text:
Product Name: Arrowhead Mills Organic Sprouted Gluten-Free Corn Flakes Cereal
What is it? Looking for a quick and nutritious gluten-free breakfast? Look no further. Arrowhead Mills just released a new gluten-free cereal, Organic Sprouted Corn Flakes. This cereal contains a whopping 33g of whole grains per serving.
Nutrition Snapshot: Made with just seven ingredients, this cereal packs a low sodium nutritional punch. Per serving, there are 3g of fiber and only 110 calories!
Ways to Enjoy: While the typical corn flake might lose its battle against the milk, this one stays nice and crunchy, yet manages to melt in your mouth (the definition of a perfect breakfast cereal, if you ask me!). Pair these delicious corn flakes with some almond milk to heighten the nutty flavor profile. Or, place the corn flakes inside a sandwich bag, smash them into small pieces and use as a coating for gluten-free chicken breasts. The possibilities are endless: cornflake and yogurt parfait, salad topper, muesli … the list goes on and on.
Where to Learn More: www.glutenfreechoices.com/products/arrowhead-mills/
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text:
Photo Gallery
Island Images: Dec. 2013, Part 3
2 of 29
Kilauea Volcano, exhibiting very little drama these days, is mostly smoking. Lava continued to burn through forested lands near Puu Oo Saturday, while Halemaumau Crater generated its own smoky pillar from its lava lake, shown here Thursday. The lake is contained within the Overlook crater, set within the larger Halemaumau. The lava-lake levels fluctuate within a range of a yard or so, but are generally about 157 feet below the floor of Halemaumau. The vent has been mostly active since it opened with a blast on March 19, 2008.
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subset: commoncrawl


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If you need Solid Wood Furniture or Furniture or even Wood Furniture in NC, look no further. www.hotfrog.com showcases more than 38 Solid Wood Furniture businesses across NC. To find more related businesses and to filter by locality, use the left navigation menu.
- 10/19/2009
We make headboards, captains beds, bunk beds, day beds, trundle beds, night stands, mirrors and other solid wood furniture
Handcrafted Solid WoodSolid WoodWood Furniture
Mebane, NC
Makers of fine solid wood furniture. Our unique, antique reproduction solid mahogany furniture has been handmade in America since 1946
Solid Wood Bedroom FurnitureLocal CraftsmanshipFormal Dining Room FurnitureMahogany Furniture
Burlington, NC
Solid Wood Bedroom FurnitureFormal Dining Room FurnitureMahogany FurnitureHandmade Furniture
Hickory, NC
Southern Designs Furniture is discount furniture retail store that offers American-made, solid wood furniture from name brand outlets such as Brown Street, Distinction Leather, La-Z-boy Kids, Sealy, Broughton Hall and m…
American Made FurnitureShaker FurnitureSolid WoodMaple Furniture
Etowah, NC
Carolina Connection, a retail store offering handcrafted country style,solid wood furniture, and home decor. Located in the mountains of western North Carolina
HutchesBarnwood FurniturePine FurnitureFarmhouse Furniture
Garner, NC
…Furniture, Furniture Repair, Furniture Restoration At Heirloom Furniture Creations we design and build solid wood furniture based on your vision for the piece with the details and features that you desire We…
Wood FurnitureBuilt In Cabinets
Lawsonville, NC
We offer a fully customizable line of furniture made from your choice of woods, (pine, antique maple, antique cherry, antique oak, and newly available centuries old reclaimed heart pine), all finished with NON-TOXIC, EA…
Farmhouse FurnitureBookcaseHand Made FurnitureCountry Furniture
Wilmington, NC
Ace Hardware, the Helpful Hardware Place. Ace Hardware has everything you need. Shop our Power Tools, Plumbing Supplies and even Solid Wood Furniture Online
Unfinished FurnitureWood Furniture
Chapel Hill, NC
… attractive styling, and affordability in every product we sale Come in and meet our friendly, knowledgeable staff and we will help you find a quality piece of furniture, that will express your own personal style
Real Wood FurnitureWoodcraft FurnitureDining ChairsDining Tables
franklin, NC
We design and handcraft rustic dining table and chair sets, children's furniture, bed frames and bedroom suites, chests and armoires, benches and barstools and much more for your enjoyment
Rustic Wood FurnitureRustic Dining TablesDining TablesCoffee Tables
Carolina Country Barnwood Furniture, offering handcrafted country style, solid barn wood furniture from 100 year old reclaimed barns. Located in the mountains of western North Carolina
hickory, nc
All of our furnishings, from our contemporary ottomans & footstools to our wrought iron table & chair dining groups, are manufactured in Hickory, North Carolina
Hardwood FurnitureDining ChairsBarstools
Pineville, NC
Tyndall Furniture has been greater Charlotte's premier furniture stores since 1984. We proudly provide the Charlotte metro area with beautiful furniture, expert recommendation & unmatched service
Coffee Tables
High Point, NC
A distinctive line of armoires, entertainment wall units and bedroom suites of solid wood construction
Solid Wood ConstructionSolid WoodWood Furniture
Conover, NC
Computer Desks: Factory direct, American made, fully adjustable, heavy duty, solid hard wood computer furniture, from $238.00
Unfinished Wood FurnitureUnfinished FurniturePine FurnitureComputer Desks
Solid teakwood, rosewood and ebony furniture. Hand-crafted oriental furniture
Oriental FurnitureTeakwood Furniture
Rocky Mount, NC
David Kent Furniture offers to you the highest quality, factory direct, solid wood bedroom furniture
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Hendersonville, NC
Barewood Furniture: Hendersonville North Carolina Unfinished Furniture Store
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Nags Head, NC
BoskoWood Unfinished Furniture Store delivers Outer Banks Furniture style throughout Nags Head, North Carolina and the Outer Banks
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Franklin, NC
Unfinished Furniture North Carolina. Whistle Stop Furniture - the finest selection of unfinished furniture North Carolina has to offer
Buy Unfinished FurnitureUnpainted FurnitureUnfinished Furniture StoreReal Wood Furniture
Archdale, NC
Entertainment ConsolesWood Furniture
Hudson, NC
Kincaid FurnitureSolid Maple FurnitureSolid Oak FurnitureSolid Wood Construction
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Lenoir, NC
Solid Wood Bedroom FurnitureKincaid FurnitureSolid Maple FurnitureSolid Wood Dining Room Furniture
Solid Wood Bedroom FurnitureWood Bedroom FurnitureSolid Oak FurnitureSolid Cherry
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Jamestown, NC
Shaker FurnitureCountry Furniture
Raleigh, NC
Lake Toxaway, NC
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text:
Press Release
Larimer County Sheriff
Larimer County Sheriff's Office deputies and Wellington Fire responded to the Owl Canyon Gliderport near Larimer County Road 7 and Hackamore Road at approximately 9:47 a.m. today on the report of a downed aircraft.
Upon arrival, deputies discovered a fixed-wing aircraft crashed approximately 75 yards west of the runway. The two occupants of the plane received only very minor injuries and refused treatment. The plane was apparently attempting to land at the gliderport when the accident occurred.
The plane, piloted by a student and his instructor, touched down on the runway, veered off the runway and struck a nearby gazebo damaging the left wing of the plane as well as the fuselage. The student and instructor were visiting the gliderport and were not members of the privately owned facility. Access to the facility is restricted.
The Federal Aviation Agency and the National Transportation Safety Board have been notified and will investigate.
A correction to the press release on the non-injury plane crash.
Update from sheriff's office:
Witnesses are reporting that the plane did NOT touch down on the runway before crashing. The plane was attempting to land and hit the gazebo then crashed.
Print This Post


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Penn Hills school construction audit complete
The $19 million that Penn Hills school board officials voted to borrow last summer has not been touched, according to a review of all construction expenses dating back to 2009.
Construction manager Dennis Russo of Russo Construction Services in Plum, along with business manager Richard Liberto, performed the audit over a roughly two-week period at the request of the school district finance committee.
During a discussion of why new tennis courts seemingly disappeared from the original design for the school's campus, Russo said he wasn't comfortable moving ahead with plans to resurface the Yuhas-McGinley Stadium track and look into building tennis courts until he'd been able to go back through all of the documents handed over by previous managers Turner Construction.
The school board voted to terminate its relationship with Turner in September 2012.
Russo said he and Liberto went back to 2009 and spent about 50 hours rebuilding the entire budget.
"There was some information that was not passed on to me when Turner left," Russo said. "There was a gap in the history that the administration had in their computer records. And the (financial) uncertainty was based on all the soft costs that I hadn't been a part of — there were services like purchase orders and engineering that I didn't have on hand."
Russo said that as of the district's last billing cycle, between $32 and $34 million remains in the construction fund, and he is comfortable going ahead with plans for the field resurfacing — which will likely take place following the Pittsburgh East Relay for Life in July, Liberto said — and exploring tennis courts further.
"What we've determined is that (post-construction), we have a $1.1 million surplus at this point, and it will take about $300,000 to $500,000 to put (courts and a new track) in," Russo said.
"I wanted to get the accounting up-to-date and that's basically what we did. So everything seems to be on the up-and-up and we're moving forward."
Russo said construction on the district's consolidated elementary center is also moving forward on schedule.
Patrick Varine is an editor for Trib Total Media. He can be reached at 412-320-7845 or firstname.lastname@example.com.
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Piglet Days!
Hallelujah! Finally … some perfect days to be out with Piglet. It's so nice not to have to bundle up from head to toe before hitting the roads … to not have to wear heavy gloves and heated liners … to be able to breathe in the warm air … and to not have my eyes water when I hit the posted MPH along my route to the station. Warm weather + Piglet = Yeah!
Sleepy-pie for Piglet
Piglet saw a lot more of the first 3 months of 2012 than this year. I'm not sure if this is a testament to how mild last Winter was … or an indication that I'm becoming more of a fair weather scooterer. I hope it's not the latter. In any event, my precious Vespa has spent the better part of the last several months under roof, attached to a battery tender, which was a gift from my husband. A battery tender is a low-current battery charger intended to maintain the charge – without overcharging – while a vehicle is in extended storage. Since the Fall of 2007, Piglet has gone through 3 batteries, so I've gotten to the point where I keep her tendered anytime she's under roof. It takes only a few seconds to connect and disconnect the leads, so it's a not the least bit of an inconvenience, especially given the peace of mind knowing she'll start right up when it's time to hit the road.


text:
Days after cleanup, trash dumped along Clackamas River
CLACKAMAS, Ore. - Hundreds of volunteers scoured the Clackamas River collecting tons of trash last week but just days later someone used the river as their personal garbage dump.
The Clackamas County Sheriff's Office worked to clean up the new dump site but they can't get to it all.
The dump site is just upstream from Riverside County Park. Whoever dumped the trash threw it off a nearly vertical cliff from the road above. They parked in a narrow turnout on Clackamas River Drive and heaved it over the edge.
The sheriff's office had to use its boat to get to the tricky spot.
They found all sorts of trash: paint, diapers, a fishing tackle box and just tons of junk. Deputies found some clues about the culprit in the trash.
"We have an address of where we think this came from, but we don't know if this is the person who cleaned it up, the person who was living there, someone contracted to do it, if it was a pass-off of cleaning service, that's what the investigation is going to reveal," said Sgt. Adam Phillips, spokesman for the Clackamas County Sheriff's Office.
It is the same stretch of river that more than 400 volunteers with We Love Clean Rivers scoured just last week. Unfortunately, a lot of this new round of garbage will have to stay put. It's caught up on the side of the sheer cliff where it's too dangerous to get to.
There was another cleanup Wednesday on the Willamette River as well. Crews with the city of Milwaukie pulled about 20 Oregonian newspaper boxes out of the river after someone dumped them there.
There is a positive to all this. We Love Clean Rivers sponsors an art show - art that it is made out of things they pull from the Clackamas River. That art is on display this weekend in Oregon City on Main Street from 4 p.m. to 9 p.m. starting Friday and ending Sunday.


text:
- Filed Under
The Marion County Council on Aging will host Caregiver Connections on May 2 with special guest speaker Dr. Lynn Ritter.
Caregiver Connections is a quarterly meeting sponsored by the Marion Council on Aging, to provide support and education for local caregivers and others interested in issues of caregiving and dementia.
Ritter is with the Northwest Ohio Chapter of the Alzheimer Association and her topic will be Extreme Home Make-Over. She will address common-sense home modifications such as lighting, furniture placement, food safety, signage and more, with the intent of making the home safer for someone dealing with dementia.
Ritter conveys her message with energy, humor and compassion for the caregiver, according to a news release.
There is no charge to attend, but reservations are limited and necessary. This event will be at the Council on Aging office, 125 Executive Dr. The meeting takes place 11:30 a.m. to 1 p.m., and lunch will be provided by Kingston Residence of Marion.
For reservations, call the Council on Aging at 740-387-0401 no later than April 26.


text:
Madagascar delays electionsComment on this story
Antananarivo - Madagascar's crunch presidential elections, expected to end a long-running political crisis sparked by a 2009 coup, has been postponed by more than two months, the electoral agency announced Tuesday.
"Operational difficulties" prompted the decision to move the vote from May 8 to July 24, the electoral commission said in a statement. A second round, if necessary, will be held on September 25, it said.
The United Nations, which is providing technical support for the polls, endorsed the postponement.
Electoral commission chief Beatrice Attallah told reporters: "We are committed to face the challenge of the election. Hopefully the outcome of this process will be beneficial for Madagascar."
One of the key challenges is funding for the polls, with the international community expected to stump up a large part of the estimated cost of $71 million (52 million euros).
Bitter rivals in the country's political crisis, Andry Rajoelina and Marc Ravalomanana, have opted out of the race, saying their absence would favour a smooth transition.
But analysts warn the pair will likely pull strings from behind the scenes and return to the stage in the 2018 elections.
Rajoelina, a 38-year-old former disc jockey, ousted the Indian Ocean island's elected president Ravalomanana with the army's backing in March 2009.
Ravalomanana is 63. - Sapa-AFP
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text:
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Date: 7 years ago
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Not all about swallowing but if you are going to suck cock take the load in your mouth!
Damn, I hope you don't waste it like that all the time.
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what a blow job i have a friend name colleen
that sucks cock just like that
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this is sampled from a free vid' released by a semi-pro...Anne, i think she calls herself. Been online for years, anyway.
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This.....is how it should be done
Dynamite money shot!!!


text:
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If for any reason whatsoever you are not completely satisfied with your Razor Mate, send it back to us and we will give you a complete refund.
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text:
Most employed people over 16 years of age make social insurance contributions. The amount you pay is based on your earnings and the type of work you do. For this reason it is called Pay Related Social Insurance (PRSI).
The law makes your employer responsible for PRSI, though you may have to pay an employee's share. The amount of PRSI paid by you and your employer depends on your social insurance class. Your social insurance class is in turn determined by your earnings and the type of work you do. You can find more information in our document on social insurance classes.
Your employer deducts your PRSI contribution directly from your wages. It is then collected by Revenue and a record of your contributions is kept by both your employer and the Department of Social Protection. Under the Payment of Wages Act 1991, you must be given a pay slip when you are paid. The PRSI contribution deducted should be noted on your pay slip.
Your employer also pays PRSI contributions for you. This contribution is not deducted from your pay. It is your employer's contribution to the Social Insurance Fund. Again, this is collected by Revenue and a record of the contribution is kept by the Department of Social Protection. The Department publishes information for employers - Employers' guide to the Pay-Related Social Insurance (PRSI) contribution system - SW 3
It was announced in Budget 2016 that a new weekly tapered PRSI credit of €12 is being introduced for employees insured at Class A whose earnings are between €352.01 and €424 in a week. A similar PRSI credit will apply to Class E and Class H employees with weekly earnings between €352.01 and €422 or €424, respectively.
The maximum PRSI credit of €12 per week applies to gross weekly earnings of €352.01. A person earning €352.01 pays €14.08 PRSI (4%). After the €12 credit is deducted they will pay PRSI of €2.08.
For people earning between €352.01 and €424, the credit of €12 is reduced by one-sixth of earnings over €352.01. For example, a person earning €377 would get a PRSI credit of €7.83 – this is one-sixth of the difference between €377 and €352.01 subtracted from the maximum credit of €12. There is no PRSI credit once earnings exceed €424 (January 2016).
The lower 8.5% Class A rate of employer PRSI will apply to weekly earnings up to €376. This threshold will increase from the current €356 (January 2016).
Your employer needs your Personal Public Service Number (PPSN) to make PRSI contributions for you. If your employer does not make the correct PRSI contribution, your employer will be held responsible for the cost of the entire contribution and any arrears that may be due. Failure to meet this responsibility can mean that PRSI which has not been paid can be recovered in court as a debt to the State.
If your employer does not make PRSI contributions on your behalf they may be required to repay to the Department certain social welfare payments you may have received illegally while working for them (these benefits include Illness Benefit, Jobseeker's Benefit, Jobseeker's Allowance, Pre Retirement Allowance, Invalidity Pension, and Family Income Supplement). Your employer may also be liable for any benefit you have lost as a result of them not paying PRSI contributions on your behalf or for repayment of social welfare assistance paid to you in lieu of lost benefit.
Every year, inspectors from the Department of Social Protection visit many employers. The purpose of these inspections is to find employers that:
The Department's inspectors have power to inspect records and there are penalties for people who obstruct or refuse to supply information, or fail to keep or produce records and documents.
In some cases, PRSI contributions from employees who are insurable under Irish social security legislation but do not come within the PAYE system are collected by the Special Collection System operated by the Department of Social Protection.
EU Regulations and bilateral social security agreements allow posted workers, who are sent by their employer on short work assignments from Ireland to the other country, to remain subject to Irish PRSI instead of transferring to the social security system of the other country. For example, if you are employed in Ireland and your employer sends you to work temporarily abroad in another EU state you will remain subject to the Irish PRSI system and will continue to pay PRSI for 24 months. Your employer must complete an A1 form (formerly E101) certificate (available from the PRSI Special Collection Unit) for inspection by the social security authorities in the member state where you are sent to work. More information is available on the Special Collection System (SW 63).
If an employer sends you to work temporarily in a country not covered by EU Regulations or to a country with which Ireland does not have a Bilateral Social Security Agreement, you remain liable for Irish PRSI contributions for up to 12 months. If the employment exceeds 12 months, it may be possible to remain liable for Irish PRSI for longer than 12 months. Employees working abroad who are not subject to the Irish PRSI system may opt to pay voluntary contributions.
A worker who is ordinarily resident in a country not covered by EU Regulations, or with which Ireland does not have a Bilateral Social Security Agreement and who is sent to work temporarily in Ireland by an employer who is not ordinarily registered in Ireland nor has their principal place of business in Ireland, Northern Ireland, Great Britain or the Isle of Man, may be exempt from paying Irish PRSI contributions for up to 12 months.
If your employer pays you when you are sick, you will continue to pay PRSI. Some employers will continue to pay you when you are out of work due to illness provided you hand over to your employer any Illness Benefit payment you get from the Department of Social Protection. In this case, your employer only pays a PRSI contribution on the amount of sick pay minus the amount of Illness Benefit.
If your employer pays you when you are ill and allows you to keep your Illness Benefit payment, your employer's PRSI contribution is paid on the full amount of the sick pay, ignoring the Illness Benefit payment. If your employer does not pay you when you are absent from work through illness, no PRSI contributions are due.
The amount of PRSI both you and your employer pay will depend on your earnings and the social insurance class you are insured under.
For Class A employees in 2015:
If you are earning less than €352 gross per week (before tax is deducted), you will not pay any social insurance. This does not mean that you are not getting a contribution. You are still covered by Class A social insurance. Your employer is paying social insurance on your behalf. Your employer will pay a contribution of 8.5% on your earnings. (The rate was halved to 4.25% from 1 July 2011 and was restored to 8.5% from 1 January 2014.)
If you earn over €352 per week, you will pay 4% PRSI on all your earnings. Your employer pays 8.5% on your earnings up to €356. Your employer pays 10.75% on your earnings over €356.
More information can be found about the amount of PRSI paid in each
Your employer deducts your PRSI contribution from your wages. As an employee who pays PRSI contributions, you are entitled by law to inspect the PRSI record kept by your employer about you. You can also get a statement of your record from your employer every three months.
If you believe that your employer has not complied with social insurance regulations you can ask to fill in form IA49 at your local social welfare office or Intreo centre.
Form IA49 is a declaration stating that you believe your former employer did not comply with social insurance regulations. As a result of this declaration, there will be an investigation into your employer's payment of PRSI.
You can also contact the Records
Section in the Department of Social Protection to check your PRSI
Department of Social Protection
McCarter's Road
Tel:(01) 471 5898
Locall:1890 690 690
Homepage: http://www.welfare.ie/
Department of Social Protection
Social Welfare Services Office
Cork Road
Tel:(01) 471 5898
Locall:1890 690 690
If you have a question relating to this topic you can contact the Citizens Information Phone Service on 0761 07 4000 (Monday to Friday, 9am to 8pm) or you can visit your local Citizens Information Centre.


text:
hey guys, a newbie here from NZ. I see a few familiar people on here though from various other forums.
I'm fortunate enough to own this wacky Mitsubishi that is the Eterna. It was imported into NZ with just 33,000kms in 1987 where it was owned by one owner until I picked it up with 128,000kms on the clock.
It features the same 2L G63B ECI Turbo engine the early jap spec Starions have, with a 5 speed manual gearbox, IRS and 4 wheel vented disc brakes. Inside it's got electric pillarless windows, electric fender mirrors, working air con, and even an alarm clock haha.
Here's how I picked it up:
I drove it around as it was like that for a few months, but I just had to start toying with it
new suspension:
Did a couple trackdays like that, and my last trackday with a TC06 and E39A VR-4 intercooler:
But that's going to change soon, as I'm going to go DOHC and do the suspension properly:
Evo TME TD05 I'll use:
Evo III manifold and AFM:
So yeah, that's about where I'm up to now.
Comments welcome yo


text:
Xenotropic murine leukemia virus (MLV)-related virus (XMRV) was initially identified in prostate cancer (PCa) tissue, particularly in the prostatic stromal fibroblasts, of patients homozygous for the RNASEL R462Q mutation. A subsequent study reported XMRV antigens in malignant prostatic epithelium and association of XMRV infection with PCa, especially higher-grade tumors, independently of the RNASEL polymorphism. Further studies showed high prevalence of XMRV or related MLV sequences in chronic fatigue syndrome patients (CFS), while others found no, or low, prevalence of XMRV in a variety of diseases including PCa or CFS. Thus, the etiological link between XMRV and human disease remains elusive. To address the association between XMRV infection and PCa, we have tested prostate tissues and human sera for the presence of viral DNA, viral antigens and anti-XMRV antibodies.
Real-time PCR analysis of 110 PCa (Gleason scores >4) and 40 benign and normal prostate tissues identified six positive samples (5 PCa and 1 non-PCa). No statistical link was observed between the presence of proviral DNA and PCa, PCa grades, and the RNASEL R462Q mutation. The amplified viral sequences were distantly related to XMRV, but nearly identical to endogenous MLV sequences in mice. The PCR positive samples were also positive for mouse mitochondrial DNA by nested PCR, suggesting contamination of the samples with mouse DNA. Immuno-histochemistry (IHC) with an anti-XMRV antibody, but not an anti-MLV antibody that recognizes XMRV, sporadically identified antigen-positive cells in prostatic epithelium, irrespectively of the status of viral DNA detection. No serum (159 PCa and 201 age-matched controls) showed strong neutralization of XMRV infection at 1:10 dilution.
The lack of XMRV sequences or strong anti-XMRV neutralizing antibodies indicates no or very low prevalence of XMRV in our cohorts. We conclude that real-time PCR- and IHC-positive samples were due to laboratory contamination and non-specific immune reactions, respectively.
Prostate cancer (PCa) is the most frequently diagnosed noncutaneous malignancy among men in industrialized countries [1]. Although early detection using tests for prostate-specific antigen and improved treatment have emerged as important interventions for decreasing PCa mortality, there is potential for improved prognosis through detection of genetic risk factors. Indeed, a positive family history is among the strongest epidemiological risk factors for PCa, and a number of genetic mutations have been implicated in PCa. For example, an R462Q polymorphism in the RNase L protein, which impairs the catalytic activity of an important effector of the innate antiviral response, has been implicated in up to 13% of unselected PCa cases [2].
Xenotropic murine leukemia virus (MLV)-related virus (XMRV) was first identified in PCa tissues, particularly those with the homozygous RNASEL R462Q mutation [3]. Genetic analysis identified XMRV as a xenotropic gammaretrovirus, closely related to those found in mice [4,5]. This suggested that XMRV represented a zoonotic transmission from mice to humans. When compared with exogenous and endogenous MLV sequences, XMRV appeared to have a unique, conserved 24 bp deletion in the gag leader region [3]. However, this deletion has recently been found in endogenous MLV proviruses in a variety of mice [6]. Initially, immuno-histochemistry (IHC) and FISH analyses suggested that only prostatic stromal fibroblasts were infected with XMRV [3]. Subsequently, Schlaberg, Singh and colleagues reported the expression of XMRV antigens in 23% of PCa and an association of XMRV infection with higher grade tumors [7]. Contrary to the initial study, Singh's study found viral antigen-positive cells primarily in malignant prostatic epithelium, independently of the RNASEL polymorphism [7]. It is notable that this study found many immuno-histochemistry-positive samples which did not have detectable XMRV DNA [7]. Another study found 11 (27.5%) of 40 PCa patients with XMRV neutralizing antibodies [8]. Importantly, there were correlations between serum positivity and nested PCR results, FISH, or the R462Q RNASEL mutation [8]. In sharp contrast, several recent reports found no or very low prevalence of XMRV (DNA, RNA or antibodies) in PCa samples [9-12].
If the role of XMRV in PCa is confirmed, detection and prevention of XMRV infection could provide a novel intervention strategy for early diagnosis and treatment of PCa. However, the conflicting epidemiological data have made it unclear whether XMRV plays a role in PCa and have questioned whether the virus is truly a human pathogen. In this study we have sought to address the association between XMRV infection and PCa, PCa grades and RNASEL R462Q polymorphism by testing prostate tissues for the presence of XMRV. In addition, to determine the correlation between PCa and seroprevalence of XMRV, serum samples from patients with PCa were compared with age-matched controls for detectable anti-XMRV antibodies. Our study found no XMRV sequences and no XMRV-neutralizing antibodies in 150 prostate tissues (110 PCa and 40 benign/normal) and serum samples (159 PCa and 201 age-matched controls), respectively, indicating no or very low prevalence of XMRV in our cohorts. We did detect MLV sequences in 6 samples, but these samples were also PCR positive for mouse mitochondrial DNA suggesting DNA contamination as a source of the MLV. We were therefore unable to confirm the links between XMRV infection with PCa, PCa grades or RNASEL mutation.
Prevalence of XMRV proviral DNA in PCa
We have previously developed a real-time PCR assay for detection of XMRV gag sequences [13,14]. Tests using the XMRV infectious molecular clone plasmid, pcDNA3.1(-)/VP62, could detect a single copy of the XMRV genome in 1.0 μg of total cellular DNA (approximately 1.4 × 105 cells). The primers and the probe used in this assay were designed to detect most MLV-related sequences from mice. Using this sensitive real-time PCR assay, we screened DNA from 150 prostate tissues (110 PCa and 40 benign/normal controls). One out of 40 high grade PCa (Gleason score 8-10), 4 out of 70 intermediate grade PCa (Gleason score 5-7), and 1 out of 40 benign/normal prostate tissues (Gleason score <4) were repeatedly positive by this assay (Table 1). The viral DNA copy numbers ranged from 0.5 to 11 copies per 1.0 μg DNA (average of 4 reactions). As one diploid cell contains approximately 7.1 pg of DNA, we estimate that PCR-positive clinical samples had 0.5 to 11 copies of proviral DNA in 1.4 × 105 cells.
Table 1. Prevalence of XMRV and tumor grade
To confirm the real-time PCR results, we screened the same DNA samples by nested PCR for XMRV/MLV gag sequences. In order to establish consistency and to minimize the risk of contamination during the procedure, three individuals independently performed the nested PCR experiments using independently aliquoted DNA samples. Four out of 6 real-time PCR positive samples (#15, 51, 52 and 112) were consistently positive by the nested PCR analysis, while the other two positive samples from intermediate grade PCa (#53 and 103) were shown to be nested PCR-positive twice in the first three attempts. Further analysis confirmed that these two samples were nested PCR-positive for viral DNA. The 144 real-time PCR-negative samples were also found to be negative by nested PCR.
No statistical link between the presence of viral DNA and prostate cancer or higher tumor grade
We then sought a correlation between viral DNA detection and the presence of PCa. There was no statistical difference between the frequency of PCR positivity in PCa and in benign/normal controls (Table 2). We also examined a link between PCR positivity and tumor grade as measured by the Gleason score. Using total of 110 DNA samples from PCa, 4 out of 70 intermediate grade (Gleason score 5-7) and 1 out of 40 high grade (Gleason score 8-10) were positive by real time PCR (Table 3). These data were not statistically significant by chi-square (x2) as indicated in Table 3, suggesting no correlation between the prevalence of viral DNA and higher tumor grade in our samples.
Table 2. Statistical analysis of XMRV positivity in controls and PCa
Table 3. Statistical analysis of XMRV prevalence and tumor grade
No correlation between viral DNA detection and RNASEL R462Q mutation
In order to consider the association between RNASEL mutation and viral infection, we amplified part of the RNASEL gene by PCR and determined the status of the R462Q RNASEL polymorphism. Of 150 prostate tissues, 20 cases were found to be homozygous for RNASEL R462Q (Table 4). However, these samples were all negative for viral DNA by real-time PCR. Thus there was no linkage between viral DNA detection and RNASEL R462Q in our clinical samples (Table 5).
Table 4. RNASEL genotyping and tumor grade
Table 5. Statistical analysis of XMRV prevalence and RNASEL genotyping
Phylogenetic analyses of MLV-like sequences in prostate tissue DNA
XMRV has been PCR amplified from prostate cancer samples in a number of studies [3,8,12] as well as in blood samples from patients with chronic fatigue syndrome (CFS) [15]. Furthermore, a recent study reported a high frequency of MLV that was distinct from XMRV by PCR in patients with chronic fatigue syndrome [16]. To examine the viral sequences identified in our PCa samples, we cloned the PCR-amplified DNA bands from four viral DNA-positive patient samples (#15 [GenBank no. JF288880, JF288881], #51 [GenBank no. JF288878, JF288879], #52 [GenBank no. JF288882, JF288883] and #112 [GenBank no. JF288884]) and determined their nucleotide sequences. We were able to identify two independent sequences from each of patients #15, #51 and #52 and a single sequence from patient #112. To compare these sequences to XMRV and to previously published MLV sequences from mice and patient samples, we reconstructed Bayesian phylogenies (Figure 1). None of the gag gene sequences amplified from our clinical samples belonged to the clade formed by previously reported XMRV sequences; instead, they clustered with known polytropic murine leukemia virus (PMLV), modified polytropic murine leukemia virus (MPMLV) or xenotropic murine leukemia virus (MLV-X) endogenous sequences of mice (Figure 1). Importantly, one of the patients (#52) appeared to be infected with two independent MLVs, one from the modified polytropic MLV clade and one from the xenotropic MLV clade. A similar result was seen when a maximum likelihood phylogeny was constructed using the software RAxML [17] (not shown). In each case, BLAST analysis of the amplified sequences identified at least one endogenous MLV sequence in the mouse genome with very high (>99%) similarity (Table 6). Two of the five fragments were identical to known endogenous proviruses and the other three were greater than 99% similar. These proviruses exist in multiple locations within the mouse genome.
Figure 1. Bayesian maximum clade credibility phylogeny of endogenous murine MLV sequences, 22Rv1 cell line and patient derived MLV gag gene sequences. Sequences derived from PCa samples in this study are colored red. Sequences from [16] are colored blue. The tree is rooted against the Moloney MLV sequence. Bayesian posterior probabilities above 0.50 are indicated on the corresponding branches. The scale bar represents the number of nucleotide substitutions per site.
Table 6. Comparison of MLV sequences amplified from patient samples with mouse genomic sequences
Because the sequences we amplified were similar to the MLV sequences detected in CFS patients [16], we also analyzed the sequences reported in that study. The sequences amplified from CFS patients also fell into both polytropic and modified polytropic clades of endogenous MLVs (Figure 1). They were also very similar (98-100%) to known endogenous MLV proviruses in mice (Table 7). In fact, the differences between the amplified sequences and the endogenous sequences are consistent with known error rates of Taq polymerase or could also be explained by polymorphisms between mice [18-20].
The similarity between the patient amplified sequences and known endogenous MLV provirus sequence in mice suggests that the MLVs may have been amplified from samples that had been inadvertently contaminated with mouse DNA. To examine this possibility further in our samples we tested each positive PCa sample for the presence of mouse mitochondrial DNA by PCR. Strikingly, all of the clinical samples that were positive for MLV were also positive for mouse mitochondrial DNA (Figure 2). When the amplified DNA fragments were cloned and sequenced, they were 100% identical to Mus musculus cytochrome b gene sequence. Thus, the MLV sequences detected by sensitive PCR methods in patient samples likely originated from contaminating mouse DNA encoding endogenous MLV proviruses.
Detection of XMRV antigens in PCa tissues
Previous IHC studies found XMRV antigen-positive cells in prostatic stromal fibroblasts [3] or in malignant prostatic epithelium [7]. Importantly, Schlaberg, Singh and colleagues also showed frequent detection of viral antigen-positive cells in PCR-negative tissues [7]. In order to seek viral antigen-positive cells in our clinical samples, we prepared prostate tissue sections and performed IHC analysis. We used the rabbit anti-XMRV antibody, which was used in the previous study by Schlaberg et al. [7]. We also used a goat anti-MLV p30/gp70 antibody, which can detect XMRV precursor Gag, CA, and Env proteins in XMRV transfected cells [13,14]. Both antisera showed clear and reproducible staining of 293T cells transfected with the infectious XMRV clone VP62 (Figure 3A) or XMRV-producing 22Rv1 cells (data not shown). No specific staining was seen when uninfected control 293T cells were stained with these antisera (Figure 3A). We prepared tissue sections of four PCR-positive tissues (Gleason scores 6 and 8) as well as two PCR negative tissues (Gleason scores 6 and 8, real-time/nested PCR-double negative), and analyzed them with the two antisera. The anti-XMRV antibody sporadically detected antigen-positive cells, exclusively in prostatic epithelium, in the sections of tissues (Figure 3B, upper middle panel with FITC). Similar results were observed with a different secondary antibody conjugated with Texas Red (Figure 3C). In contrast, no signal was detected with the anti-MLV p30/gp70 in any of the tissue sections (Figure 3B). Importantly, the anti-MLV serum did not stain the cells, which were shown to be IHC-positive by the anti-XMRV serum, in the serial sections of the same tissue (Figure 3B, upper panels). It was also notable that the anti-XMRV serum found antigen-positive cells in PCR-negative tissue sections (Figure 3C), suggesting that this serum also recognizes a non-viral protein. Similar results were recently reported by Switzer et al [21]. Considering the data obtained using the anti-MLV serum, we conclude that we cannot detect XMRV in prostate cancer tissues and that the antibody described by Schlaberg, Singh and colleagues recognizes non-viral proteins in addition to XMRV.
Figure 3. Detection of XMRV in prostate cancer tissues. (A) Specificity of anti-XMRV antiserum and anti-MLV antibody. 293T cells transfected with XMRV infectious plasmid (pcDNA3.1(-)/VP62) were stained with either rabbit anti-XMRV or goat anti-MLV. No positive staining was observed in control uninfected 293T cells. (B) Serial tissue sections from qPCR positive samples, including #51 (Gleason score (GS) 8) and #103 (GS 6) were immunostained with either anti-XMRV or anti-MLV antibody. H&E staining from each sample is also shown. (C) Serial tissue sections from qPCR positive (#112, GS 6) and negative (#47, GS 8) samples were immunostained with anti-MLV antibody, followed by TexasRed-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories, Inc., 1:200).
Absence of XMRV antibodies in patients with PCa and age-matched controls
Serological testing was performed with a recently developed XMRV neutralizing assay which measures viral neutralizing activity using a GFP-encoding XMRV and flow cytometry [14]. Positive seroreactivity was defined as 100% block of XMRV-GFP transduction with a 10-fold diluted serum sample. We randomly sampled 159 PCa cases out of 933 patients who are consenting, age 50-70 and have a clinical Gleason Score of 6 or 7 (most common) in the Mayo Clinic Prostate SPORE Biospecimen files. 201 sera from age-matched patients without PCa or any known urological disorders were included as non-PCa controls. As positive controls, we used anti-XMRV sera from XMRV-infected wild mice, Mus pahari [14]. Sera from XMRV-infected mice diluted 10-fold completely blocked XMRV-GFP infection (Figure 4A). In contrast, none of the clinical samples showed strong anti-XMRV activity at 10-fold dilution (Figure 4B). Two out of 159 PCa (Figure 4C) and five out of 201 non-PCa (Figure 4D) sera marginally reduced the XMRV infectivity (over 80% block of XMRV infectivity at a 10-fold dilution). However, by Western blot probing cell lysates from XMRV-infected and uninfected cells, these patients' sera failed to detect XMRV Env, Gag or p30 Capsid (data not shown). To rule out the possibility that these patients' sera cannot detect denatured XMRV proteins by Western blotting, we also performed the indirect immunofluorescent assay using HeLa cells (control) and XMRV-infected HeLa cells as antigens. None of the sera could detect XMRV antigens in HeLa cells at 50- and 200-fold dilutions (data not shown). We, therefore, conclude that XMRV antibodies are absent from our patient population.
Figure 4. Neutralization activity of patient sera. (A) XMRV infected 293T cells (XMRV+ control) and XMRV-infected and treated with anti-XMRV sera at a dilution of 1:10 [14] are shown. (B) Data from non-XMRV infected 293T cells is shown as a control. Patients samples which did not show positive neutralization reaction (Patients with non-prostate cancer (NonPCa)-1, Patients with prostate cancer (PCa)-1) are shown. (C) Two samples that showed positive reaction from patients with prostate cancer (PCa-2 and -3) are shown. (D) Six samples that showed positive neutralization reaction from patients with non-prostate cancer (NonPCa-2 to -6) are shown. 1:10 dilution of sera were applied for all the experiments. Percent GFP positive and percent neutralization are indicated within the gated areas and below the flow data, respectively. The percent neutralization was calculated as the reciprocal of infectivity, with a maximum infectivity being determined by incubation of the virus with an uninfected mouse serum. n.a., not applicable.
In this study, we have examined the prevalence of XMRV in patients with or without PCa at Mayo Clinic. We were unable to find XMRV sequences or anti-XMRV antibodies in our patients, most of whom are from the mid-west area of the USA, indicating that there is no or very low prevalence of XMRV in this region. Moreover, we were unable to confirm the correlation between XMRV infection and PCa, higher tumor grade or RNASEL R462Q mutation.
A high prevalence of XMRV has been reported in patients with PCa and chronic fatigue syndrome (CFS) in the USA [3,7,8], but similar studies in Europe have failed to detect XMRV [10-12]. It has been suggested that geographical differences might explain this striking variation in XMRV prevalence [11] but our results, as well as recent US studies that also find no evidence for XMRV [9,21], appear to rule this explanation out. In this regard, it is notable that previous studies to identify XMRV in patients with PCa or chronic fatigue syndrome have relied on very sensitive PCR detection methods. Because of the high similarity between patient associated XMRV/MLV and endogenous MLV sequences and the striking discordance between studies, it has been suggested that PCR-positive results might be attributed to unintentional detection of contaminating mouse DNA in human specimens [6,22-24]. It is notable that Lo et al. [16] detected polytropic and modified polytropic MLV sequences, but not XMRV, in blood samples from chronic fatigue patients (Figure 1). These authors were unable to identify the samples as contaminated using mouse mitochondrial PCR. In our study, real-time PCR and nested PCR identified 6 of 150 samples as positive for MLV. However, the amplified sequences were closely related to known endogenous MLV proviruses, rather than XMRV. In fact one patient sample (#52) contained two independent MLV sequences. This might be interpreted as evidence for evolution of the virus in the patient but closer analysis reveals that one of the sequences is identical to a known endogenous modified polytropic sequence whilst the other is a single nucleotide different from a known mouse endogenous xenotropic MLV. This, therefore, suggests either infection of this patient with two independent MLVs or PCR contamination with mouse DNA as a source. As all of the MLV PCR-positive samples contained detectable levels of mouse mitochondrial DNA, we conclude that the amplified sequences originated from mouse DNA that somehow contaminated the study samples.
In order to confirm that the viral sequences were amplified from endogenous MLV in mouse genomic DNA, but not replicating MLV in human tissue, we attempted to determine viral integration sites. We first used the protocol described by Kim et al. [25] but failed to amplify DNA sequences containing the partial XMRV LTR. We then designed universal primers to recognize LTRs from XMRV and endogenous and exogenous MLVs [26], as well as a series of primers specific for the viral sequences identified in our clinical samples. Unfortunately, we were not successful, likely due to low viral copy numbers in the clinical samples. Very recently, Robinson et al. [23] and Oakes et al. [22] reported similar observations; all XMRV PCR-positive specimens contained detectable levels of mouse mitochondrial or endogenous retroelements (IAPs). Together with our data, these findings highlight the difficulty of avoiding DNA contamination in clinical samples and the risk of testing contaminated samples as XMRV-positive by sensitive PCR detection assays. As a possible source of contamination, Sato et al. [24] demonstrated that a commercially available hot-start PCR enzyme contained mouse DNA. We used several enzymes and obtained similar results. Thus, it is unlikely that the contaminating mouse genome originated from a PCR kit. Since we could amplify the viral sequences from multiple aliquoted DNA samples, they appeared to be contaminated before or during the DNA isolation step, most likely during tissue sectioning on a microtome.
XMRV antigen-positive cells have been detected in prostatic stromal fibroblasts [3] or in malignant prostatic epithelium [7]. Our IHC study using two different antisera showed conflicting results. The goat anti-MLV antibody found no viral antigens in clinical samples, while the rabbit anti-XMRV antibody used in the study by Schlaberg, Singh and colleagues [7] detected antigen-positive cells in prostatic epithelium. Strikingly, the goat anti-MLV serum did not stain the cells, which were IHC-positive by the anti-XMRV rabbit serum, in serial sections of the same tissue. The rabbit antiserum also found antigen-positive cells in PCR-negative sections, confirming the observations of Schlaberg and colleagues who reported frequent detection of IHC-positive samples in PCR-negative tissues [7]. Importantly, both the rabbit and goat antibodies detected XMRV in experimentally infected cells with high sensitivity (Figure 3). Together, these observations strongly suggest that the rabbit antiserum is detecting a non-viral antigen sporadically expressed by tumor cells in the tissue section. We conclude that our PCa samples do not have XMRV antigen-expressing cells that are detectable by IHC.
We recently reported that Mus pahari mice elicit potent XMRV-specific humoral immune response upon XMRV infection [14]. At a serum dilution of 1:640, antisera from infected animals almost completely blocked XMRV infection [14]. Similarly, an animal study using XMRV-infected rhesus macaques and sensitive ELISA detection assays showed that infected animals rapidly develop antibodies against XMRV proteins, including gp70 (Env), p15E (transmembrane), and p30 (CA) [27]. These results indicate that XMRV is strongly immunogenic in these animals. In contrast, we were unable to detect strong XMRV-specific neutralizing antibodies in our 360 patients, age 50-70, with or without PCa. This observation further suggests a lack of XMRV in our cohorts. It is possible, although less likely, that XMRV is not immunogenic in humans or that XMRV-specific immune response might have disappeared in these relatively elderly patients.
In our study population of patients with or without PCa from the USA, we found no evidence of infection with XMRV using PCR, IHC and serological tests. Our negative results are in accordance with previous studies using sensitive PCR, ELISA and Western blot assays, which failed to detect PCR or seropositive samples in a large number of blood donors, HTLV- and HIV-infected, or patients with or without CFS [9-12,21,27-31]. Our results indicate the possible false-positive detection of XMRV/MLV-related sequences or antigen-positive cells through laboratory contamination or non-specific immune reaction respectively, and underscore the need for careful validation of previous and future studies.
Materials and methods
Prostate tissues and plasma samples from patients
Prostate tissues and plasma samples were obtained from Mayo Clinic Biospecimen Core with an approval from the Institutional Review Boards. Frozen sections of prostate cancer tissues (10 μm) were identified as 1 through 150 in duplicates. These samples included 40 normal/low grade Gleason score, 70 intermediate (Gleason score 5-7), and 40 high grade (Gleason score 8-10) with men aged between 50-70 years old. For plasma analysis, total of 360 plasma samples from 50-70 year old male patients including 159 prostate patients (Gleason score 5-7) and 201 patients with no prostate cancer or urological disorders were used in this study.
TaqMan qPCR
Total cellular DNA was extracted by PureLink Genomic DNA Mini Kit according to the manufacturer's protocol (Invitrogen). All samples were eluted in 50 μl of elution buffer, and the concentration and quality of the DNA were determined by a NanoDrop Spectrophotometer. For the real-time PCR assay, TaqMan Universal PCR Master Mix (Roche) was used along with 2 μl of each sample. Primers were used at a range of 230 nM to 300 nM final concentration. TaqMan probe #51 from Roche Universal Probe Library was used for XMRV-gag at 100 nM final concentration. A standard curve was created by using serially diluted XMRV plasmid (pcDNA3.1(-)/VP62). The assay was analyzed by the ABI 7300 Real-Time PCR System using the default thermal cycling conditions for the two-step RT-PCR method and FAM reporter [13].
RNASEL genotype was determined by nested PCR amplification using outer primers 5'-CTGGGGTTCTATGAGAAGCAAG-3' and 5'-TGAGCTTTCAGATCCTCAAATG-3', and inner primers 5'-GAGAGAACAGTCACTTGGTGAC-3' and 5'-CAGCCCACTTGATGCTCTTATC-3' with pfx polymerase (Invitrogen). Final PCR products were purified with QIAquick PCR Purification Kit (Qiagen) before sequence analysis.
Neutralization assay
The neutralization assay was carried out using GFP-encoding XMRV as described previously [13,14]. Briefly, 293T cells were transfected with pcDNA3.1(-)/VP62 and a GFP-encoding retroviral vector using FuGene 6 (Roche). Serum samples were heat inactivated at 56°C for 30 min. A mixture of plasma samples and 2.5 × 104 infectious units of GFP-carrying XMRV were incubated at 37°C for 30 min before infecting 293T cells (5 × 104). Three days post-infection, cells were resuspended, fixed with 4% paraformaldehyde and analyzed by flow cytometry (BD FACScan). The percentages of GFP-positive cells were measured using CellQuestPro software [14].
Western blot analysis
For Western blot analysis of XMRV proteins, cell lysates of prostate cancer (PC-3) cell line (ATCC) and PC-3 infected with XMRV were harvested in 1.0 ml of RIPA lysis buffer. Cell debris was removed by centrifugation, and the supernatant was diluted with Laemmli sample buffer containing β-mercaptoethanol. After heat-denaturation at 95°C for 5 min, 10 μl of proteins were subjected to SDS-PAGE with a 4-15% gradient gel (Bio-Rad), and transferred to a polyvinylidene diflouride membrane at 0.7 mA/cm2 for 40 min. Membranes were blocked in 5% milk/PBS, then stained with patient's plasma samples diluted to 1:250, followed by anti-human IgG (1:1000, Jackson ImmunoResearch Laboratories, Inc.).
Immunohistochemistry was performed on tissue samples from patients with or without prostate cancer. Sections were fixed with 4% paraformaldehyde for 20 min and treated with 0.3% Triton X100 for 15 min at room temperature. They were then blocked with 5% FBS/PBS for 30 min and immunostained with rabbit-anti XMRV (kindly provided by Dr. Ila Singh, University of Utah) or goat-anti p30/gp70 (NCI HD625 CAT No. 04-0109, LOT No. 81S000262, Quality Biotech, kindly provided by Dr. Yasuhiro Takeuchi, UCL) at a dilution of 1:500 for 4 h at room temperature. FITC-conjugated anti-rabbit IgG (1:500; Amersham) or DyLight 488-conjugated anti-goat IgG (1:500; Jackson ImmunoResearch Lab) were applied for 2 h at room temperature. Nuclei were then counter-stained with 4'-6-Diamidino-2-phenylindole (DAPI), and analyzed by confocal microscopy (Zeiss).
Nested PCR and sequence analysis of proviral DNA
Sequence analysis was performed as previously described [14]. Briefly, DNA was extracted by PureLink Genomic DNA Mini Kit (Invitrogen). Nested-PCR was performed for XMRV gag (primers for outer gag: 5'-ACGAGTTCGTATTCCCGGCCGCA-3' and 5'-CCGCCTCTTCTTCATTGTTC-3', primers for inner gag: 5'-GCCCATTCTGTATCAGTTAA-3' and 5'-AGAGGGTAAGGGCAGGGTAA-3') with platinum Taq polymerase (Cat. no. 10966-034, Invitrogen). The resulting PCR products from a total of 4 patient samples (#15, #51, #52 and #112) were cloned into the TOPO vector (Invitrogen). Sequences from the two patient samples #53 and #103 were not analyzed. From patients #15, 51, 52, 112, we sequenced 2, 2, 4, 1 clones, and got 2, 2, 2, 1 different sequences, respectively. They were analyzed by DNADynamo (BlueTractorSoftware).
Phylogenetic analyses
Seven unique gag gene sequences (255 to 528 nt), amplified from our clinical samples (GenBank no. JF288878, JF288879, JF288880, JF288881, JF288882, JF288883, and JF288884), were manually aligned with previously described murine leukemia virus gag gene sequences (n = 79), 22Rv1 cell line derived gag sequences (1605 nt; n = 15), XMRV gag sequences apparently amplified from prostate cancer and CFS samples (n = 7) [6], as well as 6 MLV virus gag sequences isolated from chronic fatigue syndrome samples [16]. Bayesian phylogenies were reconstructed as previously described [6]. The Markov chain Monte Carlo search was set to 10,000,000 iterations, with trees sampled every 1000th generation, and with a 20% burn in. The phylogeny of the aforementioned sequences was also reconstructed by maximum likelihood (ML) inference under the general time reversible model of nucleotide substitution, with gamma-distributed rate heterogeneity and proportion of invariable sites, using the program RAxML (data not shown) [32]. The ML topology was assessed by neighbor joining bootstrapping with 1000 replicates using the program PAUP*.
A semi-nested mouse-specific mtDNA PCR
We used a PCR assay for mouse mitochondrial DNA reported to be able to detect 2.5 fg of mouse DNA in the presence of 35 ng human background DNA [16]. Using this assay, we tested whether our samples were contaminated with mouse DNA. DNA from PCR positive samples were PCR amplified with KOD Hot Start DNA Polymerase following the manufactures instruction (Novagen) as described [16]. The resulting PCR fragments were further cloned into the TOPO vector and the sequences were confirmed to be identical to the mouse cytochrome b gene by DNA BLAST.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TS, SH, KAS, JMT, and PRB performed experiments. TS, SH, GT and YI designed the experiments, analyzed the data and wrote the paper. All authors read and approved the final manuscript.
Rabbit-anti XMRV and goat-anti p30/gp70 were kindly provided by Dr. Ila Singh and Dr. Yasuhiro Takeuchi respectively. This work was supported by the National Institute of Health (AI093186), Mayo Clinic Career Development Project in Prostate SPORE grant CA91956-080013, the Mayo Foundation (YI), Wellcome Trust senior fellowship WT090940 (GJT) European Community's Seventh Framework Programme (FP7/2007-2013) under the project 'Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)', grant agreement no. 223131 (SH) and the National Institute of Health Research UCL/UCLH Comprehensive Biomedical Research Centre (GJT).
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J Virol 2011, 85:1205-1213. PubMed Abstract | Publisher Full Text
Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, et al.: Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome.
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Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, Wang R, Alter HJ: Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors.
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Stamatakis A: RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models.
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Oakes B, Tai AK, Cingoz O, Henefield MH, Levine S, Coffin JM, Huber BT: Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences.
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Robinson MJ, Erlwein OW, Kaye S, Weber J, Cingoz O, Patel A, Walker MM, Kim WJ, Uiprasertkul M, Coffin JM, McClure MO: Mouse DNA contamination in human tissue tested for XMRV.
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Kim S, Kim N, Dong B, Boren D, Lee SA, Das Gupta J, Gaughan C, Klein EA, Lee C, Silverman RH, Chow SA: Integration site preference of xenotropic murine leukemia virus-related virus, a new human retrovirus associated with prostate cancer.
J Virol 2008, 82:9964-9977. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
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Groom HC, Boucherit VC, Makinson K, Randal E, Baptista S, Hagan S, Gow JW, Mattes FM, Breuer J, Kerr JR, et al.: Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome.
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Hong P, Li J, Li Y: Failure to detect Xenotropic murine leukaemia virus-related virus in Chinese patients with chronic fatigue syndrome.
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Swofford DL: PAUP*. Phylogenetic analysis using parsimony (* and other methods). Volume 4. Sinauer Associates, Sunderland, MA; 1998.


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Like all special education instructors, Cami Burton aims to help her students with autism and developmental disabilities master real-life skills that will allow them to become more independent.
But traditional teaching methods often fail to give these students the practice and reinforcement they need to embed the important skills that can enhance their long-term life outcomes.
Now, a promising study Burton conducted at Brigham Young University using iPads combined with a proven video instruction technique has helped children with autism develop and retain important math skills.
The research was conducted by Burton, a graduate student in the David O. McKay School of Education, and faculty members Darlene Anderson, Mary Anne Prater and Tina Dyches.
While many studies have shown the benefits of VSM or video self-modeling — filming students as they successfully solve a problem or complete an assignment — in regular educational settings, few studies have investigated the use of VSM in teaching core skills such as math and science to students with autism and intellectual disabilities.
In the BYU study, several junior high school-age students with autism used an iPad that showed a video of themselves accurately and independently solving a practical math problem: Estimating the amount of money they needed to pay for an item as well as how much they should get back in change.
They were then asked to solve similar problems using the VSM as a guide. Gradually, over time, the students were asked to figure out the math problem without the VSM.
The results: The students were able to independently solve the math problems with an accuracy of between 80 and 100 percent.
And, best of all, they enjoyed it. All student participants indicated that they liked having a video made of them in class and stated that they enjoyed using the iPad and watching a video of themselves.
As one student stated, "It was cool and fun."
Children with autism have difficulty generalizing, Burton explained.
"We want them to be able to generalize what they've learned and apply it to new skills. Anything visual will help heighten their awareness of the task," she said.
"We're not always able to capture their potential," said Anderson. "These new technologies can make that possible."
The BYU researchers also observed that, without exception, all of the students seemed enthusiastic and excited to participate in the study each day.
"We noticed an immediate and abrupt change in student performance each time the video model was introduced," Burton said. "Students with serious behavior issues — such as aggression and fidgeting — seemed to remain on task to a greater extent than when the iPads weren't used."
Said Anderson, "The students were clearly focused and engaged."
The research, published in the journal Focus on Autism and Other Developmental Disabilities, documented the positive functional relationship between viewing the VSM and student performance.
Burton, who completed her master's thesis while working as a full-time teacher, was motivated by her desire to use her research to help the students she works with every day.
"Our research indicated that video self-modeling via an iPad may be an effective way to deliver academic content to adolescent students with autism and intellectual disability," said Burton, who continues to successfully use the iPad/VSM method in her classes.
Future research should be easy to implement, since, thanks to ever-improving iPad applications, the video models are simple to create and are easily demonstrated and measured, Anderson said.
"I believe this model could transfer to anything you're willing to apply it to," said Burton. "And it's very socially appropriate and easy for these students to carry an iPad around with them."
In addition to its educational value, this research highlights how BYU graduate and undergraduate students and faculty members are working together to provide significant research.
To view the entire study online, visit bit.ly/17PnHRR. For more information, contact Cami Burton at (801) 489-2892 or Darlene Anderson at (801) 422-7603.


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Jessica Alba's Pregnancy Worries
What Was Your Pregnant Worry?
by Moms
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Movie star and mama-to-be Jessica Alba recently told Extra that she's already dreaming about her baby. She said:
"It had to do with breastfeeding, which is the only thing I'm paranoid about, more than giving birth," she revealed, adding that she's trying not to gain too much weight during the pregnancy."
Did you fret over something specific while expecting?
Jessica Alba
Celebrity Babies
POPSUGAR, the #1 independent media and technology company for women. Where more than 75 million women go for original, inspirational content that feeds their passions and interests.
veronicaraye 7 years
my biggest prego worry was getting pregnant...No children for me, YET!
stina829 7 years
I was worried about having an emergency c-section... And I ended up having one. My son was born this past Halloween, almost a week late. I was induced on the 30th... And nothing happened! They sent me home and told me to come back the next day and we would try it all again! So, Halloween morning I was induced again and the dr had to break my water... We waited and waited and then at 8:30 that night, after TWO epidurals that did not work, my blood pressure went up to 210/170, my doctor came in, saw that and said, no way, it's c-section time. My doctor did a wonderful job on my c-section. It's been almost three months and now you can barely see the scar and it's maybe the length of my palm. Jonathan weighed 8 lbs 3 oz and was 20 inches long... Now he weighs 12 lbs and is almost 25 inches long!
Jessamyn 7 years
PS: A lot of women do get diarrhea when the labor starts. It's because the guts and the uterus are so near together. If the womb is starting to cramp, the guts often do it, too. So lots of women do empty their bowels anyway ;)
Jessamyn 7 years
aembry, don't worry about pooping ;D When I gave birth to my daughter, it didn't happen. OR, I didn't feel it ;P When I gave birth to my son, the midwife argued that I'd better get an enema. I DIDN'T want it! But she talked and talked, and in the end she really got on my nerves and I said "OK". I shouldn't have done it, because I pooped haha. Doesn't matter, I just felt it, noone said anything. I know that they clean it up asap so noone will know/see or smell it ;P Don't worry! And: no need for an enema, if you don't feel like it. Then again, I wouldn't want to give birth in the birth tub and seeing this thingie floating on the water hahaha ;D
Jessamyn 7 years
I was worried about a c-section but never had one, luckily. It was more "fun" to have a natural birth. That was all I ever wanted, and I had it two times. Of course, it's a lot of pain and my body will never be the same again. But it was wonderful, me and my husband loved it! My biggest fear before I gave birth for the first time, was an Episiotomy. And then they cut me. And it didn't hurt?! Because they cut me while I've had a contraction. I pushed for more than 1 hour, and it hurt so much! So they said they could help me if they would cut me. Before that, I said I NEVER would want to get cut. But after 1 hour, I screamed "cuuuuuuuuut!!!", haha. And 5 pushes later, my daughter was born. When being in labor with my son almost 3 years later, they had to cut me again. It was a way smaller cut. But I've had a bad midwife and she cut me when the contraction was OVER! It hurt sooo much!!!! But ah well, he was born very fast after that, and it healed pretty quickly. It was worth it :)
KAEB06 7 years
I seriously was having nightmares where I would take a dump while pushing! Out of all of the things to be worried about I was actually having ISSUES regarding this....I ended up being totally fine in that area during delivery but I SHOULD have been more worried about the PAIN!
abqmama 7 years
I was so scared that something would be wrong with my babies and it would be my fault. Like, that time I had a few too many drinks with my first before I knew I was pregnant would mess him up for life. I also worried in the last month or so about not feeling them kick for awhile. Fortunately, they both came out perfectly healthy. Oddly enough I was never too concerned about the pain of childbirth even though I planned to do it naturally. I expected it to be really bad and I was prepared for that so when it wasn't quite as bad as I expected, I felt great about it.
lickety-split 7 years
with #1 i was freaked about the pain. totally freaked. i would stay up late worrying about it in the weeks before my first was due. in the end i got the drugs i asked for and it was pretty easy. then with the second i worried i'd deliver on our moving day (which i did and big deal everything got done). with the third i had no worries and was looking forward to the 2 days of peace and rest, lol. and that was the difficult delivery. if #3 had been #1 i'm not sure there would have been a #2.
Black-Velveteen 7 years
With my first, I was worried with just about everything, pain, labor, the baby's safe delivery and health, etc. With my second, I was worried I was gonna poop while pushing :rotfl:. Intense pushing can really do a number on the perineal floor!
aembry396 7 years
I know that at the time I won't care, but it just seems so mortifying!!!! It is just a shock. Thanks for making me feel better!
Greggie 7 years
I agree with Mykie - don't worry at all about puking or pooping. And a lot of times, unless the nurse is crass enough to tell you, you won't even know you pooped anyway. But they've seen MUCH worse, and shouldn't be phased by it. If they are, they're not very good at their job.
Mykie7 7 years
aembry, you shouldn't worry about either thing. I'm rather ashamed to admit that pooping during delivery happened to me, and while I was mortified, the nurses didn't even bat an eye. They just told me, "You'd be amazed how much it happens. For every woman that ADMITS that it happens, 4 more don't." And as to puking, if you are being induced, DON'T EAT! I made the mistake of having a "light lunch" of a sandwich and soda. Well, the drugs from being induced brought it right back up. But again, my delivery team was awesome. The biggest thing you need to do is be comfortable with your doc, and the delivery team. If you have that, everything else will work out. I've had two kids, and it really isn't that bad. AND my first labor was 42 hours. AND I'M A BIG WUSSY TOO! LOL
Mykie7 7 years
Along with the health of my babies, I was concerned about getting back into shape afterwards. Which I would venture a guess that MOST moms are worried about whether they admit it or not.
Dana18 7 years
I had to have a c-section and I worred about it. But I had no choose because I was having twins and they where breach. I focused on them coming out healthy and they are fine.
aembry396 7 years
YOU PUKE DURING LABOR!!!!! oh geez. I just learned about the pooping...but that too?!?!?!
mfern64 7 years
With my first, I had a reaccuring dream that my baby would be wierd looking. Superficial, I know, but my husband an I are from different ethnic backgrounds and I didn't know how we would mix. My second pregnancy I didn't have any worries, but my baby was stillborn at 22 weeks, so my third pregnancy, I worried about everything the whole 40 weeks!
luckyme 7 years
auntyb - I have never heard that. Holy hell, I would have been freaking! I puked so many times during labor (until the angel nurse gave me anti-nausea medication in my IV...that sweet, sweet woman!).
auntyb 7 years
Vomiting during delivery, becauseI had read an article saying that could be a sign of amniotic embolism (!). Of course I DID end up doing it but no embolism thank goodness. Just plain old nausea.
thatgirljj 7 years
I'm worried about life after the baby comes. How is childcare going to work out? How isolated will I feel during maternity leave? How are my husband and I ever going to have sex again, much less sleep?
fleurfairy 7 years
Jessica Alba is the most insecure person and nobody realizes it. She is obsessed with her weight.
rgrl 7 years
I was afraid of the unknown... like anything can happen. Luckily things were good!
Tullia 7 years
health of baby, and the pain-I am utterly terrified by any kind of pain, and hearing about other woman about how painful child birth is, it makes me even more scared.
kurniakasih 7 years
I'm the same w/ kellyswife. I didn't have any worry, I wasn't concerned about ...basically...anything, I just expect that it'll be painful (the birth and the after life :lol: ). My hubby was the one more concerned about the health of the baby, if it's going to come out alright, etc. Fortunately, our son is healthy, happy and nutty baby boy :D ...well, he's a toddler now. Very active and healthy.
kellyswife 7 years
I must be crazy because I really had no worries. I didn't expect labor to be easy, or pain-free, I didn't expect raising a baby to easy or pain free either. I wasn't worried about any of it because I knew it wouldn't be great.


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Steampunk Emporium Bazaar presented by the Sacramento Steampunk Society is February 22 and it is free.
This is a family event and it is a fun way to explore the past and dream of the future.
Generally "steampunk" is a retro or vintage term for items of the past which were newest inovations of the time period. As the name implies, this was during the period when trunks were used and the necessary items of travel were invented for a generation of travelers no longer willing to put up with Middle age technology or lack of.
New and appealing items might be on hand to ease your life or just allow the mind to travel into a fantasy of innovation. There will be both newer "artisy" as well as old antique items on display and for sale.
The time and place will be Great Escape Games at 1250 Howe Ave suite 3A in Sacramento California. The day is 2-22 a Saturday from 11 A.M. - 5 P.M. This is a free event but there will be vendors for refreshments and such.
www.sacsteam.org is the website for the Sacramento Steampunk Society which also takes trips in custome for those reliving the innovative past in full style.


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Sight - Hold the glass towards the light. Note the color, clarity and brilliance of the wine. If the wine is cloudy or discolored that indicates something wrong with the blend. Older red wines will often have more orange tinges on the edges than younger red wines. Older white wines are darker than younger white wines when comparing the same varietal at different ages.
Swirl - Swirling the glass sends oxygen through the wine and releases the wine's aromas. Swirl the wine in your glass for a solid 10 to 12 seconds by lightly holding the stem of the wineglass and rotating your wrist. Notice how the wine hits the sides of the glass. This will give you clues as to how full-bodied the wine is.
Smell - Place your nose just over the edge of the wine glass, enabling the aromas to bounce off the edge. Keep your mouth open. Close your eyes to take in the full impact of the bouquet. A wine's aroma is an excellent indicator of its quality and unique characteristics.
Sip - Take a sip and let the wine roll over your taste buds for several seconds. Consider the texture and flavor of the wine. On the second sip, try swishing the wine around in your mouth. Some tasters "chew" the wine.
Swallow - Exhale through your nose as you swallow, allowing your taste buds and sense of smell to work together. After you swallow, note the finish and length of the wine. The finish is the aftertaste and the length is the period of time that it lingers.
Stemware - The type of wine glass you use can affect the flavors and the aromas of the wine. A well-designed glass creates a "chimney" so that the aroma of the wine is concentrated and wafts toward the nose. It is generally accepted that a white wine glass has a longer stem and narrower bowl, while a red wine glass has a shorter stem and wider bowl. For an optimal wine-drinking experience, Mount Pleasant recommends Riedel glassware. Riedel has designed a different glass to meet the unique characteristics of each varietal of wine. A glass of Missouri's Norton sings in your mouth when served from Riedel's Norton glass.


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Prism present the women's London Round Sunglasses. Inspired by edgy London street style, the sunglasses feature curved and oversized frame with winged detailing. Inset with graded Carl Zeiss lenses, the pair offer 100% UV protection and boast Prism logo to the inner and outer temples. Complete with protective glasses case. - K.N.
Express Delivery - if ordered before 9pm, delivered by courier next working day. More Info »
Please remember to add postal time (2-3 working days) to obtain a complete estimate of delivery to your door.
You will receive an email to confirm when your item has been sent.
You can also check the status of your order and individual items by accessing My Account details and following the prompts.
Please allow 10 working days from dispatch of your order before notifying us of any late deliveries.
It may be advisable to check with your neighbours to see if a parcel has been left with them, check any outhouses you might have where it may be left if it cannot fit through your letterbox and contact your local sorting office to see if the item has been returned to the depot as undelivered and awaiting collection.
Please see our returns policy.
Prism Women's Capri Ombre Cats Eye Sunglasses - Matt Black
Was: £255.00
Sunday Somewhere
Sunday Somewhere Kiteys Sunglasses - Mint
Was: £180.00
Versace Embellished Cat Eye Women's Sunglasses - Havana
Versace Icons Women's Sunglasses - Havana
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Project Grants
Grants Opportunities
Dumbarton Oaks makes a limited number of grants to assist with scholarly projects in Byzantine Studies, Pre-Columbian Studies, and Garden and Landscape Studies. Support is generally for archeological investigation, as well as for the recovery, recording, and analysis of materials that would otherwise be lost. The normal range of awards is $3,000–$10,000. Eligible projects may include, but are not limited to, the following:
- non-destructive investigation, excavation, and/or on-site study of a (Byzantine, Pre-Columbian, Garden) site or component of one
- surveying or photographing monuments and objects that are at risk (e.g., architecture, gardens, paintings, mosaics, and sculptures in situ), as well as objects that have already been collected but are largely unrecorded and endangered.
Funding may be used only to cover direct costs for which original documentation must be submitted at the conclusion of the project. Examples of eligible expenses include transportation (excluding students), meals, housing, vehicle rental, workmen's wages, and technical analysis (as part of the overall project). Grants are not normally made for the purchase of computers or other equipment; or to cover the salaries of the principal investigators. Project grants are not given purely for the purpose of travel, or for work associated with a degree, library or archival research, catalogues, nor for conservation and restoration per se. Priority is given to applicants who have not previously received support from Dumbarton Oaks.
Pre-Columbian project awards are intended only to survey, excavate, and/or document sites, landscapes, or objects that are in imminent danger. Applicants should provide evidence that such sites, landscapes, or objects are under immediate threat.
Garden and Landscape grants are intended to support primary research of a specific site. Project grants may be used for a broad array of projects including field research, site analysis, botanical surveys, heritage conservation and restoration planning, with the goal of promoting the preservation and understanding of historic gardens and other significant designed landscapes.
For Byzantine project awards, priority is given to small projects, as well as start-up support for new projects, or for the final push for completion. Funding is non-recurring and will be awarded for one season only per project.
Funds can only be disbursed to individuals, not academic or other institutions, and only on a reimbursement basis, upon submission of receipts, and after the final report has been submitted. Funds are available after July 1. The final report and receipts must be received by the relevant Director of Studies before June 1.
Qualifications and Conditions
Project grants are limited to applicants holding a doctorate or the equivalent, and are awarded on the basis of:
- ability and preparation of the principal project personnel (including knowledge of the requisite languages) and
- interest and value of the project to the specific field of study.
Individuals currently affiliated with Dumbarton Oaks are not eligible. Applications are reviewed by an external committee of scholars in the relevant field. Awards are made for projects conducted within the fiscal year (July 1, 2016, through June 30, 2017).
Before applying, applicants must contact the appropriate Director of Studies, no later than October 1, to determine if the project is within the purview of Dumbarton Oaks. Completed applications must include three letters of reference posted directly by the recommenders to the online application.
The Directors of Studies may be reached by contacting their departments below:
Upon completion of their projects, grant recipients will be asked to submit brief reports of their work suitable for dissemination on the Dumbarton Oaks website. For projects involving photography, Dumbarton Oaks requests that successful applicants provide a set of digital images to document their work. Reports, no longer than 2,000 words, should be submitted as both Microsoft Word and PDF documents, with one to three illustrations sent separately in .tif or .jpg format. All photographs and illustrations for documentation and for project grant reports must be submitted in digital format, and as separate high resolution files—not embedded in another type of document. All images must be of sufficient quality for print publication—at least 7" wide at 300 dpi, or about 2100 pixels wide. Maps, graphs, and line drawings should be supplied as vector graphics, such as Adobe Illustrator .ai or .eps. Byzantine project grant recipients may also be asked to submit longer reports covering more than one season for publication in Dumbarton Oaks Papers; while it does not guarantee publication, Dumbarton Oaks Papers requests first consideration of reports growing out of funded fieldwork.
Please read and follow the online instructions. Applications, in English, must be submitted electronically via the Embark application system by November 1.
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Holiday Book GuideThursday, December 11th, 2008 - by: Brian Herzog, Head of Reference
The Youth Services staff of the library put together this list of books that would make nice gifts this holiday season. These books are divided up into type of book and age groups.
Following this listing, there are links to book guides from other sources around the internet to provide additional ideas.
Pop-Up Books
Picture Books
Ages 5-7
Ages 8-10
Ages 10-12
All Ages
Here are a few lists from other sources on the internet, listing their top book picks for the holiday season:
Other Book Gift Guides
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