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Internal/Actual Title: Integration Analyst City: Philadelphia State: Pennsylvania WHAT WE DO: Synovos is a rapidly growing leader and global provider of comprehensive supply chain services that drive increased productivity and profitability for our clients, including: Supply7/20/2016 Philadelphia, PA Save This Job
Location:United States - Houston Department:Engineering Operations Description Alert Logic, the leader in security and compliance solutions for the cloud, provides Security-as-a-Service for on-premises, cloud, and hybrid infrastructures, delivering deep security insight and8/22/2016 Houston, TX Save This Job
Location: Multiple CountriesType: Full TimeMin. Experience: Mid Level Sales Analyst, Data Analyst, Client Analytics Analyst, Sales Forecasting, Analyst, Business Requirements We are currently seeking applicants for the Professional Services Graduate Training that begins on7/29/2016 Altamonte Springs, FL Save This Job
Location: Corporate Office - Leesburg, VA Full Time Requisition Number: req258 Summary: Senior Business Analyst will be responsible for providing support to internal business units and project support requirements. They will provide innovative and creative solutions to support8/22/2016 Leesburg, VA Save This Job
Business Analyst (Groton, CT United States) Responsibilities Gather integration requirements and source data Responsible for facilitating small and medium projects from initiation to closure by working with internal and external teams Define and manage plans and timelines Manage7/23/2016 Groton, CT Save This Job
Business Analyst East Lansing, MI, United States Innovative, detail-oriented, and strategically minded? Business Analysts are part of our Product Management team and help envision the future and make those visions reality by understanding customer needs, staying close to7/27/2016 East Lansing, MI Save This Job
Location: Apple Valley, MNDepartment: QAType: Full TimeMin. Experience: Mid Level Quality Assurance Analyst ? Test Engineer Position Summary: The Quality Assurance Analyst-Test Engineer upholds and continually improves Questar?s Quality Assurance and Testing standards and7/9/2016 Apple Valley, MN Save This Job
? Location: Waltham, MA, United States ? Department: Sales Job Description: Overview The Business Analyst will be responsible for the requirements, testing and delivery processes for the team. This role will have responsibility for discovering, analyzing, and documenting6/27/2016 Waltham, MA, MA Save This Job
Chicago, IL Full Time Mid Level Overview Snapsheet is a mobile enterprise startup that is disrupting the outdated auto insurance claims process and turning a $120B market on its head. We have locked up contracts with top insurance carriers and are entering hyper-growth8/13/2016 Chicago, IL Save This Job
Department: IT Reports To: ERP Manager DESCRIPTION: Based in Houston, Texas, the Business Analyst will play a key role in the implementation and long-term development and support of RigNet?s new ERP System, SAP Business One, which will complete global launch in7/11/2016 Houston, TX Save This Job
Senior Security and Compliance Analyst? Help Please fill in the information below and use the navigational buttons to continue. Warning: Using your browser's Back and Forward buttons may produce undesirable results. Overview Challenging and rewarding opportunity available7/4/2016 Portsmouth, NH Save This Job
Client Service (CS) San Mateo, California Apply Description Client Service Analyst Customer Success Team San Mateo The BrightEdge Customer Success Team is committed to providing the highest level of support to our existing customers. As a Client Service Analyst at8/16/2016 San Mateo, CA Save This Job
Category: Domestic Minimum Experience: 4+ years Required Education: Bachelors Degree Job Description AGI?s team delivers high quality orbit data and safety of flight information and supports and benefits customers through the integration, implementation, and use of AGI?s7/12/2016 Colorado Springs, CO Save This Job
Category: Domestic Minimum Experience: 3 - 5 years Required Education: Bachelors Degree Job Description AGI?s team delivers high quality orbit data and safety of flight information and supports and benefits customers through the integration, implementation, and use of AGI?s7/12/2016 Long Beach, CA Save This Job
AT MEDSYNERGIES, INC. Date Posted: 7/26/2016 APPLY Not ready to Apply? JOB DESCRIPTION Business Process Analyst, Senior The Business Process Analyst, Senior will perform analysis of business goals, objectives, and needs of the general business environment. In this role, you7/28/2016 Irving, TX Save This Job
Job description At Empirix the Oracle Business Systems Analyst manages the functional usage, modification and configuration of the Oracle EBS Application portfolio, collaborates with Business Systems Management on corporate strategic and operational initiatives and coordinate7/14/2016 Billerica, MA Save This Job
Unifi is the leading data acquisition & data integration platform that enables the businesses to acquire data, integrate it, enrich it, and load it into a flexible environment where agile and iterative analytics can occur. In order to support a highly available, scalable and6/29/2016 San Mateo, CA Save This Job
Product Support East Lansing, MI, United States Vertafore is looking for professionals interested in joining a high performing technology company located right here in mid-Michigan. Our customer teams have the opportunity to work with emerging technologies in a customer8/26/2016 East Lansing, MI Save This Job
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COMPANY PROFILE: InstaMed is a rapidly growing healthcare technology company that has established itself as a leader in the emerging market of healthcare payments. InstaMed?s solutions are delivered via its proprietary cloud-based platform and network of healthcare providers and8/13/2016 Philadelphia, PA Save This Job


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Boxing Forum - View Single Post - PBF will lose court case. Cant criticise a man who puts his life on line?
PBF will lose court case. Cant criticise a man who puts his life on line?
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Originally Posted by
it's not because the *****s are willing to be floyd's ***** means that Manny has to as well!
if you can't see pac winning this case then that is your opinion! who are you to say. Do you think Manny, considered as National Treasure in his country will not have the best legal advise possible? As far as I know everyone has been telling him to do something but initially he didn't want to glorify the issue but one can only take so much.
As his fans, we just want him to take a stand and not let himself to be ridiculed and maligned by these people. This case is long overdue!
For the record, Manny will take the test if it was demanded by the Nevada Athletic Commission not by Mayweather Athletic Commission. Who the hell is floyd? They're both boxers for goodness sake
Did manny say anything about the random test ordered by NSAC yesterday? NO, he was more than happy to oblige. I can guarantee you thaf if Floyd asked for it, Manny would have told him where to go!
way to go bradah!
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LaRue County sophomore Cameron Dawson finished first out of 93 golfers Tuesday to capture the 5th Region Golf Tourney at Elizabethtown Country Club with an even par 72. In 2013 he walked away with a region runner-up at South Park Golf Course in Louisville, therefore he was one of six individuals expected to win this year's tourney.
Cameron is the first LaRue County golfer to win the region since Chad Hines did it in 1997. With the tournament win, Dawson qualified for the KHSAA State Golf Tourney in back-to-back seasons.
After finding ways to lose three straight weeks, the LaRue County Hawks found a way to win. On the road, no less.
The Hawks kept the Russell County Lakers scoreless on all eight of their possessions and senior Koy Lindsey's fumble recovery on the Lakers' final offensive play gave the Hawks three turnovers as LaRue County held on, 14-10.
Junior running back Marquis Franklin broke the LaRue County Hawks Football record of longest play from scrimmage when he was able to take the ball 99 yards on the first play from scrimmage Sept. 26 at Marion County. The previous record of 96 yards from scrimmage was held by Chris Singleton and Justin Thurman during the 2005 season. Franklin also now shares the 99-yard record with 16 other players from across the state since 1972.
The LaRue County Lady Hawks inched closer to the .500 mark with a 3-0 win (25-7, 25-22, 25-8) win over visiting 18th District rival Hart County on Sept. 29.
Sophomore outside hitter Cidney Neagle led the Lady Hawks (12-13, 4-1 district) with 10 kills, four aces, one block and one dig, while junior middle hitter Chloe Owen added seven aces, six digs and five kills.
LaRue County sophomore Cameron Dawson's day at the Boys' 5th Region Golf Tournament didn't get off to a good start, but he couldn't have envisioned a better ending.
Dawson rebounded from a bogey on his first hole to fire an even-par 72 to win the tournament Sept. 30 at Elizabethtown Country Club. He became the first LaRue County golfer to win the region since Chad Hines in 1997.
LaRue 2 sets, Marion County 3 (25-22, 18-25, 20-25, 25-15, 13-15)
Keeahnna Bowen - 2 kills, 37 assists, 1 ace, 3 digs
Cidney Neagle - 8 kills, 2 digs,
Chloe Owen - 11 kills, 1 ace, 6 digs, 1 block
Kylie Rogers - 1 assist, 3 digs
Alyson Yingling - 5 kills, 4 aces, 6 digs, 3 blocks
Tessa Yingling - 9 kills, 3 aces, 6 digs,
Kenzie Bradley - 2 kills, 1 dig
Breea Kirkpatrick - 1 dig
LaRue 3 sets, Caverna 0 (25-9, 25-7, 25-5)
Girl's varsity
113 runners
Kristina Durbin, 21, 22:17
Alexis Grimes, 48, 24:01
Clair Keller, 64, 24:44
Sophe Pepper, 84, 25:53
Amanda Brewer, 93, 26:54
Samantha Martin, 98, 27:05
Celeste Menard, 111, 32:51
12th of 15 teams
Boys Varsity
131 runners
Jake Cecil, 75, 20:10
Christian Silva, 92, 21:28
William Pearman, 94, 21:39
Stiles Metcalf, 101, 22:03
The Hawks lost to the Marion County Knights Friday, 34-41.
On the Hawks' first play of the night, junior running back Marquis Franklin matched the school record for longest play from scrimmage with a 99-yard touchdown run. He finished with 143 yards on 17 carries.
The Hawks were down 33-20 at the half but took the lead 34-33 in the third quarter. Marion County scored again in the fourth quarter for the win.
LaRue 34, Marion County 41
Scoring by Quarters
LaRue 12 8 14 0.............34
LaRue 1, Grayson 0
The LaRue County Boys' Soccer team lost to Grayson in the championship game in the Trojan Classic in Glasgow last weekend. Both teams had strong defensive lines and were able to remain scoreless at the half.
Riley Whitaker, Trevor Cecil, Timmy Sutherland, and Hunter Pearman were able to shut down Grayson attempts by being fast to the ball and with precise placement of sends.
Copyright © 2016
The LaRue County Herald is your source for local news, sports, events and information in LaRue County, KY, and the surrounding area.


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I just wanted to take this opportunity to thank you and your team for the great job you've done on my windows and doors. I am very very pleased with the results. Your installers were very kind and courteous and did an excellent job, they were highly skilled and efficient and I was very impressed that they cleaned up after themselves. I will most definitely recommend Heritage to friends and family and the other condo owners with our corporation.Read more
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Heritage Renovations have completed their work in good manner and our tenants have been very pleasedRead more
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They have always dealt with us with the highest integrity and ethical standards.Read more
North Star Vinyl Windows & Doors


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The Invisible Hand
The Mad Bomber
View Member Profile
Sep 12 2011, 11:47 AM
Group: Members
Posts: 440
Joined: 12-March 07
From: My Battlefield!
Member No.: 45,405
Gender: Male
Another one of my creations, hopefully this one won`t end up as a bad. It`s steampunk with airships and what not, the world is a fictional one with one huge land mass flanked by two other continents. You can make up places yourselves, if you want your characters to come from a certain type of places. Magic can also exist, if you want it to, it really makes no huge impact on the overall setting. I will be using some names etc influenced by european history, culture in describing the Imperium. Your characters can come from all walks of life as long as they have talents making them useful to the efforts of the group, they are apart of. The empire is inspired by the German/Russian empires.
On the continent of Eurepa, the main continent, The Rechsal Empire is the most dominant nation to have emerged after almost a hundred years of bloodshed. It`s armies, technology far superior to those of it`s enemies and the empire holds the rest of the remaining nations in a noose on the continent. Most notable of these Venek, Frank and Polsik Kingdoms, who share a third of the continent amongst themselves. Yet even now the empire is engaged in a bloody war with the Franks, which has ground to a halt with no clear winner. Political tensions are rising as The Prime Chancellor of the empire calls for a truce, but the emperor refuses these suggestions. Foreign nations look on worried at the situation yet also find it an opportunity because of the sudden falt of the imperial advance, even the nations on the other continents, Aesika, Kolombia and even the relatively small island nation of Britannica have theirs set on the events at the main continent. With the war going poorly the executive commanders of the armed forces have opted to engage their enemies in a different kind of war, one of agents and assasinations.
By secret imperial degree Colonel Anton Scheller was appointed in charge of these operations, forming the imperium`s first assasination squad known as `Invisible Hand`. Scheller scoured across the imperium to assemble the most talented agents available, tough some are considered utterly ruthless, of questionable loyalties and beyond redemption by the courts. Scheller, taking command of the Regina Caelorum(Queen Of Heaven) a warship disguised as a large merchant vessel gather his band of miscreants to engage the enemies of the empire in what would be an underhanded war.
1. NO ONE-LINERS (no one liners!!! NEVER!!!)
2. Blood, death, violence and swearing allowed, but let`s keep it within reasonable limits as always.
3. Romance is fine, just nothing too sexual.
4. Be serious, people; tough a comedic relief once in a while is appriciated...
5. No god-moding.
6. No controlling other RPers characters.
7. No killing another character without permission from that character's respectful RPer.
8. To improve the depth of the characters, make sure to think of a backstory that fits the setting, how the chaos and death has shook your characters life etc. Would be great if someone even made suggestions to me about my character having met your character in the past or somehow having been involved in their life.
9. Have fun!
Regina Caelorum
1. The main idea is that our characters are a part of the Invisible Hand, a team of gifted people (a very relative term) fighting an underhanded war, but if you want to have a character outside the team etc then contact me and we`ll discuss it.
2. Your character can be anything well within the realm of steampunk... Or well... as long as they prove useful to the team in some manner, they don`t even have to be a combat expert nor have any knowledge in that area.
3. Everyone has a past, you can either reveal some of it straight off the bat or bring it along as the rp progresses, but some basic facts are always nice.
Abel Cromwell
Former Profession:
Moderately skilled swordsman, extensive knowledge of the criminal underworld and contacts scattered across the land.
Pistol sword(flintlock) as his primary armament, a stiletto as his sidearm and a small collection of smoke bombs and other tools, that help him in distracting or slowing his opponents.
Blunt, selfish, exploitative and dishonest to boot.
Is able to take on problems on as they come(tough he tends to get frustrated or even scared), logical-thinker and suprisingly care-free.
Cowardly, impatient, untrustworthy.
Abel Cromwell surfaced to the awareness of the imperial authorities, in the northern city of Holm, for having commited a theft and was eventually sentenced for a few months as there was very little proof to support, that he had actually stolen anything. Having served his months Cromwell disappeared from Holm only to appear three monts later aboard a merchant vessel at the other corner of the empire, serving as a crewmember of
The Daring
, whose reputation was stained as Cromwell`s. From that day forth Cromwell sat under the learning tree of his fellow smugglers aboard
The Daring
as they made runs all over the empire, even few across the ocean to the other continents. Managing to avoid imperial authorities for a number of years,
The Daring
was finally caught up by the imperial navy whilst carrying contraband from across the sea.
The smugglers made a run for it, but were mercilessly pounded by the imperial navy and sent crashing into the countryside. The survivors were rounded up and sent to be executed at the nearest town, Cromwell however was separated from the others just before the appointed hour and brought before Anton Scheller. It appeared, that Cromwell`s luck had not run out after all as Scheller was in fact looking someone with knowledge of covertly making runs in unfriendly territory without being targeted. Cromwell was immidiately puzzled by the fact as to why he was chosen, but chose to accept the deal without another word, choosing not to end up at the end of a noose.
I am love, I am hate, I'm an atheist with faith, Vent my anger with a smile
The Mad Bomber
View Member Profile
Oct 7 2011, 08:57 AM
Group: Members
Posts: 440
Joined: 12-March 07
From: My Battlefield!
Member No.: 45,405
Gender: Male
Before the gunshots were fired, Abel was pre-occupied with the wounded guard, who made an effort to take down his assailant. Abel was forced make split second dodge, tumbling to the floor while the guard got tripped by Abel`s feet and was sent crashing admist the crowd. The two gunshots pierced his hearing and he could only hope, that those guns belonged to Marcus. Charlotte yanking him by his arm, helped him stumble back onto his feet and to his noticable relief she had got a hold of Ghredal. Abel agreed wholeheartedly with her suggestion and did not waste a heartbeat before starting to make his way towards the back door. Struggling through the last of the crowd, Abel pushed his way to the back door and found that it lead to an abandoned kitchen, which had a direct exit to the alley. Abel lead Charlotte and their newfound `friend` out of the building to the alley.
Upon emerging outside, Abel was still clutching to his bloodied switchblade, in fact he was grasping to it so hard his knuckles had turned white. The fresh air did nothing for his pounding heart, but at least they did not stumble onto more guard, which Abel thought was either lucky or suspicious. Discarding the irrelevant thoughts, Abel glanced at Ghredal, who seemed at least grateful, but seemed to be expressing a healthy degree of paranoia.
"As you realize Mr.Ghredal we have limited time and pursuers, we need a safe place to lay low, the lengthy explenation as to why we are helping you can wait for later. Unless you prefer we continue this conversation in a jail?"
Ghredal nodded before glancing back at the kitchen door, but as none of his compatriots emerged, he chose to defer to Abel`s lead. Abel nodded, satisfied, that the man did not choose to difficult and seemed to have some degree of intellegence. The trio disappeared to the back alleys of the city, heading for a hotel where Abel had arranged rooms for them.
Having safely made their escape and made sure they were not being tailed, Ghredal`s demeanor seemed to become slightly more aggressive, but he did not say a word as they entered the hotel of Abel`s choosing. This particular hotel offered privacy and service, no questions asked, mainly due to it`s nature as a base of one dominant criminal in the city. Upon arriving in the room Abel had rented for himself, he cast aside his cap and sat down at chair by a small coffee table. The room was unspectacular at best, having only the necessities, but it was far from being uncomfortable. Ghredal glanced around the room, nervously, then at Abel as he motioned for him to have a seat.
"Abel Cromwell at your service." Abel said and added a small theatherical bow into his introduction, his face adorned with an amused expression. "You have questions no doubt Mr. Ghredal? Why am I sticking my neck out to help you? Well as you must have discerned thus far, I know things including every little detail of your life and most importantly, that you are in fact a rebel recruiter for this part of the city."
Ghredal did not seem at all impressed as he took a seat nor threatened by Abel`s smug attitude, he merely glanced at Charlotte before turning to Abel, apparently the man had already formulated an opinion of the two.
"You two are criminals." He pointed out accusingly, knowing fully well what kind of people the owners and customers of the hotel were.
"Would that not indicate you one as well?" Abel rebuked the claim with an accusation of his own.
"That`s not how I see it." Ghredal stubbornly argued.
"Nor do we." Abel said with smirk flashing across his face.
"Then pray tell, what might you be?" Ghredal inquired raising his eyebrows and turning to look at Charlotte, which Abel to turn to her as well for an answer, hoping she could present herself as a rebellious idealist or something akin to that.
As the chaos subsied, the guard squad leader, a tall man with dark hair approached Marcus, who had been spared from brutality. The guards would have had a tougher time taking down the brawlers had those shots not been fired so they owned him in a manner of speaking.
"First off I appriciate what you did for us.... secondly I`d like to know who you are and what you are doing here. Your clothing is hardly commonplace and neither are those pistols, they don`t seem exactly cheap."
The man speculated with a discerning voice.
I am love, I am hate, I'm an atheist with faith, Vent my anger with a smile
Posts in this topic
The Mad Bomber
The Invisible Hand
Sep 12 2011, 11:47 AM
Sorry for the delay, I'll post my profile as s...
Sep 14 2011, 09:58 AM
(I'm sorry!! I know they're sucky ...
Sep 16 2011, 10:00 AM
The Mad Bomber
(This is a bit of a prequel, from my characters vi...
Sep 16 2011, 01:44 PM
((Sorry it took a while to post. I've been pre...
Sep 18 2011, 06:29 PM
The Mad Bomber
(We are kicking off, those that still are going to...
Sep 22 2011, 06:20 AM
"Rotten luck? Aint that an interesting thing ...
Sep 22 2011, 05:54 PM
The Mad Bomber
Abel didn`t bother reading any deeper into her...
Sep 23 2011, 02:34 AM
"Charlotte" She mumbled, almost as if he...
Sep 23 2011, 09:36 AM
The Mad Bomber
"A charlatan?" Abel spoke, as soon as h...
Sep 23 2011, 10:13 AM
The awkward silence that followed his comment was ...
Sep 23 2011, 08:09 PM
The Mad Bomber
"Well getting a sense of humour would be firs...
Sep 23 2011, 11:44 PM
"I've no sense of humor that I'm awar...
Sep 25 2011, 11:49 AM
The Mad Bomber
"The rebels or the local garrison, not that e...
Sep 25 2011, 12:34 PM
But instead of looking insulted, Marcus actually l...
Sep 25 2011, 03:13 PM
The Mad Bomber
Abel was able to take little comfort in the fact, ...
Sep 27 2011, 06:02 AM
"A nudge? We could drop a pin and these peopl...
Oct 6 2011, 07:45 PM
The Mad Bomber
The proverbial pin drop manifested itself in the f...
Oct 7 2011, 02:29 AM
Suddenly there was a loud crash, and wood splinter...
Oct 7 2011, 07:38 AM
The Mad Bomber
Before the gunshots were fired, Abel was pre-occup...
Oct 7 2011, 08:57 AM
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New game, so new gaurds. Basically, all we know is tha drummers act as "the brain" of patrols, that gaurds will have long reloading times and will use bayonets in combat. So, obviously, this raises some questions. 1. Are gaurds still progress from fodder to mediocre to decent, or will a bullet be devastating for conner? 2. Will AC2 gaurd types return? 3. Beside guns and bayonetts, will soldiers use swords? 3. Will connor only use the weapons shown in the specific trailer or will he get swords/tomahawks/axes etc. from shops. 4. Do gaurds have a central leader besides the drummer? Is the leader on foot, carry stronger armor or weapons, or on horseback. 5. Does health sync up like AC1 or AC2?
animated .gifs and youtube videos do not make a conversation, but yes, there will be guard archetypes
Your signature isn't there in the OP but it's there in your reply to Lonesoldier...
Proud member of the UUSDG
How I got my name: http://www.youtube.com/watch?v=wG_R5n9yIPg
yes, because making scottish people the big heavy brutes isn't stereotypical at all...
i believe that if life gives you lemons, you should make lemonade... And try to find somebody who's life has given them vodka, and have a party.
LOL @ that video. The Scots WERE big heavy brutes. That's why the English used them on the front lines. So don't be surprised If some Big Scotsman In a Kilt runs at you, wanting to plant an axe In your face


text:
Epigenetics in plant tissue culture
- First Online:
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DOI: 10.1007/s10725-010-9531-4
- Cite this article as:
- Smulders, M.J.M. & de Klerk, G.J. Plant Growth Regul (2011) 63: 137. doi:10.1007/s10725-010-9531-4
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Plants produced vegetatively in tissue culture may differ from the plants from which they have been derived. Two major classes of off-types occur: genetic ones and epigenetic ones. This review is about epigenetic aberrations. We discuss recent studies that have uncovered epigenetic modifications at the molecular level, viz., changes in DNA methylation and alterations of histone methylation or acetylation. Various studies have been carried out with animals, and with plant cells or tissues that have grown in tissue culture but only little work has been done with shoots generated by axillary branching. We present various molecular methods that are being used to measure epigenetic variation. In micropropagated plants mostly differences in DNA methylation have been examined. Epigenetic changes are thought to underlie various well-known tissue-culture phenomena including rejuvenation, habituation, and morphological changes such as flower abnormalities, bushiness, and tumorous outgrowths in, among others, oil palm, gerbera, Zantedeschia and rhododendron.
Genetic changes (also referred to as somaclonal variation): changes in the DNA sequence;
Epigenetic variation: long-lasting changes in the expression of the information in the genome;
And—less relevant for this review—chimeral segregation and loss of pathogens, in particular viruses.
It is characteristic for epigenetic changes that in a population of generated plants the same aberration occurs at high frequency and can be 'reproduced' when the same conditions are imposed during the production of another population (Smulders et al. 1995). In contrast, genetic changes occur at random. It should be noted that in a somaclonal population the same genetic change may be found in many individuals but then the mutational event happened early during the tissue culture period and was clonally multiplied into a number of descendants (Brettell and Dennis 1991; Kohli et al. 2004).
Characteristics of epigenetic changes
Epigenetic mechanisms include DNA methylation and modifications of amino acids in the tails of histones. Histones are proteins that package the DNA into nucleosomes and make up the chromatin in nuclei. DNA methylation and histone modifications interact and reinforce one another. In this way, transcription can be enhanced, for example, by acetylation of the lysine on position 9 of histone 3 ('H3K9'), which is found in promotor regions of actively transcribed genes. Transcription can also be decreased, for example, by di- and trimethylation of H3K9, which is a histone mark of repeat elements in the DNA (Bernatavichute et al. 2008; Tessadori et al. 2007). Repeat elements are also characterized by methylation of cytosines in the DNA.
This regulatory system has several functions. On the one hand, it is a defence mechanism against retrotransposons. Silencing of repeats is thought to occur in all cells, regardless of their location or developmental state. It is targeted by 21- and 24-mer short RNAs. Intriguingly, the same mechanism contributes to sustain the centromere through silencing and compacting the centromeric repeats (Tessadori et al. 2007). On the other hand, epigenetic mechanisms regulate gene expression during development: genes may be methylated and switched off in some tissues, but demethylated and active in others (see Matzke et al. 2009 for a description of the mechanism and the genes involved). The clearest examples of this type of epigenetic regulation are found in mammalian embryogenesis: imprinting (where the activity of a gene in early embryo development depends on whether it is inherited from the father or the mother), X-inactivation (to achieve the proper gene dosage, one of the two X chromosomes in females is heavily methylated and transcription is largely switched off), and reprogramming (of genome-wide DNA methylation patterns in the gastrula stage). Imprinting also occurs during plant endosperm development (Hsieh et al. 2009). When 'genes' are said to be methylated and transcriptionally silenced, it is actually the promotor region that becomes methylated, as even actively transcribed genes may be methylated in the 'body' of the coding region, i.e., at some distance from the 5′ and 3′ end (Roudier et al. 2009; Tanurdzic et al. 2008).
During plant development, the epigenetic imprints provide a transient 'memory' of the developmental program and influence the cell's reactions and its future development (Roudier et al. 2009). A notable example of the memory function is vernalization, the acquisition or acceleration of the ability to flower by a chilling treatment. In Arabidopsis, vernalization involves the silencing of a group of flowering repressor genes, including Flowering Locus C (FLC; Bastow et al. 2004; Roudier et al. 2009). Silencing occurs during a cold period through an epigenetic change, viz., through histone modifications (Bastow et al. 2004; Kim et al. 2009). Interestingly, in barley it is mediated by different histone modifications at the floral activator gene VERNALIZATION1 (VRN1), allowing it to become active (Oliver et al. 2009) Another example is the phase change from juvenile to adult, which is paralleled by changes in gross DNA methylation (Valledor et al. 2007).
De Klerk (1990) and Va´zquez (2001), amongst others, noted that the distinction between genetic and epigenetic changes is an oversimplification. Molecular evidence has accumulated showing that epigenetic changes may lead to many genetic abnormalities. The transposable element Activator (Ac) in maize looses DNA methylation and becomes activated upon dedifferentiation (Brettell and Dennis 1991). The same occurs in transgenic rice plants containing Ac from maize (Kohli et al. 2004). In maize, the activation is passed on for two sexual generations (Brettell and Dennis 1991). The miniature inverted repeat transposable element (MITE) mPing becomes activated in rice, e.g., in callus culture, which is correlated with alterations in cytosine methylation (Ngezahayo et al. 2009). The retroelement Tos17 becomes active in rice suspension cultures, supposedly as a consequence of changes in the epigenetic modifications that silence it. As a result, plants regenerated from these cultures have many new copies inserted in their genome (Hirochika et al. 1996). The endogenous chromovirus LORE1a looses DNA methylation in dedifferentiated cells of Lotus japonicus, so that it can become active and transpose after regeneration. Strikingly, it only does so during pollen formation (Fukai et al. 2010). Insertions of transposable elements and retrotransposons can serve as insertional mutagens of plant genomes, whereas widespread activation can lead to a wide array of chromosomal rearrangements (Tanurdzic et al. 2008). These rearrangements can, in turn, lead to misregulation of genes, aneuploidy and new transposon insertions (Lee and Phillips 1988).
Detection of epigenetic status
For an accurate evaluation of the epigenetic status, both DNA methylation and chromatin modifications should be considered, and this should be done for particular genomic regions. Differences in genome-wide levels of DNA methylation are not very informative, as there can be massive local changes whereas overall levels are very similar. Measurement of the methylation of randomly chosen cytosines is inadequate since many changes may occur in DNA with no specific function. In addition, the methylation pattern of a stretch of DNA is important, usually not that of any particular cytosine by itself. As a consequence, changes in random sequences do not correlate with particular morphological or physiological abnormalities observed in regenerants (Jaligot et al. 2002; Smulders et al. 1995). Possible correlations between the amount of changes at the molecular level and the extent of abnormalities may be established by studying many more sites. Technically, such research is becoming possible. However, if specific stretches of DNA, e.g., in a particular promotor, affect the expression of a specific gene that leads to one type of change, an estimation of the overall level of variation would not necessarily provide a strong correlation with the specific abnormality under study.
It may be easier to study changes that occur repeatedly as the correlation of certain changes in DNA and histone modifications with the phenotypic changes can be determined and used as a selection criterion. In cancer research, this yields interesting results (certain aberrantly methylated CpG islands in the genome are biomarkers for several types of cancer). The progression of a cancer goes through several specific stages, including an initial step of massive DNA demethylation and subsequent selection for cell lines that manage to establish themselves. The changes induced by adventitious regeneration may not be so predictable, and the selection for abnormal lines not so severe, although examples exist where certain types of abnormalities occur at a relatively high level.
The methods to assess epigenetic status, either DNA methylation or histone modifications, are quickly developing (Fraga and Esteller 2002; Laird 2010; Vogel et al. 2009). For DNA methylation detection, various methods and protocols exist, including bisulfite treatment of DNA changes, which changes unmethylated cytosines into uracil but does not change methylated cytosines, and amplification after digestion of DNA with methylation-sensitive restriction enzymes (Gonzalgo and Jones 1997; Xiong and Laird 1997). Some researchers use antibodies against 5-methylcytosine (Barton et al. 2001). The methylation-dependent restriction enzyme McrBC can be used to separate methylated from non-methylated DNA, followed by detection using whole genome microarray hybridisation (Vaughn et al. 2007). Bisulfite-treated DNA can be sequenced and compared to untreated DNA, thus producing detailed information on the methylation of cytosines on both strands of the DNA (Krizova et al. 2009; Zhang et al. 2009). Histone modifications are studied using chromatin immunoprecipitation (ChIP) of associated DNA followed by polymerase chain reaction (PCR) of cDNA or whole genome microarray hybridisation (Matarazzo et al. 2004; Roh et al. 2004; Shi et al. 2003). In this way, many known sites can be probed simultaneously.
Using a combination of these approaches a whole genome analysis of chromosome 4 of Arabidopsis thaliana was done on tiling microarrays by Tanurdzic et al. (2008). They generated detailed patterns of both DNA methylation and various modifications of the associated chromatin. They compared the epigenetic patterns of an immortalised cell suspension of Arabidopsis thaliana with that of the leaf cells from which the culture had been started. The heterochromatin had undergone dramatic DNA hypomethylation, which led to the transcriptional activation of certain transposable elements in culture, while other types of elements remained silent. The elements activated in culture had increased levels of 21-nucleotide small interfering RNAs, indicating interplay of RNA interference and histone modifications in epigenetic restructuring of the genome. Euchromatin, in which active genes reside, was hypermethylated in culture, but only a small percentage of genes obtained heterochromatic marks, which might lead to long-term silencing.
Krizova et al. (2009) studied the fate of an epigenetically silenced LO1 transgenic allele in tobacco taken through cell culture and regeneration from callus. They observed partial loss of Lo1E 35S promoter methylation compared with leaf tissue in the parent plant. In comparison, when starting with plants in which the promotor was not silenced, they observed an increase in promotor methylation in some regenerated plants. The data indicate that in the cell culture process the parental imprinting state was gradually lost. Bisulfite sequencing of the promoter in callus revealed molecules with no, intermediate, and high levels of methylation, demonstrating cell-to-cell methylation diversity in callus. Among regenerated plants the DNA methylation pattern was highly variable, but within a regenerated plant most cells were very similar in DNA methylation, indicating that the callus-induced epiallelic variants were transmitted to plants and had become fixed (Peredo et al. 2006). Dedifferentiation in cell suspensions and calluses of Arabidopsis thaliana led to hypermethylation of promoters of various genes. This hypermethylation in undifferentiated cells was associated with histone hypoacetylation (Berdasco et al. 2008). These changes were associated with transcriptional silencing of linked genes.
In the near future, next-generation sequencing will enable studying the detailed pattern of DNA methylation by sequencing bisulfite-treated DNA (Hsieh et al. 2009) or studying the effect of a treatment on histone modifications by comparing the DNA sequences associated with immunoprecipitated histones. Flusberg et al. (2010) managed to detect DNA methylation directly using single-molecule, real-time sequencing. These methods will be used to produce detailed overviews of epigenetic modifications among tissues and between developmental stages, in a variety of species. For a better understanding of the tissue culture process, the challenge will be to focus on relevant regions in the genome and to link the changes to the processes of dedifferentiation and differentiation. This may require sampling and studying time series of plant material while they proceed through the tissue culture process, as mature plants, and into future generations.
In tissue culture, adventitious regeneration is one of the main developmental pathways to obtain new plants. It is based on the ability of plants to redirect development (De Klerk 2009). To regain totipotency (the capability to generate a complete new plant) or pluripotency (the capability to generate an organ such as a root or a shoot), cells erase at least a part of the previous epigenetic marks. This process is called dedifferentiation. Accordingly, differences in regeneration capacity between genotypes may be brought about by differences in how fast and how easily epigenetic marks can be erased or reprogrammed. It has been hypothesised that cell type, developmental age, and physical age may affect the speed of reprogramming, and the accuracy of the process (Grafi 2004). Thus, epigenetic changes in adventitiously regenerated plants may be caused by incorrect, possibly incomplete, resetting of the epigenetic developmental program, much like what causes abnormalities in cloned mammals. Unfortunately, the dedifferentiation process is still not well understood. Dedifferentiation of specialized mesophyll cells of Arabidopsis thaliana into undifferentiated protoplasts leads to a dramatic reduction of heterochromatin with decondensation of all major repeats regions except the 45s rDNA repeat (Tessadori et al. 2007). DNA methylation and histone H3K9 dimethylation do not change during the decondensation. The decondensation process is reversed in a stepwise way upon prolonged culturing. Tanurdzic et al. (2008) observed hypermethylation of euchromatic DNA in a long-term cell suspension of Arabidopsis thaliana that had lost the capacity to readily regenerate shoots. Law and Suttle (2005) observed rapid and reversible changes in DNA methylation and histone modifications in the course of 15 days after establishment of a potato cell suspension.
Tanurdzic et al. (2008) proposed that dedifferentiation initially involves the loss of epigenetic marks. Accordingly, the expression of maintenance DNA methylases, such as MET1 in Arabidopsis thaliana, is lower during the initial phase of a cell suspension culture. Subsequently, new epigenetic marks need to be established. Indeed, the expression of de novo DNA methylases and various histone modifying enzymes such as histone deacetylases (HDACs) is generally increased in tissue culture, while that of DNA demethylase ROS1 is lowered (Law and Suttle 2005; Tanurdzic et al. 2008). Mistargeted de novo methylation would allow the establishment and perpetuation of DNA hypermethylation of promoters, which would create epialleles, and hypomethylation of repetitive DNA, which may lead to activation of mobile elements. Krizova et al. (2009) examined the development of a tobacco cell culture into callus followed by plant regeneration. They observed the loss of parental imprinting in 25% of the regenerated tobacco plants, regardless of whether the epiallele was originally methylated or unmethylated.
In micropropagation companies, the most frequently used tissue culture procedure involves inducing outgrowth of existing axillary buds by exogenous application of cytokinin or removal of the apical bud. It is generally believed that in this way very little or no genetic variation is induced (De Klerk 1990). Peredo et al. (2009) evaluated hop cultures with molecular techniques namely random amplified DNA polymorphism (RAPD), retrotransposon microsatellite amplified polymorphism (REMAP), and methylation-sensitive amplification polymorphism (MSAP). They did not detect any genetic variation between the original plants and propagated plants even after 12 cycles (2 years) of micropropagation. It should be noted that with molecular methods only a very small portion of the genome is screened, so there may actually still be abundant genetic changes. Epigenetic changes, though, were easily detected in these plants with molecular methods. Nearly 30% of the changes present in the polymorphic loci were shared by all in vitro plants. Lower levels of epigenetic variation were detected among plants from the different subcultures. The most common difference between field plants and in vitro plants was demethylation of cytosines in some restriction targets, representing 63% of the observed changes. Smýkal et al. (2007) used various molecular methods to assess the genetic stability of long-term (24 years) propagated shoot cultures of pea. No differences were found using microsatellite, inter-simple sequence repeat (ISSR) or inter-retrotransposon amplified polymorphism (IRAP) markers. However, amplification fragment length polymorphism (AFLP) and particularly methylation-sensitive amplification polymorphism (MSAP) detected 11 and 18% polymorphism, respectively. An examination of the global cytosine methylation status revealed no significant differences. Zeng et al. (2009) examined integration and expression level of transgenes during long-term micropropagation of transgenic birch. There was a stable integration of transgenes into regenerated plants, but expression levels of the transgenes among shoots subcultured 4, 7, 9 or 15 times were significantly different: the transcriptional expression level of transgenes decreased with increasing subculture number. The silencing rate of beta-glucuronidase (GUS) in the fifth subculture plants was 22–65%. 5-Azacytidine, a drug that causes global hypomethylation, reactivated transgene expression at 50–200 μM, indicating that the decrease in expression level with increasing number of subcultures was associated with DNA methylation.
Epigenetic changes of plants propagated in vitro: rejuvenation and habituation
During their lifetime, plants pass through a developmental process referred to as 'phase change'. Immediately after germination plants are juvenile. They are unable to flower, even when the conditions are optimal. After some time, depending upon the species ranging from a few weeks to decades, plants become adult and capable of flowering. This phase change may be accompanied by morphological alterations such as changes in leaf shape and direction of stem growth. From the horticultural point of view, the change in the capability to root is important. Cuttings from the juvenile phase are usually easy-to-root while cuttings from the adult phase are recalcitrant. When adult buds are cultured in vitro, though, they often undergo the reverse change (rejuvenation) within a few subculture cycles. Rejuvenation becomes apparent as improved rooting (Webster and Jones 1989). The change in the ontogenetic phase is long-lasting: Cuttings taken from micropropagated plants that have been grown in the field, still have the improved capacity to root (Jones and Webster 1989). In birch, Jones et al. (1996) found that micropropagated trees flowered sooner than seedlings indicating that not all features of the micropropagated trees had become juvenile. The ontogenetic state (juvenile vs. adult) has been related to the methylation status of DNA. In Pinusradiata, for example, the extent of DNA methylation is higher in mature meristematic regions when compared to juvenile meristematic regions (Fraga et al. 2002). In addition, histone modifications (acetylation and methylation) play a major role (Valledor et al. 2010). Irish and McMurray (2006) observed that the cytosine methylation pattern of Pl-Blotched, an allele of purple plant1 (pl1), which encodes a myb-like transcription factor that regulates anthocyanin production in maize, in culture-rejuvenated plants resembled the hypomethylated state of seedlings, rather than the hypermethylated state in adult maize organs they were derived from.
Plant tissues usually require auxin and cytokinin for continuous proliferation in tissue culture. In 1942, Gautheret reported that strains of carrot tissue gradually lost the requirement for exogenous auxin. This is referred to as auxin habituation. It was found later that habituation for cytokinin also occurs. Habituation is now defined more generally as a stable heritable loss in the requirement of cultured plant cells for growth factors (Meins 1989). The epigenetic nature of habituation is shown by the observation that plants regenerated from habituated cell cultures have the original requirement for hormones when cell cultures are established from them. The habituation shift occurs at rates that are orders of magnitude higher than mutations rates in plants (Meins 1989). One approach towards the identification of the mechanism of habituation is a comparison of gene expression in habituated and non-habituated cell cultures. A transcriptome-based analysis of habituated and non-habituated Arabidopsis thaliana plant cell cultures revealed the differential expression of more than 800 genes, including a 19-fold up-regulation of the transcript encoding the cytokinin receptor CRE1. The alterations in gene expression found among habituated and nonhabituated callus cultures do not result simply from global hypomethylation of DNA (Pischke et al. 2006). Nevertheless it has been reported that treatment of in vitro cultured cells of a nontumorous Nicotiana glauca × N. langsdorffii hybrid with 5-azacytidine, a drug causing hypomethylation, leads to the induction of a habituated phenotype (Durante et al. 1989).
Epigenetic aberrations observed in micropropagated plants
Commercial micropropagation operates since the 1970s–1980s. The main advantages include high speed of propagation and production of disease-free, superior, clonal starting-material. During tissue culture, existing epigenetic differences may become visible, e.g., as the differential performance of buds taken from several positions on the same plant. Webster and Jones (1989) reported that two apple lines established from different buds at the same plant remained different with respect to propagation and rooting after a large number of subcultures. Similar observations were made with four lines of Helleborus sp. (G. de Klerk, unpublished observations). Baránek et al. (2010) reported differences in DNA methylation between lines established from different nodes on the same parent plant. In several crops unwanted epigenetic variation caused in tissue culture is a major drawback. Below we describe cases that have been examined in some detail.
Bushiness in Zantedeschia and gerbera
Another case that has been studied in some detail is bushiness in gerbera. Anatomical analysis of longitudinal serial sections of bushy shoots shows that the apical meristem and the axillary buds have the same potential for further development, i.e., axillary bud primordia are not inhibited and develop instantaneously (Topoonyanont et al. 1999). The type of explant has a major effect on bushiness, the main shoot axis being the most sensitive. Higher cytokinin concentrations also enhance bushiness (Topoonyanont and Debergh 2001). Interestingly, addition of imazalil reduces bushiness. Imazalil is a fungicide that interferes with cytochrome P450 enzymes. These enzymes are involved in the metabolism of many xenobiotics and plant hormones.
Tissue proliferation in rhododendron
An abnormal phenotype has been observed in container-grown rhododendron cultivars initially propagated by tissue culture. This aberration has been named "tissue proliferation" and has also been found in azaleas and Kalmia latifolia. The plants display an abnormal tumour-like growth at or near the crown of the plant, and in some genotypes occasionally on aerial plant parts. The abnormal growth appears similar in morphology to crown galls, but tissue proliferation is not equal to crown gall development as Agrobacterium tumefaciens, the causal agent of crown gall, has not been detected. In some cultivars, the crown tumours produce shoots that are smaller than normal, with short internodes and a whorled arrangement of small leaves. In many affected cultivars the disorder is only observed during the first 1–3 years after initial propagation, resulting in significant financial losses for nurseries. Brand (2000) and Brand et al. (2000) reported that plants displaying tissue proliferation have an altered hormone metabolism or sensitivity. This change is apparently stable over many years but has disappeared after some 6 years. Higher cytokinin levels in vitro lead to a greater incidence of tissue proliferation in the field (Brand 2000).
Flower malformation in oil palm
Leaf shape in begonia
Searching for a method to quantify the extent of variation, De Klerk et al. (1990) determined the standard deviation of the leaf shape (measured as the natural logarithm of the ratio of length and width) in populations of regenerated plants. Plants had been regenerated either directly from leaves or after an intermediate callus phase at a high concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). The leaf shape itself was the same in both populations but the standard variation of the leaf shape was far larger in the 2,4-D population. Addition of the chemical mutagens N-methylnitrosourea (NMU) during direct regeneration also increased the standard variation significantly. As the variation was random—in some plants the width of the leaves was increased and in others the length—, the detected variation was expected to be genetic. However, when new plants were produced by direct regeneration from the leaves of plants produced via callus, the standard variation of the leaf shape almost dropped to the value of the control. The plants produced after NMU treatment, though, displayed the same high level of the standard variation after adventitious regeneration. This indicates that the larger part of the variation in 2,4-D plants was epigenetic (Bouman and De Klerk 2001).
Is epigenetic variation caused by stress?
It was suggested above that epigenetic variation may be caused by incomplete resetting of epigenetic marks during adventitious regeneration, but there are likely additional origins. When plants experience stress, they show a multitude of responses, which may include epigenetic variation (Molinier et al. 2006). Oh et al. (2007) hypothesised that labile portions of the genome are affected by stress during micropropagation of Musa. Alterations in methylation have been reported in response to water deficiency (Labra et al. 2002), osmotic stress (Kovařik et al. 1997), heavy metals (Aina et al. 2004), and—in tissue culture—heat (Baránek et al. 2010). Some loci acquired epigenetic silencing, while sometimes silenced loci increased expression. It seems evident that 'extreme' procedures such as protoplast culture and also callus formation impose stress. However, for many species shoot culture may very well be stressful (De Klerk 2007; Desjardins et al. 2009; Van Staden et al. 2006). In this respect, it should be noted that in vitro, the tissues grow under very unnatural conditions: Plantlets have been wounded, they are incomplete (protoplasts being an extreme example), they receive sugar from the nutrient medium as a replacement of photosynthesis in the leaves, they receive high doses of auxins and cytokinins, and their water balance is disturbed by the very high humidity in tissue culture containers. Important questions needing answers are: To what extent can epigenetic variation be ascribed to 'stress'? Which of the tissue culture factors are most stressful? Answers to these questions may lead to procedures that result in a reduction of epigenetic variation. One possible improvement of the procedures is in the choice of cytokinin. In micropropagation usually benzylaminopurine (BAP) is added but another cytokinin, meta-topolin, is often just as effective with respect to the breaking of apical dominance (so resulting in a similar propagation factor) but has less negative side-effects (Bogaert et al. 2006; Valero-Aracama et al. 2009; but see Bairu et al. 2008).
Long lasting changes in the expression of DNA are increasingly documented in plant development. They have been studied in relation to the ability to flower (vernalization) and the ontogenetic development from juvenile to adult. In these cases both methylation of DNA and alterations in histones have been shown to be involved. In plant tissue culture, changes in DNA methylation and in histone modifications have also been reported in many cases, ranging from protoplast formation to axillary shoot proliferation. Such changes are believed to underlie the generation of off-types, e.g., the mantled phenotype in oil palm and bushiness in Zantedeschia. Molecular research on well-defined off-types has been limited with the exception of oil palm. In oil palm, in spite of extensive research, molecular characterization of the aberration is still unsuccessful. This lack of insight in the molecular basis of epigenetic off-types impedes progress in the development of methods to avoid or counteract these aberrations.
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text:
Nacho: I Hope Marquez Drops Pacquiao Once or Twice
By Miguel Rivera
Mexico's Juan Manuel Marquez (54-6-1, 39KOs) is placing the majority of his fight strategy on scoring a knockout victory over Manny Pacquiao (54-4-2, 38KOs) on December 8th at the MGM Grand in Las Vegas.
Marquez, 39, and Pacquiao, 33, will fight for the fourth time. Three controversial endings have forced Marquez to target a knockout conclusion, according to the Mexican champion's trainer Nacho Beristain. He would like Juan Manuel to start very quickly and do his best to take Pacquiao off his feet.
"We want to gain a little more speed. In the third fight we were able to keep up with him, and we want to use that approach again to see if we can put him down on the canvas at least once," said Beristain.
"I would like to believe, and hope to God, that we put him down once or twice - but it is a bit difficult because the Filipino is also a warrior and a very tough fighter. Last time we thought we had the fight in the bag, but look at what happened...you can never tell what the judges are thinking."
Like i said Pac has slipped. If JMM ever knockdown Pac, once or twice, then that shows how long has Pac slipped. 3 fights were JMM got good clean shots against Pac but no knockdown.Comment by jr2122 on 10-18-2012
[QUOTE=twosweethooks;12591656]Wishful thinking Pac must win by KO The judges will rob again Don't tell yer fighter he is winning so he can coast Nacho mang[/QUOTE] He got critized for telling Marquez he was winning but every trainer, specially nacho with…Comment by putok on 10-11-2012
nacho has always liked pacquio. in all his interviews I always read him giving pacquao proper respect.Comment by THE DESTROYER on 10-11-2012
[IMG]http://t2.gstatic.com/images?q=tbn:ANd9GcTvUlErK-Yb52ylXqYcUO6mms2QfLj45lricp1oXiYMAs1WKAxF[/IMG][IMG]http://t1.gstatic.com/images?q=tbn:ANd9GcQlSMb2sKdglZf9esmhR4eay5WH1LHgKBAq5fha4-c2sAV9HMouwA[/IMG][IMG]http://t1.gstatic.com/images?q=tbn:ANd9GcRR4FtkkKj7Jo0Rize-ElrccHyArDPfaCqqW_aMrYlylsYCcO3l[/IMG][IMG]http://t0.gstatic.com/images?q=tbn:ANd9GcTzVe6tar1STqLYPHTQIO02um0Y5UHwKDCO7P5BmNdVXsMuBHTjKA[/IMG] yes like this.Comment by whirlwind on 10-11-2012
[QUOTE=DhymezFMJFan;12593828]Remember Pac was saved by the bell, wobbled, and hurt at times during the first two fights...[/QUOTE] A piece of advice. Stop licking Floyd sugar balls.Post a Comment - View More User Comments (60)


text: We drove from Bordeaux to come and try the English breakfast and we had a fantastic brunch. Great staff, wonderful owner and a good English breakfast, even if they were French sausages and bacon, it till had all the right bits and hot the spot. I will be going back here again for sure.


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- Report: #927383
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Complaint Review: GLAMOUR SHOTS
GLAMOUR SHOTSStaten Island Mall Staten Island, New York United States of America
GLAMOUR SHOTS DISHONEST - RIPOFF Staten Island, New York
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Then she opened my folder and lo and behold, all my pictures were not there. I explained that I had wanted these photos as a present for my daughter's anniversary present and I was driving to here home in Maryland that weekend. I waited so long for them as it was. I was totally annoyed at this point having prepaid for everything already. Another whiny, giggly girl came out and offered to have the photo framed and mailed directly to my daughter's home in Maryland, plus give me two additional photos on a disk. I said I guess I had no choice and took her offer.
Today my daughter called me. She received the photo - with no frame and I have yet to receive the additional photos on a disk. I called them, I got another giggly girl who I could barely understand who said she would check into it and call me back. Well I won't hold my breath, but I will do my best to dissuade anyone from going to Glamour Shots in the Staten Island Mall.
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This iReport is part of an assignment:
Who taught you to love food?
About this iReport
- Posted May 30, 2014 by
- nickdan2726
Philadelphia, Pennsylvania
Philadelphia, Pennsylvania
More from nickdan2726
Who taught me to love food? Well, that is really quite simple. Just two words, in fact. My Mom. My mother was a stay-at-home mom, which was far more common back then. To give you an idea of what "back then" means, I am 45 years old. Mom cooked dinner almost every night. On the nights that she did not cook a fresh dinner, it was because there were leftovers from the night before. No problem. I, for one, love leftovers. Upon arriving home from school each day, my brothers and I were greeted by the tantalizing aromas that permeated the entire house. To smell those aromas was like getting a hug from Mom as soon as she opened the door even before we got an actual hug. We are of Puerto Rican descent and Mom fed us accordingly. While Mom never set foot in a culinary school, her dishes were genuine and true to her beloved island. She learned by watching her own mother. Rice was a must - almost always the side dish. Mom cooked the rice in a caldero, which is similar to a Dutch oven with a tight fitting lid. It should be a life requirement to smell sofrito and tocino (pork fatback) simmering away at the bottom of a caldero. It smells that good. What I did not realize at the time, was that having a fresh cooked meal each night and sharing same with one's siblings was a dying art. Today's hectic schedules do not allow for such. Thank you, Mom, for the delicious food memories which are forever in my heart.
More from this assignment - Who taught you to love food?


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Follow me on Twitter.
Pretty much every company you can think of has donated to the Clinton Foundation. So has Donald Trump. Should it matter?
Imperial Capital initiated coverage of Delta Air (DAL) stock with an 'outperform' rating late Tuesday, calling it the highest quality airline when compared to competitors.
Delta (DAL) has reportedly offered its pilots a 27% raise over four years, which is below what the 13,000 pilots had pushed for previously.
Delta Air Lines (DAL) announced plans to expand in Boston, and analysts at Wolfe Research see the move as a 'direct counterstrike' to JetBlue (JBLU).


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WGA STRIKE UPDATE: WGA Awards Nominations AnnouncedWriters Guild of America Allows Writers to Work Screen Actors Guild Awards, But What About Golden Globes? 12/12/2007 10:13:00 AM Eastern
The awards show will go on for the
, which announced the nominations for its annual WGA Awards Wednesday.
But just how the show will go on for the Golden Globes, which announces its nominations Thursday, remains to be seen.
The WGA, which has been on strike since Nov. 5, already granted a waiver to the Screen Actors Guild to use writers on its Jan. 27 awards show -- not a surprise given the friendly relationship between the two groups during the strike.
But whether that will happen with the Jan. 13 Globes on NBC is still up in the air, and questions remain as to which stars will cross the line and show up anyway.
The Feb. 9 WGA show, the guild's 60th anniversary awards ceremony, will be held in both Los Angeles and New York.
Among the television nominees:
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Jen Brea is a phenomenon. After working as a freelance writer in China and Africa, she enrolled for a PhD at Harvard in political science but, four years ago, got sick. She had a fever that lasted ten days.
Discuss the article on the Forums.
Stem Cells
Discussion in 'General Treatment' started by deb obrien, Aug 13, 2009.
Page 2 of 42
Page 2 of 42


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Barcelona - Barcelona's desire to boost revenue has the European champions charging the public to view an upcoming open training session.
Club members can invite three guests free of charge for the January 5 practice, but nonmembers will be charged €5 ($6.45) to watch Lionel Messi, Xavi Hernandez and Andres Iniesta hone their skills.
Under current president Sandro Rosell, the recent Club World Cup winners signed a €171 million ($230 million) deal with Qatar Foundation, which became the first paid sponsor to adorn the club's jersey.
The heavily indebted club has also embraced cost-cutting measures, eliminating its 80-year-old baseball team and slashing funding to 12 minority sports teams earlier this year.
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Medical Insurance
Medical Insurance is offered through Blue Cross Blue Shield of Alabama.
You and your dependents are eligible for coverage immediately upon hire. Premiums are paid by employee and employer (rates listed below).
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Results 1 to 10 of 179
Thread: [4e] Fallen OOC (Full)
Hybrid View
Tuesday, 22nd September, 2009, 02:18 AM #1
Gallant (Lvl 3)
- Join Date
- Aug 2003
- Location
- Marina Del Rey, Los Angeles
- Posts
- 1,338
ø Block Sparky
ø Friend+
[4e] Fallen OOC (Full)
Recruitment is currently closed.
[OOC] Fallen
hafrogman as... Atrius (Bard)
Walking Dad as... Caged Fury (Monk)
Kobold Stew as... Pirx (Ranger)
eblue562 as... Rodeh (Warden)
Sparky as... The Friendly GM
Fallen is a homebrewed 4E D&D campaign set in the remote past of the Points of Light setting. Arcosia and Bal Turat (Arkhosia and Bael Turath) are locked in a bitter war. Nerath's star is on the rise. The Eledin are well and truly splintered, the Eladrin retreating into the Feywild and the Elves remain behind to safeguard their places of power. The Dwarves revolt against the oppression of their Giant masters and those who escape are finding their mountainhomes taken over by interlopers and beasts. Halflings are spiritually and culturally adrift, nomadic gypsies who wander the face of the world in search of their Promised Land and new Gods to shepherd their race.
IC Posting Rules
Pretty standard stuff, some guidelines to keep the IC thread tidy and readable.
Speech - In quotes. Not a huge deal if you leave them off, but it makes a better contrast with thoughts.
Thoughts - In italics. If you choose to write your character's thoughts, they should be in italics.
Languages and OOC - Different colors per language. Both the color name and coordinate are listed (some are not default colors and will not have coordinates).
Abyssal [DarkRed, B1] - Demons, gnolls, sahauginRolls - Make your rolls on Invisible Castle and post them in the OOC section of your IC post, as appropriate.
Deep Speech [Indigo, A7] - Mindflayers, githyanki, kuo-toas
Draconic [Cyan, D5] - Dragons, dragonborn, kobolds
Dwarven [Sienna, A2] - Dwarves, azer
Elven [Green, B4] - Elves, eladrin, formorians
Giant [Olive, B3] - Giants, orcs, ogres
Goblin[YellowGreen, C3] - Goblins, hobgoblins, bugbears
Nethyr(Common) [Gray, C8] - Humans, halflings, tieflings
Primordial [DarkSlateGray, A8] - Efreets, archons, elementals
Supernal [Teal, B5] - Angels, devils, gods
Arcane [RoyalBlue, C6] - Spells for Warlocks, Wizards, Sorcerers
Divine [Gold] - Prayers for Clerics, Paladins, Invokers, Avengers
OOC Text[Font Size 1, DimGray, B8] - Rolls, actions, tactics,
questions, light table talk
Stan staggers over to the door of his cell a blade concealed along the meaty part of his forearm. He is dead drunk, at least, he wants the guard on duty to think so. "Oi!" he hollers, slumping against the bars. He drowses a bit, on the verge of passing out before shaking himself awake. "Oi!" he hollers again, this time raking his tin mug across the bars of the cell. "Guard!"
"Shaddup, ya halfpint halfwit," growls Murray from across the aisle. Murray shifts on his bunk drawing up the thin covers to better hide his nervous face. Gods, I hope this works...
(OOC: Stan uses Bluff against the Guard (assisted by Murray) pretending to be drunk. Stan also uses Thievery to conceal a dagger. Rolls)
renau1g as... Kadaj (Rogue)
Phaezen as... Followingdarkness (Assassin)
Shayuri as... The Scholar (Wizard)... dropped during the Crucible
Insight as... Q'ynn (Invoker)... dropped due to time constraints
CaBaNa as... Run'n'hide (Coward)... passed on subbing in for Insight/Qynn due to time constraints
IC - Prologue: The Crucible
IC - Chapter I: Blood and Sand
Rogue's Gallery
Invisible Castle
Last edited by Sparky; Wednesday, 18th November, 2009 at 05:57 AM.
Tuesday, 22nd September, 2009, 03:06 AM #2
Gallant (Lvl 3)
- Join Date
- Aug 2003
- Location
- Marina Del Rey, Los Angeles
- Posts
- 1,338
ø Block Sparky
ø Friend+Recruiting Info
(Recruiting is closed)
The List of PC Submissions:
Atrius (hafrogman)
Caged Fury (Walking Dad)
Followingdarkness (Phaezen)
Kadaj (renau1g)
Pirx (Kobold Stew)
Q'ynn (Insight)
Rodeh (eblue562)
Run'n'hide (CaBaNa)
The Scholar (Shayuri)
The Crucible
So, I have the DM's best problem - excellent concepts and interesting builds all around. There're just too many. I'd love to run with all of these PCs, they're all cool in different and inspiring ways.
But I'm at a loss. Each time I compose a list, I see what players/PCs aren't on it and start again. I don't want to increase the party size, so I've decided to run a streamlined RP Challenge and let posts, voting and dice do the work. It'll go like this:
1. I post a situation in the IC thread for response (with a timeframe).
2. Players post a single response for their PCs.
3. The timeframe lapses.
4. Players vote within 24 hours of the timeframe lapsing for their 3 favorite responses (vote for yourself if you want to better your chances). I will be voting as well.
5. I post an unmodified d20 roll for each Player's PC response.
6. Score the round:
- - - 1 pt for posting a response (on-the-ball point)
- - - 2 pts for the 4 top responses in the voting (chemistry points)
- - - 1 pt for the 4 top d20 rolls (destiny point)
7. Repeat.
This week we'll start with 1 situation and 1 round of voting so everyone can get the hang of things (me included).
After that we'll complete between 3 and 5 more rounds and then I'll tally up the scores and the top 4 scorers will move on to the game proper.
I'll post the first situation Monday the 21st or Tuesday the 22nd.
Tuesday, 22nd September, 2009, 03:07 AM #3
Gallant (Lvl 3)
- Join Date
- Aug 2003
- Location
- Marina Del Rey, Los Angeles
- Posts
- 1,338
ø Block Sparky
ø Friend+
I've got the game threads up, don't post into the IC thread until I give the go-ahead here.
@eblue562: Forgot to answer your questions.
Q1. Will anyone be able to vote, or is it a PC's only voting?
A: Players only will vote. I considered opening the voting up to others, but it unfairly penalizes folks who might not have a group of established friends on the boards to stuff the ballot box for them.
Q2. Will you post an IC link in the OOC thread when you have the post up?
A: I crosslink like there's no tomorrow. The OP of any game thread will have a small list of links for use in the game.
Originally Posted by CaBaNa
@Everyone: Roll call! Sound off to check into the thread.
Last edited by Sparky; Tuesday, 22nd September, 2009 at 03:36 AM.
Tuesday, 22nd September, 2009, 03:43 AM #4
Spellbinder (Lvl 16)
- Join Date
- Jun 2002
- Location
- Minnapolis, MN
- Posts
- 19,735
ø Block Shayuri
ø Friend+
Allons y!
Tuesday, 22nd September, 2009, 04:00 AM #5
Enchanter (Lvl 12)
- Join Date
- Nov 2002
- Location
- Mesa, AZ
- Posts
- 8,864
ø Block hafrogman
ø Friend+
Tuesday, 22nd September, 2009, 03:54 AM #6
Waghalter (Lvl 7)
- Join Date
- Feb 2009
- Location
- Posts
- 3,064
ø Block CaBaNa
ø Friend+
Cheers, Checking in and letting everyone know Magenta is my baby, all my characters all over, (except one I took over for another player) are in Magenta.
Thanks for your consideration
Also @Sparky, I don't mind putting thoughts in italics, but do you mind if I throw ** on the ends? example
The human balled in the fetal position seems to crawl even further in the corner, as the new pen mate is let in, *he's is HUGE! Don't notice i, don't notice i, don't notice i, don't notice i...* his rocking back and forth is accentuated by occasional mumbling, "don't...notice........i..."
Last edited by CaBaNa; Tuesday, 22nd September, 2009 at 04:07 AM.MAGIC!
Tuesday, 22nd September, 2009, 04:21 AM #7
Gallant (Lvl 3)
- Join Date
- Aug 2003
- Location
- Marina Del Rey, Los Angeles
- Posts
- 1,338
ø Block Sparky
ø Friend+
Tuesday, 22nd September, 2009, 04:30 AM #8
Waghalter (Lvl 7)
- Join Date
- Feb 2009
- Location
- Posts
- 3,064
ø Block CaBaNa
ø Friend+
So the example for Run'and'Hide should instead look like this,
The human balled in the fetal position seems to crawl even further in the corner, as the new pen mate is let in, *he's is HUGE! Don't notice i, don't notice i, don't notice i, don't notice i...* his rocking back and forth is accentuated by occasional mumbling, "don't...notice........i..." (OOC: Run'and'Hide takes the Total Defense actionMAGIC!
Similar Threads
[4e] Fallen - Chapter I: Blood and Sand (Full)By Sparky in forum Playing the GameReplies: 30Last Post: Saturday, 12th December, 2009, 03:32 PM
[4e] Fallen - Prologue: The Crucible (Full)By Sparky in forum Playing the GameReplies: 44Last Post: Monday, 19th October, 2009, 09:09 AM
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Keep a close watch
Published: Wednesday, August 27, 2008 at 3:00 p.m.
Last Modified: Wednesday, August 27, 2008 at 11:32 a.m.
For months now, a small but active group of Houma residents has spent a great deal of time and energy scrutinizing the activities of American Advanced Technologies.
And that work has paid off by turning up some paperwork issues that should
concern the whole region if not the whole state.
In the process, the resident-activists also have put added pressure on the state
Department of Environmental Quality to make sure it fulfills its public mission by
monitoring the companys compliance with state laws and regulations.
The DEQ has not been in the lead on that effort.
In fact, DEQ regulators studied AATs proposal to dump up to 80,000 gallons per
day of treated water into Munson Slip.
They said they considered the fact that Munson Slip flows into the Gulf Intracoastal
Waterway, but that the proposed dumping should be all right because Munson Slip
is downstream from where drinking water is drawn from the waterway.
Those same regulators, though, failed to account for the fact that tidal action
changes the streams flow and causes the dump site to be upstream of the water
intakes about a third of the time.
That is a huge oversight in a process that is meant, above all, to ensure a safe
supply of clean drinking water.
The people who are opposing the expansion of AATs permits pointed to the earlier
DEQ failure and other problems with AAT to argue that DEQ simply isnt up to the
job of adequately monitoring the oil-recycling plant.
While their complaints are well-grounded, we think they have served to increase
DEQs awareness of the situation.
The state agency inspected AAT on two days in January and issued several
citations for failings in the required process.
Among those was a citation for operating without a solid waste permit.
Unfortunately, the DEQ later issued an order allowing AAT to process waste without
a license until it gets the required permit.
That is an inexcusable lapse. The requirement of a permit is there for a reason
to ensure companies processing this waste are doing so in a responsible manner.
Until the company meets that requirement, there should be no permit.
Although the continuation of the waste-processing operation is disheartening, there
is reason for celebration in this tale.
As the concerned residents point out, the DEQ has taken an increased interest in
the plant and seems to be giving it some scrutiny.
The bad news, of course, is that it takes loud public action to bring about the proper
conduct from the state agency.
No one can blame local residents for being concerned about oilfield waste, its
potential impact on them and DEQs ability to protect them from that impact.
There have been past cases of DEQ allowing questionable cases to go on far too
long. Marine Shale and Campbell Wells spring immediately to mind.
We urge our local activists to stay involved in the process and to continue watching
AAT and its regulators.
More importantly, though, we urge DEQ to do its job without being asked. We all
deserve that much.
Editorials represent the opinions of this newspaper, and not of any one person.
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When this plugin is activated my site RSS feed is broken and shows the common error:
XML Parsing Error: XML or text declaration not at start of entity
Line Number 3, Column 1:<?xml version="1.0" encoding="UTF-8"?>
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PLEASE HELP - the plugin doesn't work with worpress as it should.
I am using WordPress 3.1.3


text:
- Report: #970405
Report - Rebuttal - Arbitrate
Complaint Review: WYNDHAM/WORLD MARK
WYNDHAM/WORLD MARK1331 OCEAN SHORES BLVD SW OCEAN SHORES, Washington United States of America
WYNDHAM/WORLD MARK WORLDMAK, "THE CLUB" OCEAN SHORES WASHINGTON HAS Deliberately not paid a Black-Korean General Contractor OCEAN SHORES, Washington
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If you visit we are responsible for the lobby. The manager of The Club was a former employee of ours. She did everything she could to delay our completion of the project, including having us to repaint the lobby, bathrooms and 6 times! She has an axe to grind with us, and I believe it is because it is a minority business now. We live in a small town in Ocean Shores and whenever she saw me she gave me a cold stare, since she realized I was taking over the business from NBHM.
I would have never started the project if I knew this individual was the resort manager. She delayed us at every point to complete our project. The project manager dismissed us when we were 90% complete! We completed the drilling for the pool brilliantly, and then they announced that someone else would complete the job and refused to pay us for that. They have refused to pay us for the reception desk which is beautiful, and even though it was not in the contract to reconnect the computers, we did so when the Resort manager balked.......these where all delays for us.
I believe the manager of Worldmark, the club is a bigot, because she does not like the idea of me owning the business because I am Black-Korean....You can tell when they look at you with disdain. She thought the old NBHM was doing the work, but did not realize it was me. At this point they owe us close to $20,000. for work we have already done and we have not charged them for a lot of the man hours it took for all her little changes. This is totally unfair. We have employees who have families, and it is getting close to the holidays, we have taxes, L&I, and we have our vendors which we fronted the money to in order to complete the project. Worldmark, "the club" Ocean Shores refuses to pay it's contractors.
We have done a lot of work for them in the past under the NBHM Company, but when the resort manager found out I was the new owner she sabotaged us along with the Project manager in Redmond. I would think twice before you buy a time share there. We have filed a lien against this property, and the State of Washington is issuing them a rit to pay the takes we cannot pay, because they have not paid us. Thank you, C. Harrison Services
This report was posted on Ripoff Report on 11/16/2012 06:56 PM and is a permanent record located here: http://www.ripoffreport.com/r/WYNDHAMWORLD-MARK/OCEAN-SHORES-Washington-98569/WYNDHAMWORLD-MARK-WORLDMAK-THE-CLUB-OCEAN-SHORES-WASHINGTON-HAS-Deliberately-not-paid-970405. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
Ripoff Report has an exclusive license to this report. It may not be copied without the written permission of Ripoff Report.
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- Report: #379482
Report - Rebuttal - Arbitrate
Complaint Review: Consumerbill.com
Consumerbill.comwww.consumerbill.com Internet U.S.A.
Consumerbill.com BIG RIP OFFFF Internet
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Paulsboro, New Jersey
This report was posted on Ripoff Report on 10/08/2008 04:58 PM and is a permanent record located here: http://www.ripoffreport.com/r/Consumerbillcom/internet/Consumerbillcom-BIG-RIP-OFFFF-Internet-379482. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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