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Free Weekend As National Park Service Turning 100-Years OldThe National Park Service turns 100-years old Thursday, and it's celebrating all weekend long.
Best Bodies Of Water To Visit In Northern CaliforniaSan Francisco is fortunate enough to be surrounded by the bay with easy access to the Pacific Ocean. There are also plenty of lakes, bays, ponds, creeks and marshes among others that are also in and around San Francisco. Birding, boating and bicycling are all activities that can be enjoyed at these underrated water destinations. Whether you want to spend a lazy day on the water or have a picnic by the shore, these bodies of water will have you exploring these lesser known gems.
Park Fee Proposal For Sonoma County Beaches Moves To Coastal CommissionA battle is coming to a head between beach goers and Sonoma County over proposed new fees the county wants to charge people to park at four beaches.
Aggressive Wild Turkeys Breeding Like Bunnies Around The Bay AreaBay Area ecologists are concerned about an explosion in the wild turkey population, and say some are on the attack.
East Bay Parks Buying Contra Costa County Farmland To Help Protect Threatened AnimalsThe East Bay Regional Park District's Board of Directors voted Tuesday to purchase a tract of farmland in Contra Costa County to provide habitat for threatened animals, according to district officials.
San Francisco Ballot Measure Would Set Aside City Funds For Parks San Francisco Supervisor Mark Farrell introduced a ballot measure this week that he said could generate an additional $350 million for city parks over the next 15 years.
California Beaches, Parks Turning Off Showers, Foot-Faucets To Save Water During DroughtCalifornia state beaches and parks are turning off the taps starting Wednesday.
Muir Woods Entry Fee Going Up From $7 To $10Entry for children under 16 years of age will continue to be free, said the National Park Service, which announced the price increase on Thursday.
$1.5M Spent To Renovate 2 Public Restrooms In San FranciscoRecreation and Park Department officials opened renovated public restrooms Friday along Ocean Beach in San Francisco, city officials said.
6-Fold Increase In San Francisco Homeless Citations Directly Coincides With Ed Lee's Tenure As MayorThere's been a significant increase recently in the number of citations issued in San Francisco to the homeless who camp out in the city's parks and open spaces.
Free Entrance To National Parks Offered This Weekend This weekend may be the perfect time to visit a national park, as entrance fees are being waived for the third time this year.
Sex Offenders Allowed To Live Closer To Schools, Parks Under New California RegulationsCalifornia will alter its 8-year-old ban preventing all registered sex offenders from living near schools or parks, instead imposing the restriction only on pedophiles and others whose sex crimes involved children.


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|Sounds of the game… Celtics 105, Cavaliers 94
|03.07.09 at 12:30 am ET
Before Friday's game, Celtics assistant coach Armond Hill showed an inspirational video to the team, aimed specifically at young big men Leon Powe and Glen Davis.
No, it wasn't Rocky or even Hoosiers. It was a film of the Celtics scoring basket after basket with their big men executing great interior passes. The result – the undermanned Celtics outscored the Cleveland Cavaliers 58-22 in the paint and rolled to a stunning 11-point win, 105-94, over the Eastern Conference leaders.
The Celtics didn't have Kevin Garnett but they did have Leon Powe. The Celtics didn't have Brian Scalabrine but they did have Glen Davis, at least for 17 minutes before he was ejected for a flagrant Type 2 foul with 9:09 remaining in the third quarter.
But Doc Rivers, who said at the shootaround in the morning that this would be a phenomenal win, had one of his best games of the season as head coach of the defending NBA champions. Just ask Mike Brown, his counterpart on the Cleveland bench.
Brown said the Celtics deserved all the credit for pulling together and pulling off the upset, even if it was on their home parquet.
For his part, Doc Rivers gave credit to Armond Hill for showing the video and his players for taking the lead.
Paul Pierce said Powe (20 points, 11 rebounds) and Davis (10 points) showed the depth the Celtics have.
Pierce said Davis' ejection in the third quarter fired up the Green.
Leon Powe said he knew he was going to have to pick it up when Big Baby was thrown out.
Glen Davis said his foul of Anderson Varejao was a case of being in the wrong place at the wrong time.
LeBron James said, with or without Garnett, the Celtics are a great team.
King James said it's hard to believe home team has won 15 straight in the series.
Latest from Bleacher Report
- Austin Ainge Offers Window to C's Pre-Draft Process
- Trade Possibilities for C's with Draft Approaching
- Latest Buzz Surrounding Jamal Crawford, Kristaps Porzingis, Celtics'...
- Latest NBA Trade Rumors, Buzz
- Ranking Celtics' Biggest NBA Draft Needs
- Buzz Surrounding Ty Lawson, Celtics Draft Plans and More
- Realistic Targets for Celtics to Chase During Offseason


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Tags: Samurai Pudding, Chonmage Purin, 2010, Japanese movies, Comedy movies, Drama movies, asian movies
«His new film, "Chonmage Purin" has plenty of strangeness in it, but of the sweetly whimsical, TV sitcom sort. ... The real home for "Chonmage Purin" is the small screen, where the scrumptious- looking desserts and the buff Nishikikido will naturally attract eyeballs.»
Like this film? Planning to watch it or have done already? Want to share your feedback or just give a thumbs up? Found links to intresting reviews, posters or trailers? Your participation is appreciated.
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Originally posted by illorion
Originally posted by GMan3
Amazing. The OP points out that someone got a TEMP Ban when he tried to break the terms of service of TWO different games and none of you feel sorry for this person?
Where is your heart? Where is your compassion?
Probably right where mine is, in the correct place. What the person did was a creative way of gold farming, but still gold farming no matter how you look at it and I for one am sorry that he did not receive a PERMANENT BAN for it.
Sorry OP, but no matter how you try to spin this it still comes out the same. The guy was wrong, was reported, and received punishment. In the world I was raised in that is called the RIGHT THING.
And with that... i think im done... You right... no matter how I look at it it is gold farming... thank you for showing me the error of my thoughts. Im so glad he is out of the game... who knows what kind of in game devistation that would have wrought on the economy... almost broke the whole damn game.
I am in awe at the benevolance of BW/EA in even allowing this... CRETIN... back into the game. I honestly don't know how they could allow such a person in an enviroment where children play... he should be arrested.
Crap now I have to make a new thread to rally people to arrest this crazy bastard
Sorry your troll thread didn't work for you, better luck next time. Personally, I think it is people, like you, that do not think they have to follow the rules that need to be disciplined just for the way you screw up the rest of society. But that is just my opinion.
"If half of what you tell me is a lie, how can I believe any of it?"
What was the original title of the thread. The new one makes me lol.
Originally posted by illorion
Originally posted by warmaster670
Originally posted by Mellkor
@ form title, lol really?
Hes just emo because people called him on his BS title, he seems to think its ok to have a title thats a flat out lie if it gets people to read hios thread.
How was it a lie.
He asked a guildie to help him out in WoW in exchange for some help in SWTOR.... the title read
Player gets perma banned for asking guildie to help him in WoW... i dont see the blatant lie here.
Its not like it said "SWTOR bans player and kill his dog" or something
He didnt ask anyone for help, he asked someone to trade him currency.
Originally posted by illorion
Originally posted by bartoni33
Well here isn't to much to play right now... Skyrim... thats about it... and ive sank hundreds of hours into it by now. I do enjoy being on the forums... not just this one. And I talk about this game because maybe... just maybe... if enough people tear into this AAA WoW clone then they will finally stop making them and make something different for a change.
Yeah who the fuck would want to make a successful game based on the most successful game of all time. OF ALL TIME!
The troll is strong in this one. I revoke my half-ass approval of this thread and all its contents.
I bet you are just chomping at the bit to get your hands on final fantasy 15 or 16 or 22 or whatever number they are on now... because you know... copying and pasting the same successful game over and over and over again is full of win
This post alone shows are truly clueless you are, like what has already been posting, if you knew a thing about what you were talking about you would be able to see past the little number next to the name and know that each FF game is different, and the opposite of copy pasted.
Let me guess, you also think that the phantom menace is a copy paste of a new hope? makes just as much sense as what you said.
Apparently stating the truth in my sig is "trolling"
Sig typo fixed thanks to an observant stragen001.
The new title made my morning
"It has potential"
-Second most used phrase on existence
"It sucks"
-Most used phrase on existence
Originally posted by dinams
The new title made my morning
Yeah actually the new title is all that needs to be known about the contents of the thread. First post is utter bullshit.
But like I posted eariler at least he is trolling on a new subject and not rehashing the old worn out dead horses that have clogged up these here forums for days now.
Originally posted by illorion
Not sure if this has made it on here yet. Basically this guy asked his guild mates in SWTOR if anyone wanted to swap some credits for WoW gold.
He was permanently banned
Just thought it was an interesting find.
well thats basicalyl gold trading so maybe games should take a hard stance on gold trading.
ha ha nice title.
anyway I dont see any fault in asking such kind of help
lmao at the title..
Originally posted by sassoonss
ha ha nice title.
anyway I dont see any fault in asking such kind of help
MMO are a social game it's all about interaction.
My friend regularly gave me stuff and kinah in Aion as I didn't like to grind way back and in return I used to help him in WAR/AOC
We are not gold farmers , we made the money by playing the game.
You can quote the rules and bash all the moral policing u want but, don't we all share music,games ,movies amongst friends n relatives al the time.
His mistake was he asked in guild chat to people whom he is not all that friendly.
I fell the mods should have just warned him – perma ban or suspension was too much.
I don't feel it was anything wrong. I offered my friend to try SWTOR and if he likes I would give him gold so that he can speed up in return he said u can always return to Aion and he would help me out with Kinah.
See OP this is how you should have worded your opening post. I can agree with him 100% without feeling like I'm siding with a troll.
Originally posted by sassoonss
ha ha nice title.
anyway I dont see any fault in asking such kind of help
MMO are a social game it's all about interaction.
My friend regularly gave me stuff and kinah in Aion as I didn't like to grind way back and in return I used to help him in WAR/AOC
We are not gold farmers , we made the money by playing the game.
You can quote the rules and bash all the moral policing u want but, don't we all share music,games ,movies amongst friends n relatives al the time.
His mistake was he asked in guild chat to people whom he is not all that friendly.
I fell the mods should have just warned him – perma ban or suspension was too much.
I don't feel it was anything wrong. I offered my friend to try SWTOR and if he likes I would give him gold so that he can speed up in return he said u can always return to Aion and he would help me out with Kinah.
While i can understand why you might feel that way, the simple fact remains that in almost every MMO out there the Terms Of Service clearly forbid this kind of action (and yes SWTOR and WoW both forbid it). So while you "feel" one way, it really doesn't matter since you are wrong. If you violate the rules, you do it at your own risk and have to be mature enough to accept the penalties for your actions. Plain and simple.
"If half of what you tell me is a lie, how can I believe any of it?"
I don't care what the thread is about, that title is brilliant!
I didnt even think titles could go this long you sir have won the interwebs.
Originally posted by arrisb
I don't care what the thread is about, that title is brilliant!
If someone is talking in general chat in a language you dont understand, chances are they're not talking to you. So chill out and stop bitching about it!
Shadow's Hand Guild
Open recruitment for
The Secret World - Dragons
Planetside 2 - Terran Republic
Tera - Dragonfall Server
Holy thread backfire. Very amusing.
Nice to see that EA/BW is so forgiving, it should have been a perma-ban, he got off lightly.
Originally posted by illorion
Ahh the old MMO car analogy.... Ahh the old "i see what you did there so that makes me smarter than you" move
And you only prove my point... once again I am arguing the morality of permanently banning a player or even banning a player at all for this and you are still smacking me over the head with the TOS wiffle ball bat.
I have made this joke in WoW actually... and didn't get banned for it... and in General chat to boot. Back when people where leaving to play aion I used to say... "I got a toon in aion... give your stuff and you can have all of it". No one ever took me up on the offer... but i certainly didnt get banned for it... cause that would have been stupid.
Even if someone did report it... what exactly was the person thinking who read it... "Holy shit this could break the game PERMA BAN" Cmon really?
I await your next post where you cite particular sections of the TOS as well as other games TOS's if someone hasn't already done it for you before I even finished typing this
i shall counter with two points
1) i did not use the mmo car analogy...i used a driver analogy
2) he wasnt perma banned
3) i have no argument. I am not presenting a troll post to argue. Thats the responsibility of the original poster.
Ok that was 3 points but who is counting.
1) Potato Potaato
2) It was a perma banned but he managed to get it repealed to a 10 day suspension. If he hadn't it would have been a perma ban
3) My argument is that it is "morally wrong" as in "not a good reason" to perma ban a player for something like this... that is my opinion. If you disagree with it well... let's just say never trust anyone who values "technicailities" more than "what's right"
Regarding your third point, you fail to understand what a "Lawful Good" personality type is and how they react in these situations. If something is illegal and/or wrong (which this situation certainly qualifies) they are compelled to report it to the authorities, regardless of the "technicalities" as you put it.
They would turn in their own family, and certainly some random internet guild member. You may not agree with their choice but they are not wrong in their actions.
On hiatus from EVE Online since Dec 2016 - Screw off-grid PVE boosting changes
Pouring on extra "Salt" for 2017
In my day MMORPG's were so hard we fought our way through dungeons in the snow, uphill both ways.
Don't just play games, inhabit virtual worlds™
"This is the most intelligent, well qualified and articulate response to a post I have ever seen on these forums. It's a shame most people here won't have the attention span to read past the second line." - Anon
Loving the topic title
Surprised he didn't get Perma banned in WoW as it is in their ToS that using their in game products for any bartering will result in a permanent ban. Haven't read the SWTOR close enough yet, but I am shocked that SWTOR isn't the same.
Swapping game 'gold' is against the terms of service, whether you're a regular player or a gold farmer. Player is lucky they only got a 10 day ban.
Also, the title was very informative.
I can not remember winning or losing a single debate on the internet.
A guild mate of mine got banned. He didnt want everyone knowing but he told one of the other guildies he was buying credits and next thing ya know boom! Temp ban. He played it off as "He was hacked" but some of us knew the truth and point and laugh anytime we see his name pop up for being a cheap bastard.
Honestly im glad companies punish players for this type of currency trading whether its for real money or fake money of another form. People like the guild member mentioned and to an extent the O.P are the reason so many gold farmers exist. As long as there is a market for people too lazy to farm currency themselves to trade/buy it from someone else MMO Currency Farmers will never go anywhere.
Actualy have multi game guilds too and say trade 1000 WoW gold for 1000 credits (or whatever the equvilant value might be since I don't play SWTOR) if I wanted to do that usually done over vent or on a forum only viewable by members.
This isn't a signature, you just think it is.
Yep this is our beloved BW alright. I still don't understand why all the whinning. Move along, play a real mmo and forget this loading scr ... ahem game.


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Duluth, a four-season city with 11,000 acres of green space alongside the greatest lake in the world, has miles and miles of trails that allow for travel on foot, skis, bike or snowmobile.Read more...
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Regular monthly meeting of the Planning Commission (the second Tuesday of every month).
The Human Rights Commission meets:
The Commission on Disabilities meets the first Wednesday of every month at 3:00 p.m., Room 303, City Hall.
Play Gym is a FREE opportunity for parents and their young children to gather and interact in a large, open, indoor space. Play Gym has toys, sports equipment, and art materials available for use. Program offered every Wednesday from 9:30am to 11:30am at the Duluth Heights Community Center.
For more information, please call 218-730-4322.
AARP Defensive Driving Classes for 2017
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Winter Skating Party - Amsoil Arena
Skate with the UMD Figure Skating Team!
Skating Party is FREE!
Bring your own skates.
Prizes for kids - Music Provided
*Children under 10 years of age must be accompianed by an adult
Free Open Skating
Every Wednesday that school is in session: September 14th- May24th
Bring your own skates, limited skates available for loan
Valentine's Day themed skating party featuring arts and craft activities and music!
Parks and Recreation hosts free open skating at the Heritage Sport Center (120 South 30th Avenue West). Open to skaters of all ages. Limited skates are available to use.
Free Open Skate - Heritage Sports Center
Mid-Winter Break, February 20-24th
*Bring your own skates, limited skates available for loan
Bayfront Family Center- Outdoor Skating & Winter Playground
Open 7 days a week, weather permitting, for pleasure skaters and others who want to enjoy winter at its best. Ice skates, sleds, snowshoes and snow sculpting tools are available for loan. Sliding hill coming in January!
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Call 218-730-4300 during regular business hours for more information.
Special Planning Commission meeting to discuss three items; see agenda and planning commission packet fo rmore information
The public is invited to attend the first public meeting for the Duluth Traverse Trail Management and Master Plan. The City is leading a public process to determine best practices for future management, guide final segment development and construction, and gather input on improvements to the trail system.
The ESST is holding a special meeting to discuss the outline of public input sessions.
Mayor Emily Larson is continuing her commitment to reach out to Duluth residents through City Hall in the City Community Listening sessions that are held in neighborhoods across the city. Mayor Larson started these listening sessions last year in partnership with City Councilors to ensure residents have an opportunity to speak directly to their elected officials one-on-one. City staff are also in attendance and represent Public Works, Parks, Public Safety and Imagine Duluth Comprehensive Plan. The previous year's sessions enabled residents to have open individualized conversations with the Mayor, City Councilors or City staff about various issues or topics of concern. The upcoming City Hall in the City listening sessions will occur on the third Wednesday of the month and run from 5:30-7:00 pm. These events are open to all residents. Children are also welcome to attend. The first set of dates with City Councilors and locations are as follows: February 15 – Councilor Em Westerlund at Grant Recreation Center, 901 East 11th Street March 15 – Council President Joel Sipress at Kenwood Lutheran Church, 2720 Myers Ave April 19 - Councilor Noah Hobbs at American Indian Community Housing Organization (AICHO), in Trepanier Hall, 202 West 2nd Street May 17 – Councilor Zack Filipovich at Piedmont Heights Community Center, 2302 West 3rd Street The remaining 2017 listening sessions will be announced at a later date.
The Earned Sick and Safe Time Task Force gathers information, collects public input, proposes the best options for implementing ESST policies and brings forward policy recommendations.
The Earned Sick and Safe Time Task Force gathers information, collects public input, proposes the best options for implementing ESST policies and brings forward policy recommendations.
Parents are encouraged to stop by a car seat safety clinic to have their child's car seat inspected free of charge. When a parent arrives, a certified car seat safety technician climbs into the vehicle to inspect child safety seats. Necessary adjustments will be made so the seats are properly installed to protect children in the event of a crash. The technician will also answer parents' questions or concerns about car seat safety. No appointment is necessary. People will be helped on a first come, first served basis. Questions, please call 730-4397 (Sandy McComb), 348-0173 (Laura Owen), 249-5449 (Kathryn Brown), or 730-4398 (Marnie Grondahl).
The Bike and Trails Committee of the Duluth Parks and Recreation Commission will be holding a meeting at City Hall in room 106A, on Wednesday March, 1st at 7:30pm. The purpose of this meeting is to review public comments on the Cross City Trail Mini-Master Plan.


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|CARICOM Heads discuss Zika
|Written by Administrator
|Friday, 19 February 2016 00:00
Belize remains free of the Zika Virus which has spread all around the Latin American Countries, and to 5 countries within the Caribbean. The Belizean authorities want to keep it that way, and the CARICOM Leaders want to curb the epidemic to make sure it does not get any worse. That was one of the topics discussed at Twenty-Seventh Inter-Sessional Meeting of The Conference of Heads of Government of CARICOM, which was chaired by Belize's Prime Minister, Dean Barrow.


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PHOTO: Southern Miss to wear stars-and-stripes helmet decals
Rutgers tweaked its helmets to incorporate the stars-and-stripes into their decals for its game against Army Saturday, and came away with a 28-7 win. So if a similar tweak does anything similar for Southern Miss against UTEP this weekend, there will some very happy fans in Hattiesburg.
The Golden Eagles are suffering through their worst season in decades at a miserable 0-10. But Saturday's home date with the Miners will be Military Appreciation Day, and the Eagles will still celebrate it with these special decals (from the Southern Miss equipment department's Twitter feed):
USM's Eagles are Golden rather than bald, but there's still some nice synergy here, no?
HT: UniWatch


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Enrichment with anti-cytokeratin alone or combined with anti-EpCAM antibodies significantly increases the sensitivity for circulating tumor cell detection in metastatic breast cancer patients
DOI: 10.1186/bcr2131
© Deng et al.; licensee BioMed Central Ltd. 2008
Received: 24 April 2008
Accepted: 07 August 2008
Published: 07 August 2008
Circulating tumor cells (CTCs) are detectable in most cancer patients and they can meet an existing medical need to monitor cancer patients during a course of treatment and to help determine recurrent disease. CTCs are rarely found in the blood of cancer patients and enrichment is necessary for sensitive CTC detection. Most CTC enrichment technologies are anti-EpCAM antibody based even though CTC identification criteria are cytokeratin positive (CK+), CD45 negative (CD45-) and 4'6-diamidino-2-phenylindole (nuclear stain) positive (DAPI+). However, some tumor cells express low or no EpCAM. Here we present a highly sensitive and reproducible enrichment method that is based on binding to anti-CK alone or a combination of anti-CK and anti-EpCAM antibodies.
Blood samples from 49 patients with metastatic breast cancer were processed using the CellSearch™ system (Veridex, LLC, Raritan, NJ, USA), in parallel with our CTC assay method. We used anti-CK alone or in combination with anti-EpCAM antibodies for CTC enrichment. Brightfield and fluorescence labeled anti-CK, anti-CD45 and DAPI (nuclear stain) images were used for CTC identification. The Ariol® system (Genetix USA Inc, San Jose, CA, USA) was used for automated cell image capture and analysis of CTCs on glass slides.
Our method has the capability to enrich three types of CTCs including CK+&EpCAM+, CK+&EpCAM-/low, and CK-/low&EpCAM+ cells. In the blind method comparison, our anti-CK antibody enrichment method showed a significantly higher CTC positive rate (49% vs. 29%) and a larger dynamic CTC detected range (1 to 571 vs. 1 to 270) than that of the CellSearch™ system in the total of 49 breast cancer patients. Our method detected 15 to 111% more CTCs than the CellSearch™ method in patients with higher CTC counts (>20 CTCs per 7.5 ml of blood). The three fluorescent and brightfield images from the Ariol® system reduced the number of false-positive CTC events according to the established CTC criteria.
Our data indicate that the tumor-specific intracellular CK marker could be used for efficient CTC enrichment. Enrichment with anti-CK alone or combined with anti-EpCAM antibodies significantly enhances assay sensitivity. The three fluorescent and brightfield superior images with the Ariol® system reduced false-positive CTC events.
Circulating tumor cells (CTCs) are detectable in most blood samples from patients with metastatic cancer using different technologies. CTCs are rare and need to be enriched from the patients' blood sample for better detection [1, 2]. CTC analysis has been performed mostly in breast cancer, the second leading cause of cancer death in women in the US and the Western world. Metastatic breast cancer occurs when tumor cells grow unregulated and eventually lose the ability to adhere to one another. Current models of metastasis support the idea that detached cancer cells travel in the lymphatic system, usually in axilla and intercostal spaces of the sentinel nodes, and/or in the blood system to a new site. Neo-vascularisation develops and a new tumor grows.
Bone is the most common site of metastasis in patients with breast cancer. Detecting tumor cells within bone marrow has emerged as a marker of disease recurrence or survival in breast cancer patients [3]. Braun et al. reported that 30% of women with primary breast cancer have disseminated tumor cells in their bone marrow. In a 10-year follow-up study, Braun et al. were able to show that these patients had a significantly decreased disease-free survival rate and overall survival rate when compared with patients with no disseminated tumor cells [4, 5]. However, sampling of bone marrow is painful for the patient and aspiration cannot be used routinely for breast cancer monitoring.
Detection of CTCs in blood has obvious advantages as a non-invasive sampling procedure and has better potential of being a real-time biopsy of tumors because blood can be sampled frequently. Recently, Meng et al. analyzed CTCs from the blood of patients with newly diagnosed, advanced breast cancer and from patients with recurrent breast cancer by measuring gene status in CTCs compared with cancer cells in the primary tumor tissue [6]. It was concluded that individual tumor cell analysis could provide important information for clinical trials to test the correlations between gene status data obtained from CTCs before treatment and the responses of patients to various therapeutic regimens. This might lead to diagnostic tests that could select the therapy most likely to be effective for an individual patient. This could be an opportunity to evaluate CTCs as potential non-invasive tools for improving selection of individualised therapy [7].
Today, numerous methods are available to analyze CTCs from blood. Slide-based systems are the most commonly used. Traditionally, immunocytochemistry is combined with brightfield microscopy to detect CTCs on microscope slides. In 1999, a consortium of European laboratories participated in the first multi-centre study with the objective of reaching a general consensus on the criteria for defining a circulating epithelial cell as a cancer cell [8]. Subsequently, many new methods were developed that included improved immunomagnetic separation techniques. In addition, fluorescence-based assays gained importance. A review by Fehm et al. gives a good summary of the currently available CTC enrichment methods using slide-based detection assays [2].
CTCs are rarely found in the blood of cancer patients. Therefore, relatively large volumes of blood have to be processed to increase the sensitivity of the assay. Detection of CTCs without target cell enrichment or depletion of unwanted cells is challenging. Hsieh et al. described a high-speed scanning device that allows detection of circulating tumor cells after depletion of red blood cells [9]. However, a comparison to a reference method has yet to prove its superior performance. The well-studied CTC assays available today are based on enrichment with anti-EpCAM antibodies and subsequent detection with anti-cytokeratin (CK) [10, 11]. The CellSearch™ system is one example of these assays; it is based on anti-EpCAM enrichment and is currently the only instrument with regulatory-approval that allows enumeration and characterization of CTCs in blood.
In a study by Cristofanilli et al., 177 patients with metastatic breast cancer were tested for the presence of CTCs using the CellSearch™ system. The study concluded that detection of CTCs before initiation of first-line therapy in patients with metastatic breast cancer is highly predictive of progression-free survival and overall survival [12, 13]. Riethdorf et al. validated the CellSearch™ system in a multi-centre study and concluded that the system allows the reliable detection of CTCs in blood and is suitable for the routine assessment of metastatic breast cancer patients in the clinical laboratory [14].
CK is a specific tumor cell marker and is one criterion for CTC identification. Usually, anti-EpCAM antibody is used to enrich CTCs and CK is used to identify the cells. The differential expression of CK and EpCAM on the same cell will be the key to ensure that no target cells are missed. Those tumor cells that express CK but with low or no EpCAM, or vice versa [15–19], may not be enriched by anti-EpCAM antibody. Here we describe an improved anti-CK-based method for CTC enrichment and detection from the peripheral blood of breast cancer patients. Our method uses the same antibodies to enrich and identify CTCs. To compensate for low or no expression of EpCAM and CK, we also developed an assay with a combination of anti-CK and anti-EpCAM antibodies that allows the enrichment of all types of CTCs including CK+&EpCAM+, CK+&EpCAM-/low and CK-/low&EpCAM+ tumor cells. We developed a staining method that visualizes three fluorescent labeled markers and brightfield cell morphology information for CTC identification and successfully applied it to the Ariol®, an automated image capture and analysis system that can combine the three fluorescent and brightfield images on the same cell simultaneously. The comparison results with the CellSearch™ system have proven that our method has higher sensitivity, reproducibility and better accuracy.
Materials and methods
The breast cancer cell lines MCF-7 and SK-BR-3 (ATCC, Rockville, MD, USA) were used for cell spiking experiments. Blood samples from healthy donors were obtained from Advanced Bioscience Resources, Inc. (Alameda, CA, USA). The healthy donors were between 18 and 50 years of age, and had no current or medical history of malignancy of epithelial origin. They were required to understand and sign an informed consent form that conforms to federal and institutional guidelines. Blood samples from breast cancer patients were obtained from the University of Vermont. All the metastatic breast cancer patients were over 18 years of age. All enrolled patients gave their informed consent for study inclusion and were enrolled using institutional review board-approved protocols. Patients must have had radiological or histological evidence of metastatic cancer with haemoglobin levels more than 10 gm% and haematocrit levels more than 30%. No patients were excluded from this study because of prior medical treatment.
Blood from each healthy donor was collected into EDTA tubes (BD Biosciences, San Jose, CA, USA) or CellSave tubes (Veridex, LLC, Raritan, NJ, USA). Blood samples were maintained at room temperature for different time intervals and processed within a maximum of 72 hours after blood drawing. For the methods comparison study, patient blood samples were shipped from the University of Vermont to Genetix USA, Inc. (San Jose, CA, USA) and Quest Diagnostics Nichols Institute respectively (Chantilly, VA, USA). Blood samples were processed in parallel in the blind study. A 7.5 to 10 ml sample of whole blood was transferred to a 50 ml Falcon tube (Corning, Lowell, MA, USA) and gently mixed with red blood cell removal buffer (CTC Enrichment and Detection Kit, Genetix, New Milton, UK). After approximately five to 10 minutes (when the colour of the blood changed to a transparent cherry red), cells were immediately centrifuged at 700 × g for 10 minutes at room temperature using a Beckman GS-6 centrifuge with GH-3.8 buckets. The cell pellet was carefully resuspended in 0.5 ml dilution buffer (Carcinoma Cell Enrichment and Detection Kit, Miltenyi Biotec, Bergisch Gladbach, Germany). Blocking, permeation and fixation reagents (CTC Enrichment and Detection Kit, Genetix, New Milton, UK) and enrichment reagents (Carcinoma Cell Enrichment and Detection Kit, Miltenyi Biotec, Bergisch Gladbach, Germany) were added as described in Genetix's instructions for the CTC Enrichment and Detection kit.
Cells were incubated for 45 minutes at room temperature and gently mixed every 10 minutes. Dilution buffer was added to each tube to make up a total of 10 ml in each. The tubes were centrifuged at 300 × g for 10 minutes at room temperature and the cells were resuspended in 1 ml dilution buffer. Each sample was carefully applied to the centre of one 0.5 ml dilution buffer equilibrated MS separation column (Miltenyi Biotec, Bergisch Gladbach, Germany) attached to a MiniMACS or OctoMACS separator (Miltenyi Biotec, Bergisch Gladbach, Germany). After washing three times with 0.5 ml dilution buffer, the columns were detached from the cell separator.
Target cells were eluted into a 5 ml tube with 1 ml dilution buffer. Hettich cytospin chambers (Hettich, Germany) were assembled and the eluted target cells were directly added into the cytospin reservoirs using a funnel that creates cytospins with a diameter of 8.7 mm. Target cells were deposited onto Poly-Prep™ PLL (poly-L-lysine coated) glass slides (Sigma, St. Louis, MO, USA) by centrifugation for three minutes at 800 rpm using a Hettich Universal 16 centrifuge (Hettich, Germany). The supernatant was carefully removed using a fine-tipped transfer pipette. The slides were centrifuged one more time for one minute at 1000 rpm to remove any extra liquid. The slides were taken out from cytospin and dried for 30 to 60 minutes at room temperature or 37°C using a slide warmer (Fisher, Pittsburgh, PA, USA). Target cells were fixed in 100% acetone (Sigma) at -20°C for 10 minutes. The slides were dried at room temperature for 30 minutes.
A Shandon Cadenza Immuno Stainer (Thermo Scientific, Waltham, MA, USA) was used for the target cell staining. The glass slides with fixed target cells were assembled onto the Immuno Stainer and washed with PBS buffer (Genetix, New Milton, UK) twice for three minutes. The slides were incubated for 30 minutes with Image iT FX signal enhancer (Invitrogen, Carlsbad, CA, USA). Cells were stained by either direct or indirect labeling antibodies. In the case of direct labeling, we used a set of fluorescein isothiocyanate (FITC) conjugated anti-mouse IgG1 antibodies recognising CK 8, 18 and 19 (CTC Enrichment and Detection Kit, Genetix, New Milton, UK); haematopoietic cells were stained with a DyeLight 549 conjugated anti-mouse IgG1 antibody recognising CD45 (CTC Enrichment and Detection Kit, Genetix, New Milton, UK). The specificity of the antibodies was tested by Western blot analysis and/or immunohistochemistry staining with specific tumor tissues.
The healthy donor's blood samples with no tumor cells were used as negative controls. The blood samples containing tumor cell were used as positive controls. The non-specific binding blocking reagents were used in the CTC assays.
In the case of indirect labeling, the primary antibody cocktail contained a mouse IgG2 anti-CK (CAM5.2, BD Biosciences, San Jose, CA, USA) and a mouse IgG1 anti-CD45 antibody (Lab Vision, Freemont, CA, USA). Slides were incubated for 30 minutes with directly labeled antibodies (anti-CK FITC, anti-CD45 TexasRed) or incubated for 30 minutes with primary antibodies (anti-CK and anti-CD45). They were subsequently washed twice with PBS buffer, then incubated with the secondary antibodies (goat anti-mouse Alexa 488 IgG2a for CK, goat anti-mouse Alexa 568 IgG1 for CD45; Invitrogen/Molecular Probes, San Diego, CA, USA) for another 30 minutes. After washing twice with PBS buffer, the slides were stained with brightfield staining dye (CTC Enrichment and Detection Kit, Genetix, New Milton, UK) to enable visualization of intact cell morphology under white light exposure. The 4'6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories, Burlingame, CA, USA) was used for cell nuclei staining.
Image analysis was performed using the Ariol® system. Slides of stained target cells had barcodes affixed to them and were introduced into the system via the data entry application. The Ariol® was configured for all scanning, image capture and processing to be associated with the CTC assay. The cell presentation area on the slide was fully scanned with FITC, TexasRed and DAPI fluorescence channels and in brightfield. The images of CK-FITC-positive targets were captured and presented in the image gallery. Only the cells with CK and DAPI positive, and CD45 negative [10–15, 20] were counted as CTCs. Cells were further identified by brightfield images that have smooth staining and a round shape to discriminate from debris or cell fragments.
All analyzed data was reviewed and re-classified as appropriate. A report included the selected cell images for the combined fluorescence channels (composite view), an image for each of the separate fluorescence channels and a brightfield image.
The effect of blood storage temperature and addition of a cell preservative on tumor cell stability was tested with tumor cell (GFP or DsRed MCF-7, DAPI labeled MCF-7 or unlabelled MCF-7 and SKBR-3) spiked blood samples. Samples were either drawn into regular EDTA tubes (Becton Dickinson, USA) or CellSave tubes that contain EDTA plus a cell preservative. Samples were tested immediately after blood draw or stored either for 24, 48 or 72 hours at room temperature or 4°C until processing. In total, 60 samples were processed, including 12 controls.
Our method was compared with the CellSearch™ system in a blinded experiment (CTCs were analyzed separately according to the criteria before knowing any results from CellSearch™) using blood samples from metastatic breast cancer patients. The patients' blood samples were obtained from the University of Vermont, and were drawn into two separate CellSave™ blood collection tubes. One tube of the blood was processed in our laboratory (Genetix, USA) in San Jose, CA; another tube of blood was processed by Quest Diagnostics Nichols Institute according to the CellSearch™ protocol the day after blood drawing. In total, 49 patient samples were tested using both methods. Fifty control samples from healthy donors were tested as negative controls.
Blood collection, enrichment assay sensitivity and reproducibility
Spiked MCF-7 cell recovery rates in EDTA and CellSave tubes
At 4°C
At room temperature
0 to 4 hrs
24 hrs
48 hrs
72 hrs
0 to 4 hrs
24 hrs
48 hrs
72 hrs
EDTA tube
63 ± 2
75 ± 1
73 ± 5
69 ± 7
51 ± 17
37 ± 1
29 ± 12
9 ± 5
CellSave tube
75 ± 12
61 ± 22
57 ± 9
70 ± 10
71 ± 9
59 ± 15
49 ± 3
39 ± 13
Spiked MCF-7 cell recovery rates after enrichment from blood
Day repeat
Number of repeats
Recovery (%) mean ± SD
Day 1
74.9 ± 6.0
Day 2
80.6 ± 8.2
Day 3
79.9 ± 9.8
78.9 ± 8.2
CTC detection in metastatic breast cancer patients
Method comparison for circulating tumor cell (CTC) detection from blood samples of breast cancer patients
Patient ID
CTC# (CellSearch™)
CTC # (Ariol ® )
Circulating tumor cell (CTC) distribution
35 (71%)
25 (51%*)
1 to 4
8 (16%)
16 (33%*)
≥ 5
6 (12%)
8 (16%)
Positive %
Patient 68 (Figure 2) had stage IV breast cancer with extensive metastatases to bone. Initially, the patient was treated with tamoxifen (Generic), then weekly paclitaxel when the cancer progressed. The patient went through an incomplete course of radiation therapy. Letrozole (Novartis, USA) was initiated in response to an increasing level of CA27.29 with good response. However, CA27.29 levels increased again, which were reflected in the CTC counts (time point E). Both the CellSearch™ method and our Ariol® CTC method were in concordance with the number of CTCs detected at time points A to E.
Combination of anti-CK and anti-EpCAM antibodies for CTC enrichment
We looked at the CTC detection using anti-CK (our current method) and anti-EpCAM enrichment (CellSearch™ method) in a blind comparison study. Here we also compared the CTC enrichment and detection using anti-CK alone and the combination of anti-CK and anti-EpCAM antibodies in blood samples from breast cancer patients. Figure 4 shows that in one breast cancer patient, 25 CTCs were detected (all single CTCs) in one tube of the blood using anti-CK microbeads and 30 CTCs were detected (28 single CTCs plus one two-CTC cluster) in another tube of the same patient's blood using the combination of anti-CK and anti-EpCAM antibodies microbeads (Figures 4(a), (b), (c)). A 20% higher CTC detection rate was achieved with our new strategy.
By comparing the average CK intensity of the enriched CTCs, the enrichment with the combination antibodies (anti-CK and anti-EpCAM) showed not only a higher CTC count, but also detected more CTCs with relatively low CK expression levels (Figure 4(d)). This result confirmed the theory that the method using a combination of antibodies (anti-CK and anti-EpCAM) had the highest sensitivity, followed by anti-CK enrichment and then anti-EpCAM enrichment.
The three criteria for CTCs determination are considered to be CK+, DAPI+ and CD45- [10–15, 20]. CTCs are rare and heterogeneous in blood. Many different methods have been developed to isolate CTCs from peripheral blood and disseminated tumor cells from bone marrow [2, 21, 22]. Today, available methodologies include density gradient centrifugation [23–31], immunomagnetic cell enrichment [32–39] and/or depletion [40–42], flow cytometry [43, 44], filtration [45–47] and more. Often, different methods are combined with each other, such as density gradient centrifugation and immunomagnetic cell enrichment and/or depletion.
The CellSearch™ is automated, uses standardized reagents and was recently validated in a multi-centre study [14]. The study was conducted at three independent laboratories to validate the analytical performance of the system for clinical use in patients with metastatic breast cancer. It was concluded that the system enabled the reliable detection of CTCs in blood to routinely assess metastatic breast cancer patients. One of the limitations of the CellSearch™ system is the anti-EpCAM antibody-based enrichment strategy. Several authors reported the heterogeneous expression of EpCAM in mammary carcinomas [15, 19]; downregulation of EpCAM was reported for disseminated tumor cells in bone marrow and CTCs in peripheral blood [15, 19, 48].
Most other reported CTC enrichment methods are also based on anti-EpCAM antibodies only, but the enriched CTCs are identified by anti-CK antibodies [10–15, 20]. The reason for using anti-EpCAM antibodies to enrich CTCs is because EpCAM is a cell-surface marker and has an advantage in the cell enrichment process. Since EpCAM is not equally expressed in all tumor cells [15, 19], ideally, it would make better sense to use the same antibodies to enrich and identify CTCs. In the present study, we described an anti-CK antibody-based CTC enrichment method and successfully applied it to CTC assays of patients' blood samples.
In the blind comparison study of our method compared with the CellSearch™ method, we used the same CellSave blood collection tubes, processed the same patients' blood samples simultaneously in our laboratory with our method and in Quest Laboratories with the CellSearch™ method. A higher positive CTC detection rate and a larger CTC detectable dynamic range were obtained from our method in the 49 breast cancer patients. We found that in the patients with low CTC counts, there was a good correlation between the two methods. However, there were also variations in which some patients were CTC positive by our method but not by CellSearch™, or vice versa. These variations may be a result of the low frequency of CTCs or the heterogeneity of CK and EpCAM expression in the CTCs, resulting in certain types of CTCs being missed by one or the another enrichment methods. In the 49 patient samples with CTC counts in the range of one to four, our method showed a significantly higher CTC detection rate (p < 0.01) and a larger CTC detection range than that of the CellSearch™ method. These results suggest that anti-CK antibody-based enrichment is more sensitive than anti-EpCAM antibody-based enrichment.
The most interesting results were obtained from the patients with higher CTC counts. In the patients with more than 20 CTCs, our anti-CK enrichment method showed a 15% to 111% higher sensitivity of CTC detection than that of the CellSearch™ method consistently in all four available patients. In other words, the anti-EpCAM antibody-based enrichment method could lose a portion of the real CTCs population in individual patients during the CTC assay process. The potential CTC loss by anti-EpCAM antibody-based enrichment could be up to 52.7% when compared with anti-CK antibody-based enrichment and could reduce the accuracy for patient CTC monitoring. Similar results were reported in bone marrow samples involving a total of 26 CK+ tumor cells, of which none of them co-expressed EpCAM after chemotherapy. Most soft tissue tumors and all lymphomas were EpCAM negative [15–19, 48]. More research is required to determine if those tumor markers' expression changes are related to cell growth or apoptosis [49]. In the case of CTCs that express low or no EpCAM and low or no CK, we need different strategies to enrich and target the correct cells.
CK is an intracellular protein. The biological mechanism of how this protein can be used for cell enrichment is not entirely understood. Our hypothesis is that the three reactions of cell permeation, anti-CK antibody binding to CK antigens and cell fixation are balanced in the same optimized reaction time period. Anti-CK antibodies penetrate into the cell and bind to CK inside the cell while the microbeads that are coupled to the antibodies remain outside the cell. The cells are permeabilised so anti-CK antibodies are able to bind to CK but not break the cells that will be the key to success. Usually intracellular markers can be more specific; in addition, they can be used alone or in combination with cell-surface markers for better cell enrichment. Sometimes, cell-surface markers are not available or may not be strong enough to enrich cells.
The key to the success of the current study lies with CK enrichment with the intracellular marker that when utilized alone showed equal or better sensitivity than surface-marker EpCAM, which makes the enrichment strategy using combination antibodies meaningful. When the anti-CK antibodies are added to the EpCAM enrichment process, it will be a plus effect. Because CK and EpCAM are both heterogeneously expressed on tumor cells (Figures 3 and 4(d)) [15, 19, 48], the enrichment with the combined antibodies may increase the sensitivity and reproducibility by the compensation effect of biomarkers from each other. CTCs can be classified into three classes: EpCAM+&CK-/low, EpCAM-/low&CK+ and EpCAM+&CK+. CK-&EpCAM- tumor cells are not discussed in this enrichment and detection study. None of the enrichment methods reported have the capability to enrich all three types of CTCs, either because of the enrichment antibody selection or the identification antibody selection, which will cause incomplete CTC profiling (the composition of different CTC types) and CTC downstream analysis for individual patients [6, 16, 29, 35, 50].
Our new CTC enrichment strategy uses the combination of anti-CK and anti-EpCAM antibodies and can theoretically enrich all the three types of CTCs by choosing the correct markers for detection (Figure 4(a)). Our results indicate that a higher CTC detection rate can be obtained by enriching with anti-EpCAM and anti-CK in combination than with anti-CK antibody alone. They also showed that the anti-EpCAM antibody may compensate for low CK expression, giving a better sensitivity and reproducibility for CTC detection in the new strategy (Figure 4(d)). Since most of the CTCs express both CK and EpCAM [51], this new CTC-enrichment strategy will consistently enrich most CTCs with CK+&EpCAM+, CK-/low&EpCAM+ or CK+&EpCAM-/low. Therefore, this strategy may allow enrichment of CTCs for further CTC profiling and downstream analysis and may also be beneficial for the isolation and detection of CTC clusters (Figure 4(c)), which is another advantage in the clinical application [23, 24].
During the blind methods comparison study, we found some events on the glass slide samples that meet the three CTC criteria but it was clearly not a full cell by brightfield image analysis. Reports using the CellSearch™ system describe a five CTC threshold for prognostic relevance. It is not clear that the presence of five CTCs has a clinical significance from 0 or one to four CTCs, or if the threshold is due to the sensitivity and accuracy of the CellSearch™ system. It is clear that no CTCs were detected from the blood samples of most normal individuals [10] and some CTCs are not easy to determine if the rely only on the three fluorescence channels analysis. Our method with the Ariol® system provides additional brightfield cell morphological information that can reduce false-positive CTC events (Figures 3(b), (c)); this may provide additional information to understand the reasoning for the five CTC threshold for prognostic relevance.
Most recently, Nagrath et al. described a microchip technology that detected CTCs from almost 100% of patients with various cancers [1]. It is a very exciting study result, but it does not seem to solve the issue of missing the EpCAM-/low&CK+ CTCs because their microchip uses the anti-EpCAM antibody-based enrichment method. There is no data on a direct comparison between the microchip technology and the CellSearch™ system, so it is not clear how much difference in sensitivity it will make compared with the current Food and Drug Administration cleared CellSearch™ system. To our knowledge, our method with the Ariol® system is the first one to be directly compared with the CellSearch™ system and yield superior functionality in CTC enrichment and detection. The strength of our system is a multiplex anti-CK/anti-EpCAM-enrichment protocol combined with a fully automated image analysis platform using both fluorescent and brightfield detection. This method has the capability to enrich all three types of CTCs for complete CTC profiling and downstream analysis. The advantages of our method are obvious: significantly higher detection of heterogeneous CTCs, better image quality with fluorescent and brightfield images, and better detection of CTC clusters. A fully automated system with these advantages may benefit CTC-related studies and further CTC downstream analysis (fluorescence in situ hybridization, DNA/RNA etc) [35, 50, 52–59].
The anti-CK antibody-based CTC-enrichment method uses an intracellular CK protein marker to enrich CTCs and achieve better sensitivity of CTC detection than that of the surface EpCAM protein marker in blood samples from breast cancer patients. The higher CTC detection in patients with high CTC counts using our method compared with the CellSearch™ system indicates the possible advantages of monitoring, diagnostics and complete CTC profiling. With the combination of anti-CK and anti-EpCAM antibodies, our method has the capability to enrich and detect most types of CTCs, improving the sensitivity and reproducibility for CTC enrichment and detection and providing complete CTC profiling and downstream analysis. Our method with the Ariol® system provides superior cell image quality by combining fluorescent and brightfield images that allow discrimination between intact cells and cell fragments to reduce false-positive counts. The strategy of our method can be used for enrichment of other cell types with specific intracellular protein markers.
circulating tumor cell
coefficient of variation
fluorescein isothiocyanate
standard deviation.
We would like to thank Ms. Patricia Lutton and Ms. Elaine Cahoon from the University of Vermont, College of Medicine, Burlington, Vermont, for coordinating the blood sample collection according to Federal and Institutional Guidelines. We give our thanks to Mr. Scott Artis for proofreading the manuscript and for many helpful conversations and suggestions. We also thank Mr. Jerry Williamson for his full support for this study. This study and the preparation of the manuscript were supported by Genetix.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Hotels in Lubbock
Hotels near South Plains Tech Prep School - Lubbock Texas
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Rodeway Inn Lubbock2 star hotel
Mcm Elegante Hotel & Suites Lubbock3 star hotel
La Quinta Inn Lubbock - Downtown Civic Center3 star hotel
Super 8 Lubbock Civic Center North Hotel2 star hotel
Courtyard Lubbock Downtown/University Area Hotel4 star hotel
Hyatt Place Lubbock Hotel3 star hotel
Overton Hotel And Conference Center Lubbock3 star hotel
- 2401 4Th Street, Lubbock, TX, United States, 79415 , Map
Staybridge Suites Lubbock3 star hotel
Woodrow House Bed & Breakfast Lubbock2 star hotel
Rodeway Inn Lubbock2 star hotel
Howard Johnson Lubbock Tx Hotel2 star hotel
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Koko Inn Lubbock2 star hotel
Guest House Inn & Extended Stay Lubbock2 star hotel
Baymont Inn & Suites Lubbock Near Texas Tech2 star hotel
Best Western Plus Lubbock Windsor Inn3 star hotel
Americas Best Value Inn Medical Center Lubbock2 star hotel
Econo Lodge I-27 Lubbock2 star hotel
Country Inn & Suites By Carlson Lubbock2 star hotel
- 4115 Marsha Sharp Fwy, Lubbock, TX, United States, 79407 , Map
Comfort Inn & Suites Lubbock2 star hotel
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text:
NonConsent/Reluctance Series
NonConsent/Reluctance Series
- Parts:
Cumming to AmericaWoman & her daughter pay dearly for family's trip.
Curiosity Killed KatWhen a maid goes into room she's not allowed in.
Curious GirlsSara pushes her sister's friend too far.
CurrencyThat's what she was to him - at first.
CvsNTwo single mothers try out new living arrangements.
Cynful EnticementBrie gets taken
Cynthia Didn't Ask MeHis encounter with uptight female roommate.
Dabney HunterCollege professor takes virginity from unwilling freshman.
Daddy and BabygirlChapter 1.


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It is no struggle now to cross the bridge
Where the gruesome concrete structure stands.
No wild uneven rocks push back against the bogy ridge
Where knarled and unskilled hands
Made the crossing safe two centauries or more ago
And made passable the track for pigs and sheep and cattle
Through bitter wind , rain and sloughed snow ;
Each crossing a weary and worrisome battle .
Her broken wracked and splintered boulders
Lie mourning in the stream as unrewarded memories
Of the once stout and twisted shoulders
Which fashioned them with granite rock and crude emeries
A double glazed house, like a Hollywood hacienda now proudly stands
With two triumphant tinted windowed sports trucks neatly parked.
On manicured lawns like a model's manicured hands
To champion the success they've each marked
And the little bridge is now more a moat
To give credence to the credit card culture
From where they can luxuriously gloat
And they their satisfied success can pleasurably measure .
I pass over and I know I trespass
On two and more generations , each an epoch apart..
But you know I'd rather wander thought this sparse grass
Aeons ago ; back when this bridge was built
With stout boulders and with solid heart.
© Copyright 2017 donkylemore. All rights reserved.
Status: Finished
Genre: Commercial Fiction
great work, you have wonderfully described something very old and so special to cherish!!! Love the details, such a warm delight!! Oh, thank you again for your lovely comments. surely appreciate your views. thanks!!! be in touch, Vincey.
Thanks Vincey
There was such a bridge .. an old one with the boulders and the banks fallen in.. the newer bridge was bold and brash and there were 2 suvs outside the house built on a hitherto marsh.
I felt the new structure was an attempt to stamp out the ambiance of whatever past lay there , and I resented it , But time and these people march on... but they too are ephemeral.. much more so feel than the former residents , whom I would much rather have known..
Short Story / Commercial Fiction
Poem / Flash Fiction
Essay / Memoir
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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.Got it!
Can someone please help me try to find an answer to this question, I have some credit card debt about $4,000 altogether on several credit cards. And $15,000 on one card that my father had put me on as a user. I work at CVS Pharmacy around 7 hours a week part time and go to college full-time. I just got in the mail from the Sheriff Office of Suffolk County stating they will deduct 10%/week from my check for poundage on Execution fees? For one credit card I owed for $2,888. I am trying to finish school and start to work full time career and pay off these bills, but now they are trying to take over my checking acct and deduct from my little wages i get. Can I claim bankruptcy for these debts outstanding? But will it effect me getting student loans in the near future, Thank you so much
Most likely, you can claim bankruptcy on these debts. However, you must consider whether it is worth filing bankruptcy for your debts. You may not even owe the $15,000 if your father simply added you as an additional user but your credit was not used to obtain the card. As such, you may only be an additional user but not a debtor. Thus, you are left with the $4,000 in credit card debt which most attorneys would agree is not worth filing bankruptcy for.
Regarding any student loans in the future, it depends on the type of loan. Bankruptcy normally doesn't affect your ability to get new federal loans and grants. However, PLUS loans are an exception. The government will look at prior bankruptcies in considering your creditworthiness for a PLUS loan. Also, bankruptcy will affect your ability to get a private student loan and will also affect the cost of that loan. This is because the terms of a private student loan are based on your credit score, which is decreased by a bankruptcy.
That being said, you should definitely consider alternative options before filing for bankruptcy, such as negotiating with your creditors. Please consult with an attorney licensed in your state or possibly a non-profit legal services organization to learn more about alternatives to bankruptcy.
DISCLAIMER The answer given above serves for educational purposes only and is meant to provide general information for a basic understanding of the applicable law. Take notice that the answer above does not create an attorney-client relationship as this website is not intended to provide anyone with case-specific legal advice. Further take notice that any information on this site should not be used as a substitute for case-specific legal advice. Readers should also be aware that laws and their applications frequently change. As such, any information provided on this site is general in nature and may not apply to specific factual and legal situations. Contact a professional, competent attorney licensed to practice law in your jurisdiction to receive case-specific legal advice before making any important decisions regarding your legal issue.
Deciding whether to file bankruptcy is sometimes a cost benefit analysis. In your case the only benefit to you assuming you care about the fact that your father will remain liable for the $15,000 that he put you on a as a second card holder would be $4,000.
With a bankruptcy case averaging between $1,899 (Legal Zoom rate) and $2,500 (average rate) bankruptcy does not make economic sense for you.
Yes, you can find some cheapo attorney or paralegal handle your case for less, but no experienced bankruptcy attorney would recommend bankruptcy for only $4,000 of debt. Why, because you can only file once every 8 years. Should something unforeseen happen to you in the next few years and you become 10 or 20 thousand dollars in debt, you will have not be able to file again until the 8 year period is up. Yes, you can file Chapter 13 in 4 years after a Chapter 7 case but Chapter 13 requires a minimum payment to creditors over a 3 to 5 year period which would most probably not be cost effective either.
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+myConnections: Engage your community - connect to news, events and information you care about. View more information...
View your community's facilities and features online with the new Facilities Module.
Personalize your online experience to stay up-to-date on news, events and other information.
On December 19, the Honourable Minister of Municipal Affairs Diana McQueen, announced that Chestermere would become Canada's newest city as of January 1, 2015.
Obtaining city status is a historic milestone for the community. Once the title is official, Chestermere will be the 11th largest city in the province and the only one that began as a summer village.
An official celebration will be held on Saturday, January 24th at Chestermere's annual Winterfest. More details about the celebration will be provided in January.
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text:
Four inmates who won reduced sentences under racial justice law seek to uphold the decisions
Posted Apr 16, 2014 11:50 am CDT
The North Carolina Supreme Court heard oral arguments on Monday in the cases of four death-row inmates who had their sentences reduced to life in prison under the state's now-repealed Racial Justice Act.
The North Carolina law, passed in 2009, allowed defendants to use statistics to help support claims that their death sentences were affected by racial bias.
The first person to win a reduced sentence under the law was Marcus Reymond Robinson, the New York Times reports. His case was part of a study finding that race was a substantial factor in prosecutors' peremptory challenges in 173 death penalty cases.
Robinson's lawyer, Donald Beskind, and a lawyer for the state, Danielle Elder, differed on the interpretation of the law in the oral arguments on Monday. The Times covered the arguments, along with the Associated Press and the Raleigh News & Observer.
Elder argued that the Robinson's lawyers had not shown evidence of specific or intentional racism. Elder said the judge who reduced Robinson's sentence interpreted the racial justice law "in such a way that a capital defendant can obtain relief even if that defendant has never personally experienced racial discrimination in his case at any point in the criminal justice process."
Beskind countered that the law didn't require the racism to be intentional or to be the sole reason for a death sentence. He told the court that prosecutors used challenges to eliminate half of the qualified black jurors for Robinson's trial.
The court also considered the cases of three other inmates whose death sentences were reduced to life in prison: Tilmon Golphin, Quintel Augustine, and Christina Walters. Their sentences were reduced based on the law as it was amended in 2012.
Don't procrastinate! Missing April 15 tax deadline can be costly, but the IRS offers alternatives
Man in YouTube video of US Supreme Court protest gets time served, is banned for a year
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Better understanding the biophysical basis of the biological process to transfer a viral genome to infect a cell is important to many disease related fields. Predicting the thermodynamic pressures and forces including the osmotic pressure necessary to confine DNA-a highly-negatively charged, elastic polymer-into capsids (over a 250-fold compaction) is a problem with implications not only relevant to infectious disease mechanism but in phage therapy or phage antibiotics(1) and therapeutic delivery(2). Experimental measurements of phage DNA confinement include osmotic pressure ejection-inhibition experiments(3) and single-molecule loading force measurements that provide force or pressure data for validation of theoretical models.(4, 5) Structural insight into DNA packaging is aided by cryo-electron microscopy asymmetric reconstructions done in the NCMI with our collaborator Chiu.(6, 7) Most current models of phage packing assume DNA behaves as a linearly elastic polymer that bends uniformly under stress, like the 'inverse spool'model.(8) The assumption of such spooled conformations is based primarily on interpretations of cryo-EM density maps, obtained by averaging thousands of structures(9), which show density rings, especially near the capsid surface. Recent evidence shows that during translocation packing, the DNA helix is rotated in a left-handed direction thus under twisting it.(10, 11) It Is known tha under twisting reduces persistence length by 2 orders of magnitude when strand separation occurs in sequence specific places.(12) Our hypothesis is that DNA kinking induced disorder can have a strong effect on packing and pressures. How DNA overcomes the unfavorable thermodynamic barrier to enter and pack inside a capsid depends on many different intermolecular interactions. Because phage genomes are around ten kilo-basepairs long, we will employ a multi scale technique to model the structure and consequent thermodynamics. We will refine a coarse-grained model of DNA from our previous work.(13) Preliminary simulations of unconnected DNA coarse grained polymer beads in capsid-like confinement already show ringed density distributions consistent with cryo-EM data. Connected polymer paths will be constructed consistent with data. We will produce an ensemble of entropically-driven, low free energy conformations of DNA in confinement. Ultimately, we will test hypotheses related to the amount of disorder, ion screening and the contribution of DNA-protein confinement interactions.
Understanding the biophysical basis of the biological process which transfers a viral genome to infect a cell is important to many disease related fields. Predicting the thermodynamic pressures including the osmotic pressure necessary to confine DNA in phage capsids (over a 250-fold compaction) is a problem with implications in infection, phage therapies and therapeutic delivery. We will resolve questions of the thermodynamic mechanism of DNA ejection by phages.
|Wang, Qian; Pettitt, B Montgomery (2014) Modeling DNA thermodynamics under torsional stress. Biophys J 106:1182-93
|Myers, Christopher G; Pettitt, B Montgomery (2013) Communication: Origin of the contributions to DNA structure in phages. J Chem Phys 138:071103
|Theruvathu, Jacob A; Yin, Y Whitney; Pettitt, B Montgomery et al. (2013) Comparison of the structural and dynamic effects of 5-methylcytosine and 5-chlorocytosine in a CpG dinucleotide sequence. Biochemistry 52:8590-8
|Howard, Jesse J; Lynch, Gillian C; Pettitt, B Montgomery (2011) Ion and solvent density distributions around canonical B-DNA from integral equations. J Phys Chem B 115:547-56
|Ambia-Garrido, J; Vainrub, Arnold; Montgomery Pettitt, B (2011) Free energy considerations for nucleic acids with dangling ends near a surface: a coarse grained approach. J Phys Condens Matter 23:325101
|Vainrub, Arnold; Pettitt, B Montgomery (2011) Accurate prediction of binding thermodynamics for DNA on surfaces. J Phys Chem B 115:13300-3
|Chen, Chuanying; Pettitt, B Montgomery (2011) The binding process of a nonspecific enzyme with DNA. Biophys J 101:1139-47
|Ambia-Garrido, J; Vainrub, Arnold; Pettitt, B Montgomery (2010) A model for Structure and Thermodynamics of ssDNA and dsDNA Near a Surface: a Coarse Grained Approach. Comput Phys Commun 181:2001-2007
|Howard, Jesse J; Perkyns, John S; Pettitt, B Montgomery (2010) The behavior of ions near a charged wall-dependence on ion size, concentration, and surface charge. J Phys Chem B 114:6074-83
|Feng, Jun; Wong, Ka-Yiu; Lynch, Gillian C et al. (2009) Salt effects on surface-tethered peptides in solution. J Phys Chem B 113:9472-8
Showing the most recent 10 out of 19 publications


text:
Macrosomia describes an unusually large body size. It is often used to describe an oversized fetus.
The most common cause of macrosomia is diabetes in the mother. Macrosomia can lead to trauma during birth and a greater chance of a cesarean delivery.
1000 Johnson Ferry Road NE
Atlanta, GA 30342
(404) 851-8000
201 Hospital Road
Canton, GA 30114
(770) 720-5100
1200 Northside Forsyth Drive
Cumming, GA 30041
(770) 844-3200


text:
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Hotfrog Canada provides information regarding Dr. Ference Andrew in Sherwood Park AB. Dr. Ference Andrew is located at 2018 Sherwood Dr Suite 308, Sherwood Park AB T8A 5V3 and provides Healthcare services. Contact them on (780) 464-4166.
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track list
Album The Magic of Jo Stafford
Label Music For Pleasure (England)
Released March 13, 2001
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Yes We Scan! Privacy Activists Protest Against PRISM and NSA Surveillance As President Obama Arrives in Berlin
18. Juni 2013, 15:46 in Überwachung / 2 Kommentaream
Privacy activists just held a protest at the well-known Berlin Wall crossing point Checkpoint Charlie in Berlin. As President Barack Obama prepares to arrive in the German capital, the protest criticized excessive NSA surveillance and the Prism program. The call from digital rights NGO Digitale Gesellschaft asked for people to come with surveillance equipment, posing as NSA agents and their corporate helpers.
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Tags: Datenschutz, Digitale Gesellschaft, nsa, obama, POTUS, Privacy, Surveillance, Yes We Scan!
2 Kommentare


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Blank canvas of 1/5 acre in early 90s. Developed to lush, colourful, exotic to traditional plant-packed haven. A whole new world on entering from street. Generous borders, raised exotic island, long hot exotic border, ponds, stumpery, developing prairie style border. Conservatory, grapevine. Intimate seating areas along garden's journey. Open:Wed 11 July (11-5); Sun 5, Sun 19 Aug (12-5).
Admission £2.50, Children free. Times:11-5. Telephone:01507 601004/07977 318145. Open for charity.
68 Watts Lane, Louth, Lincolnshire LN11 9DG
Wednesday 11 Jul 2012
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