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tv NBC10 News Today Weekend at 6am NBC May 1, 2016 6:00am-7:01am EDT
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right now, we're two hours away from the start of the 37th annual blue cross broad street run at 8:00 this morning. if you can't make it down, we have you covered right here at 8:00. let's look at the starting line now at broad and fisher. race time will be chilly and rainy. we'll have a look ahead. >> this is nbc 10 news today.
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it is 6:00 on this sunday. first alert meteorologist michelle rosen has a look at our weather. we're focused on the race. some spectators may want to stay home. >> yeah, that's right. maybe some of them will be enjoying it with a blanket on them. if you're not seeing the rain yet, you will see the rain. so the weather story today is rainy and cold today. as we go throughout the afternoon, we'll see temperatures into the upper 50s and low 60s. right now 49 in philadelphia, that is chilly with the rain falling. and even in the afternoon we have that easterly wind, that will keep that temperature down by 5:00. the potential for rain falling
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all day long. some of it will be heavy at times. we're tracking more showers this week. we're looking at the potential for showers, most of the ten day, with just a few breaks as we go throughout the ten day. plan on that, plan on umbrellas as we go throughout the week. the radar showing us that the rain is falling here. even though you may be seeing light showers right now, dridle from time to time, you may see heavier downpours. this is a pretty expansive storm, it will take a little while to work through. and into the early part of monday before we clear things out. tracking more showers for tuesday, and then a break. >> thank you, we'll see you shortly. >> the excitement is building for the 2016 blue cross broad
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street run. watch it right here on nbc 10. the count down clock shows that we're just about two hours away from the start of the race. katie zachary is live at broad and fisher. we hope that the spectators and fans come out, but if it is too wet for them, they can watch it here on nbc 10. >> that's right. a lot of the preparations are under way right now. you see some of our own preparations right now. we will be broadcasting the race live beginning at 8:00 a.m. and bringing you updates throughout the morning as we have been on the air throughout the morning. a lot of trucks out here helping in some of the areas where runner wills be corralled. some early bird runners like to get out here super early, hop on
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septa early, and hopefully we'll have a runner live for you coming up in the next half hour. let's look at the map. we're at the start at fisher and broad. so you can see from our map, five miles in takes you to city haul, and then five miles more takes you to the navy yard. you have spectators from all different neighborhoods of philadelphia. and personally, i have run this race several times, and that is one of the most exciting parts about the race is the people from all of the neighborhoods cheering you on. some don't have to go very far at all. they just walk out their door. some runners are worried that the rain and the weather will deter spectators. >> the pest part is all of the people that run up from north philly down to south philly with
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their signs and cheering you on. >> this is a race i would run in the rain, wind, sleet, snow, i love the energy and the atmosphere. >> those taking part will start with layers on, and then i think we will see a lot of jackets and sweatshirts being shed along the race route as it warms up. despite the cooler temperatures and rain today, it will still be cool. the nation's largest 10 miler, about 40,000 people taking part. we'll be back in the next half hour and hopefully we'll get an early bird racer here ready at the starting line. we have new information this morning on a man's body found just outside of the target store in king of prussia.
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police started searching for him after he was reported missing yesterday. police say there are no signs of foul play. >> family and friends of a murdered mother in philadelphia are calling for justice as police continue to search for her killer. operation save our city had a rally in port rich monday yesterday for stephanie whose body was found last july. her body was found in her bedroom. police believe she was shot execution style while her 2-year-old daughter stood near by. police tell us that the expect handed over a demand note around 3:00 yesterday afternoon at the branch on frankfurt
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avenue. two unions have voted down a new contract that doesn't mean a strike is coming, but it does mean more negotiations will be necessary. njt officials say all 14 other unions have ratified the new contract negotiated in march. they say they're willing to talk to the unions who have not signed on. right now there is a 60 day cooling off period in effect. that prevents a strike or a lockout. 6:07 right now on this sunday, a new endorsement for ted cruz, who is backing him? plus a check of the delegate count when we come back. and it has been nearly five years since u.s. special forces killed osama bin laden. tomorrow is the anniversary and we will be right back.
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welcome back on this sunday morning, pockets of heavier rain in the next couple hours. we're going to keep the temperatures colder. right now mainly in the lower 50s. the temperatures will not be going very far. and delaware, 48 degrees. throughout the afternoon we will see temperatures in the lower 50s. rain in new jersey, phil, all spots seeing potential for heavy rain and all spots seeing potential for thunder later this afternoon. mainly a rain event. we have rain falling.
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we have pockets here, the yellow, showing we have rain falling in some spots. it will take a little while to get through here. neighborhoods right now 49, new jersey for you. zooming in closer, neighborhood by neighborhood, pinpointing the mind to upper 40s. we'll talk more about the cool and rainy morning, and if we have any improvement in our ten day in just a few minutes. >> in decision 2016, senator ted cruz is getting more support in his bid to overtake donald trump in the republican presidential race. yesterday he picked up the endorsement of pete wilson. cruz is hoping to beat trump in the indiana primary on tuesday.
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a win there and in california would help cruise block trump from getting enough delegates to clinch the nomination. he will be talking to chuck todd on "meet the press." that is coming up right here. >> trump leads with 997, truz is second with 564, john kasich in distant third. for the democrats, hillary clinton has 2151, about 800 more than bernie standers. clinton is less than 300 delegates away from securing the nomination. >> former president bill clinton is campaigning for his wife. polls show she is leading bernie
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overnight, two people were hurt in this crash in port richmond neighborhood. a car and an suv collided here. we're working right now to find out the victim's condition. in the meantime, we can tell you that investigators are looking into the cause of this crash. and now news from overseas. iraq leaders are ordering that
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protestors be arrested. hundreds stormed the parliament building calling for an end to government corruption. iraq's prime minister ordered authorities to arrest them. tomorrow marks five years since the killing of osama bin laden. on president obama's order, navy seals stormed, shot, and skilled the al qaeda leader and four others. one of his wives identified his bo body. in keeping with islamic tradition, they buried him within 24 hours in the sea. this morning, kenya's president is sending a message to animal poachers. they set fire to thousands of elephant tusks and rhino horns.
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the ivory destroyed would have been worth $150 million to smugglers. the illegal trade in ivory must stopped. another effort to protect wild animals yesterday, this one in south africa. a group of 33 lions started new lives there last night. they were rescued from circuses in south africa. they would not survive in the wild because many have been declawed or disabling injuries, but they will live out their days in a big cat sanctuary. >> we're tracking some rain, showers, and drizzle. heavier rain in some spots. overall, it will be a dreary day. soggy sunday, the potential for rain falling most of the day. also temperatures cool in the 50s. it will will chilly right now and lots of chances for showers this week. but despite the showers, we will
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be warming up closer to normal. temperatures right now mainly in the 40s. off to a chilly start. 49 in philadelphia. same story in atlantic city. and temperatures staying put in the 50s throughout the day. we have an easterly flow and that will keep our temperatures down throughout the day, and also that brings some moisture been so we'll see some drizzle and damp conditions. 7 miles per hour in coastal, and more of the direction of the kind today that will keep us uncomfortable. we have rain falling across the area. light rain in some spots, showers and pockets of heavier rain. seeing where we have that heavier rain. we're going to continue to see that rain fall in the next several hours and the potential all throughout this sunday, the wider view showing it will take alittle while to work through
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here. and we will dry things out for just a bit on wednesday. the future cast, we'll keep that rain in the forecast through 9:00 and through 11:00, 12:00, and 1:00. so by lunch we will see rain across the area and heavier rain throughout parts of southern new jersey. pockets of heavier rain and showing you we have potential for heavier rain from time to time. 2:30, same thing, rain and thunder. most of this will be a rain event. 8:30, drier conditions. still seeing the rain and potential for heavier rain. waking up to shower activity on monday. maybe a wet one as well. weapon keep the potential for showers in the forecast on monday, and we'll bring back a little more sunshine as well. for the broad street runners
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today, they will have to face these elements here. 52 by 9:00. same story, temperatures in the low to mid 50s. we'll see hour after hour of this rain. the ten day outlook today, tomorrow, close to normal, a shower chance again on tuesday and wednesday and we will dry it out. a chance on wednesday before we dry it out once again. get the umbrella ready. >> this morning the blue cross broad street run crews, it is never too early or late to start running. nbc 10s a.pamela osborn has her story. >> pamela was not always a runner. at 55 she started with a slow
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momentum. >> she set her sights on the blue cross broad street run. >> it is such a good feeling to do it with 30,000 plus other people. >> now she is getting ready for her sixth race. >> when you run philly, you see parts of the city you never knew existed and you meet all kinds of people, that i have, running in the city. >> not just the city. >> you can go into work, you can say i ran to jersey and back this morning. >> back and forth across the ben franklin in good company. >> i started with independence blue cross 15 years ago, and they have a running club. >> they became fast friends. >> they build friendships, teams, and team work.
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>> it is friendship that keeps them going. it is part pace, part practice, and it all starts with one step. for fam, this is where she gains her stride. >> it is an accomplishment. it is another run under your belt. >> in philadelphia, alabama la oz. born nbc 10 news. >> nbc 10 has you covered for the blue cross broad street run. our coverage starts at 8:00 this morning. if you're running, dvr the race coverage to watch after you finish. >> playing alongside a master. what brought them together.
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>> 6:25 right now on this sunday. 100 celloists took the stage this weekend to play with a master this weekend. special guests yoyo ma played at the kimmel center. play ins are designed so that people of all ages can experience the music. >> and north philadelphia is helping people. there was a sixth annual shoes for life. they collected new and gently
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worn shoes for families in need. and an interactive arcade last night, visitors played locally designed games, used 3-d printers. it runs through next saturday. >> walkers and runners raised $109,000. it will go towards ms research. today is race day, runner wills be trekking across philadelphia for the blue cross broad street run. nbc 10 is your home for the race. katie zachary is live at the starting line. >> you may think that runners showing up here have nothing in
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the rain at race time. good morning, this is nbc 10 today. it is 6:30 on this sunday. let get right to meteorologist michelle grossman. she is tracking the rain. >> yes, we are tracking the potential for this rain in the past few days, and here it is. rainy and cool today, that will be your theme. potential for rain falling all day long. we're looking at it warm up and more rain into the next several hours. we're going to combine it with chilly air.
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49 right now at the jersey shore. temperatures are very chilly this morning. chestnut hill is just 46 degrees. 48 degrees in society hill. summerton coming in. a lot of the runners have to contend with that rain. still protect yourself from the rain, rain is falling, you can see heavier rain, the runner wills have to face that, too. most of this light right now, coming in in the next several hours. it will work here over the next day into the early part of monday if will not be an all-day rain later on this afternoon. we'll time it out for you. >> we'll see you shortly. the ryder cunners have their bi
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the streets are closed off. we're in the final preparation. kat katie zachary is there and dressed for the weather. >> yeah, i don't have to enter the race, i can stay in this gear, but unfortunately for so many runners that are showing up he here. how will they dress differently. what will be their mind-set and mentality when they enter the race. so two experts are joining me here. how are you? >> i'm well, i'm from new jersey. >> i'm also from new jersey. >> how many broad street runs have you done? >> my seventh in a row. >> my fifth. >> tell me about the gear you
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came dressed in, and why it is so important to stay dry. >> i m v a poncho, and warm clothes underneath. i have sneakers in here, because it is important to stay dry, your body will be cooling off after the run, stay dry and as warm as possible afterwards. >> so you planned ahead, and this is a bag you were going to put on the bus that will be for you at the race end, but some people who may not have planned ahead, should they come with extra layers to shed throughout. >> yeah, you may wear sweats and sweatshirt that you want to donate to charity, you just leave it on the side, and people will come pick it up and donate it to charity. >> what is one of the things you want to tell runners? >> be careful of the paint in the crosswalks, that is the
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slipperiest part of the road surface. >> especially for novice runners, they could injury themselves or take down other runners. good luck to you guys. >> thank you. >> we'll be back in about 15 minutes with the head of independent blue cross and why it is so significant. >> katie zachary at the starting line, thank you. good add viets from those runners. we have you covered. join us for our coverage at 8:00 this morning. if you're running or already down there. you have families running, you can go back and take a look at them. >> we have new information this morning on a body discovered just outside of a target store. a police officer found the man's
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body in a car in the store's parking lot in king of prussia last night. they started looking for him when he was reported missing last night. family and friends of a murdered mother from philadelphia march for justice as police continue to search for her killer. operation save our city held a rally yesterday for the mother. her body was found in the bedroom of her port richmond home. police believe she was shot execution style while her 2-year-old daughter sfood near by. >> my granddaughter is without a mother and it hurts me every day. rips my heart out. >> police said last summer they believe this young mother was targeted, but her friends say they do not believe she had any
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enemies. >> across the river in camden, a stop the violence rally from community leaders and residents. >> they asked if you wanted steak or fish, and a bunch our customers yof wrote in paul ryan. >> president obama gets some laughs at the white house correspondence dinner. you'll hear some of his jabs at the presidential candidates.
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all right, a good sunday morning. you definitely need the umbrella this morning. the rain is falling across the area. potential for the rain all across the day over the next several hours into the afternoon and evening hours. we're also chilly this morning. right now in south jersey with the rain falling, rain is falling there as well. north andwet, the lehigh valley. 45 in the pa suburbs. a chilly start to your day. temperatures mainly in the mid to low 40s this morning. 40 in fleetwood. 47 right now in nazareth. chilly throughout the day. an easterly wind. we have rain falling for a damp
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and dreary day. here is a look at the radar right now. light rain for the most part. you notice some spots of yellow. we can't rule that out for the next few hours. here is a look to the north. reading has yellows and heavier downpours. further down to the south, main parts of southern new jersey sees heavier reiy ieier rain as. here is a live look, a bigger view, this is why we keep the threat for rain all throughout the morning. even into your monday morning. we will track it out for you, also tracking, finally a warm up, in your ten day. we'll do that in a few few minute. now to the financial crisis in atlantic city. a $2 million bond payment will
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be due on friday. the city will likely come up with the money. no city or town in new jersey has defaulted since the 1930s great depression. expect detours for the next week and a half. a railroad crossing on the connecter road on the expressway. several on and off ramps will be closed. just tap the nbc 10 app for a link to traffic information and updates for updates on this project. the nfl draft is now history. who the eagles selected in the later round. we'll also hear from howie roseman. we'll get you inside with this story, next.
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right now, runners are getting ready for the blue cross broad street run. nbc 10's katie zachary is at the starting line and she has a special guest. >> yes, i have dan hiere with m. >> it is the 37th year, and sometimes it rains, and sometimes it is sunny, you never know. it is the best run in the country, 10 miles. >> we hold it with pride here in
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philadelphia. why is it so important? >> we're about helping people get well and stay well. we give them opportunities whether it is the walk enan lunch, the indigo bike share program, anyway we can influence people to get up, be physical, and state healthy. >> how many years have you run it, and any advice you can give to your employees? >> go slow, i will go very slow. i have probably run it about ten times. my goal today is to just drot do -- trot down broad street. >> we have a gym at independence blue cross. they have been putting in miles
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there. they train together and get ready for this. they fall slowly back into the race -- >> you have done this ten times, i have faith in you. thank you so much. we'll send it back to you. >> katie, we want to remind you you can watch the blue cross broad street run right here on nbc 10 starting at 8:00 this morning. we have reporters all across the race route. if you're running or you know someone that is running, set your dvr so you can go back and look at yourself, family, and friends. call him comedian in chief. he played that whole, he fired a
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barrage of jokes toward donald trump. >> in fairness he spent years meeting with leaders from around the world. just this week -- >> the president didn't write the jokes, but he delivered them very well. he didn't spare the democrats. he spots white house candidate bernie sanders and he could not resist. bernie, you like lie a million bucks, or in terms you understand, 37,000 donations of $27 each. >> proceeds go towards journalism scholarships and
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awards. we continue to track the rain on this sunday morning, lots of runners are excited and we will get them through the race. wet roadways here, and that is what you are encountering. so a soggy sunday, a chilly start. more showers, several chances for showers in our extended forecast, and then we're warming up into the upper 50s. right now, for this time of year, we're chilly. temperatures mainly in the 40 and 49 range. temperatures will stay down throughout the day, as we zood in closer, odessa coming in at 47, you need the layer this is morning and rain gear, the rain
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is falling, so that is the story of the temperatures. it brings you that moisture, keeps us with that drizzle, and keeping it cool, winds at 5 to 10 miles per hour. not that bad in terms of speed. doppler radar showing the rain across the area. that is the story in the next several hours. you can see yellow showing up and it is indicating heavier showers. it is mainly a rain event. . so it is pretty big and it will get through here. future cast going throughout. there is heavier rain.
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10:00 that rain is still falling. same story by 3:00, recovering from the race and rain falling in philadelphia. periods of rain throughout the afternoon and evening. still seeing rain in the future cast and clouds and potential for showers throughout your monday before we clear it out on wednesday. here is a look at your broad street run, the timeline here. 11:00, the rain will be falling pretty steady. it is a chilly rain. the next five days we will be warming up. back down to the 60s, but we will see temperatures closer to normal on wednesday and thursday. >> good morning to you, i'm
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danny pommells. the eagle take a delaware native wendell smallwood who led the big 12 in rushing. he comes with baggage though. shortly after he was drafted, he has had highly questionable activity. >> i want to tell my story, and give them the real -- let them know that is nothing anything that i would ever be involved in again. i think them getting to know me, seeing the kind of man i am, chanced that perception of what was going on outside. >> once you were drafted, people gravitated to your twitter
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account. what happened there? >> i was young, irresponsible, saying embarrassing things that don't represent who i am or what i stand for. i'm real embarrassed that anyone had to read that. >> the eegs selected a defensive tackle. defensive linemen and a line backer. here is howie rosen. >> there is not as many picks in the early going, so we felt like later in the draft, taking shots on guys in the 7th round, really having the best process we ever had became a priority for us. we're excited about that and we may have done it in terms of taking guys that could have a
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role and have athletic tools that need to be developed. >> locally owls were selected, tayvon young, matthew ioannidis. and the phillys took a four-game winning streak last night. top of the first, not wasting any time, third pitch of the game with a man on. puts one into the right-field seats. his third of the season. later, tie game, this time spotted some green grass in right. that scores a run. the phillys up 4-3. can they hang on though. chasing it, reels it in with a nice sweeping grab. the phillys with their fifth straight break them up.
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seth williams and el chavez squared off in a match. the event raised money for the urban league of philadelphia. age is just a number, it's all relative. anyone can compete in the opinion relaypenn relays, take a look. keep your eye on number 19 here. this is 100-year-old idis. she competed in the 100 meter dash. you could say that ida stole everyone's heart setting a new record at 1:17.33 as a 100-year-old runner.
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excitement is building for the blue cross broad street run. we're about an hour away. >> we're live at the beginning opponent of the independence blue cross broad street run. you see runners are beginning to file in. they're going to get set not far from now. why you want to steer clear of these painted lines along broad street. >> we are tracking rain, gray skies, and skilly air, we'll have your full forecast coming up.
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in just one hour, some 40,000 people will be pounding the pavement for the blue cross broad street run. we'll take you live along the course all morning long. it won't be hot today. the runners will have to contend with the rain. a system is moving through our area and with it will come showers. now to a live look outside, we're tracking the chilly and wet conditions along the race
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Five Pawns BLUE debuts with International Partners
Five Pawns BLUE debuts with International PartnersMay 14th, 2018
Interesting parallels between the United States and international markets.
"It was a sensory overload when walking into the Hall of Vape in Stuttgart Germany, with large booths and bright colors all working to attract the attention visitors. We heard many of the same comments from customers and shop owners who visited the Five Pawns booth. Most of the e-liquids being promoted at the show seemed to taste exactly the same. We heard repeatedly how most of the e-liquids being sampled were super sweet with fruit or dessert notes being overpowered with sucralose. It was refreshing to hear from many attendees, that Five Pawns stood out as an adult positioned brand that has always represented it's products in the most professional manner and without relying on the use of artificial sweeteners. Being different is often difficult but standing alone in our core passion is always the most exciting.
There are some great e-liquid manufacturers in our industry both here and abroad, yet when you look at the overall landscape of e-liquids companies in our industry, there seems to be a common theme…make it overly sweet.
Five Pawns has always believed in creating unique complex flavor profiles without the use of artificial sweeteners. With the launch of our original flavors in 2012, we set a new benchmark for premium natured products. With the recent addition of the previously grandfathered Blue series, we feel we've set the bar even higher. The BLUE series utilizes Artisan Sea Salts to enhance the complex flavor profiles and allows for a higher VG product offering. This isn't something Five Pawns is traditionally known for and is something different than what is commonplace today."
Gavin Tucker Partner / COO Five Pawns LLC
In this video, Gavin Tucker of Five Pawns, explains some of the attributes of the BLUE sea salted flavor blends at the Hall of Vape show in Stuttgart, Germany.

subset: commoncrawl


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Russia receives two upgraded Tu-95MS strategic bombers
Russia's Aerospace Force has received two upgraded Tupolev Tu-95MS strategic missile-carrying bombers.
The Tu-95MS strategic missile-carrying bombers have delivered to the Russia's Aerospace Force by Taganrog-based Beriev Aircraft Company.
The Tu-95MS strategic missile-carrying bomber is designated to accomplish the tasks of striking vital targets in remote military-geographical areas and deep in the rear of continental theaters of military operations, employing nuclear missile weapons. A modernized bomber is furnished with a new control system and hardpoints for Kh-101 missiles that have proven their efficiency in Syria.
The Taganrog-based Beriev Aircraft Company (part of Russia's United Aircraft Corporation) is the developer of new aircraft and aircraft prototypes and serially produces planes of various designation.


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A volcanic backdrop, superb climate and long, lean coast makes Lanzarote holiday destinations ideal for beach breaks. For such a small island, you'll find great diversity here too. From calm, timid waters to breaking waves ideal for surfing, Lanzarote beaches truly do have it all. Like all the islands in the Canaries, Lanzarote emerged from the sea as a result of volcanic activity. Yet, unlike the other islands, this one has few black sand beaches. Instead, you'll find mainly beautiful white and golden sands in this guide to the best beach holidays Lanzarote has to offer.
At the north of the island away from the biggest resorts, La Garita is popular with locals who come to the area for camping. The coast in Arrieta is protected by the spurs that form the Peñas del Chache massif, creating a warm, calm microclimate. The fine golden sands gently slope down to the sea and waves form at one end making it a good spot for local surfers. There is a beach bar but you'll find more food in the village.
One of the smallest but prettiest beaches, Playa Chica lures visitors with its calm waters and underwater scenery. It sits in a sheltered bay in Puerto del Carmen, an oasis of calm in the island's party capital. Get here early to enjoy the beach at its quietest, or dive under the surface in search of the local 50-kilo grouper fish and some of Europe's most spectacular reefs. Indeed, the area has been declared a Special Area of European Conservation.
As well as having a fun name, Playa Flamingo has a fun spirit. A local spot for those staying in Playa Blanca it provides respite from the busy, breezy beach to which the area shares its name. With plenty of parasols for everyone and great cafes and bars lining the promenade behind, Playa Flamingo is the place to sunbathe in peace. It's also easy to entertain young children and keep an eye on them in the curved bay.
A curve ball this one but if it's isolation you crave, hop on to a ferry from Órzola to La Graciosa, the Canaries unofficial eighth isle. Allegedly the setting for Robert Louis Stevenson's Treasure Island, it certainly feels like an adventure trekking across to this idyllic beach. These days it's more popular with yacht-goers than pirates, but you'll have plenty of space to breath in the clean ocean air whilst enjoying the spectacular scenery.


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Indigo Homes - Display Homes
Our Rochedale display leases have ended and the displays are now closed. Follow us on facebook and social media or watch this space for further developments by Indigo Homes.
If you have any building enquiries please contact head office.
With Indigo Homes our approach to the quality of our display homes is simple.
We want you to know that the quality you see in our display homes is the quality you'll have in the home we build for you.
The quality you see is the quality you'll get.
We can deliver it because our many years of experience building homes have helped us design - and continue to evolve - a robust quality management framework that the entire company is part of.
At Indigo Homes we pride ourselves on being able to specialise in narrow lot designs, large traditional designs, and sloping site designs to enable us to give our customers whatever design they need to fit any kind of block.
With Indigo Homes you can "Relax with Us".
St Lucia 310-Rochedale Lot 5 Finch Parade, Rochedale, QLD, 412342.52
Sierra Nevada - Rochedale 6 Finch Parade, Rochedale QLD 412342.52


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Over priced, needs re decorating! Rooms don't live up to website photos! Pool is photo shopped. Conceige is poor would not return only plus is location
Saint James Albany Paris Hôtel Spa
- Pool
- Free WiFi
- Pet Friendly
- Spa
What's nearby
- Palais Royal
- 7 min walk
- Louvre Museum
- 7 min walk
- Café Ruc
- 6 min walk
- Le Soufflé
- 6 min walk
- Paris (CDG-Roissy-Charles de Gaulle)
- 27 min drive
Choose your room
Book online or call:020 3564 4853
Your dates need to occur after 2019-10-18. Try updating your dates to see prices for your trip.
About this area
In the city centre, Saint James Albany Paris Hôtel Spa is in Paris's Paris City Centre neighbourhood, a walkable area with good shopping. Palais Royal and Place de la Concorde are notable landmarks, and some of the area's popular attractions include Moulin Rouge and Jardin des Plantes. Check out an event or a game at AccorHotels Arena, and consider making time for Aquaboulevard, a top attraction not to be missed. Our guests appreciate the hotel's central location for the sightseeing. It's also convenient for public transport: Tuileries Station is just steps away and Pyramides Metro Station is 5 minutes by foot.
What's nearby
- Palais Royal - 7 min walk
- Louvre Museum - 7 min walk
- Place de la Concorde - 11 min walk
- Orsay Museum - 12 min walk
- Opéra Garnier - 12 min walk
Getting around
- Tuileries Station - 1 min walk
- Paris Pont-Cardinet Station - 4 min drive
- Paris (CDG-Roissy-Charles de Gaulle) - 27 min drive
- Le 202 Rivoli — 1 min walk
- Café Ruc — 6 min walk
- Le Soufflé — 6 min walk
- Chez Flottes — 7 min walk
- Bar Hemingway — 9 min walk
About this property
Saint James Albany Paris Hôtel Spa
A full-service spa, a restaurant and an indoor pool are available at this smoke-free hotel. WiFi in public areas is free. Additionally, a health club, a bar/lounge and a steam room are on-site. All 200 soundproofed rooms feature free WiFi and 24-hour room service. For a bit of entertainment, LCD TVs come with cable channels, and guests will also appreciate thoughtful touches such as bathrobes and slippers.
Renovations and closures
- Swimming pool
Property amenities
- Available in all rooms: Free WiFi
- Available in some public areas: Free WiFi and wired Internet
Parking and public transport
- No on-site parking available
Food and drink
- Buffet breakfast available for a fee daily 6:30 AM–10 AM: EUR 17 per person
- 1 restaurant
- 1 bar
- 24-hour room service
Things to do
- Fitness centre
- Full-service spa
- Indoor pool
- Steam room
Family friendly
- Dry-cleaning service
- Indoor pool
- Laundry facilities
- Soundproofed rooms
- Banquet hall
- Lift
- Reception hall
Guest services
- 24-hour front desk
- Concierge services
- Daily housekeeping
- Dry-cleaning service
- Laundry facilities
- Luggage storage
- Multilingual staff
- Tour/ticket assistance
Languages spoken
- English
- French
- German
- Spanish
Business services
- Business centre
- Meeting rooms
- Conference space (7535 square feet)
- Garden
- Terrace
- 5 treatment rooms
- Body scrubs
- Body treatments
- Body wraps
- Couples treatment room(s)
- Facials
- Hydrotherapy
- Massage - deep-tissue
- Massage - hot stone
- Massage - prenatal
- Massage/treatment rooms
- Spa open daily
- Hot tub
- If you have requests for specific accessibility needs, please note them in the special requests field on the booking page after selecting your room.
- Accessible bathroom
- In-room accessibility
- Smoke-free property
Room amenities
- Heating
- Hypo-allergenic bedding
- Portable fan
- Bathrobes
- Free toiletries
- Hairdryer
- 24-inch LCD TV
- TV channels
Food and drink
- Minibar
- Connecting rooms available
- Desk
- Iron/ironing board (on request)
- Laptop-compatible safe
- Laptop-friendly workspace
- Phone
- Slippers
- Soundproofing
Nearby things to do
- Bike hire
- Hiking/biking trails
Where to eat
- Le 202 Rivoli – Restaurant with a bar. Serves breakfast, brunch and lunch daily.
We booked this room specifically because it was close to the museums and had a pool/spa. The location was great and allowed for easy walking to a lot of nearby attractions. That being said, the room was so small we would hit our hands on the ceiling if we raised our arms - including the shower. They didn't have wash cloths available for bathing. There was a set in our room when we checked in and then they said that they ran out (?) so we had to use towels as washcloths which was super wasteful and awkward. The pool is in the basement of the lobby and because of the layout of the older building, this meant walking through the restaurant, bar, lounge, and lobby in a suit to then go down to the pool. They were explicit that you had to use their supplied robe and slippers (and the robe was not big enough for any person of size). So we did not even use the pool. If you're looking for a tiny room near the museum, this is it. But if you're looking for a full hotel experience, I'd suggest another space.


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Theoretical and technical aspects of the method are examined from the viewpoint of the psychological machinery involved in the interrelationship and of methodological limitations.
The following points are covered : defining support criteria in the clinical field; teleology; observation and listening techniques; statement of need; assistance techniques.
During the practical work, the students are invited to engage in support role playacting, in small groups, on the basis of scenarios taken from actual criminological settings.
Psychological and social support is a clinical observation method used in the field of research, for individual examination and for helping others. The course seeks to provide a critical knowledge of the method and to familiarize the criminology student with the method.
The contribution of this Teaching Unit to the development and command of the skills and learning outcomes of the programme(s) can be accessed at the end of this sheet, in the section entitled "Programmes/courses offering this Teaching Unit".
Definition of interviewing. Interviewing in the field of the clinical social sciences.
Principal ideal models of reference : the psychoanalysis, the systemic theory, the cognitivo-behavioral theory, the approach of Rogers.
Chapter 1 : Contexts of talks in the criminological practices and their objectives.
Chapter 2 : Interviewing : a professional and spoken meeting; the asymmetrical professional position, the spoken communication and its misadventures, characteristics of the meeting with the child, withe the teenager, with families of different culture.
Each part of the subject is exposed with clinical illustrations.
Teorical course : 15h
A broad place(15h) is reserved for the dialogue with the students.
Talks and role plaies by small groups under the supervision of an assistant. The scenarios of the role plaies are collected near criminologists experts starting from situations really met.


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Millennials are the generation that's influencing almost everything today. From trends to which brands will be popular and which ones won't be, etc. Millennials have so much influence in our everyday lives that we aren't even aware of how significant impact they have. Our digital marketing agency Dubai concluded that if something isn't "trendy", it's because this generation decided that way. That "untrendy" company can do whatever they can, and whatever is in their power, but they can't change the fact that they're unpopular among the most important group of young people today. If your brand isn't trendy, they will not buy your products or use your services, and the decrease in your business will continue to go down. Is there anything that digital marketing can do to benefit companies that are trendy or out of trend? There are some ways to escalate trendy companies to the sky while the ones that aren't trendy can keep on fighting their way through the heavy competition.
Which Result-Focused Efforts Can Digital Marketing Implement?
Result-focused efforts that digital marketing can implement into millennial brands are vast, mostly focusing on SMART (Specific, Measurable, Achievable, Relevant, Time Related) efforts. You can read the more detailed explanation by following this article https://www.mikencube.co.uk/5-digital-marketing-objectives-examples/.
Since we need result-focused efforts, there are ways to bring them to surface, if you know how to do it. If you're not sure how to handle digital marketing strategies, contact our digital marketing agency Dubai, and we'll do what we do best to come up with the best digital marketing strategy for your company. Digital marketing has numerous methods to implement, and in this article, we'll mention some of them. The most prominent result-focused ways are:
Conversion Rate Improvement
Improving your website's conversion rate can be done by using Google Analytics. Google Analytics allows you to set a goal and track the progress. For example, you can set a goal to improve the organic traffic conversion rate by 30% by December 2019. When the goal is set, you will have reports on the conversion of your traffic landing page locations, campaigns, sources, keywords,etc.
Number Of Returned Visitors
The number of new visitors compared to the returned visitors can be calculated through Google Analytics software. This metric allows you to know the percentage difference between returned and new visitors, which gives you further insight on your website's traffic. Knowing how many visitors have returned is essential since it allows you to know more about the effectiveness of your business as well to know more about customer loyalty. With Google Analytics, you can create a compelling and measurable objective that is relevant to your specific industry.
Sales Increase
Increasing sales is the highest priority objective of digital marketing today since it's very easy to track and measure and most importantly, it improves revenues. By hiring a professional digital marketing agency Dubai, you'll allow your company to stay competitive and to increase revenue more than you've ever imagined possible. Another critical factor is KPI (Key Performance Indicators) which enables you to benchmark your progress towards measurable achievement while you'll track everything through a spreadsheet.
Reducing Bounce Rates
What is "bounce"? Bounce rate in analytics is defined as "when someone opens a page and closes it before going through the rest of the pages on your website." All of this gives you an insight into the quality of your website since every business' wish is that their visitors complete their visit with a purchase, download, or making contact. Everything below 50% bounce rate is good according to digital marketers, but that can differ from business to business.
Organic Traffic
Natural traffic from search engine results pages is also known as "organic traffic." Organic traffic is free traffic, and it's different from paid traffic. By implementing SEO to your online content, you'll be able to achieve a higher ranking on search engine result pages. Therefore, increasing organic traffic is essential for all businesses. Building quality links and implementing keywords into your content will result in better quality content, which further leads to more organic traffic.
Millennial Brands
Any favorite or trendy brands today will have a lot of organic traffic, to begin with. Millennials decide who or what is popular, and the world follows – whether it's clothing company, food chains, smartphone company, or any other industry. In the UAE, another sector is becoming very popular. The fashion industry in Dubai is on the rise, and to find more details about this topic, follow this link https://thearabianpost.com/tap/2018/02/dubai-fashion-industry-blooming-says-shaikha-hind.html.
Digital marketing agency Dubai can help any company to thrive by implementing digital marketing strategies into their business. If the specific industry is not popular among millennials, there's a good chance that it can be changed for the better.
Also published on Medium.


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Blue Jackets stun Lightning 5-1 to take 2-0 series lead
TAMPA, Fla. (AP) — The Columbus Blue Jackets are making a serious bid to knock the team with the best regular-season record out of the playoffs.
Blue Jackets take stunning 2-0 series lead over Lightning
TAMPA, Fla. (AP) — Matt Duchene had a goal and three assists, Sergei Bobrovsky made 23 saves, and the Columbus Blue Jackets stunned the Tampa Bay Lightning 5-1 on Friday night to take a 2-0...
Lightning beat Senators 5-1 for 50th win of season
TAMPA, Fla. (AP) — NHL scoring leader Nikita Kucherov had a goal and an assist to help the Tampa Bay Lightning earn their 50th victory of the season in their 66th game by beating the Ottawa...
Miller leads Lightning past Flyers for seventh straight win
PHILADELPHIA (AP) — J.T. Miller had a goal and an assist, and the Tampa Bay Lightning used a fast start to win their seventh straight, beating the Philadelphia Flyers 5-2 on Tuesday...
Miller leads Lightning past Flyers for seventh straight win
PHILADELPHIA (AP) — The Tampa Bay Lightning showed off their depth to continue their roll.
Tampa Bay was without top goal scorer Brayden Point and top defenseman Victor Hedman, and it rested...
Stamkos, Kucherov lead Lightning to 6-0 win over Stars
TAMPA, Fla. (AP) — When Steven Stamkos and Nikita Kucherov are piling up points, the Tampa Bay Lightning are tough to stop.
Stamkos had two goals and an assist, Kucherov added a goal and three assists and the Lightning beat the Dallas Stars 6-0 Thursday night.
Tampa Bay scored at least six goals for the 14th time this season against a Dallas team that had allowed the second fewest goals in the league.
Lightning top Stars 2-0 on Vasilevskiy's 2nd shutout in week
DALLAS (AP) — Andrei Vasilevskiy's second shutout in a week came with a little help from his friends on the penalty kill.
The goaltender stopped 35 shots while helping to stifle six Dallas Stars power plays, and Ondrej Palat and Mikhail Sergachev scored for the Tampa Bay Lightning in a 2-0 victory Tuesday night.
Vasilevskiy improved to 10-2-1 since his return from injury, posting his third shutout this season and the 15th of his career for the Lightning, who have an NHL-best 74 points.
Nash, Bergeron score twice, Bruins beat Lightning in Game 1
TAMPA, Fla. (AP) — Tuukka Rask tossed a broken skate blade up the ice in frustration, but hardly lost his cool.
"I didn't throw it at anybody," the Boston goaltender said Saturday. "If I threw it at somebody, I'd like to apologize, but I didn't. I just wanted to make sure that everybody saw that my blade was off."
Miller, Point help Lightning hold off Islanders, 7-6
NEW YORK (AP) — Although the Tampa Bay Lightning didn't like the way they almost gave up a four-goal lead in the third period, they were happy to get the win and move on.
J.T. Miller scored twice, Brayden Point had a goal and two assists, and the high-scoring Lightning held off the New York Islanders 7-6 on Thursday night, setting a franchise record with their 51st victory
Kings forward Dustin Brown suspended for 1 game
NEW YORK (AP) — Los Angeles Kings forward Dustin Brown was suspended for one game without pay Sunday for kneeing Tampa Bay Lightning defenseman Mikhail Sergachev.
Brown was assessed a major penalty and game misconduct for kneeing in the second period of the Kings' 4-3 loss Saturday night at Tampa Bay. The suspension will cost Brown $31,586.
Vasilevskiy leads Lightning past Blackhawks 2-0
CHICAGO (AP) — Andrei Vasilevskiy was terrific, Chris Kunitz made a clutch play and Tampa Bay's penalty killers had a big night.
The Lightning followed a familiar formula back to the top of the NHL.
Vasilevskiy made 40 saves in his league-best seventh shutout and Kunitz scored a short-handed goal in the second period, leading Tampa Bay to a 2-0 victory over the sliding Chicago Blackhawks on Monday.
Byron scores in shootout, Canadiens beat Lightning 2-1
MONTREAL (AP) — Max Pacioretty broke a long goal drought to help the Montreal Canadiens end a five-game losing streak.
Paul Byron scored in a shootout and Montreal beat the Tampa Bay Lightning 2-1 on Thursday night.
Byron was the only player to score in the shootout. Pacioretty scored in regulation for Montreal in its first victory since Dec. 22. Nikita Kucherov scored for Tampa Bay.
Pacioretty opened the scoring at 20 seconds of the second period.
Stralman, Lightning lead Lightning to 6-5 win over Avalanche
DENVER (AP) — Anton Stralman scored twice to help Tampa Bay overcame a hat trick by Colorado's Gabriel Landeskog and the Lightning beat the Avalanche 6-5 Saturday night for their seventh straight win.
Mikhail Sergachev, Victor Hedman, Nikita Kucherov and Yanni Gourde also scored for the Lightning, who have two wins against the Avalanche during the streak.
Landeskog also had two assists to go along with his second career three-goal game, and Nathan MacKinnon scored twice for Colorado.
Stralman, Lightning overcome Landeskog's hat trick for Avs
DENVER (AP) — The Tampa Bay Lightning got a timely offensive boost from its defensemen to keep their winning streak going.
Anton Stralman had two goals, and fellow defensemen Victor Hedman and Mikhail Sergachev also scored to help Tampa Bay beat the Colorado Avalanche 6-5 Saturday night for their seventh straight win.
NHL Capsules
BUFFALO, N.Y. (AP) — Andrei Vasilevskiy made 34 saves in his second shutout this season, and the Tampa Bay Lightning ended a two-game skid with a 2-0 victory over the Buffalo Sabres on Tuesday night.
Defenseman Dan Girardi and rookie Mikhail Sergachev had goals for the Lightning in the third of a four-game trip. Tampa Bay is off to the best start in franchise history (17-5-2) but had lost consecutive games for the first time this season.
Vasilevskiy gets shutout as Lightning snap two-game skid
BUFFALO, N.Y. (AP) — The NHL's highest-scoring team leaned on its defense to snap a two-game losing streak.
Andrei Vasilevskiy made 34 saves in his second shutout of the season, and the Tampa Bay Lightning got goals from defensemen Mikhail Sergachev and Dan Girardi in a 2-0 victory over the Buffalo Sabres on Tuesday night.


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Trading at binary option is no doubt the craze among modern tech savvy people who enjoy the benefits of online investment at its maximum. Whichever trading platform you choose for binary trading, there are several steps to follow in order to earn substantial rewards. In case of TopOption also, you need to maintain some basic steps which can make your options more profitable and exciting without much effort. Here we have discussed about the process to follow in order to earn money on TopOption.
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This protocol describes the rapid and highly sensitive quantification of Förster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.
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Kim, H., Han, G. H., Fu, Y., Gam, J., Lee, S. G. Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer. J. Vis. Exp. (116), e54144, doi:10.3791/54144 (2016).
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Recent improvements in Förster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 °C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.
Förster resonance energy transfer (FRET) has been widely used as a biometric sensor to detect small molecules such as sugars, calcium ions, and amino acids1-4. FRET biosensors contain fluorescent proteins, cyan fluorescent proteins (CFPs), and yellow fluorescent proteins (YFPs), which are fused to both ends of periplasmic-binding proteins (PBPs). Sugars bind to PBPs located in the middle of the FRET sensor, causing structural changes to the sensor that subsequently alter the distance and transition dipole orientation of the two fluorescent proteins at either end of the PBPs. This change enables quantitative analysis of sugar content by measuring the ratio of the emission wavelengths of EYFP (530 nm) and ECFP (480 nm). Owing to the high sensitivity, specificity, real-time monitoring capacity, and fast response time of FRET biosensors, these sensors are widely used in environmental, industrial, and medical applications5. Moreover, ratiometric measurement using FRET biosensors has important practical benefits, as it can be used to measure components in complex biological samples where the sensor concentration cannot be easily controlled and background fluorescence is always present.
Despite these advantages of FRET-based sensors for quantitative visualization, small structural changes with incomplete domain motion-transfer to the fluorescent proteins produce inherently weak signal intensity. This weak signal limits the application of FRET-based sensors for in vitro or in vivo analysis6. Consequently, most FRET biosensors require the use of expensive and highly sensitive equipment. Previously, we developed an inexpensive and portable FRET analyzer with capabilities similar to those of the existing fluorescence analyzers7. In this device, inexpensive 405-nm band ultraviolet light-emitting diode (LED) was used as the light source to cause excitation of the fluorescence signal, replacing an expensive lamp or laser. The detection system of the analyzer efficiently focuses the dissipating fluorescence signal onto two photodetectors with a silicon photodiode. In a more recent study, we showed that optimization of detection temperature at 50 - 55 °C could significantly magnify the ratiometric FRET signal8. This temperature-specific signal enhancement, along with the custom-made FRET analyzer, enables the use of FRET sensors in more general diagnostic applications with rapid and high sensitivity.
In this protocol, we demonstrated the general applicability of the FRET analyzer under optimal FRET temperature conditions by quantifying the sugar content of commercially-available beverages. This protocol provides the details of the FRET device operation, as well as a brief description of sensor and sample preparation. We anticipate that this report will promote the potential application of the portable analyzer in small-scale laboratory environments and provide a foundation for further development of an inexpensive on-site diagnostic device with FRET-based biosensors.
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1. Preparation of Biosensor
- Construct the plasmid pET21a(+)-CFP-MBP-YFP-His6 by following the previously-established protocol2.
- Inoculate 5 ml of Luria broth (LB) with a single colony of an Escherichia coli DE3 strain and incubate at 37 °C for 16 hr with shaking.
- Transfer 1 ml of the O/N culture into a 500-ml flask containing 100 ml LB and incubate at 37 °C in a shaking incubator until the optical density at 600 nm (OD600) reaches 0.5 (about 3 hr).
- Harvest the cells in a 50-ml conical tube by centrifugation at 1,000 × g for 20 min at 4 °C.
- Resuspend the pellet quickly in each tube with 50 ml ice-cold distilled water (DW) and centrifuge at 1,000 × g for 20 min at 4 °C.
- Resuspend the pellet in 50 μl of ice-cold DW with 10% (v/v) glycerol by gently swirling until the solution (electrocompetent cells) reaches an OD600 of 100.
- Place the mixture of electrocompetent cells (50 μl of the cells at an OD600 of 100) and 10 ng of the plasmid pET21a(+)-CFP-MBP-YFP-His6 in an ice-cold electroporation cuvette in an electroporation device and electroporate the mixture (18 kV/cm, 25 µF).
- Quickly add 1 ml SOC medium to the cuvette and resuspend the cells gently, followed by recovery at 37 °C for 1 hr with gentle shaking in a 15 ml round-bottom tube.
- Spread the cells on an LB plate containing 100 µg/ml ampicillin and incubate at 37 °C for 12 hr.
- Isolate a single colony using a loop and inoculate the colony in 10 ml of LB containing 100 µg/ml ampicillin at 37 °C in a shaker for 12 hr.
- Add 5 ml of the seed culture to 500 ml of LB containing 100 µg/ml ampicillin and incubate the culture in a 37 °C shaking incubator.
- Add 0.5 mM isopropyl β-d-thiogalactoside (IPTG) when the OD600 reaches 0.5 and incubate the culture in a 37 °C shaking incubator for 24 hr.
- Centrifuge the cells at 4,500 × g for 20 min (4 °C) and gently remove supernatant.
- Resuspend the pellet in 5 mL binding buffer (20 mM Tris-HCl, pH 8.0, 1 mM PMSF, 0.5 mM EDTA, and 1 mM DTT).
- Sonicate the cells on ice with six 10-sec bursts at 200-300 W, following each burst with 10 sec of cooling.
- Centrifuge the lysate at 10,000 × g for 30 min at 4 °C to pellet the cellular debris. Transfer supernatant (soluble protein) into a new collection tube.
- To achieve affinity purification of the FRET sensor proteins, load 4 ml of the cleared cell lysate onto a Ni-NTA affinity column (5-ml volume) and perform a chromatography assay using fast protein liquid chromatography (FPLC)18.
- Wash the column once with five column volumes of wash buffer I (50 mM phosphate buffer, 300 mM sodium chloride, 10 mM imidazole, pH 7.0).
- Repeat the wash step with five column volumes of wash buffer II (50 mM phosphate buffer, 300 mM sodium chloride, 20 mM imidazole, pH 7.0).
- Elute the sensor protein with five column volumes of elution buffer (50 mM phosphate buffer, 300 mM sodium chloride, 500 mM imidazole, pH 7.0).
- To concentrate and desalt the eluted sample, fill concentrator (membrane size of 10,000 MW) with up to 20 ml of sample and centrifuge for 10 min at 3,000 × g. Refill concentrator with 0.8% phosphate-buffered saline (PBS). Repeat this step twice, first filling the concentrator with 20 ml of sample, and then refilling with PBS.
- Recover the concentrated and de-salted sensor protein and store it at -80 °C.
2. Measurement of Sugar Content using the FRET Analyzer
NOTE: The details of the FRET analyzer construction were described in our previous work7.
- Prepare a detection solution of 0.8% PBS containing 0.2 µM of the sensor proteins.
- Turn on the FRET analyzer. Press the "UP" button for 2 sec to calibrate the optimal temperature. Set the temperature to 53 °C using the "UP" and "DOWN" buttons and press the "SET" button.
- For the calibration, press and hold the "UP" and "DOWN" buttons simultaneously for 2 sec. Confirm that the LED panel displays "CALIB" and press the "SET" button.
- Place a 12.5 × 12.5 × 45 mm (length × width × height) rectangular parallelepiped vessel (cuvette) containing only PBS buffer into a cuvette holder of the analyzer and press the "SET" button.
- Replace the cuvette with one containing only the detection solution (see 2.1) without sugar (maltose/sucrose) and press the "SET" button to calibrate the baseline.
- Replace the cuvette with one containing the detection solution with 10 mM sugar and press the "SET" button.
- To determine the sugar content of a beverage sample, put 1 ml beverage sample in a 1.5-ml microcentrifuge tube and centrifuge at 16,000 × g for 1 min.
NOTE: FRET sensor-based fluorescence measurement has the advantage of not requiring special pre-treatment of the sample because only 1% (v/v) of the sample is included in the total volume. However, we recommend removing any material that may affect the fluorescence measurement (e.g., cells, insoluble particles, lipid, fat, or any material with autofluorescence). In addition, if a strong acid, strong base, cleaning agent (detergent), or emulsifying agent (emulsifier) is present at a high concentration and may affect the properties of the FRET biological sensor, it should be removed using an organic solvent or a neutralizer. For example, when dairy fat and emulsifiers are eliminated from frozen snacks, the samples are centrifuged in a microfuge tube at 16,000 × g for 30 min, and the liquid between the bottom sediment and the top layer of dairy fat is extracted. An equal amount of hexane is then added, followed by centrifugation at 15,000 × g for 30 min to eliminate lipids.
- Remove the supernatant with a 1-ml syringe and filter it through a syringe filter (pore size 0.2 µm).
- Place 0.1 ml filtered beverage sample in a 1.5-ml microcentrifuge tube containing 0.9 ml PBS and vortex gently.
NOTE: It is critical to dilute the beverage sample properly. In this case, 1,000-fold dilution was performed so that the sugar concentration would fall within the dynamic range of the device. We recommend estimating the target sugar concentration in advance by referring to the sugar content in the label of the beverage.
- Add 5 µl of the diluted beverage sample (1%, v/v) to a cuvette containing 0.495 ml of the detection solution.
- Place the cuvette in a cuvette holder of the FRET analyzer and preheat the sample solution to 53 °C.
- Press the "SET" button to measure the sugar content.
Note It is possible to evaluate the FRET measurement using a multilabel plate reader or a fluorescence spectrophotometer equipped with a Peltier device for temperature control by reading the ratio at 488/535 nm7,8. For sucrose detection, follow the steps from 1.1 to 2.12 with a CSY-LH sensor2.
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Representative Results
To perform quantitative analysis of sugar content using the FRET analyzer, it is necessary to build a fitted curve estimating the target sugar concentration from the observed FRET ratio. Let r define the ratio of the emission intensity of CFP at 480 nm and the emission intensity of YFP generated at 530 nm (Eq. 1).
The dose-response curve of the FRET biosensor (CMY-BII at 53 °C) can be generated by observing the FRET ratio, r, at different sugar concentrations. The curve can then be expressed as an S-shaped sigmoidal curve as follows:
where rmax and rmin represent the signal ratio with sugar concentrations of 0 and saturated (1,000 μM), respectively; x0 represents the sugar concentration at 50% response; and p represents the slope of the response, which is close to 1 or -1. In the present study, rmax, rmin, x0, and p are 4.256, 2.672, 71.779, and 1, respectively. The concentration range from 1 µM to 1000 µM was used in the model fitting.
Using Equations 1 and 2, the sugar content of commercially- available beverages was quantified with the FRET analyzer. Two maltose FRET sensors were examined to test the signal, r, depending on various temperatures2,8. The first FRET sensor, CMY-0, is a basic FRET-based sensor consisting of CFP, maltose-binding protein (MBP), and YFP, with no linker peptides. The second sensor, CMY-BII, has a Ser-Arg linker between MBP and the two fluorescence proteins2. As Figure 1A shows, CMY-0 is not observed at measurement temperatures below 50 °C, as there is no signal difference between 0 and 1 mM maltose concentration. The signal differences of both FRET sensors were maximized between 50 and 55 °C (Figure 1)8. In order to quantify the sugar content of the three types of commercially-available beverages, a dose-response curve of the CMY-BII sensor at 53-°C was generated (Figure 2A) and the maltose content of the three samples was identified by converting the FRET ratio into the maltose concentration.
As sample A is made of grains such as rice and barley, which are important maltose sources, the sample was expected to contain relatively high maltose content (average 11.892 g/235 ml) (Figure 2A). In contrast, sample C is a sports drink that had the lowest maltose (0.29 g/250 ml) among the three beverages. These results suggest that the FRET analyzer can be used at optimal temperatures to maximize the quantification of sugar content, eliminating the need for an expensive high-end FRET-detection device.
Figure 1. FRET Signal Difference Between 0 and 1 mM Maltose using the FRET Analyzer at Various Temperatures. (A) The CMY-0 sensor showed no signal difference at different maltose concentrations at temperatures below 50 °C. (B) The CMY-BII sensor was able to distinguish the FRET signal difference between 0 and 1 mM maltose in a wide range of temperatures. In both cases, the signal difference dramatically increased in a specific temperature range (50 - 55 °C). The error bars represent standard deviation. Please click here to view a larger version of this figure.
Figure 2. Maltose Content Quantification in Three Commercially-available Beverages. (A) A dose-response curve for CMY-BII. (B) Maltose content of three beverage samples was quantified. Note that "Total sugar" indicates the quantity of all sugar (including maltose) reported by the beverage manufacturer on the beverage label. The error bar indicates the standard deviation. Please click here to view a larger version of this figure.
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This protocol allows rapid and efficient quantification of the sugar content in beverage samples, using a custom-made FRET analyzer7 at an optimal temperature for FRET sensors. The analyzer was designed with a recently-developed, inexpensive 405-nm band ultraviolet-LED as the light source and two photodetectors with a silicon photodiode. This device is more cost-effective than other comparable fluorometers. The device showed high detection sensitivity, specifically when measuring the ratio of two emission wavelength bands (530 nm and 480 nm) in an optimal temperature range for FRET sensors. Its sensitivity and intensity in detecting various sugars were superior to those of a fluorescence spectrophotometer device7.
The main goal of this protocol is to support the wide applicability of the FRET-based sensors with the custom-made FRET analyzer. While the analyzer indirectly measures sugar content via FRET sensors, it is clear that the device incorporates a number of the benefits of FRET sensors, including widely extendable genetically engineered ligand specificity, modular design, sensor concentration-independent signals, and accurate targeting of subcellular small molecules. FRET sensors are actually used to detect a wide range of small molecules, including ions9, heme10, and others. Moreover, more than 20 types of FRET constructs can be easily found and ordered through the nonprofit depository AddGene11.
Despite the wide applicability of the FRET analyzer, there are two main issues with the operation of the device. First, because operation of the device is relatively simple, sample preprocessing is the critical step that affects quality of detection, except in cases of device malfunction. In this protocol, one step (sample dilution) was sufficient to process liquid samples that were clearly transparent and contained no insoluble particles. However, other samples may require additional processing to remove insoluble materials, such as cellular or lipid components. Any autofluorescent particles that can affect the FRET signal should also be removed, as noted following Step 2.7. Secondly, quality control and connectivity interfacing with hospital information systems need to be addressed, as with all types of point-of-care testing (POCT) tools12. Since the signal quality of the FRET analyzer largely depends on the quality of the FRET sensor and on the preprocessing steps, regular quality control checks are required to ensure that measurements remain within the standard signal range for regular quality control data analysis. The FRET sensor stability and storage period, both of which are essential for further reliable applications, should be investigated during quality control checks. Creating guidelines and developing appropriate software can also address the connectivity limitation. Current versions of the FRET analyzer are equipped with RS232 connectivity for remote command line control, but wireless communication may be a feature of the next version of the analyzer, which will have an improved interface for hospital information systems.
However, FRET sensors have been engineered for substrate specificity, an approach that normally allows broader specificity2. Consequently, the FRET signal may encounter unintended interference from other ingredients, including other types of sugars in commercial beverages. Further investigations should explore how FRET sensors respond to various sugar mixtures to accurately quantify the amount of sugar. Collaboration with companies that produce the beverages will help confirm the sugar content to calibrate of the FRET analyzer.
It is anticipated that the proposed portable FRET device with various FRET sensors will be used in POCT applications. POCT is used for assessing pregnancy, blood glucose levels, biomarker proteins, infectious bacteria, and infectious viruses. POCT methods have rapid turnaround times and generally exhibit low error rates owing to the small number of processing steps. These are important advantages of POCT over the central laboratory testing approach. Hand-held portable POCT devices, such as the device described herein, have attracted increasing attention because of their potential applications in food assessment and blood sugar monitoring. In particular, glucose monitoring of blood samples in patients with diabetes requires a rapid, accurate, and cost-effective POCT method13. After the Ames research team developed the first blood glucose test strip in 1965 (using a strip that contains glucose oxidase), several technologies were proposed for blood glucose monitoring purposes12. The FRET analyzer is also available to detect glucose in blood samples with appropriate preprocessing of blood and periplasmic glucose-binding protein (MglB)14-based FRET protein.
Simple, rapid methods for food quality assessment are needed. The consumption of sugar-containing beverages is associated with a variety of diseases and syndromes, such as increased body mass index in childhood15, pediatric obesity16, and risk of stroke17. Understanding this connection necessitates accurate measurement of sugar components in beverages. Therefore, the glucose and fructose concentrations of beverages are of interest to scientists concerned with human health. This protocol demonstrates the highly sensitive performance of the FRET analyzer with optimal temperature control. The device may be used with various FRET sensors to detect various small molecules- including glucose and fructose14,15. Our portable and rechargeable device, which has a battery life of 10 - 20 hr, depending on the heating protocol, is applicable for POCT. Its simple operational protocol makes the device easy to use and eliminates the need for complicated staff training. With technical improvements, including reduction of equipment size, minimization of pretreatment steps, and identification of practical requirements for field use, this device will promote FRET-based research development in small-scale laboratory environments.
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The authors have nothing to disclose.
This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031944) and the KRIBB Research Initiative Program.
|Catalog Number
|isopropyl β-D-thiogalactoside (IPTG)
|phosphate-buffered saline (PBS)
|0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4
|2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MGCl2, 20 mM Glucose
|Resource Q
|Amersham Biosciences
|6 × 30 mm anion-exchange chromatography column
|HisTrap HP1
|Amersham Biosciences
|Quartz cuvette
|Amersham Biosciences
|a fast protein liquid chromatography (FPLC)
|Cary Eclipse
|a fluorescence spectrophotometer
|a multilabel plate reader
|E. coli JM109 (DE3)
|Electrocompetent cells
|A (Beverage)
|Korea Yakult Co. (Korea)
|Fermented drinks
|B (Beverage)
|Lotte Foods (Korea)
|Soft drink
|C (Beverage)
|Lotte Foods (Korea)
|Sports drink
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