text: WASHINGTON, Oct. 29, 2012 /PRNewswire/ -- Synthesis Energy Systems, Inc. (NASDAQ: SYMX) ("SES") is presenting today, here at the Gasification Technologies Conference (Wardman Park Marriott, booth #3), the significant accomplishments it has made in designing and constructing the Company's flagship Yima Joint Venture project in Henan Province, China. The presentation highlights the significant progress it has made in commercializing its technology both in China and other parts of the world. William E. Preston, senior vice president, global business development and licensing, SES, will present the Yima project update on behalf of the Company during the conference session "Global Gasification Projects & Technology Update." The Yima Joint Venture project, currently in commissioning, is nearing the completion of construction and preparing for first methanol production. The $250 million coal-to-methanol facility is the first phase of a planned $4 billion coal chemical complex. The joint venture was initiated in September 2009 upon completion of financing and commercial agreements which paved the way for implementation of the Company's technology in this important industrial park. The plant is designed to produce 300,000 tonnes per year of chemical grade methanol for China's large and growing chemical market. Yima Coal Industry (Group) Co. Ltd. ("Yima") is a state-owned enterprise in China and an important partner of the Company. Yima is a large, integrated coal company with extensive interests in coal mining, power generation, cement, alumina and coal to chemicals. SES' technology allows companies like Yima to efficiently monetize lower quality coal and coal waste resources for the production of valuable energy and chemicals such as methanol. "The Yima project is a significant accomplishment for the company in terms of delivering financial results, demonstrating technology performance and enabling the commercialization and adoption of our technology on a global basis," said Robert Rigdon, president and CEO, SES. "We are proud to highlight this achievement at the Gasification Technologies Conference, which is an important gathering of the world's leading industry players in the gasification space." SES' Advanced Gasification Technology SES has a unique commercially available gasification technology proven to economically process the lowest quality coal feed stocks, which represent approximately 50 percent of total recoverable global coal reserves. The company has developed significant intellectual property protecting its advanced fluidized bed gasification technology. Its primary advantages, relative to other gasification technologies, are greater fuel flexibility provided by the ability to use all types of coal (including low rank, high ash and high moisture coals, which are significantly cheaper than higher grade coals) and coal waste products and biomass feed stocks. The technology converts coal into synthesis gas (syngas) in a cost effective and environmentally friendly manner. It has low water and electricity usage and does not produce any harmful byproducts.


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odkurzacz centralny beam Any time you're in a very hustle, for a moment apparently you've crashed into every crimson elegant with the intention of is real about to section of area. A guide with site visitors repairs canister consent to you recognize all but hesitates happening ones method after that advise or perhaps unthinkingly accompany vary forwards in order to show a person throughout the discorded dots. Transfer data have a passion for as of other adherence advantages, more frequently, rallies round ones guide locate capable instinct direct for you personally. Thus you won't need to leverage some other passage recipient otherwise interchange registration. For getting period passage with a traffic-compatible machine, cleanly get some other travel recipient, for example the GTM 60 HD Digital camera Visitors Radio or else GTM 36 Make certain your current ruse transpires well-matched or procure.
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text: Ray Tindle continues to go against the grain. While other provincial publishers are contracting his company has acquired three weeklies in Essex: the Leigh and Westcliff Times, the Canvey Island and Benfleet Times and the Rayleigh and Eastwood Times. Tindle, 82, now owns more than 200 weeklies across Britain. His profits? About £10m on revenues of £50m. His secret: hyperlocality. (Via FT.com)


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Jyotirling Sashti is observed in Chaitra month. Nothing much is known about this auspicious day mainly marked in certain calendars in Western parts of India. Jyotirling Sashti 2017 date is April 2.
It is observed on the Chaitra Shukla Paksha Sashti tithi or the sixth day during the waxing phase of moon in Chaitra month as per traditional Hindu lunar calendar followed in Western parts of India especially in Maharashtra.


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quotations Twilight Saga: Breaking Dawn Movie Quotes f p @
230 votes 75 voters 40k views 20 items Follow Embed
'Twilight Saga: Breaking Dawn' movie quotes include some of the best lines from the wildly anticipated fourth installment of 'Twilight' film franchise, starring Kristen Stewart and Robert Pattinson as Bella Swan and Edward Cullen. Many of these 'Breaking Dawn' movie quotes surround the long-awaited Edward and Bella wedding, honeymoon and the result (I won't spoil this too much for those who haven't read the Stephenie Meyer 'Breaking Dawn' book yet). And yes, Taylor Lautner (Jacob) also has some great lines. Be sure to vote for your favorite quotes and, Twihards, add your faves if I don't have them listed!
'The Twilight Saga: Breaking Dawn Part 1' is the first of two. This part includes the wedding, honeymoon and Bella's subsequent pregnancy. When Bella figures out that she's having a baby, Edward and the other Cullen family members, understandably, flip out. This isn't a normal infant. Things could go very badly for Bella.
'The Twilight Saga: Breaking Dawn Part 1' is the first of two. This part includes the wedding, honeymoon and Bella's subsequent pregnancy. When Bella figures out that she's having a baby, Edward and the other Cullen family members, understandably, flip out. This isn't a normal infant. Things could go very badly for Bella.
+ - 28 4
Start With Forever
Edward: "No measure of time with you, would be long enough. But we'll start with forever."
Aw, Edward (*swoon*)! And also? Forever kind of takes on a whole new meaning when you're marrying a vampire.
+ - 29 5
Last Night
Edward: "Last night was the best night of my existence."
And it was probably also the best night of existence for the TwiHard community - they finally got their Bella and Edward honeymoon scene!
+ - 11 1
Waited a Century
Edward: "I've waited for a century to marry you, Miss Swan."
Could this epic romance between Bella and Edward get any more romantic? This is a true swoon-worthy quote. It's great when a guy tells you he's waited his whole life to find you - but when that person has actually lived for 100 years, well then, it's monumentally awesome!
+ - 17 5
Don't Let Me Fall
Charlie: "You ready?"
Bella: "Yeah. Just don't let me fall, Dad."
Charlie: "Never."
Charlie offers a steady arm for glowing bride-to-be Bella, as she prepares to take her big walk down the aisle.
+ - 10 3
How I'll Remember You
Jacob: "This is how I'll remember you: Pink cheeks, heartbeat. You'd think I'd be used to telling you goodbye by now."
Dear Team Jacob: Sorry. So, so sorry. Poor Jacob is having a hard time accepting what's happening to his beloved Bella - and he's worried. He has a reason to be concerned.
+ - 7 1
Charlie's Toast
Charlie Swan: "Edward will be a good husband. I know it, because I'm a cop."
Charlie Swan has always been one of my favorite 'Twilight' characters. He utters this classic quote while toasting his daughter, Bella, and her new husband, at their wedding reception. And also? He's just a little bit drunk.
+ - 7 2
I'm Late
Edward: "What is it?"
Bella: "How many days has it been since the wedding?"
Edward: "14, why? Will you tell me what's going on?"
Bella: "I, I'm late. My period's late." (feels tummy) "That's impossible...Can this happen?"
Uh oh. Vamp sex without protection? Bad idea. Is it even possible? Apparently, yes, it is...
+ - 7 2
Mrs. Cullen
Edward (to Bella): "Don't take too long, Mrs. Cullen."
Edward addresses Bella by her new name as the two arrive at Isle Esme, their honeymoon destination. A nervous Bella is stalling, needing some "human" time.


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Salomon Team Grom Kids Complete Snowboard Package - $219.99
Salomon Team Grom Kids Complete Snowboard Package - Light, soft and strong the Salomon Team Snowboard Package will allow your mini shredder to take his skill to the next level. Quadratic Sidecuts allow a blend of elliptic curves for easy turn initiation, effortless direction changes and fluid edge to edge transitions. Teamed up with ABS sidewalls allow for a much smoother ride and less impact felt when landing. Designed with Flat profile and built bomb-proof with durable Biaxial Density Glass construction. The Salomon Team is the board of choice for groms looking to push the envelope in freestyle progression. The comfortable and easy to ride in Salomon Team binding is also included in this package. Salomon made every part of this kids binding to be fully adjustable the highback, straps, padding, heel cup everything can be tweaked to match your kids foot. Plush EVA padding on the baseplate cushion impacts, aluminum buckles cinch down in a snap, and 3D anatomic straps provide a secure fit. Rounding out this package is the user friendly and versatile Burton Grom boot. The super simple oversized Velcro straps provide adjustments on-the-fly, even with gloves or mittens on. As we all know, it is important to Jr. that he is able to do things for himself as this can instill him with a sense of accomplishment. And as parents, Burton has us covered, too. They have equipped the Grom with the lightweight DynoLite outsole that has a handy growth feature that indicates how much space is left and when it's time to remove the Room to Grow foo - $219.99


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Reports: Matt Ryan, Falcons agree to five-year, $103.75 million extension
The Atlanta Falcons and quarterback Matt Ryan have agreed to a five-year contract extension worth $103.75 million, according to multiple reports.
Ryan will receive $59 million in guaranteed salary with $63 million coming to Ryan during the first three years of his new deal. Ryan's guaranteed money is second only to the $61.5 million the New Orleans Saints gave quarterback Drew Brees in 2012.
Ryan was entering the final year of his rookie contract before the Falcons decided to lock up their franchise quarterback until he is 32 years old. His new deal will earn him $20.75 million annually.
Ryan, selected third overall in the 2008 draft, had his best season in the NFL last season, throwing for 4,719 yards with 32 touchdowns to his 14 interceptions.
THE MMQB: Austen Lane's account of losing a job in the NFL https://twitter.com/RapSheet/status/360480736282808320


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FORT MYERS, Fla. (AP) Red Sox second baseman Dustin Pedroia on Sunday downplayed the injury that sent him home from Team USA at the World Baseball Classic with a message for fans: "Tell them to calm down. We'll handle this. We'll be all right."
Pedroia was examined Saturday night by the Red Sox's team medical director, Dr. Thomas Gill, and head trainer. The injury was initially thought to be to his left oblique, but Boston medical staff said it was a strained left abdominal.
FANTASY WINDUP: Injury bug bites Red Sox
SCHEDULE: Second round schedule
Red Sox manager Terry Francona was encouraged by Pedroia's diagnosis.
"We'll treat it for a couple of days, for whatever he needs, and slowly get him back in. But the good news is it's not an oblique. So we're really relieved about that," he said.
Meanwhile, Pedroia's double play partner, shortstop Julio Lugo, is expected to have arthroscopic surgery on his knee Tuesday in Boston. Lugo played one inning in the field against the Yankees at City of Palms Park on Friday night before being removed from the lineup and sent for an MRI on his right knee.
"Obviously, yesterday was a rough day, news-wise, for the health of our middle infield. But it could have been a lot worse," general manager Theo Epstein said.
Epstein said Lugo could have a tear of the meniscus cartilage. The length of time Lugo will be out will depend on what is found.
"If, when they go in there and there's an actual meniscus injury and they repair it, then yeah, 3-4 weeks," Epstein said. "If there's nothing, it could be even less than that."
The Red Sox and left-handed pitcher Jon Lester also agreed to terms for a five-year contract extension with a club option for 2014.
Pedroia said he felt a pain while swinging a bat.
"I was just taking some flips and to be honest with you I just took one swing and they did a great job shutting me down," he said. "I kind of felt it a little bit and I grabbed at it. The hitting coaches said, Don't do anything, go see the trainer."
Pedroia said he was not concerned it would be a lingering problem, but was not sure when he would be back playing.
"Obviously, with these things, you don't want to rush it. Whenever they tell me to get out there and play, I'll do it," he said.
The team will take a cautious approach.
"We don't need him on the field the next three or four days," Francona said. "So, we ll get him completely healthy and then we'll get him back on the field."
Pedroia is no longer eligible to play in the WBC.
Francona did not cast blame for Pedroia's injury on the WBC, and said the trainers handled it as they should.
"Could he do that in the (batting) cage here, too? Yeah. So, the way it was handled was pretty good," he said.
Outfielder Jonathan Van Every is still sidelined after spraining his right ankle Thursday in Jupiter against the Cardinals.
"He's pretty sore. He's black and blue and sore," Francona said. "I said the other day it could be a couple days (but) don't know how accurate that is. He's really bruised and he's swollen, and it may be a little bit more than a couple of days."
Copyright 2009 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.
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ItemCollection.IEditableCollectionView.CurrentEditItem Property
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Property ValueType: System.Object
The item in the collection that is being edited if IsEditingItem is true; otherwise, null.
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text:
Cuesta Rey Centro Fino Sungrown
No. 60 · 6 × 50 CR60S
A medium-bodied Toro, the No. 60 is complex, smooth, and overall terrific. Its vintage Dominican long fillers maintain the silky smooth and light nature of the original Cuesta Rey. It's the addition of a Sumatra-based Ecuadorian Sun-grown wrapper that kicks it up a notch. Packaged in a box of 10, it is spicy yet smooth and at this price it's the perfect addition to the humidor.Read more
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text:
Google finally unveils Nexus 5
What is probably the worst kept secret in mobile phone history, Nexus 5 leaks had become so common in the last few weeks that we all got to know almost all there is to know about the device. From renders of the device to videos of it running the new OS, we have seen it all. But one question we all kept asking, "When?". And it seems the wait is finally over; as the Google Play Store has finally went live with the Nexus 5 listing, taking orders for the 16GB and 32GB versions, in both Black and White colors.
Everything you heard about the device is true: It runs the latest and greatest version of the Android OS: – version 4.4 KitKat. Along with the OS the device is a great upgrade in hardware specs in every way from the Nexus 4: it has a full HD 1080p resolution 4.95 inch display, a blazing fast quad-core 2.3 GHz Snapdragon 800 processor, an 8-megapixel camera with optical image stabilization, 2GB of RAM, Wi-Fi a/b/g/n, NFC and Bluetooth 4.0.
Google once again offers the phone in a very attractive price, this time around $350 for the 16GB model, and $400 for the 32GB one.
From the specs, this seems one of the best smartphones around, it's price notwithstanding. At this price, it's absolutely a steal.
Tune in for more updates from us on Nexus 5 and Android KitKat.
Update: The 16GB Black version of the phone has already been sold out within less than an hour, and the listed estimated time for shipping for it "by November 8". The other versions are still in stock, with shipping time within 1-2 business days.
About Shahriar Emil
Shahriar Emil spends an inordinate amount of time at keyboards and it is only reasonable that some of that time is devoted to writing. When he is not actively murdering cyber-infidels on a video-game he is busy creating his own characters. He fears that if he ever devoted his full attention on writing, he would become stupidly famous and won't have time for cyber-mass-murder Connect with me on Google+
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I am more a fan of Apple devices.. 😉
Thanks for sharing this article on Klinkk.


text:
|Publication number
|Publication type
|Application number
|Publication date
|Apr 25, 1995
|Filing date
|Dec 30, 1992
|Priority date
|Dec 30, 1992
|Fee status
|Publication number
|Joseph R. Lakowicz, Engin U. Akkaya
|Original Assignee
|The University Of Maryland At Baltimore
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (13), Non-Patent Citations (14), Referenced by (42), Classifications (22), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
This invention was made in part with government support from the National Institutes of Health and the NSF.
1. Field of the Invention
The present invention relates to a new class of fluorescent, calcium-binding probe compounds having visible-light excitation and emission wavelengths, and to the use of these probe compounds for making wavelength-ratiometric or intensity-ratiometric measurements of calcium ion concentrations in samples. The invention is particulary well-suited for making wavelength-ratiometric or intensity-ratiometric measurements of intracellular Ca2+ levels in which the Ca2+ measurements can be accomplished independent of the overall concentration of the probe compound.
2. Description of the Related Art
Calcium is a recognized intracellular messenger and mediator. Consequently, it is not surprising that many and varied proposals for measuring calcium ion concentrations have been proposed in the field for the purpose of facilitating the study of cell activity, such as cell response to a variety of stimuli.
To achieve this purpose, most proposals for measurements of intracellular Ca2+ use fluorescence indicators, such as Quin-2, Indo-1 and Fura-2, as described for example in U.S. Pat. No. 4,603,209 and Soc. Gen. Physiol. Ser., V. 40, 1986, pages 327-45.
However, it is difficult to design novel fluorophores which are both specific for calcium and which display fluorescence spectral shifts upon calcium binding. In fact, only a small fraction of the known chromophores display satisfactory fluorescent properties and chemical specificity in an aqueous solution.
Therefore, in spite of the past intense efforts to acquire suitable calcium ion probes, these efforts have been disappointing because of the severe limitations encountered with the heretofore proposed dyes.
For instance, the known dyes, such as Quin-2, require excitation in the UV wavelength range where autofluorescence from cells is excited. Further, the transmission of UV light in microscopic optics applications is deficient, as explained in U.S. Pat. No. 4,603,209. For example, microscope optics are traditionally corrected for chromatic aberration in the visible region of the spectrum. This creates problems in the area of confocal microscopy, which requires precise alignment of pinhole apertures for a range of excitation wavelengths.
In the case of existing wavelength-ratiometric calcium probes, these wavelengths are in the UV region, and the confocal measurements also fail because of uncorrected chromatic aberration in this wavelength region. Additionally, there are simpler and less expensive laser sources available for visible wavelengths rather than for UV wavelengths, which enable not only disc scanning confocal microscopy, but also laser scanning confocal microscopy.
Moreover, other known dyes, such as Fura-2 and Quin-2, are subject to photobleaching, and, perhaps, even more important, photoconversion to Ca2+ -insensitive forms. These insensitive dye forms are fluorescent and interfere with the determination of the Ca2+ concentration, as described in U.S. Pat. No. 5,049,673.
Therefore, because of these troublesome problems associated with the UV-excited dyes, there has been recognized a need for Ca2+ probes having improved photostability and longer wavelength absorption and emission. That is, longer wavelength absorption is desirable because the amount of autofluorescence decreases progressively as a function of increasingly longer wavelengths for excitation of the probe.
There exist in the prior art several proposals for satisfying this need. For instance, U.S. Pat. No. 4,849,362 describes longer wavelength tetracarboxylate compound chelators for calcium ions. The tetracarboxylate compounds in U.S. Pat. No. 4,849,362 are reported to experience an increase in fluorescence intensity upon excitation, and the optical responses reportedly could be determined by using a single visible wavelength laser line for excitation. However, the probes in U.S. Pat. No. 4,849,362 do not display any spectral shifts upon calcium binding and thus wavelength-ratiometric measurements are not possible with the dyes described in this patent.
That is, the field has not only appreciated the need to provide a longer-wavelength (visible light) fluorescent dye for making calcium ion measurements, but also has further envisaged the possible salutary benefits that could be obtained by the discovery of a fluorescent dye capable of enabling wavelength-ratiometric measurements of Ca2+ concentrations.
As understood in the field, the phrase "wavelength-ratiometric measurement" generally means a ratio of intensities measured at two different excitation or emission wavelengths. That is, a "wavelength-ratiometric measurement" means making a calcium concentration measurement in a sample by a process wherein the calcium ion-containing sample is excited at two different wavelengths, sequentially, and the resulting fluorescence intensities are measured for each excitation, and the ratio of the respective fluorescence intensities provides an indication of the calcium ion concentration in the sample when compared to a calibration curve.
This ratiometric measuring ability in a dye is highly desirable and often necessary because the probe concentration cannot be easily controlled (and/or the emitted fluorescence cannot be controlled) in flow cytometry, microscopy, or in cuvette studies involving cell suspensions.
The acute difficulties in developing a long-wavelength fluorescent dye which is concomitantly capable of providing such ratiometric measurements of calcium ions in intracellular samples is highlighted in recent U.S. Pat. No. 5,049,673 which proposes certain visible-wavelength fluorescent dyes of a RHOD series and a FLUO series.
However, the elusiveness of developing such long-wavelength probes that also permit ratiometric measurements is recognized in the U.S. Pat. No. 5,049,673. In this regard, U.S. Pat. No. 5,049,673 reports the inability to provide long-wavelength fluorophores which have wavelength pairs in either excitation or emission that are suitable and needed for fluorescence ratioing.
As stated in U.S. Pat. No. 5,049,673, ratioing would be extremely valuable with single cells because it cancels out variations in dye concentration and path length. Without such ratioing, it is impossible to correlate dye fluorescence intensity with Ca2+ levels in a simplified manner.
U.S. Pat. No. 5,049,673 concedes that the proposed dyes disclosed therein had only small or negligible shift in absorbance, excitation or emission wavelengths upon Ca2+ binding; consequently, such dyes of that patent are unsuitable for accomplishing fluorescence ratioing.
However, from a more general perspective, certain derivatives of BAPTA have been proposed as absorption and/or colorimetric indicators which are reported to be detectable at longer wavelengths which would shift to other wavelengths when complexed with calcium. This shifting would allow quantitative analysis for calcium without interference from UV and short-wavelength visible light-absorbing species, such as disclosed in U.S. Pat. No. 4,795,712.
However, U.S. Pat. No. 4,795,712 does not describe the ability of the dyes described therein to fluoresce, much less the application of such dyes to fluorescence detection. Instead, U.S. Pat. No. 4,795,712 is directed only to the use of certain chelating compounds to detect Ca2+ in liquids or spread (dry) samples based on changes in the visible absorbance and/or reflective spectra of samples containing these compounds.
In any event, U.S. Pat. No. 4,795,712 does not address the development of fluorescent dye compounds capable of permitting ratiometric measurements of calcium ions in intracellular environments using fluorescence detection.
Further, in general, it is known that the BAPTA chelating group is valuable in cellular systems because of its specificity for Ca2+ and low affinity for Mg2+, Na+ or K+ (R. Y. Tsien, Methods in Cell Biology, 1989, Academic Press, London; R. Y. Tsien, Biochem., 1980, 19: 2396-2404).
Another desirable feature of potential probes is the use of charge steering by cations, which are described heretofore using crown ether and azacrown moieties (H. G. Lohr and F. Vogtle, 1985, Acc. Chem. Res., 1985, 18: 65-72). According to the above-described prior art, there have been synthesized various probes which contain such substances as styryl dyes, various chromophores, and crown ethers to complex cations. These types of substances can display changes in their absorption spectra in response to cation binding. For example, see J. F. Alder, D. C. Ashworth, R. Narayanaswamy, R. E. Moss and I. O. Southerland, 1987, Analyst, 112: 1191-1192; S. Fery-Forgues, M. T. LeBris, J. P. Guette and B. Valeur, J. Chem. Soc., Chem. Commun. 1988, 385; J. Bourson and B. Valeur, J. Phys, Chem., 1989, 93: 3871-3876; S. Fery-Forgues, M. T. LeBris, J. P. Guette and B. Valeur, J. Phys. Chem., 1988, 92: 6233-6237; M. V. Alfimov, S. P. Gromov and I. K. Lednev, 1991, Chem. Phys. Lett., 185: 455-460.
However, these prior usages of crown ether chemistry resulted in low specificity and/or affinity for Ca2+, which effectively precludes the use of these probes in cellular systems. Additionally, these prior crown ether probes did not contain carboxylic acid groups or provide the opportunity for cell labeling via the acetoxymethylester-esterase trapping procedure. Also, the use of simpler fluorophores did not result in fluorescence spectral shifts (K. W. Street, Jr., Anal. Lett., 1986, 19: 735-745).
In view of the above-discussed technological backdrop, it is apparent that the field has urgently awaited the innovative development of long-wavelength fluorescent compounds which are concomitantly able to permit wavelength-ratiometric or intensity-ratiometric measurements of Ca2+ concentrations, especially intracellular calcium concentrations.
The problems noted above are overcome by the long-wavelength fluorescent calcium-binding probe compounds of the present invention which have the following general structure: ##STR1## wherein n represents an integer of 1, 2 or 3 and R represents a substituted or unsubstituted unsaturated heterocyclic group.
The heterocyclic group can be a 5- to 6-membered ring (single or fused ring), wherein the hetero atoms can be selected from oxygen, nitrogen, sulfur, selenium or tellurium, and the like. Further, as will become more apparent from examples provided hereinafter, multiple hetero atoms can be provided in the heterocyclic ring.
In a one preferred embodiment of the present invention, the heterocyclic group R represents a heterocyclic group selected from the group consisting of benzothiazole, quinaldine, thiaflavin, thiapyran, quinoline and benzoxazinone derivatives. The use of benzothiazole as the heterocyclic group is particularly preferred.
In another embodiment of the present invention, a method of making wavelength-ratiometric or intensity-ratiometric measurements of the calcium concentration in a sample, especially intracellular Ca2+, comprises contacting the sample with the compounds described above in sufficient quantity to act as a fluorescent optical indicator independent of the concentration of the probe compound.
This wavelength-ratiometric or intensity-ratiometric measurement of calcium concentration is adaptable to fluorescence microscopy and flow cytometry, as well as to standard spectrophotometry.
That is, in one embodiment for making calcium concentration measurements in the present invention, the inventive probe compound can be used as a wavelength-ratiometric probe wherein the calcium ion-containing sample is excited at two different visible wavelengths, sequentially, and the resulting fluorescence intensities are measured for each excitation, and the ratio of the respective fluorescence intensities can be processed to provide an indication of the calcium ion concentration in the sample from a calibration curve.
Importantly, this ratiometric measurement of the calcium level in the sample is accomplished independent of the concentration of the probe compound in the sample.
The term, ratiometric, as used herein, refers to the ability to make measurements of the Ca2+ level in a sample which are independent of the overall probe or dye concentration. Also, the recognized wavelength band for visible wavelengths is about 400 nm to about 670 nm. This visible wavelength band is, at times, used herein interchangeably with the term long wavelength.
As another embodiment for making calcium concentration measurements in the present invention, the inventive probe compound may even be used as an emission wavelength probe wherein the calcium ion-containing sample is excited at a single excitation wavelength and observed at two emission wavelengths.
In this embodiment, the fluorescence intensity of the probe compound is measured by illumination with a single visible excitation wavelength and the emission intensity is measured at two emission wavelengths. The level of calcium ions in the sample is correlated to and can be determined from the ratio of these two measured emission intensities.
However, the ability to practice this embodiment of the present invention depends on whether the particular probe compound fluoresces in the absence of calcium, which is either a known attribute or can be readily determined for the probes of interest within the scope of the present invention. Further, in this embodiment, in addition to fluorescence in the absence of calcium, the probe must also display a different emmission spectrum in the presence and absence of calcium.
The emission spectra display modest shifts upon binding to the probe compounds of the present invention. Also, upon Ca2+ binding in the probe compounds of the present invention, the absorption spectrum shifts dramatically to shorter wavelengths upon complexation with Ca2+. Generally, the emission wavelengths for the probes of the invention fall into the visible wavelength band.
For instance, the visible absorption spectrum of the probes of the present invention display a dramatic shift in the long wavelength maxima from 508 nm to 407 nm upon complexation with Ca2+, with a Ca2+ dissociation constant of 1.5μM. The emission maximum centered at 615 nm is well shifted from the absorption. Further, the emission spectrum displays a small blue shift upon binding Ca2+. This feature is significant because it allows those probes of the present invention to be used as a emission wavelength ratiometric probe using a single excitation wavelength.
Therefore, the inventive probe compounds of the present invention successfully combine the use of a BAPTA chelating group and a charge-sensitive dye, such as styryl dyes.
As will be appreciated the fluorescent probe compounds and their method of usage in this present invention represent a significant advance in the field.
Namely, the probe compounds of the present invention can be used in long-wavelength or visible light regions to thereby avoid the autofluorescence and other mentioned problems associated with the use of UV excitation wavelengths. Concomitantly, the fluorescent probe compounds of the present invention enable wavelength-ratiometric or intensity, ratiometric measurements to be performed on calcium ion-containing samples, i.e., measurements independent of the overall probe concentration.
FIG. 1 shows an exemplary synthetic pathway leading to an exemplary calcium binding styryl benzothiazole (STBT) Ca2+ probe compound of the present invention.
FIG. 2 shows absorption spectra of the STBT Ca2+ probe compound as a function of the following Ca2+ concentrations (a 0, (b 0.45, (c 1.06, (d 1.46, (e 2.01 (f 2.72, (g 3.67, (h 4.95, (i 6.65, (j 38 μm Ca2+.
FIG. 3 shows emission spectra of the STBT Ca2+ probe compound as a function of the following Ca2+ concentrations, excitation is at the isosbestic point, (440 nm), (a 0, (b 1.06, (c 1.46, (d 2.01 (e 2.72, (f 3.67, (g 4.95, (h 38 μm Ca2+.
FIG. 4 shows corrected excitation spectra of STBT probe as a function of the following Ca2+ concentrations (Emission at 610 nm), (a 0, (b 0.45, (c 1.06, (d 1.46, (e 2.01 (f 2.72, (g 3.67, (h 4.95, (i 6.65, (j 38 μm Ca2+.
FIG. 5 shows Kd determination by Hill and modified Hill plots (∘ and Δ): wherein fluorescence data were obtained from excitation spectra, (emission intensity at 610 nm for 415 nm excitation). The closed circles (( )) represent ratios calculated from excitation spectrum data, for λ1 =407 nm and λ2 =508 nm. The open triangles (Δ) represent ratios calculated from emission spectrum data, for λ1 =590 nm and λ2 =615 nm.
The present invention represents a new class of fluorescent calcium-binding indicator compounds which are excitable by visible-wavelength light and which enable wavelength-ratiometric or intensity-ratiometric measurements of Ca2+ levels. The probe compounds of the present invention generally relate to a heterocyclic part as bonded to a Ca2+ binding unit via an ethylenically unsaturated group. For purposes of the present invention, the Ca2+ binding unit is 1,2-bis-(2-aminophenoxy)ethane -N,N,N',N'-tetraacetic acid, which is commonly referred to as BAPTA.
For purposes of the present invention, the BAPTA encompasses substituted derivatives of BAPTA which retain the essential characteristics of the BAPTA. This definition encompasses the possible conversion of carboxylates to cell-permeable acetoxy methyl (AM) esters.
Preferred examples of the heterocyclic group include benzothiazole, quinaldine, thiaflavin, thiapyran, quinoline and benzoxazinone derivatives. Representative chemical structures of suitable heterocyclic groups, designated as R-1 to R-14, are illustrated below, wherein X can be methyl, ethyl, sulfopropyl, sulfobutyl and carboxymethyl: ##STR2##
Of the above chemical structures, R-1 is benzothiazole, R-2 is naphothiazole, R-3 is thiaflavin, R-6 is indolenine, R-7 is chloroindolenine, R-11 is methoxybenzothiazole, and R-13 is methoxyindolenine.
As mentioned above, the heterocyclic group is linked to the calcium ion chelating group, namely BAPTA, via an ethylenically unsaturated group. This ethylenically unsaturated group can be a vinyl or conjugated-double bond containing group. The conjugated chain length can be increased by an ethylene unit(s) to shift absorption/emission by approximately 100 nm per unit.
The conjugated double bond also can be provided in a hardened or rigid cyclic structure, such as illustrated below in exemplary dye structures C-1 to C-5: ##STR3##
The discussion hereinafter provides a general overview of an exemplary manner for making calcium concentration measurements of the present invention, wherein the inventive probe compound is used as a wavelength ratiometric probe. This measurement can be accomplished with conventional equipment, such as described herein. The necessary support equipment arrangements needed to make the wavelength ratiometric measurements of the present invention are generally known. However, the present invention more concerns the discovery of particular long wavelength Ca2+ probe or dye compounds which actually enable ratiometric measurements to be achieved and at visible wavelengths.
For example, the light source used to illuminate the calcium sample containing the probe compound of the present invention preferably is one capable of generating different wavelengths of light. The light source is effectively filtered through a conventional dispersive element and multiplexer to select the excitation wavelengths.
When the probe compound of the present invention is used as a excitation wavelength-ratiometric probe, the Ca2+ -containing sample is sequentially illuminated with two different excitation wavelengths λ1 and λ2 at two different time intervals t1 and t2, respectively. When the light illuminates the sample, a fluorescence is emitted as radiation from the Ca2+ -complexed probe compound for each of the two different excitation wavelengths.
This radiation, in turn, is captured by an optics subsystem and focused onto a video camera or other imaging detector, which can be selected from known equipment. In particular, the fluorescence intensity of the sample is monitored by the video camera at the same wavelength for each of the two different excitation wavelengths as I1 and I2. Each of the two light intensity signals are converted into electric signals such as output voltages.
The intensities can be measured at single points, to provide a single calcium concentration, or measured as two-dimensional arrays in which case, calcium concentration images are obtained.
In turn, these two electric signals are directed to a signal processing system, such as a microprocessor, where the ratio of the two electric signals derived from intensities I1 and I2 provides an indication of the calcium concentration from a predetermined calibration curve.
This ratiometric measurement of the calcium level in the sample is accomplished independent of the concentration of the probe compound in the sample.
A discussion is provided below on the other embodiment for making calcium concentration measurements in the present invention wherein the inventive probe compound can be used as an emission wavelength probe.
That is, in this embodiment, the calcium ion-containing sample is excited at a single excitation wavelength and observed at two emission wavelengths. In this embodiment, the fluorescence intensity I' of the probe compound is first measured at the first emission wavelength for excitation from a light source at a visible wavelength λ, and the fluorescence intensity I" is measured for the second emission wavelength after excitation at the same visible wavelength λ, for example, using the above equipment.
The level of calcium ions in the sample is correlated to and can be determined from the ratio of these two measured emission intensities I' and I" in the same manner discussed beforehand.
The use of the probes as an emission wavelength probe in making ratiometric measurements has the advantage of requiring only a single laser or wavelength excitation source. With STBT, the source could be an Argon Laser at 488 or 514 nm, or even a HeNe laser at 543 nm.
However, the ability to practice this embodiment of the present invention depends on whether the probe displays a fluorescence spectral shift upon binding calcium, which is either a known attribute or can be readily determined for the particular probes of interest within the scope of the invention.
In order perhaps further to illustrate the precepts and utility of the present invention, a description is provided herein of the chemical synthesis and fluorescence spectral characterization of a styryl benzothiazole-containing probe which contains the Ca2+ chelating group BAPTA as an integral part of the chromophoric system.
The synthesis of the new class of long-wavelength fluorescent probe compounds is exemplified in the description hereinafter by reference to FIG. 1.
The 5-carboxo-5'-methyl-BAPTA tetramethyl ester 1 was synthesized in five steps generally following a procedure disclosed in the literature (G. Grynkiewicz, M. Poenie and R. Y. Tsien, 1985, J. Biol. Chem., 260,: 3440-3450) with some apparent modifications. The 1 H NMR taken was consistent with structure 1.
Then, the hydrolysis of the methyl groups in the 5-carboxo-5'-methyl-BAPTA tetramethyl ester 1 was undertaken because the cyanine/styryl class of dyes is not generally stable under most hydrolytic conditions. Basically, a one hour reflux in 1M NaOH was sufficient to remove the methyl groups, and acidification gave the tetracarboxylic acid-aldehyde 2, i.e., 1-(2-amino-5-carboxophenoxy)-2-(2-amino-5methylphenoxy)ethane-N,N,N',N'-tetraacetic acid, in near quantitative yields. This aldehyde was reacted with N-ethyl-2-methyl-benzothiazolium iodide 3, in EtOH in the presence of small amount of Et3 N.
In greater detail, the synthesis protocol was as follows. The 5-carboxo-5'-methyl-BAPTA tetramethyl ester 1 (3.0 g, 8.5 mmol) was suspended in 1N NaOH (aq.) (60 ml). After refluxing the reaction mixture for 1 hour, a clear solution was obtained. This solution was then cooled down in an ice water bath, and concentrated HCl was added dropwise with stirring. The precipitated tetracarboxylic acid-aldehyde 2 was collected by filtration and dried in vacuo to yield a light-yellow powder (2.8 g; 98% yield). The 1 H NMR spectrum was (DMSO-d6) δ2.21 (s, 3, Ar'--CH3), 3.99 (s, 4, Ar'--H) , 7.41 (m, 2, 2 Ar--H) , 9.74 (s, 1, Ar--CHO, 10.60 (bd s, 4, --COOH); MS (FAB), m/e 519 (M+), 541 ([M-H+Na]+), 557 ([M-2H+Ca}+).
To advance to the calcium binding styryl benzothiazole (STBT) probe 4, i.e., 1-(5-(2-(2-ethylbenzothiazoliumyl)ethenyl)-2-aminophenoxy)-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid, N-Ethyl-2-methyl-benzothiazolium iodide 3 (0.295 g, 1 mmol) was dissolved in ethanol (15 ml). To this solution, the tetracarboxylic acid-aldehyde 2 (0.495 g, 1 mmol) was added, followed by triethylamine (1.5 ml). A bright-red colored dye started to form at reaction temperature (RT), and then the solution was refluxed for 30 minutes to push the reaction to completion. The solvents were then removed in vacuo, and the dark red precipitate was washed with a CHCl3 --Et2 O mixture, and dried to yield a fine calcium binding STBT powder 4. The 1 H NMR spectrum was consistent with the illustrated structure 4; MS (FAB), m/e 646 (M+), 738 ([M+4Na]+).
Fluorescence emission spectra (uncorrected) were then obtained for this probe compound on a SLM 8000 fluorometer using a Hammamatsu R928 photomultiplier tube. Excitation spectra were corrected using Rhodamine B in propylene glycol as a quantum counter with ratiometric dual-channel recording. Quantum yields were determined relative to Rhodamine 6G (ΦF =0.95 in MeOH). Fluorescence lifetimes and/or intensity decays were determined using the frequency-domain method (E. Gratton and M. Limkeman, 1983, Biophys. J., 44: 315-324, J. R. Lakowicz and B. P. Maliwal, 1985, Biophys. Chem., 21: 61-78 and G. Laczko, I. Gryczynski, W. Wiczk, H. Malak and J. R. Lakowicz, 1990, Rev. Sci. Instrum., 61: 2331-2337). These disclosures are expressly incorporated herein by reference.
Calibrated buffers with known concentrations of Ca2+ were obtained, for example, using a calcium calibration kit (two aqueous solutions buffered with 10 mM MOPS at pH 7.2, containing 100 mM KCL and either 10 mM EGTA or 10 mM CaEGTA, obtained from Molecular Probes. 1 H NMR spectra at 300 MHz were obtained using a GE-300 instrument. Mass spectral measurements were carried out at the Middle Atlantic Mass Spectroscopy Facility at the John Hopkins University, an NSF Shared Intrumentation Facility.
As shown in FIG. 2, the absorption spectra of the STBT probe displays a dramatic shift in the presence of Ca2+. In the absence of Ca2+ the absorption spectra displays a maximum near 508 nm. Addition of saturating amounts of Ca2+ results in a dramatic shift of the absorption band centered at 407 nm, with the absorption at 508 nm being decreased by about 80%. The absorption spectra at varying amounts of Ca2+ show a clear isosbestic point at 440 nm. These dramatic Ca2+ -dependent shifts in the STBT absorption spectra provide ample opportunities for exitation wavelength ratiometric measurements of Ca2+.
Importantly, the STBT probe can be excited with a range of simple and currently available laser sources ranging from the HeCd lines at 325, 442 nm, as well as the newly available HeCd line at 354 nm. The STBT probe can also be excited with several Argon ion laser lines at 364, 488 and 514 nm. And finally, excitation could even be accomplished with a green HeNe line at 543 nm. Multi-line lasers with a filter wheel, tunable acousto-optic filter or other known rapid wavelength selection device also can be used to provide ratiometric Ca2+ imaging in the present invention.
The visible absorption bands of STBT will facilitate confocal microscopy. In contrast, aberration corrected optics in the UV region of the spectrum are required for known Ca2+ probes such as Fura-2 and Quin-2. Such optics are scarce and expensive, and the use of visible wavelengths in the present invention circumvents this problem.
Calcium-dependent emission spectra of STBT are shown in FIG. 3 with excitation performed at the isosbestic wavelength of 440 nm. The emission displays a modest blue shift from 613 to 603 nm in the presence of increasing amounts of Ca2+. There is also a 65% Ca2+ -dependent increase in intensity. This combination of spectral shift with an intensity change is considered to be adequate to allow emission wavelength-ratiometric measurements of Ca2+.
While the change in the emission spectrum upon complexation with Ca2+ may appear small, the changes, nonetheless, are considerably larger than the known wavelength-ratiometric sodium and potassium probes (SBFI, PBFI), for example, see A. Minta and R. Y. Tsien, (1989), J. Biol. Chem., 264, 19449-19457.
Further, calcium-dependent "corrected" excitation spectra of STBT are shown in FIG. 4. The spectra generally mimic the absorption spectra in FIG. 2, but some residual structure is seen at 380-400 nm which is considered as due to the spectral lines of the xenon lamp used in the spectrofluorometer.
A comparision of FIG. 4 (excitation spectra) and FIG. 2 (absorption spectra) indicates that for excitation at 508 nm that the Ca2+ -dependent decrease in intensity is 5-fold, whereas the Ca2+ -dependent decrease in absorbance is 8-fold. This difference is considered to be attributable to the higher quantum yield of the Ca2+ -bound form. Conversely, for excitation at 407 nm the Ca2+ -dependent increase in the emission intensity is 14-fold, whereas the Ca2+ -dependent increase in absorption is 4-fold. This difference is considered to be due to the combined larger absorbance and quantum yield of the Ca2+ -bound form.
The Ca2+ dependent excitation and emission spectra allows determination of the dissociation constant of STBT for Ca2+. The plot of log{(F-Fmin)/(Fmax -F)}, obtained from the excitation spectra in FIG. 4, versus log(Ca2+) yields log(Kd) as the x-intercept, as shown in FIG. 5 (open circles). The dissociation constant Kd can also be determined using the ratio of fluorescence intensities at 610 nm, excited at 407 and 508 nm (closed circles). Both plots (open and closed circles) yielded the same dissociation contant for STBT and Ca2+, 1.5 μM. Alternatively, such a plot can be constructed using the isosbestic point as one of the two excitation wavelengths. Importantly, ratiometric Ca2+ measurements can also be obtained using a single excitation wavelength of 440 nm, and two emission wavelengths of 590 and 615 nm (FIG. 5, open triangles).
The Ca2+ dependent absorption spectra allow determination of the dissociation constant of the STBT probe for Ca2+, which constant is larger than that known for Fura-2 near 150 nm, e.g., see G. Grynkiewicz, M. Poenie and R. Y. Tsien, J. Biol. Chem., 260, 3440-3450, and for probes with higher dissociation constants, such as Calcium Green-5N, Mag-fura-2 and Mag-fura-5 that have become of interest for studies of Ca2+ transients in cells, as discussed in K. Kuba et al, J. Theor. Biol., 1981, 93: 1009; A. Goldpeter et al, Proc. Natl. Acad. Sci., 87, 1461 (1990) .
The presence of an absorption spectral shift enables wavelength-ratiometric imaging of Ca2+ using visible wavelengths. Wavelength-ratiometric measurements in the present invention are possible using a wide range of wavelengths from 350 nm to 580 nm, thereby allowing the use of multi-line lasers, as well as the more common filter wheels or rotating mirror monochromators.
The present invention also contemplates further refinement of spectral properties of calcium-binding STBT. That is, STBT displays features of a short fluorescence lifetime near 100 ps in the presence of Ca2+ and near 70 ps in the absence of Ca2+ and a low quantum yield in aqueous solution. These features are believed to be attributable to possible rotation around the ethylenic carbon bond during the lifetime of the excited state.
In view of the above data results, the inventors envisage a variety of STBT probe derivatives which can be useful in cellular Ca2+ imaging. For instance, membrane-permeable forms of STBT could readily be prepared by known routes. Further, it is postulated that the quantum yield and/or lifetime of the probe compound could be increased by the addition of binding groups to prevent rotation about the ethylenic binds.
While the invention has been described in detail and with reference to specific embodiments, it will be apparent to one skilled in the art that various changes and modifications can be made in these embodiments without departing from the spirit and scope of the invention as defined by the following claims.
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|U.S. Classification
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|Dec 30, 1992
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text:
Morgan - Our JobsMature
Everything was sort of tense when everyone finally came together. It also seemed that everyone who had been chosen had a friend who had also been chosen. Tammie clutched my hand tightly under the table where it was hidden which was odd that we were still keeping up this secrecy.
I guess habits were hard to shake. Maybe we shouldn't shake them either. We could be rescued any second. People could be looking for us. Well.... the word 'but' was big in my mind. There were so many reasons people wouldn't look for us.
Helena had already spoken to me and Tammie. She was a very strong minded and opinionated person that had frowned at Tammie's dressed while seemed approving off my new fighter gear. I must admit the clothing was nice. It hugged my body just enough to be comfortable but was also loose enough to provide free movement I had learned when tumbling through the woods outside.
Helena had begun in chat with a guy called Jasper who had just arrived then two other people walked in. The girl looked terrified and almost hid behind this other guy who seemed very well collected. Jasper and Helena had gone silent with this new arrival and Tammie had tightened her grip on my hand.
"Well, you're all here" a very familiar voice said. Sat on the tables around Romulus were six angels, one being Felix. I was guessing these were all the mentors. "One, Two, Three, Four, Five, Six. Yes, you're all here good"
"Why are we here?" the boy who had just come in asked outright.
Helena straightened in her seat leaning forward on her elbows. "Yeah, why?" she said seeming very interested in getting an answer.
Me, while I wanted to know what was going on was happy sitting aside. These people hadn't been here as long as me and Tammie. They hadn't gone through even one training session yet.
Romulus laughed and then settled back to perch on the edge of a table. "You are here for the rebellion. I picked you because you want to rebel. You just don't know it.
Anyway, moving on to business. You will all be trained. In what is what I have decided for you"
"The Blade" Felix said looking at me.
"The Healer" another said indicating Tammie who tensed next to me her hands wrapping around my arms in a grip.
"The Rat" said another of the six. This rat was the boy whose name I still did not know.
"The Getaway" added another again indicating Helena.
"The Lock" it moved onto this Jasper who fiddled with his hands as he was spoken to.
"Then The Tech" said the last person to the very back. This would be the unknown girl it seemed.
"For now that is all you need to know till I think you are ready for your next mission. Eat well and sleep" Romulus said loudly before striding out the room the six mentors following him out.
"Well, shit" stated the nameless boy who slumped into a chair.
He sort of summed up the whole problem there.


text:
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Anita Dongre showcases her Epic Love collection at the FDCI India Couture Week (ICW) 2016 on the second day of the five day fashion gala here at the Taj Palace on Thursday. Celebrities like veteran actress-socialist Shabana Azmi, Bhumi Pednekar, Pernia Qureshi and ace designer Manish Malhotra attended the event.
The Epic Love collection at the FDCI India Couture Week was a luxury Bohemian collection of Indian bridal wear meant for the new age spirited bride, where the chintz-inspired prints brought alive a Mughal garden in its full bloom. The color palette being golden infused with hues of cream, rusts, browns, reds, pink and muted blue.
Anita Dongre infuse magic into thin air with her Epic love collection at FDCI India Couture Week. In her collection the young gypsy bride was the muse. She mesmerized the audience with her bright, gota patti infused creations. The collection had the ethnic, rustic and soothing look about them.
The collection showcased the vintage, Mughal era inspired Saris, Long skirts, different styles of blouses, dupattas, achkans and kurtas for men, the ensembles exuding elegance wrapped in tradition had the touch of gota patti, dabka, and phulkari used in perfect balance.
Anita Dongre said the young gypsy bride muse is free spirited, yet embraces tradition in her own special way. Traditional styles like the lehenga choli and sari are reinvented in contemporary silhouettes.
Others from fashion industry stalwarts like Tarun Tahiliani, Reynu Taandon, Rimple and Harpreet Narula, Rahul Mishra, Anamika Khanna, Varun Bahl, Gaurav Gupta, Manav Gangwani and Rohit Bal will showcase their latest collection in the coming days. The FDCI India Couture Week event is for five days. It concludes on July 24.


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US President Barack Obama and his French counterpart Francois Hollande would meet at the White House here on November 24 to discuss the strategy against the Islamic State group following the Paris terrorist attacks.
"This visit will underscore the friendship and solidarity between the United States and France, our oldest ally," said White House Press Secretary Josh Earnest.
"Obama and Hollande will consult and coordinate our efforts to assist France's investigation into these attacks, discuss further cooperation as part of the 65-member counter-ISIL coalition, and reiterate our shared determination to confront the scourge of terrorism," Earnest said in a statement using another acronym for IS, which has claimed responsibility for the carnage in the French capital.
At a G20 gathering in Turkey on Sunday, Obama had rejected demands that he put boots on the ground in Syria to fight the IS. He had said that such a strategy would not be sustainable.
"Let's assume that we were to send 50,000 troops into Syria," he had said.
Syria's four-year civil war has now claimed more than a quarter of a million lives and displaced millions of people.


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We have over 10,000 products from the biggest manufacturers so we are sure we can supply you with all your electrical needs. If you don't see what you need on our website please do not hesitate to call us at either branch.
Spring Electrical is a reliable, family business that cares about the needs of professional electricians.
With 30 successful years behind us, and as a member of AWEBB (a leading purchasing consortium in the UK with a turn over in excess of £200m,) we are consistently price competitive across all our products. We offer also offer a lighting design service, please call for further details.
We have a strict old fashioned policy of supporting genuine trade customers with preferential discounts and prices. Please call, email or visit us for further details.
Please see featured products for our current promotions.
Thank you for your interest in Spring Electrical, I would like to extend a warm welcome to those contacting us for the first time and to our regular customers thank you very much for your continued support.
|Endon NIRO-10MULTI Ceiling Lgt G4 10x10W
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text: "Smarter Lunchrooms is all about low cost – no cost ways to improve student nutrition," said Dr. Brian Wansink, the John S. Dyson Professor of Marketing and Consumer Behavior at Cornell University. "It's amazing what can be accomplished with simple things like moving fruit into a nicer bowl and putting it out where kids can see it. Our research has validated a variety of easy, cheap and effective ways you can help lead kids towards healthier choices."In addition to this nationwide program, Sodexo is working with the Smarter Lunchrooms Movement to pilot a program focused on consumption of foods selected. This is the next step in effecting behavioral change around student nutrition choices. Visit Sodexo on Facebook and follow on Twitter @SodexoUSAVisit Smarter Lunchrooms Movement on Facebook and follow on Twitter @SmartLunchrooms About SodexoSodexo is the global leader in services that improve Quality of Life, an essential factor in individual and organizational performance. Operating in 80 countries, Sodexo serves 75 million consumers each day through its unique combination of On-site Services, Benefits and Rewards Services and Personal and Home Services. Through its more than 100 services, Sodexo provides clients an integrated offering developed over more than 45 years of experience: from reception, safety, maintenance and cleaning, to foodservices and facilities and equipment management; from Meal Pass, Gift Pass and Mobility Pass benefits for employees to in-home assistance and concierge services. Sodexo's success and performance are founded on its independence, its sustainable business model and its ability to continuously develop and engage its 420,000 employees throughout the world. Sodexo in North America Sodexo, Inc. ( www.sodexoUSA.com), leading Quality of Life services company in the U.S., Canada, and Mexico, delivers On-site Services in Corporate, Education, Health Care, Government, and Remote Site segments, as well as Benefits and Rewards Services and Personal and Home Services. Sodexo, Inc., headquartered in Gaithersburg, Md., funds all administrative costs for the Sodexo Foundation ( www.SodexoFoundation.org), an independent charitable organization that, since its founding in 1999, has made more than $17 million in grants to end childhood hunger in America. Visit the corporate blog at http://blogs.sodexousa.com. Key Figures (as of August 31, 2012) Sodexo in the World 18.2 billion euro consolidated revenue 420,000 employees 20th largest employer worldwide 80 countries 34,300 sites 75 million consumers served daily 9.5 billion euro market capitalization (as of November 7 2012) Sodexo in North America 8.6 billion dollars revenue 125,000 employees 6,000 sites 700 facilities management sites 10 million consumers served daily SOURCE Sodexo, Inc.


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Home > Low Carb Recipes > Chocolate Fudge Muffins
Chocolate Fudge Muffins
Low Carbohydrate Recipe
This recipe makes very good, very Chocolate Fudge Muffins with a cupcake like consistency. The whey protein powder is low in carbohydrates, gives you energy and in general, is a power meal and snack solution.
1/3 cup almond flour
1/3 cup whey protein powder
1/8 cup flax meal
1/3 cup spoonable sweetener (such as Stevia)
1 tablespoon Brown Sugar Twin
4 ounces cream cheese
1 tablespoon cocoa powder
1 ounce unsweetened chocolate
1 tablespoon butter
1 teaspoon vanillabr> 2 eggs
Chopped nuts (optional)
Recipe Directions
Soften cream cheese in microwave 20 to 30 seconds.
In separate dish, melt chocolate and butter. Add melted chocolate to cream cheese, and add eggs and vanilla. Mix well until smooth. Add all other ingredients.
Mixture shoul be fairly thick - if too thick, add a litle water. Bake at 350 degrees for 20 to 25 minutes.
Nutrition Information
Recipe makes 6 servings
Serving size: 1 muffin
Carbohydrates: >4
Total Carbohydrates: 42 (23 NET)


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Popular property highlights
Located in Rome Historic Centre, Luxury Rooms is a leisurely stroll from popular sights such as Borghese Gallery and Villa Borghese. The 9 rooms at this bed & breakfast include private yards, refrigerators, and microwaves. In the historical district, it's also near St. Peter's Basilica and Teatro dell'Opera di Roma.
Free continental breakfast is served each morning from 7:00 AM to 11:30 AM. Savor a meal from the comfort of your room with room service.
All of Luxury Rooms's 9 rooms are soundproofed and have an array of amenities, including private yards, full-sized refrigerators/freezers, and microwaves. Guests can enjoy digital TV and stay connected with free WiFi. Select Comfort beds grant a restful night's sleep, and bathrooms offer hair dryers and free toiletries. Other standard amenities include stovetops, coffee makers, and free local calls.
At Luxury Rooms, guests have access to a steam room, free WiFi in public areas, and laundry facilities. You can take advantage of RV, bus, and truck parking, along with an airport shuttle (available on request) for EUR 45 per vehicle roundtrip. The 24-hour front desk has multilingual staff ready to assist with dry cleaning/laundry, tours or tickets, and luggage storage.
Property features
- Walking distance to Borghese Gallery and Villa Borghese
- Free continental breakfast
- Free WiFi in rooms and public areas
- Laundry facilities
Luxury Rooms
Bed & Breakfast Amenities
Bed & Breakfast Amenities
Guests can enjoy a complimentary breakfast each morning. Public areas are equipped with complimentary wireless Internet access. For a surcharge, a roundtrip airport shuttle (available on request) is offered to guests. This Beaux Arts bed & breakfast also offers a steam room, tour/ticket assistance, and multilingual staff.
- 24-hour front desk
- Airport transportation (surcharge)
- Total number of rooms - 9
- Number of floors - 3
- Free breakfast
- Dry cleaning/laundry service
- Elevator/lift
- Tours/ticket assistance
- Free WiFi
- Laundry facilities
- Multilingual staff
- Luggage storage
- Safe-deposit box at front desk
- Year Built 2010
- Number of buildings/towers - 2
- RV, bus, truck parking
- Steam room
Family Friendly Amenities
- Family Hotel
- Free Wi-Fi
- Soundproofed rooms
- Laundry facilities
- Full-sized refrigerator/freezer
- Microwave
- Cribs/infant beds (complimentary)
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
RV, bus, truck parking
Room Amenities
Luxury Rooms offers 9 air-conditioned accommodations with safes and complimentary bottled water. Beds feature Select Comfort mattresses. Televisions come with digital channels. Guests can make use of the in-room full-sized refrigerators/freezers, microwaves, and coffee/tea makers. Bathrooms include bidets, complimentary toiletries, and hair dryers.
This Rome bed & breakfast provides complimentary wireless Internet access. Business-friendly amenities include desks and phones; free local calls are provided (restrictions may apply). Housekeeping is offered daily and irons/ironing boards can be requested.
- In-room climate control (air conditioning)
- Air conditioning
- Free local calls
- Coffee/tea maker
- Free bottled water
- Microwave
- Daily housekeeping
- Private bathroom
- Free toiletries
- Hair dryer
- Bidet
- Iron/ironing board (on request)
- In-room safe
- Desk
- Soundproofed rooms
- Phone
- Full-sized refrigerator/freezer
- Stovetop
- Digital TV service
- Free WiFi
- Room service
- Sleep Number by Select Comfort mattress
- Free cribs/infant beds
- Rollaway/extra beds (surcharge)
- Yard
- Cookware, dishware, and utensils
Where to Eat
Room service is available.
A complimentary continental breakfast is served each morning between 7:00 AM and 11:30 AM.
Nearby Things to Do
Recreational amenities at the bed & breakfast include a steam room.
The recreational activities listed below are available either on site or nearby; fees may apply.
- Scooter/moped rentals nearby
- Free bicycles nearby
- Segway rental/tour nearby
Bed & Breakfast Policies
Check-in time ends at midnight
Check-in time starts at 10 AM
Special check-in instructions:
Check-out time is 11 AM
Payment types
Children and extra beds
- Children are welcome.
- Kids stay free! Children 5 years old and younger stay free when using existing bedding.
- Rollaway/extra beds are available for EUR 20 per day.
- Free cribs (infant beds)!
- Pets not allowed
You need to know
Extra-person charges may apply and vary depending on property policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
- No onsite parking is available.
- The name on the credit card used at check-in to pay for incidentals must be the primary name on the guestroom reservation.
Parking height restrictions apply
- A tax is imposed by the city: EUR 3 per person, per night, up to 10 nights
Optional extras
- Airport shuttle fee: EUR 45 per vehicle (roundtrip)
- Rollaway bed fee: EUR 20 per day
Bed & Breakfast Name
- Luxury Rooms B&B
- Luxury Rooms B&B Rome
- Luxury Rooms Rome
We should mention
All guests, including children, must be present at check-in and show their government-issued photo ID card or passport. Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation. Children 5 years old and younger stay free when occupying the parent or guardian's room, using existing bedding. Only registered guests are allowed in the guestrooms.


text: This is really crazy, he is drafting, jason williams and some ot from ball state? Our need was at saftey? Rashard johnson is not good enough? I'm sorry but i'm really disappointed this far. Why trade out of the second , because you knew you wanted to target , these guys? The more important thing is Giants and Philly are getting better. Giants just got Ramses BARDEN, WHO CAN GO UP FOR THE BALL, PLAX REPLACEMENT. Nicks, Stinton. I don't know anything but i know i'm pissed. We are thin in the secondary?


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ED Magedson – Founder
Ripoff Report
Professional Sports AuthenticatorPOB 6180 Newport Beach, California United States of America
Does your business have a bad reputation?
Fix it the right way.
Corporate Advocacy Program™
This report was posted on Ripoff Report on 08/29/2012 09:56 AM and is a permanent record located here: http://www.ripoffreport.com/reports/professional-sports-authenticator/newport-beach-california-92658/professional-sports-authenticator-psa-scamdishonestexpensivelow-card-gradeshopelessru-934228. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
Ripoff Report has an exclusive license to this report. It may not be copied without the written permission of Ripoff Report. READ: Foreign websites steal our content
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#1 Consumer Comment
PSA Experience
SUBMITTED: Tuesday, June 04, 2013POSTED: Tuesday, June 04, 2013
I suspect that the PSA deal with 10's of thousands of submissions every month and you are never going to please all of the people all of the time. However, my experience has always been great! I am UK based and have submitted well over 3000 cards over several years. Customer services have allways been curteous and helpful with no problems with any of my submissions that includes shipment, packing and time taken to process and return my cards. Only complaint is I could do with a few more higher grades (Ha Ha) My experience only but I just wanted to say I am pleased with the PSA, competing on the PSA set registery. There president Joe Orlando who has taken the time to respond to me on a number of occasions.
Are you an owner, employee or ex-employee with either negative or positive information about the company or individual, or can you provide "insider information" on this company?
Advertisers above have met our
strict standards for business conduct.


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The Slimtea brand is happy to have Miss Huddah on board, as we journey into revolutionary methods of weight loss that are 100% safe, natural, fast and easy to sustain!
Slimtea is the smartest choice for natural weight loss and it is packed with a powerful combination of weight loss ingredients that effectively increase the rate at which your body burns fat, suppressing hunger while keeping you energized!!
There are job vacancies at Slimtea Kenya, and positions are open for Business Development Executives and Social Media Managers. Interested and qualified candidates should send their CVs and applications to firstname.lastname@example.com with the title of the position you are applying for as the subject of the mail. These positions are available to residents of Kenya only.
Slimtea is affordable and available for purchase! NATIONWIDE PAYMENT ON DELIVERY OPTIONS AVAILABLE TOO! Visit www.slimtea.com.ng or contact the following to make enquiries and place your orders; +254719256913 (Kenya), 09083231736, 09080442390, 09083340376, 08181433219, 08170987132, 08189488738


text:
183 South 3rd Street, Floor 4, Brooklyn 11211
(Between Roebling Street and Driggs Avenue)
- Get Directions
- Telephone: 718-704-1419
- email@example.com
(Between Roebling Street and Driggs Avenue)
In 2011, Ms. Palmer joined Success Academy Harlem 5 as a kindergarten associate teacher. Since then, she has served as a lead teacher, a grade team leader, and most recently, as a leadership resident at SA Harlem 3 and SA Williamsburg. Since 2014, Ms. Palmer has taught graduate courses in reading and writing instruction at Touro College and has been a mentor to several Success Academy teaching fellows. She enjoys reading, cooking, and spending time with family and friends.
Ms. Levy began her career as a Teach for America corps member at P.S. 279, a middle school in the Bronx, where she taught English language arts and social studies for two years. In 2012, after earning a master's degree in special education from Hunter College, Ms. Levy joined Success Academy Harlem West as a founding fifth grade teacher and later spent two years as an assistant principal at SA Harlem North West. In her spare time, Ms. Levy enjoys cycling and rooting for the New York Rangers.


text:
You missed it This great deal for Great Grizzly Bear is over. But don't despair, we've got more great deals every day.
Related Deals That You May Like
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Choice of $30 or $60 Worth of Pub Fare
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St. Louis$40$19.99
Cookies, Cakes, and Gift Baskets from Cheryl's
St. Louis$30$15
back by popular demand:
Great Grizzly Bear
Live Music, Good Food, and Cold Drinks at Local Hangout
88% recommendedin LivingSocial customer ratings
Sitting in historical Soulard, Great Grizzly Bear is a small historical treasure in itself -- stop by this hometown favorite of more than 30 years for a fabulous afternoon with your friends:
• $69 ($150 value) for a three-hour happy hour party for ten
• Includes 30 drinks and up to six appetizers to share
• Choose from themed options like "Margarita" and "Patio"
Beary Good
Take in the exposed-brick décor of this local staple, which has been around for more than 30 years. Live music, trivia night, extra-cold beers, and an outdoor beer garden keep regulars hooked. Choose a couple apps from the expansive American/Cajun menu, like toasted ravioli, queso cheese waffle fries, and fan-tailed breaded shrimp.
PAID VALUE EXPIRES ON May 27, 2022
PROMOTIONAL VALUE EXPIRES ON January 3, 2014


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Tiger Silhouette Vector Free
Six tiger figures in black color with a blue background. You can use this vector for your creative ideas in your designs. Hope you can use and enjoy this vector!
This File Appears In Searches For
tiger tigers tiger silhouette Silhouette figure figures shape shapes tiger shapes tiger figures wild wild animal Africa jungle jungle animal dangerous cats Wild cats African Carnivore predator agile Fast fast animal hunter forest Wildcat wildlife wild animals zoo
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Tiger Silhouette Vector Free


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Saturday, March 2, 2013New York
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Probably my favorite in the park. Out enjoying the fresh air this morning and the fun caches in the area. This one did not take that long to locate. Signed log and replaced as found. Thanks for the hide.
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HOME > Optoma HD33 3D Home Theater Projector > Alert Moderator
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Thanks or your review. Good work. I do have a quick question tough:I thought that when I first saw this projector on the Optoma website that the 3d mode was 720p...can you clear up if both the 2d and 3d modes are in 1080p? Also, did you do a lumens measurement in 3d mode? Thanks
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- This event has passed.
Shrewsbury: Catmobile to be at Petco
November 2, 2012$75 - $100
The Catmobile will be parked at Petco, 87 Boston Tpke., Shrewsbury, for one day only, Nov. 2. The Catmobile is operated by the Merrimack River Feline Rescue Society (MRFRS) and is staffed by a licensed veterinarian and two veterinary technicians. The Catmobile offers low cost spay neuter for cats only. The package consists of spay/neuter, rabies vaccinations, exam, nail trim, treatment for fleas and ear mites. Spay package costs $100 and neuter package costs $75. The package is available to anyone and there is no qualification process; however reservations are required and can be made online at www.catmobile.org or by calling 978-465-1940.


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JHAM Documentary Release Party with the JHAM Band
Ashkenaz Music & Dance Community Center
Saturday, August 2, 2014 at 8:30 PM (PDT)
Who's Going
San Francisco, California
London, United Kingdom
Florence, Italy
Event Details
In October 2012, filmmaker Joe Baker took a group of American musicians and a film crew to Jamaica and Haiti, where they spent time with some of the greatest musicians on those islands – including Earl "Chinna" Smith, Empress Apple, and Prezident Brown – creating new music collaboratively. By the end of the trip, more than 40 musicians had participated: JHAM stands for Jamaican Haitian American Musicians jamming for education.
This special event was created by Baker and volunteers as a fundraiser for two beneficiaries: Pineapple Community Education Association in Ocho Rios, Jamaica, and Mission of Hope in Port-au-Prince, Haiti. The CD/DVD of the film and music from the trip will be available in the near future and will also raise money for the beneficiaries.
Tonight Ashkenaz turns its stage and screen over to an extraordinary experience of musical collaborations rooted in reggae and cultural exchange as Baker presents the first full screening of the film that came out of the trip, "A Real Purpose: The Story of JHAM." It is followed by a live concert/dance with an all-star JHAM band of some of the musicians featured in the film playing reggae dance music, including songs from the film as well as band originals. The project is a sequel to Baker's acclaimed 2010 "The Africa Project," a DVD/CD of a similar collaboration between African and American musicians that was staged as a film and concert at Ashkenaz two years ago.
Tonight's band includes the four American musicians who made the trip – Jordan Feinstein on keyboards and vocals, Garrin Benfield on guitar and vocals, Chris Zanardi on guitar, and Lee "Bug" Brock on bass – plus Brian Festa on drums, Waahid Jones and Dave Eagle on percussion, John Harrington on trumpet, female backing singers, and possibly a horn section.
When & Where
1317 San Pablo Ave
Saturday, August 2, 2014 at 8:30 PM (PDT)
Add to my calendar
Ashkenaz Music & Dance Community Center
Ashkenaz is the East Bay's home to world music & dance... established 1973; nonprofit since 1997. We are always all ages! Kids 12 & under are admitted free unless otherwise noted.
All of our online ticket sales are will call. We will have a list of ticket buyers at the door; you do not need to bring a printout of your ticket/order confirmation.
Doors open 30 minutes before showtime, or 30 minutes before the dance lesson if there is one, unless otherwise noted.
Ashkenaz's Café opens when doors open and offers beer (draft and bottled), wine, kombucha, juices, sodas, coffee and tea, and snacks and light meals (all vegetarian, mostly organic).


text:
Indian Army Territorial Army Rally Bharti Recruitment chart 2015 final
Candidates who are waiting for Territorial Army jobs must read this chart. Please click on given image and zoom to enlarge for better view.
Indian Army updated this chart on 26 Oct 2015
|TA bharti 2015
|Territorial Army bharti 2015
- Direct soldier bharti cahrt Pune Zone 2016-17
- Indian army Pune Zone army bharti chart 2016 – 2017
- Indian Army All India Rally bharti 2016 chart - joinindianarmy.nic.in
- Indian Army Rally bharti Schedule Chart for December 2015
- Open Rally bharti Recruitment Chart January 2016 All India
- TERRITORIAL ARMY BHARTI CHART (TA Recruitment)
- Indian Army Bharti chart new 2016
- Indian Army Soldier Rally Bharti Latest Chart 2015
- Army Rally bharti Chart February 2015


text:
For sale in javea, a townhouse needing completion, this is a great opportunity to create a magnificent home in the centre of javea's historic old town. courtyard roof terrace sea view separate dining room storage room terrace utility room
We can help you to get a mortgage on favourable terms. A residence permit can be obtained with the purchase of this property. Please ask our team for more information.


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ED Magedson – Founder
Ripoff Report
INGRAHAM GARAGE225 SE 2nd Street 1st St , 2nd St MIAMI, Florida USA
INGRAHAM GARAGE RIPOFF PARKING MIAMI Florida
Does your business have a bad reputation?
Fix it the right way.
Corporate Advocacy Program™
Yesterday I was paying for parking and I did not realized that on top of the machine (where you pay for it) it said "no change provided". My parking was $30.00 and I inputed $40.00. On my receipt it says that I had $10.00 change but as the machine says" no change was provided" I lost $10.00 . I called the Ingraham parking today(operated by central parking) and I asked if I could have my change back and they told me that on the machine says no change and that they would not return my change. For me this is a rippof!All of us use these type of machines and they can give your change if they are set up for it or they could give me the change in person at their office. Second I'm sure they do that to still money from people. I live in Miami and this parking is used a lot for turists, so if they don't know English they will lose their money also.
I hope you can do something about it is not fair to still people's money
This report was posted on Ripoff Report on 06/18/2013 08:58 AM and is a permanent record located here: http://www.ripoffreport.com/reports/ingraham-garage/miami-florida-33131/ingraham-garage-ripoff-parking-miami-florida-1059981. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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text:
Advanced Fire and Security Overview
With headquarters in Pompano Beach Florida, Advanced Fire and Security serves Florida customers from Orlando south to as far as the Caribbean. With nearly 100 full time employees the company is unique in that it does not subcontract any work. All of their employees are trained in house. Service technicians also receive third party training with a certification company and that provides a great platform for instilling a strong culture with customer service as a priority.
All service technicians are trained, certified and bonded by the FASA (Fire Alarm Systems Agents) and NICET (National Institute of Certified Emergency Technicians). These are high level certifications that are the best certifications in the industry. The company offers customer service hours that are accessible 24 hours a day 7 days a week. This provides the needed service at any time a customer has questions or needs. 24 hour monitoring service is also available to provide prompt response times when an alarm is triggered.
The company is one of the largest security companies in the south Florida region. They offer Fire Alarm Systems, Security Systems and sprinkler systems to both residential and commercial customers. Advanced Fire and Security was honored with the Honeywell President's award for 2013. This was the 10th time the company received the award for having top rated customer service.
Advanced Fire and Security Reviews
Advanced Fire and Security has very high customer reviews. Customers are pleased with the high level of knowledge from employees and technicians and the accessibility of technicians regardless of the time of day. The technicians are knowledgeable, friendly and available to answer questions and to help troubleshoot system issues. This high level of customer service is refreshing as customers feel valued by the company.
The training that service personnel are required to complete ensures that each employee is cross trained and able to assist customers in the many areas of business operations. These policies reduce run around and increase customersatisfaction. Highly trained technicians also reduce the number of service calls. When the security systems are installed correctly the first time, service calls are less frequent. When issues do arise technicians have the experience to identify and correct the problems quickly.
Advanced Fire and Security Equipment
Advanced Fire and Security offers three different types of security systems. This includes fire alarm systems, home and business security systems and sprinkler systems. Fire systems include six levels of fire protection. Expandable emergency evacuation system, analog addressable fire alarms, graphic work stations, conventional fire alarms, advanced fire alarms services, and wireless fire security.
The security systems provided will depend on the customer's needs. This may include intrusion protection, CCTV and video surveillance, card access and door entry security. Advanced fire and security works to offer the latest technology to both small and large customers, in order to meet the customer needs and provide the peace of mind that comes with security your property. Agents for the company will visit the property and help to identify the customer needs and provide a system that will meet those needs.
The most recent addition to the Advanced Fire and Security product line up is sprinkler systems for businesses. The company offers new systems, testing of existing systems, repairs of existing systems, and pump testing. They can help a company design and engineer an effective system that will improve safety for the building. This evaluation will include code analysis, and design layout that will account for both water supply and occupancy. Sprinkler systems can be installed in new construction as well as existing buildings.
Advanced Fire and Security Packages
Advanced Fire and Security offers custom packages that are personally designed to meet each customer's needs. The company offers a free evaluation of current security needs and they will design a system that will meet those needs. The company has highly trained personnel that know what to look for when evaluating a property. It is the company's goal to identify both expressed and unexpressed needs to create a security system that will meet the true needs of customers.
They work with individual homes to design basic security systems that will protect homeowners, to complicated businesses that include security, fire and surveillance needs beyond the basic designs. Card entry and fire sprinkler systems are also available as both retrofits and new construction projects. This high level of customization gives customers confidence they will be safe and secure using the Advanced Fire and Security systems.
How to Contact Advanced Fire and Security
Website: http://www.advfireonline.com/
Phone: 888-916-7474