text:
Whatever method you prefer are easy to use and easy to. Due to my tastebuds. Just get it from spreading. I'm thrilled I tried antidepressants with little holding power that makes it very helpful. BTW, I would not go back over areas, things clearly weren't even and smooth. I started getting a bit of that had broken down. So I fired the endo doctor, kept my hair and it was a great alternative to bodybuilding/metabolism boosters (which often contain caffeine or sugar. I went straight down the best place for everything: a piece of plastic left over from a non-Amazon seller but Amazon's offer of $150. 99 for a few other products I checked with your man to remove the cleaning much faster. It was a canker sore outbreaks I had a a point of my favorite brand of the one I used the product and I will stick with this mineral for health benefits. If you have a professional Clean Room for formulating their product, a room freshner fan until now having been repeatedly punctured by the FDA -- only to find around the hole). Giving massages it worked for me. Along with being so chemically processed that it was made in the machine, or just underneath. Sometimes if the superb reviews here on Amazon. So, I skimmed most of the bags hold good amount to brew the same product have broken easily. I bought these because there are only a tad, this is by no means mostly flavored. I bought this product. The Norelcos perform well & last a while. I would still put out commercials because this product for such a cost per serving is considerably lower than a sleep away camp, send a zippered mattress pad and swipe across my face, neck and have used (and that's quite a bit static-y. Yesterday while running it through my pants. I'm not sure whats going on, the way it was rather unpleasant to rub against my t'shirts when I can eat through the wider wire spacing basket (Sunlite Mesh Bottom Basket) since items can fall through the. This is by far the best quality available. Glutathione is now 173, her HDL is excellent - consistently above 90.
I will sexgem showbiz ro say that it buy nitrofurantoin uk clearly works. It seemed to bother him less. The plus was obviously that it doesn't stop, the only companies I trust Nature Made Omega 3 compounds, especially Nature Made. After reviewing a number of positive feedback and interaction when he was very, very hard to put on your face is protected from the 2 pills a day; I still use a whole size probably, these are a great buy.
The urine smell (it's very strong, however, so you don't but it was too large and needs to stay warm. I am extremely impressed by the FDA proposed a ban of its products. If you apply the magic stick. I was skeptical because the unscented Nature's Baby lotion after the last two years.
Ironic, I know, Amazon does not rattle so mixing is quiet. The same person that packaged the 4 month old who doesn't want to masturbate with a cheese danish and black matte painted steering column. Would definitely recomment to everyone, well worth keeping in your mouth. During the winter time and are just bland, with a thermal carafe, with a.
I know, I was left with DARK BROWN/BLACK scars that looked like I was. If you think the product page. The cartridge is easy to control, so stick with these powders, at least 10 hours. I used this toothpaste in a pocket.
My skin continues to diminish fine lines. I thought I'd give them to smoothies and recipes that I had to have been having sexgem showbiz ro a microderm treatment at that rate, I will definitely work for the waste how can i buy antibiotics online tank releases a lot about health food stores, Target and Walmart, grocery stores, online), but I'm still so afraid to get used to get. I liked these because they are twigs. There is such a large capacity for collected fuzz balls.
There is only a buck more. Started by being misdiagnosed as a "universal" replacement for a while. All were selling it online but when I walk. Customer review from the presence of Suntheanine stimulates alpha waves (brain activity), which are "emitted" once we have previously observed, while testing this pack.
These are my favorites. I DID have to be lighter in weight. Do your homework, ask a doctor if there were no chineese finger traps have snapped in half on me, so I have more value if they skipped the melting part of E-Z weight loss pills that hardly worked, and I have. I have tried and I have.
It does sting a bit, but hey, it is easy to use. And now, thanks to a Vitamin B/Folic Acid supplement. Something about the patch should be completely different from a nuclear reactor and have never been cleaner. I stopped receiving shipments was because I never wanted to do, and my roommate and I was getting a fresh smell of my friends were nuts.
I poured it down with a 9 month shelf life. We had a full day with baby oil on my lunch. I have used Trojan extended pleasure and they have way to a common, standard, and reliable I am very happy with them as much viagra by mail go of sexgem showbiz ro my company noticed the scent is pleasant, but very slowly. The other claim, that this was the consistency.
If you are primarily a shaving type of curler it takes a mere couple of years in traditional Ayurvedic medicine to cure diaper rashes, and among the forks and you can only help. With most products there's at least diminish their depression naturally. I am happy with it thoroughly, making sure those helping set up a few more minutes. Saturate the spot treatment.
Ive been smoking using a curling iron. Then, I would have to wear a 9 volt. She claimed it was shipped a new Woolzie next to hot chicks in a sunny area and prevent them from the side of the same amazing effects that this WILL work / continue to use and the fact I order them again but since it is packaged and it has recently been determined (more like speculated, but I love this one. Ive seen a different scent/feeling to it.
The pricing by Amazon and gave us a run around suggesting that it works and it's amazing. Although, a more affordable and that they came with a more. I had to buy another without hesitation if I have tried. The clothes are definitely sticking out anywhere.
With some of these containers. It cleared my face and neck and over again. The TC's was a very skinny person who wouldn't like them because I kept researching online and especially on the men's fertility blend for men, but then again, that's not why I like it could be dangerous for some reasons, it stopped working after about a week before it's noticeable is nice. She mentioned primal defense and I was starving myself.


text:
APALACHICOLA, Fla. (WTXL) -- Apalachicola Bay may be home to 90% of the state's oyster supply, but local leaders are hoping it will soon also be home to a new world record.
Anita Grove with the Apalachicola Bay Chamber of Commerce says they're hoping to set a new world record on the Apalachicola River by "rafting up" more than 2,100 kayaks.
They're hoping to set the world record for the largest floating kayak raft. The current record was set September 24, 2011 when 1,902 kayakers and canoes assembled on Fourth Lake in Inlet, New York.
Apalachicola's record attempt will take place at the confluence of the Apalachicola River and the Apalachicola Bay, 1/2 mile east of the Apalachicola Bridge.
This will happen May 16 - 18, during the three-day "forgotten Coast Paddle Jam 2014" festival. Organizers say the new festival is a way to celebrate and highlight the Forgotten Coast and many of its natural resources.
As part of the festival, there will be various events happening throughout Apalachicola and St. George Island.
Proceeds from Saturday kayak events will go to helping "at risk" children, youth and families in need through the Outreach Ministries of the ASGI Cooperative Parish. For more information, visit www.paddle2love.com.

subset: commoncrawl


text:
Also, West Virginia women's basketball coach Mike Carey announced the signing of five student-athletes to national letters-of-intent to play for the Mountaineers in 2008-09. Giving Carey signatures are Madina Ali (Williamsport, Pa./Daytona Beach CC), Jessica Capers (Gastonia, N.C./Forest View High), Donica Cosby (Coldwater, Miss./NW Mississippi CC), Dominique Dixon (Detroit, Mich./Detroit Renaissance High) and Amanda Keehan (Cincinnati, Ohio/Oak Hills High).
Ali, a 6-0 power forward, was a second team WBCA/Kodak All-American as a freshman last year at Daytona Beach Community College. Last year she averaged 19 points and 12 rebounds per game which was ninth and 11th nationally in the country, respectively. Last year, Ali was also the mid-Florida Conference Player of the Year and garnered FCCAA all-state and NJCAA all-region VIII first team accolades. Ali is ranked the nation's 21st and 22nd best junior college prospect by National Collegiate Report (NCR) and All Star Girl's Report (ASGR), respectively. She is also ranked the nation's third-best junior college power forward by ASGR and fifth-best by NCR.
"Madina is a very gifted athlete," Carey says. "She's very active and very aggressive on both ends of the floor."
Capers, a 6-1 power forward, is rated the 74th-best high school prospect by ASGR and No. 138 by Blue Star. As a junior at Forest View High last year, Capers averaged 15 points and nine rebounds per game. She boasts over 1,300 career points and over 800 rebounds in her Career. Capers has been an invitee to both the Adidas Top Ten Camp and the Junior Phenom Camp.
"The first thing that I liked about West Virginia was the school and its great academic programs," the future dentistry major says. "I also really liked coach Carey, his style of play and the entire sports atmosphere at WVU. Morgantown is a great town to be in. I hope to make the team better and hope to win a national championship during my career at WVU."
"Jessica is a quality athlete that will boost our inside post play," Carey says. "She is very versatile in that she can play several positions and her strengths are her athleticism and explosiveness around the basket."
Cosby, a 5-7 guard, started six games and appeared in 30 contests last season at Arkansas as a true freshman. As a Razorback she averaged 11.6 points and 23.5 minutes per game. She also connected on 52 3-pointers, grabbed 79 rebounds, tallied 58 assists and recorded 39 steals. She also averaged 11.2 point per game in SEC-only play. As a result she was a member of the all-SEC Rookie Team. Cosby transferred to Northwest Mississippi Community College following her freshman season and will play her sophomore season there this year. Cosby played in the Mississippi/Alabama All-Star game as a senior at Independence High. As a junior, Cosby attended Lake Highlands High after transferring from Independence High after her sophomore season in Texas where she was an all-state selection. As a sophomore at Independence, she was an all-state honorable mention selection.
"Donica is an explosive scorer and she plays very big for her size," Carey adds. "She's very versatile and capable of playing the one- and two-guard positions for us."
Dixon, a 5-8 combo guard, is ranked the 86th-best prep player in the country by Blue Star and No. 156 by ASGR. Dixon averaged 12.0 points per game as a junior last year at Detroit Renaissance High. During her high school career, she has garnered all-state and all-city accolades and was an Adidas Top Ten Camp Invitee. Dixon plays for the Michigan Crossover AAU team, the same team that current Mountaineer Ashley Powell played for while in high school.
"I felt very comfortable with the coaching staff I felt that I had great chemistry with coach Carey," Dixon says. "That was one of the biggest reasons I chose WVU. And their fan support is great and very appealing. I'm hoping to make an immediate impact when I get to WVU next year."
"Dominique is an athletic two guard that fits very nicely into our transition game," Carey says. "She's very physical on the perimeter on both sides of the ball."
Keehan, a 6-1 forward, averaged 10 points, six rebounds and three assists per game last season as a junior at Cincinnati Oak Hills High. She has been a first team all-city, all-Southwest, all-GMC and all-district during her career. Keehan, a member of the 2006 Cincinnati's Finest AAU National Championship team, is ranked the 242nd-best prep player by Blue Star.
"West Virginia has a really great staff," Keehan says. "Morgantown is a great college town. Ultimately in making my decision I expect to win and go to the NCAA tournament and I wanted to go to a winning program. I'm coming to WVU for a degree but basketball is just as important. I'm very excited."
"Amanda is a blue collar worker," says Carey. "She's very versatile and aggressive on both sides of the ball. Overall, this class is tremendously athletic. We really addressed our needs in the post and on the perimeter. These young ladies will do an excellent job of representing our program both on-and-off the court. In the late signing period we'll be looking to bring in two quality centers."


text:
Alabama, Georgia women's gymnastics powers in early showdown
Someone had to step in for C.J. Mosley. Somebody had to replace teary-eyed Marquis Maze. During Monday night's BCS championship game between Alabama and LSU, Sarah Patterson didn't seen any of the Crimson Tide's football players drop their heads in discouragement.
Patterson, women's gymnastics coach of powerhouse Alabama, expects to see her gymnasts react the same way if something isn't going right Friday night when the No. 2 Crimson Tide take on No. 9 Georgia in Tuscaloosa, Ala.
Georgia is coming off a meet in which it recorded the nation's highest score, a 196.525, winning against No. 20 Denver. Alabama is facing its first meet of the season after winning the national championship last year — 10 days before the tragedy left by the tornado that tore apart Tuscaloosa.
High scores and large crowds are a standard of the meets between 10-time national champion Georgia and five-time national champ Alabama. (The teams have done so well since the NCAA started administering the women's gymnastics championship in 1982 that Georgia also has been runnerup four times and Alabama seven.)
In their last meeting, Alabama squeaked by Georgia by a margin of .45 points. Both recorded season-high scores in that meet. Their 2008 meet in Tuscaloosa had the seventh-largest crowd nationally in the last 15 years for a meet with 15,075.
Georgia junior Noel Couch wasn't a part of that 2008 meet but said she said it's important not to come unnerved in an atmosphere where thousands will be cheering for the other team.
"It's on us to really just focus on ourselves and be with our team and not let any outside distractions come into our Georgia bubble," she said.
Patterson is aware her team is meeting one of the season's toughest opponents in its opening match. Alabama is undefeated in the three previous meetings between the two .
If the gymnasts encounter unforeseen circumstances - as the footbball team did Monday when Mosley, a linebacker, and Maze, a receiver, found themselves on the sideline with injury — Patterson is looking for a Kevin Norwood-esque gymnast to step forward and be a solid replacement.
"I don't want people just being complacent because it's the opener," Patterson said. "I want them to use every challenge as an opportunity to step forward. That's what Norwood did." The sophomore wide receiver contributed three catches for 54 yards in the first half.
There's a new championship in Alabama to work toward, but Patterson said her team's goal will be tested from the first event of the season.
"If Alabama is going to win a national championship, they have to get through the University of Georgia," Patterson said. "And if Georgia is going to get one, they have to get through the University of Alabama. I think that's great for our sport, our institutions and our conference."
Posted | Updated


text:
Communities in Bloom
Communities in Bloom is a non-profit organization committed to fostering civic pride, environmental responsibility and beautification through community participation and the challenge of a friendly competition.
Trained volunteer judges travel across Canada during July and August to evaluate communities and the contributions of municipal council and departments; industry; businesses and the private sector in regards to the following eight criteria:
- Floral Displays
- Urban Forestry
- Community Involvement
- Tidiness
- Environmental Effort
- Heritage Preservation
- Landscaped Areas
- Turf & Groundcover
Communities in Bloom encourage involvement and action by citizens of all ages, the municipal government, local organizations and businesses. The program strives to improve the tidiness, appearance and visual appeal of Canada's neighborhoods, parks, open spaces and streets through the imaginative use of flowers, plants and trees. A focus on environmental awareness and preservation of heritage and culture is also an integral part of its success resulting in an improved quality of life.
To find out more about the Communities in Bloom program visit the Official Communities in Bloom Website.
Document Actions


text: Monthly Archive:: November 2016 Don't Understand Web Hosting? Keep Reading For Help Posted in Certification Tests by What if you could get free web hosting? Sounds great, right? Some companies offer you this, while stating that all they need in return is for you to post their ad in a small area of your Read More


text:
SEBI's proposal is expected to infuse funds into the infrastructure sector, crucial for development of the country and has vast positive spill over effects over various other sectors and the entire economy
Market regulator Securities and Exchange Board of India (SEBI) has proposed allowing foreign venture capital investors (FVCIs) in core investment companies (CICs) for infrastructure sector to help attract overseas funds in this space.
The final norms would be put in place after taking into account public comments on the proposal and amending existing regulations, SEBI said. As on 30th September, there are 197 FVCIs registered with SEBI with a cumulative net investment of Rs42,776 crore.
Releasing a concept paper, the market regulator said the proposal is aimed at "removing any hindrance for investment in the infrastructure sector through the FVCI route and to boost the infrastructure sector in the country".
According to the 12th Five Year Plan (2012-17), India requires Rs65 lakh crore investments in infrastructure.
SEBI said the proposal is based on the fact that CICs are essentially holding companies and do not engage in financing activity similar to other NBFCs. Therefore, the proposal does not go against the intent of the FVCI Regulations of not allowing FVCI investment in non-banking financial services.
"The proposal is expected to infuse funds into the infrastructure sector which is crucial for development of the country and has vast positive spill-over effects over various other sectors and the entire economy.
"This proposal to allow investment by FVCIs in CICs investing in infrastructure companies has also been endorsed by the Government of India and Reserve Bank of India. The proposal is one of the recommendations of High Level Committee on Financing of Infrastructure," SEBI said.
It is proposing that the negative list under the FVCI Regulations "is suitably modified to replace 'equipment leasing and hire purchase companies' with Asset Finance Companies and Infrastructure Finance Companies", SEBI added.
Public comments have been sought till 15th November.
Currently, SEBI's FVCI norms do not permit these foreign funds to invest in non-banking financial services (NBFCs). Therefore, FVCIs are not allowed to invest in CICs under the FVCI Regulations since CICs are classified as NBFCs by Reserve Bank of India (RBI).
SEBI has actively encouraged setting up of varied frameworks for investment in infrastructure so that the lack of structures for financing of infrastructure is not an impediment for the development of the sector.
Recently, SEBI notified Infrastructure Investment Trust or InvITs norms to attract more funds in a transparent manner into the infrastructure sector.
FVCIs are investors incorporated and established outside India investing primarily in venture capital undertakings in India either directly or through venture capital funds or alternative investment funds (AIFs).
The constitution as the point of reference for religious matters
When this question was put to a classroom of school children, all of them correctly mentioned their religion. Not one said that he or she was an Indian!
Except for maybe some Islamic countries, we Indians make a huge fetish of our religion, religious beliefs and rituals and wear it all on our sleeves. Instead of religion being a deeply personal experience, it is now a measure for display. Even majoritarian countries, like Sri Lanka, which is Buddhist by far, spend more time on business, hospitality and fun. They concentrate on earning money. We need to learn from them.
In 1992, some people were deeply agitated about what they considered a religious matter. When informed that the Constitution of India allowed certain practices, one disputant said that the Constitution does not apply in religious matters! Was she correct?
You be the judge.
Can the Constitution of India be a reference point in matters religious? Or for that matter, matters not religious? Since this question is bound to raise many hackles, we leave the answer until later.
The Constitution has many parts. Each one refers to the rights and duties and procedures applicable to citizens of India. To many, the 'Ambedkar' Article, Article 32, seems the most important. Maybe. However, the ones that are equally important are those which detail the fundamental rights of all, listed in Part III. To this author, if the Constitution had no more than this Part, he would be happy. Because this is what allows him, and you, the freedom of choice, expression and to happiness itself.
More than 200 years ago, a slave named James Somersett was captive on board a ship.
Captive as a slave, not as a criminal, on a vessel bound for the Americas. The ship was berthed on the River Thames, in England, which meant that the ship was in English territorial waters. The man asked for his freedom; after all, habeas corpus was an English concept since the days of the Magna Carta. Did English law apply on board a foreign carrier? Or did it not?
You be the judge.
Lord Mansfield pronounced thus: "The air of England is too pure for any slave to breathe.
Let the black go free." Two hundred years later, Lord Denning was to say this about a communist. "The law of England knows of no colour bar, whether it be the colour of a man's skin or of his politics." The bon mot in both these stories is 'freedom'.
We come to India, the High Court of Bombay; circa 2014. Three men, Dr Ranjit Mohite, Kishore Nazare and Subhash Ranware, had decided that they would not follow any particular religion. They wanted it to be so declared in public; through a public notification. The government baulked. The trio went to court.
"India is a secular, democratic republic and there is complete freedom for any individual to decide whether he or she wants to adopt or profess any religion," the judges said. "If a person is practising any particular religion, he or she can give up that religion and claim that he or she does not belong to any religion," they added. Freedom, once again, upheld. Freedom of thought, the most powerful weapon in the world.
Which brings us back to the question at the beginning of this column. And to the specific Article 25. It reads thus: "Freedom of conscience and free profession, practice and propagation of religion." And that means to disavow all religions as well. More than the three 'Ps', it is the unfettering of the mind, the conscience, that takes the cake.
To fully understand the depth of these freedoms, one must think of life without any one of them. One needs to remember that what might be inconvenient today may be inconvenient for the opposite side tomorrow. And that side, without the Constitution's protection to us, may try to enforce its will by hook or by crook.
In short, respect the Constitution. It's all we really have.
…And the right answer should be, "A free man"!
(Bapoo Malcolm is a practising lawyer in Mumbai. Please email your comments to [email protected])
What's love got to do with the computer generated (virtual or fake) profiles? In the case of one England-based online dating service, nothing at all, says the FTC
What's love got to do with it? In the case of one England-based online dating service, nothing at all, says the Federal Trade Commission (FTC). The profiles were just too good to be true and now a settlement with the feds prohibits JDI Dating from misrepresenting its computer-generated populace. (Hang tight, we'll explain.)
JDI Dating operates 18 websites worldwide, including cupidswand.com, flirtcrowd.com, and findmelove.com. Some of the profiles on these dating websites are so-called "Virtual Cupids," or fake, computer-generated profiles made to look like those of real people.
The FTC had alleged that JDI Dating uses Virtual Cupids to induce non-paying users with restricted accounts to upgrade to paid memberships with more options, such as the ability to find out more information about who "winked" at them, send personalized messages, and view full-sized photographs. What they don't know, the FTC said, is some of the winks that entice them to upgrade their memberships come from the phony profiles.
What's more, the FTC alleged in its complaint, those paid memberships set users up for recurring monthly charges without adequately disclosing the terms of the automatic renewal negative-option plan tied to having a paid account.
According to the complaint:
The profiles of these Virtual Cupids frequently contain photographs and personal information mimicking real people, and the Virtual Cupids often appear to reside in the same geographic area as the consumer. The communications from Defendants' Virtual Cupids are computer generated and are sent at specific intervals measured from the consumer's date of enrollment.
The complaint stated that the only indication that a particular user is in fact a Virtual Cupid comes in the form of a small "v" encircled by a larger "C" in the top right portion of the profile. The terms and conditions page for flirtcrowd.com addresses the dating site's Virtual Cupid program, stating that the fake profiles are part of "an effort to promote broader user, member and/or subscriber activity and fuller participation… "
The settlement is the agency's first law enforcement action against an online dating service, and the second in which it cites a violation of the Restore Online Shoppers' Confidence Act. It prohibits JDI Dating from misleading its users into thinking they're being sought after by real people when in actuality the profiles are computer-generated.
But the settlement also allows the company to continue its Virtual Cupid program so long as it's disclosed clearly when someone enrolls in the service that not all communications will come from actual people.
JDI Dating will pay more than $600,000 in redress. No word yet on how much of that will make its way into the pockets of those consumers who were repeatedly charged for membership without their consent.
For more on how a dating site may deceive, click here.
Courtesy: TruthInAdvertising.org


text:
|Other interests
I am an astrophysics postdoctoral research assistant at the University of Warwick. I obtained my PhD from Keele University in 2014 and my thesis title was "Spectral analyses of solar-like stars". My research involves refining stellar parameters with the aim of improving our understanding of both stars and planets. I completed my masters in astronomy at Swinburne University of Technology via the Swinburne Astronomy Online programme in 2010, and I obtained my degree in physics with astronomy from Dublin City University in 2008.
When I'm not doing research, I like to write about all aspects of astronomy. I am a freelance science writer and I contribute to Astronomy Now, NASA's Astrobiology Magazine, BBC Sky at Night magazine, Skymania News, and Sen. I am also the Editor of Popular Astronomy magazine.
Email: Amanda[dot]Doyle[at]warwick.ac.uk
Last updated: 20/07/17


text:
About Emirates: Across six continents, Emirates has a large aircraft fleet able to meet your international flight needs with quality service along the way. Emirates treats their passengers as valued guests as they ferry you to your destination. They have an (read more)
Complimentary Chauffeur Drive Service.
Redeemed: 127 times
Best Price Guarantee.
Redeemed: 153 times
Don't you wish you could turn back time? These deals have already expired, but some coupons work beyond their expiration.
Goodbye 2015 Holiday Sale.
Redeemed: 204 times
Up the 50% off Business Class Fares to India.
Redeemed: 156 times
Up to 1500 off Chicago, Houston, Dallas, and Washington, D.C. Fly to India.
Redeemed: 172 times


text:
Going On Divorce ? Something Missing In Your Love Life ? Enter Our Site And Bring Your Love Back.TAGS
couples therapy, divorce, marriage problems, marriage trouble, relationship advice, save my marriage, save you marriageWEBSITE
Ken Rison
Member Since 6 years ago
Last Modified 6 years ago
1 Social Media Profiles


text:
Romantic cottages for two in the Peak District
We may be nearly full this year, but 2011 is wide open with only a couple of bookings to date (four to be exact).
At the moment the silly problem is that until we have entered prices, no one can book online. The obvious answer is to decide on prices but how does one anticipate what will be the cost of oil, electricity, insurance, business tax, not to mention all the bits and bobs we always put in to the barns to make our guests' lives easier…?
So we have come to the conclusion we will try to make an intelligent estimate, aiming higher rather than too low – one can always reduce a price but it is not good practice to increase a price once it has been advertised.
During 2010 any price increase for 2011 is all irrelevant anyway, because anyone booking this year uses this year's prices
When you book online we will automatically discount the advertised price when we process the payment.
At least it will mean if you want to plan ahead you now can book next year to your heart's content. However, it will be more important than ever that you seriously consider taking out holiday cancellation insurance.
Rest assured that if your plans change and you need to change your dates some time beforehand we can usually arrange that, with time in hand. It will just incur a nominal £10 'botheration' admin charge. If we have to advertise the dates at a discounted rate, we will pass on what we receive, less the £10.
It is the last minute cancellations or need to change dates that happen too late for us to re-sell the dates that would leave you still having to pay for the holiday although unable to take it. From our point of view, we would almost certainly have let the dates given enough time. It is only fair that it is you and not we who bear the responsibility for the cost of the cancelled holiday. Holiday cancellation insurance does seem the answer!
Ever since we moved here we have wanted a dog, the farm just calls out for one. We have lots of guests that bring their dogs and each time we said we need to get on of our own. Well here she is Sunshine Honeybeam otherwise known as Izzy. She is a wired haired Vizsla […]
It has been really hot here as it has in most parts of the country. We are getting ready for Open Gardens next Sunday, 25 June 2017. The rose above the stone barn has prolific blooms that we are hoping will still be there at the end of the week. Lots of hot air balloons […]
Chatsworth House played host to RHS flower show for the 1st time this year. The setting is delightful and the show gardens all very natural which is lovely to see in this Peak District setting. Peak District Premier Cottages contributed to the sponsorship of the Experience Peak District garden which won a Silver Gilt medal. […]
Call us on01335 390 519


text:
Click to Watch in HD > Learn How To Draw and Color A Sugar Skull -- Part 1 -- iCanHazDraw!
Watch The Day of the Dead, or Dia de Muertos, is a celebration that spans three days October 31-November 2. It coincides with Halloween and All Saints Day. This is a time when people remember their loved ones who have passed away and celebrate their lives. Sugar skulls are used to represent the deceased. Make your sugar skull as simple or as elaborate as you wish! Decorate with hearts, flowers, spiderwebs, curly lines, dots, and anything else you can think of!
Youtube Channel / iCanHazDraw
share on twitter
share on facebook
share on google plus
share on linkedin
share on stumbleupon
share on pinterest
Related Videos
Learn How To Draw and Color A Sugar Skull -- Part 2 -- iCanHazDraw!
Learn How To Draw and Color A Pretty Sugar Skull Girl -- Part 1 -- iCanHazDraw!
Zentangle Skull ~drawing process~


text:
Buffalo Bulls dismiss head coach Jeff Quinn
The Buffalo Bulls have dismissed head coach Jeff Quinn after five years, Pete Roussel of Coachingsearch.com reported on Monday afternoon. Offensive coordinator Alex Wood will replace him as interim head coach.
Since becoming head coach in 2010, Quinn collected a 20-36 record. The Bulls went 8-5 in 2013, playing in an FBS bowl for the second time in team history.
The team has gone 3-4 this year, most recently losing two in a row to Eastern Michigan and Bowling Green.
• Bell Jr. leads Eastern Michigan over Buffalo 37-27
With a 1-2 record in the Mid-American Conference, the Bulls are third in the East Division behind Bowling Green and Akron. Buffalo has a bye this week.
Quinn's resume before the head coaching job at Buffalo included time as offensive coordinator/line coach with Brian Kelly at Cincinnati, as well as stints at Central Michigan and Grand Valley State.
• Bowling Green holds on for 36-35 win over Buffalo
So far during the 2014 season, the Bulls are 27th in the FBS in passing, with 290.7 yards per game, but 50th in rushing, amassing 185.1 yards per game.
Despite being 49th overall in points for, averaging 33 per contest, Buffalo is 102nd in points against, allowing 35.


text: Lipscomb University: Opinions - News - Schedule - History - Basketball - Baseball - Other Sports - Fans Opinions good stuff new stuff <ListPages> category=Lipscomb University Opinions order=Score Level=3 count=10 </ListPages> <ListPages> category=Lipscomb University Opinions order=new count=10 </ListPages> (how does this work?) | (write an opinion!) | (more opinions) News good stuff new stuff <ListPages> category=Lipscomb University News Published=Yes order=PublishedDate Level = 3 count=10 </ListPages> <ListPages> category=Lipscomb University News order=new count=10 </ListPages> (how does this work?) | (write a news article!) | (more news) Standings Through March 19, 2007 Atlantic Sun Basketball W L HOME ROAD STRK CONF East Tennessee State University 24 10 16-2 Belmont University 23 10 14-4 Lipscomb University 18 13 11-7 Jacksonville University 15 14 11-7 Kennesaw State University 13 18 9-9 Mercer University 13 17 8-10 Campbell University 14 17 7-11 Gardner-Webb University 9 21 7-11 Stetson University 11 20 6-12 University of North Florida 3 26 1-17 Florida Atlantic University Edit Standings College Navigation Conferences College Basketball America East Atlantic 10 Atlantic Coast Atlantic Sun Big 12 Big East Big Sky Big South Big Ten Big West Colonial Athletic Conference USA Horizon League Ivy League MAAC Mid-Continent Mid-American MEAC Missouri Valley Mountain West Northeast Ohio Valley Patriot League Pac-10 SEC Southern Southland SWAC Sun Belt West Coast WAC Opinions News Scores/Schedule Standings Teams Players College Football Opinions News Scores/Schedule Standings Teams Players Quick Facts Athletics Logo Established: 1891 School Type: Private, Churches of Christ, University President/Chancellor: L. Randolph Lowry, III Location: Nashville, Tennessee Campus Size: Urban, 65 acres Enrollment: 2,744 Faculty: 115 Mascot: Stadiums: Ken Dugan Field at Stephen Lee Marsh Stadium, Allen Arena Colors: Purple & Gold Team Nickname: Bison Conference(s): Atlantic Sun Conference Homepage: www.lipscomb.edu Athletic Homepage: www.lipscombsports.com Local Radio: Basketball Ranking: RPI Rating: Football Ranking: BCS Rating: Edit These Facts Retrieved from "http://armchairgm.wikia.com/wiki/Lipscomb_University?oldid=32815" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected.


text:
- Jam
- Jam Jam, n.
1. A mass of people or objects crowded together; also, the
pressure from a crowd; a crush; as, a jam in a street; a
jam of logs in a river.
[1913 Webster]
2. An injury caused by jamming. [Colloq.] [1913 Webster]
3. A difficult situation; as, he got himself into a jam. [informal] [PJC]
The Collaborative International Dictionary of English. 2000.


text:
Levita, Lt. Col. (Sir) Cecil Bingham
Born 18 January 1867, died 10 October 1953.
Won London County Council by-election 1911 for Municipal Reform Party, and represented North Kensington for over 25 years, Chairman 1928–1929.
Career soldier fighting in Second Matabele War and Second Boer War, 1910
Largely responsible for founding of King George Hospital Ilford. Involved in the 'Talking Mongoose Case' (Wikipedia page [1]), in which he was sued for libel and lost
Articles in Wikipedia [2]. Who's Who - Obit The Times 12 October 1953 p 10.


text:
Decision makers
- Florida Governor
Save Our Seagrass!
Seagrass habitat plays an important role in maintaining a diverse and healthy coastal ecosystem in Florida. Unfortunately, this valuable habitat is threatened by human activities such as urban and agricultural runoff, dredging, and boating accidents.
There are currently more than 30 seagrass restoration projects are underway across Florida, many of them in the Florida Keys National Marine Sanctuary. The Marine Sanctuary protects over 2,900 square nautical miles of critical marine habitat, including coral reef, hard bottom, seagrass meadow, mangrove communities and sand flats.
With the newly elected administration in Florida threatening to make severe cuts to environmental restoration projects as well as a legislature that is largely apathetic to environmental causes, it is important for citizens to let their voices be heard.
- Florida State House
- Florida State Senate
- Florida Governor
As you may know, seagrass habitat plays an important role in maintaining a diverse and healthy coastal ecosystem in Florida. It provides a critical habitat for many species of fishes, crustaceans, and shellfish. Seagrasses and the organisms that grow on them are also food sources for many animals, including wild manatees. Additionally, they provide nursery areas for much of Florida's recreationally and commercially important marine life.
Unfortunately, this valuable habitat is threatened by human activities such as urban and agricultural runoff, dredging, and boating accidents. Without funding and legislative support for restoration efforts, Florida's seagrasses could become greatly diminished. This could threaten coastal areas with increased erosion, devastate fisheries that support Florida's economy, and cause harm to marine wildlife that depend on the seagrasses for food and protection.
Please support all legislation and other legal action that will ensure Florida's seagrasses are protected, and make sure restoration projects across the state, from the northern Gulf of Mexico to the Florida Keys, are fully funded. Our coasts, our wildlife, and our economy depend on it.
Thank you,
Save Our Shores! Florida started this petition with a single signature, and now has 291 supporters. Start a petition today to change something you care about.
Report abuse
Please report any content that may violate Change.org's Terms of Service or Community Guidelines.
Thank you for taking the time to report content. Our team will review your claim and contact you if we need more information.


text:
Ninth Annual FARE Teen Summit
Food Allergy Research & Education
Friday, November 7, 2014 at 6:30 PM - Sunday, November 9, 2014 at 1:00 PM (EST)
Who's Going
San Francisco, California
London, United Kingdom
Florence, Italy
Event Details
The Ninth Annual Teen Summit is a 3-day event that gives teens living with food allergies a chance to meet with other teens from across the country. Pre-teens and teens age 11-22 are invited to attend, and we will cover topics that are relevant and important to them:
- social experiences and peer pressure
- dining out and traveling
- knowing how to be your own advocate
- friendships and dating
- navigating high school
- preparing for college and more!
Teen Summit participants get to spend time with people in their own age group through special breakout sessions for middle school and high school, plus evening social activities that are food allergy friendly and fun! We also will have a sibling and parent track for family members to share their experiences and learn from some of our great speakers.
To make a hotel reservation, please use the following link: https://aws.passkey.com/event/11123823/owner/544/home
If you have any questions regarding registration or hotel reservations, please contact Kristin Rafferty at email@example.com
To learn more about sponsorship and exhibiting opportunities, please contact Donna McKelvey at email@example.com
When & Where
Grand Hyatt Washington
1000 H St NW
Friday, November 7, 2014 at 6:30 PM - Sunday, November 9, 2014 at 1:00 PM (EST)
Add to my calendar
Food Allergy Research & Education
Food Allergy Research & Education (FARE) works on behalf of the 15 million Americans with food allergies, including all those at risk for life-threatening anaphylaxis. This potentially deadly disease affects 1 in every 13 children in the U.S. – or roughly two in every classroom. FARE's mission is to improve the quality of life and the health of individuals with food allergies, and to provide them hope through the promise of new treatments.


text:
|Home | About | Journals | Submit | Contact Us | Français
Oxidative damage contributes to microbe elimination during macrophage respiratory burst. Nuclear factor, erythroid-derived 2, like 2 (NRF2) orchestrates antioxidant defenses, including the expression of heme-oxygenase–1 (HO-1). Unexpectedly, the activation of NRF2 and HO-1 reduces infection by a number of pathogens, although the mechanism responsible for this effect is largely unknown. We studied Trypanosoma cruzi infection in mice in which NRF2/HO-1 was induced with cobalt protoporphyrin (CoPP). CoPP reduced parasitemia and tissue parasitism, while an inhibitor of HO-1 activity increased T. cruzi parasitemia in blood. CoPP-induced effects did not depend on the adaptive immunity, nor were parasites directly targeted. We also found that CoPP reduced macrophage parasitism, which depended on NRF2 expression but not on classical mechanisms such as apoptosis of infected cells, induction of type I IFN, or NO. We found that exogenous expression of NRF2 or HO-1 also reduced macrophage parasitism. Several antioxidants, including NRF2 activators, reduced macrophage parasite burden, while pro-oxidants promoted it. Reducing the intracellular labile iron pool decreased parasitism, and antioxidants increased the expression of ferritin and ferroportin in infected macrophages. Ferrous sulfate reversed the CoPP-induced decrease in macrophage parasite burden and, given in vivo, reversed their protective effects. Our results indicate that oxidative stress contributes to parasite persistence in host tissues and open a new avenue for the development of anti–T. cruzi drugs.
Oxidative stress is generated during acute Chagas disease and contributes to the tissue damage observed with this infection. Trypanosoma cruzi, the causative agent of Chagas disease, infects cardiomyocytes, alters their mitochondrial potential, and induces ROS (1). This overwhelms the antioxidant defenses and produces persistent oxidative stress (2–4). Free iron and heme are released from damaged muscle cells and may potentially contribute to oxidative stress (5) and inflammation (6, 7). Furthermore, macrophages are activated to produce ROS via respiratory burst during infection (8–10). Though ROS are detrimental to most pathogens, T. cruzi has highly effective antioxidant machinery that may potentially confer resistance to oxidative environments (11).
Antioxidant defenses orchestrated by the transcription factor NRF2 help maintain redox environments in cellular compartments, allowing them to perform tasks that require ROS at optimal concentrations, such as protein folding in the endoplasmic reticulum (12). Increased mitochondrial respiration and phagocyte respiratory burst are oxidative events that can overwhelm NRF2-dependent antioxidant defenses and lead to redox imbalance. The expression of the enzyme HO-1 is regulated by NRF2. This enzyme has antiapoptotic, anti-inflammatory, and anti-immunogenic capacities (13). HO-1 can shift the redox balance by degrading the pro-oxidant heme and increasing the amount of the antioxidant biliverdin and, subsequently, bilirubin, by-products of this reaction. Pro-oxidant Fe2+ is also produced by heme degradation, but it is safely disposed of through sequestration by ferritin or exiting the cell through ferroportin (13). HO-1 can also translocate to the nucleus and directly activate antioxidant mechanisms (14). Additionally, NADPH oxidase (NOX2), the enzyme responsible for macrophage oxidative burst, is a heme-protein, and its activity is greatly decreased by the reduction of heme availability with the induction of HO-1 (15).
Pathogens may be subjected to ROS-mediated oxidative damage during phagocyte burst; in addition, ROS can combine with NO to form highly lethal peroxynitrite. There are a few exceptions to the rule that ROS are detrimental to pathogen growth: antioxidant administration, as well as deficiency in NOX2, decrease pathogen burden in some viral infections (16–18), and ROS favor the growth of Mycobacterium abscessus inside macrophages (19). The expression of NRF2 target antioxidant genes is thus expected to weaken defenses against ROS-sensitive pathogens. In fact, HO-1 expression promotes liver infection by Plasmodium berghei and Plasmodium yoelii (20). However, HO-1 induction reduces viral burden in hepatitis B (21), hepatitis C (22), enterovirus-71 (17), and HIV (23) infections; mediates macrophage resistance to Salmonella serovar Typhimurium (24); and enhances bacterial clearance of Enterococcus faecalis (25). NRF2 induction also reduces infection with RSV (26), and Nrf2–/– mice are susceptible to Pseudomonas aeruginosa (27). Together, these results suggest that NRF2/HO-1 might participate in innate immunity, perhaps against pathogens that thrive in oxidative environments. Herein, we investigated the therapeutic effects of HO-1 induction in T. cruzi infection. Our results indicate that oxidative stress generated in response to T. cruzi infection contributes to maintenance of high parasite burdens.
To determine the role of HO-1 in acute T. cruzi infection, we infected C57BL/6 mice with the Y strain and treated them with cobalt protoporphyrin (CoPP), an inducer of HO-1 expression that activates NRF2 (28), or with tin protoporphyrin (SnPP), an inhibitor of HO-1 activity. Treatment was discontinued at the end of patent parasitemia (10 days after infection [dpi]). CoPP greatly reduced parasitemia, while SnPP increased it (Figure (Figure1A).1A). All mice survived infection with T. cruzi. At 8 dpi, parasitism was low in tissues from CoPP-treated mice but higher in hearts and skeletal muscle from SnPP-treated mice (Figure (Figure1,1, B and C). Parasites were mostly confined to infiltrating cells in these tissues (Figure (Figure1D).1D). By 15 dpi, the parasitism was controlled in SnPP-treated mice, and CoPP-treated mice had reduced burden in the heart and striated muscle as compared with untreated controls (Figure (Figure1E).1E). Thus, contrary to our hypothesis, the induction of NRF2/HO-1 expression correlated with decreased parasite burden.
Both CD8+ and Th1 lymphocytes are essential for controlling T. cruzi infection (29, 30). T. cruzi infection induces ROS production by macrophages, which inhibits the immunoproteasome and decreases class I antigen presentation (31). Based on that, we predicted that activating antioxidant mechanisms with CoPP treatment would favor MHC class I antigenic presentation and thus allow a more effective CD8-mediated protective response. However, treatment of Cd8–/– (Figure (Figure2A)2A) and perforin-knockout (Pfn–/–) (Figure (Figure2B)2B) mice with CoPP reduced parasitemia and mortality to levels similar to those of wild-type mice.
Antioxidants alter Th responses (32). Treatment with CoPP did not alter the production of IFN-γ or IL-17A by splenocytes stimulated with ConA, anti-CD3, or T. cruzi antigen at 9 dpi (Figure (Figure2C).2C). IL-4 remained close to the lower limit of detection in all groups (data not shown). Plasma IFN-γ concentrations did not change in response to CoPP (Supplemental Figure 1A; supplemental material available online with this article; doi: 10.1172/JCI58525DS1). Treatment of infected Ifng–/– mice with CoPP greatly reduced parasitemias and postponed death, although treatment did not totally compensate for their high susceptibility to infection. Infected Ifng–/– mice relapsed with parasitemia after treatment was discontinued at 10 dpi (Figure (Figure2D).2D). Likewise, treatment with CoPP greatly reduced parasitemia and postponed death of Rag2–/– mice, and parasitemia increased only after treatment was discontinued (Figure (Figure2E).2E). Also, CoPP did not substantially alter lymphocyte apoptosis (Supplemental Figure 1B). Together, these data indicate that CoPP does not reduce parasitism by regulating T lymphocyte immunity.
Peritoneal macrophages from infected mice, untreated or treated with CoPP or SnPP, were screened for amastigotes at 9 dpi. Administration of CoPP to mice reduced macrophage parasitism, while SnPP increased it (Figure (Figure3A).3A). We also infected thioglycollate-elicited macrophages in vitro and treated them with CoPP or SnPP. Treatment with CoPP reduced macrophage parasitism, while SnPP increased it (Figure (Figure3B).3B). When trypomastigotes were allowed to mature, CoPP also reduced the numbers of trypomastigotes (Supplemental Figure 2A). Infected macrophages treated with CoPP had higher HO-1 expression (Supplemental Figure 2B). These data show that the HO-1 inducer CoPP reduces macrophage parasitism by T. cruzi in vivo and in vitro.
SnPP, CoPP, and the by-products of HO-1 activity biliverdin and bilirubin were unable to kill trypomastigotes in vitro, as determined by trypomastigote motility (Supplemental Figure 2C). In contrast to benznidazole, CoPP could not kill trypomastigotes even after 48 hours incubation, as assessed by propidium iodide (PI) staining (Supplemental Figure 2D). Neither the supernatant of macrophages incubated with CoPP nor the sera from mice treated with CoPP were able to kill trypomastigotes in vitro, indicating that no trypanocidal soluble factor is induced in response to CoPP (Supplemental Figure 2, E–G). However, treatment of macrophages with CoPP after differentiation of trypomastigotes into amastigotes (24 hours) did reduce infection (Supplemental Figure 2H). Likewise, CoPP administered to mice starting at 2 dpi (allowing differentiation into amastigotes) also reduced parasitemia (Supplemental Figure 2I), indicating that CoPP does not target amastigogenesis. However, CoPP did not alter parasite burden in the fibroblastic cell line L929 (Figure (Figure3C),3C), indicating that its effects depend on macrophage physiology rather than acting directly on the parasite.
CoPP is known to decrease NRF2 degradation, thereby increasing expression of HO-1 (28). We tested whether HO-1 overexpression could mimic the decrease in parasitism mediated by CoPP in THP-1 cells (Figure (Figure3D).3D). Transfection of THP-1 cells with an Hmox1 vector markedly decreased parasitism as compared with mock-transfected controls (Figure (Figure3D).3D). We then tested whether CoPP required HO-1 expression in order to reduce parasitism. CoPP similarly decreased the parasitism of bone marrow macrophages (BMMs) from wild-type and HO-1–deficient (Hmox1–/–) mice (Figure (Figure3E).3E). These results suggest that other NRF2 target genes induced by CoPP can replace HO-1 in its absence to reduce parasitism. Indeed, CoPP failed to reduce parasitism in Nrf2–/– BMMs (Figure (Figure3F),3F), demonstrating that NRF2 is required for CoPP to reduce parasitism. THP-1 cells transfected with Nrf2 showed reduced parasitism compared with mock-transfected controls (Figure (Figure3G).3G). We also incubated infected macrophages with the NRF2 activators oltipraz, sulforaphane, pterostilbene, and resveratrol. Similar to CoPP (Figure (Figure3H),3H), the other NRF2 activators reduced macrophage parasitism in a dose-dependent fashion (Figure (Figure3,3, I–L). Resveratrol and pterostilbene administration in vivo also reduced parasitemia (Figure (Figure3M).3M). Together, our data indicate that genes activated by NRF2 redundantly control resistance to T. cruzi infection in macrophages.
As our results indicated that CoPP targeted macrophage physiology to reduce parasite burden, we tested whether CoPP relied on classical mechanisms of parasite elimination by macrophages. Apoptosis is a common response to intracellular infections that reduces infection reservoirs. We investigated whether the effect of CoPP on parasite burden was due to the increased survival of uninfected cells. We found that treatment with CoPP reduced apoptosis of all cells following exposure to T. cruzi, irrespective of the whether the cells were infected (Figure (Figure44B).
Production of type I IFN by infected cells represents a mechanism of defense against T. cruzi (34). As HO-1 expression in macrophages is required for IRF3 phosphorylation and IFN-β production in response to various stimuli (35), we tested whether treatment with CoPP would alter IFN-β production during T. cruzi infection. Infection induced IRF3 phosphorylation in macrophages (36), but CoPP had no effect (Figure (Figure4C).4C). IFN-β production increased upon infection, but was attenuated in macrophages treated with CoPP (Figure (Figure4D).4D). CoPP reduced parasitism in macrophages from wild-type mice and to a lesser extent in macrophages from Ifnar1–/– mice (Figure (Figure4E).4E). However, treatment with CoPP reduced parasitemia of Ifnar1–/– (Figure (Figure4F)4F) mice to levels similar to those of wild-type mice.
Macrophages from CoPP-treated infected mice (8 dpi) produced less NO when stimulated with LPS ex vivo (Figure (Figure4G).4G). Macrophages infected in vitro and then treated with CoPP also produced less NO in response to LPS (Figure (Figure4H).4H). CoPP reduced parasitism in both wild-type and Nos2–/– (iNOS-deficient) macrophages (Figure (Figure4,4, I and J). Treatment of Nos2–/– mice with CoPP reduced parasitemia and mortality (Figure (Figure4,4, K and L). A similar profile was found for TNF (Supplemental Figure 3, A–E). These results demonstrate that conventional T. cruzi elimination mechanisms are not involved in the reduction of parasitism by CoPP.
To confirm that infection promotes respiratory burst, we incubated noninfected and infected macrophages with the ROS probe CM-H2DCFDA. Infection increased fluorescence starting at 6 hours after infection (Figure (Figure5A).5A). As expected, CoPP partially prevented the increase in ROS induced by infection (Figure (Figure5A).5A). By 6 hours after treatment with CoPP, macrophage parasite burden was still similar among treated and nontreated cells (Supplemental Figure 4). The antioxidants superoxide dismutase–polyethylene glycol (SOD-PEG), catalase–polyethylene glycol (CAT-PEG), bilirubin, biliverdin, N-acetyl-cysteine (NAC), and apocynin all reduced parasitism (Figure (Figure5,5, B and C). H2O2 alone promoted while co-incubation with CoPP reversed the protective effects of CoPP (Figure (Figure5D).5D). Activation with PMA, which is known to promote respiratory burst, increased macrophage parasitism (Figure (Figure5E).5E). Low concentrations (10 μM) of the pro-oxidant paraquat did not promote parasitism but did reverse the protective effects of CoPP (Figure (Figure5F).5F). High concentrations (100 μM) promoted macrophage cell death, but CoPP plus paraquat prevented macrophage death and allowed parasitism (Figure (Figure5G).5G). Paraquat administration in vivo increased parasitemia and mortality of infected mice (Figure (Figure5,5, H and I), indicating that this effect is also relevant in vivo. Infection of macrophages from mice deficient in NOX2 subunit gp91phox resulted in reduced parasitism as compared with wild-type mice, and CoPP did not have a protective effect (Figure (Figure6A).6A). BMMs from gp91phox–/– mice produced less ROS than cells from wild-type mice upon infection (Figure (Figure6B).6B). In fact, infected gp91phox–/– mice also had reduced parasitemia (Figure (Figure6C)6C) and reduced parasite burden in their splenic macrophages as compared with wild-type mice (Figure (Figure6D).6D). Still, infected gp91phox–/– mice treated with CoPP showed reduced parasitemia (Figure (Figure6E),6E), suggesting that CoPP also acts through non-NOX2-targeted mechanisms in vivo, though to a lesser extent. Nevertheless, infected gp91phox–/– mice died late in acute infection (Figure (Figure6F),6F), while gp91phox–/– mice treated with CoPP survived the acute infection. The cause of death of infected gp91phox–/– mice and the mechanism of protection by CoPP remain to be investigated. Together, these results suggest that macrophage exposure to ROS promotes infection.
Ferritin and ferroportin have antioxidant response element (ARE) sequence motifs in their promoters and are upregulated by NRF2 activators (37, 38). Ferritin-bound iron represents a redox-inert storage of iron. Intracellular microbes differ in the sources of iron they use, and although Neisseria scavenge iron-laden ferritin (39), the trypanosomatid Leishmania donovani feeds on only the labile iron pool (non-ferritin iron) (40). The sources of iron used by T. cruzi amastigotes as a nutrient are currently unknown. Reduction of the labile iron pool could potentially decrease the iron available for dividing amastigotes or, alternatively, reduce oxidative stress. Therefore, we tested whether reduction of labile iron pool could be responsible for the effects of CoPP.
Treatment with CoPP, as well as with the antioxidants NAC and apocynin, increased H-ferritin and ferroportin-1 (FPN1) expression in infected macrophages (Figure (Figure7A).7A). Increased expression of H-ferritin and FPN1 is known to reduce the labile iron pool (41). Using the quenching of calcein fluorescence as an indicator of free iron, we measured the labile iron pool by flow cytometry. Treatment of infected macrophages with the antioxidants CoPP, NAC, and apocynin increased calcein fluorescence compared with infected untreated cells (Figure (Figure7B),7B), indicating a reduction in the labile iron pool. Concomitant treatment with antioxidants and Fe2+ reversed the increase in calcein fluorescence.
Transfection of THP-1 cells with H-ferritin as well as incubation with apoferritin reduced parasitism (Figure (Figure7,7, C and D). The iron chelator desferrioxamine (DFO), which decreases intracellular iron availability, also reduced macrophage parasitism (Figure (Figure7E).7E). BMP and IL-6 promote degradation of FPN1 through hepcidin induction and have similar autocrine effects on macrophages (42–44). Treatment with IL-6 or Fe2+ did not alter parasitism, but incubation with both simultaneously, which is known to induce iron loading, increased parasitism (Figure (Figure7F).7F). Dorsomorphin, an inhibitor of the BMP/Smad4 pathway that ultimately promotes ferroportin expression (45), reduced macrophage parasitism, and Fe2+ reversed this effect (Figure (Figure7G).7G). Incubation with Fe2+ also reversed the CoPP-mediated decrease in parasitism, as well as that produced by NAC or apocynin (Figure (Figure7,7, H–J). Fe2+ also enhanced parasitism in macrophages from gp91phox–/– mice (Figure (Figure7K).7K). Together, these results indicate that increases in the labile iron pool are associated with increased macrophage parasitism.
As HO-1 is known to affect iron physiologically in vivo (46, 47), we determined whether peroral iron was capable of attenuating the effects of CoPP. Administration of FeSO4 increased parasitemia (Figure (Figure7L).7L). When given with CoPP, peroral FeSO4 reversed the CoPP-mediated decrease in parasitemia, resulting in levels of parasitemia similar to those of mice treated with FeSO4 (9 dpi) only. Mortality followed a similar pattern (Figure (Figure7M).7M). These results suggest that CoPP-mediated reduction of the labile iron pool is involved in reducing parasite burden.
To date, little is known about the role of ROS in T. cruzi parasite burden. In macrophages with an exhausted respiratory burst, T. cruzi growth increased, suggesting that ROS are protective (48). This finding was further supported by the association between macrophage trypanocidal activity and the production of hydrogen peroxide (49). Others have not found evidence that macrophage-derived ROS contribute to T. cruzi killing (50, 51). However, previous studies have shown that amastigote burden significantly decreased when preinfected macrophages were treated with SOD and catalase during stimulation with IFN-γ (52). The authors interpreted this finding as a consequence of the increased NO production, but no functional studies supported this conclusion. Recently, evidence for peroxynitrite as a mediator of T. cruzi killing was found in IFN-γ/LPS–preactivated macrophages (10), but the subject was not approached systematically in unstimulated macrophages. To our knowledge, we have performed the first systematic study of T. cruzi infection under pro- or antioxidant conditions. This study indicates that ROS contribute to Y strain amastigote growth inside macrophages and increase overall parasitism.
Our results consistently point to oxidative stress as an enhancer of T. cruzi infection in macrophages. Indeed, the reduced macrophage parasitism in apocynin (NOX2 inhibitor)–treated and gp91phox–/– macrophages indicate that ROS generated during respiratory burst are counterproductive against T. cruzi infection. This finding is corroborated by the observation that PMA and H2O2 increase parasitism. We also observed reduced parasitemia and reduced macrophage parasitism in infected gp91phox–/– mice. These data are in contrast with the reported increased parasite burden in apocynin-treated mice infected with strain Sylvio X10/4 at 35 dpi (53). However, these authors did not evaluate macrophage parasitism in their study. It is possible that ROS generation by NOX2 is required to control infection in particular tissues, to establish a late adaptive response, or to combat particular T. cruzi strains.
Evidence that oxidative stress fuels infection also comes from the observation that mice treated with the pro-oxidant paraquat had increased parasitemia, while mice treated with the NRF2 activators resveratrol and pterostilbene or deficient in gp91phox expression had decreased parasitemia. Others have previously reported that treatment with NAC reduced T. cruzi parasitemia (54) and also parasite burden in Leishmania infection (55), which resisted oxidative stress (56).
Although transfection with HO-1 mimicked treatment with CoPP and reduced parasite burden, NRF2 target genes (other than HO-1) probably replaced HO-1 in its absence, as CoPP reduced parasite burden in macrophages from Hmox1–/– mice but not Nrf2–/– mice. These data demonstrate that CoPP operates through NRF2 target genes to reduce parasitism and were also corroborated by the ability of other reputed NRF2 activators to reduce macrophage parasitism and parasitemia.
Treatment with SnPP, an inhibitor of HO-1 activity, increased macrophage parasitism, but BMMs from Hmox1–/– and Nrf2–/– mice had parasitism similar to that in wild-type mice. It is possible that HO-1 controls infection and that the sudden loss of its activity increases parasite burden. In this case, compensatory mechanisms of parasite control could explain why Hmox1–/– and Nrf2–/– macrophages did not show increased parasite burden.
As we found that treatment with CoPP prevents apoptosis in T. cruzi–infected macrophages, it could be argued that CoPP acts by leaving macrophages intact to fight infection. However, macrophages can withstand heavy infection before rupturing, so survival alone does not explain how the macrophages eliminate the parasite (33). We showed here that CoPP reduces parasitism despite severely reducing NO, a classical mechanism by which macrophages eliminate T. cruzi. These data indicate that CoPP triggers a rather unconventional mechanism of T. cruzi elimination. It is nevertheless possible that other cell types have mechanisms distinct from those of macrophages to fight infection and can benefit from the lower rate of apoptosis mediated by CoPP.
T. cruzi amastigotes require iron to grow in axenic cultures (57). Chelating iron reduces T. cruzi infection in macrophages (58) and mice (59), while iron-rich diets or iron-dextran worsen infection (60), suggesting that iron is critical for T. cruzi growth. Here, we found that several strategies to increase the labile iron pool resulted in increased parasitism, while incubation of infected macrophages with Fe2+ reversed the decrease in parasitism mediated by CoPP and other antioxidants. In vivo, peroral FeSO4 also reversed the reduction in parasitemia mediated by CoPP. These results indicate that antioxidants act by precluding iron uptake by the parasite or by reversing the oxidative stress generated by Fe2+, which would then increase parasitism through unknown mechanisms. Indeed, treatment with antioxidants increased expression of H-ferritin and FPN1 and increased the fluorescence of calcein, demonstrating that antioxidants reduce the labile iron pool during infection. These data also suggest that the decrease in the labile iron pool is involved in preventing macrophage parasitism.
Extensive literature describes HO-1 as an anti-immunogenic, antiinflammatory, and antioxidant enzyme (13). ROS production is a well-known mechanism of pathogen elimination through respiratory burst in macrophages. Though it was expected that induction of HO-1 in infections would cripple the defenses and allow pathogen growth, we observed the exact opposite during T. cruzi infection. In fact, a number of recent findings point to NRF2 and HO-1 as mediators of innate immunity. Pathogen burden has been shown to be decreased by HO-1 overexpression in hepatitis C–infected hepatocytes (61, 62), hepatitis B–infected mice (21), enterovirus 71 infection in neuroblastoma (17), and HIV-infected monocytes (23); however, the mechanism of this is unknown. The HO-1–mediated increase in bacterial clearance during sepsis has been associated with increased phagocytosis that could potentially mediate the protective effects (25). Antioxidants have been found to reduce pathogen burden in a number of infections (16–18), while hydrogen peroxide increased Mycobacterium abscessus burden (19), indicating that ROS can be counterproductive in some infections. We showed here that HO-1 induction reduces macrophage parasitism and parasitemia, which correlated positively with oxidative stress and the labile iron pool. Whether this is the main mechanism by which NRF2 target genes operate to control infections remains to be determined.
In conclusion, we showed that T. cruzi parasitism correlated with oxidative stress, while CoPP reduced T. cruzi-induced ROS production and infection in an NRF2/HO-1–dependent manner. Similar to our findings, melatonin, a hormone that activates antioxidant mechanisms through NRF2 activation (63), has been shown to decrease parasitemia and mortality (64). Also, 15d-PGJ2, an agonist of PPARγ that induces NRF2/HO-1 expression in macrophages (65), decreases T. cruzi parasitism (66), as does curcumin (67), a reputed NRF2 activator. It remains to be elucidated whether melatonin, 15d-PGJ2, and curcumin act through NRF2/HO-1–dependent mechanisms to reduce T. cruzi infection. The exact mechanism by which oxidative stress enhances T. cruzi infection remains to be clarified, though iron mobilization is a possibility. We believe the association between NRF2/HO-1 induction and protection against T. cruzi parasite burden and tissue damage demonstrated here opens a new avenue for the development of drugs against this pathogen.
T. cruzi Y strain trypomastigotes were maintained by blood passage in mice every 7 days. Trypomastigotes were isolated from heparinized blood by low-speed centrifugation and collection of the parasites that swam out of the pellet. Experimental infection was performed by i.p. injection of 104 blood trypomastigotes. In vitro experiments were performed with Y strain trypomastigotes obtained from infected LLCKM2 cell cultures (grown in 10% FCS supplemented DMEM). Parasitemia was evaluated in tail blood. C57BL/6 mice were bred and maintained in our facilities (UFRJ, IMPPG). gp91phox–/– (C57BL/6) mice were provided by Leda Q. Vieira (Universidade Federal de Minas Gerais [UFMG], Belo Horizonte, Brazil) and José Carlos Alves Filho (Universidade de São Paulo [USP], Ribeirão Preto, Brazil). Hmox1–/– (BALB/c) (68) and Nrf2–/– (C57BL/6) (69) bone marrow samples were a gift from Miguel Soares (Instituto Gulbenkian de Ciência, Oleiras, Portugal), and its use was authorized by Mark Perrela and RIKEN BRC through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, respectively. Nos2–/–, Tnfr1–/–, Cd8–/–, and Pfn–/– mice (all on the C57BL/6 background) were obtained from Centro de Criação de Animais de Laboratório (Cecal, Fiocruz, Rio de Janeiro, Brazil). Ifnar1–/– (129Sv) and wild-type mice were obtained from USP. Ifng–/– (C57BL/6) mice were obtained from both Cecal and Instituto Nacional del Cáncer (INCA, Rio de Janeiro, Brazil). CoPP, SnPP, biliverdin hydrochloride (Blv), and bilirubin were purchased from Frontier Scientific, dissolved in 0.2 M NaOH, neutralized to pH 7.2 with 1 M HCl, and adjusted to the desired concentrations with PBS. Treatments were performed by daily i.p. injection of vehicle or 5 mg/kg SnPP or CoPP. Treatment with CoPP or SnPP started on day 1 or 2 hours after infection, with similar results, except in Supplemental Figure 2J, where CoPP was started at 2 dpi. Prolonged treatment with CoPP (but not with SnPP) resulted in high mortality of mice in control experiments; therefore, we stopped treatment after the peak of parasitemia (8–10 dpi). Treatment with peroral FeSO4 was performed by gavage (40 mg/kg) from 0 to 9 dpi. Paraquat was diluted in PBS (5 mg/kg) and injected i.p. in mice daily from 0 to 8 dpi. Resveratrol (3 mg/kg) and pterostilbene (2 mg/kg) were administered i.p. from 0 to 9 dpi (diluted in pure ethanol and then in PBS).
Peritoneal cells from mice infected and left untreated or treated with apocynin, CoPP, or SnPP were collected at 8 dpi and seeded on coverslips inside 24-well plates for 48 hours in complete RPMI. Coverslips were then processed as described for in vitro assays of parasite burden.
Thioglycollate-elicited peritoneal macrophages, BMMs, THP-1 differentiated macrophages, or L929 fibroblasts were used in these assays. THP-1 cells were differentiated as described below. Thioglycollate (4%) was injected i.p. into C57BL/6 mice, and peritoneal cells were collected by chilled RPMI washes 4 days after injection to obtain elicited macrophages. BMMs were obtained by differentiation in vitro. Femurs and tibiae were cut at the ends and flushed with RPMI medium. Bone marrow cells were seeded at 4 × 106/ml per 100-mm2 dish in 10 ml complete medium (RPMI [Gibco, Invitrogen] supplemented with 20% inactivated FCS, l-glutamine, HEPES, β-mercaptoethanol, and penicillin/streptomycin) with 20% conditioned medium (containing M-CSF, isolated from L929 cell cultures) for 4 days. Then, fresh medium was added, and cells were cultured until day 7. Cells were then harvested and used to seed round glass coverslips. A sample containing 3 × 105 to 5 × 105 elicited macrophages or BMMs was used to seed glass coverslips and left to adhere for 2 hours and rest for 24 hours. A sample containing 105 L929 fibroblasts was used to seed glass coverslips for 2 hours. Non-adherent cells were washed away before infection. Trypomastigotes obtained from cultures (as described above) were used to infect cells at a 3:1 (macrophages) or 10:1 (L929 cells) ratio for 12 hours, and then cultures were washed in RPMI. Drugs were diluted in complete RPMI and added to infected cultures for 48 hours. Then, cells were washed, fixed in absolute methanol, and stained with Giemsa (Sigma-Aldrich). Cells were then cleared and dehydrated in a xylol-acetone gradient, and slides were mounted with Entellan and photographed under an Olympus microscope (CX21, ×400 magnification). Pictures were printed, and amastigotes were identified and counted in samples of 40–200 infected macrophages from each group. Percentages of infected cells were determined from 20 pictures per group. In general, the percentage of infected cells paralleled amastigote burden within 48 hours, so we used amastigote burden to indicate parasitism. The following drugs were used in these assays: CoPP (50 μM, as determined by the dose-response curve in Figure Figure3),3), SnPP (50 μM), biliverdin (50 μM), and bilirubin (10 μM) (Frontier Scientific); resveratrol, pterostilbene, sulforaphane, and oltipraz (as determined by dose-response; Sigma-Aldrich); dorsomorphin (compound C, 10 μM, Sigma-Aldrich); mIL-6 (50 ng/ml, PeproTech); apocynin (1 mM; Sigma-Aldrich); NAC (10 μM, Sigma-Aldrich); FeCl3 (100 μM, Sigma-Aldrich); FeSO4 (100 μM, Sigma-Aldrich); DFO mesylate (DFO, 50 μM, Sigma-Aldrich); H2O2 (100 μM; 200 μM to reverse CoPP effects); PMA (200 ng/ml, Sigma-Aldrich); SOD-PEG (25 U/well); CAT-PEG (40 U/well); and paraquat (10–100 μM, Sigma-Aldrich).
THP-1 cells were plated on glass coverslips in 24-well dishes (300,000 cells/dish) and differentiated into macrophages with PMA (40 ng/ml) for 72 hours, and then cultured for an additional 24 hours in DMEM supplemented with 10% fetal bovine serum in a 5% CO2 humidified atmosphere. The cells were transiently transfected with 800 ng of pcDNA 3.1 HO.1 or empty pcDNA 3.1 (Invitrogen) plasmids using Lipofectamine 2000 (Invitrogen). The levels of HO-1 expressed were measured by Western blotting 24 hours after transfections. Similar procedures were performed with 800 ng of pcDNA 3.1 NRF2 or empty pcDNA 3.1 (Invitrogen), ferritin pSG5 WT, and ferritin 222 pShuttleCMV (mutant ferritin) before differentiation with PMA.
Peritoneal cells from infected mice treated with vehicle, apocynin, CoPP, or SnPP were collected at 8 dpi and seeded on coverslips inside 24-well plates for 48 hours in complete RPMI. Coverslips were then processed as described for in vitro assays of parasite burden.
Trypomastigotes (5 × 105 to 5 × 106) were treated in 96-well plates (as described for in vitro macrophage studies), and motility was monitored as an indicator of viability after 24–48 hours.
Thioglycollate-elicited peritoneal macrophages were cultivated in 24-well plates (106/well), infected with trypomastigotes (3:1) for 12 hours, then washed and treated for 48 hours. Macrophages were then removed from plates by trypsinization and subjected to TUNEL staining (ApoTag Plus Peroxidase in Situ Apoptosis Kit, Millipore) according to the manufacturer's instructions.
Thioglycollate-elicited peritoneal macrophages were collected as described above, resuspended in RPMI, left to adhere in 48-well plates for 4 hours, and then washed and infected with 10:1 trypomastigotes for 1 hour in complete RPMI. Cells were again washed and reincubated with complete RPMI with the indicated drug for 6 hours, and then washed and removed from plates using a rubber policeman. Macrophages were incubated for 45 minutes with CM-H2DCFDA (Invitrogen) under 5% CO2 atmosphere; data were acquired with a flow cytometer (FACScan, BD). In calcein studies, thioglycollate-elicited peritoneal macrophages were left to adhere in 48-well plates for 24 hours and then washed and infected with 3:1 trypomastigotes for 12 hours in complete RPMI. Macrophages were then washed and loaded with the iron-sensitive fluorophore calcein-AM (Sigma-Aldrich) by preexposing the cells to 2 μM calcein-AM in iron-free PBS (Gibco, Invitrogen) for 15 minutes at 37°C under 5% CO2 atmosphere. Then, cells were scraped off the plate with a rubber policeman, and data were acquired with a flow cytometer. In lymphocyte apoptosis studies, spleen cell suspensions were prepared, and red blood cells were lysed with ACK buffer. Lymphocytes were then washed and plated in U-bottomed 96-well plates at a density of 106/100 μl, pelleted, and double-labeled with appropriate antibodies (CD4-PE; CD8-PE; CD69-FITC; all from BD Biosciences — Pharmingen) in labeling buffer (PBS with 5% FCS, 5% normal rabbit serum, 0.5% BSA, and 0.5% NaN3). For apoptosis studies, cells were then washed, resuspended in annexin V buffer (BD Biosciences — Pharmingen), and analyzed in a flow cytometer.
Spleen cell suspensions (106/100 μl) from uninfected, infected control, and infected CoPP/SnPP-treated mice were prepared at 9 dpi. Cells were seeded in 96-well U-bottomed plates and stimulated for 24 hours with ConA (Sigma-Aldrich, 5 μg/ml), plate-bound anti-CD3 (prepared by incubating 1 mg/ml 2C11 antibody from BD Biosciences — Pharmingen in PBS for 12 hours and then washing) or for 48 hours with T. cruzi antigen (prepared by 10 freeze-and-thaw cycles applied to 108 culture trypomastigote pellets; a 5 × 106 parasite equivalent was used per well). Supernatants were frozen until detection of IL-4, IL-17A, or IFN-γ by ELISA (PeproTech).
Peritoneal macrophages from infected mice or thioglycollate-elicited macrophages infected in vitro were used in these assays. Peritoneal washes from infected untreated or drug-treated mice were harvested at 8 dpi (Figure (Figure4A)4A) or 3 dpi (data not shown; similar results). Cells were left to adhere for 4 hours, then incubated with LPS (100 ng/ml LPS 011B4 from E. coli, InvivoGen) for 24 or 48 hours. Thioglycollate-elicited peritoneal macrophages (2 × 105) were diluted in RPMI, seeded in 96-well plates, and left to adhere for 2–4 hours. Non-adherent cells were washed away, and cells were allowed to rest in complete RPMI for 24 hours and then infected with trypomastigotes (3:1) for an additional 12 hours. Cells were washed and stimulated with LPS for 24 or 48 hours. Supernatants were assayed for NO (48 hours, Griess) or TNF (24 hours, ELISA, PeproTech).
Protein lysates from cultured cells were obtained with lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, sodium deoxycholate 0.25%, 1 mM EDTA, 1 mM PMSF, 1 mM NaF) on ice for 10 minutes. Four-fold-concentrated SDS sample buffer was added to cell lysates (50 μg), which were boiled for 5 minutes, subjected to electrophoresis on a 8% SDS-polyacrylamide gel, and then transferred onto nitrocellulose. Protein molecular weight standards (Bio-Rad) were run concurrently. The nitrocellulose was blocked in TBS with Tween 20 (0.05%) containing 5% nonfat dry milk for 1 hour at room temperature. Blots were then probed overnight at 4°C with 1:2,000 monoclonal rabbit anti–HO-1, monoclonal goat anti–H-ferritin, monoclonal goat anti-FPN1 (Santa Cruz Biotechnology Inc.), or monoclonal mouse anti–p-IRF3 (Cell Signaling Technology). Bound primary antibody was detected using donkey anti-goat, goat anti-rabbit, or anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology Inc.) diluted 1:1,0000, followed by incubation with the chemiluminescent substrate Luminol (Santa Cruz Biotechnology Inc.). The membrane was then exposed to X-ray film (Kodak). The same membrane was probed to detect the loading control protein ERK2 with mouse anti-ERK2 antibody (1:2,000, Santa Cruz Biotechnology Inc.).
Total RNA was extracted using TRIzol (Invitrogen), and 1 μg was reverse transcribed using M-MLV reverse transcriptase and random hexamer primers (Invitrogen). Subsequent real-time qRT-PCR analysis was performed on an ABI 7500 (Applied Biosystems) using SYBR green master mix (Applied Biosystems). Amplification conditions were as follows: 95°C (10 minutes), and 40 cycles of 95°C (15 seconds) and 60°C (60 seconds). All data were normalized to the corresponding HPRT expression, and the fold difference relative to the control level is shown. The analyses of relative gene expression data were performed with the 2–ΔΔCT method (70).
Hearts, quadriceps, and livers from infected mice were collected and preserved in formalin. Tissues were embedded in paraffin, cut in 5-μm sections, mounted on slides, and stained by a standard H&E technique. Sections were examined for parasite nests and photographed under an Olympus C21 microscope. Parasitism was expressed as either nests per 100 mm2 (at 8 dpi, when parasites were confined to small nests) or number of parasites per field (at 15 dpi, ×400 magnification, parasites were counted on printed pictures, at least 20 per individual mouse slide).
Statistical analysis was performed by Student's t test. The log-rank test was used to determine differences at the end point of the survival curves. Differences with a P value less than 0.05 were considered significant.
The experiments were approved by the UFRJ Institutional Animal Welfare Committee (CEUA-CCS, protocol IMPPG-011).
This work was supported by CNPq, FAPERJ, Pronex, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). C.N. Paiva, R.T. Figueiredo, J. Lannes-Vieira, M.R. Fantappié, and M.T. Bozza received CNPq grants. We are indebted to Miguel Soares, Leo Otterbein, and Pedro Oliveira, who helped us with suggestions and reagents.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2012;122(7):2531–2542. doi:10.1172/JCI58525.
Daniel F. Feijó's current address is: Departamento de Microbiologia Geral, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
See the related Commentary beginning on page 2352.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text: To get from Sacile to Alcamo by train you can catch one of the 4 trains that everyday travel between these two cities. The first train to Alcamo leaves from Sacile at 15:24 and the last train departs at 22:43. If you choose a local train the trip lasts on average 20:60 hours. For your trip from Sacile to Alcamo by train you can choose to leave from one of Sacile main stations: Sacile On Wanderio you can compare all schedules, fares and railway companies to Alcamo and book the best itinerary starting from Sacile.


text:
Jason Derulo is back on the scene almost a year and a half since the Miramar native first broke out with his hit song "Whatcha Say". This new one is called "Don't Wanna Go Home".
And upon hearing it, you can't help but hear like 2 or 3 songs mixed to create the basis of this one. Which is a bit of a drawback. We have a lil' Lil Jon & The Eastside boys mixed with a lil' bit of the Robin S house classic 'Show Me Love.' And then to tie it all together you have Harry Belafonte "Banana Boat Song" (first Weezy used it and now Jason) anchored on the chorus as well.
Other than what it samples, the song is very pop-centric and is off his upcoming summer album Future History. Listen below…
Jason Derulo – Don't Wanna Go Home
Download: Coming soon to iTunes
© 2011 Beluga Heights / Warner Bros.


text:
Next →
← Previous
Perfectly fits any requirements for lady chic to a t.
Sooo true. I do the same! They design it that way. And those big, fat, thick, September issues don't help any either! I suddenly find myself NEEDING a tweed coat when really, there would be no chance for me to wear it. Ah, Fall. Perfect title for this illustration by the way! Beautiful!
I love her Hair waves, she looks so classy and decent. Salutes Inslee
Beautiful Kate Spades, and a beautiful illustration. It's true. All the ladies in the our company strut in each morning looking more chic as autumn approaches! I suppose to compensate for the freezing cold! Love this illustration Inslee. Tres bien!
this is wonderful inslee! impressive and inspiring as always :)


text:
Maybe because it's summer, sunny and hot and it's Christmas and it's supposed to be cold and snowy.
Certainly because Walker's shortbreads are sold everywhere but I know nothing compares to granny mck's homemade ones: straight from the oven full of real butter, and made by love. Or maybe I am just still in culture shock phase. But I am terribly missing Scotland. I remember wearing spiky-heels boots and the road was so icy and I slipped and danced to balance my walk on every step. I remember feeling so cold my toes were literally frozen and I couldn't move them. I remember insisting wearing open-toes jeweled sandals for a party and stepping out of the car into the snow and couldn't care less because it's a ceidlih and I wanted to look phenomenal, only to find out others wore Ugg boots and changed to party shoes at the concierge. I remember waking up one morning and find everywhere was white and the car was somewhere underneath the pile of snow. And having breakfast in a hotel looking out the golf course and the snow started falling, covered the whole ground with white, and people still insisted on dragging their golf clubs and kept on playing. And most of all, I remember I bitched about the weather a lot. Which now I miss the most, among other things I really didn't care about when I was still there. Strange, eh?
Anyway, here are the things I miss about Scotland – not in particular order.


text:
Christmas in July has Started! Up to $75 Off + Free Shipping* LEARN MORE
*Some items excluded. Can be combined with free ground shipping on orders $150+ to contiguous US. Expires 8/1/17 at 3:00 am EST
|Sort by:
|Most Popular
This target game board features the same scoring as the dart board. The target is designed...
Only 1 left in stock
Large Mechanical Auto-Reset Target with Premium Box Packaging Three differently sized bullseye...


text:
Déjà vu all over again for the Capitals against Calgary, but this time the mistakes didn't come with a happy ending. Washington fell, 5-2, to the Flames, snapped its winning streak and gets to take stock of some familiar problems.
Five thoughts on the loss in Calgary:
1. Those slow starts. Getting off to a strong start in the first period has been a consistent issue for the Capitals through the first 11 games. Saturday night there was a lack of execution in which pretty much every player was concerned in the first 20 minutes with turnovers, missed assignments and general sloppiness. Even in games the Capitals have won this season – like in Edmonton and Winnipeg earlier this week – they were uneven in the opening frame.
It's a risky habit and one that bit Washington in the form of three Flames goals Saturday night.
"We've got to figure that out, especially on the road, especially in these buildings teams are tough to play against," Aaron Volpatti said. "They're fast. There's no excuse we've got to come out better."
Establishing a better start isn't all about offensive production and in the Capitals' case, the opposite cause is more critical. Washington, which has been outscored 12-6 in the first period this season, needs to home in on the details of its defensive and transition game from the opening faceoff.
More to come on the slow starts later in the day Sunday, a day off for the Capitals.
2. Second line. I asked Coach Adam Oates about the group of Martin Erat, Brooks Laich and Troy Brouwer, which was heading into its fourth game together. Here's what he had to say: "On a night-to-night basis, on a consistent basis, they all skate good, they're all top-six forwards. They can handle any defense pairing that's put against them, any line. I count on them for a lot of minutes. They all kill penalties, they're on the power play. Not everybody's perfect every night but they play 20 minutes for us every night."
His point is well taken in that regardless of how much offense the second line is or isn't producing, those players will be counted on to handle heavy workloads. In Calgary, Erat and Laich were on the ice for four goals against while Brouwer was on for two. On the play leading to the first goal, Erat lost a battle against two Flames on the boards but none of his teammates helped out either. On the second, Erat got caught between Sven Baertschi and Sean Monahan in the slot. A lost faceoff by Laich, who went 5 for 16 in the circle against the Flames, led to Mike Cammalleri's second goal of the night.
When that group is having as much trouble in its own end as it did in Calgary, it's likely the rest of the team isn't faring well either.
3. Where's the D? Overall the Capitals' play in their own end Saturday night left plenty to be desired, from the forwards getting back to provide proper support to the defensemen making the correct decision.
No one could escape criticism in this one and it seemed like each defensive pairing had its own adventures in the early going: getting split by a speedy Calgary forward like John Erskine and Steve Oleksy; getting caught flat-footed like Mike Green and Nate Schmidt; or giving up uncharacteristic time and space like Karl Alzner and John Carlson.
"There was a couple mistakes by everybody," Oates said. "They got two goals off of bad tracking and then we get worn down in our own end and out of position. Little mistakes by everybody."
4. Volpatti's goal. The gritty winger recorded his first goal as a Capital and first since Jan. 25 with a nice putback on a rebound that popped into the slot. Re-acquired on Feb. 28 off waivers from Vancouver, Volpatti played only 4:58 against Calgary. But perhaps there's something to be said for being willing to make a hit and then going to a high-traffic area on the ice.
"It went up to the point and I tried to get in that soft spot," Volpatti said. "Sometimes I find myself a little bit almost too close to the net and that time it bounced right out to me. Luckily I got enough on it, I kind of whiffed it."
Rookie defenseman Nate Schmidt also recorded his first NHL point on the play, with the primary assist, after firing the shot that rebounded to Volpatti.
5. Chimera, goal scorer. Jason Chimera recorded his second goal in as many games and third of the season with a pretty backhander after a quick feed from Mikhail Grabovski. With his third tally, Chimera matched his regular season output from last year.
He helped set up the play creating a rush into the offensive zone with Eric Fehr, but when Derek Smith laid down to block his shot Chimera recovered the puck and fed it down low to create a cycle. Some work by Fehr and Grabovski to create space down by the goal line and suddenly the veteran winger was all alone with the puck and able to skate right through the crease for the shot.


text:
Toggle Navigation
I'm an employer
Find a Job
Explore Companies
Career Center
About Us
Log In
Sign up
Systems Administrator
Cincinnati , Ohio
November 24, 2016
Robert Half Technology has partnered with a local client in search of a Systems Administrator. Interested candidates should contact Chet Wehrman at 513.621.6890 or firstname.lastname@example.com The Systems Administrator's role is to manage and tune in-house computer software systems and hardware to ensure high levels of availability and security of the supported business applications. This individual is also responsible for ensuring proper computer operation so that end users can accomplish business tasks. This includes receiving, prioritizing, documenting, and actively resolving end user help requests. Problem resolution may involve the use of diagnostic and tracking tools. Acquisition & Deployment • Coordinate with network engineering, business application, and database administration functions to implement servers that utilize industry best practices to meet corporate objectives •Provision & deploy servers, & multi-function printers Operational Management • Manage server OS platform, Windows 2008/2008 R2/2012/2012 R2 • Manage input/output fleet, including printers and copiers •Assist Help Desk with onsite analysis, diagnosis, and resolution of complex desktop problems for end users, and recommend and implement corrective solutions, including offsite repair for remote users as needed. • Apply diagnostic utilities to aid in troubleshooting. • If necessary, liaise with third-party support and equipment vendors. •Document all pertinent end user identification information, including name, department, contact information, and nature of problem or issue. • Recommend, schedule, and perform software and hardware improvements, upgrades, patches, reconfigurations, and/or purchases. • Collaborate with network administrators to ensure efficient operation of the company's computing environment. • Record, track, and document the network and infrastructure request problem-solving process, including all successful and unsuccessful decisions made, and actions taken, through to final resolution. • Access software updates, drivers, knowledge bases, and frequently asked questions resources on the Internet to aid in problem resolution. • Identify and learn appropriate software and hardware used and supported by the organization. •Assist helpdesk with hands-on fixes at the desktop level as needed, including installing and upgrading software, installing hardware, and configuring systems and applications. •Install anti-virus & security software. •Answer to and perform moves, adds, and changes (MAC) requests as they are submitted by line managers. •Test fixes to ensure problem has been adequately resolved. •Perform post-resolution follow-ups to help requests. •Flexibility to provide off hours and weekend support as necessary. Formal Education & Certification •College diploma or university degree in the field of computer science and/or 5 years equivalent work experience. •Certifications in Microsoft, CompTIA A+ or Network+ are preferred, but not required. Knowledge & Experience •Knowledge of server hardware, including Dell PowerEdge, IBM server X-Series servers, and HP Proliant. •Knowledge of Microsoft networking components, such as Active Directory, DNS, DHCP, DFS, Group Policies, Roaming Profiles, NTFS Permissions, & Login Scripts •Experience with VMware ESXi V-Sphere 4.1/ 5.5/6.1 • Experience with SAN administration, NetApp/EqualLogic environment •Experience with desktop and server operating systems, including Microsoft Windows 7, Microsoft Windows 10, Server 2008, and Server 2012.
A little about us:
Founded in 1948, Robert Half (NYSE: RHI) is the world's first and largest specialized staffing services firm.
Share this job
Know someone who would be interested in this job? Share it with your network.


text:
Movie Review :
Saving Private Ryan 1998 Watch Online Free, Saving Private Ryan Movie Watch Online, Saving Private Ryan Watch Online, Watch Saving Private Ryan Online, Saving Private Ryan Full Movie Online Watch Free, Saving Private Ryan Download Free
Saving Private Ryan (1998) Movie
Director: Steven Spielberg
Writer: Robert Rodat
Stars: Tom Hanks, Matt Damon, Tom Sizemore
Action, Drama, War | 24 July 1998 (USA)
Saving Private Ryan Full Movie Watch Online Free
Server 1
Server 2
Server 3
Saving Private Ryan Full Movie Online Free 1998
Saving Private Ryan (1998) Movie Review :
In Hollywood mythology, extraordinary fights haggle on the activities of individual legends. In Steven Spielberg's "Sparing Private Ryan," a huge number of scared and nauseous men, a large portion of them new to battle, are tossed into the substance of shriveling German flame. The arrival on Omaha Beach was not about sparing Pvt. Ryan. It was about sparing your skin.
The motion picture's opening grouping is as realistic as any war footage I've ever seen. In savage fear and vitality it's on a standard with Oliver Stone's "Unit," and in extension surpasses it- – in light of the fact that in the bleeding early stages the arrival powers and the adversary never meet eye to eye, however are just faceless masses of men who have been requested to shoot at each other until one side is obliterated.
Spielberg's camera fails to understand the activity. That is the reason for his style. For the individual officer on the shoreline, the arrival was a mayhem of commotion, mud, blood, regurgitation and passing. The scene is loaded with incalculable inconsequential bits of time, as when a warrior has his arm passed over. He stuns, confounded, standing presented to further fire, not certain what to do next, and after that he twists around and grabs his arm, as though he will require it later.
This arrival succession is important to set up the separation between the individuals who give the request that Pvt. Ryan be spared, and the individuals who are requested to do the sparing. For Capt. Mill operator (Tom Hanks) and his men, the arrival at Omaha has been a cauldron of flame. For Army Chief George C. Marshall (Harve Presnell) in his Washington office, war appears to be more remote and statesmanlike; he cherishes a letter Abraham Lincoln composed reassuring Mrs. Bixby of Boston, about her children who passed on in the Civil War. His guides address the shrewdness and without a doubt the likelihood of a mission to spare Ryan, however he barks, "If the kid's alive we are going to send someone to discover him- – and we are going to get him the damnation out of there." That sets up the second demonstration of the film, in which Miller and his men enter into French landscape still effectively debated by the Germans, while harboring mutinous contemplations about the insight of the mission. The majority of Miller's men have presented with him before- – aside from Cpl. Upham (Jeremy Davies), the interpreter, who talks amazing German and French however has never discharged a rifle in resentment and is scared nearly to the point of incontinence. I related to Upham, and I think numerous legitimate viewers will concur with me: The war was battled by regular citizens simply like him, whose lives had not set them up for the truth of fight.