text:
This convenience store in the historical Saint-Jean-Baptiste district is popular with beer connoisseurs from around the globe. Over 400 types of beers from Québec, across the Americas and elsewhere can be found here. We have it all: hopped, malted, full-bodied, pale, dark, etc. Our knowledgeable staff can help you find just the right beer, either an old favourite or a new brew. La Duchesse d'Aiguillon is a beer lover's paradise!
Hours: Sunday to Tuesday, 11 a.m. to 9 p.m. Wednesday, 10 a.m. to 11 p.m. Thursday to Saturday, 11 a.m. to 11 p.m.
Schedule and rates are subject to change without notice. Visit their website or contact them for up-to-date information.
469, rue Saint-Jean
Québec, QC
Borough of La Cité - Limoilou / Downtown
Enlarge map
Parking: Free Outdoor on Site, Outdoor on site for a fee, Bike Parking: Free Outdoor on Site
English, French


text:
The Magic of the Carousels
Enjoy the laughter, music and magic of the Heritage Carousel and the new Winter Wonderland Carousel located along Memorial Drive at North Bay's Waterfront.(across from the former Marina store)
The Winter Wonderland Carousel
The Winter Wonderland Carousel opened in Grand style on July 1st, 2005 during a well-attended Canada Day celebration.
The Winter Wonderland carousel will be located for approximately 3 years alongside the original Heritage Carousel at the Waterfront until the rail lands are prepared for use. It will be housed on a cement floor under a large blue and white striped tent reminiscent of the circus tents of days past.
This carousel is 28 feet across ... 6 feet larger than anticipated, so the original 14 carvings will need to be increased to 16 animals. For the time being, 2 spare horses from the first carousel will be used, but they will eventually be replaced with a lynx and a calf moose.
The Carvers and Artists ...
Thank you to the carvers and the artists who have brought
this dream to life!
The Animals ...
Whimsical Beauty
The animals of the carousel are whimsical and beautiful ...
reminiscent of all the creatures found in our Northern Ontario rivers, lakes and forests. Thousands of hours of volunteer labour went into creating these remarkably unique art pieces. The fine details and intricate design make every one special in it's own way.
Our Winter Wonderland Animals

subset: commoncrawl


text:
I usually try to go to the ceramics showcase at Bussière Badil in the spring, when about 20/30 potters exhibit, and about 2 years ago I saw some delicate porcelain pots with what looked like engraving or carving. I asked the potter how he got this effect, and he was a little cagey, as you would be, if you had been working up a technique. He said it was done by painting the pot with a special paint, then wiping with a wet sponge. And that is basically it. The pot is thrown and turned, and while still green a design is painted using, in my case, button polish.
Care must be taken not to get the water all over the place, and not to wet the pot too much, or to wipe back the surface until the pot is too thin and collapses. And it is difficult to get a uniform thinness, to allow uniform translucency.
I have tried the same technique now with stoneware, a very different clay, I have ended up spraying the pots with a water jet, which is much more of a performance art.


text:
Watch Movies OnlineWatch Zero Effect 1998 Online
The world's greatest detective Daryl Zero aided by his associate Steve Arlo investigates a complex and mysterious case of blackmail and missing keys for shady tycoon Gregory Stark who is less than forthcoming about what is really happening!
Watch Zero 2016 Online
Strange events start happening to a woman after marriage.
Watch Zodiac 2007 Online
A San Francisco cartoonist becomes an amateur detective obsessed with tracking down the Zodiac killer.
Watch Z 1969 Online
Following the murder of a prominent leftist, an investigator tries to uncover the truth while government officials attempt to cover up their roles.
Watch Zero Focus 2009 Online
One week after their marriage, Kenichi Ubara (Hidetoshi Nishijima) bids farewell to his wife Sadako Ubara (Ryoko Hirosue) prior to departing for Kanazawa on a business trip. The couple met on a blind date through a matchmaker before marrying. Kenichi promises to return to shortly, but never returns. Sadako then travels to Kanazawa to uncover his whereabouts. There she meets two women. Sachiko Mur…


text:
VLADIVOSTOK (Russia), Sept 5 ― Malaysian Prime Minister Tun Dr Mahathir Mohamad said he could not exclude discussing the MH17 crash with Russian President Vladimir Putin at their meeting later today, reported Sputnik news agency.
"Well, if the matter is mentioned, then of course," the prime minister said during an interview session with Sputnik when asked if the crash could be discussed.
The two leaders will be meeting on the sidelines of the Eastern Economic Forum (EEF) in Russia's Vladivostok.
To another question, Dr Mahathir said Malaysia would welcome Putin's visit.
"We would certainly welcome president Putin if he were to make a visit to Malaysia. It would be a continuation of the dialogue we have been having between me and president Putin in various other forums" the prime minister said ahead of his meeting with Putin on the sidelines of the Eastern Economic Forum (EEF) currently held in Russia's Vladivostok.
The meeting will take place later today. ― Bernama


text:
Charlo Jet Kimono Short
$109.00 $159.00
Charlo Jet Kimono Short CHL2401S
Our signature short length Kimono that finishes approx top of thighs, split sleeve cuffs and binding around the edges. Fabric in a chiffon navy base with deep purples and dark pink in a large floral print.
The Rock Box is here to offer you a 24/7 fun stress free shopping experience! We hope you love your purchase but know that mistakes can be made when shopping online when you cannot try on the garment before buying it. If you are unhappy with your purchase we can offer you a shop credit, unfortunately we cannot offer you a refund if you simply change your mind. Refunds are only offered in case of manufacturing faults (see below).
Please take care when trying on your new garment for the first time. You should always remove your makeup and jewelry and be unperfumed before trying on..watch out for self tanner as well. We will only exchange or issue you a store credit if the garment is returned in it's original clean condition..unmarked, unworn, unwashed.
Please note: All SALE items and ACCESSORIES are FINAL SALE.. no exchanges, credits or refunds.
Returns and Exchanges...
- Step 1 : Contact us within 24 hours of receiving the goods by email or phone
- Step 2 : We will issue you a Returns Authorization Number
Step 3 : Box up your return/exchange in it's original packaging along with the receipt. Items must be UNWORN with Original Tags attached. Customers are responsible for the cost of postage to return items back to The Rock Box. We must receive the item back to us within 7 days of you first receiving it. Please use a track and trace shipping method, we will not be responsible for any items damaged or lost in transit.
- Step 4 : Address the package to :
The Rock Box
244 Hardy Street
Morrison Square
NELSON 7010 New Zealand
Step 5 : When the package is received we will assess the garment and send you an email confirmation within 3 working days, not including weekends or holidays.
Due to the popularity of some items we cannot always guarantee that exchanges will be possible as sizes may sell out quickly. If this is the case you'll be issued with a store credit to be used on the The Rock Box website and be notified via email.
The Rock Box team does a thorough inspection of all goods before being shipped to our customers to ensure they are free of any faults. Any item that is returned as faulty will be inspected and a decision will be made as to whether it's a manufacturing fault or normal wear and tear.
If it is deemed to be a manufacturing fault we will meet our requirements under the NZ Consumer Guarantees Act. A full refund will be issued if the item cannot be repaired or replaced first.
Thank you for shopping with us and we hope you enjoy your purchase !
Deane & Jennifer
Post Haste Track and Trace
Once payment has been received in full, we will dispatch your item within 24 hours and email you a tracking number.


text:
Abby Winters Review
Girls Out West is a site featuring all natural Aussie amateur girls having fun, showing off their bodies and getting themselves off alone or with their boy and girl friends. These are just a few of the girls who are sick of their vibrators and want a REAL cock to please them tonight. If you like teasing and nude erotic pin ups give this one a shot!


text:
In South Dakota and Pennsylvania, Hunter Education Goes Online
The South Dakota Game, Fish and Parks Department recently approved the new online HuntSAFE program, an online education program that be used as in place of attending in-person classroom instruction for the state's hunter safety certification course.
After students pass the online test, they will then be required to participate on an in-person HuntSAFE field day. The day lasts for four to six hours and includes a written final exam.
"After passing the online portion of the course, students are equipped and eligible to participate in the required, in-person HuntSAFE field day. Overall, the HuntSAFE course does a quality job of teaching the information and skills to students, to ensure that they will be safe, legal and responsible in the field," said Jason Kool, GFP program specialist.
Videos are also available online that present safety information to viewers, including topics such as the transportation of firearms, the importance of blaze orange clothing and how to properly match ammunition.
The course features interactive animations and learning modules. Progress is automatically saved, allowing users to work at their own pace, from a home computer, smartphone, tablet, e-reader or Internet TV.
While education in the state is considered useful for all hunters, South Dakota law requires any hunter under the age of 16 to complete a hunter education course. Students must be at least 11 years old.
The online portion of the course will cost students $15, although the classroom alternative is free.
A similar online program has been created by the Pennsylvania Game Commission for first-time hunting license buyers in Pennsylvania, who will not be required to complete their hunter education entirely online.
The full course can be completed in about six to eight hours and is available for students age 16 and over.
"In recent years we've upped the number of traditional, classroom courses we offer and have offered many more courses at peak times in the fall when more people are thinking about hunting and hunter education, and record numbers of new hunters have become certified as a result. But demand still exists, and some folks simply can't make the time to attend a traditional classroom course," said Andy Hueser, a hunter-education specialist for the commission.
The curriculum for the online course is the same as that taught in the traditional classroom course, covering topics such as tree-stand safety, lawful trapping of furbearing animals and firearms safety, among others.
The online course is 11 units long and requires students to spend a certain amount of time on each page to ensure they are not simply skimming the information. A quiz must be passed at the end of each unit in order to move on to the next. A final exam is completed at the end.
Those who pass the test can print out a certification card that can be instantly used to purchase a hunting license.
02 26, 2015 Kristin Decarr


text:
Pantheon One Hour Guided Tour
Rome, Italy
From USD 37.81
Office phone number: +39 338 884 7405
Pantheon One Hour Guided Tour
Rome, Italy
Culture shock rating
Worried we're going to shock your senses? Relax! We believe part of the fun of travel is immersing yourself in the destination and its culture, meeting the people, and learning what makes the place tick. We've got a wide range of tours with something for everybody. The cultural shock rating ranks how different the experience is from most Western cultures. But no matter the level, don't worry, you'll have a local, in-the-know guide with you every step of the way.
Consider these tours your 101 intro to a place. Transportation might be private or a very comfortable public option, and the activities are usually visits to iconic sites and locations that are familiar to most Western cultures – but that will still give you fantastic insight into a destination.
Expect to rough it for parts of this tour, whether that's on a packed public bus or in a local market off the tourist trail. There might be a few language barriers or unfamiliar cultural customs, and you'll get an experience different from what you're used to at home.
You're out there in the global community! We're going to take you down streets you'd rarely explore on your own, introduce you to local customs and languages, and take you for a ride in whatever transport is available. Get ready to take it as it comes, whatever comes. There might be a shock, but oh man, it's worth it.
Physical rating
Worried our tours are too tough? Relax! From leisurely strolls to muscle-burning treks, we've got a wide range of tours with something for everybody. The physical grading gives you an idea of how much huffing and puffing you can expect on the tour.
Slow and steady is all you need here. These tours have very limited physical activity, such as walking relatively flat streets, sites, or markets, and climbing in and out of the transport provided.
Not too hard, not too soft, these tours are just right! You can expect a bit of physical activity, but nothing overly challenging – perhaps walking up and down hills, riding a bike for up to 30 kilometers along mostly flat terrain, or jumping in a kayak for a gentle paddle on flat water.
Get ready for a workout! These tours are our most challenging and involve intense walking, hiking, kayaking, swimming, or bike riding. You could be making steep climbs by foot or pedal, or working your core in the water. We recommend you have a good level of fitness to join this tour.
Tour snapshot
Join a local expert guide for a whistle stop one-hour tour around the Pantheon, one of the world's most spectacular and best preserved Roman buildings. Get the lowdown on the history of this majestic edifice and find out how the exquisite architecture in front of you was carefully pieced together. This early morning tour offers you the opportunity to stroll around the interior of the Pantheon without the tourist hordes getting in your way, allowing you to get lost in the majesty, if only for a moment, of this ancient marvel of engineering.
- Visit the Pantheon, one of the Seven Wonders of the Ancient World, with a local guide by your side
- Find out the history behind one of the planet's best preserved Roman buildings, now more than 1,300 years old
- Learn about the different types of stone, granite and marble used to build this beautiful structure
- Visit early in the morning so that your precious holiday snaps are not filled with selfie-stick-loving tourists!
- Discover the stories of some of the famous artists and personalities who are said to be buried inside
Schedule details
- Duration: 1 hour
- Meeting point:
Information desk inside the Pantheon situated on the right-hand side. Your guide will be wearing Urban Adventures branded clothing or holding an Urban Adventures sign.
Address: Pantheon - Piazza della Rotonda 00186 Roma RM
- Starting time: 9.00 AM
- Ending point:
Main exit of the Pantheon
Language: English
Inclusions: Local, English-speaking guide.
Exclusions: Food and drinks, souvenirs and personal shopping, tips/gratuities for your guide.
Upon arrival at the Pantheon please make your way to the reception area inside the main entrance, where your local guide will be waiting for you.
As you walk into this most exquisite of all Roman buildings, you may notice the brass doors, which are unusual because most of the brass plaques and doors that were originally included here have been stripped and melted down over the centuries and used to build structures in the Vatican. The most famous object made from the 'borrowed' brass is the Baroque-sculpted bronze canopy that resides over the high alter in Saint Peter's Basilica, designed by Bernini.
As we walk around we'll talk about the building's origin as a temple for the gods commissioned by Marcus Agrippa and its subsequent rebuilding by Emperor Hadrian in the 2nd century AD. You will learn about how this miraculous structure was built and the different guises it took on over the centuries.
We will discover side chapels that contain the tombs of the Kings of Italy and other famous names such as Raphael and hear some of their stories.
We'll spend time examining the remarkable dome that, unbelievably, is bigger than the dome at St Peter's. The dome itself represents the heavenly sphere of the gods and has been studied by some of the world's greatest architects, including Brunelleschi and Michelangelo.
We will also take a peak at the Pantheon from the outside too, where you will see the original level the Pantheon was built upon. In the ancient world, the city of Rome was closer to sea level than it is now.
Your tour ends where it began and hopefully you will leave with a better understanding of ancient Roman architecture and culture. Before you go, don't forget to ask your local guide for tips on what else to see and do during your stay in Rome.
Additional information
Inclusions: Local, English-speaking guide.
Exclusions: Food and drinks, souvenirs and personal shopping, tips/gratuities for your guide.
Dress standard: Please ensure that your shoulders are covered and you are adhering to a respectful dress code (no short shorts/skirts, vests, etc.), as the Pantheon is still a consecrated church.
Your Trip: For your Urban Adventure you will be in a small group of a maximum of 12 people.
Confirmation of booking: If you have your voucher, your booking is confirmed. We'll see you at the start point. Get in touch if you have any concerns or require more information via the email address or phone number (business hours only) on your voucher.
Child Policy: This is a child-friendly tour. Children between the ages of 6 and 11 inclusively are permitted on this tour at the rate listed above. Please select 'child' above when booking. Children under the age of 6 are permitted to join this tour free of charge. Please inform us at the time of booking if you'll be bringing a child under the age of 6. You can do so in the special request box on the checkout page.
Local contact
Office phone number: +39 338 884 7405
Email address: email@example.com


text:
Mukoko set to hoist Zimbabwean flag high
Zimbabwe Peace Project is proud to announce that Ms Jestina Mukoko, our National Director, has been selected to represent Zimbabwe in the 5th Edition of the Leadership Forum for Strategic Impact to be held in Yale University and Washington DC
It Never Rains But Pours
ZPP March 2019 MMR
MDC Intra-Party Violence Regrettable
MDC Intra Party Violence Regrettable 2 April 2019 The Zimbabwe Peace Project (ZPP) condemns the intra party violence that has characterised the preparations for the Movement for Democratic Change (MDC) elective congress scheduled for May 2019. ZPP has received reports
ZPP condemns misinformation programmes on disability on ZBC
11 December 2018 The Zimbabwe Peace Project is appalled by the National Broadcaster, Zimbabwe Broadcast Corporation (ZBC) for airing views that promote hate and perpetuates stereotypes against people with disabilities in violation of the spirit of the Constitution and also
Commemoration of the 2008 Enforced Disappearances and Abductions culminating Into the International Human Rights Day
23 November 2018 As the world prepares to commemorate the International Human Rights Day on 10 December 2018, (with 2018 also marking the 70th year anniversary for the Universal Declaration of Human Rights), Zimbabwe Peace Project (ZPP) will remember and
Impact of Enforced Disappearances and Abductions: Not One More!!
5 December 2018 70 years after the Universal Declaration of Human Rights in 1948, the global annual average of enforced disappearances and abductions is still high. The Worldwide Movement for Human Rights in a statement in August 2017 reported that
Alert: Police and civilian clashes in Gweru
21 August- The Zimbabwe Peace Project has noted the violence that has erupted in the Gweru Central Business District where police who were trying to evict vendors from that area were attacked by the resisting vendors. In the past such
ZPP June MMR 2018
ZPP June MMR 2018
Statement: ZPP condemns MDC-T Violence
The Zimbabwe Peace Projects strongly condemns incidents of intra-party violence reported at the MDC-T Bulawayo offices over the weekend. The violence comes three months before the elections and a week after violence rocked the same party in Buhera at the


text:
MARQUETTE, Mich. (WLUC) - February is heart month. This is an opportunity for organizations such as the American Heart Association to highlight the importance of cardiovascular health.
At U.P Holistic Medicine, Dr. Scott Doughty is doing just that through a clinical trial. Focusing on secondary prevention, his goal is to reduce heart attacks, stokes, and death using chelation therapy.
"The clinical trial that we are involved with is called 'TACT – 2' and TACT stands for trial to assess chelation therapy. It's the second trial of its kind. Thus the number two," said Dr. Doughty. "What chelation therapy is, is the binding of heavy metal. In or in this case, specifically lead and cadmium and some other toxins that have been shown to have an impact on blood vessel health and there for the clogging of arteries."
This worldwide trial is even using 5 local patients.
"There are seven clinical sites in the state of Michigan. These include Henry Ford Hospital, Wayne State, the University of Michigan, and little ol' U.P. Holistic Medicine, which is looking like the 'Little Engine That Could,' because we are right behind Henry Ford Hospital in the number of participants."
Dr. Doughty, who's also the vice president of the International College of Integrated Medicine, always stresses to his patients that in order to keep the heart healthy, you need to keep your entire body active.
"Exercise is probably one of the best forms of medicine, not just for heart health, but for overall," Dr. Doughty said.
Dr. Doughty will be heading downstate next month for a cardiovascular conference to show what he's learned and to represent the Upper Peninsula.


text: The Night Kobe Bryant Turned Into R. Kelly Brandon Caldwell March 7, 2012 Sports 1 Comment For the record, let me state that I've never been a proponent of Kobe Bryant. Just an honest critic. After all, up until Lindsay Lohan decided to blow up the spot, Kobe Bryant moved the needle the most here. Because after his fifth ring, we questioned why was he so polarizing a figure in sports. Did we use the "hate" word? Yes, but you have to admit that people truly dislike the guy. And he could care less. Last night did KB24 no favors as well as he opted to go with the black protective mask instead of the clear one to shield his healing nose. With the black mask? 1-6 shooting. Clear mask? 7-20. Percentage wise, the clear one won out even though the black one kept Kobe from sweating and agitating him. While Kobe obviously used the Kobe System to trick the Lakers head trainer into thinking he wouldn't look foolish last night in Detroit, the Pistons got the last laugh in giving the Lakers yet another road loss in OT, 88-85. Let it be a lesson to Kobe, don't go to a dangerous place and either show up like a crime fighter or wanting to shoot another remix to "Hotel". Rodney Stuckey Scores 34 As Pistons Top Lakers In Overtime | AP Share this:TweetShare on Tumblr


text:
When Capetonian Andrew Patterson was retrenched it seemed his world had come tumbling down around him, but instead of curling up in a heap of despair, he walked up Table Mountain every day for a year to raise money for charity.
Speaking after taking some time out after his final walk on Dec 31, Patterson told News24 that when he heard the axe was going to fall at his job at a big beverage company, he was terrified.
"It was nerve wracking. It was the worst time of my life," said Patterson.
Deep down he knew he was not getting anymore satisfaction out of the corporate world, but he was stuck in a cycle of just working to keep his salary.
To fill his soul he had started volunteering for charities, and had spent the previous three years leading small groups up the mountain in his spare time.
Stuck in traffic near the Good Hope Centre, with eight days left to final severance in 2017, he had an epiphany.
He decided he was going to walk up Table Mountain every day, and he was going to get people to sponsor him for charity while he did it.


text:
Q: Can Monthly Operating Reports (MORs) be uploaded to CMDP?
A: All structured operational data required per the federal rules are covered in the Operational Samples Category of CMDP: CFE, IFE, EPRD, DSRD, TTHM/HAA5 summary, LCR WQP, Chlorine Dioxide/Chlorite, TOC. Please note that, as shown in the CMDP User Manaul, LCR, WQP, Chlorine Dioxide/Chlorite, Bromate and TOC are not yet mapped to SDWIS/State, but many of these can be reported using Chem/Rad screens as a workaround.
CMDP Operational Data Template
Primacy agencies that have additional reporting requirements for operational sample types could use the CMDP to report Federal requirements and to have State requirements added as an attachment to the CMDP Sample Job.


text:
Development update no. 5
Development update no. 5 - Global : Surprise! We're early with our update this week! We have made a number of bug fixes live on the iSpot website this week. Here's a roundup of what's changed… Bug fixing progress
Surprise! We're early with our update this week!
We have made a number of bug fixes live on the iSpot website this week. Here's a roundup of what's changed…
Bug fixing progress
- Duplicate observations being posted – We've added a "Please wait" spinner to the add/ edit observation wizard. This disables buttons on the form while slow operations like uploading of photos occur. We hope this will reduce or eliminate the incidence of duplicate observations. This is a situation where we're eliminating a probable cause of the bug (duplicate observations), which may reveal further issues, in which case we will investigate the issues and provide further fixes.
We've tested the spinner across a range of devices. In the Chrome browser on iOS (iPads, iPhones), there is an issue where the spinner does not appear but the text "Please wait" does. All other device/ browser combinations appear to work as expected. (fix now live)
- We have fixed the peculiar "1970″ date that was appearing if the first photograph used in the add observation wizard contained no EXIF data (https://en.wikipedia.org/wiki/Exchangeable_image_file_format). For those of you who are interested you can read https://en.wikipedia.org/wiki/Unix_time to find out why the erroneous date was the 1st January 1970! (fix now live)
- There was another date-related bug, namely dates "disappearing" when you went back to edit an observation or project. This was a puzzler, however in the end the fix involved updating a third party module. (fix now live)
- Some strange text was appearing at the bottom of the add interaction wizard – on the location step. This turned out to be "debug" text (stuff added by developers to help them solve a problem). It took a little while to find where the text was being introduced, then remove it. We know the interaction functionality isn't working properly, and we're looking at this issue now. (fix now live)
- We've reduced the size of pages sent to browser by stripping out non-essential HTML code/comments. (fix now live)
- Southern Africa localities are not working post-migration - We've worked out the cause of the "missing" location title auto-complete options for our southern African cousins. The problem stems from significant differences between the global and old ZA iSpot sites. There are data and even tables that aren't present in the legacy ZA site, because it only contained one community and species dictionary. We think we'll need significant down-time to fix this issue, having tested a fix which took 4 hours to run, so we're discussing how best to tackle this whilst minimising disruption.
What will happen next week
- MySQL 5.7 migration work will be picked up again, hopefully we're not too far off completing this work. We anticipate this change will make a significant difference to the speed of the site: it will provide us with a critical feature called "row-level locking" (in place of current table-level locking), which will remove a massive bottle-neck and improve performance. The way the current incarnation works is, any time any user carries out any interaction with the site, Drupal creates a "write" request to the database (even though no writing is required!!), and our MariaSQL database then locks an entire table until the request has been dealt with. The slow performance you're seeing is iSpot waiting for a queue of requests to clear before it gets to your one.
The challenge for us making this change of database engine is to ensure we maintain the geo-spatial database capabilities that iSpot requires, whilst making the most of the feature improvements.
- Investigate why Southern Africa localities are not working post-migration (in progress)
- Fix "add interaction" functionality (in progress)
- Begin testing implementation of OU network performance enhancements through central IT department
Investigate and fix why indexes for projects are not refreshing (in progress)
We hope that these fixes help you enjoy iSpot and reduce the frustrations. Thank you for your patience, and your apparently inexhaustible enthusiasm!
19 Feb 2015


text:
(Borrowed from Group) Hey bookworms! I decided to get back in the blogging swing this week with Top 5 Wednesday post. Honestly with the stress of life and job hunting, what better way to get my mind off of things … Continue reading
Tag Archives: NA Books
My 24 Hour Readathon TBR: AKA That Time of Year Where I Go Crazy
Hey Bookworms! Guess What…? Yep… I've decided to lose my mental state even more and join the Dewey's 24 Hour Readathon! For those of you that don't know what this readathon is: It is exactly what the title implies. It … Continue reading


text:
Anna Maria Island has a lot of little old houses with little old windows. I live in one of them. The windows are the old jalousie style, which crank open like blinds, in horizontal sections. The seals on the metal frames around these old windows are poor. The fit of the window sections into the frame is not always tight. In one case, I've even used tape to cover a gap between the window and window frame. The glass is thin and weak. It's time for new windows!
Ideally, my new windows would meet the standards of the Miami-Dade building code, which is the gold standard for all windows in hurricane territory. They would pass the large missile test, namely, they would not shatter if a six-foot-long two-by-four hit the window at 34 miles per hour. And they would pass the small missile test, enduring ten ball bearings hitting the window repeatedly at 50 miles an hour.


text:
- Yazdır
- 07 Haz 2016
- empelhof-Schöneberg Belediye Başkanı Angelika Schöttler
- 1663 Okunma
- 26 Yorum
Opt for the truly useful options the moment selecting and start this sort of replica baggage. You should be great to make sure you assess just for superior quality and therefore the type for the Replica Handbags prior to when buying these consumers. Still, any time you're looking for [url=http://www.replicas-rolex.biz]Rolex Replicas[/url] for this states history the software for the variance arent which usually exceptional that's why could quite possibly distribute as being a definite basic Replica [url=http://www.replica-rolex.co/submariner_rolex-watches.html]Replica Rolex Submariner Watches[/url] just for person whos not likely ****ply trained for [url=http://www.replica-rolex.co]Replica Rolex Watches[/url] devices. The next [url=http://www.replica-rolex.co/daytona_rolex-watches.html]Replica Rolex Daytona Watches[/url]really are mindfully put together to make sure you survive many, many years about all the [url=http://www.replicas-rolex.biz/rolex_gmt.html]Replica Rolex GMT Watches[/url] depreciation.
Oy verebilmek için lütfen üye olun ya da üye girişi yapın.


text:
TOP 10 searching results for Gucci Sunglasses Repair as seen on April 1, 2020
Gucci Sunglasses Repair - Similar Shopping Results
Gucci Sunglasses Repair Video Results
Gucci GG 3512-S Sunglasses, How to replace the Lenses
How to replace sunglass lenses in Gucci GG 3512-S Sunglasses sunglasses or any make and model of sunglass using lenses from https://www.thesunglassfix.com/manufacturer. Get replacement lenses for gucci gg 3512-s sunglasses here https://www.thesunglassfix.com/index.php?route=product/search&search=...
- Likes: 1
- View count: 1350
- Dislikes: 0
- Comments: 0
Alba's hinge repair on her Kenneth Cole Sunglasses
Watch as I fix the hinge on Alba's Kenneth Cole sunglasses. Email any questions to [email protected]
- Likes: 29
- View count: 5599
- Dislikes: 2
- Comments: 20
How To Fix Broken Glasses Arm
once you apply super glue leave it over night to dry
- Likes: 468
- View count: 145184
- Dislikes: 61
- Comments: 70
Lens Popped Out of Glasses - Repair Framefixers Long Version
This video is about Lens Popped Out of Glasses - Repair Framefixers Long Version Need your glasses repaired? I can help: please visit me at http://www.framefixers.com or call 1-866-FRAME-FX. Just send us your broken glasses. I'll fix them and get them back to you in no time: http://framefixers....
- Likes: 33
- View count: 20169
- Dislikes: 22
- Comments: 5
View Best Deals for Gucci Sunglasses Repair from Every Store at KingdomOfTheSun.net Price Comparison Engine. Shop Now Top Fashionable Gucci Sunglasses Repair with Fast & Free Delivery to U.S. of America, UK, Australia, Canada, Europe, and Worldwide in KingdomOfTheSun.net Price Comparison Engine.
KingdomOfTheSun.net Store Recommends Also Salt Lake City Creek, Mens Eye Frames and Victoria Justice Style - Shop Now For Sale Prices at Amazon, Walmart, Ebay. Enjoy your shopping for discount prices? Want to compare prices on Most Popular Sunglasses online before making a purchase? Looking for ways to save money? All of this and more is possible at KingdomOfTheSun.net - your premier source for online comparison Sunglasses shopping: compare prices on 300 000+ models from leading stores Glasses, Lookmatic, Ray-Ban, WayFair, Steven Alan , Target, Warby Parker, BestBuy, Dior, SunglassHut, SunglassesShop and other.


text:
Remarkable methodological advances in the past decade have expanded the application of liquid chromatography coupled with mass spectrometry (LC/MS) analysis of biotherapeutics. Currently, LC/MS represents a promising alternative or supplement to the traditional ligand binding assay (LBA) in the pharmacokinetic, pharmacodynamic, and toxicokinetic studies of protein drugs, owing to the rapid and cost-effective method development, high specificity and reproducibility, low sample consumption, the capacity of analyzing multiple targets in one analysis, and the fact that a validated method can be readily adapted across various matrices and species. While promising, technical challenges associated with sensitivity, sample preparation, method development, and quantitative accuracy need to be addressed to enable full utilization of LC/MS. This article introduces the rationale and technical challenges of LC/MS techniques in biotherapeutics analysis and summarizes recently developed strategies to alleviate these challenges. Applications of LC/MS techniques on quantification and characterization of antibody biotherapeutics are also discussed. We speculate that despite the highly attractive features of LC/MS, it will not fully replace traditional assays such as LBA in the foreseeable future; instead, the forthcoming trend is likely the conjunction of biochemical techniques with versatile LC/MS approaches to achieve accurate, sensitive, and unbiased characterization of biotherapeutics in highly complex pharmaceutical/biologic matrices. Such combinations will constitute powerful tools to tackle the challenges posed by the rapidly growing needs for biotherapeutics development.
Biotherapeutics, especially therapeutic monoclonal antibodies (mAb), have become one of the primary focuses for the pharmaceutic industry worldwide (van den Broek et al., 2013). Sensitive, accurate, and high-throughput analytical methods that deliver high-quality quantitative data for pharmacokinetic (PK), pharmacodynamic (PD), and toxicokinetic studies, are critically important to the development of these agents (Nowatzke et al., 2011; Geist et al., 2013b). Traditionally, ligand-binding assays (LBA), such as enzyme-linked immunosorbent assay (ELISA), are the primary means for quantification of therapeutic proteins, which are often considered to afford sufficient sensitivity and throughput for PK, PD, and toxicokinetics studies (Urva et al., 2010; Shah and Balthasar, 2014). Nevertheless, LBA methods may fall short in that they are often matrix and species dependent (e.g., methods developed in one matrix/species cannot be readily transferred to another), and the quantitative accuracy and specificity may be compromised by interferences from biomatrices, mAb modification/degradation, and anti-mAb antibody, especially when highly specific critical reagents are not available (Damen et al., 2009; Hoofnagle and Wener, 2009). Moreover, the method development is often time consuming and costly, which is particularly problematic in the phases of discovery and early development (Savoie et al., 2010).
Liquid chromatography coupled with mass spectrometry (LC/MS) has emerged as a promising alternative to LBA for quantitative characterization of biotherapeutics (Heudi et al., 2008). Since the late 1990s, LC/MS has been a powerful tool for sensitive, accurate and rapid analysis of small-molecule drugs, metabolites and biomarkers (Trufelli et al., 2011). More recently, various LC/MS techniques have been developed for the quantification of proteins of interest in complex biologic matrices (Qu et al., 2006; Pan et al., 2009). Although it is possible to quantify proteins by LC/MS on both intact-protein and proteolytic-peptide levels (Kippen et al., 1997; Pan et al., 2009; Duan et al., 2012a; Rauh, 2012; van den Broek et al., 2013), the vast majority of LC/MS-based protein quantifications are performed at peptide levels for several important reasons. First, the sensitivity of MS is far superior at the peptide level than at the protein level (Blackburn, 2013); second, in a biologic system, intact proteins often carry a cohort of posttranslational modifications (PTM), which shift the masses of the proteins and introduce considerable analytical variability; per contra, when protein is being quantified at the peptide level, the quantification is usually based on the selected peptide domains where modifications are not likely to occur, and thus ensuring high reliability and reproducibility (Hopfgartner et al., 2013); third, the upper m/z limits of most MS analyzers are often too low to analyze the multiply charged precursor ions of a relatively large protein such as a therapeutic mAb, whereas the m/z of most peptide precursors can be readily detected by almost all MS analyzers (Blackburn, 2013).
For protein quantification at peptide level, selected-reaction monitoring (SRM) operated on a triple-quadrupole MS is by far the most commonly used technique. Briefly, the first quadrupole analyzer selects a specific peptide precursor ion from the complex matrix, which is then fragmented in a downstream fragmentation chamber filled with collision gas; the second quadrupole analyzer then monitors a specific fragment from the target peptide (Qu and Straubinger, 2005). Compared with other tandem MS techniques, SRM-MS exhibits higher sensitivity, better quantitative accuracy, and a wider dynamic range for targeted protein quantification, and can be easily multiplexed (i.e., quantification of multiple analytes in one LC/MS analysis) by quickly switching among different precursor/product transitions (Qu and Straubinger, 2005). When the excellent specificity of SRM is combined with sufficient LC separation, the LC/SRM-MS constitutes a versatile and powerful tool for the quantification of proteins in complex matrices. A typical procedure for LC/SRM-MS-based quantification includes sample treatment/cleanup, digestion using enzymes, and quantification of the target proteins based on selected signature peptides (SP) derived from the target. Stable isotope labeled (SIL) SP surrogate or SIL full-length-protein is used as the internal standard (IS). Extensive reviews on this technique can be found in Lange et al. (2008) and Liebler and Zimmerman (2013).
LC/SRM-MS has several attractive features over LBA for analysis of biotherapeutics, including fast-method development and validation, high specificity, and small sample consumption per analysis (Jemal and Xia, 2006; Fernandez Ocana et al., 2012; Liu et al., 2013; van den Broek et al., 2013). Furthermore, many proteins can be simultaneously quantified in one LC/SRM-MS analysis (Xiao et al., 2014), and a method developed in one matrix or species can often be readily transferred to another (Savoie et al., 2010; Pendley and Shankar, 2011; Bronsema et al., 2012; Duan et al., 2012a; Li et al., 2012; Geist et al., 2013b; Jiang et al., 2013). Finally, LC/MS techniques can be employed to obtain critical information on the molecules of biotherapeutics that pose a daunting challenge for LBA, such as chemical degradation (e.g., oxidation or deamidation of residues) (Huang et al., 2005).
With an emphasis on the LC/SRM-MS technique, this article will compare LC/MS techniques versus LBA for quantification of antibody biotherapeutics, and then discuss the technical challenges of LC/SRM-MS and emerging approaches to alleviate these challenges. Finally, recent applications of LC/MS techniques to the analysis of biotherapeutics are summarized.
Comparison of LC/SRM-MS versus LBA
For quantification of biotherapeutics, the sensitivity, specificity, and robustness achievable by LBA are heavily dependent on the quality of critical reagents (often antibodies) raised against specific epitopes of the target proteins (Lee and Kelley, 2011; O'Hara et al., 2012). By comparison, usually the LC/SRM-MS method does not require target-specific critical reagents, and thus may provide the following prominent advantages over LBA.
First, the process to develop a LBA method for quantification of therapeutic proteins in complex pharmaceutical matrixes (e.g., plasma and tissues) with sufficient sensitivity, selectivity, and accuracy is often both time consuming (e.g., 6 to 12 months; Savoie et al., 2010) and costly. This process typically includes the development of optimal critical reagents, the extensive examination of endogenous interferences, and rigorous method validation in samples from various sources (Nowatzke et al., 2011; Pendley and Shankar, 2011). In contrast, LC/SRM-MS-based approaches do not require critical reagents, so these methods can be developed more rapidly (e.g., in 2 to 3 weeks) at substantially lower cost (Pan et al., 2009).
Second, the critical reagents used for LBA are generally produced via biologic processes, which are inherently prone to variability arising from such factors as PTM, variations in different batches of reagents, and biologic interferences (Lee and Kelley, 2011; O'Hara et al., 2012); as LBA does not employ an internal standard to correct quantitative bias and variation, stringently controlled operations are required to prevent deterioration of assay performance (Pandya et al., 2010; Lee and Kelley, 2011). Consequently, it is often challenging to maintain high interbatch/interlaboratory consistency, which is necessary to correlate results among batches and to transfer validated methods between laboratories (Ezan and Bitsch, 2009). On the contrary, analytical variation is frequently much less a concern for LC/SRM-MS approaches because critical reagents are not required, and isotope-coded peptides or proteins are prevalently used as IS, which effectively corrects analytical variations introduced by LC/MS analysis and matrix effects (Pan et al., 2009; Bronsema et al., 2012; van den Broek et al., 2013; Nouri-Nigjeh et al., 2014). In practice, the performance of a developed and validated LC/SRM-MS method is quite robust so long as instrument maintenance and quality control are performed properly.
Third, measurement of the distribution of biotherapeutics in different matrices (e.g., plasma or tissues) and across different species is important for the development and evaluation of these agents (Lin et al., 1999; Garg and Balthasar, 2009; Deng et al., 2011; Shah and Betts, 2013) but is difficult to achieve with a single LBA method. The specificity of LBA is profoundly affected by the matrix: in different matrices or species, the extents of interferences and cross-reactions by matrix components vary considerably (Pendley and Shankar, 2011), rendering it difficult to transfer a LBA method among matrices (e.g., from plasma to a tissue or among different tissues) (Damen et al., 2009; Ezan and Bitsch, 2009; Hoofnagle and Wener, 2009). Conversely, as LC/SRM-MS minimizes the matrix effect by employing isotope-coded IS and sufficient chromatographic separation (Qu and Straubinger, 2005), the methods are often readily transferrable among different matrices. For example, recently we applied the same LC/SRM-MS method for the quantification of therapeutic mAbs in mouse plasma and tissues, such as brain, heart, liver, spleen, kidney, and lung, with rapid and simple verification and revalidation in different matrices (Duan et al., 2012a).
Fourth, although it is very challenging to develop a LBA method capable of quantifying multiple proteins in one analysis, LC/SRM-MS approaches can be multiplexed for hundreds of targets in one run (Li et al., 2012; Shi et al., 2012; Xiao et al., 2014). Such multiplexing capacity has been widely used in biomarker and proteomewide mechanism studies (Sakamoto et al., 2011; Percy et al., 2014; Xiao et al., 2014). The multiplexing method is also highly valuable for the research and development of biotherapeutics such as simultaneous quantification of an mAb and its circulating or tissue-specific targets (Li et al., 2009; Kawakami et al., 2011; Ohtsuki et al., 2012; Chambers et al., 2014) or multiple drug candidates in the same sample obtained from administration strategies such as cassette dosing (Jiang et al., 2013; Li et al., 2013). The cassette-dosing strategy, which doses multiple drug candidates together to one subject to enable rapid screening, has long been employed in PK profiling and metabolite screening for small-molecule drugs (Bayliss and Frick, 1999; Korfmacher et al., 2001). This approach also substantially reduces the time and resources required for the initial screening and development of biotherapeutics (Liu et al., 2008). A multiplexed LC/SRM-MS approach enables the simultaneous albeit specific analysis of different mAb candidates—even the sequences are only slightly different (Geist et al., 2013a; Li et al., 2013)—and thus is the method of choice for cassette-dosing studies.
Other merits of LC/SRM-MS over LBA include low sample consumption and high operational robustness (Ouyang et al., 2012; Liu et al., 2013). For example, in our recent studies, each LC/SRM-MS analysis only used peptide digests from ∼0.1 µL of plasma or ∼0.1 mg of tissue (Duan et al., 2012a,b; Nouri-Nigjeh et al., 2014), which is much lower than the sample amount required by a typical LBA method.
The main disadvantage of LC/SRM-MS compared with LBA is that an expensive MS instrument is required. Moreover, for these well-established biotherapeutics with industry-grade LBA methods that have been already been developed and validated, sensitive and high-throughput quantification can be achieved following a relatively straightforward LBA procedure (Ezan et al., 2009). Consequently, LBA remains the preferred choice for these targets (Damen et al., 2009).
Challenges of LC/SRM-MS and Strategies
Despite of the merits of LC/SRM-MS and its rapidly growing utility in the quantification of biotherapeutics, a number of significant technical challenges still exist. This section discusses the challenges and recent efforts to address them.
Method Development.
Until now, the optimal strategy for the development of an LC/SRM-MS method has remained elusive (Pan et al., 2009; Duan et al., 2012a). The key aspect for method development is the discovery of optimal SP derived from the target biotherapeutics that ensures sensitive, specific, and robust quantification. Currently, in silico prediction approaches are popularly used that employ tools such as PeptideAtlas, Skyline, and MRMaid, to identify and validate the SRM transition with minimum wet laboratory labor (Cham Mead et al., 2010; Halquist and Thomas Karnes, 2011; Stergachis et al., 2011; Rauh, 2012). Nevertheless, this approach may not accurately predict the most sensitive, stable peptides or matrix-dependent parameters such as chemical interferences (Cao et al., 2010; Duan et al., 2012a). Discovery of optimal SP by experimentally evaluating many proteolytic candidates in the target matrix (e.g., plasma or tissue digest) is the most reliable approach (Cao et al., 2010; Duan et al., 2012a); however, to experimentally evaluate these candidates, it is necessary to obtain optimal SRM conditions (e.g., the optimal parent/product transitions and the declustering/collision energy) for each of the many candidates in a digest mixture, which is challenging. Moreover, it is important to choose stable peptides as the SP (Cao et al., 2010; Duan et al., 2012a) to prevent quantitative variation and bias arising from poor peptide stability, which has been often overlooked. Finally, most methods use a lone SP for the quantification of a mAb, which may carry a significant risk of error where the mAb could be truncated biologically outside the SP domain or certain residues within the SP domain could be biologically modified (Hoofnagle and Wener, 2009).
To address these issues, we devised a new pipeline to facilitate a high-throughput and accurate method development (shown in Fig. 1). Instead of using an in silico method to predict the best SP and optimal SRM conditions, we employed an experimental strategy to discover and optimize many SP candidates, and then evaluate these candidates in target matrices prior to SP selection (Cao et al., 2010; Duan et al., 2012a,b; Nouri-Nigjeh et al., 2014). Briefly, the pool of SP candidates was generated by a data-dependent LC/MS experiment following a stringent filtering step to remove peptides that are not unique to the target, containing labile amino acid (cysteine residues were not excluded as a number of studies showed cysteine-containing peptides may be used in a reliable quantification) (Keshishian et al., 2007; Picotti et al., 2009), known modification or miss cleavage. To evaluate these candidates, the target protein was spiked into the blank matrices (e.g., plasma or tissue extract) and then prepared and digested. The optimal LC/MS conditions of all SP candidates were accurately obtained by a high-throughput and on-the-fly orthogonal array optimization (OAO) procedure (Cao et al., 2010; Duan et al., 2012a,b), which has the capacity to develop the SRM conditions for >100 candidates within one single LC/MS run with high accuracy and reproducibility. Using the developed LC/MS conditions, all candidates were thoroughly assessed for stability and signal-to-noise ratios (S/N) in the matrix digest. Among the stable peptides, two peptides with the highest S/N were selected as the SPs. The use of two SP from different domains of the same protein provides a versatile gauge for the reliability of quantitative methods and results. Details can be found in the following references (Cao et al., 2010; Duan et al., 2012a,b).
Recently, Furlong et al. (2012) described the use of a "universal surrogate peptide" derived from the constant Fc region of human antibody for quantification of human antibodies in nonhuman animal models. This method may greatly simplify and expedite the method development for the study of human antibodies in preclinical animal models.
Sample Preparation.
To achieve a sensitive and accurate quantification with LC/SRM-MS, it is critically important to achieve efficient and reproducible sample preparation, including effective sample cleanup, high and quantitative recovery of protein and efficient, and reproducible peptide recovery (Qu et al., 2006; Pan et al., 2009). So far, a universal and optimal preparation procedure for the quantification of biotherapeutics in pharmaceutic matrices has yet to be established, largely because the tissue and plasma samples are highly complex with numerous proteins and small-molecule compounds and the structure of a typical mAb renders it resistant to enzymatic digestion (Ouyang et al., 2012; Yuan et al., 2012). Moreover, it is challenging to prepare tissue samples for quantification of biotherapeutics, owing to the generally low drug concentrations in tissues and the lack of a quantitative and high-throughput protein extraction procedure that is compatible with LC/SRM-MS analysis (Duan et al., 2012a).
Recently, we developed a gel- and filter-free procedure that achieved effective protein denaturation and sample cleanup, and high, quantitative peptide yields from plasma and tissue samples (Duan et al., 2009, 2012a). Briefly, plasma or tissue samples were treated or extracted with high concentrations of detergent cocktail, which not only effectively solubilized proteins in the samples to ensure a high recovery but also completely denatured the proteins to achieve an efficient reduction, alkylation, and digestion (Duan et al., 2009; Tu et al., 2013); the mixture was then cleaned up by precipitation with cold organic solvents, which effectively removed the detergents and significantly reduced nonprotein matrix components such as lipids and fragmented or small-molecule nucleic acids that may negatively affect the robustness and consistency of LC/SRM-MS analysis (Qu et al., 2014). After precipitation, an on-pellet-digestion approach was employed without dissolving the protein pellet. This approach consists of two phases: under active agitation, the short phase-I digestion brings the pellets into solution by cleaving the pelleted proteins into soluble albeit large tryptic peptides; these incompletely cleaved peptides were then subjected to an overnight phase-II digestion for complete cleavage. Compared with other preparation methods used for LC/SRM-MS-based protein quantification, the detergent-aid precipitation/on-pellet-digestion provided higher digestion efficiency and much cleaner sample than in-solution digestion (Duan et al., 2009; Cao et al., 2010; Yuan et al., 2012) while affording higher and more reproducible peptide yields than in-gel digestion (Olsen et al., 2006) and filter-aided sample preparation (FASP) methods (Manza et al., 2005; Wisniewski et al., 2009). Thus, this procedure can be used for high-throughput quantification of mAb in plasma and tissues with excellent analytical sensitivity and robustness.
Recently, a number of techniques were developed to enable rapid digestion of proteins, such as digestion assisted by microwave (Lesur et al., 2010), ultrasound (Priego-Capote and de Castro, 2007), and infrared radiation (Wang et al., 2008a), and accelerated digestion with immobilized trypsin (Krenkova et al., 2009; Yamaguchi et al., 2009; Yuan et al., 2009). Some products using immobilized trypsin have been commercially available, such as Perfinity Flash Digest Kit (Perfinity Biosciences, West Lafayette , IN) for rapid and efficient digestion (Rivera-Burgos and Regnier, 2012). The performance of these newly emerged techniques for quantification of biotherapeutics remains to be extensively evaluated.
Although LC/SRM-MS is considered a highly sensitive technique, insufficient sensitivity is often a prominent concern for quantification of therapeutic mAb, largely for two reasons: 1) the signal response of LC/MS depends on the molar rather than mass amounts of the analyte, so the large molecular weights of mAb pose a considerable disadvantage; 2) owing to the very high protein contents in plasma or tissue samples (Tu et al., 2011), it is often necessary to dilute the samples to a large extent before analysis (Dams et al., 2003; Chambers et al., 2007; Cao et al., 2010; Duan et al., 2012b; Yuan et al., 2012).
To improve sensitivity for targeted protein analysis, we have developed a robust nano-flow LC/SRM-MS strategy (Cao et al., 2010; Duan et al., 2012a,b) that typically lowers the limit of quantification (LOQ) by ∼30- to 50-fold compared with a conventional-flow LC/SRM-MS. Another approach to improve sensitivity is to enrich target proteins or peptides before LC/SRM-MS analysis. For instance, Dubois et al. (2008) achieved a LOQ at 0.02 µg/ml for quantification of a chimeric mAb in human serum samples with an enrichment procedure; Lin et al. (2013) used immunoprecipitation enrichment before LC/SRM-MS analysis to achieve a LOQ of 10 ng/ml for mAb analysis.
A variety of other techniques have been developed to increase the sensitivity for LC/SRM-MS-based protein quantification, although these have yet been applied in quantification of biotherapeutics. To give several examples, the stable isotope standards and capture by antipeptide antibodies (SISCAPA) technique was developed to enrich signature peptides using polyclonal antibodies (Anderson et al., 2004). More recently, Neubert et al. developed a series of affinity-based methods for quantitative enrichment of target proteins and/or SPs in plasma, achieving ultrasensitive quantification of circulating biomarkers in plasma (Ocaña and Neubert, 2010; Neubert et al., 2013; Palandra et al., 2013). Furthermore, except to increase sensitivity, the affinity capture–based method can quantify specific targets, such as free or total mAb (Fernandez Ocana et al., 2012), which will be reviewed in the application section. Other approaches to improve the sensitivity of targeted quantification include strong cation exchange fractionation (Keshishian et al., 2009), high-pH fractionation before LC/MS analysis (Shi et al., 2012), and the use of long columns to obtain high S/N of target peptides (Shi et al., 2013).
Quantitative Accuracy.
The correct measurement of drug concentrations in plasma and tissues is essential for the research and development of biotherapeutics; consequently, highly accurate quantification methods are important (Wang et al., 2008b). SIL-IS is prevalently used for LC/SRM-MS quantification of therapeutic proteins, which greatly enhances the analytical reproducibility (Pan et al., 2009; Li et al., 2012; Nouri-Nigjeh et al., 2014). However, insufficient quantitative accuracy frequently represents a daunting problem. Most current works on LC/MS-based protein quantification employ synthesized peptides as the calibrator and SIL peptides as IS (spiked after digestion) (Bronsema et al., 2012; van den Broek et al., 2013). Such peptide-level calibration approaches enable straightforward development of quantitative methods, and both the calibrators and SIL-IS are readily available from commercial sources. However, the use of a SIL peptide IS only addresses variations/biases caused by LC/MS analysis but not the upstream steps such as sample preparation and digestion (van den Broek et al., 2013); furthermore, these approaches assume nearly 100% efficiency of the preparation and digestion procedures, which may not be true (Cao et al., 2010); for example, tryptic digestion is rarely complete and can be partially nonspecific (Picotti et al., 2007). Our recent investigations showed severe negative biases by the peptide calibration approaches (Duan et al., 2012b; Nouri-Nigjeh et al., 2014), and the quantification using two different SP from distinct domains of the same mAb resulted in profoundly discordant quantitative results.
To address problems related to digestion efficiency, the extended-peptide calibration approaches, which use synthesized extended-peptide containing the SP sequence and (typically) three to six flanking residues extended from both the N- and C- termini, were introduced. A SIL extended-peptide is used as the IS, which is spiked before digestion (Ocaña and Neubert, 2010; Rauh, 2012; Neubert et al., 2013). This approach may help to compensate for the bias and variation introduced in the digestion step (e.g., missed cleavage or peptide degradation; Ocaña and Neubert, 2010). Neubert et al. recently demonstrated that the extended-peptide calibration approach enabled accurate and sensitive quantification of a small protein biomarkers (e.g., nerve growth factors) in plasma (Neubert et al., 2013; Palandra et al., 2013). Our recent study showed that the extended-peptide calibration method still resulted in considerable negative bias when quantifying a much larger protein (mAb) (Nouri-Nigjeh et al., 2014).
Protein-level calibration methods that employ full-length protein calibrator with SIL protein IS can correct errors and variations in all preparation and analytical steps, so they are considered the gold standard for accurate quantification (Heudi et al., 2008; Li et al., 2012). However, SIL proteins are costly to produce and may be impractical for many classes of proteins. Our laboratory and others demonstrated accurate quantification of regulatory proteins and protein drugs in plasma and tissues using "hybrid" calibration strategies (e.g., protein calibrator with SIL peptide or SIL extended peptide IS) (Cao et al., 2010; Duan et al., 2012a,b; Jiang et al., 2013), provided that reproducible sample preparation and digestion are achieved. Our recent study suggested the hybrid strategies may provide a cost-effective means for accurate quantification without the costly SIL protein (Nouri-Nigjeh et al., 2014). The quantitative biases by protein, extended-peptide, and peptide level calibrations and hybrid methods for the quantification of the same mAb in plasma are shown in Fig. 2.
Applications of LC/MS in the Analysis of Biotherapeutics
Quantification of mAb in Plasma and Tissues.
LC/SRM-MS-based strategies have been widely applied to the quantification of mAb in plasma to support PK studies. Some representative works are exemplified here. Heudi et al. (2008) developed and validated an accurate quantitative method for a candidate mAb in marmoset serum by use of postdigestion Solid Phase Extraction (SPE) cleanup and SIL full-length-protein as IS. The method was applied to PK analysis in marmosets at a dosing level of 150 mg/kg. It was discovered that the concentrations by LC/SRM-MS were higher than those obtained via a parallel ELISA assay, which might reflect the fact that ELISA measures the free form of mAb whereas LC/SRM-MS measures the total mAb (Heudi et al., 2008). Li et al. (2012) developed a universal LC-SRM/MS approach for quantification of a variety of therapeutic mAb based on the use of a full-length SIL mAb as the common IS; such a method is valuable for preclinical studies.
Hagman et al. (2008) developed an LC/SRM-MS method to quantify a human mAb in the serum of cynomolgus monkeys. The study showed that the analytical sensitivity was significantly increased by an albumin-depletion procedure before LC/MS analysis. Ouyang et al. (2012) described the combination of on-pellet digestion with LC/SRM-MS for reproducible analysis of a mAb drug candidate in monkey plasma. Through an extensive comparison with other preparation methods, the investigators showed that on-pellet digestion was the optimal technique as it permitted a straightforward and efficient preparation of mAb.
Yang et al. (2007) established a quantification method for somatropin and a therapeutic human mAb in human and rat serum samples that employed a solid-phase extraction for cleanup and bovine fetuin as IS; the method was applied in a rat PK study at a dosage of 10 mg/kg. Lu et al. (2009) employed albumin depletion, protein A capture, and antibody capture coupled with LC/SRM-MS for sensitive quantification of a mAb candidate (CNTO736); the investigators concluded that all LC/SRM-MS-based methods evaluated in the study provided adequate sensitivity for PK study.
Fernandez Ocana et al. (2012) established a LC/MS strategy to quantify free and total anti-MadCAM mAb (PF-00547,659) in human serum, which captured free mAb with a biotinylated anti-idiotypic antibody followed by enrichment with streptavidin magnetic beads; the total target mAb was enriched by protein G magnetic beads. The strategy was successfully applied in a clinical PK study.
Our laboratory described a sensitive nano-LC/SRM-MS method for quantification of a chimeric mAb (cT84.66) in mouse serum (Duan et al., 2012b). Owing to the high sensitivity and selectivity achieved, the method was successfully applied to the preclinical PK study with a subcutaneous dosing at 1 mg/kg. The high sensitivity achieved in the work made it feasible for PK study at even lower dosages and/or over a longer period after administration.
Although the determination of the levels of biotherapeutics in tissues is critical for PK studies, such works have rarely been reported due to technical challenges such as the low drug concentrations and problems associated with tissue matrices, as discussed earlier. Using a sensitive nano-LC/SRM-MS, effective sample preparation, and a high-throughput method optimization strategy, we described sensitive quantification of two mAb (8c2 and cT84.66) in seven tissues with lower LOQ in the range of 0.156∼0.312 µg/g tissue (Duan et al., 2012a). The method was applied to the investigation of steady-state tissue distributions of 8c2 in various mouse models. Similar distribution characteristics were observed among the wild-type animals and those deficient in FcγRIIb and FcγRI/RIII; by comparison, the 8c2 tissue levels in the FcRn α-chain deficient group were significantly lower, as expected due to the absence of FcRn-mediated protection of antibody from catabolism. The work demonstrated that LC/SRM-MS is a promising alternative to radiolabeling strategies, which may fall short in problems related to assay accuracy and specificity, degradation of labeled protein, and radiation exposure to investigators and animals (Duan et al., 2012a).
Application in Cassette-Dosing.
As discussed previously, LC/SRM-MS is capable of simultaneous quantification of multiple targets, and therefore enabling cassette-dosing study of drug candidates. Though cassette-dosing has been prevalently used to screen small molecule candidates (White and Manitpisitkul, 2001; Smith et al., 2007), it was found to be even more suitable for preliminary investigation of mAb candidates (Li et al., 2013), because 1) studies of multiple mAb usually do not carry the risk of drug-drug interaction, and the PK of proteins is not affected by CYP450 and transporters (Zhou and Mascelli, 2011; Li et al., 2013), and 2) it is fairly straightforward to find an optimal, common formulation for multiple mAb (Dani et al., 2007; Spencer et al., 2012). Jiang et al. (2013) developed and validated an LC/SRM-MS method for simultaneous quantitation of two coadministrated mAb, which showed good sensitivity, reproducibility and accuracy for both targets. The method was successfully applied to toxicokinetic study in monkeys. Li et al. (2013) reported an analytical method to quantify four mAb after subcutaneous cassette-administration with LOQ of 0.1∼0.5 µg/ml in plasma.
Characterization of Intact mAb.
The recent technical advances and increasing availability of high-resolution MS instruments have resulted in rapid growth in using LC/MS to characterize intact mAb. Although most of these studies do not directly quantify antibodies, such works greatly facilitate assay development and/or PK/PD studies by affording detailed physicochemical information on these agents. In most cases, a top-down strategy employs a high-resolution analyzer to directly analyze intact proteins. Despite the top-down techniques generally being less sensitive than the bottom-up methods, they can provide overall information and accurate PTM mapping of the target protein (Peng et al., 2013).
One important paradigm is the characterization of the critical reagent for LBA (Geist et al., 2013b). Such studies can substantially improve the management of critical reagents and contribute to the development of a robust LBA method by providing essential information such as Fab/Fc sequence, enzymatically produced PTM, and charge state. Details on the use of LC/MS approaches for troubleshooting LBA methods and critical reagent quality control can be found in a previous publication (Geist et al., 2013b).
Another prominent utility of top-down technique is the quality control of mAb products. Thompson et al. (2014) performed a top-down profiling of glycosylation on mAb. Wang et al. (2005) established a series of top-down and bottom-up electrospray ionization time-of-flight MS (ESI-TOF-MS) methods to comprehensively investigate N-terminal pyroglutamate formation, cleavage of C-terminal lysine, glucosylation, and deamidation of a recombinant mAb. Dillon et al. (2006) developed a LC/MS method with a TOF analyzer to obtain accurate mass and unique terminal ladder sequences of a recombinant antibody, which provided the glycosylation profile and heterogeneity information of the molecule. In another study, a LC-ESI-TOF method was developed to determine glycosylation pattern of a therapeutic mAb (siltuximab) after immunoaffinity purification (Geist et al., 2013a). The work quantified therapeutic mAb produced in two different host cell lines. Xie et al. (2010) developed a LC/TOF-MS method for comprehensive comparison between innovator mAb versus a biosimilar, in terms of intact protein mass, sequence, and peptide mapping, which provided valuable information for assessment of biosimilars.
Characterization of Antibody-Drug Conjugates (ADC).
Antibody-drug conjugates (ADC), which use mAb for targeted delivery of highly potent small-molecule drugs, constitute a new class of biotherapeutics for effective targeted therapy (Doronina et al., 2003). Drug-to-antibody ratios (DAR) and drug-load distribution are critical parameters that profoundly determine the in vivo efficacy and toxicity of an ADC (Ducry, 2013; Kaur et al., 2013). For example, even a small decrease in DAR (e.g., loss of conjugated drug in circulation) will lead to a significant change of exposure at the targeted site (Xu et al., 2011); thus, determination of DAR is essential for ADC development and PK/PD studies.
LC/MS with a high-resolution analyzer has become a promising technology for characterization of ADC in recent years. An affinity capture capillary LC coupled to quadrupole-TOF MS has been employed to analyze anti-MUC16 THIOMAB–drug conjugate (TDC), which obtained both in vitro and in vivo DAR information (Xu et al., 2011). Valliere-Douglass et al. (2012) developed a native LC/MS method for the determination of DAR via analysis of intact protein.
Recently, a more sensitive method was reported by Chen et al. (2013), which employed a native nano-ESI-TOF analysis in conjunction with limited digestion by cysteine protease to obtain DAR information. This method also demonstrated substantially higher sensitivity than the traditional hydrophobic interaction chromatography.
Conclusion and Future Perspectives
LC/MS represents a promising alternative to traditional LBA methods for the analysis of biotherapeutics because 1) it can be readily adapted to quantification in plasma and tissues and across various species; 2) it provides extraordinary specificity and high reproducibility with low sample consumption and low interlaboratory/batch variance; and 3) it is capable of simultaneous quantification of multiple proteins (e.g., biotherapeutics and/or their targets) in one analysis. Furthermore, the development and validation of a LC/MS method is rapid at a relatively low cost, which is a highly desirable feature that facilitates the rapidly growing developments of biotherapeutics. Nonetheless, LC/MS methods still face challenges associated with sensitivity, sample preparation, method development, and quantitative accuracy. Recent technical advances helped to overcome these problems; to name a few, the use of target-specific enrichment and low-flow-LC/MS have substantially improved analytical sensitivity; robust and efficient preparation methods are emerging; and hybrid calibration methods have been demonstrated to provide high quantitative accuracy without using expensive SIL protein IS.
In spite of the drastically increasing role of LC/MS in biotherapetuics analysis, most likely it will not fully replace traditional methods such as LBA in the foreseeable future. First, in the event that a well-developed LBA method is available for the target molecule, LBA may be advantageous over LC/MS because it may be more sensitive (de Dios et al., 2013) and does not require expensive LC/MS instruments; moreover, LBA can be developed to quantify free or total mAb whereas LC/MS only detects the total mAb unless specific affinity capture enrichment is employed (Lee et al., 2011). Second, the combination of LC/MS and traditional biochemical strategies provides a powerful tool to acquire critical information on biotherapeutics in highly complex pharmaceutic and biologic systems. For instance, immunocapture and immunodepletion have been demonstrated to greatly enhance the quantitative sensitivity of LC/MS; size-exclusion, immunoprecipitation, or native gel separation before LC/MS analysis will provide important information on the target protein, such as binding, aggregation, and degradation states.
In a broad sense, antibody capture of a target molecule from a biologic matrix followed by LC/MS analysis may be considered a new form of LBA method, with LC/MS as the means of detection. As LC/MS affords much higher selectivity and sensitivity than the spectrophotometers conventionally used for LBA, it will greatly facilitate and expedite methods development; for example, development of critical reagents may be markedly faster because the requirements for selectivity and sensitivity of the critical reagents are far less stringent when LC/MS is used as the downstream detection approach.
In summary, LC/MS is a versatile and powerful tool for analysis of biotherapeutics. It can be used either alone or as the detector downstream of various biochemical procedures. We expect its application in the research and development of biotherapeutics to continue to expand rapidly in the future.
Authorship Contributions
Wrote or contributed to the writing:` An, Zhang, Qu.
- Received May 3, 2014.
- Accepted August 28, 2014.
This work was supported, in part, by National Institutes of Health Eunice Kennedy Shriver National Institute of Child Health and Human Development [Grants U54 HD071594, HD075363], National Institute on Drug Abuse [Grant DA027528], National Institute of Allergy and Infectious Diseases [Grant AI060260], and National Heart, Lung, and Blood Institute [Grant HL103411], by the Center for Protein Therapeutics, and by American Heart Association (AHA) award [12SDG9450036] (all to J.Q.).
- antibody-drug conjugates
- drug-to-antibody ratio
- enzyme-linked immunosorbent assay
- electrospray ionization
- internal standards
- ligand-binding assay
- liquid chromatograph
- limit of quantification
- mAb
- monoclonal antibodies
- mass spectrometry
- pharmacodynamic
- pharmacokinetics
- posttranslational modifications
- stable isotope labeled
- signal-to-noise ratios
- signature peptide
- selected-reaction monitoring
- time-of-flight
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics


text:
Original article can be found here
Watch it on Youtube: Buy Hatch Baby Rest Review
FBA vs Retail Arbitrage vs Private Label Vs Wholesale:
Amazon FBA is dead:
Video: amazon fba
Lonely Planet's top budget choice for Cancun in '10, '11, '13, and 2014! **
We are an awesome Hostel! You won't regret making us your Home Away from Home while in Cancun!
At Hostel Ka'beh we believe in slow travel. You're on vacation! Why should we wake you & take your bed away if it's not necessary? Why put a restriction on your breakfast time? Why should you stress about having to adapt to us? Relax! You're at home. Your home away from home!
(PLEASE NOTE: If you want to stay for just one night, just drop by. We'll definitely find a way to accommodate you.)
Positive Approach
Milton Keynes Hospital Charity
Cambridge Weight Plan
Setfords Solicitors
Director and Solicitor at Purcell Solicitors
Neves Solicitors LLP
Avant Garde Corporate Gifts & Promotional Items
Sarah-Jane Smyth Jewellery
Woodfines Solicitors
Travel Treats
Yellow Yoyo
Flowers 2 Impress
Willen Hospice
Independent Financial Adviser, Odyssey Wealth
Headspace for Business
Sue Malleson
MK Community Foundation
MK Box Self Storage
Hewitsons LLP
Lesley Arkin Optometrists
Watch it on Youtube: Market My Business Online Milton Keynes Market My Business Online Milton Keynes
Your email address will not be published. Required fields are marked *
Watch it on Youtube: Bloggii Bonus


text:
Price: $59.99
Quantity: 1 available
Item/Description: Millefiori (thousand flowers) design and playing cards suits silhouettes art glass paperweight.
In addition to the marvelous millefiori pattern, interspersed throughout that pattern are three of the four playing cards suits: hearts, clubs, and spades only (NO diamonds), as well as stars, flowers and other symbols in scrumptious, rainbow hues of ruby red, lemon yellow, emerald green, grassy green, sapphire cobalt blue, sky blue, pumpkin orange, snowy white, and jet black.
Not only is this exquisite work of glass art desirable as an addition to the collection of any discerning paperweight aficionado, but for anyone whose hobbies or avocation includes playing cards, this would make a quite apropos gift, given its charming "playing cards suits" motif.
***You may view additional &/or enlarged photos of this item in our Etsy shop: https://www.etsy.com/shop/VintageTreasuresAZ?ref=shop_sugg
Age/Period: Vintage - ca pre-2000s (ca 1990s)
Manufacturer/Identification/Markings/Country of Origin: Unknown/Unspecified
Material(s)/Format: Glass
Size/Measurements: Approximately 3" H x 3" in Diameter
Quantity: One (1)
Condition: Colors remain exquisitely vibrant. There is one tiny air bubble noted. There is also a tiny pin prick in the glass.
***Please note that what appears to be "spots" on the paperweight are merely nothing more than camera glare reflections!
***COMBINED SHIPPING is always available for multiple purchases when the stated "Shipping Included" method is the same for each item purchased AND each item can be safely packaged together. Otherwise, multiple purchases without the same shipping method listed AND which require separate packaging & shipping will be shipped separately at NO ADDITIONAL CHARGE TO YOU (to ensure the greatest possible degree of protection for each item during transit).
Additionally, because the shipping methods that are included within each listing do vary from item to item, it is important to be aware that delivery transit times will likewise vary per item (kindly refer to "Store Policies" for detailed information regarding all shipping methods & delivery transit times applicable to each respective method).
Thus, should you desire a more expedient shipping method than that which is already included for any particular item, upon checkout simply select the expedited shipping option that best suits your needs (Priority Mail, Priority Mail Express, Federal Express)!
Title: Millefiori (Thousand Flowers) Motif and Playing Cards Suits (Hearts, Clubs, Spades) and Other Designs/Symbols Glass Paperweight *****USPS PRIORITY MAIL SHIPPING INCLUDED – DOMESTIC ORDERS ONLY!*****
Categories: Paperweights ,
lbs: 2.00 1 lbs
Seller ID: Playing Cards Suits Paperweight 1 of 2
Keywords: Art and Collectibles; Art Glass Paperweights; Boss's Day Gifts; Clubs; Collectibles; Diamonds; Father's Day Gifts; Hearts; Home and Living; Home Decor; Millefiori Paperweights; Office Collectibles; Office Decor; Paperweights; Paper Weights; Playing Cards Suits; Spades; Stars; Vintage Millefiori; Vintage Paperweights; Vintage Paper Weights; Vintage Treasures AZ; VintageTreasuresAZ,


text:
Nudges to the Finish Line
Experimental Interventions to Prevent College Late Departure
Recent evidence suggests that many college students who withdraw from their institution complete most of the credits they need to graduate before dropping out.
Complex information about course sequences that lead to a degree and limited access to advising may contribute to this phenomenon. The purpose of our proposal is to investigate whether low-cost, behaviorally-informed nudges that provide personalized information to students about how they can finish their degree improve college completion rates at open-enrollment institutions.
Our partnerships include the following postsecondary systems and universities: City University of New York, with almost 270,000 degree-seeking students; Texas Higher Education Coordinating Board, with 1.5 million students statewide; Virginia Community College System, with over 250,000 students enrolled each year; Ohio University, with nearly 40,000 students; and University of Washington–Tacoma, with almost 5,000 students. These partners represent a range of institution types, student groups, and geographic contexts in which we will conduct this study.
In the Development phase of the project we will target actively or recently enrolled students at broad access two- and four-year institutions who have completed at least half of the credits typically required for graduation at a 2- or 4-year college, respectively. In the Efficacy phase, we will target students who exhibit risk factors for late departure that emerge from our analysis of the Development phase. Our samples will consist of 4,000 students at 8 institutions in the Development phase, and 25,500 students at 17 institutions in the Efficacy phase.
Students will receive personalized text messages containing information, nudges, and guidance about concrete steps they can take to earn their degrees. In some partner sites, students will also have access to dedicated advisors who will provide further customized support. The text campaign content will provide customized guidance about courses students need to complete to finish their programs; generate positive social norms around using campus-based resources, and provide planning and implementation prompts to help students plan and execute steps necessary to earn their degree.


text:
The Grey Knights of the Inquisition somehow got word that the Eremoi caste of Dark Angels has been dabbling in warp science and were dispatched to – er – dispatch them.
Tom & I got in a great game of Kill Team this evening using my new Sector Fronteris board.. He fielded his psykers and I my heavy-hitting Unforgiven. Terror Tactics was the mission.
After Tom advanced on my forward position and psybolted my heavy bolter (heavy specialist), I brought down his Demon Hammer-wielding combat specialist on turn one. Following that early action we turned out attention to the center of the board and began to duke it out.
I charged the aggressor, who had survived a double tap from my tactical sergeant (sniper) firing both profiles on his combi-plasma gun. I kept my intercessor sergeant farther from his grey knight to avoid being hit with a mind bullet.
As we fought for control of the center of the board, our gunlines sustained fire behind the melee.
But wait! The stalwart Grey Knight Andros not only survived six attacks from a power sword and killed my plasma gunner, he fell back and made a run for the edge of the board! With two flesh wounds, he advanced right out of the game, giving Tom the two points that locked up the win for the Inquisition.
We had a fun time putting two good-looking teams onto to some nice terrain and slaughtering one-another's space marines. Things looked like a cake-walk for Tom early on, but proved fairly competitive as the game continued. We made a few mistakes we'll try to correct next time, but overall the exciting and fast paced game played out well.
Hope you have time in your weekend for some great gaming with friends!


text:
Slack lines
Blog, news and reviews from Snowdonia Fly Fishing Guides
Fishing in Snowdonia
A stunning day's fishing yesterday. The weather was perfect, warm, bright and just a gentle breeze.
I started with an hour on a mountain lake chasing wild brownies before moving on to the river. The peaty brown coloured lake holds a good head of small wild brownies that are wary and very fast but rise readily to a dry fly.
Late morning I moved down to the river, there was little visible movement and no rise forms. Spiders and nymphs did nothing so decided to try a dry. I tied on a single size 16 IOBO Humpy, just as a few March Browns started to hatch. The next 2 hours was fantastic with several fish rising to the fly, unfortunately the grayling don't realise they are out of season so several were caught, but quickly released.
Leave a Reply.
A well known fly fishing guide and instructor John can often be found fishing or guiding on the Welsh Dee or passing his knowledge to others at game fairs and country shows.
June 2020
guided fly fishing in North Wales
Guided fly fishing trips on rivers, streams and upland lakes in Snowdonia. and surrounding areas of North Wales.
Fishing for summer & winter grayling and wild brown trout.in the spectacular, tranquil countryside that is Snowdonia National Park.
Fly fishing lessons in North Wales
Fly fishing and casting lessons for beginners. Casting fault analysis, single handed spey, slack line and presentation casting tuition for more experienced fly fishers.
Develop your watercraft skills on freestone rivers and upland lakes.


text:
Can I buy a car with no credit?
Whether you have good credit, bad credit, or no credit, Gerald Subaru of North Aurora has several financing options to accommodate all of our customers' circumstances.
Maybe even a calm indifference?
But haven't you seen people who seem to coast into good things, like the farmer who found the Hope Diamond? I've known people like that. In fact, after I'd been in Japan for a while and had set up a "channel" for business to flow through, I could just think about receiving more money, and I'd get an immediate surge of business within hours.
- This pattern went on for 16 or 17 years.
- So it wasn't an occasional fluke.
- No matter what term you use, it's basically the same thing.
- Achieving their dream doesn't appear to have a lot to do with burning desire.
How does that feel?
How do you affect those around you? How do they feel? Absorb every aspect of this path that you can take today if you so choose. Notice everything that you need to know about what it will be like if you carry on with the same behaviour, putting off change. Drift back to where the path splits. Step Five: Now take a step out on to the path to the right. This is where you create powerful, progressive change.
- Notice the sense of freedom in your thoughts.
- They often mean leaving the perception of security in order to discover your personal freedom.
- These are the changes that will bring happiness and satisfaction into your life. Just go there now.
- You have the freedom to be how you want to be.


text:
All Kinds of people are foster parents. They may be younger or older, have young children, adult children, or no biological children at all. They may own their own home or rent, be a one or two income family, or even retired. Foster Parents are just like you!
Becoming a Foster Parent with Camelot Community Care is an opportunity to be part of a team of dedicated professionals caring for children who through no fault of their own, have suffered the trauma of abuse and neglect. Camelot Community Care provides two types of foster care programs, Traditional Foster Care and Treatment Foster Care.
Camelot Foster Parent Handbook
To learn more about fostering, please complete this form and a recruitment specialist will contact you.


text:
At Iceland Airwaves festival Icelandic singer and songwriter Emiliana Torrini was awarded the latest 'Inspiration Award'.
The Keychange Inspiration Award honours and celebrates female pioneers who have made an extraordinary contribution to music and are role models for the next generation and for the Keychange network of 60 talented female artists and industry professionals. Keychange partners select an Inspiration Award winner in each of their countries.
Upon receiving the Inspiration Award, Emiliana Torrini said,
"I didn't know how to take getting an inspiration award, to be picked from an ocean of inspiring women is humbling. I'm doing what I do today because of the people who have inspired me. To be giving that back somehow means so much. Now is a very exciting time for women, a time to move forward through the old tar. Our journey has created many inspiring voices that empower us to put ourselves out there, to do the things we thought we couldn't do. These voices bring changes and create spaces where once we were not welcome. We're taking the credits we deserve, challenging the doubts within and supporting each other.
Something like Keychange helps empower women within our industry and like with the beginning of change in so many things in life the arts have to take the lead… so lets."
Vanessa Reed, CEO of PRS Foundation and founder of Keychange said:
"Congratulations to Emiliana on being presented with this Inspiration Award today. Emiliana is a fantastic artist and songwriter whose impressive career has spanned more than two decades, nine albums, numerous collaborations and featured performances. She is a brilliant role model for female music creators demonstrating that it is possible to sustain a successful 20-year career in this industry."
As well as being an artist in her own right, Emiliana has written for artists including Kylie Minogue and has been nominated for a Grammy Award.


text:
All posts tagged "lone star"
Lone Star Brewing: Contributing to Texas Communities for Hurricane Harvey Relief
In efforts to contribute to Hurricane Harvey relief in Texas, Lone Star Brewing has created the Lone Star...
5 Brilliant Ways to Showcase The Lone Star at Home
No matter how a Texan decides to show off Texas' most beloved symbol, the Lone Star is an...
Live Without These 5 Items? Not Us Texans
Choosing things we can't live without is difficult for us Texans, but here's five to start the list.
5 Great Reasons Texas is the Proud Prodigal Son
Here are a few great reasons as to why the state of Texas is in the state that...
Conan O'Brien Messes with Texas, But Does Texas Win?
In this clip from Conan O'Brien's late-night show Conan, he pits Texas against its arch enemies: California, Florida,...
10 Thoughtful Ways to Showcase the Texas Star in Your Home
In the sky, or on a small Texas town's city marker, or in the homes across the vast state you...