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These sources have minimal bias and use very few loaded words (wording that attempts to influence an audience by using appeal to emotion or stereotypes). The reporting is factual and usually sourced. These are the most credible media sources. See all Least Biased Sources.
Factual Reporting: HIGH
Notes: The Sun Journal is the daily newspaper in New Bern, North Carolina, United States. The New Bern Sun Journal reports news with very low bias and is factually sourced. The editorial content has a slight lean to the left based on topic selection, but not enough to justify moving this source from the least biased category. (6/12/2017)
Source: http://www.newbernsj.com/
Latest from New Bern Sun Journal
Today in history: Nine members of the Milwaukee police department and two civilians were killed when a bomb exploded inside a police station.
Source: Flynn lawyers make a break with Trump team
Single dad seeks joyful Christmas for children
Retailers battle it out as Black Friday sales begin
Trump tells troops 'We're winning' against foes


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Hello there~ Seems you somehow came across my profile! Welcome!
I'm GalaxySky, SkySilvally on most other sites.
God you don't need me
But somehow You want me
Oh how You love me
Somehow that frees me to open my hands up
and give You control
~Control by Tenth Avenue North, one of my fave songs by my absolute most favorite band ever <3
I enjoy playing Pokemon, Smash Bros, and other Nintendo games.
My fave movies are Fantastic Beasts and Where to Find Them / Priceless~
I am hopelessly addicted to teriyaki sauce, plz halp
Thanks for visiting!
This super amazing drawing of Jojo by Raythenerd on DA

subset: commoncrawl


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2017's Top Rated Baby Monitors
Dorel Juvenile Infant Girl's Cute As A Hoot Amble Quad Travel System - Polka Dot
Product Description
The Carter's Amble Quad Travel System by Safety 1st is 3-piece system with car seat, stay-in-car base and stroller that takes you from car to strolling and back again. With the Quad stroller, you can give your baby a bit of shade with the retractable canopy during the ride and still easily check on them through the peek-a-boo window. You can also easily get baby comfortable with the multi-position seat recline, and keep your phone and drink handy in the convenient parent tray with two cup holders and storage compartment. Larger items fit nicely into the under stroller storage basket. The car seat is sturdy and very lightweight, making it easier to transfer from stroller to car. You'll appreciate the Side Impact Protection and secure 5-point harness that keep your baby safer and snug during the ride. Complete with all the features you and your child need to make every journey more enjoyable, this versatile stroller and extra-light infant car seat combo will be a welcome addition to your family. see more...
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Dorel Juvenile Infant Girl's Cute As A Hoot Amble Quad Travel System - Polka Dot
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Côte d'Ivoire banking sector preparing for Basel II and III
Banque d'Abidjan (BDA) became the new lender to open its doors in Côte d'Ivoire, bringing the total number of financial players in the country to 28 on September 5.
Its launch took place against a backdrop of sweeping reforms being implemented across the sector as Côte d'Ivoire prepares to meet the capital standards' requirements of Basel II and III.
BDA is a subsidiary of the Société Ivoirienne des Finances Holding, a joint venture for Senegal's Banque de Dakar Group, which has an 80% stake in the lender, and the country's national-owned mail carrier, La Poste, which owns the remaining 20%.More >
Useful tips for travellers to Nigeria
Relationships are very significant within the business community. Meetings typically open with a handshake, followed by a casual conversation about family, health and travel. In the Muslim north, wait for a woman to extend her hand before offering yours. The business discussion begins at the host's indication.
DRESS: Business formal is the required dress code for foreigners in any type of conference, while locals tend to opt for either Western or traditional attire. In a lot of work spaces, Nigerians will wear traditional garments on Fridays. Appearance is generally very significant and indicative of social and economic status.More >
Quattara Commends Buhari Over Nigeria's Recession Exit
President Alhasan Quattara of Cote d'voire on Monday in Abuja commended President Muhammadu Buhari for taking Nigeria out of recession.
Mr. Quattara gave the commendation at the same time as he addressed National Home correspondents next a closed-door conference with Buhari at the Presidential Villa.More >
- Key Facts
West Africa, as well called Western Africa and the West of Africa, is the westernmost region of the African continent. In line with the current membership of the Economic Community of West African States (ECOWAS),
Benin, Burkina Faso, Cape Verde, Gambia, Ghana, Guinea, Guinea-Bissau, Ivory Coast, Liberia, Mali, Niger, Nigeria, Senegal, Sierra Leone and Togo
Development priorities of the Republic of Guinea2017/09/24
Guinea Prime Minister, Mamady Youla: "The Government of Guinea, starting with the President and myself, is fully committed to supporting the DU's initiatives, as the objectives are in line with the development priorities of the Republic of Guinea."
Top 10 Most Attractive Investment Destinations In Africa2017/08/20
Africa's feverish increase has decelerated in recent years and a lot of nations have buckled under the pressure of falling resource prices, security disruptions, fiscal imprudence and adverse weather conditions.
Africa's Relationship With China Is Ancient History2017/07/02
In 2002 South Africa's Parliament unveiled a digital reproduction of a map - of China, the Middle East and Africa - that some speculated could be the initial map of the African continent. The Da Ming Hun Yi Tu - the Comprehensive Map of the Great Ming Empire - was drawn up around 1389 during the Ming Dynasty, according to historian Hyunhee Park.
Economic Overview2017/05/31
IMF and private support help cope with financing needs
Côte d'Ivoire Global leader in cocoa production2017/05/14
Côte d'Ivoire
Global leader in cocoa production
Climate change laws around the world2017/05/14
There has been a 20-fold increase in the number of global climate change laws since 1997, according to the most comprehensive database of relevant policy and legislation.
The database, produced by the Grantham Research Institute on Climate Change and the Environment and the Sabin Center on Climate Change Law, includes more than 1,200 relevant policies across 164 countries, which account for 95% of global greenhouse gas emissions.
Economic Overview Over-reliance on the primary sector causes exposure to exogenous factors2017/05/08
Burkina Faso
Concerns about domestic stability and regional security
A mouth-watering array of African foods2017/01/09
African cuisine is as diverse as the hundreds of different cultures and groups that inhabit the continent. This diversity is reflected in the many local culinary traditions in terms of choice of ingredients, style of preparation and cooking techniques. Many of the dishes are also affected by the subsistence nature of living in many parts of the continent – you find farmers, herdsmen and fishermen everywhere. The crops they grow or the animals they keep thus affect the popular dishes in their regions.
President of 22 years defeated2016/12/03
Economic and Financial Strategies against Africa2016/11/23
Other than the divisive military-security perspective and 'dark scenario' forecasting that's been hitherto applied for understanding African geopolitics, there's also the more unifying and positive approach of analyzing the continent's financial, economic, and integrative institutions.
Six reasons to invest in Africa2016/10/03
The conversation about Africa is shifting from one of "deficits" and "gaps" to one about opportunities, prospects, ventures and creativity. That's not news to companies that have paid close attention to the continent and invested there. The fast growing youth populations, the urbanization expected to drive over 50% of Africans to cities by 2050, and Africa's formalising economy are all well known. These trends and other developments have driven a half century or additional of increase in Africa, and will continue to do so.
Top 10 most competitive Sub-Saharan African economies2016/10/02
A ten-year decline in the openness of economies at all stages of development poses a risk to countries' ability to grow and innovate, according to The Global Competitiveness Report 2016-2017, which is published today.
Sub-Saharan Africa: importance of institutions for developing food and agriculture value chains2015/12/05
Despite its huge food and agricultural potential, Sub-Saharan Africa's agricultural production has barely kept up with people increase as productivity increase has lagged behind. Over the completed decades the region has become a net food importer. Food and agricultural trade deficits are on the rise, posing risks to food security. Conference this challenge requires the region to develop its role in food and agricultural price chains.
It should move from predominantly smallholder production for informal markets and export of (agricultural) commodities to adding additional price at different stages in the price chain. Possibilities for doing so depend crucially on climatological and soil circumstances and on the presence of enabling factors. The degree to which nations provide an enabling environment differs largely across the region, and is strongly linked to nations' in general institutional development, as is shown by investigating a set of relevant indicators.
President Alassane Ouattara is steadily putting the Ivoirian crisis behind and laying the groundwork for a brighter future2015/09/16The West African national is back on the international stage, strengthening its foreign relations, economic sectors and public-private partnerships to expedite increase and development for its people.
A retrospective on the first cases of the outbreak2015/03/31
Ebola at 6 months
On 26 December 2013, a 2-year-old boy in the remote Guinean village of Meliandou fell ill with a mysterious illness characterized by fever, black stools, and vomiting. He died 2 days later. Retrospective case-finding by WHO would later identify that child as West Africa's first case of Ebola virus disease. The circumstances surrounding his illness were ominous.
His Excellency Dr. Ernest Bai Koroma President of the Republic of Sierra Leone EU Ebola Conference Brussels 3 March 20152015/03/04
We are honored to be here to chart ways forward on the current Ebola Outbreak in our nations.
The EU has always been a great promoter of its core price of social solidarity and human advancement. Ebola tested the durability of these values, but the EU and its member states, inclunding the United Kingdom showed great commitment to these values by supporting our nations to tackle this virus. We deeply appreciate your commitments.
Nigeria Year in Review 20142015/01/23
Falling oil prices and increased instability in the north impacted Nigeria's economic increase in 2014, though a rebasing of GDP means that whoever wins the forthcoming presidential elections in 2015 will take the helm of Africa's major economy.
Ghana Year in Review 20142015/01/23
Although room for fiscal manoeuvring has shrunk in Ghana in recent months, the government's subsequent efforts to tighten spending and boost revenues is being welcomed by investors and lenders. Cuts to public expenditures and lower oil gain will likely further slow increase in 2015, but strong output from the agriculture industry and improving performance from service sectors should help ensure a favourable medium-term outlook.
Belt tightening
Building for a lifetime, lasting through generations, Ghana2017/08/17
Ibrahim Bah, Managing Director of Regimanuel Gray Limited (RGL), discusses the performance of the real estate sector in Ghana and how RGL strives to be the market leader particularly in terms of technology or advancing construction techniques.
Mr Ghassan Yared, CEO of Forewin Group2017/08/11
In this interview with The Worldfolio, Mr. Ghassan Yared, CEO of Forewin Group, discusses the role of the private sector in Ghana's economic increase, how Forewin Group has managed to diversify into one of Ghana's biggest conglomerates and their new venture with Actis, "The Exchange" project.
Mr. Emmanuel Kofi Nti Commissioner General of Ghana Revenue Authority2017/07/02
In this interview with The Worldfolio, Emmanuel Kofi Nti, Commissioner General of the Ghana Revenue Authority, discusses the importance of domestic revenue mobilization for the sustained socioeconomic development of the country, and how Ghana is moving from an 'up-front tax to back-room tax', allowing businesses to become additional competitive
Like the rest of the world, Africa remains uncertain about what is approaching next world geopolitical shifts like Donald Trump's election in the US and the upcoming Brexit. How can Africa take chance of this world landscape and gain investor confidence?
Africa: Making Things Happen at the Bank - 'Not a Talk Shop' - Akin Adesina2017/07/02
Dr. Akinwumi Adesina is focusing on five areas to achieve the African and world goals for a prosperous continent since becoming president of the African Development Bank - Africa's major public financial institution in September 2015. He was a keynote speaker at this month's Corporate Council on Africa's U.S.- Africa Business Summit in Washington D.C. and moderated a lively panel with five African government ministers. He as well received the Gene White Lifetime Succcess Award from the World Child Nutrition Foundation. This week, he was named the 2017 recipient of the World Food Prize, a prestigious honor that includes a $250,000 award. In an interview in Washington, DC, Adesina discussed the Development Bank's ambitious schedule and his vision for attracting the increase capital Africa needs. Posting questions for AllAfrica was Noluthando Crockett-Ntonga.
Benin designs a digital future2017/06/24
In December 2016, Benin announced its economic transformation plan, including 45 flagship projects addressing the major components of the west African country's economy. Six of the projects are specifically focused on constructing a digital economy. Serge Adjovi, director of Benin's Digital Economy Agency, speaks to Joy Macknight about the laying the foundations for progress.
Georges Philippe Ezaley, Mayor of Grand-Bassam, on the citys significance and potential2017/03/14
Which sectors offer the majority potential for economic increase in Grand-Bassam?
GEORGES PHILIPPE EZALEY: As the major tourist destination in Côte d' Ivoire for both the local and international community, Grand-Bassam offers an array of tourism attractions.
Francis Okoh, Commercial Director, Dolphin Telecom2014/01/01
Francis Okoh the Commercial Director at Dolphin Telecom is a round peg in a round hole. The man, who is responsible for implementing sales and marketing strategies inclunding managing customer relations across West Africa, is a guru with business agreements with operators.
Philip Sowah, CEO Airtel Ghana2014/01/01
You can't deny the fact that Mr Philip Sowah is a thorough bred in his field. The amiable Managing Director of Airtel Ghana Ltd is a definite goal getter. His track records show so easily. Prior to joining Airtel Ghana, Sowah was the Chief Officer of Onetouch (presently Vodafone Ghana) where he championed its aggressive expansion from 160,000 to 1.3 million subscribers.
Fredrik Jejdling, Head of Region for sub-Saharan Africa, Ericsson2014/01/01
Full of brains, highly energetic Fredrik Jejdling has held his current position since April 2013. He joined Ericsson in 2006 next having before been Director and Chief of Managed Capacity, Vice-president and Chief of Sales and Finance for Business Unit World Services.
CEO explains success of ASKY Airlines2013/08/12
Speaking in an interview with the Ghana news Agency (GNA) in Accra, Mr. Awel , CEO of the Gambia based ASKY airlines, said the airline's major goal was to connect all capital cities in Africa with daily flights in order to relieve movement within the continent and it was making great strides in that regard.
ASKY, he noted, currently served about 22 destinations, mainly in West and Central Africa with two or three weekly flights.
The destinations include Benin, Burkina Faso, Cameroun, DR Congo, Chad, Gabon, Ghana, Liberia, Mali, Republic of Congo, Niger, Nigeria, Senegal and Sierra Leone.
Mo Ibrahim is chair of the Mo Ibrahim Foundation2013/05/18
Being an optimist about Africa's economic prospects was once a pretty lonely cause. But I am thrilled to presently find myself in a very crowded field.
The continent's strong and sustained increase – evolution amount the additional remarkable given the severe problems in the world economy– has been one of the success stories of the last decade.
Andreas Karlsson is the Chief of Supply for Ericsson in sub-Saharan Africa2013/04/25
Andreas Karlsson is the Chief of Supply for Ericsson in sub-Saharan Africa & Country Manager for Ericsson Ghana. Ericsson is Africa's leading provider of communications technology and services and employs over 3,000 professionals who support additional than 100 customers in 43 nations across sub-Saharan Africa.
Bornman du Toit, MTN Group's General Manager for Products, Services and Innovation2013/03/31
There is no doubt that Africa is a hot bed for Mobile Banking and Mobile Money. In this issue, glObserver catches up with Bornman du Toit, MTN Group's General Manager for Products, Services and Innovation, to get a lowdown on mobile telecom industry in Africa.
Mo Ibrahim - founder and chair of the Mo Ibrahim Foundation.2013/01/14
The founder of Celtel talks about the role businesses can play in transparency and discusses trends in African governance
As entrepreneurial success stories go, few come bigger than Mo Ibrahim's. In 1998, with a telecoms boom in full force, the Sudanese-British entrepreneur hatched a plan to create a pan-African mobile operator, and to do so without paying bribes.
Lapodini Marc Atouga2013/01/14
Since its inception in 2005, where does the implementation of the Common Agricultural Policy (ECOWAP) stand today?
A large step has been made towards the effective implementation of ECOWAP since 2009 with the development and implementation of National Agricultural Investment Programmes (NAIPs) and the Regional Agricultural Investment Programme (PRIA).
Kofi A. Annan2013/01/13
As Secretary-General, Mr. Annan has given priority to revitalizing the UN through a comprehensive programme of reform; strengthening the Organization's traditional work in the areas of development and the maintenance of international peace and security; advocating human rights, the policy of law and the universal values of equality, tolerance and human dignity; restoring public confidence in the Organization by reaching out to new partners and, in his words, by "bringing the United Nations closer to the people".
Strive Masiyiwa, philanthropist and founder of Econet2015/11/23
As a global business leader, African philanthropist, anti-corruption activist, and mentor to thousands, Strive Masiyiwa is a vibrant example of someone who has little doubt about what motivates him, what he hopes to accomplish, and what sort of example he wants to set for others along the way. His is a character forged by struggle and perseverance against corruption, and shaped by a deep faith that guides him on the world stage as he looks to bring about positive change to his home continent. We had the pleasure of speaking with Masiyiwa in London during a brief pause in a schedule bursting with business and philanthropy interests.
(Economic Community of West African States2012/12/16
ECOWAS (Economic Community of West African States) is a 15-member regional bloc founded in 1975 to promote cooperation and trade within West Africa. We would like to use resources at the disposal of ECOWAS– an institution which often commits funds to developmental projects within member nations–to further Niger's capacity for sustainable economic and social growth. PAN and ECOWAS's vision for the region are shared in many respects, thus cooperation with ECOWAS would be relatively easy should PAN's policies be adopted.
- BOTSWANA: Namibia Scraps Visas for Africans
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- NIGERIA: Credit facilitates economic growth
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- Americas Africa RelationMore >
- UNITED STATES: What Africa Needs to Boost Trade With the U.S.
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- AFRICA: Africa Tourism Report
- NIGERIA: Useful tips for travellers to Nigeria
- WaterMore >
- AFRICA: Renewable water resources
- West Africa News
- NIGERIA: Small firms the focus as Nigeria launches action plan to improve business conditions
- ABIDJAN: Côte d'Ivoire banking sector preparing for Basel II and III
- NIGERIA: Useful tips for travellers to Nigeria
- GUINEA: Red Cross 'outraged' over pilfering of Ebola aid millions by its own staff
- NIGERIA: Quattara Commends Buhari Over Nigeria's Recession Exit
- NIGERIA: Nigeria Has Gender Problem in the Workplace
- Trending Articles
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- BOTSWANA: Ecobank launches mVisa across 33 African Countries
- UNITED STATES: Paradise Papers: Apple shifted billions offshore to avoid tax
- GOVERNMENT: Mugabe Meets Army Boss, Envoys Amid Calls for His Resignation
- HEALTH: Red Cross 'outraged' over pilfering of Ebola aid millions by its own staff
- GOVERNMENT: Resigned Lebanon PM Saad Hariri Says He Is 'Free', Will Return To Beirut
- AGRICULTURE: Zimbabwe: Racist Land Policies Fatal for Economy, Warn Analysts
- OTHER: Ecobank launches mVisa across 33 African Countries
- COMPANY: Paradise Papers: Apple shifted billions offshore to avoid tax


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Interactive Treat Dispensing Dog Toy
This Interactive Treat Dispensing Dog Toy does it all. Traps treats with its inner ridges and tips over to keep dogs entertained and challenged. Interlock the two sizes (sold separately) to make playtime over the Toppl. Perfect for smaller dogs, bigger dogs and clever dogs. Like all of our Zogoflex® dog toys, TopplTM is recyclable, BPA-and-phthalate-free, FDA compliant (meaning it's safe to eat off of), and Guaranteed to Last.
The newest Zogoflex® of the bunch, TopplTM is the treat-keeping toy that active dogs of all sizes have been waiting for. Made in large and small for all sized mouths, Toppl corrals treats, doling out sweet rewards one nip at a time. With endless playtime possibilities, Toppl falls over tipsily, rolls around clumsily, or combines with its other size to create a one-of-a-kind puzzle plaything! Owners will love watching their dogs chase Toppl just as much as their pups love playing with it! And unlike other toys that are perpetually plastered with yesterday's treat leftovers, Toppl slides safely into the dishwasher for quick, simple cleanup that lets pups get back to playing-pronto.
Toppl is not just a treat toy; it's a treat experience. With two different sizes (for dogs big and small!) and an interlocking feature (for dogs clever and cunning), Toppl has a multitude of ways to make playtime easy or challenging, from eager puppies on up to wise ol' dogs.
Picky pups? No problem. Owners can create their own brand of Toppl tastiness by choosing a tasty treat and wedging it into the inner cage. And since Toppl slips easily into the dishwasher, owners can dole out a different snack morning, noon and night. Perfectly customizable for dogs that delight in dry food, wet food, most-loved doggie treats, or rarely-doled-out table scraps (or all of the above).
Toys R UsOur Toys"R"Us Pets Fetch 'N' Treat Toy stimulates your dog's retrieval instincts and inspires natural fun and exercise. When your dog retrieves the ball and drops it in the Fetch 'N', he's instantly rewarded with a treat. This toy is sure to add some flavor to playtime-keeping your pet happy, healthy and entertained. Only at PetSmart. Features: Stimulates your dog's retrieval instincts Includes: 1 Fetch 'N' Treat Toy Intended For: Dogs Activity: Hide, Learning Color: White/Blue Pet Size: Medium Caution: Intended for dogs only. Not a child's toy. Advice for Use: Ideal for use with dog's favorite kibble or hard bite-sized treats. Not for use with sticky or soft treats. Toys are meant for you and your pet to enjoy together. Observe your pet's behavior with the toy for suitability. Some toys may be inappropriate for your pet. Remove and replace the toy if it becomes damaged. Instructions for Use: 1. Remove all plastic wrapping and discard properly. 2. Place the toy on a flat surface with ample room for your dog to access it. 3. Fill the toy with up to 2 cups of kibble or hard treats. The maximum size of the kibble or treats is 0.5 inch (12.5 mm) ; anything larger will not flow out of the toy. During the initial play session, demonstrate how to drop the ball inside and let him see that this causes the ball to launch and treats to be dispensed. 4. Give the ball to your dog and encourage him to drop it into the toy. When he's successful, offer praise and let him enjoy the treats. 5. Be patient as learning takes time. Be sure to praise your dog for each successful "fetch." Size: Medium. Color: White.$29.99
Hugs Pet ProductsHugs Pet Products Trick Treat Interactive Treat Dispensing Toy for Cats Interactive Treat dispensing toy Cage balls with bells excite natural instincts Mount to wall and fill with treats Offers a level of difficulty to challenge your cat's abilities$7.99
The KONG Wobbler Cat Treat Dispensing Toy is an entertaining, beneficial treat and meal dispenser. Though cats have been domesticated for a long time, they still have an instinctual desire to chase and capture prey. The KONG Wobbler for Cats helps satisfy these natural hunting urges and creates a positive outlet for the indoor cat by providing rewards for the successful hunter. Cats are naturally curious about things that are in motion. The unpredictable wobble action of this toy, along with the fun, floppy, catnip-stuffed tail, will grab the attention of most cats. Additionally, the translucent top lets cats see the movement of the treats inside, which further piques their curiosity. Key Features: Dispenses food and treats for hours of fun Twist off, easy fill dispenser Prevents rapid eating$12.99
DOSKOCIL MANUFACTURING CO.,INCJW (R) Pet Treat N' Squeak Treat Dispensing Dog Toy The JW (R) Pet Treat N' Squeak Treat Dispensing Dog Toy is an interactive treat dispensing natural rubber dog toy that is easy-to-load and easy-to-dispense. This squeaky fetch toy rewards your dog when retrieved. Interactive dog toys help to stimulate your dog's mind as well as strengthen the bond between you and your dog. Features: Durable, natural rubber Easy-to-stuff with your dog's favorite dog treats Squeaks for additional enjoyment Helps to stimulate your dog's mind Helps to strengthen bond between you and your dog Item Specifications: Color: Assorted (Red/Blue/Green) Please allow us to pick for you Size: One Size: (3.5"" L x 3.5"" W x 7.2"" H)$6.49
DOSKOCIL MANUFACTURING CO.,INCJW (R) Pet Treat Tower Treat Dispensing Dog Toy The JW Pet Treat Tower Treat Dispensing Dog Toy is an interactive toy designed to keep your dog stimulated and entertained. Simply insert dog treats and watch your dog go to work. As a learn-and-play dog toy, the Treat Tower difficulty level can be adjusted as your dog learns. Available in two sizes to accommodate most size dogs. Features: Interactive dog treat toy dispenser When your dog wobbles the Treat Tower, treats are dispensed Difficulty level can be adjusted as you dog learns Treats are easy to load Available in two sizes to accommodate most size dogs Item Specifications: Color: Multi Size: Small: Recommended for small and medium size dogs Large: Recommended for medium and large size dogs$10.49
STARMARK PET PRODUCTSStarmark Treat Dispensing Snoggle Dog Toy Starmark Treat Dispensing Snoggle is a perfect combination of plush and treat dispensing toy. Extremely durable, but soft in your dog's mouth, the Snoggle is ideal for feeding your dog his meals or for retrieving fun. Special easy-fill, slow-release opening allows you to fill with treats or kibble of your choice. Snoggle provides hours of challenging play, which helps reduce anxiety and boredom. The puzzle releases treats as your dog paws, nudges, and carries it around. You can regulate the difficulty by adjusting the opening and rate at which the contents release. Your dog will not be able to get enough play time with the Snoggle. Features: For all dogs Holds a variety of treats and kibble Provides hours of challenging fun Helps reduce anxiety and boredom Regulate difficulty of puzzle Item Specifications: Size: 11""L x 8.5""H x 6""D$12.49
THE KONG COMPANYThe Kong Wobbler is a Kong-shaped food dispensing toy and feeder that provides a new way to entertain and challenge your dog. The Wobbler sits upright until pushed with dogs paw or nose, then wobbles, spins and rolls, dispensing treats through a Kong shaped hole near the top. Made in the USA of durable, FDA food-approved, high-strength polymer. It unscrews for quick filling and easy clean-up. Top drawer dishwasher safe. For dogs 25 lbs and over. Measures 7 1/4"" H x 4 1/2"" at widest part.$17.99
The PetSafe Busy Buddy Waggle Treat Dispensing Dog Toy keeps your pet occupied with tail-wagging fun. The Busy Buddy Waggle features the patented Treat Meter; reservoirs in the balls at each end dispense tasty morsels throughout your do$7.99
PET SAFEBusy Buddy (R) Barnacle Multi-Chamber Treat Dispensing Dog Toy The Busy Buddy (R) Barnacle takes fun for your dog to a whole new level! This interactive treat dispensing toy feature three patented Treat Meters that randomly dispenses your dogs's favorite treats like Buddy-Ohs! and Buddy Berries dog treats. Trim the Treat Meter prongs with scissors to increase the rate at which the treat come out. The bone- shaped grooves can be filled with your dog's favorite smearable treat such as peanut butter and cheese for an extra treat. The Busy Buddy bounces wildly to keep your dog moving and happy! Features: Interactive treat dispensing dog toy Three patented Treat Meters randomly dispensing you dog's favorite treats like Buddy-Ohs! and Buddy Berries (sold separately) Treat Meters can be trimmed to increase rate at which the treats come out. Bone-shaped grooves can be smeared with your dog's favorite smearable treat such as peanut butter or cheese. Unpredictable bounce keeps your dog on the move 100% dishwasher safe (top rack only) Comes in three sizes to accommodate all breed sizes Item Specifications: Size: X-Small: For dogs 10 lbs and under Teacup and Toy Size Breeds Holds Buddy-Ohs! and Buddy Berries Small: For dogs 10 to 25 lbs Breeds such as Jack Russell, Pug, and Boston Terriers Holds Buddy-Ohs! and Buddy Berries Medium: For dogs 25 to 50 lbs Breeds such as Beagle, Spaniel, and Border Collies Holds Buddy-Ohs! and Buddy Berries Large: For dogs 50-90 lbs Breeds such as Retriever, Sheppard, and Boxers Holds Buddy-Ohs! and Buddy Berries Click here to watch the product video.$14.95
PetSafeSmall, 4.5; L X 3.5; W, Treat dispensing activity ball designed to keep your dog busy for hours. Helps redirect potentially destructive behavior into positive playtime. Unscrews for easy loading & quick clean-up.$11.24


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British Values: 'King Alfred Entertains The Vikings.'
by Michael Redwood
"Legend has it, that when the Vikings over ran Saxon Britain, King Alfred went amongst the viking camps as an entertainer to understand his enemy better. What he discovered as his secret weapon, was tolerance and over the next 40 years these tribes merged to build the foundation of our modern country. Let us create this story with your pupils as Alfred joins the cheeky Splats circus troop as they travel the land entertaining all who will watch."
Recommended for: British Values, Drama, Viking and Saxon History, King Alfred, Historical Fiction.
Ages: Key Stage 2 and above – See filter on the gallery of days below for age group and type of day.
Amount of pupils: Up to 140 in one day (more on request) max 35 per class.
Availability: All year – UK wide. Advance booking preferred but can help with last minutes plans.


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The Amor replacement coils are 100% authentic in quality and brand! These coils perform with maximum capabilities and assist in enhancing flavors from your favorite E-Liquids. The wicks on these coils are made of Japanese organic cotton so they'll absorb the finest ingredients from the juice. Looking for massive vapor production? The Amor replacement coils give you another reason to make these your go-to product. With a resistance of 0.2Ω, these coils are ideal for vaping anywhere between 20W-75W on your mod.
The WS01 Triple replacement coils are specially designed for the Amor Plus Tank, Amor Mini Tank, SinuousSW, P80, Predator and RX Mini
Package Contents:
5 x Wismec Amor Replacement Coils
1 x Authenticity Scratch-Off Sticker on Retail Box
Product Specifications:
Vertical Coil Head
Resistance: 0.2Ω (triple coil)
Japanese organic cotton wick
Anti-spitback mesh screen
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When you purchase a digital subscription to Cake Central Magazine, you will get an instant and automatic download of the most recent issue.
12x12 cut in half for tractor body and 6x6 cut in half for cab. This was for a 78th birthday of a man that used to build John Deere Tractors.
Hay and Wheels are made of Rice Krispie treats and covered in fondant.
John Deere
John Deere 1St Birthday
John Deere Tractor
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Ben Turner and the team with the latest breaking news, weather and travel via our Old Hall street news centre
Here's the latest amid Storm Aileen. The Met Office say very strong winds will continue during rush hour near the Lancashire and Merseyside coast, though winds are slowing down inland, and they will gradually ease everywhere.
Blustery showers, some heavy and thundery, will come and go all day. Maximum temperature 14 °C.
Tonight: Blustery showers will continue during the evening, though winds will be much lighter. Rain will stop later in the night and skies will clear, allowing it to become quite cold. Minimum temperature 6 °C.
Also read: UK braced for raw Arctic blast as bitter winds sweep Britain
Source: liverpoolecho.co.uk


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First Transit Portland
Officers & Contracts
Your Officers
Kathleen Brown
Liaison Officer, Region 1[restrict level="all-members"]
First Transit Region 1
2800 NE Nela Street
Portland, OR 97210
Phone: (503) 819-9065
Cathy Redwine
Liaison Officer, Region 2[restrict level="all-members"]
First Transit Region 2
2055 SW Merlo Ct
Beaverton, OR 97006
Phone: (503) 332-6133
Matt Leist
Liaison Officer, Region 3[restrict level="all-members"]
First Transit Region 3
3705 SE 99th Avenue
Portland, OR
Phone: (503) 454-8280
Property News
The latest news impacting First Transit Portland
(From First Transit Portland, the 757, National, and International)
President Shirley Block's Letter to International President Hanley
Thank you for your letter dated November 30, 2016 in which you state that you do not support, nor think it is wise for the members of Local 757 to seek an election to sever [...]
Letter from International President Hanley
ATU International President Larry Hanley's letter to Shirley Block concerning the challenges confronting Local 757 resulting from the local union's large number of bargaining units spread over a large geographic area. Read International President Hanley's letter in it's [...]
From the International: Videos on Bathroom Breaks and Bus Driver Blind Spots
Every day our members face the stress of ensuring riders get to their destination safely, meeting schedules, dealing with attacks by passengers and fare beaters, weather, and traffic congestion. On top of these our members [...]
ATU International Video on Operator Assaults
As you know, the International Union has an ongoing campaign to expose and stop assaults on our members. There are many facets to their ongoing campaign. Here is a video showing the real life experiences [...]
Four Kelowna bus drivers assaulted in a day, Local calls for action
In an unprecedented day of violence on Kelowna's bus system, four drivers were attacked in separate incidents last Friday, May 20. Local *1722* has demanded immediate action from the police and transit system to address [...]
Bay Area bus driver saves, reunites lost child with family
AC Transit bus driver Ari Megaro was on his normal route when he noticed a small boy riding a scooter alone. Sensing a potentially dangerous situation, the Local 192 member immediately brought his coach to [...]
Local Web Admins:
Contact your Officers or your local web admin to post stories to the ATU 757 website or to your Property's news feed.
Admin Needed[restrict level="all-members"]firstname.lastname@example.com[/restrict]
Your Contract
Contract Details
First Transit, Region 1
2800 NW Nela
Portland, OR 97210[restrict level="all-members"]Contracts (PDF):
First Transit R1[/restrict]
First Transit, Inc. Region 2/Tri-Met Lift
16400 SW Merlo Rd
Beaverton, OR 97006[restrict level="all-members"]Contracts (PDF):
First Transit R2[/restrict]
First Transit, Inc. Region 3
3705 SE 99th Avenue
Portland, OR[restrict level="all-members"]Contracts (PDF):
First Transit R3
Represents about 383 members[/restrict]
Contract Suggestions
[restrict role="0″]Are you an ATU Property member? Sign in here to submit your contract requests: Login[/restrict] [restrict level="all-members"]Members of this property can submit contract suggestions with this form:[/restrict]
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If you don't see the suggestion form, and you are a member of this Property, please contact your liaison officer or local website admin.[/restrict]


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324 herbicides
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52 fish
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42 environment
39 Seeds
37 agriculture
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33 Genotoxic
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31 salt
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18 Cytotoxic
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15 Cytotoxicity
14 Rapeseed oil
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13 Chloride
13 lipid peroxidation
13 Serine
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13 Milk
13 Fluometuron
|'Obama Express' food store caught trafficking food stamps
|Heal yourself in 15 days by transforming your blood (part four)
|Getting back to nature
|Is Dr. David Gorski mentally deranged because he was brain damaged by vaccines?
|REPORT: Legal marijuana would be a $10 billion industry for Canada
|Kickstarter campaign launched to develop Personal Drone Detection System
|Scientists link pesticide exposure to premature childbirth
|Antipsychotic drugs can cause heart disease
|The 8 most insane and absurd US laws regarding food, water, and medicine
|Grassroots movement affirming right of individuals to deny vaccines gaining momentum across US
|EU Food Safety Authority can't hide the names of 'experts' who change pesticide risk assessment rules, court decides
|Chemical Used on Crops could Make You Fat
|New York cop illegally trespasses private property, arrests woman for filming suspicious traffic stop
|Deadly 'superbugs' escaped hospitals, now infecting homes
|Why the California water crisis will lead to a housing collapse, municipal bankruptcies and a mass exodus of climate refugees


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EU leaders shun Ukraine over Tymoshenko case
UKRAINE faced growing isolation yesterday as five presidents and the EU Commission cancelled visits over the suspected treatment of Yulia Tymoshenko, its jailed former prime minister.
Jose Manuel Barroso, the EU Commission's president, and Viviane Reding, its justice commissioner, said they would boycott the Euro 2012 tournament.
There were also reports that Angela Merkel, the German chancellor and an avid football fan, may snub the tournament over the suspected maltreatment of Mrs Tymoshenko, Ukraine's opposition leader, who is reported to be on hunger strike in prison.
The presidents of Germany, Austria, the Czech Republic, Italy and Slovenia yesterday informed Kiev of plans to miss the Yalta meeting of central and eastern European presidents, on May 11 and 12.
European leaders and officials have previously complained that Mrs Tymoshenko's jailing last year was unfair, but disapproval grew last week when pictures emerged of her showing bruises, which she claimed were the result of a beating when she was forcibly taken from prison to visit a clinic on April 20.
In October she was sentenced to seven years in prison for abuse of office, in what her supporters claimed was a show trial orchestrated by the president, Viktor Yanukovych.
Mrs Tymoshenko's daughter, Eugenia, said the rebuffs were "the last diplomatic warning to the regime".
Mrs Merkel has yet to confirm her position. But a spokesman for Ukraine's foreign ministry said: "I would not like to think that the statesmen of Germany are capable of reanimating the methods of the Cold War and trying to make sport a hostage to politics."
Mr Barroso said he would skip the opening ceremony of Euro 2012, "unless there is a swift improvement in the human rights situation". (© Daily Telegraph, London)
Irish Independent


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Kyle Lafferty's Northern Ireland blow opens the door for postman Conor Washington
- Norwich striker Kyle Lafferty limped out of Northern Ireland training
- Michael O'Neill insists Lafferty is fine and he is likely to play against Poland
- But strike partner Conor Washington is ready to take the scoring burden
- Washington was in non-league working as a postman during Euro 2012
- 24-year-old had never even set foot in Northern Ireland at that time
- QPR striker is Northern Ireland's answer to England's Jamie Vardy
No sooner had a thunderstorm gathered over Northern Ireland's training base at Saint-Georges-de-Reneins on Tuesday than Kyle Lafferty gave his country its first injury scare of Euro 2016.
Manager Michael O'Neill insisted that Lafferty is 'fine' and the Norwich striker is expected to be fit to face Poland in Sunday's Group C opener in Nice.
But there was no disguising the concern that gripped the Northern Ireland camp as their top scorer in qualifying and talisman was helped off the pitch after tweaking his groin in training.
Players and staff gather round Kyle Lafferty to check he is okay after he injured himself in training
Manager Michael O'Neill (centre) said the striker was 'fine' despite suffering injury while making a tackle
Lafferty limps off the training pitch before being taken to an ice bath to help his recovery
For Lafferty's strike partner Conor Washington, on the other hand, it appears as though nothing can cast a cloud over his career at the moment.
Four years ago, Washington watched Euro 2012 on television at home while juggling non-League football with Cambridgeshire's St Ives Town in the Southern Football League with his job as a postman. He had never set foot in Northern Ireland.
Washington was ready to follow his brother into the RAF until his football career suddenly took off. After helping Newport County reach the Football League, he scored 33 goals in 94 games for League One Peterborough to earn a £2.5million move to QPR in January.
Strike partner Conor Washington (left) is on a high as Northern Ireland prepare to face Poland
Washington (right) was playing non-league and working as a postman during Euro 2012 four years ago
Poland, Stade de Nice, 4pm, June 12
Ukraine, Stade de Lyon, 4pm, June 16
Germany, Parc des Princes, 4pm, June 21
The 24-year-old has a Scottish mother and English father, but his agent alerted O'Neill to the player's eligibility for Northern Ireland through his grandmother in Belfast.
Washington was drafted into the squad in March, and after two goals in four appearances for his adopted country, he is still coming to terms with the transformation in his life.
'I don't pinch myself sometimes, I pinch myself all the time about where I am now,' said Washington after training on Tuesday. 'Just having an open training session today in front of hundreds of local people is a new one for me. I've never experienced that before, but I'm relishing it.
'It's so surreal to think where I was four years ago, but it's also surreal for a lot of people back home to see me on TV scoring goals against international teams. In 2012, I was playing part-time for St Ives and delivering post. Up at 5am and we'd be done for midday or 2pm at the latest.
Washington (right) qualifies for Northern Ireland through his grandmother and is now a first-team regular
QPR striker Washington faces the media on Tuesday ahead of Northern Ireland's first Euro 2016 game
'I was turning up to some FA Vase games on a Saturday, the biggest games of my career at the time, and I literally had to run my round.
'I probably shouldn't say this but I had to bring my mate in. There was late post, it was probably delivered to the wrong address, parcels going missing! One of the last games of the season, I turned up three minutes before kick-off. It was just carnage.'
In many respects, Washington is Northern Ireland's answer to Jamie Vardy — a striker who rose through the ranks the hard way. The England man has inspired others further down the ladder.
'It's opened a lot of doors for players like me,' said Washington. 'The lower leagues are being respected a bit more now and people are starting to realise that with a bit of coaching here and there they can mould international footballers like Vardy.
'Maybe I didn't have the rub of the green when I was younger to get into an academy, but I appreciate the way it's happened for me.'
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deCODE Genotyping Laboratory Receives College of American Pathologists Accreditation
News May 21, 2009
deCODE genetics has announced that its CLIA-registered DNA isolation and genotyping laboratory, which processes the company's deCODEme™ personal genome scans and risk assessment diagnostic tests for several common diseases, has been accredited by the American College of Pathologists (CAP) following a recent inspection.
The U.S. Centers for Medicare and Medicaid Services (CMS) has granted the CAP Laboratory Accreditation Program deeming authority, and its accreditations can also be used to meet many state certification requirements.
"We believe that testing for genetic risk factors for common diseases is going to play a central role in refocusing our healthcare system on prevention and early intervention. deCODE has led the way in discovering validated genetic risk factors for diseases with a major impact on public health, and in bringing to market products that put this knowledge in the hands of individuals and their doctors. Quality – in our world-leading science and in-house genotyping and data analysis – sets us apart from our competition in the field of personal genomics. CAP certification serves to emphasize this advantage and will enable us to provide our products to an ever wider public," said Kari Stefansson, CEO of deCODE.
Protein "Intentionally" Terminates own Synthesis by Destabilizing Synthesis Machinery — the RibosomeNews
A research group has discovered that a protein, during its synthesis, may destabilize the structure of the ribosome and end its own synthesis prematurely, and found that this phenomenon is used for adapting the cell to its environment.READ MORE
What Makes Cancer Cells Metastasize?News
Metastatic spread of tumor cells from one location in the body to another is the cause of 90 percent of cancer-related deaths.READ MORE
Revolutionary Imaging Technique Uses CRISPR to Map DNA MutationsNews
The new high-speed AFM method can map DNA to a resolution of tens of base pairs while creating images up to a million base pairs in size. And it does it using a fraction of the amount of specimen required for DNA sequencing.READ MORE
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3rd Annual NGS Data Analysis and Informatics Conference
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3rd Annual Genome Editing & Engineering Conference
Feb 08 - Feb 09, 2018


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R | | Comedy, Romance
Lester is an occasional substitute teacher and he's very jealous. He is jealous about the last boyfriend of Lester's slightly wacky current partner Ramona - arrogant best-selling author ... See full summary »
On The Man Who Shot Liberty Valance marquee that appears in the film, a quote ("a classic") is attributed to G Brown. The critic in question is Georgia Brown, famed Village Voice film critic and mother of writer/director Noah Baumbach.
In the last paragraph Dashiell quoted from Gustave Flaubert's The Sentimental Education, "In every parting there comes a moment when the beloved is already gone from us."
Numerous occurrences. At one point, not only is the mike visible, the *boom* is visible.
After the final credits Lester, Vince and Lint are shown in an additional scene in the diner. They apparently have been playing dominos and Lint is somehow offended and is packing up his set while accusing Vince of still having a 20-sided die from their D&D days.
$30,732 (USA) (7 June 1998)
$287,752 (USA) (19 July 1998)
25 April 2017 12:39 PM, -05:00
Noah Baumbach Tells All: 7 Things We Learned About His Craft During a Candid Tribeca Talk
22 July 2015 10:01 AM, -05:00
The Playlist
Exclusive: Listen To 3 Tracks From Dean Wareham & Britta Phillips' Score To Noah Baumbach's 'Mistress America'
Tim Kash discusses actors turned director, including Michael B. Jordan, and Kerri Doherty talks about "Grey's Anatomy," the ground-breaking 300th episode, and the career of its creator.
Dive deep into everything superheroes, Star Wars, Fall TV, and more.


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Title: CAE Development Engineer
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Silkworm posterior silkgland is a model for studying intracellular trafficking. Here, using this model, we identify several potential cargo proteins of BmKinesin-1 and focus on one candidate, BmCREC. BmCREC (also known as Bombyx mori DNA supercoiling factor, BmSCF) was previously proposed to supercoil DNA in the nucleus. However, we show here that BmCREC is localized in the ER lumen. Its C-terminal tetrapeptide HDEF is recognized by the KDEL receptor, and subsequently it is retrogradely transported by coat protein I (COPI) vesicles to the ER. Lacking the HDEF tetrapeptide of BmCREC or knocking down COPI subunits results in decreased ER retention and simultaneously increased secretion of BmCREC. Furthermore, we find that BmCREC knockdown markedly disrupts the morphology of the ER and Golgi apparatus and leads to a defect of posterior silkgland tube expansion. Together, our results clarify the ER retention mechanism of BmCREC and reveal that BmCREC is indispensable for maintaining ER/Golgi morphology.
In eukaryotic cells, diverse endomembrane compartments communicate with each other through different kinds of vesicles, including clathrin-coated and coat protein I (COPI)4/COPII vesicles. The COPI vesicle is coated by the COPI complex, which is composed of seven coatomer subunits: α-, β-, β′-, δ-, ϵ-, γ-, and ζ-COP (1). COPI vesicles are well characterized for the retrograde transport from the Golgi apparatus to the ER network. Luminal proteins with ER retrieval signal, such as KDEL, are recognized by transmembrane receptors and retrogradely transported by coat protein I (COPI) vesicles (2). The deficiency of COPI transport dramatically impairs the architecture of the endomembrane system and secretory pathway (3–6).
Intracellular transports are driven mainly by microtubule-based motor proteins, including dynein and kinesin (7, 8). Human and mouse Kif5b belong to the Kinesin-1 subfamily and are the major anterograde kinesins involved in transporting protein complexes, vesicles, and organelles (9–11). In the posterior silkgland (PSG) cells of silkworm, Bombyx mori, an abundant amount of fibroin, a major component of silk, is synthesized and secreted into the glandular lumen. This highly efficient protein synthesis and secretory pathway is guaranteed by a complicated cytoskeletal network and extensive endomembrane system (12, 13). Recently, in silkworm PSG, we identified a novel member of the Kinesin-1 subfamily, BmKinesin-1, and proposed its roles in transporting fibroin-containing secretory granules and exuperantia-containing RNA-protein complexes through its cargo binding domain (CBD) (14). Therefore, we proposed silkworm PSG as a model for studying kinesin-driven intracellular transport (14).
BmCREC (previously known as Bombyx mori DNA supercoiling factor, BmSCF) belongs to the CREC protein family, and was reported to supercoil DNA in vitro (15, 16). Its homologous gene in Drosophila encodes two isoforms, with one in the nucleus and the other in the cytoplasm (17). The mammalian CREC members include calumenin, reticulocalbin-1, reticulocalbin-2 (ERC-55), reticulocalbin-3, and Cab45, whose subcellular localizations are radically different (18).
Here, using the PSG model, we identify BmCREC as a luminal cargo of BmKinesin-1-driven COPI vesicles and find that BmCREC is recognized by the KDEL receptor (KDELR), and retrieved to the ER through COPI retrograde transport. The ER resident BmCREC is indispensable for ER/Golgi morphology maintenance, as well as PSG tube expansion.
Silkworm strain and BmN cells were used as reported previously (14).
The CBD of BmKinesin-1 and full-length BmCREC were inserted into the vector pGEX-6P-1 (GE Healthcare). BmCREC-EGFP (C-terminal enhanced GFP-tagged) was inserted into the pFastBac-1-based pFastBac-hr5/IE1 vector as reported previously (4, 19). EGFP-BmCREC (EGFP inserted after the signal peptide), EGFP-BmCREC# (without HDEF), CFP-BmCREC and YFP-KDELR, with the fluorescent protein inserted after the signal peptide of BmCREC or KDELR, were inserted into the pFastBac-hr5/IE1 vector. See supplemental Table S1 for primer information.
For BmCREC rescue, the first 300 nucleotides of BmCREC coding sequence were mutated to ATGCTACGATTCTTTTTCGTCTTGCTCGCTATATCCATGGTATTTGCCGGCGTTCCTACCAACGAGGAAACAAAAAGACTAATGGACCACTTATCGGACGCAGAGCACTACCGGAACGAACACCACAATAAACAATTCGACCACGATGCTTTCCTAGGCGAAGATCAAGCGAAGACGTTTGACCAGTTGTCTCCGGAGGAGAGCAAACGACGGTTGGGTGAGATCGCTGATAAAATTGACTCCGATCAAGACGGATTCATTACATTGGTAGAACTAAAGGACTGGATTCGGTACACCCAA, with the underlined nucleotides being mutated.
Isolation of total RNA and quantitative real-time PCR were performed as described previously (4). See supplemental Table S2 for primer information.
Anti-BmCREC and anti-EGFP rabbit polyclonal antibodies were produced and purified according to a previous report (14). Anti-silkworm α/β′/γ-COP (4), anti-BmKinesin-1 (14), anti-calnexin (Abcam), anti-EGFP (MBL), anti-Drosophila GM130 (Abcam), anti-KDELR (Abcam), anti-lamin (20), and anti-tubulin (Sigma) antibodies were also used.
Pulldown assay with silkworm PSG homogenates was performed as reported previously (14). For immunoprecipitation in PSG, the homogenates were incubated with the affinity-purified antibodies in lysis buffer (20 mm HEPES, 320 mm sucrose, 120 mm NaCl, 2 mm EGTA, 1 mm MgCl2, pH 6.8). The homogenates were then incubated with precleaned protein A-Sepharose beads, followed by extensive washing and sample preparation for SDS-PAGE separation. For immunoprecipitation in BmN cells, cells were washed in lysis buffer (20 mm HEPES, 120 mm NaCl, 5 mm EDTA, 1% Triton X-100, pH 6.8) and subjected to antibody incubation.
For identifying proteins from SDS-PAGE, the protein bands were separately excised and digested with sequence-grade modified trypsin (Promega). These samples were subjected to LCQ Deca XP Plus Analyzer liquid chromatography-tandem mass spectrometry (LC-MS/MS; Finnigan). The endogenous BmCREC band was also excised for Edman N-terminal amino acid sequencing using the Procise 491 Protein Sequencer (Applied Biosystems).
For determining the secretion ratio of BmCREC, BmN cells were maintained at serum-free TC-100 medium (Sigma) for 24 h. The total cell lysates and the corresponding serum-free conditioned medium were collected 48 h after transfection and were compressed to the same volume (50 μl). One fifth of the total sample (10 μl) was loaded and subjected to Western blotting analysis. Western blotting and band intensity calculation were performed as described previously (21).
For nuclear-cytoplasmic fractionation, the BmN cells were lysed in hypotonic buffer (10 mm HEPES, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT, 0.05% Nonidet P-40, pH 7.9) by lysing with ~20 strokes in a glass Dounce homogenizer and were then subjected to centrifugation at 3,000 × g for 15 min to pellet the nuclei. The nuclear and cytoplasmic fractions were then subjected to Western blotting analysis.
Cell transfection and immunostaining were performed as reported previously (14).
For RNAi, dsRNAs of BmCREC (1–300 nucleotides), β′-COP (661–961 nucleotides), BmKinesin-1 (2493–2793 nucleotides), KDELR#1 (32–291 nucleotides), KDELR#2 (300–561 nucleotides), and EGFP (1–300 nucleotides) (served as control dsRNA) were synthesized and extracted using a MEGAscript Kit (Ambion). See supplemental Table S3 for primer information. For knockdown in silkworm larvae, 1–2 μg of dsRNAs was injected into the hemolymph of the third-instar larvae as described previously (4, 22). One week after injection, PSG were dissected and photographed, and the diameters of both glandular lumen and PSG tube were calculated by ImageJ (National Institutes of Health). For the pupation and cocoon analysis, dsRNAs were injected into silkworm larvae twice at the fourth and fifth instars. After pupation, the pupae were taken out, and the cocoons were collected and weighed.
Immunostaining and FRET assay were conducted as reported previously (23). For Pearson correlation analysis, cells immunostained with BmCREC and different markers were photographed with ZEISS LSM 710 confocal microscopy, equipped with a 100×/N.A. 1.4 oil-immersion objective lens (Zeiss). The photographs were then analyzed using the colocalization finder plugin of ImageJ software as reported previously (24). In each case, >15 cells were measured.
Cryosection and the following immunostaining, and electron microscopy were performed as reported previously (4). For Golgi morphology analysis in silkworm PSG, the numbers and areas of the Golgi structures were calculated using ImageJ. The diameter of the ER tubule was measured similarly.
Silkworm cDNA sequences were obtained mainly from Silkworm Genome Database (25, 26) and SilkBase (27). The phylogenic tree was constructed using MEGA 3.1 (28). The domain architecture, especially the signal peptide, was predicted by SMART (29). Statistical data were evaluated by unpaired two-tailed student's t test.
To identify novel cargo molecules of BmKinesin-1, we performed pulldown analysis in silkworm PSG homogenates using prokaryotically expressed GST-BmKinesin-1-CBD. We identified many potential BmKinesin-1-CBD-interacting proteins by LC-MS/MS assay (Fig. 1A). BmSCF was one of these candidates (Fig. 1A and supplemental Fig. S1A), and it was previously shown to be a DNA supercoiling factor in vitro (16). To investigate the in vivo characteristics of BmSCF, we produced a rabbit polyclonal antibody against the full-length BmSCF. The antibody specifically recognized a protein band of ~47.5 kDa in PSG homogenates as expected by Western blotting analysis (supplemental Fig. S1B). Both immunostaining and nuclear/cytoplasmic extraction results showed that BmSCF was not a nuclear protein (Fig. 1, B–D, and supplemental Fig. S1C), suggesting that BmSCF does not function to supercoil DNA in the nucleus as previously proposed (15, 16). As BmSCF does not belong to any subfamily of CREC proteins (supplemental Fig. S1D), we renamed BmSCF as BmCREC (GenBank accession number AEO79985).
Quantitative real-time PCR and Western blotting analysis showed that BmCREC was ubiquitously expressed in various silkworm tissues, including brain, Malpighian tubule, fat body, silkgland, and midgut, and was relatively higher in midgut in terms of both mRNA and protein levels (supplemental Fig. S1, E and F). The expression levels of BmCREC during the seven developmental days of the fifth-instar PSG were also determined. Although the mRNA levels of BmCREC were relatively higher from the 1st to the 3rd developmental days (supplemental Fig. S1E), the protein levels were stable, except for the last 2 days (supplemental Fig. S1G), possibly due to programmed cell death of PSG cells caused by pupal metamorphosis (30).
To confirm the association of BmCREC with BmKinesin-1, we performed pulldown analysis again with PSG homogenates, and the endogenous BmCREC was detected bound to GST-BmKinesin-1-CBD (Fig. 1E). Immunoprecipitation assay further confirmed the interaction of BmCREC with BmKinesin-1 in the PSG (Fig. 1F). Taken together, these data suggest that BmCREC is not a nuclear protein, but a novel associating protein of BmKinesin-1.
To unravel the detailed localization of BmCREC, we coimmunostained BmN cells for BmCREC and ER or Golgi marker. The results showed that BmCREC partially colocalized with the cis-Golgi marker GM130 (31) (Fig. 2A), whereas the majority of BmCREC was colocalized with the ER marker calnexin in BmN cells (Fig. 2B).
Next, using immunoelectron microscopy, we showed that the gold-labeled BmCREC proteins were preferentially localized on the membrane of both the ER and Golgi apparatus in PSG cells (Fig. 2, C and D), suggesting that BmCREC may attach to the membrane, possibly through interaction with some transmembrane proteins.
Based on the ER localization of BmCREC, its membrane attachment, and the bioinformatic prediction by SMART (29) that the N-terminal 16 amino acids of BmCREC were signal peptide, we assumed that BmCREC is an ER luminal protein. To explore this, we purified the endogenous BmCREC from PSG homogenates by immunoprecipitation with anti-BmCREC antibody (Fig. 2E) and determined its N-terminal amino acid sequence by standard Edman degradation. Matured BmCREC protein started from position 17 of its deduced amino acid sequence (Fig. 2F and supplemental Fig. S2A), implying that the N-terminal 16 amino acids of BmCREC were cleaved as a signal peptide. This result was consistent with the bioinformatic prediction. To further confirm this, BmN cells were permeabilized with Triton X-100 or digitonin before immunostaining. Triton X-100 permeabilizes both the plasma membrane and the intracellular membrane, whereas digitonin selectively permeabilizes the plasma membrane (32). The signal of BmCREC was detected only after Triton X-100 treatment (supplemental Fig. S2B). Collectively, these data suggest that BmCREC is localized in the ER lumen.
To identify interacting proteins of BmCREC, we performed immunoprecipitation with anti-BmCREC antibody using PSG homogenates and identified α-COP by LC-MS/MS assay (Fig. 3A and supplemental Fig. S3A). Besides, β′-COP was also detected by LC-MS/MS assay in the pulldown precipitates of GST-BmKinesin-1-CBD (supplemental Fig. S3, B and C). As both α-COP and β′-COP are subunits of the COPI complex (33), we hypothesized that BmCREC is associated with the COPI complex. To this end, we performed GST-BmKinesin-1-CBD pulldown experiments again, and the results revealed interaction of COPI subunits with BmKinesin-1-CBD (Fig. 3B). Immunoprecipitation analysis further confirmed that both BmCREC and BmKinesin-1 were associated with COPI subunits (Fig. 3C). Consistently, immunostaining showed partial colocalization of BmCREC with α-COP in BmN cells (Fig. 3D). Together, these data suggest that BmCREC is associated with the COPI complex.
The major function of COPI vesicles is to retrieve escaped ER proteins from the Golgi apparatus back to the ER (2, 33). The association of BmCREC with COPI prompted us to investigate whether BmCREC is retrogradely transported by COPI vesicles. To address this, we first examined whether disruption of the COPI retrograde transport affects the subcellular localization of BmCREC. In normal BmN cells, only a little portion of BmCREC was secreted into the conditioned medium (Fig. 3E). However, knockdown of β′-COP resulted in a dramatic increase of secreted BmCREC protein level and a decrease of intracellular BmCREC protein level (Fig. 3F), with no significant alteration of its mRNA level (Fig. 3G). Moreover, immunofluorescence images showed that knockdown of β′-COP induced punctum accumulation of BmCREC in BmN cells (Fig. 3, H and I). However, these enlarged cytoplasmic puncta of BmCREC were not colocalized with the cis-Golgi marker GM130 (supplemental Fig. S4), although COPI knockdown facilitated BmCREC entering the secretory pathway (Fig. 3F), which might lead to an increase of its localization in the Golgi apparatus. Thus, the identity of these BmCREC puncta needs to be further defined. To sum up, these data suggest that COPI deficiency leads to the failure of BmCREC to be transported back to the ER and the subsequent increase of its secretion.
The endogenous BmCREC was localized in the ER lumen, but we found that a large proportion of BmCREC-EGFP (C-terminal EGFP-tagged) was secreted to the extracellular space (Fig. 4, A and B). The C-terminal sequences of BmCREC are HDEF, which is proposed to be a retention signal to retrieve target proteins to the ER (34, 35), although it is believed to be inefficient (36). Accordingly, we speculated that the C-terminal EGFP tag might mask the HDEF retention signal and subsequently suppress the retrograde transport of BmCREC by COPI vesicles and increase its secretion. We then examined the secretion of EGFP-BmCREC and EGFP-BmCREC# (without HDEF), in which the EGFPs were inserted after the signal peptide (Fig. 4A). Compared with EGFP-BmCREC, a much higher amount of EGFP-BmCREC# protein was detected in the conditioned medium (Fig. 4C), indicating that HDEF motif is crucial for the ER retention of BmCREC.
The HDEF motif resembles the consensus ER retention signal KDEL, which is recognized by the KDELR and confers ER localization of target proteins (37). To study whether KDELR also recognizes the HDEF motif of BmCREC, we coimmunostained BmN cells for BmCREC and KDELR and showed their partial colocalization (Fig. 4D). We further used a FRET assay to confirm their interaction in BmN cells. The fluorescence intensity of CFP-BmCREC (donor) was increased after YFP-KDELR (acceptor) bleaching, and CFP-BmCREC and YFP-KDELR revealed approximately 15% FRET efficiency (supplemental Fig. S5), suggesting a direct interaction of BmCREC with KDELR. Furthermore, both β′-COP and EGFP-BmCREC, but not EGFP-BmCREC# (without HDEF), were immunoprecipitated with KDELR-HA (Fig. 4E), indicating that KDELR mediates the association of BmCREC with COPI, and the recognition of BmCREC by KDELR is mediated by its HDEF motif. Moreover, knockdown of KDELR enhanced the secretion ratio of BmCREC (Fig. 4F). Also, less β′-COP was immunoprecipitated with BmCREC in KDELR knockdown cells than that in control cells (Fig. 4G). Taken together, these data indicate that BmCREC is recognized by the KDELR through its C-terminal HDEF motif and subsequently captured by the COPI vesicles.
To investigate the physiological function of BmCREC, we knocked down BmCREC in BmN cells by dsRNA transfection (Fig. 5A). In BmN cells, there are two types of Golgi apparatus distribution patterns. The Golgi apparatus in most cells was dispersed throughout the cytoplasm, whereas in approximately 20% of cells it accumulated around the nucleus (Fig. 5, B and C). Knockdown of BmCREC significantly increased the proportion of cells with dispersed Golgi apparatus (Fig. 5, B and C). Intriguingly, only BmCREC, but not BmCREC# (without HDEF), recovered this phenotype (Fig. 5D and supplemental Fig. S6A). These results imply that HDEF-mediated ER retrieval of BmCREC is responsible for the morphology of the Golgi apparatus.
To investigate further the physiological function of BmCREC in vivo, we knocked down BmCREC by injecting its dsRNA into silkworm larvae (Fig. 5E and supplemental Fig. S6, B and C) and examined the subcellular structures in PSG cells by electron microscopy. After BmCREC dsRNA treatment, the ER network appeared indistinct and disordered (Fig. 5F), and the diameter of the ER lumen decreased markedly (Fig. 5G). Moreover, the bubble-like Golgi structures in the BmCREC-knockdown PSG cells became fewer and smaller compared with those in the control PSG cells (Fig. 5F). We measured the numbers and areas of the Golgi structures (>0.1 μm2) and further confirmed that knockdown of BmCREC reduced both the population and the size of Golgi structures in PSG cells (Fig. 5H). Collectively, these data suggest that BmCREC is indispensable for maintaining the morphology of the ER and Golgi apparatus.
A previous report showed that a collapsed endomembrane system causes a defect in PSG tube expansion (4), prompting us to examine whether the PSG with deficient BmCREC is abnormal in tube expansion. Knockdown of BmCREC resulted in significantly narrower PSG glandular lumens (Fig. 5I and supplemental Fig. S6D). Further, we measured the diameter of both the glandular lumen (arrow a in Fig. 5I) and the PSG tube (arrow b in Fig. 5I), and determined the ratio of a to b (a/b) and found that it decreased dramatically after BmCREC knockdown (Fig. 5J), further confirming that BmCREC is required for PSG tube expansion. Knockdown of BmCREC also led to reduced cocoon weight after pupation (supplemental Fig. S6E), possibly due to the decreased secretion of fibroin (38). Therefore, we conclude that BmCREC is crucial for maintaining the morphology of the ER and Golgi apparatus, and its deficiency triggers a defect in PSG tube expansion (Fig. 6).
Silkworm is one of the best characterized model organisms, due to its suitability for large scale culture, available genome/cDNA database (26, 27), and the use of various well established techniques, such as RNAi (39). Using the PSG, silkworm kinesins were cloned, some cargo molecules of BmKinesin-1 were identified (14), and the roles of the COPI complex in Golgi morphology and PSG tube expansion were unraveled (4), suggesting that the silkworm PSG is a model for studying intracellular trafficking and key developmental processes (14, 40). Here, we identify BmCREC as a novel cargo protein of COPI vesicles driven by BmKinesin-1 in PSG and reveal its role in ER/Golgi morphology maintenance and PSG tube expansion. This study further strengthens that the silkworm PSG would facilitate identification of cargo molecules of motor proteins and serve as a model for studying intracellular trafficking.
Our data show that BmCREC is localized mainly in the lumen of the ER, whereas it was previously showed to supercoil DNA according to an in vitro experiment (15, 16). It is possible that another BmCREC isoform produced by alternative splicing may enter the nucleus to supercoil DNA. This is supported by an early report that DmSCF (the BmCREC homologue in Drosophila) encodes two isoforms of about 30 and 45 kDa. The 30-kDa isoform is transported into the nucleus to exert its function as a DNA supercoiling factor (17). Therefore, it is possible that there is another alternatively spliced isoform of BmCREC, which enters the nucleus to supercoil DNA in silkworm. However, we did not amplify any more transcripts from the PSG cDNA, possibly due to its relatively limited mRNA expression or the lack of this isoform in PSG cells.
COPI vesicles are reported to be transported by the microtubule-based motor protein Kif5b (41), and COPI deficiency leads to disrupted ER/Golgi morphology (4). However, ER/Golgi morphology remain normal in Kif5b-knock-out mouse cells (42), suggesting that Kif5b is dispensable for the Golgi-to-ER transport. Recently, a report of the role of Kinesin-2 in COPI-mediated recycling (43) reconciled the controversy, indicating that Kif5b and Kinesin-2 might synergistically transport COPI vesicles. Here, we show that BmCREC is transported by the Kinesin-1-driven COPI vesicles, but whether Kinesin-2 is involved needs further investigation.
Previous studies showed that HDEF is served as an ER retention signal (34, 35), but it is believed to be inefficient (36). Our results indicate that the C-terminal tetrapeptide HDEF of BmCREC is necessary for its ER retention of BmCREC, suggesting that HDEF is sufficient to direct BmCREC to the ER. However, although HDEF resembles the consensus ER retention signal KDEL, it is unclear whether the HDEF motif is also recognized by KDELR or other unknown receptors. Here, we find that BmCREC is associated directly with KDELR, and this association is mediated by the HDEF motif of BmCREC, suggesting that HDEF is also recognized by the KDELR and plays an important role in retrieving proteins back to the ER.
The ER and Golgi apparatus are two major members of the endomembrane system, whose morphology and homeostasis are strictly regulated to guarantee the normal physiology of a cell. Our data show that BmCREC is crucial for maintaining ER/Golgi morphology. BmCREC belongs to the CREC protein family and contains seven EF-hand domains that usually bind calcium ions (18). In mammalian cells, CREC family members were reported to regulate calcium homeostasis by various pathways such as SERCA2 and SPCA1 (44, 45). Previous reports showed that an increase in cytosolic calcium ion concentration may induce Golgi fragmentation and vesiculation (46) as well as change in ER morphology (47). Also, the ER network formation is reported to be correlated with rapid ER calcium efflux (48). Therefore, it is possible that deficiency of ER resident BmCREC induces a disorder of calcium homeostasis, which results in disrupted ER/Golgi morphology. Nonetheless, all of these hypotheses require further investigation and verification.
We thank Prof. Guozheng Zhang and Prof. Zhifang Zhang (the Sericultural Research Institute of the Chinese Academy of Agricultural Sciences) for silkworm strain and artificial diet, Prof. Chuanmao Zhang (Peking University) for lamin antibody, Prof. Li Yu (Tsinghua University) and Prof. Zhengfan Jiang (Peking University) for discussion and suggestions, and Dr. Xiaolei Su and Prof. Ian C. Bruce for reading the manuscript.
*This work was supported by the National Natural Science Foundation of China Grant 31271424, the Major State Basic Research Development Program of China (973 Program) Grant 2010CB833705, and the China Agriculture Research System Grant CARS-22-ZJ0305.
This article contains supplemental Figs. S1–S6 and Tables S1–S3.
4The abbreviations used are:
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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IBM Corp. (IBM - Free Report) recently announced its collaboration with Dream Payments to help U.S. financial institutions in faster deployment of different electronic payment solutions.
The partnership targets to address the regulations and legacy systems related problems in the payments sector that slow down the launch of products by different financial institutions. Dream Payments will be providing the partnering solutions via IBM cloud backed by VMware (VMW - Free Report) virtualization solutions.
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