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Discover spirulina – an algae that's been consumed since ancient times and which now enjoys star status among nutritionists worldwide.
Studies suggest that spirulina could indeed be a "superfood", but research is ongoing on the benefits of these blue-green microscopic algae, which grow in naturally alkaline lakes. Not only does spirulina provide protein, it's also an excellent source of B-vitamins and iron.
The spirulina sold in health shops nowadays comes mostly from commercial spirulina farms.
Spirulina shouldn't be taken without first consulting your doctor, especially in the case of children and expectant mothers. If you're allergic to seafood, steer clear of spirulina.
History and discovery
Spirulina grows in both fresh and salt water and has been dried and eaten since ancient times in countries as diverse as Chad and Mexico. It's said that Hernando Cortez saw in 1519 that the Aztecs were eating these algae. It's thought that spirulina dried into cakes was the primary source of protein for the Aztecs for hundreds of years.
Explorer Pierre Dangeard published a report in a journal in 1940 on this seemingly nutritious algae eaten by people living around Lake Chad.
Twenty years later, botanist Jean Leonard made the connection between the algae and the green cakes sold in the markets in this area. He remarked that 70% of the food people ate in this area was accompanied by a sauce made from these cakes.
The first spirulina commercial production plant was set up by the French in 1969 close to Lake Texcoco in Mexico. It isn't easy to grow as the ecology of the pond/lake in which it is growing has to remain stable and uncontaminated by things such as chemical fertilisers.
It grows in warm climates and needs bright sunshine, a pure water source and a pollution-free environment. It yields over 20 times more protein than soybeans, utilising the same space.
Spirulina easily absorbs the nutrients from the water in which it grows, so it should never be harvested from water that is polluted, or contains heavy metals.
There are many different spirulina species, but the two used most frequently used in commercial products are Spirulina maxima and Spirulina Platensis.
Despite many claims that spirulina is an excellent source of protein, studies have found that it's no better than meat or milk, but its production costs are about 30 times higher.
Its weight-loss properties remain unproven, but it may be effective for the treatment of pre-cancerous mouth lesions.
Despite a range of claims on the health benefits of spirulina, researchers are yet to prove the following benefits conclusively:
• It reduces arsenic levels in the body.
• It can relieve allergy symptoms in adults.
• It may reduce blood sugar levels in diabetics.
• It may lower cholesterol levels.
• It lowers anxiety and depression in menopausal women.
How to include it in your diet
Spirulina is available in either tablets or powder. If taken as a powder, it's recommended that it be taken with water and not milk or juice or other beverages.
Powdered spirulina is usually less expensive than spirulina in tablet form, as the production process is simpler. Powder is easier to digest than tablets.
If you're taking spirulina tablets, make sure that you follow the instructions on the packaging carefully.
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The comedian sticks as religiously to her theme as a dancer sticks to a diet.
Sticking with a marriage. That's true grit, man.
I like suits that are fitted. And I usually stick to grays, whites, and blacks.
I think the great sketch shows, like 'Python' and 'Mr. Show,' they didn't stick around for very long. There's something kind of cool about that.
My preference is that, that day when someone sticks a tripod in front of you with a camera on the top, it is not day one.
The trick is after a workout you're supposed to have gummy bears or some candy to get your veins to stick out. Of course, it's all about protein, too, but right before you're filming a shirtless scene, you have a little bit of sugar to pop the veins.
Damn your principles! Stick to your party.
My eyes are too big, my nose is too flat, my ears stick out, my mouth is too big and my face is too small... my body is thin as a clarinet and my ankles are so skinny that I wear two pairs of bobby socks because I don't want people to see how thin they are.
When you think about such fine actors as Maggie Smith or Michael Gambon, they do all mediums. I think it would be quite sad and a bit dull just to have to stick to one. I like all of them.
If we just stick to one kind of music, our creativity is limited. We wanted to extend the audience for the cello, especially the younger people, and to show them how cool and how powerful and how diverse the cello can be.
Why the fairy tale of Willie Mays making a brilliant World Series catch, and then dashing off to play stickball in the street with his teenage pals. That's baseball. So is the husky voice of a doomed Lou Gehrig saying, 'I consider myself the luckiest man on the face of this earth.'
A lot of people don't like bumper stickers. I don't mind bumper stickers. To me a bumper sticker is a shortcut. It's like a little sign that says 'Hey, let's never hang out.'
I got the stern end of the stick. It was kind of that iron fist. I am who I am today because of my mother.
Writers of fiction should stick to writing, not pop up on panel shows or as a talking head.
It would be ugly to watch people poking sticks at a caged rat. It is uglier still to watch rats poking sticks at a caged person.
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10 Little-Known Uses for CBD - Pt. 2w/
Today, we will continue our discussion of the lesser-known uses of CBD.
Evidence is continuing to mount about the medicinal benefits of CBD for Post-traumatic Stress Disorder (PTSD). CBD's anti-anxiety, antipsychotic, and anti-inflammatory effects can produce a calming effect, which helps provide a stable mental environment for those who suffer from PTSD and allows them to overcome anxiety and stress. A German study from 2012 and published in the journal Translational Psychiatry compared the antipsychotic Amisulpride and CBD in 45 patients who suffered from schizophrenia, and found that while both treatments were effective, the fewer side effects of CBD made it preferable to the pharmaceutical medication.
CBD could also benefit bowel diseases, and research shows cannabis' anti-inflammatory properties, along with CBD and THC's ability to control gut function in the body, offers relief for Crohn's disease patients. Using animal models and cell cultures, scientists from the Cajal Institute found CBD reversed inflammatory responses and provided protection from multiple sclerosis (MS). Within 10 days of beginning CBD treatment, mice had superior motor skills and showed improvements in their condition. Lastly, CBD provides a non-habit forming sleep aide that can help those who suffer from insomnia.
This concludes our post about CBD's little-known uses. This information has been provided by High Times Magazine and approved by our Chief medical officer.


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Not just any old limousine, we have one very unique Audi Q7 S line Stretched Limousine with only a couple confirmed in the UK this is one of the best looking Limousines around.
Now available to arrive in style for your Birthday, Prom, Stag, Hen, Wedding or any special occasion in Newcastle and the surrounding areas in the north east. As there is only 1 audi q7 limo please book early to avoid disappointment. Very spacious luxury leather seating complete with 2 mini bars, dvd screens, ambient and disco lighting to go with any mood, along with a Pioneer sound system you can also connect your own device to enjoy on the journey.
Wedding Cars
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You may not have seen the badge on your wedding car or even had any idea of the manufacturer; however, as soon as you set eyes on our luxury fleet, you will know which vehicle will suit you on your wedding day.
We focus on providing a bespoke chauffeuring service which are represented not only in our fleet of vehicles, but is also reflected in our levels of service and attention to detail on your service requirements.
We have the widest selection of prestige vehicles in the North East such as Bentley, Chrysler 300c Mercedes & Rolls Royce to ensure you have the right car, whatever the occasion.
Prom Cars
Do you really want to be dropped off by Dad on your Prom night? We think not… the only way is Autograph!
We have Luxury Bentley, Chrysler 300C or Mercedes if there are up to 4 passengers or for something different why not book one of our Supercars, imagine turning up at your prom in our Lamborghini or Aston with the Stig from Top Gear!
We have a large selection of prestigious cars which include the amazing Rolls Royce Phantom, Rolls Royce Ghost, Bentley, Mercedes S Class, CLA, Chrysler 300C, Range Rover, and of course our Supercar selection, Ferrari, Lamborghini, Aston Martin Audi R8 and Porsche. Call us today to reserve your prom car.
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The unmistakeable presence of an Aston Martin, Audi R8, Bentley, Ferrari, Range Rover or Lamborghini makes special days even more special.
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Click Through our Supercar Section to view the full selection
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Autograph Chauffeurs Newcastle provide an extremely professional service to the VIPs of your company when you need to make more of an impression than an ordinary taxi or hire car.
Your business will benefit from professional and prompt service as our experienced chauffeurs deliver a service which is second to none.
Once a booking is made, we ensure we are fully prepared for the journey with the route planned ahead and being up to date with latest roadwork's and traffic congestion spots.


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The fourth graders at Pinehurst Elementary School gathered in the auditorium for their celebratory Assembly at the end of the Pet Responsibility Program. Pet Responsibility Committee (PRC) volunteer Barb Ross hosted the event. Although the classes were held at the very end of the school year, the students concentrated and were very good listeners, excellent learners and exceptionally polite; they were attentive and serious about their role as animal advocates. During Lesson 5, all the fourth graders received "You Are Pawsome" wrist bands to remind them that they are advocates for animals. At the Assembly, students were joined by four very special guests – the PRC mascot, Speuter, Lieutenant Robert Williams of the Southern Pines K9 Police and JAS Dog Training, Leigh Morgan and Jake, a service dog trained by Lt. Williams. Lt. Williams talked to the students, teachers and parents about the work of the Southern Pines Police Department K9 Unit. He also talked about his private dog training work. He trains dogs for people who have challenges. Leigh and Jake demonstrated what a well-trained dog can do. The partnership between a person and a dog helps the person live a fuller, happier life. Service dogs (and therapy dogs) are advocates for people! Lt. Williams talked about training dogs with positive reinforcement. Everyone appreciated the work Lt. Williams does in the SPPD and how his special dog training helps many people.
The fourth graders who wrote entries for the Speuter Story Contest proved their love for pets by speaking up for those who have no voice. Rowan, Wyatt, Kayla, Dahlia and Addison all wrote excellent stories and were recognized with Good Work! certificates of appreciation. Layla, Addy, Cina, Emma Rose, Taylor and Peyton were all announced as the Speuter winners for 2018 and were awarded framed certificates by the PRC Mascot, Speuter. They also received colorful paw print "dog" tags. Because of their efforts, two dogs, Beau Blue Bayou and Scarlet, and one cat, Kit-Kat, and three other pets will be spayed or neutered to help prevent pet over-population. Winners' families attended the Assembly and were very impressed by how much the students knew about being responsible pet owners. Congratulations to all the Pinehurst fourth graders for being part of the team that makes life better for all pets!


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ADM Live Garage
Live House
- Website:
- http://www.adm-rock.com
- Address:
- GS Haimu B1F, 1-22-1 Higashi-Ikebukuro
- Area:
- Ikebukuro
- 住所 (日本語):
- Map link:
- http://www.adm-rock.com/access.html
- Closest stations:
- Ikebukuro, Higashi-Ikebukuro
GS Haimu B1F, 1-22-1 Higashi-Ikebukuro From the east exit of JR Ikebukuro station, find your way to Sunshine dori and head towards Tokyu Hands. At the intersection with Tokyu Hands and SEGA, turn right. About 30m ahead, you'll find ADM in the basement on the right.
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Published: 18Dec2015
Here is my 20th annual column of the past year's heroes and zeros.
Hero Legendary Dykes to Watch Out For cartoonist Alison Bechdel's autobiographical graphic novel Fun Home won five Tony Awards, including Best Musical.
... Hero Out TD Asset Management CEO Timothy Thompson, who for five years will donate an annual scholarship of $20,000 to a student at McGill University's Desautels Faculty of Management who demonstrates leadership within the LGBT community. The first recipient will be named in September 2016.
Read full article: Fugues, December 16, 2015


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these things are silly
communication can be so misconstrued
we say what we don't mean
and we lead on to other conclusions
or perhaps we say what we really feel and our minds want to take it back
i can't decide which is true (or more true)
was there an expression of regret?
or rather a way of smoothing things over
was there a genuine want of something else?
or friendship only without the underlying tension
i mean, i think you're cute
do you feel the same?
regardless, what of this other?
is that really what should be?
regardless again, i am still in the same boat
but the weather is nice
the seas are calm
and i am confortable where i am (as i should be)
if you would like to stear close, my course is set
set yours accordingly
if you want to board, ill oblige
you came by after i passed through the storm otherwise this would habe appeared to be another example of the futility of male-female relationships
but because of the timing it is an encouragement, and maybe there actually is something there
if not, it wasn't at the beginning of the week so i am no worse off
it still made me smile
both then and now
so don't feel hated, you're not
don't feel that you're a "sucky person," you're not
and don't feel like you can't get to know me still, because i want you to
Hopefully A Freudian Slip
these things are silly


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New to Rise Chapel?
Come out and enjoy some great coffee, dessert, and fellowship with Pastor Joe and his wife.
Dessert w/ the Pastor is a chance to see where God has brought Rise from and where He is going. This is the time to bring all of your questions about the who, what, where, when, why & how of Rise Chapel.
Sign-ups are available in the Church Lobby.
** Space is limited as these only happen every other month.


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Gov. Abiola Ajimobi of Oyo State has stated that no other president in the country has managed the nation's economy better than President Muhammadu Buhari.
The governor spoke in Ibadan on Friday evening while receiving members of the National Congress Committee of APC at the Government House.
The News Agency of Nigeria(NAN) reports that the committee led by Alhaji Musa Aliyu were in the state for the ward congress of the APC scheduled to hold on Saturday.
"I read Economics and Finance, and there is no economic theory that we have not read. The most important part of all the economic theory is leadership. Read more


text: Please send Norman a message and he will personally respond! You may also submit an order for personally autographed CD versions of any of Norman's albums here. Peace and Disco Beats. Name: Email: Comment: Which of Norman's recordings do you own?: ON THE REBOUND - Norman Kelsey A TALENT FOR LOVING - Norman Kelsey THE DANCE DIVINE - Pacific Soul Ltd. RECOGNIZE - Bat Country ENTERTAINMENT NOW - Bat Country DECLARING THE TRUTHS - Idle Kings Have you seen Norman in Concert?: Yes No Anxiously awaiting the next show


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Diabetes is hard to manage. You can get a comprehensive understanding of the disease here and get the information about the latest treatments and get professional advices. read more
Micro-Chinese Medicine Osmotherapy is a brand-new therapy based on traditional Chinese herbal medicines dating back thousands of years ago. It combines the essences of herbal medicines read more
Hormonal therapy is commonly used to treat Kidney Disease in clinic. It refers to use one or more than one hormone medicines to suppress the over-reactive immune reaction read more
Patients are suggested to treat kidney disease first before they get pregnant read more
If you are diagnosed with Kidney Disease, you should talk to your doctor about starting an exercise read more
Foods and Drinks play an important role in Kidney Disease patients read more
Kidney Disease News and Events read more
In every week, a famous experienced renal medicine specialist will be invited to help diagnose and evaluate inpatient's disease condition, offering detailed therapeutic schedule. read more
Provided by Expert Zhang Youkang on 2013-03-13 14:17
Creatinine is the byproduct of muscle and is filtered out of body by kidneys normally.So creatinine level keeps at constant level in right condition.Creatinine level is an indicator of how your kidneys are functioning. The following article will introduce some herbal medicines for high creatinine.
Chinese rhubarb
Chinese rhubarb is a traditional Chinese herb and has been used to treat a variety of conditions.This herb can help clear blood stasis, promote blood circulation and increase the excretion of toxins from body. By increasing blood flow to kidneys, it can help relieve renal ischemia and anoxia.
Salvia is one of common herbal medicines for high creatinine. Some researches show that it can increase the rate f which creatinine is filtered and evacuated from the body. It also can enhance the circulation to the kidneys as well.
Cinnamon is commonly used to treat kidney problem, especially in diabetics with kidney disease. It can help enhance the filtration process and improve renal function, thus increasing the excretion of wastes from body.
Cordyceps sinensis
Cordyceps sinensis can regulate immune system and control inflammatory response in kidneys. So it can delay kidney disease progression, improve renal function and reduce the damage caused by toxins in body to kidneys. It is one of commonly used herbal medicines for high creatinine.
Chamomile is one of herbal medicines for high creatinine. A study found that decreased levels of creatinine appeared in those drinking chamomile tea. It is due to high levels of antioxidants present in chamomile.
The above are common herbal medicines for high creatinine. However, if they are not used rationally, they may be toxic to kidneys. So you should never use medicines without consulting a herbal medicine practitioner.
If you send us email, please leave your phone number or WhatsApp/Viber (with country code) in email so that our renal expert can communicate with you timely and directly. We will do utmost to help you solve the problems.
What else you want to know:
Make An Appointment Beijing Tongshantang Hospital of Traditional Chinese Medicine.
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8pm PREP
Read Isaiah 58:1-14
So why are we doing this? For many of you, this will be your first fast. As we prepare for our fall kick-off, we prayerfully seek God's guidance for our God Talks series beginning on September 3. We pray that people who are far from God, will be moved with a new desire to be in relationship with him. We pray that people who have drifted from God, will come back to him. We pray for our children's relationship with him. We pray for the release of sin that so easily traps us.
- Moses fasted for 40 days at least two times. Jesus fasted just before his earthly ministry began.
- Fasting will help restore the loss of your "first love." (Rev. 2:4)
- It's a biblical way to humble yourself before God. (Psalm 35:13, Ezra 8:21)
- It enables the Holy Spirit to reveal your true condition, resulting in repentance and transformation.
- It can transform your prayer life, worship, and strengthen your relationship with God.
It begins! The writer of Hebrews said in Hebrews 12:1-2 "…let us run with endurance the race that is set before us," (NKJV).
Let's begin our fast with an inward focus. What is going on in your life that you need supernatural strength to beat? Is it an addiction? Is it anger? Bitterness? Maybe you're just struggling to get through. As children of God, we're not called to survive, we're called to thrive! But you have to get sick and tired of being sick and tired! That means with God's help, you can run this race with endurance.
When I ran my first half marathon, I made the rookie mistake of buying new running shoes, and taking them on a half marathon for their inaugural run! Even my running coach said not to. "Nothing new on race day!" She said. But I'm the type of person, if I'm going to do something new, I probably need new shoes! By mile 5, I realized this was a mistake! I started developing a blister. Now I'm 5 miles from the beginning, and 8 miles from the end! So what did I do? I persevered. I finished the race, with all the Texas hills, complete with blisters, and limped through the finish line.
Winston Churchill said, "A pessimist sees the difficulty in every opportunity; an optimist sees the opportunity in every difficulty."
Now is the time to shed the sin that so easily traps you. If you're truly going to live for God, confess your sins, and lean on his supernatural strength to get you through. Just like that half marathon, blisters and all, one foot in front of the other! You can do this!
If my people who are called by my name humble themselves, and pray and seek my face and turn from their wicked ways, then I will hear from heaven and will forgive their sin and heal their land. (II Chronicles 7:14)
When Jesus had been fasting for 40 days, just before his earthly ministry began, he was led into the wilderness by the Spirit to be tempted by the Devil. Did you ever wonder why the Spirit would lead him to be tempted by the Devil? The writer of Hebrews tells us why. "For we do not have a high priest who is unable to sympathize with our weaknesses, but one who in every respect has been tempted as we are, yet without sin." (Hebrews 4:15)
We had an amazing church service on August 14! It was a time where the Spirit of God was moving long before the first song. And at the end of the service, three people made decisions for Christ!
Isn't this why we have church? To introduce people to Jesus, and help them follow him?
Etta James made famous a song called A Sunday Kind of Love. She sings of a love that lasts past Saturday night. Many of us treat Christ as a Sunday kind of love. But we don't need Christ only on Sunday! We need him on the 35 freeway on Monday. We need him in our office meetings on Tuesday. We need him in our kid's soccer match on Saturday.
Then the devil left him, and behold, angels came and were ministering to him. (Matthew 4:11 ESV)
At Grace Place, our vision is simple. Love God, love people. How are we going to do that? Our mission is to Gather to worship Jesus. Grow to know Jesus. Go to preach Jesus.
"On your tombstone will be two numbers. When it all began, and when it all ended. My question for you is, 'what are you going to do with the dash?" –Dave Ramsey
If 2016 were to be the last year of your life, what would you want it to be known for? We were made for a purpose! There may be accidental parents but there aren't accidental kids. God formed you on purpose, for a purpose.
Our purpose is to live a life of such significance, that people want to know what makes us tick. Hint… It should be Jesus!
So then the Lord Jesus, after he had spoken to them, was taken up into heaven and sat down at the right hand of God. 20 And they went out and preached everywhere, while the Lord worked with them and confirmed the message by accompanying signs. (Mark 16:19-20)


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Incentive Pay Program Drives Staff Performance & Productivity
Executives at Notre Dame FCU (NDFCU) are very satisfied with results from FMSI's Teller Management SystemTM (TMS), seeing a front-line productivity increase of 17%. While the CU's tellers are proud of that improvement as well, they are particularly pleased about what has helped them reach that figure: the FMSI incentive pay program.
Along with the TMS workforce optimization application, the $440 million credit union has incorporated FMSI's default incentive pay program that rewards tellers based on the number of transactions they process.
"The incentive program complements TMS," said Tim Martin, VP Branch Administration. "I see the results in the numbers, and I see the results in the faces of our tellers. It motivates them to work harder and more efficiently, driving them to reach their numbers. We are asking our staff to do more, and they are always willing. But making the extra effort is much more palatable when you get something extra for it."
Implemented six months after the workforce optimization software was deployed, the incentive program produced quick, noticeable results, helping lift transactions per hour from 16 — where the CU stood before TMS — to 18.7 today. "We saw further improvement immediately when we introduced the incentive program," said Tim, adding the incentives have also made front-line staff — all skill levels — much more willing to work Saturdays. "They know those extra hours give them the opportunity to earn more money."
No More Spreadsheets and Guessing
The incentive program combined with the workforce optimization tool has reduced labor costs per transaction by 11%, and excess labor costs are down 25%, for a monthly savings of $6,496. The increased productivity and savings have added up, allowing the South Bend, Indiana-based credit union to lower its overall operating expense ratio. Tim stated that the improvements are a direct result of being able to "dig into the data" of front-line activity to understand staffing needs and correctly deploy tellers to meet member demand. "We had to get away from the spreadsheets that really did not tell us what we needed. Prior to TMS, we would look at manually assembled data from a spreadsheet — basically transaction reports — guess, and go from there."
Solving an Understaffed Branch Issue
The TMS workforce optimization application has streamlined the scheduling processand helped NDFCU adjust retail branch staffing levels to be more in line with member traffic patterns."This does not always mean the credit union has to cut back." Tim pointed out. TMS has helped by revealing where the credit union is understaffed. At one branch, NDFCU quickly learned that more employees were needed to keep member service high. "Not having enough tellers can be more harmful than having too many," Tim explained.
Gaining a clear understanding of member traffic patterns has helped NDFCU make better use of part-time employees, as well. "It takes a little time to really move that metric," stated Tim, who said part-time utilization has improved 23%. "You have to be patient and as employees leave, you can make adjustments with part-timers."
Best Practices from the VP of Branch Administration at NDFCU
- Spend time looking at the data. The more you look at the activity reports, the more you learn about your staffing needs, which translates into better member service. The reports are very clear and consistent, which makes it even easier to spot needs as the months go on. I spend a fair amount of time reviewing the reports to fully understand what is happening in our branches.
- We have two floating tellers, which has given us flexibility to react to the TMS data more effectively. We have 11 locations and many branches are close enough together that we don't make a big impact on an employee's commute when we send him or her to different offices. We can even shuttle floaters between branches on the same day.
The benefits of TMS are recognized throughout NDFCU, from management to the front line. TMS delivered what the credit union asked of it, and the incentive program pushed results higher. "Our main goal was to increase teller transactions per hour, and we have achieved that," noted Tim. "Much of the success can be credited to the data, but the incentive program has played a key role — not only in the numbers but in the attitude of our employees."


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Temperature for July 2011
In July 2011 the average high temperature in Bettles, AK was 68.9°F, and this was 2.2°F cooler than the average of 71.1°F. The hottest day in July 2011 was 9 July when the temperature reached 79.0°F. Overnight the average temperature in July is 49.1°F and in 2011 the average overnight temperature was 2.4°F cooler at 46.8°F.


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The 2016 NDHSAA State Class B One-Act Play Contest was held Monday and Tuesday, November 21 and 22 in Jamestown.
The top 8 places awarded at the finals were as follows: 1st Larimore, "Shut and Bar the Door";
2nd Enderlin, "In A Grove: Three Japanese Ghost Stories"; 3rd Central Cass, "The Amish Project"; 4th Richardton - Taylor, "AND"; 5th Edgeley High School, "Nora's Lost"; 6th Thompson, "The Survivor and Other Urban Legends"; 7th New Salem-Almont, "Cyrano de Bergerac"; 8th Burke County, "The Struggles".
Superior Actor Awards were presented November 21 at Jamestown High School to: Alexis Waldren - Washburn; Cassidy Neidviecky - Central Cass; James Seibel - Harvey/Wells County; Robert Wright - Glenburn; Annah Greenmeyer - North Sargent; Daniel Young - New Salem - Almont; Lexi Mahlum - Burke County; Katelynn Holmgren - Kulm; Shelby Tilton - Larimore
Superior Actor Awards were presented November 21 at University of Jamestown to: Maria Mack - Rugby; William Egan - Enderlin; Ben Helfrich - Lewis & Clark; Arcilla Davis - Richardton-Taylor; Evan Braaten - Wyndmere; Sage Friedt - Carrington; Jake Wink - Kenmare; Grace Wolf - Center Stanton; John Paul Snyder - Thompson
Summer Steinwand Edgeley
Schools qualified for State from one of 10 regions statewide with contests held in early to mid-November. Participating were: Burke County, Carrington, Center-Stanton, Central Cass, Edgeley, Enderlin, Glenburn, Harvey-Wells County, Kenmare, Kulm, Larimore, Lewis & Clark, New Salem-Almont, North Sargent, Richardton-Taylor, Rugby, Thompson, Washburn, and Wyndmere.


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The UKMVA nominations are in and Black Dog Films are in the running for six awards including best new director.
The 10th annual ceremony, which takes place at the Roundhouse London and is presented by Adam Buxton, celebrates the breadth of creativity in music video and live film-making from across the globe.
The Black Dog nominations are as follows…
Best New Director – Thomas James
Best Alternative Video – alt J- In Cold Blood. Directed by Casper Balslev.
Best Live Concert – Rammstein Paris. Directed by Jonas Åkerlund.
Best Animation in Video & Best Rock/Indie Video (International) – Father John Misty – Things It Would Have Been Helpful to Know Before the Revolution. Directed by Chris Hopewell.
Best Urban Video – Mura Masa –Love$ick. Directed by Yoni Lappin.
Best Animation in Video – Katie Melua – Perfect World. Directed by Karni & Saul.
The UK Music Video Awards editorial director, David Knight, says "It's been a remarkable year for music videos in many ways and the nominations at this year's UK Music Video Awards prove it. Music videos are so important as a medium of expression for music artists now that we're seeing the conventional idea of the music video being tested and stretched by some of the world's most popular artists."
Well done to everyone involved. Check out the full list of nominations HERE. Roll on the awards.


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© 2000-2010
John B. Deitz
Build: 100521.2
Saturday, 13 August 2005. Trenton, GA to Roanoke, Virginia
While I was up and out of the room by my usual 6:30-7:00 am, Mike slept late, and we did not get on the road until nearly 9:30 am. The morning was damp and foggy -- and a day where mountaintop viewing was likely to be blocked by clouds. But we set out for Lookout Mountain taking SR 136, then the Lookout Mountain Scenic Highway (SR 189). on the fringes of the Chickamauga and Chattanooga National Military Park -- the first and largest of the four National Military Parks. On these fields and hills, Union and Confederate armies clashed during the fall of 1863 in some of the hardest fighting of the Civil War. The prize was Chattanooga, key rail center and gateway to the heart of the Confederacy. Lookout Mountain was key to the Confederate defenses with its commanding view of Chattanooga and the Tennessee River valley. The trip to the top of the mountain took us along a ridge which apparently is a favorite of hang gliders. Much of the mountain itself appears to be developed and has been incorporated as the City of Lookout Mountain, GA.
After coming down off the mountain, we get on I-75 and then I-81 north. I've been on I-81 many times, but not in the last few years. It has become a much more busy highway. I remembered it pleasantly as an easy and beautiful ride. But it's now, quite obviously, a major north-south trucking route.
We stop in Roanoke, VA for the night. After a brief rest, head into the city for dinner. I remember Roanoke as a sleepy factory town, with almost no nightlife. But "old" downtown now has a vibrant few blocks of restaurants, galleries, museums and shops. Lots of motorcycles about. Had o.k. pub fare at a pleasant local bistro.
Then decided to find out what Saturday night might be like at the gay bars in town. While Roanoke is reported to have a sizeable gay population, I felt like I was being transported back 30 years. One bar, Backstreet, was truly sleazy -- now I normally don't mind sleazy bars, but this was a place where you only drink bottled beer and wash your hands when you leave. The other, the Park, was a dance club with a cover. Both were in dingy industrial areas. We were going to go into the Park, but when we parked our bikes there was this man, obviously drunk, soliciting us. I got nervous about the safety of the Road King, and decided to leave. Oddly, a few years ago this would not have bothered me, but I guess I'm getting too old (and tire to early) for this stuff.
|Distance: About 396 miles
|Accommodation: Days Inn (WiFi)
|Cost: $65.95
23 October 2007


text: You'll find yourself so energized by your surroundings that you might try jet skiing on a house WaveRunner. Or stroll 10 minutes north along the pristine, silky-sand Playa Mal Pasos to San Pancho (population 10,000). Each has a distinctive personality and provides an up-close perspective of life in small-town Mexico, surprisingly good restaurants and shopping. And San Pancho even has a full-fledged adventure activity center with kids' club, kayaking, horseback riding, whale-wathcing excursions and much more. With two Suburbans at your disposal, you might even want to explore the resort mecca of Puerto Vallarta just 45 minutes away.


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Michael Jelenic
Teen Titans Go! to the Movies Trailer Streamed
DC Entertainment has updated their official Youtube channel with a new trailer for Teen Titans Go! to the Movies. The latest trailer features at the premise for the film and features a look at Deathstroke who will played by Will Arnett. Teeen Titans Go! To the Movies will premiere in theaters on July 27.
Teen Titans Go! to the Movies Teaser Trailer Streamed
The official DC Youtube channel has updated with a teaser trailer for the upcoming film for Teen Titans Go. For those who haven't been following, the long running Cartoon Network series will be receiving a film called Teen Titans Go! to the Movies. The film will premiere in theaters on July 27, 2018.
Scooby-Doo! & Batman: The Brave and the Bold Clip Streamed
IGN has posted a trailer for the upcoming DC animated film titled, "Scooby-Doo! & Batman: The Brave and the Bold," on their official Youtube channel. For those who are unaware, last October IGN revealed that the next animated DC film will feature a crossover between Batman & his friends from Batman: The Brave and Bold and the Scooby gang from Scooby Doo. Scooby-Doo! & Batman: The Brave and the Bold will be released on DVD and Digital tomorrow.
Scooby-Doo! & Batman: The Brave and the Bold DC Animated Film Announced
IGN has posted an official trailer for the next DC animated film, which will feature the Scooby gang meeting Batman and the Justice League based on their appearances from Batman: Brave and the Bold. The film titled, "Scooby-Doo! & Batman: The Brave and Bold," will feature familiar faces such as Aquaman, Plastic Man, The Question, and Martian Manhunter. Some of the villains both teams will face include the Joker, Penguin, Catwoman, Riddler, Poison Ivy, and Harley Quinn. However, Batman's rogue gallery will not be the only famous villains that be making an appearance in this film. TheRead More
Batman: The Caped Crusaders vs. Two Face Animated Film Trailer Streamed
IGN has posted the official first trailer for the upcoming Batman: The Caped Crusaders vs. Two Face animated film. The film is a sequel to the Batman: Return of the Caped Crusaders film that was released in 2016. The trailer features the caped duo fighting off against the villainous Two-Face voiced by William Shatner and more. The film will be released in a Blu-Ray/ DVD combo pack on October 17. Seeing as this is one of Adam West's final performances I hope the movie turns out to be as good if not better than Batman: Return of theRead More


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2 large eggs
2 ¼ C all-purpose flour
1 tsp baking soda
3/4 cup brown sugar, packed
3/4 cup granulated sugar
2 cups mini semi-sweet chocolate chips
1 tsp salt
1 tsp vanilla extract
1 cup chopped pecans
1 cup butter
Preheat oven to 375F.
Combine flour, baking soda and salt in a small bowl. Beat butter, granluated sugar, brown sugar and vanilla extract in large mixer bowl until creamy. Add eggs, one at a time, beating well after each addition. Gradually beat in flour mixture. Stir in chocolate chips and nuts. Drop teaspoon sized portions onto ungreased baking sheets.
Bake for 9-11 minutes or until golden brown. Cool on baking sheets for 2 minutes; remove to wire racks to cool completely.


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This event has passed.
"Special Needs Night"
March 11 @ 5:00 pm - 7:00 pm
This event is ongoing. Held every 2nd Monday of the month.
Welcome to a fun night out for families of children with special needs! Held from 5-7pm, enjoy reduced numbers of visitors, space and freedom to explore at your own pace, adapted exhibits, a quiet area, fun craft & sensory activities and the opportunity to connect with other families. Snacks provided by Menchie's Frozen Yogurt! Special Needs Night is a FREE event held every 2nd Monday of the month. Call 920-458-4263 for more information.


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A chiropractor will address any physical discomfort over the course of two appointments at this Parnell Square clinic
Price: €29.00
Value: €150.00
Discount: 81%
You save: €121.00
The Deal
- €29 for a chiropractic examination and treatment (81% off)
Those seeking relief from back, neck or joint pain can find solace with a visit to Abbey Chiropractic. During a 45-minute appointment, a chiropractor will perform a full consultation including orthopaedic and neurological exams. Patients will then be presented with a report of the findings and undergo a treatment during a 30-minute follow-up appointment.
Dublin 1
46 Parnell Square West
Terms and Conditions
Expires 4 months after purchase. Limit 1 per person, may buy 1 additional as gift. Booking by phone on 01 8734363; quote coupon code when booking and present Groupon on arrival. 24-hour cancellation policy. Valid Mon-Fri 9am-8pm. New customers only. Under 18s must be accompanied by adult. Consultation inclusive. Full refund will be provided if unsuitable for treatment. Therapist-client confidentiality privileges apply excluding intent to harm. Verified using our merchant's website on 14 Sep 2016.


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It's almost time for the second ELEAGUE Street Fighter V Invitational! The esports tournament has announced its panel of analysts that will give us the play-by-play during the brackets. In addition, we're welcoming a new host to the desk — but otherwise, expect lots of familiar FGC faces.
As usual, we're going to see the unstoppable duo, Steve "Tasty Steve" Scott and Stephen "Sajam" Lyon, joined by Reepal "Rip" Parbhoo and Zhi "Zhieeep" Liang Chew. They compose the panel of experts. Michele Morrow is the new host. As for the interviewer who will keep us connected to the competitors, Malik Forté returns from last year's ELEAGUE.
The action starts very soon, in a little over one week: the tournament begins Friday, June 1st, at 11:00 PM ET on TBS's channel. However, live coverage will start on their Twitch channel that day at 3:00 PM.
However, there is also a preview show airing on TBS this Friday, May 25th, at 11:00 PM ET/PT. This segment will showcase all the ELEAGUE competitors "as well as catch fans up on what makes the FGC unique, along with its history." There will also be a highlight of JB's victory through The Challenger reality TV show.
Check out a sneak peek of the show below:
Source: ELEAGUE [press release]


text:
Dharaa Mobility Pvt. Ltd. ("Jiffy Cars") is the owner and publisher of the cyberspace resource www.jiffycars.in and it's mobile website 'Jiffy Cars' (together hereinafter referred to as the, "Website"). Jiffy Cars Website is a tech platform for showcasing licensed (licensed car for hire ) self drive cars of certified vendors for purpose of hiring for a consideration.
All terms and conditions framed in this document / links are framed as per the Jiffy's Certified Vendors T&C and hereby adhered to Rules and Regulations framed by Its Certified Vendors
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The terms and conditions prescribed below are enforced by the vendors who have listed their vehicles in accordance to Rent a Cab Scheme of Motor Vehicle Act,1989. The User should follow ensure the following terms and conditions are followed while hiring the car from Jiffycars.
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If a User has any questions concerning Jiffy Cars, the Website, this Agreement, the Services, or anything related to any of the foregoing, Jiffy Cars customer support can be reached at the following email at firstname.lastname@example.com or via the contact information available from the following hyperlink: www.jiffycars.in
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5 Seater
6 Seater
7 Seater
"User" shall mean any person who has agreed to become a registered member of Jiffycars.in by providing personal information such as Name, Mobile, E-mail, Copy of driving license and ID proof
The user of Jiffycars should be 22+ years of Age to use the car rental service
The user should have valid National or International Driving permit and should also hold Identification card issued by government body. (Adhaar Card/ Voters ID/ Passport)
User of our self drive car should also hold credit/debit card for making payment online. No cash transactions are allowed
The terms and conditions prescribed below are enforced by the vendors who have listed their vehicles in accordance to Rent a Cab Scheme of Motor Vehicle Act,1989. The User should follow ensure the following terms and conditions are followed while hiring the car from Jiffycars.
Jiffy Cars
222, Nurturelabz,
Indiranagar Double Road,
Domlur 2nd Stage, Indiranagar,
Bangalore - 560071
Yes, if you are 22 years old and hold a valid driving license. You need to furnish an ID proof which should be issued at least one year before the pick-up date.
As per the law of the land, you need to provide a photo copy of the original driving license and a valid proof of address and it can be ONE of the following documents:
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Yes, since our tariff doesn't include fuel cost. This needs to be borne by whoever is renting the car. We also expect our customers to return the vehicle with the same level of fuel as the time of pick-up.
All our self drive cars can be driven anywhere across India. However, the inter-state road taxes and road tolls should be paid by the customer while crossing the border of each state.
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Yes, your safety is our main priority. You may call us on +91 85480 01000 for any kind of assistance during any unforeseen event.
You may travel with your furry friend at an additional cost of Rs. 1,500 per trip. Additional charges may apply if the car interiors are damaged or returned in a dirty condition. (Refer Schedule of Charges to understand more).
You may extend your booking by calling our customer care
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We do give our customers 1 hour as buffer time to return the self drive car at the end of the reservation period. One Hour Buffer time is applicable only for cars dropped by Customer. Any subsequent hours of delay will be charged at actuals with 50% surcharge
The maximum liability for damages will be up to the total Security Deposit paid by the customer. However, it is subject to the terms and conditions mentioned in the Terms of Use document.
For any further questions/enquiries, please write to us at email@example.com or you may reach us at +91 85480 01000.
Yes, for all cities except Cochin. For Cochin, Unlimited Kilometres drive is available for bookings over 48 hours.


text:
- Research
- Open Access
Studies in the mouse model identify strain variability as a major determinant of disease outcome in Leishmania infantum infection
- Filipe Marques†1,
- Sílvia Vale-Costa†1, 2,
- Tânia Cruz†1,
- Joana Moreira Marques1,
- Tânia Silva3,
- João Vilares Neves1,
- Sofia Cortes4, 5,
- Ana Fernandes6,
- Eduardo Rocha7,
- Rui Appelberg3,
- Pedro Rodrigues3,
- Ana M. Tomás3 and
- Maria Salomé Gomes3Email authorView ORCID ID profile
© Marques et al. 2015
- Received: 16 September 2015
- Accepted: 11 December 2015
- Published: 18 December 2015
Visceral leishmaniasis is a severe and potentially fatal disease caused by protozoa of the genus Leishmania, transmitted by phlebotomine sandflies. In Europe and the Mediterranean region, L. infantum is the commonest agent of visceral leishmaniasis, causing a wide spectrum of clinical manifestations, including asymptomatic carriage, cutaneous lesions and severe visceral disease. Visceral leishmaniasis is more frequent in immunocompromised individuals and data obtained in experimental models of infection have highlighted the importance of the host immune response, namely the efficient activation of host's macrophages, in determining infection outcome. Conversely, few studies have addressed a possible contribution of parasite variability to this outcome.
In this study, we compared three isolates of L. infantum regarding their capacity to grow in the organs of mice, the way they activate the host's macrophages and other components of the immune response and also their capacity to cope with host's antimicrobial mechanisms, namely reactive oxygen and nitrogen species.
We found that the three parasite strains significantly differed regarding the degree to which they induced nitric oxide synthase (NOS2) and arginase expression in infected macrophages and the pattern of cytokine production they induced in the host, resulting in different degrees of inflammatory response in infected livers. Additionally, the three strains also significantly differed in their in vitro susceptibility to reactive oxygen and nitrogen species. This variability was reflected in the capacity of each strain to persist and proliferate in the organs of wild-type as well as NOS2- and phagocyte oxidase- deficient mice.
The results obtained in this study show that parasite strain variability is an important determinant of disease outcome in L. infantum visceral leishmaniasis, with relevant implications for studies on host-pathogen interaction and also for leishmanicidal drug development.
- Nitric Oxide
- Visceral Leishmaniasis
- Leishmaniasis
- Arginase
- Granuloma Formation
Protozoa of the genus Leishmania are transmitted through the bite of phlebotomine sandflies and cause a range of human and canine diseases with varying severities. Visceral leishmaniasis, caused by Leishmania donovani and L. infantum, is the most severe of these diseases. Once symptomatic, the infection is often fatal if not treated, being responsible for the death of 20,000 to 40,000 people every year. In certain areas, such as East Africa, devastating epidemics of visceral leishmaniasis occurred recently, with very high mortality rates. Moreover, leishmaniasis are endemic in almost 100 countries around the world and HIV infection increases susceptibility to visceral disease [1].
Interestingly, not all individuals infected with visceralizing species of Leishmania develop pathology [2]. In their mammalian hosts, Leishmania reside and proliferate mainly inside macrophages. Successful control of Leishmania parasites is believed to require the differentiation of type 1 T helper cells, capable of producing IFNγ and allowing a "classical" activation of macrophages, enhancing their antimicrobial capacity [2]. As incomplete or inappropriate activation of this immune response will result in disease, the observed differences in pathology have been attributed mostly to host factors. However, a few previous studies have suggested some variability among Leishmania isolates [3–6]. Such variability may also have a high impact on disease outcome, since different strains may differ in resistance to the host's effector mechanisms or in their capacity to activate infected macrophages.
Activated macrophages can eliminate pathogens by different means, including their exposure to highly toxic oxygen species such as superoxide, nitric oxide and hydroperoxide [7, 8]. Phagocyte oxidase (phox) and nitric oxide synthase 2 (NOS2), the enzymes responsible for the generation of superoxide (O2• − ) and nitric oxide (NO•), respectively, are thus among the most important antimicrobial systems of macrophages [7–10]. Both enzymes have been shown to be fundamental for the control of cutaneous forms of leishmaniasis, namely in the context of mouse experimental infection with L. major [11, 12]. However, their relevance for the control of infections caused by visceralizing Leishmania is not so clear. Two previous studies addressing the role of NADPH oxidase and NOS2 in the control of L. donovani replication in mouse tissues gave conflicting results [11, 13], which we can now hypothesize were due to strain variations. A better understanding of the contribution of host leishmanicidal mechanisms for the control of visceral leishmaniasis deserves further investigation, as it can have a high impact on improving the management of the disease.
In this work, we used three strains of L. infantum isolated from different sources in the Mediterranean region to investigate their comparative infectivity patterns in mice and how these related to the parasite's capacity to i) influence macrophage activation, and to ii) withstand the toxicity of reactive oxygen and nitrogen species. We found a high heterogeneity among L. infantum strains in the tested parameters, highlighting the importance of parasite variability in determining the course of visceral leishmaniasis disease.
Characterization of L. infantum strains used in this work
Clinical manifestation
Adult; HIV negative
Visceral leishmaniasis
Child; HIV negative
Visceral leishmaniasis
Adult; HIV negative
Cutaneous leishmaniasis
For each experiment, parasites were re-isolated from the spleens of infected mice. Promastigotes were differentiated from spleen amastigotes by culturing at 25 °C in complete Schneider's medium (Sigma-Aldrich Co., St Louis, MO, USA) supplemented with 10 % heat-inactivated fetal bovine serum (iFBS), 100 U/mL penicillin, 100 μg/mL streptomycin (all from Gibco, Life Technologies, Carlsbad, CA, USA), 2 % human urine, 5 μg/mL phenol red (Sigma-Aldrich Co.) and 5 mM HEPES sodium salt (Sigma-Aldrich Co.) pH 7.4. Promastigote cultures were expanded at 25 °C, for a maximum of 5 passages, in RPMI 1640 GlutaMAX™-I medium (Gibco, Life Technologies), containing 10 % iFBS, 50 U/mL penicillin, 50 μg/mL streptomycin and 20 mM HEPES sodium salt pH 7.4. Promastigote differentiation from the exponential to the stationary phase was promoted by culture at 25 °C for 5 to 7 days without medium renovation.
In vitro susceptibility to oxidative stress
Synchronized mid-log phase promastigotes were seeded at 105/well in 96-microtiter plates. Hydrogen peroxide (H2O2), menadione or diethylaminenonoate sodium salt (NONOate) (all from Sigma-Aldrich Co.) were diluted in complete RPMI medium and added to parasite-containing wells at different concentrations (10–150 μM for H2O2,1–15 μM for menadione and 625–4000 μM for NONOate). Each concentration was tested in duplicate. After 18 h at 25 °C, 2 mM resazurin (Sigma-Aldrich Co.) was added and the cultures were incubated 72 additional hours. The concentration of resorufin, the product of reduction of resazurin, in the Leishmania cultures was then determined by reading the fluorescence (560 nm excitation, 590 nm emission wavelengths) in a BioTek®SynergyMx (BioTek, Winooski, VT, USA) fluorometer. Wells containing untreated parasite cultures, culture medium with each concentration of the compounds, medium alone and resazurin alone were included as controls.
The concentration of each compound that caused a fifty percent decrease of promastigotes viability (IC50) was calculated using a nonlinear regression of variable slope and four parameters in GraphPadPrism 6 (GraphPad Software Inc., La Jolla, CA, USA). Three independent assays were performed and the mean and standard deviation of the three were determined. One-way ANOVA with Bonferroni post-hoc test was performed to detect significant differences between parasite strains.
Mice deficient in the p47 subunit of the NADPH oxidase complex, on a C57BL/6 background (p47phox −/− ), were bred from a breeding pair purchased from Taconic (Lille Skensved, Denmark). Mice deficient in the nitric oxide synthase 2, on a C57BL/6 background (Nos2 −/− ), were bred from a breeding pair kindly provided by Drs. J. Mudgett, J. D. MacMicking and C. Nathan (Cornell University, New York, USA). C57BL/6 (wild-type), as well as p47phox −/− and Nos2 −/− mice were bred and housed at Instituto de Biologia Molecular e Celular (IBMC; Porto, Portugal) animal facilities under specific pathogen free conditions and fed ad libitum. The p47phox −/− mice were administered trimethoprimsulfamethoxazole (Bactrim; 600 mg L−1) in the drinking water, as prophylactic treatment against bacterial infection. This treatment was ceased when infection experiments began. Animals for each experimental infection were age and sex matched. Mice were euthanized by isofluorane anesthesia followed by cervical dislocation.
Ethics statement
All animals were handled in strict accordance with good animal practice as defined by national authorities and European legislation EEC/86/609. The experimental animal procedures were approved by the Local Animal Ethics Committee of IBMC and licensed by the Portuguese Directorate-General of Food and Veterinary Medicine (DGAV, Ministry of Agriculture, Rural Development and Fishing) with reference 520/000/000/2006.
Macrophage infection
To obtain peritoneal macrophages, naïve female C57BL/6 mice were sacrificed and the peritoneal cavity was washed with sterile PBS. The cells were centrifuged at 1200 rpm during 10 min at 4 °C and re-suspended in DMEM medium, containing 10 % iFBS, 50 U/mL penicillin, 50 μg/mL streptomycin, 2 mM L-glutamine and 10 mM HEPES sodium salt pH 7.4 (all from Gibco, Life Technologies). The suspended cells were seeded at 4 × 105 cells/well in 24-well plates and incubated at 37 °C and 5 % CO2 during 24 h. The promastigotes were added to the culture at an infection ratio of 10 parasites per cell. After 3 h of co-incubation, non-internalized parasites were removed by gently washing with Hank's Balanced Salt Solution (Gibco, Life Technologies) and fresh medium was added to the cells. The percentage of infected macrophages at 16 h and 72 h was determined by Giemsa staining and microscopic inspection (Leica DM E), to control experiment variability. After 16 h or 72 h, the cells were lysed and total RNA was isolated using RNeasy mini kit (QIAGEN, Hamburg, Germany), according to the manufacturer's protocol. Total RNA quantification was performed using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham MA, USA), quality was assessed by visualization in a denaturating agarose gel and 0.35 μg of each sample were converted to cDNA by VILO SuperScript kit (Invitrogen, Carlsbad, USA), according to the manufacturer's protocol. Arginase and Nos2 mRNA expression were determined by quantitative real-time RT-PCR, as described below.
In vivo infection
For in vivo infection studies, C57BL/6 (wild-type), as well as p47phox −/− and Nos2 −/− mice were injected in the lateral vein of the tail with 2 × 107 L. infantum stationary promastigotes in 200 μL of phosphate buffered saline pH 7.4 (PBS). At 15 and 60 days after infection, the animals were euthanized and tissues were collected and homogenized in aseptic conditions. Parasite load in each organ was estimated using a limiting dilution approach, as described previously [16, 17]. Briefly, serial dilutions of the homogenized tissue suspensions were performed in quadruplicate. After 7 to 14 days at 25 °C, the wells were examined for viable promastigotes. The reciprocal of the highest dilution that was positive for parasites was considered to be the number of parasites per mL of suspension and was used to calculate the number of parasites per organ. Statistical analysis was carried out using GraphPad Prism 6 (GraphPad Software Inc.). For multiple group and/or time point comparisons, the one- and two-ways ANOVA tests with post-hoc Bonferroni multiple-comparison test were used.
Histological analysis
Liver samples were collected from each infected mouse, by cutting similarly located and similarly sized longitudinal sections from the left side lobe. These were fixed in 3.7 % buffered formaldehyde and routinely processed for embedding in paraffin. Four micrometer tissue sections were obtained using a RM2155 Leica microtome (Leica Microsystems, Wetzlar, Germany) and were stained with hematoxylin-eosin using standard procedures. An Olympus BX50 light microscope equipped with a DP21 Olympus digital camera (Olympus, Center Valley, PA, USA) was used to examine the liver sections and obtain representative pictures.
To assess the percentage of liver area (equivalent to the relative volume) occupied by granulomatous lesions, a virtual slide microscope scanner Olympus VS110 and VS-ASW software (Olympus) were used. In each tissue section, granulomatous areas were delineated by a trained operator and measured using the microscope software. The percentage of liver area occupied by granulomatous lesions was calculated from the ratio between the total area occupied by granulomas and the total area of the whole section. The median and the range of the values obtained from the (5 to 8) mice in each experimental group was determined. Statistical analysis of the results obtained in histological analysis was performed using the software STATISTICA, version 12 for Windows (StatSoft, Inc., Tulsa, OK, USA). Due to non-homogeneity of variances, nonparametric methods were applied: Kruskal-Wallis test, followed by Mann–Whitney U test with the Bonferroni with sequential correction for the p-value, in case of significant results.
Animal tissue RNA isolation
Liver and spleen samples were collected, snap frozen in liquid nitrogen and stored at −80 °C until further use. Total RNA was isolated from tissues with the PureLink RNA Mini Kit protocol for animal tissues (Invitrogen) with the optional on-column PureLink DNase treatment (Invitrogen), according to the manufacturer's instructions. Total RNA quantification was performed using a NanoDrop 1000 spectrophotometer (Thermo Scientific). For all samples, 1.25 μg of each were converted to cDNA by Thermoscript™ and an oligo (dT) 20 primer (Invitrogen), for 40 min at 50 °C, according to the manufacturer's protocol.
Gene expression measurement using quantitative real time RT-PCR
Relative levels of arginase (Arg1), nitric oxide synthase 2A (Nos2), interferon gamma (Ifng), tumor necrosis factor alpha (Tnf), interleukin (Il)-10 and Il-12p40 mRNAs were quantified by real-time PCR analysis using an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules CA, USA). One μL of each cDNA sample was added to a reaction mix containing 10 μLiQ SYBR Green Supermix (Bio-Rad), 8 μL of ddH2O and 250 nM of each primer (Additional file 1: Table S1), making a total volume of 20 μL per reaction. The cycling profile was the following: 94 °C for 3.5 min, 40 cycles of 94 °C for 30 s, 59 °C for 30 s and 72 °C for 30 s. A melting curve was generated for every PCR product to confirm the specificity of the assays and a dilution series was prepared to check the efficiency of the reactions. Hypoxanthine-guanine phosphoribosyltransferase1 (Hprt1) was used as the housekeeping gene. The comparative CT method (2-ΔΔCT method) based on cycle threshold (CT) values for Arg1, Nos2, Ifng, Tnf, Il-10, Il-12p40 and Hprt1 was used to analyze the expression levels of each non-housekeeping gene. Statistical analysis was carried out using GraphPad Prism 6 (GraphPad Software Inc.). Data normality was checked by performing Kolmogorof-Smirnoff test and Student's T-test was used for estimating statistical significance. Multiple comparisons were performed with One-way ANOVA and post hoc Tukey test. A p value of less than 0.05 was considered statistically significant.
Impact of L. infantum strains on macrophage activation
This experiment demonstrated that the different strains of L. infantum differed in the way they interact with macrophages, namely in the degree to which they induce the antimicrobial mechanisms of these cells.
Expression of genes involved in host's immune response in the livers of mice infected with different strains of L. infantum
Survival and growth of different strains of L. infantum in mouse tissues and impact of host's reactive oxygen and nitrogen species
In order to evaluate how the inter-strain diversity observed so far impacts on the course of in vivo infection, we infected C57BL/6 wild-type mice as well as mice deficient in the antimicrobial enzymes phagocyte NADPH oxidase (p47phox −/−) and nitric oxide synthase 2 (Nos2 −/−) with each of the described L. infantum strains and the number of parasites in their livers and spleens were quantified after 15 and 60 days of infection.
The three strains of L. infantum also differed in the way their survival and growth was affected by host's phox and NOS2. IMT-151 parasites were partially eliminated in mouse liver and maintained its numbers in the spleen, irrespective of the expression of p47phox and Nos2 (Fig. 3c and d). Parasites of the strain ITMAP-263 grew more in mice lacking p47phox, most notably in the spleen, but not in mice lacking Nos2 (Fig. 3a and b). Regarding strain IMT-202, the absence of host's p47phox and Nos2 had no impact in parasite survival in the liver (Fig. 3e). However, in the spleen, the parasite burden was significantly higher in mice lacking Nos2 both at 15 and 60 days after infection, whereas it was only transiently affected by the lack of p47phox (significant difference in parasite load only at day 15) (Fig. 3f).
These results showed that different isolates of L. infantum clearly differ in survival and growth capacity in the organs of infected mice, which may be related to the previously shown differences in their patterns of immune response stimulation. Additionally, the three strains differ in the degree to which their in vivo growth is affected by host's phox and NOS2.
Histological alterations in the livers of mice infected with different strains of L. infantum
Percentage of liver area occupied by granulomatous lesions in mice infected with different strains of L. infantum for 60 days
L. infantum strain
Min - Max
p47phox −/−
Nos2 −/−
p47phox −/−
Nos2 −/−
p47phox −/−
Nos2 −/−
In C57BL/6 mice, strain IMT-202 induced the largest granuloma response, followed by strain ITMAP-263, while IMT-151 caused the lowest response. The extent of granuloma formation also varied among mouse genotypes, with p47phox −/− showing a lower granuloma response and Nos2 −/− mice developing the largest granulomatous involvement, as compared to C57BL/6 wild-type mice. Overall, a significantly larger extent of the liver tissue was involved in granulomatous lesions in Nos2 −/− mice infected with L. infantum IMT-202 than in any other experimental group (Table 2 and Fig. 4).
In vitro susceptibility of L. infantum to reactive oxygen species
Susceptibility of promastigotes of L. infantum to oxidative and nitrosative stress
L. infantum strain
IC50 (μM)
43 ± 11
2.6 ± 0.4
92 ± 15
3.9 ± 0.2 (*)
2190 ± 360 (*)
71 ± 29
4.7 ± 0.2 (*)
1560 ± 530 (*)
Overall, these results suggest that L. infantum strains have significant variability, not only regarding the way they activate host's antimicrobial mechanisms, but also the way they cope with host's generated oxidative stress.
In face of a lack of bona fide virulence factors that could discriminate between high virulence and low virulence strains, L. infantum (and Leishmania species in general) have usually been considered genetically uniform and the observed range of severity in clinical manifestations of infection attributed mostly to host factors. In this work, we show that different isolates of L. infantum can exhibit very different infectivities, pathogenicities and resistance to host defense mechanisms.
One key aspect of Leishmania-host interaction is the activation of macrophage's antimicrobial mechanisms. Mouse experimental infections with Leishmania contributed decisively to identify phagocyte oxidase (phox) and especially nitric oxide synthase 2 (NOS2) as fundamental antimicrobial weapons of the macrophage [20, 21]. However, while the role of NOS2 and phox in infection by L. major has been unequivocally shown by several authors [12, 21–23], its role in infection by visceralizing Leishmania species is not so clear. A first report by Murray and Nathan in 1999 demonstrated that both NADPH oxidase and NOS2 were involved in the early control of L. donovani replication in the liver, whereas NOS2 alone was sufficient to resolve late infection [13]. In contrast, a subsequent study showed that these antimicrobial mechanisms had no influence on the outcome of L.donovani infection in the liver, though NOS2 exerted moderate protective effects in the spleen at late phases of infection [11]. Among the three strains included in this work, only IMT-202 showed increased proliferation in Nos2 −/− mice, indicating once again that NOS2 is not universally involved in the control of Leishmania growth in mice. Interestingly, we found that NOS2 was involved in the control of IMT-202 in the spleen but not the liver, which is in line with a recent report by Nascimento et al. showing increased parasite load one week after infection in the spleens but not the livers of Nos2 −/− mice, with a strain of L. infantum different from those presented here [24].
We also observed that strain ITMAP-263 had increased survival in the livers of mice deficient in the phagocyte oxidase (Fig. 3a). Additionally, both ITMAP-263 and IMT-202 showed increased survival and proliferation in the spleens of phox-deficient mice, indicating that for some L. infantum strains phox is important for growth inhibition in this organ. Relatively few studies have evaluated the role of phox in the visceral growth of Leishmania, but Blos et al. have reported that it was necessary for the control of L. major infection in the spleen and not in other organs of mice [12].
The different in vivo infection outcomes observed in this study with three L. infantum strains could result either from differences in immune response activation or in resistance to the toxic reactive oxygen and nitrogen species produced by the host. Our results show that both these aspects are important and additional parasite characteristics may also play a role.
Strain IMT-151 showed a limited capacity for growth in mouse organs. Interestingly, this strain was not particularly sensitive to ROS and RNS, with IC50s for hydrogen peroxide, menadione and NONOate comparable to that of IMT-202 (Table 3) and to other values reported in the literature [25–27]. Consistent with this finding, it did not grow more in the organs of phox −/− or Nos2 −/− mice (Fig. 3c and d). Interestingly, this strain did not significantly induce the production of pro-inflammatory cytokines in vivo which correlated with a low inflammatory response as evaluated by histological analysis. Moreover, when incubated with macrophages in vitro L. infantum IMT-151 led to the early induction of Arginase rather than Nos2 expression, although in vivo a significant induction of Nos2 in the liver could be seen at 60 days post-infection. Overall, the data gathered with this parasite strain indicates that it does not strongly activate the host's immune response, but might have a low intrinsic capacity to grow in the organs of infected mice, because of defects in nutrient uptake or other survival mechanisms.
Strain IMT-202 exhibited some features of increased virulence as compared to the other two strains, both because it had a higher survival and proliferative capacity in wild-type mouse organs (Fig. 3) and because it induced a more extensive liver pathology and inflammatory response (Figs. 2 and 4 and Table 2). Of note, the IMT-202 strain was associated with the highest induction of both Ifng and of Il-10 in infected mice (Fig. 2). While the first cytokine is necessary for an efficient granuloma formation [19], the second is associated with parasite persistence [19, 28].
Granuloma formation in the liver in response to Leishmania requires the recruitment of CD4+ T cells and the release of IL-12, IFNγ and TNF, among other cytokines [18, 19]. The proximity between IFNγ-producing T cells and infected macrophages is thought to be important for macrophage activation and parasite killing [18, 19]. Thus, granuloma formation is believed to be necessary for the containment of Leishmania parasites in this organ [19]. In the present work, no correlation was found between the extension of granuloma formation and the containment of parasite growth. The persistence and growth of L. infantum IMT-202 in spite of extensive granulomatous response could be due to the concomitant expression of Il-10 (Fig. 2). It has been previously reported that chronic infection with visceralizing species of Leishmania is accompanied by the development of CD4+ T cells of the IFNγ/IL-10 double producer phenotype [28–30]. The contribution of IL-10 for parasite persistence and proliferation has been suggested to be due to decreased expression of Nos2 in infected macrophages [28]. However, what we observed with L. infantum IMT-202 was concomitant expression of Il-10 and Nos2. Future studies analyzing the cell types involved in cytokine production and the temporal correlation between Nos2 and Il-10 expression in the liver will help elucidate this apparent paradox.
It is interesting to note that Nos2 −/− mice infected with L. infantum IMT-202 had 30 times more liver tissue occupied by granulomas than wild-type mice infected with the same parasite strain (Table 2). This observation, suggesting an inhibitory effect of NOS2 on granuloma formation has been reported before with other pathogens [31]. Several mechanisms may contribute to explain this effect. Nitric oxide has been demonstrated to inhibit T cell proliferation and recruitment during granuloma formation [21, 32] and the activity of nitric oxide synthase may also delay granuloma formation by interfering with collagen synthesis [33].
ITMAP-263 is a reference strain, used by different investigators around the world [30, 34]. We found that this strain has a high resistance to nitric oxide (Table 3) as suggested by previous reports [34], while being more sensitive to ROS than the other two. This resistance/sensitivity profile was consistent with the growth of ITMAP-263 in the two knock-out mice models. In fact, while the parasite growth was not affected by Nos2 expression, it was increased in the absence of p47phox (Fig. 3).
The high variability in sensitivity to ROS and NO found in this study suggests that field isolates vary in resistance to these antimicrobial molecules. This can have a high impact not only in their interaction with the host but also in their response to chemotherapy. In fact, several lines of research are engaged in the development of anti-leishmania drugs which act through the release of reactive oxygen or nitrogen species [35–37]. Additionally, previous reports showed a correlation between sensitivity to nitric oxide and to antimonials, both in L. infantum and L. braziliensis [34, 38], which were underlined by subtle differences in the expression of enzymes involved in basal metabolic pathways of Leishmania [34]. Varying resistance to antimonials has also been reported among L. donovani isolates, although no correlation with resistance to oxidative stress was found in this study [39]. We thus suggest that the inclusion of several parasites strains is crucial when screening new drugs for anti-Leishmania effect.
The "microdiversity" among isolates within a single pathogen species has been increasingly acknowledged in the case of Mycobacterium tuberculosis, another important human pathogen which can give rise to highly variable disease outcomes [40, 41]. In the case of L. infantum, although a few previous studies failed to identify major differences between strains in terms of serum sensitivity, macrophage infectivity and other markers of virulence [5, 42], a number of other reports indicate significant genetic diversity among strains [6, 15, 43]. A study done in French healthy blood donors suggested that subtle genetic differences exist between L. infantum parasites found in asymptomatic carriers as compared to leishmaniasis patients [4]. Moreover, a comparison between 3 isolates of L. donovani in India revealed different sensitivities to oxidative stress, as well as different capacities to survive and grow in mouse tissues [39]. More recently, three L. infantum strains isolated in Spain were shown to have distinct profiles of macrophage activation and intra-macrophagic survival [3]. In this work, we clearly show that L. infantum isolates differ regarding the degree to which they are controlled in vivo by both NOS2 and phox. This diversity is due both to differences in susceptibility to killing by reactive oxygen and nitrogen species and to differences in activation of the host immune response.
The body of data collected in these studies allows us to conclude that it is important to increase awareness about the diversity among the agents of visceral leishmaniasis and refrain from generalizing assumptions on host-pathogen interactions and on drug effectiveness based upon studies with one or a few reference strains.
The authors would like to thank Prof. Lenea Campino, from the Institute of Tropical Medicine and Hygiene in Lisbon for helpful discussions and the critical review of this manuscript; Carolina Caldas and Ana Carolina Moreira, from the Iron and Immunity group and all the staff of the IBMC Animal Facility for their technical assistance with mouse experiments.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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The past fifteen years I've gotten mail everywhere but in my own mailbox. Much of it has to do with my traveling ways. It's hard to blame the post man for not filling your letter box if you're never around – and don't own a mailbox.
It's something I finally got around to correcting.
I've spent a good deal of the past 15 years traveling the world by boat, mule and bike. Between journeys, I'd find a place to stay – for a few months to a few years – then carry on.
There were the semi-stationary addresses – strings of rented barn apartments, regular apartments and hunt boxes (horse stalls below, digs above) – mostly in Southern Pines, North Carolina. There were sea level addresses (thanks Keith and Melinda) and others on mountain tops (howdy Kristin and Grant).
There were even government addresses with tiny brass doors – that's called a PO Box.
There were times of no address at all. On the road I got mail the old fashioned traveler's way – General Delivery. General Delivery Tecumseh, Oklahoma while traveling by mule and pack pony across America. General Delivery Keyes, Oklahoma heading south to Mexico with a yellow wagon. Overseas, in French territories like New Caledonia, it sounded even grander – "Post Restante".
This getting mail all around drove the search engines bug house nuts.
Hell, over the years I've had so many addresses when I searched my address online I learned I lived in St Thomas, USVI. Oh, right… In the late 1990s I ran aground a few months in the Caribbean. (I was giving riding lessons so I could earn enough money to sail on to the Pacific – which I did).
Of course all of this spun through my head as I attached the trace chain that attached the locust log to mule.
Say what?
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Where this story happened:


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What if we stopped calling it Obamacare or Trumpcare and just started calling it Medicaid Plus or something that does not prejudice an entire population of people? And what if those that don't qualify for Medicaid (but still don't make enough to purchase a policy) just pay a little something into Medicaid Plus on a sliding scale? Those who can afford to pay for their own private plans continue to do so, or pay for Medicaid Plus until we figure out a way to deregulate or regulate, fix or undo whatever is not working in the system itself...Will that, at least, stop the fighting, because the stress is killing us...Literally!
You may not want to continue reading if you like playing the blame game. Most of us intelligent and intuitive beings, though, know as a fact that going to single payer or staying as we were will not stop the cost of medical care from escalating. That will continue to rise and threaten to bankrupt states if we don't all step up and take personal responsibility for what we are contributing to the problem and the cost. Whether we are a patient or payer; a doctor or hospital administrator; an employee of the FDA, big Pharma, a school or the government; a parent, lobbyist, farmer, rancher, food manufacturer, or business owner; technology and product developers, even a member of the press, owners of television stations and creators of the programming...Anyone and everyone, including ourselves, who has an influence on our bodies, psyches, sickness, health, and wellness have a participation in what has gotten us into this mess. And every single one of us has the responsibility of helping us get out.
Unhealthy lifestyle choices and the stress that comes with them cost us trillions of dollars in medical costs, lost productivity, increases in crime, traffic accidents, relationship woes, divorce, loss of income, loss of happiness, alcoholism, drug addiction, sex addiction, spousal abuse, unemployment, disability, hostility, mental illness, depression, suicide, childhood issues that lead to adult disease, death, and so much more, as if that is not enough.
So as we all keep complaining about what others aren't doing to fix this problem, who among us is actually taking responsibility for their own body as well as their children's? How about diet, exercise, and your physical, emotional, mental, and spiritual state of being? Who is taking the time to learn about hidden dangers that surround us? How about natural cures and our body's innate ability to self-heal? The power of the mind to hurt or help us? Energy and negativity that perpetuates itself?
Who is making a commitment to themselves and their family to do EVERY LITTLE THING they can to be the healthiest they can be? And if you are a person in a position to create positive change that will affect many people, are you doing the one thing you can do to help, or are you going with the flow of ignorance? Are you staying in a mindframe of blame, fault, and ridicule for what everyone else is doing or not doing?
I'm standing to unite us on this one issue: The primary reason our healthcare costs are escalating is that we are using the system too much! Prevention is the key to this mess we are in, and yes, fear, greed, laziness, and entitlement contribute; that is an absolute, but we "buy" into it, which is why it perpetuates itself. There are many simple solutions; things we can do right now! Are we smart enough, or should I say, sick and tired enough, to want to finally pursue them?