The first thing to do is reduce the moisture content in the wood, to less than 20% moisture. Moisture meters can be used to determine moisture level in the wood. Central heat, vapor barriers and good ventilation can help control moisture. Rough-cut lumber should be dried in a kiln to kill all stages of the beetle. Reducing moisture may not be enough to completely control powder post beetle infestations.
Sanded and varnished wood will not be as attractive to adult beetles. They can not penetrate or find cracks and crevices in the wood surface in order to deposit their eggs.
Don't store valuable items in sheds and barns. Wood boring beetles are common in these areas.
Infested furniture that is unfinished (unvarnished, not painted, not waxed or not sealed) can be treated with Tim-bor or Boracare. Boracare and Timbor will prevent newly hatched larvae from entering the wood. Infested furniture that can not be treated with a product like Tim-bor and Boracare can be treated in fumigation chambers. Fumigation chambers are used to fumigate infested furniture. Look for pest control operators that are licensed for fumigations.
If practical, remove infested wood. If not, use residual borate insecticides such as Boracare or Timbor. Due to its long life cycle (12-18) months in the wood, you may see emerging beetles emerge even after treatment. The eggs and coming larvae will come in contact with the insecticide to kill them.
Timbor is a powder that mixes with water. One l.5 lbs pouch is mixed with one gallon of water and sprayed to the surface area of infestation. When sprayed it penetrates the entire wood, where it will remain for several years.
An alternative to Timbor is: Boracare. Boracare is a liquid borate that penetrates faster initially than Timbor for the first few hours, but is equal after that. Timbor is considerably cheaper per gallon to use.
Note when using insecticides: Powderpost beetles life cycles vary from 3 months to a year. Emerging larvae could be maturing with adult beetles emerging for up to a year. If the wood is too dry (less than 15%), depth of penetration with the Boracare may only occur to the top 1/4 inch. To solve this, spray the wood first with water to increase moisture.
Fumigation may be advisable in cases of severe powder-post beetle damage, especially where other methods have failed or where rapid elimination of the insects is critical or desired. Fumigating is advantageous where it is hard to apply borate treatments such as cramped crawlspaces. Fumigation must be done by a certified pest control operator with a fumigation license. Fumigation is non residual and will not last. Because of this, fumigation will not prevent future re-infestation if the wood is exposed to adult powder post beetles looking to lay their eggs.
Alternatives to Fumigation describes the benefits and drawbacks to borate liquid insecticides instead of fumigation.
The first step to management is deciding if there is an active infestation. You may be inspecting old damage. An active infestation will have borings in piles that are accumulated on the floor below or near the holes. You may also hear a ticking sound made by the larvae. If the damage is old and not currently active, you do not have to treat.
Inspect periodically all exposed wood surfaces and probe them for evidence of internal damage. Evidence of attack is more common in attics, crawl spaces, unfinished basements and storage areas.
Again, make certain that the infestation is active(not old damage or old frass), there should be fresh frass the color of newly sawed wood, or live larvae or adults in the wood.
Most of the procedures which will prevent attack on wood before it is used are the responsibility of those who harvest, mill or store the wood. However those who use the wood should take precautions to reduce the chances of building an infestation into structures and furniture.
Using properly kiln or air-dried wood.
Using chemically treated wood(wood preservatives or insecticides).
Using good building design such as ventilation, drainage and proper clearance between wood and soil will reduce the moisture content of wood creating less favorable conditions for beetle development.
Central heating and cooling systems also speed up the wood drying process.
The larvae of these beetles reduce timbers to a mass of very fine, powder-like substance. The adults do very little damage In this section, we will discuss four types of Powderpost beetles in four families: Lyctidae, Bostrichidae, Anobiidae, and Cerambycidae. Adults do little damage, and it is the larvae(woodworms)that does the major part of the damage.They go through a complete metamorphosis: adults, eggs, larvae and pupae.
The adults are tiny, less than 1/4" in size. They are flattened and reddish-brown to black in color. Larvae (woodworm)are white, and cream colored, shaped with dark brown heads. Larvae create tunnels in the wood and become pupae. As adults, they bore out through the wood, pushing a fine powdery dust out.The shape of their holes is round,about 1/32-1/16 pinholes.
The diet of these beetles is sugar, starch and protein found in the sapwood of hardwood. They attack woods with high moisture. Moisture content of less than 6% is ideal for keeping these beetles from attacking. In the United States, the True Powderpost Beetle is the most common. They are often found in structures that have been built from infested lumber. They can re-infest.
They attack hardwoods depositing their eggs. True Powder Powderpost Beetles breed in dead and dried hardwoods such as the dead branches and limbs of trees. Their presence is overlooked until they are discovered in stored lumber, rafters, joists, finished wood, and furniture products.
Many times the Powderpost Beetle (Lyctidae) enters lumber that is stored or cured. It later emerges afterward. Old wood antiques are frequently attacked by these beetles. Hardwoood floors such as ash, hickory, oak, walnut and cherry are frequently attacked. These hardwoods that are damaged have starch-rich sapwood and are large-pored.
Presence of extremely fine, flour like powder falling from the surface holes. The frass left by other wood borers usually contains pellets and has a course texture and a tendency to stick together. When inspecting damage, be sure to distinguish old damage from active beetle infestations.
Recently formed holes and frass(sawdust like) are light in color and clear in appearance; old holes and frass are dark in color.
Since the False Powderpost Beetles are larger than other families of powderpost beetles, their exit holes are larger. These holes do not contain frass, but the galleries contain the frass. The frass is tightly packed, tends to stick together and is meal like( contains no pellets).
The adults are 1/8-to 1-inch long, cylindrical, and reddish brown to black. The adults bore into the wood in order to lay eggs, leaving a hole larger that 1/8 inch, usually in wood less than 10 years old.
The larvae are curved and wrinkled. Their diet is dependent on the starch in the wood, they are more common in softwood ,but can attack hardwoods. They require 6-30% moisture content in the wood, and complete the average life cycle in one year.
Most of the hardwoods attacked are not those commonly found used for interior floors,woodwork or trim. Most of this species does not re infest wood after it is seasoned, so the damage is limited to that inflicted by one generation. However the speed of the damage can be considerable.
They are often found in oak, firewood and furniture.
The furniture beetle is found mostly in the eastern half of the United States and it infests structural timbers as well. The Death-watch beetle is found throughout the United States. It attacks building timbers in poorly ventilated areas where moisture tends to collect. These beetles are heard at night making an audible ticking sound. It picked up the nickname,"Death watch," from this behavior.
Various anobiid beetles attack seasoned wood in the United States. These beetles range in size from 1/32- to 3/8-inch long; however, those that attack structures are 1/8- to 1/4-inch long.
They have highly variable body forms but most are elongate and cylindrical. The first body segment (pronotum) is hood-like, hiding the head when viewed from above. The last three segments of the antenna are lengthened and expanded into a club.
The furniture beetle, Anobium punctatum, is 1/8- to 1/4-inch long, cylindrical, and red-brown to dark brown in color. It has a series of pits in rows that run lengthwise on the wing covers. The pits can be seen through the fine yellow hairs that cover the body. The last three segments of the antenna are longer than the first eight combined.
The adult deathwatch beetle, Xestobium rufovillosum, is 1/4- to 3/8-inch long and is gray-brown with patches of pale hairs on the back of the body.
It does not have the rows of pits on the wing covers and their 11-segmented antenna end in three elongated segments that are as long as the previous five segments.
The larvae form tunnels in both softwoods and hardwoods. They require 13-30% moisture content.
Their holes are round,1/16-1/8 inches. They can digest cellulose from the wood. They are inclined to the softwoods ,for this reason they are common in crawl spaces and basements infesting the pine used as framing lumber.
The powder outside the holes (frass) is fine to coarse, many times with small pellets. The life cycle averages 1-3 years.
They commonly re-infest crawl space areas that are poorly ventilated and humidity is absorbed in the wood.
Furniture beetle adults emerge in the spring from cells just below the surface of the infested wood. Soon afterward, mating occurs, and egg laying begins. The female lays 20-60 eggs in old emergence holes or cracks and v in the wood. Eggs hatch in six to 10 days. The larvae feed for about one year before pupating for two to three weeks. The wood moisture content required for larval development is 13-30%. When development is complete, the adult bores directly to the surface of the wood, emerging through a round hole 1/16- to 1/8-inch in diameter. Development under ideal conditions can be completed in one year; however, two to three years is more common. The adults are active at night. Some species are attracted to light.
These beetles commonly infest seasoned sapwood of hardwoods and softwoods. They attack structural timbers, lumber, cabinets, and furniture. These beetles re-infest, and the females usually lay eggs in the wood from which they emerged. The larvae typically follow the grain of the wood when feeding ad fill their tunnels with wood frass. The frass is a fine powder with long pellets loosely packed into the galleries.
Determine if the infestation is active before initiating treatment. Wood in structures and furniture infested by these beetles may go unnoticed until the round adult emergence holes appear in the surface. The characteristic pellets found in the frass and the consistency of the frass are useful in determining what species is infesting the wood. Infested wood can be removed and replaced with treated wood. Reducing the wood moisture content to approximately 12% slows the development of the larvae.
The surface of unpainted or otherwise unprotected wood can be treated, and the galleries injected with disodium octaborate tetrahydrate such as BoraCare or Timbor. These products kill exposed larvae and prevent re-infestation when the eggs hatch and immature larvae begin to penetrate the wood. However, the most effective way to eliminate anobiid powder post beetle infestations is to fumigate using sulfur fluoride or methyl bromide.
Most representatives of this family infest and feed on dead or dying trees. However there are many that feed on living trees. There are some of these species which begin their development in dying trees, logs, or unseasoned lumber and then complete the development as the wood seasons.
A common source for these beetles indoors is from firewood brought indoors.
A sighting of these beetles can lead to a false assumption of a structural attack. Firewood that is brought in should be used very soon after it is brought inside.
The beetles of this family lay their eggs in cracks or crevices in the bark or on the surface of rough sawn timbers. The larvae are wood borers.
Mature larvae are large, varying from 1/2 to 3-4 inches long. The body is long and narrow and a light cream color. The rear portion of the head is partly drawn into the body so that only the mandibles and other mouthparts are easily seen.
Adults vary in size from 1/2 inch to 3 inches long. They can be easily distinguished from other beetles by their long, thin antennae which may be longer than the body; hence the adults are called long-horned beetles.
The adults of these boers will emerge from the wood after it has been incorporated into the structure. They will not re infest the wood because of its dryness, but they are of great concern to property owners who find them or evidence of their activity.
The Old House Borer is one of the most common from this family, with it's larvae hollowing out galleries in seasoned softwood(pine). It is found in older buildings, but is more frequent in newer buildings,(in houses less than 10 years old).
It is well established along the Atlantic Coast, but infestations have been reported as far as Louisiana and Minnesota.
The adults are brownish-black to black, slightly flattened and about 3/4-1 inch long.
The life cycle of the old house borer ranges between three to twelve years. Because this beetle has a very long life cycle, it can make re infestations of the same piece of wood. It may be many years before serious structural damage is recognized. The exit holes of emerging adults do not occur in very large numbers until the infestation has been established for several years. This, along with the fact that larvae will do extensive feeding without breaking through the surface of the wood, make it necessary to inspect infested wood very carefully to detect old house borer damage.
Rough wood being examined should be probed or struck to detect weakness or the presence of boring dust. If exit holes are present, they will be broadly oval and about 1/4-3/8 inch in diameter.
When wood has been infested with fungi, the larval development is faster. Their powder (frass) in the tunnels are like sawdust, tightly packed.


(6.5 million annually). Among those, 6 people experience a major life disruption. These 6 people account for more than 250,000 people coping with the devastating effects of suicide loss.
Suicide deaths have exceeded the number of homicides every year since 1981. Married persons have lower suicide rates than single, never married individuals. The highest rates occur among widowed and divorced persons.
According to Badge of Life study, Officer.com, law enforcement officer suicides took more lives (108) than gunfire and traffic accidents combined (97) in 2016. For every one police suicide, almost 1,000 officers continue to work while suffering the painful symptoms of PTSD.
In 2015, suicide rates were highest in the West (14.9), followed by the South (14.3), the Midwest (14.0) and the Northeast (10.8). Eleven U.S. states, nine in the West, had age-adjusted suicide rates in excess of 19: Alaska (27.2), Wyoming (26.8), Montana (26.3), New Mexico (24.0), Idaho (21.7), Utah (21.0), Oklahoma (20.2), South Dakota (20.2), Colorado (20.0) Arkansas (19.4) and Nevada (19.3). Three locales had age-adjusted suicide rates lower than 9 per 100,000: New Jersey (8.8), New York (8.4) and the District of Columbia (5.1).
In 2015, the highest U.S. suicide rate (20.0) was among American Indians and Alaskan Native and the second highest rate (17.0) was among Whites. Much lower and roughly similar rates were found among Hispanics (5.8), Asians and Pacific Islanders (6.4), Blacks (5.6).
For many years, the suicide rate has been about 4 times higher among men than among women. In 2015, men had a suicide rate of 22.5, and women had a rate of 6.3. White males accounted for 7 of 10 suicide deaths in 2015 and the number is highest in middle age men.
In 2015, the highest suicide rate (20.3) was among people 45 to 54 years old. The second highest rate (19.4) occurred in those 85 years and older. Younger groups have had consistently lower suicide rates than middle-aged and older adults. In 2015, adolescents and young adults aged 15 to 24 had a suicide rate of 12.5.
The Centers for Disease Control and Prevention (CDC) collects data about mortality in the U.S., including deaths by suicide. Suicide is the 10th ranking cause of death in US. Homicide ranks 16th. In 2015 (the most recent year for which data are available), 44,193 suicide deaths were reported. In that year, someone in the U.S. died by suicide every 11.9 minutes.


text:
US Geoengineers Turn to Balloon for Planet-Cooling Test
By Live Science Staff 17 July 2012
A new project will investigate the feasibility of one so-called geoengineering technique: the idea of simulating volcanic eruptions that release small particles into the stratosphere, which then reflect some of the incoming solar radiation back into space, with the effect of cooling the Earth. Not all scientists agree this could work.
(Image: © Hugh Hunt, SPICE project)
A U.S. geoengineering project aimed at using a balloon to spray sun-reflecting particles comes just a few months after the cancellation of a similar British experiment.
The new plan would test a planet-cooling technology for fighting rising temperatures related to climate change, according to The Guardian. Inspiration for the idea comes from how Earth's volcanoes have historically had a cooling effect on the planet by spewing sulfate particles into the atmosphere.
Two Harvard engineers plan to launch the balloon on a flight over Fort Sumner, New Mexico. One of the engineers, Keith David, also happens to head a geoengineering research fund provided by Microsoft founder Bill Gates. [Changing Earth: 7 Ideas to Geoengineer Our Planet]
David opposed the cancelled British geoengineering effort, called SPICE, because he said it would not have contributed to better understanding the impact of geoengineering. But he plans to draw lessons from the British effort as he and his colleague, James Anderson, prepare for their own experiment within a year.
Geoengineering ideas have usually tried to counter global warming by either reducing heat-trapping carbon dioxide (CO2) or reflecting sunlight back into space. But the idea remains controversial among scientists who argue the cure could prove worse than the disease, so to speak — a European study recently suggested that planet-cooling efforts based on reflecting sunlight would slash the amount of rainfall in North America and northern Eurasia.
This story was provided by InnovationNewsDaily, a sister site to LiveScience. Follow InnovationNewsDaily on Twitter @News_Innovation, or on Facebook.

redpajama_set_name: RedPajamaCommonCrawl


text:
May 12, 2008: 2:15 PM EDT
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Sam Zell's smooth Newsday move
You might have thought that Sam Zell, facing hundreds of millions of dollars in loans due, was in a weak position entering negotiations to sell Newsday. You'd be wrong.
By Devin Leonard, senior writer
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NEW YORK (Fortune) -- Sam Zell is in a difficult spot. The real estate mogul turned newspaper baron borrowed $8.4 billion last December to finance his leverage buyout of the Tribune Co., owner of the Chicago Tribune and the Los Angeles Times. Now he's discovered the newspaper business is harsher than he expected. Zell is trying to sell some of his new company's assets to keep his creditors happy.
But Zell isn't acting like he's desperate. On Monday, the Tribune CEO announced his company was selling a 97% stake in Newsday, its Long Island daily, to Cablevision (CVC, Fortune 500) in a deal valued at $650 million. For weeks, it appeared that he was rushing to cut a similar deal with Rupert Murdoch's News Corp. (NWS, Fortune 500) for $580 million. At the last minute, Zell convinced Cablevision to give him everything Murdoch promised - including a joint-venture structure that will save Tribune an estimated $245 million in taxes - for $70 million more than News Corp. had offered.
It was a stunning victory was a guy whose back is supposedly up against the wall. In hindsight, Zell orchestrated the Newsday sale flawlessly. Early on, he lined up three potential bidders. Two of them - News Corp. and New York Daily News owner Mort Zuckerman - were publishers who were interested in Newsday for strategic reasons. News Corp wanted to cut costs at the New York Post by combining the tabloid's printing and distribution operations with those of Newsday. Zuckerman wanted to do the same by creating a joint venture between the Daily News and Newsday.
The third bidder, of course, was Cablevision. The Long Island-based cable company didn't own any newspapers. But the Dolan family - Cablevision's controlling shareholders - seemed to like the idea of owning the biggest paper in their area.
The trick was for Zell to turn this into a bidding war. That was difficult at first. The three interested parties acted as if they had the upper hand. Cablevision did some tire kicking, but the Dolans didn't make an offer. Zuckerman reportedly made a lowball bid.
Zell turned up the heat by entering into negotiations with News Corp. to accept $580 million for a majority stake in Newsday. Murdoch clearly felt he had the inside track. He began courting Long Island's political leaders whose support he would surely need to get the deal approved by the FCC in Washington. That's because News Corp. already owns the Post and two New York City television stations.
It now appears Zell was using News Corp.'s offer to establish a floor for the bidding. Zuckerman soon matched News Corp.'s offer. Then Cablevision did what non-strategic bidders often do in such situations. It offered to pay a higher price than either newspaper publisher.
Murdoch misread the situation. As recently as News Corp.'s conference call on Thursday, he acted as if he had a deal with Zell: "I trust Mr. Zell absolutely. He's famous for being a man of his word, and we think everything is in hand." Murdoch insisted there was no need to sweeten his company's Newsday bid. "We're certainly not in the business of getting into an auction here," he said.
But, of course, that's exactly what Zell was conducting. The Dolans understood this perfectly. They offered everything Murdoch did at a higher price. Soon, Cablevision will be Newsday's majority owners.
The Newsday sale is an important victory for Zell. After all, Tribune needs cash to pay down its debt. The newspaper publisher has to repay $650 million in December. There's another $750 million payment looming in May 2009.
So Zell will surely have to sell more assets. Next up is the Chicago Cubs franchise. Analysts say the Tribune CEO could get as much as $1 billion for the baseball team and related assets like Wrigley Field if he handles the sale right. The last thing Zell needs is to look desperate right now. Anybody who thinks he's an easy mark after the Newsday sale is in for a disappointment.
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We all know that having a nice lobby attracts customers and makes them feel welcomed and nice. Let's also consider the impact that decor and environment have on employee behavior.
Commercial interiors and commercial art are crucial to the overall work environment. Your interior artwork and decor effect pride, quality, morale, efficiency, and performance? In fact, industrial design studies show that the first step in changing corporate culture and behavior is with changing the environment to be in alignment with the corporate culture goals. Of course, the same goes for negative impact from a non-optimal commercial interior. If your corporate culture is one of young and fresh, does the interior design and art reflect that? Bright colors and abstract shapes might be what is needed. Is your company high tech? Then aluminum and glass should be prominent. Are you stable and established? Rich wood finishes and leather might convey that.
The bottom line is this – we as humans adapt to our environment, and the art we are surrounded by along with the interior decor of our work environment defines our behaviors and approaches to our jobs.
What direction is your commercial interior pointing your employees?


text:
5 x reasons why you want to get a haircut at this barber
No! The deer park on the Koekamp and in the Zuiderpark must disappear
Flag faux-pas: Belgians accidentally hang Dutch flag upside down
EU passes new law, crickets now allowed as ingredient in supermarket foods
153 hours later: Dutch team breaks world record for longest podcast
16 October 2022, by Victoria Séveno
Have you ever found yourself listening to the latest instalment of your favourite podcast and wished the episode would last forever? Well, a team of broadcasters and podcasters from the Netherlands successfully managed to turn that dream into a reality, breaking the world record for the longest uninterrupted podcast episode during Dutch Media Week.
The Netherlands breaks record for longest audio-only live stream
Working together, a team of podcast fanatics and experts planned and carried out what can only be described as a podcast marathon, recording a total of 153 hours of content without any breaks.
Taking place in Hilversum from October 1 to October 9, various podcasts – including De Leeuw lult verder, Dit was de radio, PodBast, Han Peekel 75 and Mand – were recorded live and back-to-back in order to achieve the 153-hour record. It's a seriously impressive feat, as the previous Guinness World Record for the longest audio-only live stream was just 53,1 hours, set in July 2020 by two podcasters from the UK.
Sadly, if you are curious to hear what the final product sounds like, all the content is in Dutch, so you might want to brush up on your language skills first. Alternatively, check out our list of some of the best podcasts for expats in the Netherlands.
By clicking subscribe, you agree that we may process your information in accordance with our privacy policy. For more information, please visit this page.
The Hague news


Melanie over at Big Mama did a post a few weeks ago about her top 10 most angsty songs she lip synced as a teen. I read her list and loved it. I decided it would probably be best for me to copy her and write one of my own.
Although I can't really be accused of the whole hairbrush-as-a-microphone thing, I can be accused of plenty of horrible dance moves and embarrassing faces made in the mirror above my dresser. There was an abundance of that business going on. I didn't need a mic, y'all. As if.
This has to be on every girl's list. It's just so good. And nobody can resist that key change.
A breathy voice, easy lyrics, and a repetitive melody made for the perfect pop hit. It sucked me in from day one. Oh, Donna. You're only one that can sing, "Never stop, never whatever," and it not sound stupid.
When I realized that this song was made up of 3 chords that I could play, my life changed. And so it began - realizing every pop hit from then on would also be made up of 3 chords.
My love for this song runs deep. I've gone through so many lyric changes as I've gradually understood more and more of the words coming from Stephan's mouth. But don't worry, I still don't have them all. Actually, I've never looked them up on purpose. I want to keep singing my version. Maybe someday I'll post them for you.
First of all, anyone whose name is Robyn spelled with a 'y' is awesome. The bridge of this song is still one of my favorite bridges of all time.
Someone please tell me why this guy is not famous still. And please, someone tell me you were just as in love with this song as I was (am). His voice is like butter, the melodies are lovely, and the words are exactly what my teenage heart wanted some guy to say to me. If you haven't listened to this song in a while, do it now.
Not only did I spend time dramatically singing this song, but I spent time dramatically playing this song on the piano. I will always love Enya.
N'Sync must have also spent a little extra time on naming that song. I mean, gah lee, how long does a title of a song have to be?? And if you just say "God Must Have Spent" it sounds dumb. So it's either all or nothing.
Perhaps this was a favorite song of mine during the same time of life when my parents were worried I was turning goth. Guys, I don't know why, I just liked wearing black. I made no connection to the other kids in my middle school that also loved wearing black while doing really inappropriate things. Like smoking cigarettes, wearing too much eyeliner, and painting their nails black. I was oblivious.
Threw you for a loop on this one, huh? My high school boyfriend got in my car one night and had his ears blasted off by Evanescence when I turned the key. He said something like, "This is the last genre of music I would've ever expected you to like and listen to." And he was right. It was a bit off my musical map. But who can resist shouting, "I can't wake up!!"? No one. No one.
The amount of dances that were choreographed to this song by my sister and me is embarrassing.
This song - on my Discman - on repeat - as I fell asleep. I lived many a night like this. If you need a refresher, this was the "band" (nobody played any instruments) that Puff Daddy (before the P. Diddy nonsense) created on "Making the Band." I know Eden's Crush was short lived, but I would totally go see them in concert if they ever decided to put a reunion tour together. But they totally wouldn't because Nicole Scherzinger is way too busy doing all of her ridiculous songs.
At one point, I was dead set on this song being in my wedding in some form or fashion. Unfortunately (or maybe fortunately), it never made the cut.
Did you really think I'd get to the end of this list before talking about my favorite? This song is perfect in every way and I still get a wonderfully magical feeling anytime I hear it. Sarah knows how to write 'em.
haha number 8 was so mine!


Maths deals with developing reason and logic. In Singapore, Maths is fundamental to doing well in school. A student will likely have to continue Maths later on in Secondary and JC (or Integrated Programme) and so a firm grasp of Maths early on will give him/her the foundation to continue to get better grades. Maths also opens the door to many career choices.
The Study Room believes a child learns the most through having fun and uses real-world experiences and role-play, maths puzzles and activities to teach the student the complexities of the subjects in the simplest way.
The Primary 5 & 6 student will be exposed to challenging Olympiad training to help them ace their PSLE and boost them through their secondary school years.


text:
Q: What does `$this` mean when used outside class in PHP 5? I'm migrating an application from a PHP 5 server to a PHP 7 server for a client. I've come across some nonsensical code that works (in the sense that it does not throw errors or warnings) on the PHP 5 server but crashes on the PHP 7 server. I'm trying to understand the semantics of $this in PHP 5 that would help me decipher what the code is doing:
if ( !is_object( $this ) )
{
    require_once '' . '/model/Model.php';

    $me = new Controller( false );
    $me->invoke();
}
else
{
    require_once 'model/Model.php';
}

The offending statement is of course if ( !is_object( $this ) ); $this is illegal outside of a class.  My guess is the code is attempting to instantiate some kind of singleton and can only assume the semantics of $this in PHP5 resolved $this as nullish when used outside of a class. I am having trouble confirming this.
Did PHP ever allow this kind of usage? Or was there some configuration option that enabled it to be used this way? What did $this mean when used this way?

A: No, using $this in global scope won't have ever done anything useful*
It was actually PHP 7.1 that made the change - specifically the Fix inconsistent behavior of $this variable RFC. Before that, PHP just treated it the same as any other undefined variable. Now you'll see a fatal error instead, see https://3v4l.org/b4Wte
I'd assume that this code was copied from a class method, where it looks like it was trying to run from both a static and object context.
The really quick change would be to swap out the is_object check for isset, which can still run safely with $this. But since the line will always be true, you might as well just replace the whole thing with
require_once '' . '/model/Model.php';

$me = new Controller( false );
$me->invoke();

* Strictly speaking, all the way back in PHP 4 you could have redefined $this to point to whatever you want, but I'm guessing that probably doesn't apply here.

redpajama_set_name: RedPajamaStackExchange


text:
Showing: June, 2006
June 22, 2006 | 01:35am
Fact Check: Senate Democrats Have Killed Standard Deduction Bills for Years
A state senator said he was very surprised when he first heard Senate Democrat leaders taking credit for an increase in the state's standard deduction, because for years Senate Democrats have blocked Republican efforts to raise the standard deduction for Oklahoma taxpayers.
"Senate Democrats killed my bill to increase the standard deduction during the 2006 regular legislative session, so I was surprised to hear them take credit for this issue in the historic tax cut agreement," stated Sen. Clark Jolley, R-Edmond.
Sen. Jolley says Democrats have killed Republican standard deduction bills for years.
Sen. Wilcoxson Calls Superintendent's Reaction to State Graduation Report Misleading
State Sen. Kathleen Wilcoxson called a recently released report on high school graduation rates alarming. She said she is disappointed that State Superintendent Sandy Garrett has failed to explain to the public how high the drop-out rates in Oklahoma actually are and how this impacts the entire state.
The newly released report by the Editorial Projects in Education Research Center showed that Oklahoma does slightly better, with 71 percent of students graduating in 2002-2003, compared to a national rate of 69.6 percent.
Sen. Wilcoxson discusses report on OK graduation rates.
Sen. Leftwich Pushes Cost-of-Living Increase for Retirees
As legislative subcommittees put the final touches on the 2007 state budget, Sen. Debbe Leftwich wants to ensure retirees get a much needed cost-of-living increase. Leftwich is calling on fellow lawmakers to make sure retired state employees, teachers, police, firefighters and state law enforcement officers such as highway patrol troopers all get a cost-of-living increase.
Sen. Leftwich wants cost of living increases for retirees.
Senator Coates Calls New Animated Film 'Cars' a Windfall for Oklahoma
State Sen. Harry Coates says the new Disney and Pixar film "Cars" could be a real boon for tourism in Oklahoma. That's because one of the main characters in the film is based on a constituent in his district and her Route 66 Rock Café in Stroud.
"I've known Dawn Welch for many years and she's a huge promoter for Stroud, Route 66 and our entire state," said Coates, R-Seminole. "It's a windfall for all of Oklahoma that Disney and Pixar decided to make a movie about Route 66."
Dawn Welch discusses how she became a character in Cars.
Sen. Coates says Cars movie great for Oklahoma.
Morgan: Republicans Insatiable Need to Cut Taxes, Democrats Resolve Equals Victory for Middle Class
Senate President Pro Tempore Mike Morgan said Friday that the tax cut and budget framework agreed to by state leaders is an historic victory for working and middle class families in Oklahoma.
The agreement includes raising the Oklahoma standard deduction to the level of the federal standard deduction over the next four years – a provision that will historically and dramatically change the tax structure in Oklahoma.
Sen. Morgan discusses budget framework.
Senator Capps Says License Announcement Is Great News for Oklahoma's Aerospace Industry
Assistant Majority Leader State Sen. Gilmer Capps said Oklahoma's aerospace industry took a major step forward Tuesday with the approval of an operator's license for the Oklahoma Spaceport in Burns Flat.
It creates the first inland spaceport in the nation and allows the Oklahoma Spaceport/Clinton-Sherman Airport to serve as a site for the launch and recovery of sub-orbital reusable space vehicles.
Sen. Capps discusses the latest development in Oklahoma's aerospace industry.


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text:
β-arrestin- but not G protein-mediated signaling by the "decoy" receptor CXCR7
Sudarshan Rajagopal, Jihee Kim, Seungkirl Ahn, Stewart Craig, Christopher M. Lam, Norma P. Gerard, Craig Gerard, and Robert J. Lefkowitz
PNAS January 12, 2010 107 (2) 628-632; https://doi.org/10.1073/pnas.0912852107
Sudarshan Rajagopal
Jihee Kim
Seungkirl Ahn
Stewart Craig
Christopher M. Lam
Norma P. Gerard
Craig Gerard
For correspondence: Craig.Gerard@childrens.harvard.edu lefko001@receptor-biol.duke.edu
Robert J. Lefkowitz
Contributed by Robert J. Lefkowitz, November 9, 2009 (sent for review October 1, 2009)
Ubiquitously expressed seven-transmembrane receptors (7TMRs) classically signal through heterotrimeric G proteins and are commonly referred to as G protein-coupled receptors. It is now recognized that 7TMRs also signal through β-arrestins, which act as versatile adapters controlling receptor signaling, desensitization, and trafficking. Most endogenous receptors appear to signal in a balanced fashion using both β-arrestin and G protein-mediated pathways. Some 7TMRs are thought to be nonsignaling "decoys" because of their inability to activate typical G protein signaling pathways; it has been proposed that these receptors act to scavenge ligands or function as coreceptors. Here we demonstrate that ligand binding to the decoy receptor CXCR7 does not result in activation of signaling pathways typical of G proteins but does activate MAP kinases through β-arrestins in transiently transfected cells. Furthermore, we observe that vascular smooth muscle cells that endogenously express CXCR7 migrate to its ligand interferon-inducible T-cell alpha chemoattractant (ITAC), an effect that is significantly attenuated by treatment with either a CXCR7 antagonist or β-arrestin depletion by siRNA. This example of an endogenous "β-arrestin-biased" 7TMR that signals through β-arrestin in the absence of G protein activation demonstrates that some 7TMRs encoded in the genome have evolved to signal through β-arrestin exclusively and suggests that other receptors that are currently thought to be orphans or decoys may also signal through such nonclassical pathways.
biased receptor
G protein-coupled receptors
seven-transmembrane receptor
Seven-transmembrane receptors (7TMRs) are the most common class of receptors in the genome and the most common target for medical therapeutics (1). Classically, these receptors were thought to signal exclusively through heterotrimeric G proteins and be desensitized by β-arrestins; therefore, they are frequently referred to as G protein-coupled receptors. It is now appreciated that β-arrestins act as multifunctional adapter proteins that mediate signaling in their own right and also control receptor desensitization and trafficking (2). Most endogenous 7TMRs are thought to signal through G protein- and β-arrestin-mediated pathways in a balanced fashion (3). However, some receptors can be made to signal in a biased fashion toward β-arrestin or G protein signaling by ligand modifications (4), so-called biased ligands (5), or by mutation of key receptor residues involved in coupling to G proteins or β-arrestins, resulting in G protein- or β-arrestin-biased receptors (6). To date, no endogenously expressed biased 7TMRs have been described. We speculated that candidates for such receptors are the "decoy receptors," which do not appear to signal through G proteins and are thought to scavenge or internalize ligands or act as coreceptors for other chemokine receptors (7).
Seven-transmembrane decoy receptors that have been demonstrated to be unable to activate typical G protein-mediated signaling pathways include C5L2, Duffy antigen receptor for chemokines, D6, and CXCR7 (7). Some of these receptors contain variants of highly conserved residues that are thought to play an important role in signaling to G proteins (8). The decoy receptor CXCR7 (9, 10), which binds the ligands (Stromal-derived factor) SDF-1α/CXCL12 and ITAC/CXCL11, does not activate typical Gαi pathways of a chemokine receptor that would result in GTP hydrolysis or calcium mobilization (9, 11–13). It is currently thought that CXCR7 acts primarily to modulate signaling of CXCR4, an alternate receptor for SDF-1α. A number of mechanisms underlying this function have been proposed. Some propose that CXCR7 may scavenge or sequester SDF-1α, thereby generating gradients of SDF-1α that lead to differential signaling by CXCR4 (12, 14). It has also been proposed that CXCR7 acts as a coreceptor for CXCR4 (11, 13), as the two receptors form heterodimers in the context of overexpression in transiently transfected cells. It has also been proposed that ligand binding to CXCR7 may result in crosstalk with CXCR4 mediated by intracellular signaling molecules (15). More recently, it has been demonstrated that CXCR7 interacts with β-arrestin in a ligand-dependent manner (15–17), resulting in ligand internalization, but without any assessment of changes in signaling. Given these findings we set out to test whether CXCR7 can signal through β-arrestins to determine whether this receptor acts as an endogenous β-arrestin-biased receptor.
CXCR7 Recruits β-Arrestin in a Ligand-Dependent Fashion.
Previous studies have demonstrated the inability of CXCR7 to generate typical G protein responses such as an increase in intracellular calcium after treatment with either SDF-1α or ITAC (9, 11–13). However, we did observe recruitment of β-arrestin to the plasma membrane upon treatment with both ligands in HEK293 cells transiently transfected with CXCR7 and GFP-labeled β-arrestin2 (Fig. 1A), consistent with recent reports of ligand-dependent interactions between these two molecules (15, 16). Notably, we did not observe such recruitment in mock transfected HEK293 cells, which express endogenous CXCR4, an alternate receptor for SDF-1α (Fig. 1B). Recruitment of β-arrestin was rapid with translocation to the plasma membrane by 2 min, but at late time points (30 min) there was a distinct difference in the pattern of recruitment between those cells treated with SDF-1α or ITAC. In those cells treated with SDF-1α for more than 10 min, β-arrestin2 was localized to cytoplasmic vesicles, referred to as a "class B" pattern, whereas in those cells treated with ITAC, β-arrestin2 continued to localize to the plasma membrane, referred to as a "class A" pattern (Fig. 1A) (18). These patterns of β-arrestin recruitment for SDF-1α and ITAC suggest a functional difference in response to these two ligands, because class A interactions are transient in nature and result in rapid recycling of receptors, whereas class B interactions are stronger and result in slow and relatively low levels of recycling (19).
CXCR7 recruits β-arrestin resulting in MAP kinase activation in transiently transfected HEK293 cells. (A) β-arrestin recruitment in live HEK 293 cells transiently transfected with CXCR7 and GFP-labeled β-arrestin2 before and 30 min after treatment with SDF-1α and ITAC. (B) Thirty minutes after treatment with either ligand, there is a pattern of pERK formed in CXCR7-transfected cells. Fixed cells were labeled for CXCR7 (blue) and pERK (red) after transfection with CXCR7 and β-arrestin 2-GFP (green). In cells treated with SDF-1α, there is clear localization of all of these components in cytoplasmic vesicles on merged images (white), whereas this change is not as pronounced in those cells treated with ITAC.
CXCR7 Recruitment of β-Arrestin is Associated with phosphoERK Formation.
We observed these same patterns of recruitment in fixed cells that were analyzed for MAP kinase activation, in which we stained for phosphoERK1/2 (pERK) after 30 min of ligand stimulation (Fig. 1B). β-arrestin-mediated pERK formation by class B receptors is a well-characterized response that differs from G protein-mediated pERK phosphorylation in several respects: G protein-mediated activation occurs maximally at short times (2–5 min) and β-arrestin-mediated activity at longer times (≥10 min) (20,21); G protein-mediated pERK distributes evenly in the cytoplasm and nucleus whereas β-arrestin-mediated pERK localizes to cytoplasmic vesicles (21). After 30 min of treatment with SDF-1α, the majority of β-arrestin is present in cytoplasmic vesicles with strong colocalization of CXCR7, β-arrestin-2, and pERK on merged images (Fig. 1B). In cells treated with ITAC, most β-arrestin still localized to the plasma membrane with the class A pattern. These patterns of pERK in transfected cells are consistent with previously characterized β-arrestin-mediated processes, in terms of both spatial and temporal localization (21).
Rat Vascular Smooth Muscle Cells Express CXCR7.
We next sought to address a functional β-arrestin-mediated response by CXCR7 in an endogenous system. Based on analysis of 7TMR mRNA expression in the mouse (22), we chose to study vascular smooth muscle cells (VSMCs), which are predicted to express high levels of CXCR7 and relatively low levels of CXCRs 4 and 3, typical chemokine receptors for SDF-1α and ITAC, respectively, that could confound our analysis (Fig. 2A). Similar to the mouse, we found that rat VSMCs (rVSMCs) expressed orders of magnitude more CXCR7 than CXCR4 and CXCR3 at the mRNA level (Fig. 2B). As expression of CXCR7 at the protein level does not always correlate with its mRNA level (15), we next sought to demonstrate its expression by anti-CXCR7 antibody binding and its unique signature of chemokine binding. At the protein level by immunoblot analysis, we did detect both CXCR7 and CXCR4, but not CXCR3 (Fig. 2C).
Expression of chemokine receptors for SDF-1α and ITAC in rVSMCs. (A) CXCR7 shares ligands with the typical chemokine receptors CXCR4 and CXCR3. Other ligands for CXCR3 that are not shown are IP10 and Mig. (B) mRNA expression levels of CXCR7 (R7), CXCR4 (R4) and CXCR3 (R3) in passage 0 rVSMCs by semiquantitative RT-PCR at 30 and 40 cycles. (C) Immunoblot analysis of receptor expression in rVSMCs using antibodies against CXCR7, 4, and 3.
Radioligand binding studies of rVSMCs demonstrate that unlabeled ITAC displaced 125I-SDF-1α, a profile of chemokine binding unique to CXCR7 and not CXCR4 or CXCR3 (Fig. 3A). To assess whether expression of CXCR4 or CXCR3 was at a sufficient level to be functional for G protein activation in rVSMCs, we determined activation of Gαi by pulldown with an antibody against the active conformation of Gαi. Treatment with SDF-1α significantly increased amounts of active Gαi, implying activation of CXCR4, whereas ITAC had no effect on these levels compared to untreated controls, consistent with the absence of CXCR3 in these cells (Fig. 3B). Treatment of rVSMCs with either SDF-1α or ITAC did not result in intracellular calcium flux, a response that would be expected for activation of either CXCR4 or CXCR3 (Fig. 3C) (compared to a positive control of angiotensin, a well-characterized Gαq-coupled receptor) (23). Thus, we conclude that ITAC binds CXCR7 alone without any activation of G proteins, and SDF-1α binds to both CXCR4 and CXCR7, resulting in Gαi activation, albeit with minimal signaling through a typical Gαi-coupled pathway.
Chemokine receptor expression and lack of G protein activation by ITAC in rVSMCs. (A) Treatment with unlabeled ITAC or SDF competes off radiolabeled SDF-1α from rVSMCs. (B) Activation of Gαi in SDF-1α-treated rVSMCs (SDF) but not in untreated (UT) or ITAC-treated (ITAC) samples. The negative control was incubated with GDP (GDP) and the positive control incubated with GTP (GTP) for 90 min prior to immunoprecipitation. Data shown are mean ± SEM from three independent experiments. (C) Absence of calcium influx as assessed by change in Fura-2 fluorescence emission ratio upon stimulation of rVSMCs with SDF-1α (blue) or ITAC (red) compared to Angiotensin II (black) from a representative experiment from three independent experiments.
Rat VSMCs Migrate to ITAC in a CXCR7- and β-Arrestin-Mediated Process.
We next addressed whether activation of endogenous CXCR7 in the rVSMCs could result in a functional β-arrestin-mediated response. We first assessed for MAP kinase activation that we had observed in the transiently transfected HEK293 cells, but we were unable to detect significant increases in pERK upon treatment with either SDF-1α or ITAC over the background high basal activation in the rVSMCs even after more than 48 h of serum starvation (Fig. S1). We also did not observe a mitogenic response to these ligands (Fig. S1), suggesting that CXCR7 couples to different signaling pathways in rVSMCs than it does in the context of overexpression in HEK293 cells. We next assessed rVSMC migration to these ligands as β-arrestin-mediated chemotaxis is a well-described phenomenon in a number of 7TMR systems (24–27). We found that SDF-1α and ITAC resulted in significant rVSMC migration across a range of concentrations tested (Fig. 4A), with a response similar to that of other ligands known to induce rVSMC chemotaxis, such as PDGF (28).
rVSMC migration in response to ITAC is a CXCR7- and β-arrestin-dependent process. (A) Migration of rVSMCs to 1–100 nM of SDF-1α and ITAC compared to PDGF positive control. Shown is representative data from at least three independent experiments. (B) Effects of CCX704 (inactive control), CCX733 (CXCR7 antagonist), T497 (CXCR3 antagonist), and AMD3100 (CXCR4 antagonist) on ITAC-stimulated migration. Only treatment with CCX733 resulted in a significant inhibitory effect (∗ p < 0.05). (C) Significant inhibition of ITAC-stimulated rVSMC migration by β-arrestin2 depletion (∗ p < 0.05) but not β-arrestin1 depletion. Data shown are mean ± SEM from at least four independent experiments.
To avoid possible confounding with CXCR4-mediated processes, we then further characterized the response of rVSMCs to ITAC alone. To assess whether this migration response was mediated by CXCR7, cells were treated with selective chemokine receptor antagonists. Treatment with the CXCR3 antagonist T487 (29) or the CXCR4 antagonist AMD3100 (30) had no significant effect on migration, however, the CXCR7 antagonist CCX733 had a significant inhibitory effect on migration, whereas a structurally related compound which does not bind CXCR7, CCX704, had none (Fig. 4B). Therefore, we conclude that the majority of the migration response to ITAC is mediated by CXCR7 and not the typical chemokine receptors CXCR3 and CXCR4. To determine whether this CXCR7-mediated migration was β-arrestin-dependent, we depleted β-arrestins by siRNA knockdown. Although depletion of β-arrestin1 had no statistically significant effect on migration, depletion of β-arrestin2 resulted in a marked attenuation of rVSMC migration in response to ITAC compared to control siRNA-transfected cells (Fig. 4C). Although it is impossible to exclude signaling by the dozens of different Gα and Gβγ subunits (31), we did find that this response was not sensitive to treatment with pertussis toxin and thus independent of Gαi (Fig. S2). The specificity of CXCR7 antagonism and effect of β-arrestin depletion demonstrate that CXCR7-mediated rVSMC migration is a β-arrestin-mediated process and that CXCR7 acts as a β-arrestin-biased receptor.
Although virtually all reports in the literature have agreed that CXCR7 does not couple to G proteins, functional roles for the receptor that belie a signaling event suggest a nonclassical positive signaling role for this receptor. In the initial characterization of CXCR7, its expression increased the adhesion of cancer cells to endothelia and enhanced their survival in vitro and in vivo (9). In the zebrafish, polarized expression of CXCR4 and CXCR7 is essential for primordial germ cell migration (12, 14). These reports suggested that CXCR7 may scavenge the CXCL12 in the stationary, nonmigrating cells. Prostate cancer cells expressing CXCR7 suggest a role for this receptor in adhesion, invasiveness, and survival, where activation of Akt is linked to CXCR7 and results in the regulation of a number of genes associated with the NF-κB and MAP kinase pathways (32). Our current data, in which CXCR7 is an active receptor capable of signaling through β-arrestin, provide a mechanistic basis for understanding these earlier studies.
For the vast majority of 7TMR signaling, it is thought that there is a balance between G protein- and β-arrestin-mediated pathways (Fig. 5A). Agonist binding typically results in signaling mediated by G proteins and β-arrestins accompanied by receptor desensitization and internalization mediated by β-arrestins. This is in contrast to a system with biased signaling, where signaling is mediated selectively through only one of these two pathways. Two limit cases of biased signaling can be considered. In the case of a biased ligand, treatment of a balanced receptor with a biased agonist results in a biased response (Fig. 5B), whereas, in the case of a biased receptor, binding of a balanced agonist results in a biased response (Fig. 5C). Before the present study, receptors that have been demonstrated to be biased have been genetically engineered from balanced receptors by mutation of key residues involved in G protein coupling, such as mutations of the highly conserved DRY motif of the AT1AR to generate the variant AT1AR(DRY/AAY) (33) or mutations of three highly conserved residues in the β2 adrenergic receptor to generate the β-arrestin-biased β2AR(TYY) (6). Surprisingly, similar mutations are observed in decoy receptors such as CXCR7, which lacks a typical sequence surrounding the DRY motif (7), suggesting that biased receptors have evolved in our genome.
Balanced and biased signaling by 7TMRs. (A) In a balanced system, signaling is mediated by G proteins and β-arrestins, while β-arrestins also control desensitization and receptor trafficking. (B) Treatment of an unbiased receptor with a biased agonist results in a biased response, in this case, β-arrestin-mediated signaling only. (C) In the case of a β-arrestin-biased receptor, treatment of the receptor with an unbiased ligand results in β-arrestin-mediated signaling only.
β-arrestins were initially discovered as negative regulators of G protein-mediated signaling by 7TMRs (34), but it was subsequently discovered that they themselves were capable of positively regulating cell signaling (35, 36). The discovery of an endogenous β-arrestin-biased receptor has important implications for our basic understanding of 7TMR biology. It inverts the classical paradigm of signaling from the view that all signaling by 7TMRs is mediated by heterotrimeric G proteins, to one where receptors may only signal through non-G protein-mediated mechanisms. This suggests that other receptors that have been considered to be "nonsignaling" may also signal through such nonclassical pathways. Many drug discovery and deorphanization strategies are based solely on assays for G protein-mediated actions and may not recognize β-arrestin-mediated processes. In the future, a more complete understanding of 7TMR pharmacology can only be gained by looking at G protein and β-arrestin activity in concert.
A plasmid encoding CXCR7 in a modified version of pcDNA3 (pFIRES) was a gift from Chemocentryx (9). Recombinant chemokines were obtained from R&D Systems. Anti-CXCR7 monoclonal antibodies were obtained from R&D systems, anti-rat CXCR4 and anti-rat CXCR3 antibodies from Abcam, antiphosphoERK antibody from Cell Signaling Technologies, and antitotalERK antibody from Upstate Biotechnology. Chemokine inhibitors CCX704 and CCX733 were obtained from Chemocentryx. T487 was obtained from Tularik, and AMD3100 was purchased from Sigma.
Transient Transfection.
HEK-293 cells were maintained in MEM media supplemented with 10% FBS and 1% penicillin/streptomycin. For DNA transfection, FuGene transfection reagent (Roche Applied Science) was added at a ratio of 5-μl to 1-μg plasmid DNA to a solution of plasmid DNA in 500-μl serum-free MEM. This transfection mixture was incubated for 30–45 min prior to addition to HEK293 cells of 50% confluence. For a 10-cm cell culture dish, 5 μg of plasmid DNA encoding CXCR7 was used. Cells were split and used for assays 48 h after transfection. For rVSMCs, 80–90% confluent early passage (< 3) VSMCs were transfected with siRNA using Lipofectamine 2000 transfection reagent (Invitrogen) according to the modified manufacturer's instructions (37). The siRNA sequence targeting β arrestin1 was 5-AGCCUUCUGUGCUGAGAAC-3, corresponding to position 431–459 relative to the start codon. The siRNA sequences targeting β arrestin2 were 5-GGACCGCAAAGUGUUUGUG-3 and 5-CCAACCTCATTGAATTCGA-3, corresponding to positions 150–168 and 1115–1133 relative to the start codon.
Confocal Microscopy.
HEK-293 cells were split into 35-mm glass-bottom dishes (MatTek) and transfected with 1 μg of CXCR7 and 0.2 μg of β-arrestin-2-GFP. Forty-eight hours after transfection, cells were starved for at least 5 h in serum-free media prior to treatment with ligand. For live cell imaging, cells were maintained at 37 °C with a heating plate while confocal microscopy was performed. SDF-1α and ITAC were added to a final concentration of 100 nM and images were taken from 0 to 30 min. For immunostaining, cells were treated for 30 min with 100 nM ligand, followed by aspiration of serum and fixation. Samples were then washed followed by permeabilization and blocking. Cells were then incubated overnight with primary antibody and then washed prior to incubation with secondary antibody at 1∶500 dilution. Samples were then washed and visualized.
PCR.
RT-PCR primers for rat CXCR7, CXCR4, and CXCR3 were obtained from Qiagen. Total RNA was extracted from passage 0 rVSMC by phenol-chloroform extraction using Ultraspec reagent (Bio-X). Qiagen RT-PCR kit was used for 30 and 40 cycles for semiquantitative RT-PCR. Samples were run on 1% agarose gel and stained with ethidium bromide prior to digital imaging.
Active Gαi Pulldown Assay.
rVSMCs were starved for 48 h prior to 5 min of stimulation with SDF-1α or ITAC (positive control). After 5 min, the cells were placed on ice, washed with ice cold PBS and lysed, solubilized for 1 h, and then incubated for 1 h with an antiactive Gαi antibody and Protein A-agarose beads (New East Biosciences). Beads were washed with lysis buffer three times and incubated with SDS-PAGE buffer at 95 °C for 10 min and run on a 4–20% gradient polyacrylamide gel, transferred to nitrocellulose membrane, and incubated with anti-Gαi antibody.
Intracellular Calcium Flux Assay.
rVSMCs were split into glass-bottom dishes at least 12 h before experiments. Smooth muscle cells (SMCs) were loaded with 5 μmol/L Fura-2/acetoxymethyl ester (Invitrogen) for 30 min. Intracellular calcium levels were quantitated by the Fura-2 excitation ratio at 340 and 380 nm on an epifluorescence microscope. Agonist-stimulated calcium release was calculated as the change in the Fura-2 excitation ratio from baseline.
Radioactive Ligand Binding.
Cells were grown in 24-well plates until confluent. Binding was assessed by incubation with 0.02 nM 125I-SDF-1α or ITAC in 25 mM Hepes, pH 7.5, containing 140 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, and 0.2% BSA for 2 h at 22 °C in the presence of 0–300 nM unlabeled ligand. In some experiments, AMD3100 (50 mM) or CCX733 (1 mM) were also included. Cells were washed three times with binding buffer containing 0.5 M NaCl, solubilized with 0.3 mL of 0.5 M NaOH, and counts per minute bound determined by gamma counting.
VSMC Migration.
Rat aortas were stripped of adventitia and endothelial cells, and then digested with 0.1% collagenase II and 15 U/mL elastase in the presence of 0.1% soybean trypsin inhibitor. Released SMCs were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin (Life Technologies). SMCs were used during passages 1–2. The migration of serum-starved SMCs was assessed using Transwell™ membranes (Costar; 8-μm pore). Filters were coated with 10 μg/mL fibronectin (in PBS overnight at 4 °C), rinsed once with PBS, and placed in 24-well dish wells that contained serum-free DMEM supplemented with agonists. SMCs suspended in serum-free DMEM containing 0.1% BSA were added to the upper chamber (1 × 105 cells/well). Cells were allowed to migrate for 5 h at 37 °C. Nonmigrated SMCs were removed from the top filter surface with a cotton swab. Migrated SMCs, attached to the bottom surface, were fixed in 4% paraformaldehyde and dyed with crystal violet. Dye intensity was quantified by densitometry. For rVSMCs transfected with fluorescent siRNAs, images were obtained on an epifluorescence microscope and the number of cells quantified.
We thank Thomas Schall and Juan Jaen for valuable discussions and advice. We thank Donna Addison and Elizabeth Hall for secretarial assistance. We thank Chemocentryx for a construct encoding CXCR7 and selective CXCR7 antagonists. This work was supported in part by National Institutes of Health (NIH) Grants HL16037 and HL70631 (to R.J.L.). R.J.L. is an Investigator with The Howard Hughes Medical Institute. S.R. is supported by NIH T32 training Grant HL07101-34. Drs. Gerard are supported by NIH Grants HL69511 and 36162.
1To whom correspondence may be addressed. E-mail: Craig.Gerard{at}childrens.harvard.edu or lefko001{at}receptor-biol.duke.edu.
Author contributions: S.R., J.K., S.A., N.P.G., C.G., and R.J.L. designed research; S.R., J.K., S.C., C.M.L., and N.P.G. performed research; S.R., J.K., S.C., and N.P.G. analyzed data; S.R., C.G., and R.J.L. wrote the paper.
This article contains supporting information online at www.pnas.org/cgi/content/full/0912852107/DCSupplemental.
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You are going to email the following β-arrestin- but not G protein-mediated signaling by the "decoy" receptor CXCR7
Sudarshan Rajagopal, Jihee Kim, Seungkirl Ahn, Stewart Craig, Christopher M. Lam, Norma P. Gerard, Craig Gerard, Robert J. Lefkowitz
Proceedings of the National Academy of Sciences Jan 2010, 107 (2) 628-632; DOI: 10.1073/pnas.0912852107


Skipper Rio Ferdinand was ruled out only 24 hours after arriving in South Africa with a knee ligament injury. His replacement, Ledley King, suffered a groin problem during the opening clash with the United States and was substituted at half-time.
"Hopefully I'll play, Ledley did his groin early on and did well to battle on to half-time but it's a squad thing now," said Carragher.
"We have a couple of injuries in that area of the team. If I can get a chance, I would love to take it. That's one of the reasons I came back.
"I wasn't stupid. I knew I was coming here as a squad player, that I wasn't a starter, and so I was delighted to get on against USA and hopefully will get another chance.
"To play in another World Cup for England is a great thing for me and I'm proud of that from a personal point of view.
"The main thing now is that hopefully I can get a run in the team and we can get through the group."