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Now that Signing Day 2010 is here, bitter disappointment over Stanford's last minute loss of admitted 4-star linebacker Jordan Zumwalt as well as the earlier losses of Louis Young and Brandon Bourbon leaves a bad taste in the mouths of many Cardinalmaniacs. As of this writing, a class that had been touted all year as one of the best classes in the Pac-10 checks in on the Scout.com team recruiting rankings as the 24th best class in the nation and just the fifth best class in the Pac-10. Nonetheless, the hype and media attention surrounding Signing Day exaggerates the importance of any single late decision. Ultimately, the class that Stanford did sign and its strengths and weaknesses are what matter.
In assessing the 2010 class for Stanford, any disappointment over the end game must be weighed against the clear progress Stanford has made in its recruiting operation under Jim Harbaugh and his staff. If Stanford's class ends up in the top 25 in the nation, it will be the second most highly ranked class of the last nine years. Coming on the heels of the most highly-touted class of that period, the 2010 class helps confirm an upward trend in Stanford recruiting under the Harbaugh staff that has long been apparent to close observers of Stanford Football. Taken along with the 2009 class, today's haul solidifies the improved overall quality of talent and depth promised by last year's group. Additionally, the departing class of fifth-year seniors came in as the #38 ranked class in the nation, so at least on paper, Stanford got better today as the 2010 class essentially replaced the 2005 class.
Moreover, despite the disappointment that characterized the last days of the recruiting cycle, the quality of the class should not be judged against the lofty expectations of a congenitally optimistic staff or fan base but instead against base line goals for success that we would have outlined at the outset of the year. A year ago, a list of priorities for the 2010 recruiting class may have read:
1. Recruit good quality and quantity at offensive line to address the problematic depth recruited over the previous three years. On a related but slightly broader level, Stanford needed to recruit 260+ pound recruits better than it has in recent years in order to provide sufficient quantity of options at positions traditionally manned by players naturally that size, including the offensive line but also defensive tackle.
2. Recruit good quality and quantity at linebacker to address the problematic depth recruited over the previous three years. Stanford recruited linebacker lightly in 2009 and has had numerous players from the previous two years not pan out at linebacker. With most of Stanford's contributing linebackers from this past season either out of eligibility or only possessing one year of eligibility left, new options will have to emerge. The 2010 class needed to give Shayne Skov some company in the pipeline.
3. Attract potential difference makers. While any individual recruit can turn into a superstar, the likelihood of producing all-league performers and other standouts increases as teams recruit consensus elite prospects. As Stanford begins to aspire to compete at the top of the conference and gain berths in the Rose Bowl or other BCS bowls, true difference makers arguably become even more important.
4. Improve the depth on the roster. The rosters of the teams that languished under Buddy Teevens and Walt Harris invariably included a handful of players who were elite recruits in high school and others who went on to play in the NFL. But the teams could not sustain any level of success for whatever reason. To the extent that the failures of that era owed to poor position coaching or overall leadership, Stanford fans hope and believe the Harbaugh staff represents a significant improvement in that regard. However, the failures were also due in part to rosters that lacked quality depth. Injuries to starters proved devastating. Practices often pitted some quality Pac-10 players against teammates with considerably less ability. Perhaps most significantly, weak links at certain positions emerged and prevented the quality efforts at other positions from propelling Stanford to overall team success.
Stanford's 2010 class should be judged against these metrics to determine how well it addressed the priorities facing the program in attracting talent.
Recruit good quality and quantity at offensive line
Stanford ended up with four offensive linemen in the 2010 class: Dillon Bonnell, Cole Underwood, David Yankey, and Cameron Fleming. All were evaluated by Scout.com as offensive tackles and are clustered among the #37 to #52 offensive tackle prospects in the nation in Scout.com's estimation. The group reportedly includes sufficient athleticism among the various members for projection to either guard or tackle. The specific position projections matter less than that Stanford signed four quality, sought-after, and versatile offensive linemen who provide options across the offensive line.
In terms of quality, the group appears to fit the bill for Stanford. Bonnell ranks among the highest 3-star recruits in the nation and, in fact, slipped out of the top 300 overall prospects only recently and does merit 4-star status on another website. In Scout.com's view, all are legitimate candidates for top 500 overall players in the nation and, extrapolating based on the 30 top 300 offensive tackles in the nation, Bonnell, Underwood, and Yankey almost certainly would rank in the top 500.
For those who place more credence in the evaluation of college coaches than recruiting services, Yankey had arguably the most elite recruitment of any Stanford signee this year, picking Stanford over offers from Florida, Georgia, Tennessee, UCLA, and many others. Bonnell had offers from at least half of the Pac-10 as well as many other schools, including Georgia Tech, Nebraska, Tennessee, and Utah. Underwood also had double digit offers, including Nebraska, TCU, and Wake Forest. Fleming was long committed to TCU and had a more under-the-radar recruitment but also had double digit offers, including Oklahoma State, Arkansas, and Missouri in addition to the Horned Frogs.
By any measure, Stanford recruited very good quality at offensive line this year. Another website ranks Stanford as the 17th best offensive line haul in the nation. However, Stanford's ability to rank even higher was constrained by its inability to sign an impressive quantity of offensive linemen. Four offensive linemen is not a big group. Given Stanford's extremely acute needs in the offensive line pipeline due to an entire class in 2007 that has yet to produce a Pac-10 lineman and classes the next two years that only combined for five linemen, four was at best a bare minimum for Stanford to recruit in 2010. Thus this class fits the bill in terms of the need for quality at offensive line but is more dubious in terms of the need for quantity. Ultimately, Stanford faced a severe need at offensive line this year and ended up signing four quality players at those positions but likely heads into the next recruiting cycle still needing bodies at offensive line. That is far from ideal since the 2011 class is likely to be smaller in size than the last two, but the quality of Stanford's 2010 offensive line haul should push any complaints somewhat to the margins.
On the related need of Stanford needing to recruit 260+ pound recruits better, Stanford continues to recruit physically small classes. One of Stanford's offensive line recruits reports a weight below 260 pounds, none report a weight above 280 pounds, and 16 of the 21 recruits who have signed Letters of Intent report a weight of 250 pounds or less. That leaves Stanford with only 11 recruits in the last three recruiting classes who reported weighing 260 pounds or more coming out of high school and one of those, defensive tackle Padric Scott, is no longer at Stanford. Stanford has clearly struggled to recruit physically big recruits under the Harbaugh regime, strongly prefers smaller athletes, or both. In assessing this from a depth perspective, offensive line remains a concern, albeit much less of one than it was a year ago now that Stanford has added quality at those positions, and defensive tackle is by far the biggest problem facing the program going forward. Stanford will need to recruit the position strongly in the next year and hopes that Eddie Plantaric becomes a fixture there.
Recruit good quality and quantity at linebacker
The loss of Zumwalt stings in this regard. He would have joined Blake Lueders as a consensus top 300 overall player joining the class at a position of need for Stanford. Stanford should be thrilled to have Lueders, the #79 recruit in the nation according to Scout.com and the crown jewel of Stanford's class, but he may have equal projection for defensive end as he has for linebacker. Thus the loss of Zumwalt could seriously impair Stanford's ability to meet the need at linebacker with this class.
In the end, Stanford signed four probable linebackers, nabbing 3-stars Joe Hemschoot, Cleophus Robinson, and A.J. Tarpley in addition to Lueders. Hemschoot was an under-the-radar recruit until the end of the year, when his recruitment blew up after the late bloomer sent his senior year highlight tape to schools. A year younger than most of his peers, Hemschoot was nonetheless one of the best players in Colorado this year as do-everything quarterback and linebacker. Stanford won his services in a frenzied late competition with Oregon and Colorado. Despite not having any recruiting profile for much of the year, he ultimately garnered recognition from Scout.com at the #34 outside linebacker prospect in the nation, a high 3-star and top 500 overall kind of prospect.
While Robinson and Tarpley do not have the same recruiting profiles as Lueders or Hemschoot, Stanford hopes they also contribute to improved depth at linebacker for the Cardinal. Robinson reportedly suffered from the same lack of exposure as Hemschoot insofar as he did not get his highlight tape to coaches until late in the process. He has been a solid player in the South New Jersey area the past two years as a pass rushing linebacker/defensive end, combining for 27 sacks in those two years. He chose Stanford over Louisville and Temple. Tarpley has long been in the fold for Stanford and won Gatorade Player of the Year for Minnesota. He chose Stanford over offers from Central Florida and Kansas State.
From a recruiting profile standpoint, Stanford's 2010 linebacker haul is a mixed bag now that Zumwalt is not in the class. Two of the signees, Hemschoot and especially Lueders, were highly sought-after recruits with different, potentially complementary skill sets. Lueders is a long-established, active, big athlete with an undeniably high recruiting profile as Stanford's only U.S. Army All-American in the class. Hemschoot is a much smaller athlete known more for his late development and potential for growth. The other two signees, Robinson and Tarpley, are more unknown quantities who Stanford will hope develop beyond their recruiting profiles.
Whether this group fills Stanford's need at linebacker is an unknown at this point. It is possible that, combined with last year's haul of Shayne Skov and Jarek Lancaster, this group provides Stanford a solid core of underclass linebackers to compete for positions and fill a quality starting linebacker group. However, much depends on a major wildcard looming over the program: what defense will new defensive coordinator Vic Fangio run on The Farm?
Fangio is a highly respected football coach with close associations to mentors Jim Mora (the senior version and former Stanford assistant) and Dom Capers. His experience coordinating defenses for those men and also his more recent experience coaching linebackers for the vaunted Baltimore Ravens defense endow him with substantial experience running a 3-4 defense. It is possible that Stanford's personnel prompts Fangio to install a different system than the one he has coached for the bulk of his career as a defensive coordinator. Equally likely, though, is that Stanford undergoes a fundamental transformation in its defense under Fangio. If Stanford does move to a 3-4 defense or something closer to it than the base defenses Harbaugh has installed under Ron Lynn and Scott Shafer, any analysis regarding the 2010 linebacker haul must be conducted with that in mind.
If Stanford is moving to a 3-4 defense, the quartet in this class and the duo in the previous class would probably be incapable of producing sufficient depth at linebacker without position switches. As with any position, the likelihood of injury or bust exists for any of these recruits and Stanford needs sheer numbers to be able to populate a 2-deep in a 3-4 defense. But the possibility for position switch does exist. Indeed, a move to a 3-4 defense would necessitate a reformulation of numerous roles. On the current roster, somebody like Chase Thomas would be forced to adapt to a new role as an outside linebacker/rush end. In the current recruiting class, defensive end Alex Turner would presumably join the aforementioned quartet in the linebacker corps if Stanford did indeed move to a 3-4 defense. Safety Ed Reynolds may be another outside linebacker candidate.
At this point, whether the 2010 class meets Stanford's needs at linebacker remains an open question, subject to how the players in the class develop, what defense Stanford runs, and what players in this class end up at linebacker.
Attract potential difference makers
Any member of a recruiting class can turn into a difference maker. Stanford's most decorated returning player in terms of all-league recognition is fullback Owen Marecic, a lightly recruited linebacker/fullback coming out of high school. Toby Gerhart was one of the jewels of his class from the get-go, but a year as a consensus All-American and Heisman runner-up surely qualifies as exceeding high 3-star or low 4-star expectations. Conversely, elite national recruits can fall short of difference-making contributions. Stanford has seen its share of 4- and 5-star recruits this decade never become all-league performers for a variety of reasons (bust, injury, supporting cast, etc), including Michael Craven, Trent Edwards, Ekom Udofia, Will Powers, and others. Nonetheless, research shows a correlation between the prominence of one's recruiting profile and all-league or NFL accomplishment.
The star of the class in terms of recruiting profile is Blake Lueders, the #79 recruit in the country according to Scout.com, #226 recruit in the country according to Rivals, and #113 recruit in the country according to ESPN. He is also the only Army or Under Armour All-American in the class after playing in the Army game, where he led the East team in tackles and impressed observers as a potential linebacker or defensive end.
Running back Anthony Wilkerson was ranked by Scout.com as the #158 prospect in the nation and by ESPN as the #150 prospect in the nation. Rivals evaluated him as a 4-star and the best running back outside of the top 250.
Quarterback Brett Nottingham was ranked by Scout.com as the #253 overall prospect and by Rivals as the #122 overall prospect. ESPN evaluated him as a 4-star and the sixth best quarterback outside of the top 150.
Defensive back Devon Carrington was ranked by Scout.com as the #236 prospect and by ESPN as the #140 prospect. Rivals evaluated him as a high 3-star.
Those four represent the consensus elite players in Stanford's class. Another elite national prospect is running back Ricky Seale, the third best 3-star running back in the nation according to Scout.com, just outside of the top 300, while Rivals views him as a 4-star and the fourth best running back outside of the top 250.
In addition to these recruits, Scout.com also gives 4-stars to quarterback Dallas Lloyd. The five Scout.com 4-stars falls short of last year's haul of one 5-star and nine 4-stars, but most Stanford fans will welcome it as simply a consolidation of high-end talent above and beyond what Stanford has brought in the past decade prior to Harbaugh. Aside from last year, the 2010 class represents the highest number of Scout 300 players Stanford has signed in any of the nine years covered by the Scout database, tying the four 4+ star players in the 2003 class and edging out the four 4+ players of the 2005 and 2008. Many Stanford fans are understandably disappointed that the much-discussed 2010 class only arguably surpasses the 2003 and 2005 classes in terms of recognized elite prospects and does not match the 2009 haul despite an 8-win Sun Bowl season.
Improve the depth on the roster
Stanford fans will, however, find more to rejoice in the class' probable strengthening of the depth of the roster. The 2010 recruiting class may be the deepest in the history of Stanford football. It certainly qualifies as the deepest of the decade, when recruiting classes can be imperfectly but somewhat systematically compared across years, but many long-time Stanford observers note that it may be unparalleled in Stanford's entire history. In previous years when Stanford may have recruited elite players better than it has this year, it nonetheless typically filled the class with less-desired prospects that can only be charitably be described as hoped-for diamonds in the rough but in many cases were simply reaches. In 2010, Stanford has signed essentially full of players it has either long wanted or fell in love with late in the cycle after seeing film. Almost every recruit has a recruiting profile of some repute.
Numerically, the outstanding depth of the class is represented by 20 Letters of Intent signed by 3+ star prospects according to Scout.com. By itself, that illustrates the profound improvement in depth this class should represent once added to the significant improvements in depth promised by the 2009 class, which also included twenty 3+ star signees. (To put those numbers in perspective, the 2004 and 2005 classes combined for nineteen 3+ star signees.]
But that actually understates how good the depth of this class is. With approximately 1000 3-star recruits per year, there is enormous variation in the evaluation and of course, college production, of 3-star recruits. A three-star recruit in the top 500 overall would be one of the top 200 3-stars nationally and clearly more elite in the evaluators' (admittedly imperfect) estimation than somebody ranked among the top 1300 recruits nationally, who could also nominally have 3-stars. Looking more closely at Stanford's class, the singular advantage of the 2010 class is that loads up on 3-star recruits at the high end of the spectrum. In addition to the five players ranked by Scout.com among the top 300 recruits nationally, Bonnell, Seale, Underwood, and defensive end Henry Anderson all rank among the highest-rated 3-stars in the nation, top 400 overall kind of candidates. Plantaric, Yankey, Turner, Hemschoot, athlete Keanu Nelson, and tight end Davis Dudchock are all probably top 500 kind of players. Kickers are harder to evaluate, but Jordan Williamson is ranked as the #5 kicker in the nation, an elite designation among specialists. In all likelihood, Stanford has as many as 15 players in the top 500 nationally and certainly a vast majority of the class in the top half of 3-stars or higher. The depth of the class is staggering, probably the best in the history of the Stanford Football program.
This depth is important to give Stanford an opportunity to minimize the chance that a major weak link or injury to an individual player torpedo a season. While offensive line and linebacker were the principal needs in the class, a healthy pipeline needs balance across position groups every year. Stanford only signed one defensive back last year but signed four this year, assuming one out of Nelson or Seale will end up on defense, and that group includes one of Stanford's most highly-touted defensive back recruits ever in Devon Carrington. Stanford may have whiffed at wide receiver this year, although quarterback Darren Daniel or Nelson may have some projection there, but signed three receivers last year and will likely make it an area of emphasis in 2011. Moreover, Stanford has even continued to stock up at positions of already-impressive strength in order to further bolster depth and increase the likelihood of difference-makers emerging. After signing three 4-star running backs last year, Stanford went and got Anthony Wilkerson and Ricky Seale as two of the most prominent members of this class. After signing an army of tight ends and defensive ends last year, Stanford added guys like Lueders, Dudchock, Anderson, Turner, Plantaric this year. While not all of those guys will be able to see the field at, for instance, defensive end, the sheer quality of big athletes may give Stanford options going forward. In particular, the weakest area of recruiting the last two years, defensive tackle, may find some help in this group. Indeed, Plantaric looks like a lock to be in the mix for Stanford at defensive tackle going forward.
Stanford's recruiting class, while very disappointing to those hoping to get a slew of elite national recruits or hoping to end strongly rather than dropping in national rankings, helps the Stanford Football program. It ranks in the top half of Pac-10 classes. It adds very good quality, albeit not very good quantity, at the absolutely critical area of offensive line, by far the biggest need in the class. It adds a promising but mysterious group at linebacker, the second biggest need. It brings in potential difference makers with the same frequency as Stanford's better years in recent experience, although not on par with last year or expectations for this year. And, in its most crucial feature, it complements the 2009 class nicely in substantially improving the likely depth of the roster in future years.
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Sliceandrice (View their submission) · 255 days ago
Awh crap sorry bout that also this is made with game maker
dos (View their submission) · 255 days ago
I don't see the connection with the theme ;_; I waited for a punchline, like, this is the block from the game that Maks could have lost if he didn't catch his computer when he fell, or something!
JamSnack · 256 days ago
Hey cool! Say, what program did you use to make this?
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subset: commoncrawl


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Arsenal have been heavily linked with Barcelona striker David Villa but he Catalans are not willing to let the Spain striker leave this month. While Atletico Madrid's Adrian Lopez has been a long-term target, the Gunners are interested in Stevan Jovetic from Fiorentina.
Wenger is on the look out for a new striker despite having Oliver Giroud and Theo Walcott upfront. France striker Giroud scored twice while Walcott netted the crucial third goal in their 3-2 win over Brighton to enter the fifth round of the FA Cup.
Wenger nearly pulled out of the race to sign Adrian from Atletico after the Spanish giants slapped a £15m valuation for the star striker while David Villa was left frustrated after being benched for their 2-4 win over Malaga in the Copa Del Rey.
Villa, 31, has been lacking first-team football with the Catalans and his growing frustration is being monitored by the Gunners. Although Barcelona are not letting Villa leave this month, Arsenal are ready to bid nearly £15million for the World Cup winner.
Wenger is also interested in signing a midfielder in West Ham's Mohamed Diame, who is also a target for Tottenham and QPR, and Wigan Athletic's James McCarthy, who is targetted by Everton.
Diame, 25, has a release clause of £3.5million in his contract but his £75,000-a-week salary could threaten the deal. However, Wenger will be keen to replace Emmanuel Frimpong, who was sent on loan to Fulham, and Mikel Arteta, who is injured.
Meanwhile, Real Madrid are planning to replace Jose Mourinho with Wenger. The Frenchman had already denied two such offers from Real president Florentino Perez in the past but Spanish champions are aware of Arsenal's position in the league and their fans growing frustration.
OneIndia News


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Search the skies and flag down a passing ship
I did my best to set up some kind of communicator to attract other worlds. The trouble is it's hard to exercise your brain fully whilst taking all these anti-psychotic drugs that I found in the Medi-Store. I did manage to get someone's attention... but I hadn't put any Anti-Spam filters on the communicator and so I got picked up by Seleshantor Intergalactic PLC. I'm with them now. They've been trying to sell me: double-glazing; a three-day break on Earth; an actor reading "Better Eyesight Without Glasses" such that it feeds straight into your brain; home insurance; life assurance; holiday insurance to cover a three-day break on Earth and insurance to cover the potential brain-damage caused by an actor reading "Better Eyesight Without Glasses" straight into your brain.
I need help. I'm not going to get it here: these people are crazier than me! I have asked them to drop me off on the nearest planet with an S3 atmosphere but they say that's that horrible prison I've just released myself from and I'll have to stick with them for a while. Truth be told I'm not that bright so I wouldn't know if they're lying or not.
They want me to prepare every night to be a Brain Choc salesman. I'm so embarrassed. I'm a bit shy around people, I now realise, and the last thing my self-confidence needs is being rejected by tribe after tribe on Geldimoss or wherever we're going as I try to sell them the idea that what they really need is some expensive chocolate which will allagedly improve their mathematical ability. I mean, how can that possibly work? Anyway, suppose there's some tribal leader reigning happily over her subjects and some silly blond boy turns up offering to help her multiply 11 by 15 more rapidly and claiming she should eat more fattening, carb-ridden food? Silly. Silly.
To be honest the main thing I'm dreading isn't approaching total strangers, although that's scary enough - my superior indifference was, I realise, a defence mechanism so I didn't have to get close to anybody - it's being told, "No", "No", "No" by everybody. I hate the training I'm being given. They keep clapping me on the back. I'm not very tactile and I don't like people getting into my personal space like that but I feel a bit awkward saying anything. I hate having to sound "loud and confident" and having to "project" and "share my thoughts with the folks at the back of the tribe" and all that stuff. I HATE it! And all that, "Who knows what one of THESE is?" (And you have to whip a Brain Choc bar out of your pocket on the word 'THESE'). I mean I don't talk like that. No-one talks like that. Suppose they don't believe what I'm saying? Suppose one of the tribes sends a report to Seleshantor saying what a failure I am and that I'm the worst salesman they've ever seen? Suppose they send a video to everyone I've ever met of me with them while the entire tribe laughs at me? Suppose they torture me for insulting them? Suppose they have the death penalty and I get executed for dumbing down or something? I mean that's exactly what they might hate, isn't it?
Hold on: I need to take some more medicine. Hold on. Hold on.


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Desperate MemoriesMature
Salleem danced through the crowds of the sand ridden streets of Asyut. Behind him two men, one wielding a scimitar dawned in thin metal plating and the other, a merchant wildly flailing a loaf of bread above his head and screaming at the top of his lungs ran after him. Many people were shoved to the dusty cobble ground in the confusion as the two very out of place men forced their way through the busy narrow street. Salleem, twelve years old, peered down at the soft loaf of bread in his left hand and smiled widely. Today he would eat.
He looked over his shoulder as he ran. Salleem new the city well enough to be sure he wasn't going to run into anything. The bread merchant and his guard had gotten caught up by a group of rowdy cloaked men who were looking for a fight after being shoved aside during the pursuit. Now was his chance. Salleem quickly ducked into the alleyway beside him and hid himself behind a large pile of trash. Although Asyut was considered one of the nicer villages in the region, it was not clean. He carefully leaned over the rounded corner of the alleyway just enough to get a look at the two men confusedly running down a different street. He laughed to himself before taking a bite of bread. At the far end of the alleyway against a weathered wood gate sat an old homeless man balled up in a rough hide blanket. After finishing his bread, Salleem couldn't help but notice the head of cabbage lying beside him. As far as morals went, Salleem fall a little short, but even he had qualms about stealing from the homeless. But hunger is a powerful thing. And since the bread he had just gotten away with he had yet to eat in four days. So with great caution, he slowly made his way towards the rotting wooden gate.
He planned to grab the cabbage and run as fast as he could from the man so that he might escape the guilt that followed the pleading gaze so many others had given him after a successful theft. What was about to happen was going to be much worse. The old homeless man lied motionless only a meter or so in front of him. "Is he dead?" Salleem thought to himself. As quickly as he could, he plunged his hand toward the cabbage. Then it happened. The decrepit hand of the homeless man shot up and grabbed Salleem by the wrist. Salleem let out a quick squeak before stopping himself. He tried to pull himself away but the old man's grip was so strong he might as well have been chained to the wall. There was no escape.
"You dare steal from me?!" The old man's head shot off from the ground and glared into the face of the boy before him. "Foolish boy, you've made a mistake you're going to deeply regret." With his other hand the man brought out a dagger that had been concealed within his blanket. Slowly he raised it towards the boy's chest. Tears streamed from Salleem's eyes and he couldn't get out a scream before the man leaned forward and with a grunt the dagger dove itself deep into his chest. "Never will you be able to appreciate the here and now! Never will you live a normal life!" The man screamed as Salleem fell to the ground. Everything went black.
Salleem woke up in a cold sweat within the prison cell. He gasped for air and stared blankly at the ground before him. Had that been just a dream? No, he had never been so fortunate. He hadn't ever gone a month without that man cluttering his dreams. Through the bared window the first rays of sunlight flooded cell and awoke some of the other prisoners. A few new members of the cell remained asleep against the wall. They had probably been captured sometime in the night.
"Looks like you three are up. Good." The woman from the day before gave them a wild, sadistic grin. In her hand several keys dangled loosely from a chain.
Salleem's mind raced. He had been tortured in a prison before. However, the cold look the lady was giving him said that sometimes those that enter her torture chamber don't leave. He knew he had to get out. Incredibly aware that these might be his last moments, he tried to phase his arms out of the shackles. No good, they weren't ordinary cuffs. Behind his back, just as his wrists were half way out of the shackles, they flashed a bright blue for an instant and his arms snapped back into place. Beside him against the wall, the armored man stared wide eyed for only a moment before concealing his interest. It seemed as though he might have been the only one to see Salleem's attempt at escape. The thief lay panting on the ground, defeated.


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Wedding is, undoubtedly, the most beautiful moment of your life. This extraordinary occasion must be celebrated with exhilaration and a perfect planning. You must start early and make suitable plans. From engagement to vows, a well-defined plan can transform every moment into a lifetime memory that you will cherish forever. In this regard, there are certain things that can save your day.
- Make Early Plans and Check Availability:
Once you have finalized the date of your wedding, it's time to pull up your socks and start the execution of your plans. Check the availability of venue, decorators, caterers, photographers, photo booth companies, et al. The list seems endless but you can save your efforts by hiring a proficient event planner in Ireland.
- Plan Your Budget:
Before you start planning your special day, it is necessary to set aside a budget and follow it religiously. Your budget should be appropriate. It implies that your wedding budget should neither be too petite nor exorbitant. Include every important arrangement while making a budget to avoid unforeseen expenses at the last minute.
- Make a List Of Invitees:
It is understandable that you wish to invite the whole city to your wedding. But this list should not be inflated with too many names. An overstated list of invitees can crash your budget and lead to several problems. Also, consider the size of the venue before preparing this list because an overcrowded venue can mar all the arrangements. Hence, the number of invites must be limited to your friends, family, relatives, and co-worker's.
- Hire the Professional Wedding Planners for a Perfect Irish Wedding:
You need not be a pro in making wedding arrangements. All you should do is hire the professional event planners in Ireland. They are acquainted with the latest trends and can incorporate them impeccably in your wedding plan. Apart from that, they possess the experience of handling the last minute glitches with ease and dexterity. Hence, hiring them can be the best thing that you can do.
However, before hiring the event planners in Dublin or any other Irish city, you must check the services that they provide. It is futile to hire multiple companies for wedding photography, dance floor hire, wedding décor, uplighting, and photo booth hire in Ireland. Rather, you must consider hiring a single company to finish all these jobs within the stipulated time. They must be professionally managed and possess agility to perfectly plan every aspect of your wedding.
The writer is an experienced wedding author writing on bridal trends and tips for event hire in Ireland since many years. She has ample exposure and familiarity with the international wedding industry. She absolutely loves to share this knowledge with her readers.


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Product Pathways - Tyrosine Kinase / Adaptors
Phospho-c-Abl (Tyr89) (61A6) Rabbit mAb #3098
PhosphoSitePlus ® protein, site, and accession data: ABL1
|100 µl ( 10 western blots )
|现货查询 购买询价 防伪查询
|carrier free & custom formulation / quantity
|email request
|MW (kDa)
|135 (c-Abl); 210 (Bcr-Abl)
|Rabbit IgG
Species cross-reactivity is determined by western blot.
Applications Key: W=Western Blotting,
Species predicted to react based on 100% sequence homology: Mouse,
Specificity / Sensitivity
Phospho-c-Abl (Tyr89) (61A6) Rabbit mAb detects endogenous levels of c-Abl only when phosphorylated at Tyr89. This antibody may cross-react with other tyrosine-phosphorylated proteins.
Phospho-c-Abl (Tyr89) (61A6) Rabbit mAb兔单抗能检测内源性Tyr89位点磷酸化的c-Abl蛋白。这种抗体可能会与其他酪氨酸位点磷酸化的蛋白发生交叉反应。
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr89 of human c-Abl.
Western Blotting
Western blot analysis of extract from CML-T1 leukemic cells with Phospho-c-Abl (Tyr89) (61A6) Rabbit mAb (upper and middle) or c-Abl Antibody #2862 (lower). The phospho-specificity of this rabbit mAb was verified by treating the membrane with calf intestinal phosphatase (CIP) (middle and lower) before antibody probing.western blot方法检测细胞提取物:用Phospho-c-Abl (Tyr89) (61A6) Rabbit mAb (上图和中图)和c-Abl Antibody #2862 (下图)处理的CML-T1白血病细胞。在使用抗体检测之前,先用牛小肠碱性磷酸酶处理转印膜已验证这种特异位点磷酸化的兔单克隆抗体。
The c-Abl proto-oncogene encodes a nonreceptor protein tyrosine kinase that is ubiquitously expressed and highly conserved in metazoan evolution. c-Abl protein is distributed in both the nucleus and the cytoplasm of cells. It is implicated in regulating cell proliferation, differentiation, apoptosis, cell adhesion and stress responses (1-3). c-Abl kinase activity is increased in vivo by diverse physiological stimuli including integrin activation, PDGF stimulation and binding to c-Jun, Nck and RFX1 (2,4). The in vivo mechanism of regulation of c-Abl kinase activity is not completely understood. Tyr245 is located in the linker region between the SH2 and catalytic domains. This positioning is conserved among Abl family members. Phosphorylation of Tyr245 is involved in the activation of c-Abl kinase (5). In addition, phosphorylation of Tyr412, which is located in the kinase activation loop of c-Abl, is required for kinase activity (6).
c-Abl原癌基因编码一个非受体蛋白酪氨酸激酶,该激酶在多细胞动物中普遍表达且高度保守。c-Abl蛋白分布于胞核以及胞质中,参与调节细胞增殖、分化、凋亡细胞粘附和应激反应(1-3)。在体内,通过整合素激活、PDGF刺激以及结合到c-Jun、Nck 和RFX1 蛋白上(2,4)等各种生理刺激,能使c-Abl激酶的活力提高。调节c-Abl蛋白激酶活力的体内机制目前尚不完全清楚。Tyr245位点位于SH2和催化区之间的连接区域,Abl蛋白家族在这个位点处都是保守的。Tyr245位点的磷酸化涉及到c-Abl激酶的活化(5)。另外,位于Abl蛋白的激酶活化环处的Tyr412位点,对激酶的活性也是必需的(6)。
Phosphorylation of c-Abl on Tyr89 was identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's LC-MS/MS platform for phosphorylation site discovery as well as another publication using MS technology (7). For additional information please visit PhosphoSitePlus®, CST's modification site knowledgebase, at www.phosphosite.org.
- Wang, J.Y. et al. (2000) Oncogene 19, 5643-5650.
- Van Etten, R.A. et al. (1999) Trends Cell. Biol. 9, 179-182.
- Danial, N.N. et al. (2000) Oncogene 19, 2523-2531.
- Shaul, Y. et al. (2000) Cell Death Differ. 7, 10-16.
- Brasher, B.B. et al. (2000) J. Biol. Chem. 275, 35631-35637.
- Pluk, H. et al. (2002) Cell 108, 247-259.
- Meyn, M.A. et al. (2006) J. Biol. Chem. 281, 30907-30916.
Application References
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- Western Blotting
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- 12630 SignalFire™ Plus ECL Reagent
- 12757 SignalFire™ Elite ECL Reagent
- 2861 Phospho-c-Abl (Tyr245) Antibody
- 2862 c-Abl Antibody
- 2864 Phospho-c-Abl (Thr735) Antibody
- 2865 Phospho-c-Abl (Tyr412) (247C7) Rabbit mAb
- 2868 Phospho-c-Abl (Tyr245) (73E5) Rabbit mAb
- 3009 Phospho-c-Abl (Tyr204) (C42B5) Rabbit mAb
- 6883 SignalFire™ ECL Reagent
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 7727 Biotinylated Protein Ladder Detection Pack
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text:
Casa Histórica no quintal Bastille
Nº de hóspedes:8
Casas de banho2
Tamanho140 m²
Andar no.4
- Cozinha
- Conveniente para Família/criança(s)
- Máquina de lavar/secar
- Varanda/terraço
- Internet
Descrição da propriedade
|Rue du Faubourg Saint-Antoine, 75011 Paris, França
|Tipo de propriedade
|ID da Propriedade
|Taxa de aceitação
|Tempo de resposta
|Umas horas
|Calendário atualizado
|Alguns meses atrás
Localizado em um pátio encantador e tranquilo com flores, árvores e lírios, esta antiga casa histórica tem um monte de personagem que lhe dará uma experiência diferente durante a sua estadia em Paris. Este alojamento 140 metros quadrados tem 4 quartos, mais um Mezzanin. É acomoda 4-8. É equipado com uma máquina de lavar, secador, um acesso ilimitado à Internet de alta velocidade, um telefone, um aparelho de som, uma TV a cabo, beamer e um DVD.
O acesso à casa é através de um pequeno jardim privado orientado para sul e recentemente renovado.
Entrada / sala de jantar
No primeiro andar, você tem uma grande sala de jantar. Próximo a ele é um quarto com mezanino.
A cozinha metro quadrado 12-americana, com um hard-madeira-mesa de bar para 4 está aberta para a sala de estar / jantar, para não tirar a sensação de espaço. A cozinha é funcional, com um forno, um refrigerador, uma cafeteira, uma máquina de lavar e secar roupa.
Sala de estar do primeiro andar
Equipado com uma televisão HD e videocassete, assim como fast-internet hook-up, você poderá desfrutar de teto alto, design sóbrio e estátuas exóticas. Quartos e um banheiro confortável com chuveiro e banheiras estão disponíveis.
Um porão independente dá mais um quarto com banheiro privativo. O volume surpreendente de modo silencioso.
A área animada no bairro Bastille, com diárias coloridos mercados ao ar livre, um porto agradável e barcos bonitos. Rue de Lappe, rue de la Roquette são ruas repleta de bares e restaurantes também estão próximos à casa. Em apenas andando lá você vai se sentir um verdadeiro parisiense.
A subida beco está equipado com um código de segurança.
Regras da Casa
|Preço semanal para até 8 hóspedes
|1504 €
|Preço mensal para até 8 hóspedes
|4167 €
|Check-in depois das
|Por acordo
|Check-in antes
|Por acordo
|Check-out antes das
|Por acordo
|Política de Cancelamento
Comentários de outras propriedades deste anfitrião
I can recommend the studio. The host is very friendly and open to help. The room is very small but everything you need is there.
A very good area, near Bastille, underground and Velib parking lot. Just on the corner of a boulangerie with just baked croissant. An apprtement is apparently constructed from a garage, it has a nice kitchen, a small table with two chairs, and a bed. The internet is very fast. A matress is not very comfortable, and I and my husband were sleeping in the middle of it 'cause it was sinking. A toilet doesn't have a door, so the appartment is for a very close family:) An owner was late for a check-out time for 20 minutes, so we had to leave without saying goodbye to her personaly, we had a plane to catch.
All in all, appartment is good for the money you pay))
very nice studio, fully equipped and a very nice host.
The host cancelled the booking, this review is automated.
The stay at Francois Studio was really nice! We were in Paris for a week and the little studio has everything you need. A big wardrobe ( garderobe ) was perfect for our clothes and books to feel like home.
When you book this studio you have to know that the bathroom is really small but there is everything you need. The small place to cook is really good for little meals after a nice trip. Only that the fridge was off and closed wasn't nice because the circuit.
The first contact to Francois was a little bit difficult because he didn't answer to us really soon.
But after the first contact worked Francois answered really soon to all our questions and that really kind!
All in all the stay was really comfortable even for a small studio. If we ever will be again in Paris we are looking forward to book this studio again.
Have a nice stay Lisa
The apartment is very nice, cozy, warm and clean, ideal for a short break in Paris; the owners are friendly and helpful for everything. The study is very near to the metro and buses, which lead to the main attractions of the city.
In the neighborhood there are all the necessary amenities (supermarkets, lavandere, bakeries, etc.) and in the evening there are many nice pubs and restaurants.
I hope to return soon
I loved my stay in Francois's Studio. The studio was well setup, with fridge and cooking facilities so i could make use of the interesting fresh food from the local markets. Location was ideal. I regularly made use of Bus69, which has a stop about 50mtrs from the Studio. The bus travels through Paris to Eiffel Tower. The studio would be perfect for a romantic couples holiday.
I have been staying in the studio for three weeks and really enjoyed my stay in Paris. Before and at arrival, Francois was very kind and helpful and hand-over of keys was uncomplicated. In the studio, space is used in an optimal way; kitchen and bathroom were clean and the studio was equipped with all necessary objects. Heating is well functioning, so that temperature in the studio was comfortable. The studio fits perfectly for one person and is in a quiet and safe location in a nice courtyard. Public transport connection is very good, next metro stations are easy and fast to reach. I really liked living in the quarter of Bastille which is quite lively with its nice restaurants, cafés and small shops. Absolutely recommendable!
Thank you for the lodging! I spent here 3,5 months and the studio fits well one person. Good location, compact studio and vibrant area. There are several "laveries" nearby. The landlord is helpful and understanding. Good English as well!
Some things to improve, such as heating (cold in the floor and hot in the roof area) and humidity (circulation of air not efficient enough). Fixing the excess humidity problem would not only improve coziness, but also make the few bugs disappear. I guess they are already working on it...
we really enjoyed our stay in francois apartment, it was perfect for the 3 days, cute and clean :) francois was really helpful when we got there.
- Qualidade do alojamento garantida
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text:
By tailoring dashboards to show the latest competitor data and market insights on a single screen for different user groups, companies have been able to increase the effective communication and use of their competitive intelligence. The best competitive intelligence dashboards combine text, information feeds, images, video and interactive elements. They can range from basic displays of information to the more interactive, dynamic formats where users can combine different data sets and even conduct predictive modeling.
We have witnessed how dashboards are energizing intelligence teams to become more active in communicating with their internal audiences.
Competitive intelligence dashboards that enjoy high usage rates by key stakeholders share five core common characteristics.
1. Well defined target audiences
2. Flexibility in formats and functionalities
3. Commenting and alerts features
4. Support from key stakeholders
5. Regular reviews and audits
The target audience for each dashboard must be well defined, and the purpose of each dashboard considered and linked to a key business processes or decisions. Usage rates tend to increase when several different perspectives from important stakeholders are included.
Stakeholders have different preferences, so the ability to change the look and feel as well as the different functionalities on specific stakeholder dashboards is important.
Dashboards that are constantly updated attract more users. In fact, the more they mimic social media platforms, the better. For example, ensure that your users get to see other users' contributions displayed as part of a landing page on their dashboards, or in sections such as "latest comments", "top rated items" and "most read articles".
It is always helpful to identify users, preferably senior stakeholders, who will lead by example and be active commentators. So invite your senior management to start interesting discussions on key dashboards; this will trigger other users to take part.
Things change over time, and so do the needs of your users. You can only keep your competitive intelligence system valuable to its users if you can monitor their usage rates and conduct an audit of their needs from time to time. This will impact how you upgrade your dashboard from time to time.
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text:
The Use of FTIR Spectroscopy and Chemometrics for Rapid Authentication of Extra Virgin Olive Oil
- First Online:
- Received:
- Revised:
- Accepted:
DOI: 10.1007/s11746-013-2370-5
- Cite this article as:
- Rohman, A., Che Man, Y.B. & Yusof, F.M. J Am Oil Chem Soc (2014) 91: 207. doi:10.1007/s11746-013-2370-5
- Citations
- 1.3k Downloads
The authenticity of high value edible fats and oils including extra virgin olive oil (EVOO) is an emerging issue, currently. The potential employment of Fourier transform infrared (FTIR) spectroscopy in combination with chemometrics of multivariate calibration and discriminant analysis has been exploited for rapid authentication of EVOO from canola oil (Ca-O). The optimization of two calibration models of partial least square (PLS) and principle component regression was performed in order to quantify the level of Ca-O in EVOO. The chemometrics of discriminant analysis (DA) was used for making the classification between pure EVOO and EVOO adulterated with Ca-O. The individual oils and their blends were scanned on good contact with ZnSe crystals in horizontal attenuated total reflectance, as a sampling technique. The wavenumbers of 3,028–2,985 and 1,200–987 cm−1 were used for quantification and classification of EVOO adulterated with Ca-O. The results showed that PLS with normal FTIR spectra was well suited for quantitative analysis of Ca-O with a value of the coefficient of determination (R2) > 0.99. The error, expressed as root mean square error of calibration obtained was relatively low, i.e. 0.108 % (v/v). DA can make the classification between pure EVOO and that adulterated with Ca-O with one misclassified reported.


text:
John Micklethwait leaving The Economist to join Bloomberg
By Janina Oles
12th December 2014
The Economist's editor John Micklethwait is leaving to become editor-in-chief of Bloomberg. Micklethwait has been editor of The Economist since April 2006. He will begin work at Bloomberg's global headquarters in New York City shortly after the New Year.
A replacement editor is expected to be confirmed before the end of January; under the Articles of Association, his successor must be supported by the newspaper's four independent trustees.


text: Research Funding Office Last updated on 1/4/2017 Print this page Welcome to the Research Funding Office website. We are here to help you and to ease some of the anxiety and pain when putting together a research proposal. We are friendly, approachable and very keen to give you the support you require. Please feel free to contact us.


text:
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Blake was the captain of the ship Lady Sefa. He was murdered by Sister Ulicia.
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text:
The festive season is upon us, which gives our politicians a chance to show us how creative they are with their Christmas cards. But this year has been a pretty boring one, with David Cameron and Ed Miliband both choosing fairly dull family scenes. Only Nick Clegg gets into the spirit of Christmas, with a card that his children designed. The urge not to embarrass or offend with a misguided or off message winterval theme means that we are often met with a disappointingly tame selection.
Can you show our politicians how a Christmas card should look? Do you go all out with reindeer, Santa and snow or do you prefer a classic nativity scene? Send us your own best yuletide artwork, or a photo of your favourite home-made card from this year.


text: LAWRENCE, Mass., Nov. 22, 2011 /PRNewswire/ -- Taking control of your dialysis and finding the information you need about home hemodialysis with the NxStage ® System One ™ just got easier for end stage renal disease (ESRD) patients with the launch of the NxStage Find a Doctor directory. The on-line directory created by NxStage Medical, Inc. (Nasdaq: NXTM), a leading manufacturer of innovative dialysis products, makes it easier for patients to identify a nearby nephrologist with experience caring for home hemodialysis patients using the NxStage System One. The Find a Doctor directory can be found under the " Home Hemodialysis" tab of NxStage's website. (Logo: http://photos.prnewswire.com/prnh/20110503/MM94799LOGO ) Today, the large majority of dialysis patients in the US receive conventional dialysis, which consists of treatment on a Monday/Wednesday/Friday or Tuesday/Thursday/Saturday schedule. This conventional in-center schedule leaves a 2-day gap between the last treatment of the preceding week and the first treatment of the following week, during which time toxins and fluids build up and may stress the heart and body. Recent publications have shown this long gap between treatments is associated with increased patient hospitalization and mortality. "With such an extensive pool of published data showing that, when compared with conventional thrice-weekly treatments, daily home hemodialysis can lead to improved health, quality of life and survival outcomes, the Find a Doctor Directory is an especially timely resource to encourage dialogue between patients and physicians who are knowledgeable about this treatment, empowering patients to explore all options for their kidney care," said Robert S. Lockridge, Jr., M.D., medical director of the UVA Home Dialysis program. "Dialysis patients should be informed of all treatment options and have access to the treatment that is right for them, including frequent home hemodialysis." "Many patients and their families have told us that simplifying access to home hemodialysis would be very beneficial," said Jeffrey H. Burbank, Chief Executive Officer, NxStage Medical Inc. "As a patient focused organization, we believe that our new 'Find a Doctor' directory is a convenient tool to help interested patients identify a local nephrologist and pursue more frequent home hemodialysis. We thank the many nephrologists that have chosen to collaborate with us in this important effort, and we look to continue to expand this in the future." To search for a nephrologist, users provide their zip code to access a list of doctors within a designated radius, including contact information. With this information, users can reach out directly to the nephrologist's practice to learn more about more frequent home hemodialysis with the System One. In addition to the Find a Doctor directory, users can also search the Find a Dialysis Center directory to locate a dialysis center offering training for the NxStage System One. Disclaimer The Find a Doctor and Find a Dialysis Center directories are not intended as tools for verifying the credentials, qualifications, or abilities of any physician or center contained therein. The use of either directory by any entity or individual to verify the credentials of listed physicians or center is prohibited. Inclusion in either directory does not imply recommendation or endorsement by NxStage nor does omission in either directory imply NxStage disapproval.


text:
Doing the Word (5 of 11) by Joe Alain
This content is part of a series.Doing the Word (5 of 11)
Series: James
Joe Alain
James 1:22-27
Here's a spiritual evaluation question: How much of God's word do you practice? Consider that we are commanded to pray (1 Thess. 5:17); to study (2 Tim. 2:15); to love (Jn. 13:35); to forgive (Eph. 4:32); to imitate God (Eph. 5:1); to hunger for righteousness (Matt. 5:6); and to abide in Christ (Jn. 15:4), just to name a few things. How are you doing? Are you a doer of the word?
Doing the word begins first with hearing the word properly (i.e., quickly, quietly, and calmly, v.19). This kind of hearing allows the word of God to be planted within our lives (v.21) so that we "naturally" become "doers of the word." In our passage, James cautions us to make sure that we don't stop with just hearing of the word. James says, "But be doers of the word and not hearers only, deceiving yourselves" (v.22).
1. The Command (1:22)
Doing the word is stated as a command (v.22). James says it is possible for us to hear the word of God and feel as if we are doing the word. But in reality, he says that we are only "deceiving ourselves." It's possible for believers to be deceived into thinking that they are doing the word when actually they might be disobeying the word of God. We are "deceiving ourselves" if we believe that listening to sermons and attending Bible studies is the sum of what we are to do as Christians. We are "deceiving ourselves" if we believe that our talking about ministry constitutes the same thing as doing ministry, or that our giving of our finances is a substitute for the giving of our selves. We are "deceiving ourselves" if we think that increased knowledge about God is all that God requires. We are "deceiving ourselves" if we believe God is pleased more with ritual than obedience.
The command is quite clear, "be doers of the word and not hearers only." After the command, James provides . . .
2. The Illustration (1:23-25)
(1) The Hearer Only (1:23-24)
The one who hears the word ...
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text:
The living microarray: a high-throughput platform for measuring transcription dynamics in single cells
Current methods of measuring transcription in high-throughput have led to significant improvements in our knowledge of transcriptional regulation and Systems Biology. However, endpoint measurements obtained from methods that pool populations of cells are not amenable to studying time-dependent processes that show cell heterogeneity.
Here we describe a high-throughput platform for measuring transcriptional changes in real time in single mammalian cells. By using reverse transfection microarrays we are able to transfect fluorescent reporter plasmids into 600 independent clusters of cells plated on a single microscope slide and image these clusters every 20 minutes. We use a fast-maturing, destabilized and nuclear-localized reporter that is suitable for automated segmentation to accurately measure promoter activity in single cells. We tested this platform with synthetic drug-inducible promoters that showed robust induction over 24 hours. Automated segmentation and tracking of over 11 million cell images during this period revealed that cells display substantial heterogeneity in their responses to the applied treatment, including a large proportion of transfected cells that do not respond at all.
The results from our single-cell analysis suggest that methods that measure average cellular responses, such as DNA microarrays, RT-PCR and chromatin immunoprecipitation, characterize a response skewed by a subset of cells in the population. Our method is scalable and readily adaptable to studying complex systems, including cell proliferation, differentiation and apoptosis.
A central challenge in the post-genomic era is determining how gene expression is regulated during complex biological processes. Hybridization and sequencing-based technologies such as DNA microarrays and RNA-seq have played a valuable role in identifying and characterizing the components of such processes on a comprehensive scale. Moreover, the combination of these technologies with high-throughput methods for studying protein-DNA and protein-protein binding has enabled us to glean insights into global networks of interactions
However, since existing methods of measuring transcription provide discrete measurements of a transcriptional response obtained from large populations of cells, they suffer from two major drawbacks. First, quantifying transcription dynamics using microarrays at multiple time-points is expensive when long processes are under study. Second, despite improvements in assay sensitivity, these approaches typically involve pooling mRNA from thousands of cells. The averaged response measured in this way is adequate for classifying different cell or tissue types, but it is not well-suited for studying processes that show cell-to-cell variation, such as cell division, differentiation, or drug responsiveness. Recent developments in cell-based assays combined with advances in reporter technology allow us to address these limitations, since expression levels can be repeatedly assayed in single cells. Here we describe a method in which we specifically transfect hundreds of clusters of cells with fluorescent reporter constructs and measure single-cell fluorescence changes using automated microscopy.
Reverse transfection arrays were first described as a method to introduce mammalian cDNA constructs into adherent cells at defined locations
Construction of the Living Microarrays
Living Microarrays use fluorescent reporter constructs to measure the expression of individual promoters in live cells. High throughput parallelization is achieved using a reverse transfection protocol
Figure 1
Living Microarray method
Living Microarray method. (A) Platform Workflow. The Living Microarrays method allows for highly parallelized measurement of reporter gene expression at the single-cell level. Synthetic promoter cassettes are cloned into the VC5 reporter construct and spotted with transfection reagent onto glass slides. Transfected cell clusters are imaged iteratively using automated microscopy and analyzed to generate normalized single-cell measurements of transcriptional activity. (B) Whole chip view of a Living Microarray. HEK-293T cells were reverse-transfected at 600 spots with varying ratios of pEGFP-N3 and Lipofectamine2000 and fixed cells were imaged at 10 μm resolution using a microarray scanner. Maximal transfection conditions are indicated by the white box and shown at higher magnification. (C) Reverse transfection reporter microarrays using human, mouse and rat cell lines originating from various tissues transfected with pEGFP-N3. Scale bars = 350 μm.
In our experiments, the array is created using spots with 6.7 nL transfection complex; these have a 400μm diameter and typically result in 75 transfected cells (293T cells). Using a square pattern we can spot 600-1000 spots within an 8.6 cm2 chambered coverglass slide (Figure
To test the suitability of our platform with other cell lines, we tested 14 adherent cell lines commonly used as tissue models for disease. We successfully transfected a constitutively active fluorescent reporter (pEGFP-N3) into lines originating from a variety of tissues, including fat, muscle, liver and bone (Figure
Additional file 1
Supplementary Data. Contains additional figures and tables referenced in the manuscript.
Click here for file
While the positions of dried spots can be easily located on the microscope, these boundaries disappear once the slide has been flooded with cells. We therefore developed a method of registering slides on the microscope using the positions of dried spots relative to invariant features at the edges of the slide. To avoid using autofocus routines that are time-consuming and unnecessarily expose cells to potentially damaging light, we also developed a method to determine accurate focus positions for all 600 transfected clusters by fitting a subset of 45 manually-focused areas to a third-order polynomial function that closely approximates the surface of our slide (Additional file
Development of a dual-fluorophore reporter
To measure dynamic changes in transcriptional activity, we used the Venus-NLS-PEST fluorescent reporter
Figure 2
Measurement of transcription using a dual-fluorophore vector
Measurement of transcription using a dual-fluorophore vector. (A) Time-lapse images of (GRE)x3-AdMLP induction by dexamethasone in 293T cells using nuclear and cytoplasmic reporters. The first two rows represent the same spot transfected with the p(GRE)x3-AdMLP-VC5 vector and imaged in ECFP (top) and Venus (middle) channels. The induction profile using the Venus reporter is comparable to that of the cytoplasmic destabilized EGFP reporter, p(GRE)x3-AdMLP-d2EGFP, shown in the bottom row. Moreover, the use of the ECFP channel enables the tracking of transfected cells before induction has taken place. Scale bar = 100μm. (B) Map of the Venus-ECFP5 (VC5) plasmid.
We automatically measure single-cell expression values by segmenting each frame to identify nuclear boundaries and measure the total pixel intensity within each segmented region (Additional file
To test our Venus reporter, we modified the well-known pGL3 reporter system by replacing the luciferase gene with a dexamethasone-inducible Venus-NLS-PEST ((GRE)x3-AdMLP-GV3). 293T cells were co-transfected with this plasmid and a second reporter containing a constitutively active, nuclear-localized dsRed (pCMV-dsRed-nuc) to control for transfection efficiency. Single-cell segmentation of cells imaged in Venus and dsRed channels allowed us to measure a normalized upregulation of cellular fluorescence of 11.0 fold (+/- 0.7) after 24 hours (Additional file
Conventional transfection-based reporter strategies typically rely on co-transfected plasmids to normalize signal for variations in plasmid copy number, for instance co-transfection of a plasmid constitutively expressing
We therefore cloned downstream of Venus a cassette containing ECFP driven by the human EF1α promoter (Figure
Single-cell transcriptional measurements of inducible promoters
To test our system, we subcloned three inducible promoters into the multiple-cloning site of Venus-ECFP5 (VC5, Figure
We observed induction of Venus transcription for all three synthetic reporter constructs, while the expression levels from the normalization channel remained relatively constant (Figure
Figure 3
Global expression changes
Global expression changes. 293T cells were reverse-transfected with inducible constructs, each arrayed over 75 replicate spots, and fluorescent cells were imaged over 24 hours. Single-cell measurements at each spot were averaged for the Venus (orange) and ECFP (blue) channels and are shown over time for (A) p(GRE)x3-AdMLP-VC5 following induction with 1 × 10-7M dexamethasone, (B) pTetRE-VC5 following induction with 2.2 × 10-6M doxycycline, (C) p(RARE)x3-AdMLP-VC5 following induction with 1 × 10-6M all-trans retinoic acid and (D) the promoterless VC5 reporter. The mean of all replicate fields is drawn in red for Venus signal and black for ECFP signal.
Our measurements of gene expression were obtained using data from 75 replicate spots. To determine the minimum number of replicate spots that would accurately measure the sample mean and variance, we used a permutation strategy in which the data was segregated into subsets of a fixed number of randomly chosen spots and repeated this process 1000 times for subsets of between 1 and 20 spots. The distribution of means from these subsets at the 15 hour timepoint was then compared to the actual distribution for 75 transfected spots. We found that samples with 5 replicates had a standard deviation that was within 0.5 standard deviations of the population standard deviation.
These endpoint measurements are analogous to existing methods that average responses between cells, such as RT-PCR, DNA microarrays and reporter assays. However, in addition to being able to measure average expression changes, single-cell measurements provide a wealth of information that can be used to better characterize signal variability between cells. Using the signal from our ECFP cassette, we were able to study the single-cell distribution of responses with respect to transfection efficiency (Figure
Figure 4
Single-cell measurements of promoter activity
Single-cell measurements of promoter activity. Single-cell measurements are plotted at 0 (blue) and 24 hours (red) post-induction for (A) EF1α-VC5 and (B) p(GRE)x3-AdMLP-VC5. (C) Multi-dimensional view of inducing cells on a single spot. Spatial movement of cells from one field is shown where each cell is represented by a different color and the intensity of normalized Venus fluorescence is proportional to the size of the dot. (D-F) 293T cells transfected with p(GRE)x3-AdMLP-VC5 were tracked over 24 hours after induction with 1 × 10-7M dexamethasone. Fluorescence measurements for single cells having induced over 3 standard deviations above the initial mean are displayed for (D) ECFP and (E) Venus. (F) Single-cell expression profiles normalized for ECFP signal. Darker lines represent the distribution of normalized cell intensities at 1 and 15 hours following treatment, illustrating cellular heterogeneity in the transcriptional response.
Single-cell tracking of inducing cells
The single-cell population endpoint measurements could also be obtained using other technologies, including two-color flow cytometry. A distinct advantage of the Living Microarray system is that expression changes in individual cells can be tracked over time by linking single cells in consecutive frames. We observed that fluorescent proteins tend to concentrate within sub-nuclear regions, such that each cell has a characteristic appearance that remains relatively constant between frames. We exploited this feature to track each cell over time based on its appearance and position (Additional file
These steps result in a set of tracked cells per field, where each track contains information about the cell's position, intensity and shape over time that can be visualized in many ways. For instance, each cell's position and intensity from a single field can be imaged as 3D trajectories inside a spot-centred space-time volume (Figure
The ability to track single cells allows us to extend population measurements by evaluating the dynamics with which a given cell will change its expression in response to a stimulus (Figure
Figure 5
Single-cell tracking identifies different profiles of transcriptional activation
Single-cell tracking identifies different profiles of transcriptional activation. 293T cells transfected with p(GRE)x3-AdMLP-VC5 were induced with 1 × 10-7M dexamethasone and tracked over 24 hours. (A, C) Tracks of cells inducing (A) and remaining unchanged (C) over 24 hours. Each cell track is represented by a line where each dot represents a different timepoint and the color describes the time of induction from 0 (blue) to 24 hours (red). The lower teal line represents the mean of the starting population, while the upper teal line shows the mean + 3 standard deviations. Single-cell time-lapse images are shown for cells that have similar starting ECFP expression levels, but whose response to dexamethasone is either high (B) or remains unchanged over time (D).
We have described the development of a high-throughput platform capable of measuring transcriptional dynamics for many genes in large numbers of single cells exposed to the same experimental conditions. Existing methods for measuring gene regulation, such as DNA microarrays and chromatin immunoprecipitation, have excelled at identifying members of transcriptional networks and the interactions that occur between them at specific points in time. However, these techniques acquire data from homogenized samples derived from populations of cells and cannot provide accurate temporal and spatial resolution at a scale appropriate to characterize time-dependent transcriptional responses
Here we used a high-throughput cell-based assay to specifically transfect hundreds of constructs into adherent cell lines and tracked each fluorescent cell over 24 hours. We adapted the technique to use reporter constructs, so that changes in transcription from different promoters could be assessed at single-cell resolution. In its current form, 600 spatially distinct clusters of cells can be specifically transfected in a single chamber. This removes many sources of well-to-well variability that are frequently associated with high-content screening, such as differences in seeding density, ligand concentration and temperature.
Using the ECFP channel provides an internal control that enables single-cell normalization of the target promoter activity levels without requiring co-transfection. Tracking uninduced cells was also possible using this channel, such that we could investigate each cell's history during the experiment. As with the Venus reporter, destabilizing the ECFP protein by fusing it to a PEST domain could provide a more sensitive method of detecting loss of the plasmid and relating this to variability in the Venus channel.
Despite identifying several cell lines suitable for our platform, we determined that a subset of lines (such as F442-A, AtT-20, MCF-7 and HepG2) either did not grow as monolayers or could not be transfected at high enough levels to be used (Additional file
The measurement of single-cell expression offers much potential and has been studied using other methods, such as the dynamic proteomics approach
The Living Microarray platform can be applied to many systems. Expanding the number of synthetic reporters could provide parallel measurements for the activities of dozens of transcription factors in response to various stimuli, as has been shown with pooled measurements of sequencing and luciferase-based reporters
Gene expression is an inherently stochastic process, both within single cells and among cells of a population, owing to the many sources of intrinsic and extrinsic noise
The advent of high-throughput methods for measuring changes in gene expression has facilitated the study of biological systems as a whole (i.e. gene interaction networks), rather than on the discovery and characterization of its individual components (i.e. genes)
Plasmid construction
A detailed description of the cloning steps involved in plasmid construction is contained in Additional file
Additional file 2
Supplementary Methods and Data (Plasmid Construction). Contains methods used to construct the plasmids used in the manuscript, including vector maps.
Click here for file
Reverse transfection
Transfection complexes containing reporter plasmid, transfection reagent and fibronectin were prepared as previously described
Standard transfection
Transfections were performed in triplicate in 6-well dishes (Corning, Lowell, MA) seeded the previous day with 1 × 106 293T cells grown in stripped serum. Transfection complexes were prepared with Lipofectamine 2000 (Invitrogen, Burlington, ON) as per the manufacturer's instructions, except that we used half the recommended amount of transfection reagent. Cells were treated with 1 × 10-7M dexamethasone (Sigma-Aldrich, Oakville, ON) and were incubated for 24 hours at 37 degrees before imaging.
Imaging setup
Imaging of the Living Microarray is performed using an inverted fluorescence microscope (TE2000, Nikon, Melville, NY) with a 20X objective (numerical aperture = 0.75) and an ultra stable close-loop feedback mercury light source (X-Cite exacte, EXFO, Mississauga, ON). The microscope is equipped with an incubator maintaining the cells in a humidified environment at 37°C and 10% CO2 (Solent Scientific, Segensworth, UK). The motorized stage includes encoders with accuracies of 0.5 μm and 0.02μm in the X/Y and Z axes, respectively (ProScan™II, Prior Scientific, Rockland, MA). Fast switching (<50 ms) excitation and emission filter wheels used in combination with a multiple band dichroic mirror (filter set 86006, Chroma, Bellows Falls, VT) allows for rapid switching between channels. Image capture is performed using a back-thinned electron multiplier CCD camera (C9100-12, Hamamatsu, Bridgewater, NJ). A custom adapter was designed to hold the Labtek Chamber slides onto the stage. The stage, filter wheels and camera are controlled using custom software based on the micro-manager package
Slide registration
Deviations in printed spot positions are detected by imaging each printed slide using a digital camera. After correcting the image for lens distortion (pincushion type) as previously described
Focus interpolation and acquisition settings
The focus positions of 45 spots evenly distributed across the array are manually determined. The focus positions (z) of the remaining spots are subsequently interpolated by fitting these 45 coordinates to a third degree polynomial surface:
We used RANSAC outlier detection for added robustness. After each pass of acquisition, these focus positions are corrected using a global offset estimated from autofocused features. The autofocus routine uses an autocorrelation-based focusing algorithm
In our experiments, each field was imaged once with the ECFP filter (gain: 90; exposure: 0.027 s) and at two different EYFP gain settings to maximize the dynamic range of the system (gain: 60, 120; exposure: 0.027 s). Each spot was therefore exposed for at most 150 ms, minimizing fluorescence-induced cytotoxicity. One pass of 600 spots took 20 minutes and cells were imaged in this manner for 24 hours in three independent replicate experiments.
Image Segmentation and Tracking
Watershed segmentation is prone to over-segmentation when applied to raw images. To avoid this we filter the raw images using a Fast-Fourier Transform in combination with a disk shape frequency domain filter
Cell tracking is accomplished using a two step approach. The first step (strict linking) links two cells from consecutive frames that are less than 10 pixels (8 μm) apart and that have similar appearances. We calculate the appearance score as the average squared difference between normalized cell images (between 0 and 1) and minimize this score by rotating the images relative to each other. The chosen score threshold where cell links are kept is 0.0155. In the cases where multiple putative links have acceptable distance and appearance changes, the link with the best score is chosen. No link is established if there are discrepancies in the scores.
The second phase (flexible linking) establishes potential links between tracks from the first step based on distance. We determine the distribution of intensity differences between potentially linked cells across the slide and remove links where this difference is greater than 3 standard deviations. Links with the smallest intensity differences are then kept. Additionally, to allow for transient loss of tracking, for instance because a cell leaves the field or is occluded by another cell, we iteratively consider links over five successive time frames.
Authors' contributions
SR conceived, designed and carried out all biological experiments and optimization, partly wrote the software for hardware control, generated some of the figures and wrote the manuscript. HD assembled and in some cases built the hardware, performed the microarray spotting, developed and implemented the routines relating to slide registration, focus interpolation and image analysis, generated some of the figures and edited the manuscript. HCPD cloned a subset of the reporter constructs and performed the Venus-dsRed validation experiments. RS and TJH contributed to the study design and provided supervision, intellectual contribution and manuscript revisions. All authors read and approved the final manuscript.
The authors would like to thank B. Bodmann (University of Houston), H. de Sterck and J. Wan (University of Waterloo) for their helpful discussions in image analysis, as well as M. Lemire (Ontario Institute for Cancer Research) for advice on statistical analysis. We also thank E. Nagoshi and U. Schibler (University of Geneva) for providing the Venus-NLS-PEST plasmid. This work was supported by Genome Canada, Genome Quebec, the Canadian Institutes for Health Research and the Ontario Institute for Cancer Research. SR is a recipient of a William Ian Mackenzie Turner Jr. Research Fellowship from the Research Institute of the McGill University Health Centre (MUHC). RS is a recipient of a Chercheur-boursier award from the Fonds de la Recherche en Santé du Québec and a 175th Anniversary Award from the Research Institute of the MUHC and the Montreal General Hospital Foundation. TJH is the recipient of a Senior Investigator Award from the Ontario Institute for Cancer Research, through generous support from the Ontario Ministry of Research and Innovation.
Transcriptional regulatory code of a eukaryotic genome
A systems approach to mapping DNA damage response pathways
Empirically controlled mapping of the Caenorhabditis elegans protein-protein interactome network
The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line
Microarrays of cells expressing defined cDNAs
Microarray transfection analysis of transcriptional regulation by cAMP-dependent protein kinase
Transfection microarray of human mesenchymal stem cells and on-chip siRNA gene knockdown
Freeze-drying of cationic polymer DNA complexes enables their long-term storage and reverse transfection of post-mitotic cells
Localized transfection on arrays of magnetic beads coated with PCR products
Microarrays of lentiviruses for gene function screens in immortalized and primary cells
A novel reverse transduction adenoviral array for the functional analysis of shRNA libraries
Atelocollagen-based gene transfer in cells allows high-throughput screening of gene functions
RNAi microarray analysis in cultured mammalian cells
Atelocollagenmediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo
High-throughput RNAi screening by time-lapse imaging of live human cells
An RNAi screening platform to identify secretion machinery in mammalian cells
Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes
A Reporter System for Reverse Transfection Cell Arrays
Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells
A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications
Generation of destabilized green fluorescent protein as a transcription reporter
Automated three dimensional image analysis methods for confocal microscopy
Bioluminescence imaging for assessment and normalization in transfected cell arrays
Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system
Use of the human EF-1alpha promoter for expression can significantly increase success in establishing stable cell lines with consistent expression: a study using the tetracycline-inducible system in human cancer cells
Ca2+ but not H2O2 modulates GRE-element activation by the human mineralocorticoid receptor in HEK cells
Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors
Imaging in systems biology
Quantitative microscopy and systems biology: seeing the whole picture
Dynamic Proteomics of Individual Cancer Cells in Response to a Drug
Cellular arrays for large-scale analysis of transcription factor activity
Transcriptional regulation during the mammalian cell cycle
Regulation of transcription by proteins that control the cell cycle
Transcriptional control by cell-cycle regulators: a review
Transcriptional regulation and function during the human cell cycle
Gene profiling of cell cycle progression through S-phase reveals sequential expression of genes required for DNA replication and nucleosome assembly
Identification of genes periodically expressed in the human cell cycle and their expression in tumors
Microarray analysis of gene expression during the cell cycle
Intrinsic and extrinsic contributions to stochasticity in gene expression
Stochastic modelling for quantitative description of heterogeneous biological systems
Transcriptome-wide noise controls lineage choice in mammalian progenitor cells
Regulated cell-to-cell variation in a cell-fate decision system
Dynamic patterns of growth hormone gene transcription in individual living pituitary cells
Dynamic Changes in Prolactin Promoter Activation in Individual Living Lactotrophic Cells
Dynamics of stimulus-expression coupling as revealed by monitoring of prolactin promoter-driven reporter activity in individual, living mammotropes
Dynamic organisation of prolactin gene expression in living pituitary tissue
Systems Biology and New Technologies Enable Predictive and Preventative Medicine
Systems Biology: A Brief Overview
Applying and removing lens distortion in post production
Autofocusing in computer microscopy: Selecting the optimal focus algorithm
Survey of image denoising techniques


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Sherwood Anderson: Short Stories(1962)
A collection of stories by Sherwood Anderson
Sherwood Anderson was cited by Hemingway, Fitzgerald, Cather, and others as a major influence in the writing of short stories. "Winesburg, Ohio" is a landmark work many of us encounter as students. Anderson, in many ways, is best experinced in audio. He believed stories were meant to be told, as they are in the stories themselves. These nine are his best: "Brothers", "The Door of the Trap", "The Dumb Man", "The Egg", "Motherhood", "The New Englander", "The Other Woman", "Seeds", and "War." He covers the great American themes of ambition, cheerfulness, big dreams that don't work out, feeling imprisoned by limitations one can't escape, and repressed sexuality - especially among women due to the code of conduct imposed upon them.
Used availability for Sherwood Anderson's Sherwood Anderson: Short Stories
See all available used copies of this book at Abebooks UK or Abebooks US
Paperback Editions
January 1962 : USA Paperback
|Title: Sherwood Anderson: Short Stories
Author(s): Sherwood Anderson
ISBN: 0-8090-0052-0 / 978-0-8090-0052-4 (USA edition)
Publisher: Hill & Wang
Availability: Amazon Amazon UK Amazon CA
Audio Editions
March 2012 : USA Audio edition
|Title: Sherwood Anderson Short Stories
Author(s): Sherwood Anderson
Publisher: Simply Media
Availability: Amazon
March 2012 : UK Audio edition
|Title: Sherwood Anderson Short Stories
Author(s): Sherwood Anderson
Publisher: Simply Media
Availability: Amazon UK
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Get an overview of this hotelWaterfront condo with an indoor pool
Popular property highlights
This Brigantine condo is on the beach, 1.7 mi (2.7 km) from Links at Brigantine Beach, and within 6 mi (10 km) of Senator Frank S. Farley State Marina and Delta Basin. Snug Harbor and Atlantic City Miniature Golf are also within 6 mi (10 km).
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Room AmenitiesInformation missing or incorrect? Tell us!Opens in a new window
Legacy Vacation Resorts Brigantine Beach
Condo Amenities
- Air conditioning
- Total number of rooms - 108
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- Bar/lounge
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- Indoor pool
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- Airport nearby
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Check-in time starts at 4 PM
Minimum check-in age is 21
Check-out time is 10 AM
Payment types
Children and extra beds
- Children are welcome.
- Kids stay free! Up to 6 children 18 years old and younger stay free when using existing bedding.
- Cribs (infant beds) are available.
- Pets not allowed
You need to know
Extra-person charges may apply and vary depending on property policy.
Government-issued photo identification and a credit card are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
No rollaway/extra beds available
- Deposit: USD 100.00 per stay
- Resort fee: USD 3.99 per accommodation, per night
Condo Name
- Legacy Vacation Resorts Beach
- Legacy Vacation Resorts Beach Condo Brigantine
- Legacy Vacation Resorts Brigantine Beach
- Celebrity Resort Brigantine Beach
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We should mention
Up to 6 children 18 years old and younger stay free when occupying the parent or guardian's room, using existing bedding.