a significant exception: The quality (and quantity) of the SEALs' tattoos in the film was not up to his standards.
David Granger
David Granger has been Esquire magazine's Editor in Chief since 1997.
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In Arabidopsis thaliana, DES1 is the only identified l-Cysteine desulfhydrase located in the cytosol, and it is involved in the degradation of cysteine and the concomitant production of H2S in this cell compartment. Detailed characterization of the T-DNA insertion mutants des1-1 and des1-2 has provided insight into the role of sulfide metabolically generated in the cytosol as a signaling molecule. Mutations of L-CYS DESULFHYDRASE 1 (DES1) impede H2S generation in the Arabidopsis cytosol and strongly affect plant metabolism. Senescence-associated vacuoles are detected in mesophyll protoplasts of des1 mutants. Additionally, DES1 deficiency promotes the accumulation and lipidation of the ATG8 protein, which is associated with the process of autophagy. The transcriptional profile of the des1-1 mutant corresponds to its premature senescence and autophagy-induction phenotypes, and restoring H2S generation has been shown to eliminate the phenotypic defects of des1 mutants. Moreover, sulfide is able to reverse ATG8 accumulation and lipidation, even in wild-type plants when autophagy is induced by carbon starvation, suggesting a general effect of sulfide on autophagy regulation that is unrelated to sulfur or nitrogen limitation stress. Our results suggest that cysteine-generated sulfide in the cytosol negatively regulates autophagy and modulates the transcriptional profile of Arabidopsis.
Cys occupies a central position in plant primary and secondary metabolism. Beyond its importance as an amino acid, Cys is a precursor for a large number of essential biomolecules. Many plant defense compounds produced under biotic and abiotic stress derive from Cys (Rausch and Wachter, 2005) and contain sulfur moieties as functional groups. Recently, it has been suggested that Cys itself is an important determinant of the antioxidative capacity of the cytosol in Arabidopsis thaliana (López-Martín et al., 2008a, 2008b). The biosynthesis of Cys involves the sequential reaction of two enzymes, Ser acetyltransferase (EC 2.3.1.30), which synthesizes the intermediary product O-acetylserine from acetyl-CoA and Ser, and O-acetylserine(thiol)lyase (OASTL; EC 2.5.1.47), which incorporates the sulfide derived from the assimilatory reduction of sulfate with O-acetylserine to produce Cys. Plant cells contain different Ser acetyltransferase and OASTL enzymes in the cytosol, plastids, and mitochondria, resulting in a complex variety of isoforms and different subcellular Cys pools. In the model plant Arabidopsis, five Ser acetyltransferase (Howarth et al., 2003) and nine OASTL genes (Wirtz et al., 2004) have been identified.
In recent years, much progress has been made in understanding the most abundant OASTL enzymes in Arabidopsis, with a primary focus on their involvement in the primary sulfate assimilation pathway. We recently investigated DES1, a minor OASTL-like protein located in the cytosol, and showed that DES1 catalyzes the desulfuration of l-Cys to sulfide plus ammonia and pyruvate, rather than promoting Cys biosynthesis; thus, DES1 is a novel l-Cys desulfhydrase (EC 4.4.1.1) (Álvarez et al., 2010). A role of DES1 in plant metabolism was evidenced by the phenotypes of the T-DNA insertion mutants des1-1 and des1-2, which exhibit premature leaf senescence along with increased expression of senescence-associated genes and transcription factors. Disrupted DES1 function also significantly reduces the total Cys desulfuration activity in leaves, with a concomitant increase in the total Cys content (Álvarez et al., 2010). In response to plant pathogens, des1 mutants were found to behave as constitutive SAR mutants, exhibiting high resistance to biotrophic and necrotrophic pathogens, salicylic acid (SA) accumulation, and WRKY DOMAIN CONTAINING TRANSCRIPTION FACTOR54 and PATHOGENESIS RELATED1 induction (Álvarez et al., 2012). Taken together, these data highlight the importance of DES1 in the signaling and regulation of plant responses to various processes.
Hydrogen sulfide has increasingly been recognized as an important signaling molecule, of comparable importance to NO and CO in mammalian systems. The list of biological roles of H2S in various systems of the human body has rapidly expanded (Łowicka and Beltowski, 2007; Gadalla and Snyder, 2010; Kabil and Banerjee, 2010). Hydrogen sulfide is weakly acidic and dissociates in aqueous solution. Under physiological pH conditions (pH 7.4), one-third of the H2S present is undissociated, and the remaining two-thirds dissociate into H+ and HS−. HS− may subsequently dissociate into H+ and S2−, but this dissociation requires high pH conditions. Similar to NO and CO, H2S is lipophilic and permeates plasma membranes freely, although the ionized form HS− cannot permeate membranes (Kabil and Banerjee, 2010). H2S is endogenously produced in mammalian tissues by enzymatic reactions of l-Cys, primarily via two cytoplasmic enzymes, cystathionine-γ-lyase (EC 4.4.1.1) and cystathionine-β-synthetase (EC 4.2.1.22), which both use pyridoxal-5′-phosphate as a cofactor, with ammonia and pyruvate as by-products (Gadalla and Snyder, 2010). To our knowledge, DES1 is the only l-Cys desulfhydrase present in the Arabidopsis cytosol that catalyzes the desulfuration of l-Cys to sulfide plus ammonia and pyruvate and requires pyridoxal-5′-phosphate as a cofactor (Álvarez et al., 2010). DES1 may therefore be responsible for the release of metabolically regulated sulfide in this cellular compartment. The l-Cys desulfurases (EC 2.8.1.7) also catalyze Cys desulfuration required for iron-sulfur cluster, thiamine, biotin, and molybdenum cofactor synthesis, but they instead generate l-Ala and elemental sulfur (Van Hoewyk et al., 2008).
Emerging data over the recent years suggest that H2S may be a signaling molecule equally important to plants as NO and H2O2. H2S has been found to mediate increases in tolerance and protection against certain plant stresses. For example, sulfide alleviates the inhibitory effects of copper and aluminum stress on wheat (Triticum aestivum) germination and is associated with antioxidative defense (Zhang et al., 2008, 2010). Similarly, the inhibitory effect of boron on cucumber (Cucumis sativus) root elongation is substantially reduced by H2S treatment, which upregulates the cell wall–associated proteins and expansins (Wang et al., 2010). Moreover, sodium hydrosulfide pretreatment improves heat tolerance in tobacco (Nicotiana tabacum) suspension cultured cells, requiring the involvement of calcium and calmodulin (Li et al., 2012). It has also been reported that sulfur fertilization increases plant resistance against fungal pathogens, and it has been proposed that H2S is involved in the mechanisms of the sulfur-induced resistance, or sulfur-enhanced defense, phenomenon (Rausch and Wachter, 2005). A field experiment demonstrated that Brassica napus is able to react to fungal infection and releases H2S as a result of increased l-Cys desulfhydrase activity (Bloem et al., 2004). We recently demonstrated that cytosolic Cys plays a role in the establishment and signaling of the plant response to pathogens. This function could be due to Cys itself or to its function as generator of a particular sulfur compound (Álvarez et al., 2012). Hydrogen sulfide has been recently discovered as a component of the abscisic acid signaling network in guard cells (García-Mata and Lamattina, 2010; Lisjak et al., 2010). It has also been shown to modulate photosynthesis in spinach (Spinacia oleracea) seedlings by regulating the expression of genes involved in photosynthesis and thiol redox modification (Chen et al., 2011).
In this work, we observed senescence-associated vacuoles and an induced autophagy phenotype in des1 mutants. Additionally, we found that these phenotypes could be rescued by the exogenous application of H2S. An in-depth study of the effects of exogenous H2S revealed a specific role of sulfide as a general repressor of autophagy and a transcriptional modulator in Arabidopsis.
We previously demonstrated that a mutation in DES1 causes premature leaf senescence, as evidenced by increased transcript levels of senescence-associated genes (Álvarez et al., 2010). To further dissect this particular phenotype, we aimed to determine how senescence was induced at the cellular level in this mutant. Plants contain different types of vacuoles, and senescence-associated vacuoles (SAVs) are formed de novo during leaf senescence. SAVs are small in size and exhibit a lower pH than the central vacuole (Zouhar and Rojo, 2009). des1-1 mutant plants and their respective wild-type Columbia-0 (Col-0), were grown for 4 weeks side by side under long-day photoperiod and nutrient-sufficient conditions. The identification of vacuolar compartments associated with leaf senescence was performed in mesophyll protoplasts using Lysotracker Red, a fluorescent marker for acidic organelles (Otegui et al., 2005). After incubating the protoplasts prepared from des1-1 and wild-type leaves, small fluorescent structures were clearly visible in the cytoplasmic periphery around the plastids in des1-1 protoplasts but not in wild-type protoplasts (Figure 1A). Further evidence indicating that this fluorescence signal was due to the mutation in DES1 was the presence of these structures, albeit at lower abundance, in a second mutant allele, des1-2, and their absence in Nossen-0 (No-0) plants, the respective wild-type ecotype (Figure 1A).
Because cellular components are degraded and the released nutrients are mobilized for reuse during leaf senescence (Lim et al., 2007), we evaluated whether the SAVs observed in the des1 mutants were related to an autophagic mechanism. Proteins involved in autophagy (ATG proteins) have been used to monitor autophagic activity in plants, such as ATG8 accumulation and lipidation. The ATG8 protein is covalently conjugated to phosphatidylethanolamine (PE), and its lipidation is required for the formation of the autophagosomes, which is the most remarkable feature of autophagy (Nakatogawa et al., 2009). We used polyclonal antibodies raised against the recombinant ATG8 protein from Chlamydomonas reinhardtii, which has been shown to be functionally conserved and therefore useful as a molecular autophagy marker in Arabidopsis (Pérez-Pérez et al., 2010). Four-week-old des1 mutant plants and their respective wild-type ecotypes were grown as described above, leaf tissues from these plants were homogenized, and total protein samples were subjected to immunoblot analysis (Figure 1B). The antibodies detected two groups of ATG8 proteins, with a protein-banding pattern similar to those previously observed in Arabidopsis (Phillips et al., 2008; Chung et al., 2010). The slower-mobility group corresponds to unmodified protein forms, and the group with faster mobility includes ATG8 proteins conjugated with phosphatidylethanolamine. The SDS-PAGE profiles of the ATG8 proteins revealed increased accumulation in both des1 mutant alleles relative to the wild-type plants, predominantly for the modified ATG8 forms (Figure 1B). Collectively, these results suggest that deficient DES1 protein function promotes accumulation of SAVs and autophagy activation.
DES1 catalyzes the enzymatic desulfuration of l-Cys to sulfide, and disruption of DES1 provokes a 20 to 25% increase in the amount of Cys (Álvarez et al., 2010), but it may also reduce the capacity of the cytosol to release H2S. To assess the latter, we used an H2S-selective electrode to measure endogenous H2S concentrations in leaf extracts from des1 and wild-type plants grown in soil for 4 weeks under a long-day photoperiod (Figure 2). Both des1 alleles showed a 30% reduction in endogenous sulfide compared with their respective wild-type plants, although the endogenous H2S concentration in leaves is dependent on the ecotype in Arabidopsis, with a 45 to 49% higher sulfide content in No-0 than Col-0. When plants grown under physiological conditions were treated exogenously with 200 μM Na2S for 10 d, we observed an increase in the endogenous H2S content that was limited to ∼13 pmol/mg fresh weight (FW) in Col-0 and des1-1 plants and ∼15 pmol/mg FW in No-0 and des1-2 plants (Figure 2). No phenotypic damage was observed during the period of the treatment, whose length is similar to conditions used in plants by other groups (Chen et al., 2011; Zhang et al., 2011; Dawood et al., 2012). Therefore, the quantified sulfide values do not exceed the sulfide toxicity thresholds that the plant can tolerate without compromising viability.
To determine if exogenously applied sulfide could completely or partially rescue the phenotypic characteristics observed in the des1 mutants, 20-d-old plants grown with sufficient sulfur nutrition were treated for 10 d with 200 μM Na2S and compared with untreated plants grown adjacent to them. The sulfide treatment clearly eliminated the phenotypic differences of the des1-1 mutant. After mesophyll protoplasts were prepared from the leaves of sulfide-treated plants and analyzed for the presence of SAVs, yellow fluorescent structures corresponding to SAVs stained with Lysotracker Red were not detectable in any of the high number of des1-1 protoplasts analyzed (Figure 3A). We also observed a reduced accumulation of lipidated ATG8 forms in the Na2S-treated des1-1 plants, suggesting that exogenous sulfide addition can inhibit the autophagy process (Figure 3B).
Studies assessing the role of hydrogen sulfide in mammalian systems typically involve the administration of exogenous H2S generated in vitro from both Na2S and NaHS at micromolar concentrations (Szabó, 2007). Because the NaHS compound is more widely used as the hydrogen sulfide donor, we performed experiments similar to those described above on 20-d-old plants treated with different concentrations of NaHS. We observed that exogenous addition of NaHS rescued the autophagy activation phenotype of the des1-1 mutant. Immunoblot analysis of ATG8 showed that even with a low NaHS concentration of 50 μM, the significant accumulation of modified ATG8 was lost; the protein banding patterns were similar at all NaHS concentrations (Figure 4).
To confirm that the observed phenotype of the des1-1 mutant plants was indeed due to the disruption of the DES1 gene and a reduction of cytosolic endogenous sulfide, complementation analysis was performed using the full-length DES1 cDNA fragment. We first measured and compared the endogenous H2S concentrations in leaf extracts of the complemented des1-1:P35S-DES1 line, the wild type, and the des1-1 mutant. The wild-type and complemented line contained similar levels of endogenous sulfide, 10.5 and 10.1 pmol/mg FW, respectively, but the des1-1 mutant displayed a reduced level of 6.8 pmol/mg FW. Additionally, the des1-1:P35S-DES1 line showed the same pattern of ATG8 accumulation observed in the wild type. The ATG8-phosphatidylethanolamine adducts observed in the des1-1 mutant were strongly reduced, especially considering the higher protein loading in the lanes corresponding to the wild type and complemented line (Figure 5). Therefore, complementation of des1-1 with the P35S-DES1 construct resulted in wild-type levels of endogenous sulfide and also reversed the autophagy-induced phenotype.
Further confirmation that the absence of functional DES1 in the cytosol specifically provoked the accumulation of SAVs and the induction of autophagy was the inability to detect SAVs in oas-a1.1 mesophyll protoplasts and the observation of the same patterns of ATG8 lipidation in wild-type and oas-a1.1 mutant plants (see Supplemental Figure 1 online). The oas-a1.1 mutant previously described is deficient in the most abundant OASTL isoform located in the cytosol, which dominantly contributes to the Cys biosynthesis in this compartment (López-Martín et al., 2008a). The oas-a1 mutants showed a phenotype opposite to the des1 mutants, with a significant reduction in the amount of Cys and opposite responses under pathogen attack (Álvarez et al., 2012).
In plants, autophagy is recognized as a key process in nutrient remobilization when the environmental nutrient supply is limited. Several studies have reported autophagy induction by carbon and nitrogen starvation, which is observed in many cases as a substantial increase in ATG8 protein levels (Hanaoka et al., 2002; Xiong et al., 2005; Chung et al., 2009). As indicated above, increased ATG8 protein levels were observed in des1 mutants when the plants were grown with a sufficient C, N, and S nutrient supply; thus, the effect of sulfide treatment does not appear to depend on the nutrient supply of the plant. To ensure that sulfur depletion was not a contributing factor in our plant system, we analyzed the transcript levels of genes in the sulfate assimilation pathway that are regulated by sulfur availability. Col-0 wild-type plants were grown for 20 d under the same conditions used for the previous experiments and irrigated with water or 200 μM Na2S for the next 10 d. The expression of sulfate transporter genes and APS reductases was analyzed. The transcript levels of the four sulfate transporter genes and the three APS reductases analyzed by quantitative RT-PCR (qRT-PCR) were similar for both sulfur nutrition conditions (see Supplemental Figure 2A online). These results suggest that inhibition of autophagy mediated by sulfide (as measured by ATG8 protein accumulation and modification) is unrelated to sulfur limitation.
To further reinforce our conclusion that the autophagy phenotype of the des1 mutants is completely unrelated with nutrient limitation, we analyzed the transcript levels of genes in the nitrate assimilation pathway. No significant differences were observed in the transcript levels of both nitrate transporter genes, NTR1.1 and NTR2.1, or in the expression level of NIA1 gene, encoding the minor nitrate reductase. However, we did observe in the des1-1 mutant a significant but slight induction of the NIA2 gene, encoding the major nitrate reductase (see Supplemental Figure 3 online), whose mRNA level showed to rise in response to nitrate treatment (Campbell, 1999; Wang et al., 2003).
Previous studies have shown that ATG8 mRNA accumulates when detached leaves or whole plants are placed in extended darkness to deplete the available sugars (Sláviková et al., 2005; Thompson et al., 2005; Xiong et al., 2005). To test if the effects of sulfide as an autophagy inhibitor were observable under conditions unrelated to sulfur nutrition, wild-type and des1 mutant plants grown in soil were placed in darkness to induce carbon starvation and then allowed to recover. In the absence of sulfide, enhanced chlorosis of the cotyledons and leaves was evident after the dark treatment in both the wild-type and des1 mutant plants. By contrast, sulfide addition during the dark treatment and the recovery period apparently reduced the extent of chlorosis in all plant lines, resulting in a healthier phenotype (see Supplemental Figure 4A online). Consistent with the data previously described, ATG8 protein levels, primarily the conjugated ATG8 form, increased in Col-0 wild-type plants under dark-induced carbon starvation compared with plants grown under normal physiological conditions; in the des1-1 background, a further increase in ATG8 protein accumulation was not observed (Figure 6A). Remarkably, the addition of sulfide not only reduced the levels of ATG8 proteins in the des1-1 plants grown under physiological conditions but also reduced ATG8 protein accumulation in both Col-0 and des1-1 plants under carbon starvation (Figure 6A). Nearly identical results were obtained in wild-type No-0 and des1-2 mutant plants. Sulfide also reduced the accumulation of ATG8 proteins in carbon-starved No-0 and des1-2 plants and physiologically grown des1-2 plants to the levels observed in wild-type No-0 grown under physiological conditions (Figure 6B). Further analysis of the transcript levels of four sulfate transporter genes and three APS reductase genes in Col-0 wild-type plants subjected to carbon starvation in the absence and presence of sulfide revealed no significant differences for the two sulfur conditions (see Supplemental Figure 2B online). This finding further supported that the effect of sulfide was not related to sulfur limitation.
Since DES1 catalyzes the desulfuration of l-Cys to sulfide plus ammonia, we performed the dark-induced carbon starvation experiments in the presence of ammonium to confirm the specific role of sulfide as a repressor of autophagy. Unlike sulfide, ammonium was unable to reduce chlorosis in any plant lines (see Supplemental Figure 4B online) and was unable to reduce the enhanced accumulation of ATG8 proteins in both Col-0 and des1-1 mutant plants under carbon starvation (Figure 7).
Using the Affymetrix ATH1 GeneChip, we performed a comparative transcriptomic analysis on leaves of des1-1 and Col-0 plants. Total RNA was extracted from the rosette leaves of plants grown for 20 d in soil under identical long-day conditions, with three biological replicates for each genotype. These samples were used to prepare complementary RNA, which was then hybridized to the chips (Gene Expression Omnibus repository GSE19244).
The normalized data from the replicates revealed differential expression of only 16 genes in the des1-1 mutant, with all of them upregulated and none downregulated by more than twofold (see Supplemental Table 1 online). The 16 induced genes were classified into functional groups using MapMan categorization, and the most abundant group corresponded to proteins that respond to abiotic and biotic stimuli. This group included the PLANT DEFENSIN PROTEIN 1.2B (PDF1.2B) and PDF1.2A genes and CATION EXCHANGER3 (CAX3), a gene encoding the vacuolar H+/Ca2+ antiporter. Two genes closely associated with senescence in rosette leaves, SENESCENCE-ASSOCIATED GENE21 (SAG21; At4g02380) and the NAC transcription factor NAP (At1g69490), were also induced in des1-1 at this growth stage.
In another experiment, total RNA was extracted after 30 d of growth under identical conditions, and the des1-1 transcriptional profile in rosette leaves changed dramatically at this later growth stage (Gene Expression Omnibus repository GSE32566). The normalized data from the replicates showed differential expression of 1614 genes in the des1-1 mutant, with 701 genes downregulated and 913 genes upregulated by more than twofold. These differences between the transcriptional profiles of young and old des1-1 plants were clearly visible when the MA plots [differential expression of all genes as logRatio (M) plotted against their logSignal (A)] were compared (Figures 8A and 8B).
The 1614 genes with altered transcript levels were classified into 23 functional groups using MapMan categorization (see Supplemental Figure 5 online). The most highly induced genes were a Cys-type endopeptidase with unknown function (At2g27420) (71-fold induction), SAG29 (60-fold induction), which integrates environmental stress responses into the process of senescence, and LATE ELONGATED HYPOCOTYL (LHY) (50-fold induction). LHY encodes a MYB transcription factor involved in the circadian rhythm along with the MYB transcription factor encoded by CCA1, which was also induced at this stage (10-fold induction). The genes upregulated at the earlier growth stage were induced at the later growth stage with even higher levels of induction; for example, the vacuolar H+/Ca2+ antiporter gene CAX3 exhibited a 12-fold induction at the later growth stage.
Excluding genes with no functional assignments, the category "proteins" was the most important functional group and included 103 upregulated and 119 downregulated genes. Of the genes within this functional group, 41 encode proteins associated with ubiquitin- and autophagy-dependent degradation (see Supplemental Figure 6 and Supplemental Table 2 online). The autophagic recycling of intracellular constituents requires the ubiquitin-like ATG8 and ATG12 proteins (Chung et al., 2010). The genes encoding the ATG8B and ATG12A isoforms were induced in des1-1 at the later growth stage, corresponding with the induced autophagy phenotype observed in the mutant at this stage of growth. Furthermore, most of the deregulated genes in this functional group are related to ubiquitin-dependent degradation. Genes encoding proteins of the ubiquitin-proteasome system deregulated in des1-1 include three genes associated with the ubiquitin-conjugating E2 enzyme, 24 RING domain E3 ubiquitin ligase genes, and components of the SCF ubiquitin ligase complex. Among the upregulated RING domain E3 ligase genes, we identified the gene encoding the XERICO protein, which promotes abscisic acid accumulation and drought tolerance and has been identified as a DELLA target (Davière et al., 2008). The SCF ubiquitin ligase complex genes include SKP1 (At5g57900), the protein product of which functions as an adaptor to bind F-box proteins in the SCF complex, and 11 F-box genes, including AFR, which mediates the turnover of a repressor of phyA signaling (see Supplemental Table 2 online).
The effects of sulfide observed in the des1 mutants, namely, the reversal of the early senescence phenotype evidenced by the presence of SAVs and ATG8 accumulation and modification, were also observable at the transcriptional level. Total RNA was extracted from rosette leaves of des1-1 and wild-type plants grown for 20 d and treated with sodium sulfide for 10 additional days, with three biological replicates for each genotype (Gene Expression Omnibus repository GSE32566).
A comparison of the transcriptional profiles of des1-1+Na2S and Col-0+Na2S revealed that exogenous sulfide reversed differences at the transcript level between the mutant and the wild-type lines, analogous to the reversion of autophagy observed in previous experiments (Figures 8B and 8C). The transcriptional profile of the treated plants was similar to the profile at the earlier growth stage, with only six genes upregulated and nine genes downregulated by more than twofold (see Supplemental Table 3 online). The induced genes included PDF1.2B and PDF1.2A, which were also induced in untreated plants at the earlier growth stage (Figure 8A).
We performed real-time RT-PCR on a subset of genes to validate the expression data of the microarray analysis performed in plants at the late growth stage. Genes with altered expression were selected, including those with the largest fold changes in the des1-1 transcriptional profile of rosette leaves from plants grown for 30 d. The qRT-PCR analysis was performed in both mutant plants, des1-1 and des1-2, and compared with the wild-type Col-0 and No-0, respectively. Overall, there was qualitative agreement between the qRT-PCR results and the microarray analysis. The des1-1 mutant showed the same changes in gene expression; the des1-2 mutant also showed the same pattern of regulation, but with some quantitative differences (see Supplemental Figure 7 online). We also performed qRT-PCR analysis in plants grown for 20 d and treated with sodium sulfide for 10 additional days to validate the results of the transcriptomic analysis. In both mutants, we observed a reversal of the transcriptional changes of the selected genes to the expression levels observed in the wild type. Therefore, the microarray data and the results of the qRT-PCR analysis in the des1-1 and des1-2 mutants were strongly correlated (see Supplemental Figure 7 online).
The effect of the sulfide treatment on the transcriptional regulation of genes in both mutants was reinforced by the qRT-PCR analysis of the senescence-associated genes. In untreated plants, SAG21 showed low levels of transcript abundance, with higher levels in des1-2 than in des1-1, while SAG12, NAP, and PR1 had high transcript levels, and these levels were highest in des1-1. Sulfide strongly reduced most of the transcript level changes in both mutants, supporting the role of sulfide as a transcriptional regulator in the des1 mutants. SAG12, NAP, and PR1 transcripts levels were significantly reduced by sulfide treatment in both mutant plants, and SAG21 transcript level decreased in des1-2 and des1-1, but only slightly in the latter (Figure 9). Furthermore, the observed transcript level accumulation of the SA-responsive defense marker PR1 in both mutants in the absence of sulfide correlated with an increase of SA. We measured 1.6- and 1.9-fold increases in SA levels in the des1-1 and des1-2 mutants, respectively, compared with their corresponding wild-type ecotypes (see Supplemental Figure 8 online).