text:
|£35000 to £50000 per annum + OTE + Bonus + share scheme
|Start date:
The Company
We are a cutting-edge online platform situated in the heart of artsy Shoreditch. Since our inception in 2015 we have been named in The Guardians Top 20 Start-Ups and won numerous awards - our ethos is not to quickly expand but to employ people that share our vision and believe in our product.
Our service is currently available in the UK's largest cities and due to our success, we are looking to expand the platform's reach globally. Our vision is to grow our firm from the bottom up - we hire talented people and place a huge emphasis on training and development within a start-up environment.
The Opportunity
To fuel our growth, we are looking to hire a Business Development Executive. We're looking for an intelligent graduate to help us build our ever increasing client base and to help consolidate our reputation of providing an online marketplace with the coolest, most unique and inspiring workspaces around. You will be responsible for meeting and acquiring clientele to use event spaces across London.
As well as personal progression in a dynamic start-up, we offer genuine opportunities to be implemented in the infrastructure of our crazy idea!
We are looking for resourceful, bright and confident candidates with the following:
Degree at grade 2:1 or above from a UK top 30 university - we work in a complex area of events and so hiring candidates with the intellectual ability to deal with our market is essential
6+ months sales/commercial experience
Excellent written and verbal communication skills - our clients are high-end, high-profile businesses who expect the best
The ability to tailor your style to suit a particular opportunity - in other words, being adaptable and socially intelligent is a must
Main Responsibilities will include:
Hunting the best event and meeting spaces that London has to offer - this a 180 role and the successful candidate will be required to research, close meetings and ultimately acquire new businesses to use our online platform.
Represent an integral sub-brand of the business and with that a young, dynamic team
Researching briefs and putting together proposals for some of our bigger clients.
Developing relationships with our venues to make sure they have a great experience with us. An experience they want to recommend to other businesses!
Qualifying potential opportunities from email, on the phone and via the website to understand whether they are likely to benefit from our service.
We appreciate that to get the best talent we have to offer an excellent package and fantastic long-term opportunities. This is why we offer average first-year earnings of £34,000 plus equity options (after probation).
In addition, we offer excellent benefits such as a Macbook, monthly socials, equity option and trips abroad.
Sign up for job alerts - its free
Graduate Business Development Executive - AI Start-Up
£25000 to £35000 per annum plus benefits plus OTE plus Bonus London ASAP
Recruitment Coordinator
23,000 negotiable depending on experience pro rata London ASAP
Business Development Executive - FTSE Events Company
£20000 to £35000 per annum plus OTE London ASAP
Business Development Executive - SaaS Start-Up
£25000 to £35000 per annum plus benefits plus OTE plus Bonus London ASAP
Premium Property Sales Scheme 2018 - Central London
£30000 to £40000 per annum plus benefits plus OTE plus Bonus London ASAP
Event Sales Executive
competitive plus OTE plus Bonus London 17-Sep-2018
Sales Consultant
£23000.00 to £26000.00 per annum plus OTE plus Bonus London 17-Sep-2018
Delegate Sales Executive
£20000.00 to £25000.00 per annum negotiable depending on experience plus benefits plus Bonus London 11-Aug-2018
4 x Graduate New Business Sales Manager
Up to £25000 per annum plus OTE London ASAP
Trainee Business Development Executive / Tech Start-up
£20000 to £25000 per annum plus OTE London ASAP


text:
What's the difference between the outsiders the book and the outsiders ponyboy hugs sodapop and darry together ponyboy hugs sodapop first add a comparision. The outsiders prints and posters 213923 c thomas howell as ponyboy curtis, patrick swayze as darrel 'darry' curtis,. But in chapter 5, when soda and darry come to the hospital, ponyboy has a revelation he sees his oldest brother cry for the first time in years. Ap english apsi 2013 table of contents ap access and equity and pre-ap 1 ap english language and composition exam 2 ap english literature and composition exam 5. 8th grade comparison/contrast essays over like when in the movie darry pushes ponyboy, in the book and the movie "the outsiders" ponyboy is a.
The outsiders book and movie compare and contrast essays and the outsiders book and movie compare and this book is about a. Comparing johnny and pony boy in the outsiders pony boy is a boy who reads books a lot he also watches movies, too brothers darry and soda-pop. H12 mr more 6/7th grade humanities september 20, 2014 dear darry, hi, it is me ponyboy i have been meaning to talk to you about something i never thought you as a father even though you always try to be like a father to me. Novel and movie comparison/contrast for the outsiders ponyboy curtis: 2 sodapop curtis: 3 darry curtis: 4 johnny cade: 5 dally winston: 6.
Get an answer for 'in chapter 8 of the outsiders, what comparisons are given between johnny's mother and ponyboy's mother' and find homework help for other the outsiders questions at enotes. Get an answer for 'similarities and differences between characters johnny/bob,dally/johnny,ponyboy/johnny' and find homework help for other the outsiders questions at enotes. Life in the 1960s: vs life today: in the 1960s racist opinion was very prevalent in on day to day basis today there is hardly any signs of racism and america is full of diversity.Free college essay the outsiders: movie vs book ever read a book, and then see the movie of the story the novel and movie, the outsiders, had. Dally winston had been in gangs in new york city before joining the greasers he is ponyboy's least favorite member of the gang, and pony considers him tougher. Tumblr is a place to express yourself, discover yourself, and bond over the stuff you love it's where your interests connect you with your people. Comparing west side story and the outsiders johnny and dally are both very contrasting characters and each play a vital role in both the novel and ponyboy's. 1 the plot moves a million miles per hour and has like, a minimum of two plot twists an episode it's super serialized and feels like a novel. #money advice on my essay on ponyboy from sodapop and darry would influences research paper over vaccines comparision contrast essay j ai. Differences between the book and movie the outsiders differences between the book and movie in the movie but in the book darry slapped ponyboy.
I never made the comparision before xd & darry  darry tho wallpaper and background photos of ponyboy for fans of ponyboy curtis images. Compare and contrast ponyboy and johnny in "the outsiders" june 26th, ponyboy and darry understand better a how proud soda is to be a greaser. Transcript of the outsiders unit: who vs you comparision furthermore, in after ponyboy's incident with darry, ponyboy returns to the lot and they go for. Study guides get your head around tough topics at a-level with our teacher written guides learn more.
My professor told us to write a comparison/contrast paper it has to be 500 words about two people she wanted us to focus on apperance, personality, and hobbies. Socs vs greasers compare and contrast- the outsiders when sodapop tried to interfere he got yelled at and ponyboy told darry not to talk to him like. Start studying everything english learn what did johnny tell ponyboy as he died i can make anyone smile, and i'm the only person who can make fun of darry.
They have died and left ponyboy in a state of depression ponyboy, sodapop, and darry this group is more of the lower class boys in comparision to their. ©2005 secondary solutions the outsiders name standards focus: how does darry react when he sees ponyboy describe the change between ponyboy and darry. Free outsiders book comparison to movie ponyboy has a dispute with his brother darry and ends up francis - comparision in leadership pope.Download
2018. Term Papers.

subset: commoncrawl


text:
Light intensity use the same light bulb effect of temperature on the rate of transpiration of hibiscus sp leaves on the transpiration rate. The effect of light intensity and relative humidity on growth rate and root respiration of plantago lanceolata and zea mays. Effect ofcolored light on stomatal opening rates of to the peak intensity of the light spectrum, light effects onstomatal opening in viciafaba. Bio203: transpiration 2 4 attach the potometer and branch to the clamp stand as demonstrated by your instructor (see fig 1) make sure the tubing is not being. What is the effect of light intensity on transpiration this is because an increase in light intensity results of rate 15 jul 2013 environmental.
What relationship is there between stomatal density and rate does light intensity density of stomata and the effect this has on transpiration. Effect of the environment how the environment effects transpiration is the faster the rate of evaporation and therefore transpiration light intensity. To investigate whether light intensity affects the how does the light intensity affect the rate of transpiration to determine the effect of. Because of this, the transpiration rate is increased by an increase in light intensity the rate of transpiration can be affected by several factors.
#41 effect of light intensity on the rate of photosynthesis this is the rate of photosynthesis at that particular light intensity. Effects of relative humidity, light intensity and leaf temperature on transpiration of tomato scions. Class practical in this activity the rate of water uptake, due to transpiration, of an h-shaped potometer stomata when the light intensity is insufficient.Lab 3: transpiration in high light/high wind and low light/low wind habitats variables include light intensity a transpiration rates in light versus dark. This helps regulate transpiration and further effect on the rate of photosynthesis for of light intensity on the rate of photosynthesis. How light intensity effects transpiration rate in mangrove shoots biology eei task. When the relative humidity in a plant's environment rises, its rate of transpiration lowers, and a decrease in humidity causes the transpiration rate to. Transpiration, interesting mechanism of plants harmful effects of evaporation to plant so when the light intensity increases transpiration rate too. Effects of silicon on transpiration and leaf conductance in rice time after changes from high to low light intensity e, transpiration rate of whole plant. What is the effect of increasing and decreasing light intensity on the rate of transpiration of a plant and why thanks x. A brief review of light intensity or brightness of some effects of light intensity on plant this reduces the rate of light absorption and the rate of.
Transpiration and factors affecting it 35 / 5 higher light intensity increases rate of transpiration lower light intensity lowers rate of transpiration. Free an experiment to investigate the effect of light intensity the effect of light intensity on the rate of and rates of transpiration in. Q1 some students investigated the effect of light intensity on the rate of photosynthesis they used the apparatus shown in diagram 1 diagram 1.
Quizlet provides factors that affect the rate of transpiration positive correlation between light intensity and transpiration humidity effect transpiration. The rate of transpiration under the effects of certain atmospheric i predict that if the wind speed temperature or light intensity increases the rate of. Placing the cuttings near a high intensity (but cool) light there will be a loss in mass that will over-estimate the rate of transpiration nuffield foundation.Download
2018. Term Papers.


text:
A leader of the Black Axe confraternity in Ijebu-North, Ogun State has told the police they killed a young man, Adebisi Oriade and his lover, Julian Imaga, both students of Olabisis Onabanjo University (OOU) because he neither gave the cult money nor recognised them.
The deceased were gruesomely murdered at about 11pm, on May 28, while driving home in the man's Honda Crostour car marked FST350FA after visiting his friend identified simply as Akogun.
It was gathered that the suspects, Muritala Babatunde, 29, Taiwo Rasak, 18, and a certain Sodiq, still at large, blocked Oriade's vehicle, pinned the victims down with their guns and drove the vehicle to the bush.
Babatunde, it was gathered, planted his lover and alleged armourer, Yetunde Seun, to trail Oriade and feed him back with his location to enable them strike him.
Babatunde and Rasak were arrested on June 19 by operatives of the Inspector-General (IG) Intelligence Response Team (IRT), Ogun and Oyo States Federal Special Anti-Robbery Squad (FSARS) following a compliant from the deceased's relative, Rafiu Oriade.
"On the receipt of this complaint, police swooped on the hoodlums and two of the principal suspects were arrested. An iPhone X with etisalat simcard and two pairs of shoes belonging to the deceased were recovered from Babatunde.
"The decomposing corpses of the deceased were recovered on June 19 and deposited at the General Hospital morgue Ijebu-Ode for autopsy. The car was also recovered and parked at Awa Division, Ogun state and their operational guns were equally recovered. Investigation continues," said police spokesman, Jimoh Moshood, an acting Deputy Commissioner.
According to the police, Babatunde confessed he masterminded the murder, adding that the ordered Oriade to unlock his i-Phone x mobile phone, which he kept for himself.
The police said: "Babatunde is a university dropout and native of Oru-Ijebu in Ijebu-North LGA. He said he was initiated into Black Axe (Aiye) cult while he was in school and was currently the number one of the cult group in Ijebu-North axis.
"He had in his possession two locally made pistols which he and his gang used for crime.
"On the incident of May 28, he said had nursed evil intentions towards Oriade and so, he noticed his movement along Oru-Ijebu junction that day, called his gang, who assisted in the kidnap of the victims.
"He said they rode a motorcycle and blocked the victims, who were in Oriade's car, kidnapped them and took them to a bush along Ijebu-Ode Awa/Ibadan Expressway where both of them were killed.
"He confessed that before Oriade was killed, he ordered him to deactivate his password on his iPhone X. He took the phone and two pairs of shoes from Oriade's car and thereafter abandoned the car on the expressway having dumped their lifeless bodies in a bush at the back of an uncompleted building where they were murdered.
"He said the late victim, whom he said was a Yahoo boy, was giving money to a rival cult group and was not giving him money. He said the victim did not recognis him as the overall cult leader of that area."
Although Babatunde tried to cover up his lover, Rasak, it was learnt, told the police that Seun was the one who spied on the deceased and brought the weapons used to kill them.
In his confessional statement, Rasak, a native of Imota in Ikorodu, said Babatunde told him that Oriade took his money and for that reason, he wanted to kill him.
He said: "I was initiated into Aiye when I was 15-year-old. I have been staying with our leader, Babatunde.
"On that day, he called myself and Sodiq to meet him at Oru junction. When we got there, he told us he wanted to kill Oriade because he parted with his money.
"It was Babatunde's girlfriend, Seun Yetunde that monitored Oriade's movement and she also brought a bag containing two guns and a cutlass to us.
"Our leader gave me one of the guns, held one and gave the cutlass to Sodiq while we waited patiently for Oriade to come.
"Few minutes later, Oriade and his girlfriend we spotted and we based them down with the gun. Babatunde took over the steering, while Sodiq and I held the victims at gunpoint at the backseat.
"We drove off the junction and entered the bush where we killed them with planks and gun."


text:
Country music torrents
Music - Country / Western Torrents - SeedpeerClick to see more from the latest Country Guitar Lessons lessons (90 in total).Get the latest Country Music News and Videos on your favorite Artists.
Download Country Music (Videos) torrent or any other torrent from the Video Music videos.
Smithsonian Collection Of Classic Country Music (1981
All New Album Releases | AllMusic
100 Greatest Country Albums - Rate Your MusicThe CMA Awards is the longest running, annual music awards program on network television.
download Country Music torrents free - best torrent free
To share this torrent use the code below and insert it into comments, status messages, forum posts or your signature.The complete set includes 2 FREE BONUS CDs of sweet country ballads by Eddy Arnold, Johnny Cash, Lefty Frizzell,.
New Releases in Country - amazon.comThe Digital Music Index is a report produced by Musicmetric, a data and analytics company that maps the trends and preferences of music fans around the world.Download Top 500 Country Music Songs torrent or any other torrent from the Audio Music.
Download country music Torrents - Torrentz.CD
Country Guitar Lessons
Disqus - country music collection download torrentUsing Torrent Websites you can Download Music torrents, albums,. 15 Best Torrent Download Sites.
CMT : TV Shows : Watch Full Episodes Online : Featured
Country / Western Music torrents - btscene.unblocked.camAnnual ranking of the most popular country songs, based on radio airplay audience impressions as measured by Nielsen Music, sales data as compiled.
Discover top country songs from your favourite country music stars.
Country Music: Top Country Albums Chart | Billboard
Country Karaoke - Instrumental MP3 - MP3 KaraokeTop Country Albums ranks the most popular country albums of the week, as compiled by Nielsen Music, based on multi-metric consumption (blending traditional album.
VA – Classic Country Music: A Smithsonian Collection (1990
IUMA: US Classic Country Music - Internet Archive
Top Country - Top Country Songs, Country Music Playlists
Fast and Clean downloads from joutorrent.com a free public file sharing platform.About New Releases in Country These lists, updated hourly, contain bestselling items.
RoughStockThe Free Music Archive offers free downloads under Creative Commons and other.Are there any actual trackers (private or not) that specialize in country music torrents.
Visit CMT.com to watch Full Episodes of your Favorite Country Music Television Shows Online.
Homepage - 2017 CMA Awards
Hot Country Songs - Year-End 2015 | BillboardNoStream has Shut Down. Jan. 9. 2017. NoStream was a Private Torrent Tracker for MUSIC.
VA – Ultimate Country: 4CDs of Great Country Music (2015
Country Music - download free torrents| loadtorrents.net
Torrent search for Pirate Bay, Extratorrent, RARBG, Kickass Torrents and many private torrent trackers.
Music - Private Torrent Trackers & File Sharing
Download country music Torrents | 1337x
Jamendo is a website with a revolutionary model that allows artists to promote and publish their music. country, popularity. 5 Ways To Download Torrents.


text:
A. By default, you can capture images that ADS can use by rebooting the server to be captured, then rebooting it through Preboot Execution Environment (PXE). PXE runs a set of automated steps and captures the content of the local disk, then uses the ADS deployment agent to create an image file. Sometimes you might want to manually create images by booting from an alternative medium, such as a WinPE boot CD-ROM or by using WinPE over a network. To let you manually create images in this way, Microsoft provides the imgdeploy.exe command, which is typically used to capture and restore server images but works for imaging any type of disk partition. To use imgdeploy.exe, you need these three files:
- imgdeploy.exe
- imglib.dll
- adssupport.dll
The imgdeploy.exe and imglib.dll files are located by default in the C:\program files\microsoft ads\bin folder. (Adssupport.dll is installed only with the full installation of ADS.) Place these files in the same folder. Then, to capture a partition, run the command
imgdeploy /capture /p <partition to capture> <location for the captured file> "<a comment about the captured image>"
For example, you might type
imgdeploy /capture /p c: m:\images\xppro.img "Windows XP Professional image"
(The command wraps to two lines here because of space constraints.) In this sample command, the /p switch compresses the disk image--which contains the entire content of the C drive--and copies it to an image file called xppro.img in the \images folder on network drive M. As the command executes, you'll see a capture-progress message like the figure at Figure.You can deploy the image file to other servers or use it as a backup copy of the partition.


text:
Frequently Asked Questions
Gift Certificates
Purchasing Gift Certificates
How to send Gift Certificates
Buying with Gift Certificates
Redeeming Gift Certificates
When problems occur
Product Customisation
Paper Types
Aging Effects
Leather Colours
Construction Techniques
Leather Finishes
Construction Techniques
When ordering our leather goods you can choose which construction method is best for you. This allows varying levels of authenticity for the product you are purchasing. You can choose from the following:


text:
Bi Writers & Poets, Musicians & Singers, Artists & Photographers, & Comedians: express yourself & be visible! Everyone else is invited to listen in. Bring friends. Themes are bi visibility and celebrating bisexuality, so be your fierce bi self! Sign up sheet to perform at the door: first come, first serve. Everyone has 5 minutes. Host Sheela Lambert is director of the Bisexual Book Awards and the Bi Writers Association, the event sponsors. She is the editor of Best Bi Short Stories, the only book of bisexual short stories in the world. Best Bi Short Stories got the triple whammy: shortlisted by the Lammys, Rainbow Book Awards and Bisexual Book Awards. She also runs the Bi Book Club which meets monthly at the Bureau.
Equipment: If you need anything besides a mic, contact Sheela at the Bi Writers Assoc. as soon as possible and let me know what equipment you need and why.
RSVP: Bi Meetup Performers can post a comment about what they'll perform on Bi Meetup too.
Note: Our definition of bi or bi+ includes bisexual, pansexual, fluid and many other identity words that imply emotional and/or sexual attraction to more than one gender. Questions? Write us.
@BiBookAwards https://www.facebook.com/biwriters


text:
Post # 1
- bobbypinpearls
- 9 years ago
- Wedding: July 2010
Ugh, we need invites, but i don't know were to start. I've seen some online that i like, but then there are outragious prices or not the colors i want. I've also thought about making my own, but i'm worried i won't have the attention span for and i'll procrastinate.
Where did you get your invites? any pictures?or advice?
thanks girls!


text:
Call at Kindle customer service number and get instant solution for your. On reading responces. A thorough Kindle Paperwhite, Kindle Keyboard, side- by- side feature comparison of Amazon' s Kindle, Kindle Fire Kindle Fire HD. Kindle: $ 79 A good, cheap E- Reader.
Even if you don' t have a Kindle, you can still download free Kindle ebooks to read on your PC. Touchscreen display that reads like paper, no screen glare even in bright sunlight. Kindle 6" glare- free touchscreen display designed as a dedicated e- reader. The Nook Sony EReader, iPad , Kindle other eBook readers can display Project. How to download books to kindle using wifi. It was announced on September 25 is. 6" high- resolution display with built- in light- - provides crisp, laser quality text with the highest resolution of any ereader.
Seeking online Kindle fire technical service help for any kind of Kindle related problem. How to download books to kindle using wifi. For the unacquainted the Amazon Kindle is an e- ink portable reader that allows you to download bring over a thousand books with you wherever you go.
This is a web app that connects to your Amazon Kindle account and lets you read your eBook collection from any PC. Buy Kindle Wi- Fi Touch E- Reader - White at Argos.
Dec 30 · Project Gutenberg is not just for your desktop notebook computer! Kindle Cloud Reader: read eBooks from anywhere. Get the latest Apps reviews Apps buying guides Apps prices from the knowledgeable experts at PCWorld.
Dear Sir Shortcuts Guide". Kindle Keyboard WiFi: $ 99 w/ offers this thing is pretty cheap. The Fire HDX formerly named Kindle Fire HDX is the high- end model in Amazon' s Kindle Fire line of tablet computers. Our best- selling Kindle.
Learn how to buy kindle books in Singapore and Malaysia for your kindle, kindle Paperwhite or kindle fire. This is a FREE tutorials with step- by- step guide!
Buy Amazon Kindle Wi- Fi Voyage E- Reader at Argos.


text:
Tengku Irfan, 20 year old Malaysian, started his piano lessons at age 7 and developed an interest in composing shortly after. He started composing piano pieces and later branched out with chamber pieces and larger orchestral works. Irfan has performed with orchestras worldwide with conductors Claus Peter Flor, Neeme Järvi, Kristjan Järvi, Robert Spano, George Stelluto, Sydney Hodkinson, Larry Rachleff, Jeffrey Milarsky, Timothy Weiss, Stephen Burns, Evan Rogister, Nicolae Moldoveanu, Darrell Ang, Dariusz Mikulski, Claudio Cruz among others. The New York Times described his playing as "eminently cultured" and possessing "sheer incisiveness and power". His compositions have been premiered by highly acclaimed orchestras/ensembles and won international awards.
His debut major performance was at age 11, as a soloist performing Beethoven's Piano Concerto in E flat (Wo04) with the Malaysian Philharmonic Orchestra under the baton of music director Claus Peter Flor, when he improvised his own cadenzas for all three movements. He continued to improvise his own cadenzas during subsequent performances of Mozart piano concerti.
Amongst his other onstage improvisation was in 2010, when he improvised on the piano to music simultaneously improvised by Neeme Järvi and the orchestra, at an event jointly organised by the International Arbo Valdma Summer Master Class for Pianists and Neeme Järvi International Master Class for Conductors. This led to future reengagements with Neeme Järvi and collaborations with Kristjan Järvi.
Previous performances include with the Juilliard Orchestra, solo recital at the la Virée classique Festival Montreal (on invitation from Kent Nagano), AXIOM, Singapore Symphony Orchestra, Sao Paulo State Youth Orchestra, Estonian National Symphony Orchestra, Malaysian Philharmonic Orchestra, Aspen Chamber Symphony, Peoria Symphony Orchestra, MDR Sinfonieorchester and Aspen Philharmonic Orchestra, amongst others.
At the Aspen Music Festival 2012, Irfan performed a four-hand piano concert with Yoheved Kaplinsky, where he learned and performed a repertoire previously unknown to him in mere two days upon being called at short notice to replace Choong Mo Kang, who injured his hand just before the concert.
He is also often chosen to premier new compositions (in performances and studio recordings) due to his speed and ease in learning new and demanding repertoire, and also for his interpretation of composers' works.
After winning the first prize in the Aspen Music Festival 2013 Prokofiev Piano Concerto No.2 Competition, Irfan continued to perform this concerto worldwide.
In 2014, Irfan was chosen as resident pianist for the Aspen Contemporary Ensemble, led by composer/conductor Sydney Hodkinson, and later by conductors Donald Crockett, Timothy Weiss and Stephen Burns. 2017 saw Irfan serving his fourth consecutive year in the Aspen Contemporary Ensemble. Irfan's chamber performances include collaborations with Esther Heideman, Edgar Meyer, David Halen, Robert Chen, James Dunham, Bruce Bransby, amongst others.
Irfan won both the 2012 ASCAP Morton Gould Award and the 2012 Charlotte Bergen Award for his 'String Quartet'. His orchestral piece Sahibul Hikayat Fantasy Overture, which was commissioned and premiered by the Malaysia Philharmonic Orchestra for their 2013-14 Season Opening & 15th Anniversary Gala, won the 2014 ASCAP Morton Gould Award.
His composition, an orchestral piece titled Keraian, was premiered by the New York Philharmonic at Avery Fisher Hall, with Case Scaglione conducting. The Singapore Symphony Orchestra world premiered Meditation in Singapore & Kuala Lumpur. Another orchestral piece titled 'Vivacity' was commissioned and performed by Kristjan Järvi & the MDR Simfonieorchester at the Leipzig Gewandhaus as part of the Leipzig Bachfest 2016 - 'Vivacity' won Irfan his third ASCAP Morton Gould Award (2017).
His other compositions which have been premiered in New York include 'Twelve-Tone Variations' (piano), 'Vier Miniaturen' (clarinet & piano), 'Introduction and March' (trumpet & piano), 'Symphony No.1' (full orchestra), 'Nocturne' (string orchestra), 'Suite for Wind Quartet', 'Melody' (violin & piano) and 'Moment Musical No.2' (bassoon & harp). Irfan's choral piece 'Tragedi Dewi Suria' was premiered by choral conductor Harold Rosenbaum and the New York Virtuoso Singers.
Irfan made his conducting debut with the MusicaNova Orchestra in April 2015, for the premiere of his string orchestral piece Nocturne. He conducted the premiere of his piece Pengembaraan Minda dan Kenyataan in New York, and recently conducted the Malaysian Philharmonic Youth Orchestra for the world premiere of his orchestral piece What Does It Take to Dance? in Kuala Lumpur and Singapore.
Amongst the venues worldwide which he has performed include the Central Music School of the Moscow Tchaikovsky Conservatory, Dewan Filharmonik Petronas (Kuala Lumpur), Kuala Lumpur Convention Center, Nokia Concert Hall (Estonia), Pärnu Concert Hall (Estonia), The Benedict Music Tent (Aspen), Harris Hall (Aspen), Ho Chi Minh City Opera House (Vietnam), Hanoi Opera House (Vietnam), Alice Tully Hall (Lincoln Center, New York), Peter Jay Sharp Theater (Lincoln Center, New York), Merkin Concert Hall (Lincoln Center, New York), Esplanade Concert Hall (Singapore), Sala Sao Paulo (Brazil), Teatro Polytheama (Brazil), Suhl Centrum (Germany), Leipzig Gewandhaus (Germany), Tokyo Opera City Hall (Japan), Hong Kong and Washington DC.
He has performed on live telecasts on national television in Malaysia, and on popular U.S. classical music radio programs 'From the Top' (when he was introduced by Emanuel Ax), WQXR, WGBH Classical New England's 'Drive Time Live', Europe's Klassikaraadio, Mitteldeutscher Rundfunk (MDR.de), and Deutschland Radio.
Irfan was a double major in piano & composition in Juilliard Pre-College (under Yoheved Kaplinsky & Ira Taxin). He is currently in Juilliard School as a double major in piano & composition under Yoheved Kaplinsky and Robert Beaser respectively, and also studying conducting with George Stelluto. Irfan was a High Honors high school student at the Professional Children's School, served as Teaching Artist Intern for the New York Philharmonic Composer's Bridge Program, and a proud recipient of the Juilliard School Kovner Fellowship Award.


text:
In-House Validation and Comparison of Two Wheat (Triticum aestivum) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR
Common wheat is one of the most important staple food crops worldwide. However, unlike other important staple crops such as maize or soybean, genetically modified (GM) wheat is not yet present in the global food market. Nonetheless, in the recent past, the adventitious presence of GM glyphosate-tolerant volunteers was reported in open wheat fields in the USA. The European Union Reference Laboratory for GM Food and Feed (EURL-GMFF) was therefore called to develop a strategy to detect such unauthorised GM wheat in wheat samples by using both taxon-specific and screening tests. Two candidate common wheat taxon-specific real-time PCR methods were suggested, one targeting ssII-D gene coding for starch synthase and the other targeting waxy-D1 gene, coding for granule-bound starch synthase. In the present study, the two above-mentioned real-time PCR taxon-specific methods were in-house verified and compared, proposing droplet digital PCR (ddPCR) as a new tool for supporting the application of the European Network of GMO Laboratories (ENGL) established method performance criteria. Preliminary performance data of waxy-D1 and ssII-D methods in ddPCR format are shown too to give a contribution to the bridging process from the consolidated to the emerging quantitative PCR methodology.
KeywordsValidation Wheat Real-time PCR Droplet digital PCR Taxon-specific Endogenous reference gene Genetically modified organism GMO
Common wheat (Triticum aestivum) is one of the major food crops in the world. Belonging to the Triticum genus, it is one of the most widespread staple cereals in the food industry, and consists of several cold-resistant cultivars allowing its cultivation in frigid climate areas too. The genus Triticum includes very complex polyploid genomes, symbolised by the letters A, B, D and G. Common wheat (T. aestivum) is hexaploid AABBDD (1C value = 17.33 pg; Bennett and Smith 1976) and Triticum durum is tetraploid AABB.
Betting on its potential impact in the food sector, the public research biotech industry has been striving for decades to put genetically modified (GM) wheat on the market by trying to incorporate desired traits such as herbicide tolerance, disease resistance and improvements in the quality of this crop (Leckband and Lörz 1998; Anand et al. 2003; Hu et al. 2003; Abouseadaa et al. 2015; Shin et al. 2008). Within this line of research, in the 90s, Monsanto, one of the major agrobiotech companies, launched a programme for the development of genetically modified common wheat tolerant to glyphosate herbicide, and some promising GM "events" (such as MON71800) were authorised in the USA to be tested in field trials. However, after completion of the experimental phase, no GM wheat has ever been marketed. Nevertheless, in 2013, the presence in Oregon (USA) of unexpected glyphosate tolerant wheat was noticed and then confirmed and reported by the United States Department of Agriculture (USDA). This event was ascribed to MON71800 volunteers, most likely originating from the GM wheat field trials (United States Department of Agriculture (USDA) 2015). Moreover, in 2016, a similar situation was found in Washington state due to the adventitious presence of a MON71800 sister GM event named MON71700 (United States Department of Agriculture–USDA news 2016). As this GM crops are not authorised in the EU, in both cases, the US Animal and Plant Health Inspection Service (APHIS) informed the European Commission and the member states of the European Union of the event and the European Commission urged the member states to start testing consignments of common wheat at import from the USA for the presence of unauthorised GM wheat.
The authorisation process for introducing GM products in the EU market is ruled by Directive 2001/18/EC (European Parliament and Council of the European Union 2001), for the deliberate release into the environment of genetically modified organisms, and by Regulation (EC) 1829/2003 (European Parliament and Council of the European Union 2003) for GM food and feed. According to this legislation, in addition to the data concerning risk assessment, the applicant must provide a method for the detection and quantification of the transformation event under authorisation. The method has to be validated by the European Union Reference Laboratory for GM Food and Feed (EURL-GMFF), established at the Joint Research Centre and assisted by the European Network of GMO Laboratories (ENGL), according to the document "Definition of minimum performance requirements for analytical methods of GMO testing-MPR" (European Network of GMO Laboratories (ENGL) 2015).
Quantitative methods are required for enforcing EU labelling and traceability rules for food/feed containing authorised GM material >0.9% with respect to the specific ingredient. The quantitative analysis consists of a relative quantification of the GMO event to the plant species itself (taxon reference system). In this context, the selection of the species-specific reference system is crucial for the reliability of the quantitative analytical results obtained.
Real-time PCR represents the gold standard for GMO analysis, and the MPR guidelines refer exclusively to this technique. Nevertheless, digital PCR (dPCR) represents one of the more recent and promising quantitative PCR techniques. In dPCR, the sample is distributed across a large number of partitions containing zero, one, or more copies of target DNA. The absolute number of target DNA is then calculated by taking into consideration the ratio of positive (or negative) partitions to total partitions and using Poisson distribution model. According to this model, λ is the average number of target copies per partition, and it is determined by using λ = −ln(1 − k/n), where n is the total number of partitions detected and k is the number of positive partitions detected (Pinheiro et al. 2012).
Digital PCR platforms currently available are essentially classified by the way partitioning of reactions is obtained, either making use of microfluidic chambers or running individual reactions in droplets generated in a water-oil emulsion. The number of references to dPCR and its possible application in the field of GMO quantification have dramatically increased in recent years (Burns et al. 2010; Corbisier et al. 2010; Morisset et al. 2013; Demeke T. et al. 2014; Dobnik et al. 2015; Koppel and Bucher 2015; Dobnik et al. 2016; Félix-Urquídez et al. 2015; Gerdes et al. 2016; Zhu et al. 2016).
In the lack of an event-specific method for the identification of the above-mentioned unauthorised GM wheat events, the European Union Reference Laboratory for GM Food and Feed (EURL-GMFF) developed a strategy to detect GM wheat in wheat samples by using both taxon-specific and screening tests. This strategy was then described in a guidance document published on the EURL-GMFF website (European Union Reference Laboratory for GM Food and Feed 2013a), together with a comprehensive review and related bioinformatics analysis on all available relevant literature on taxon-specific real-time PCR systems for the identification of T. aestivum (European Union Reference Laboratory for GM Food and Feed 2013b). The results of the study suggested that the method described by Matsuoka et al. (2012), targeting ssII-D gene coding for starch synthase, and the one described by Iida et al. (2005), targeting waxy-D1 gene, coding for granule-bound starch synthase, may represent good candidates to specifically identify common wheat even in complex food samples. With regard to the latter method, what was reported in the above-mentioned EURL-GMFF guidelines had to be carefully considered, i.e. "according to in silico analyses conducted by the EURL GMFF, the primer sequences described in Iida et al. 2005 are not correct. The primers (Wx012F/Wx012R) and probe (Wx-Taq 1) sequences reported by the same authors in the following article: "Imai et al. (2012), Food Hyg. Saf. Sci. 204 Vol. 53 203–210 and the patent EP2180051" are recommended as reference.
In 2016, EURL-GMFF verified an endpoint real-time PCR method submitted by Monsanto Company, for detecting event MON71800 in wheat, but the wheat-specific system of choice was designed on T. aestivum acetyl-coenzyme A carboxylase, Acc-1, reference target (Genebank AF029897.1), although it is unable to discriminate between T. aestivum and T. durum (European Union Reference Laboratory for GM Food and Feed 2016). Given the importance of T. durum in food matrices, such an evident lack of specificity was considered to be a major drawback, and therefore, Acc-1 targeting method was not deemed appropriate for the aim of our study. The two above-mentioned methods (waxy-D1 and ssII-D), which had been previously recommended by EURL-GMFF, were then analysed and compared to select a unique taxon reference system for the identification and the quantification of wheat.
In order to evaluate possible within-species copy number variation of the two above-mentioned target sequences (waxy-D1 and ssII-D), a representative number of wheat cultivar samples were analysed in parallel using both real-time PCR and digital droplet PCR platform. The two taxon-specific systems, waxy-D1 and ssII-D, were compared for several validation parameters in accordance with "Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing" document (European Network of GMO Laboratories (ENGL) 2015).
Materials and Methods
Plant Materials
Seeds from the following 16 wheat cultivars, provided by Consiglio per la Ricerca in agricoltura e l'analisi dell'Economia Agraria (CREA) seed testing laboratory, were used in the study: (A) Aquilante, (B) Palesio, (C) Bologna, (D) Blasco, (E) Aubusson, (F) Antille, (G) Bandiera, (H) Alatamira, (I) Pandas, (L) Ora, (M) Albachiara, (N) Laria, (O) Mieti, (P) Bisquit, (Q) Sagittario and (R) Bramante (Table S1 in supplementary material).
Sample Preparation and DNA Extraction
Seed samples were milled (GM200 RETSCH, Haan, Germany) and the ground material was used for DNA extraction. Total genomic DNA was extracted using the ION FORCE FAST DNA Extraction Kit for processed food and feed complex material (GENERON, Modena, Italy) from 2 g of each cultivar, according to the manufacturer's instructions. DNA concentration was calculated from the absorption at 260 nm measured with a UV spectrophotometer (Biophotometer, Eppendorf, Hamburg, Germany). The DNA purity was checked by both calculating the ratio of the absorption values at 260/280 nm and carrying out a real-time PCR (inhibition run) targeting waxy-D1 encoding gene on two different amounts of each extracted DNA (300 and 75 ng, i.e. 1:4 dilution). As the theoretical ΔCq value is 2 for 1:4 dilution, the following acceptance criterion was considered: 1.5 < ΔCq < 2.5.
Oligonucleotide Primers and Probes
Primers and probe used in this study (real-time PCR and ddPCR)
Gene name
Primer/probe name
Real-Time PCR
Real-time PCR reactions were performed in optical 96-well PCR plates and run on the ABI PRISM SEQUENCE DETECTION SYSTEM 7900HT (Thermo Fisher Scientific Waltham, MA USA), in a final volume of 25 μL. Real-time PCR mixture for both primers and probe set (waxy-D1 and ssIID) contained the following amounts: 200 ng DNA, 0.5 μM primers pairs, 0.2 μM probe and 12.5 μL Universal Master Mix (Thermo Fisher Scientific Waltham, MA USA). Real-time PCR runs had the following thermal profile: 2′ 50 °C, 10′ 95 °C; [15″ 95 °C, 60 °C 60″] × 50 cycles. Amplification plots were visualised in the logarithmic graph for manual setting of the fluorescent threshold value. This threshold was chosen in the middle of the linear phase of the PCR plots; the baseline was evaluated in the linear graph by manual setting too. After manual adjustment of these parameters, a regression curve for each DNA dilution set was obtained by plotting all of the Cq values, from each concentration level, against the log of the DNA copy number, obtained by ddPCR (see protocol below), at the corresponding concentration level. The regression curve Cq = a log(copy number) + b was used to calculate the experimental copy number corresponding to each specific Cq value.
Droplet Digital PCR
ddPCR reaction mixture contained 4 μL of DNA template (20 μL total reaction volume); final primers and probe concentrations in ddPCR mixes were identical to the real-time PCR conditions used in this study. Droplets were generated in 8-well cartridges, using the QX100 droplet generator (Bio-Rad, Pleasanton, CA). Water-in-oil emulsions were transferred to a 96-well plate and amplified in a PCR thermocycler GeneAmp PCR System 9700 (Thermo Fisher Scientific, Waltham, MA, USA) with the following thermal profile: 10 min at 95 °C, followed by 45 cycles of two steps of 30 s at 94 °C and 60 s annealing/extension at 60% ramp rate 60 °C; after amplification, products were denatured at 98 °C for 10 min, then cooled to 4 °C. Plates were then transferred to the QX100 droplet reader (Bio-Rad, Pleasanton, CA). The threshold was set manually, using the fluorescence amplitude versus event number (1D amplitude). Data generated by the QX100 droplet reader were analysed by Quantasoft 1.7.4.0917 software rejecting PCR reactions (single wells) with a too low number of droplets (<8000).
With regard to ddPCR-specific quality criteria (i.e. amount of rain and peak resolution R s ), the calculation procedures indicated in Lievens et al. (2016) were adopted.
Experimental Design for the Real-Time PCR Verification and Comparison Study
Evaluation of Within-Taxon Allelic Variation of the Target Sequences
For each cultivar, a DNA dilution set was prepared (D1 to D6) from 160 to 0.13 ng (see Table S2 in supplementary material). Each dilution set was tested on the same plate with waxy-D1 and ssII-D systems, both in real-time PCR and ddPCR (four replicates for D1 to D4 and eight replicates for D5 and D6). A total of 16 real-time PCR runs and 16 ddPCR runs were carried out and analysed. Cq values attained by real-time PCR were plotted against the log of the copy number estimated by ddPCR (for each corresponding concentration level) to create 16 regression curves for waxy-D1 system and 16 regression curves for ssII-D system.
Precision—Relative Repeatability Standard Deviation
In order to evaluate precision of waxy-D1 and ssII-D real-time PCR methods, five DNA dilutions (1:4) of two varieties, Aquilante (A) and Palesio (B), from S1 (300 ng) to S5 (1.17 ng) were performed and then tested in ddPCR for both endogenous systems.
Two different real-time PCR plates were run, one for waxy-D1 and one for ssII-D (Table S3 in supplementary material). In each plate, cultivar A was used as calibrator. Dilutions from S1 to S5 (three replicates each) were loaded three times in order to have three calibration curves in the same plate. Cq values, from each concentration level, were plotted against the corresponding log copy number, as estimated by ddPCR. An identical dilution set from cultivar B was loaded three times on the same plate as unknown samples. For each concentration level, 27 data points (9 by each dilution set) were generated to calculate percent relative standard deviation of repeatability (RSDr%).
Limit of Detection and Limit of Quantification
A subset of DNA dilution sets (D1 to D6) prepared as described above from 5 out of 16 cultivars (Aquilante, Palesio, Bologna, Blasco, Albachiara) were exploited in order to obtain 20 data points for D1–D4 and 40 data points for D5 and D6.
For improving the limit of quantification (LOQ) resolution, a second test was carried out on a new dilution set (i.e. 1400, 700, 350, 175, 90, 45, 23 and 11 theoretical copy numbers) from Aquilante cultivar DNA. For each level, 20 data points were obtained.
In order to check the capacity of the methods to remain sufficiently unaffected by deliberate deviations, they were tested at different concentrations of master mix components (less/more 30% primers and probes and less/more master mix by addition or subtraction of 1 μL), as well as with a different real-time PCR instrument (FAST REAL TIME PCR SYSTEM 7900HT, Thermo Fisher Scientific, Waltham, MA, USA). Robustness was tested on 50 copies of the target (waxy-D1 and ssII-D) in six replicates at each condition.
Sequence Analysis of the waxy-D1 and ssII-D Target Sequences
Target sequences of both waxy-D1 and ssII-D PCR systems were amplified for all 16 wheat varieties, by using external primer pairs, conveniently designed (Primer-BLAST) on the sequences referred to on the respective original papers, to be specific for the corresponding flanking sequences from all wheat varieties. The amplicons obtained for both waxy-D1 and ssII-D from all 16 wheat varieties were sequenced by ABI Prism 3500 genetic analyser (Thermo Fisher Scientific, Waltham, MA, USA). All the sequences were then aligned by using Sequencing Analysis Software (Thermo Fisher Scientific, Waltham, MA, USA). Primers used in this study for sequence analysis (see Table S4 in supplementary material) have been selected on the corresponding reference sequences by using Primer-BLAST online resource (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).
Results and Discussion
Selection of the Two Taxon-Specific Real-Time PCR Methods and Alignment of the Thermal Profiles
Specificity—"property of a method to respond exclusively to the characteristic or analyte of interest". The annealing/extension temperature at 60 °C was in both cases increased by comparison with the original settings. The specificity of both PCR assays was then expected to be increased and, in any case, not decreased by this shift. We looked at the rather extensive specificity tests conducted in the two original papers as well as at the independent and exhaustive EURL-GMFF survey before deciding that such a parameter was already covered by the existing literature. So we just checked some GM CRMs (ERM and AOCS) available in our laboratory for routine tests (data not shown), but the specificity test was considered redundant;
Trueness—"the closeness of agreement between the average value obtained from a large series of test results and an accepted reference value". Trueness was not assessed due to unavailability of wheat CRM in copy number and to the lack of clarity r in the scientific community regarding the use of dPCR data as "true value" for this purpose. In absence of an "accepted reference value", this parameter was not included in the study;
All the other MPR guideline performance parameters were evaluated in this study.
Use of ddPCR Versions of waxy-D1 and ssII-D Real-Time PCR Methods in This Study and Some Performance Data
In this study, digital PCR was mainly used for supporting the evaluations and comparisons on the corresponding real-time PCR systems. Droplet digital PCR is a fairly new PCR format, and its performance criteria are still being defined and harmonised. With this regard, a recent paper (Lievens et al. 2016) has shed new light on ddPCR performance parameters, paying special attention to the GMO context. It recognises some performance parameters adopted for qPCR, such as applicability, practicability, specificity, dynamic range and trueness, to be directly transferable to the digital PCR. Nevertheless, some new ddPCR-specific criteria are suggested to ensure robust classification of positive and negative droplets, as this aspect shows to be of crucial importance for obtaining an accurate and reliable quantification of target copy number in ddPCR. In particular, it is recommended to check that (i) a single amplification product is present, (ii) the amount of rain (droplets having an intermediate fluorescence level between negative and positive populations) is lower than 2.5% and (iii) the peak resolution (R s ) is at least 2.5. According to the authors, the optimal conditions for evaluating such parameters could be achieved with control samples having a λ of approximately 0.7 as at this concentration, approximately half the partitions are positive and half are negative for the target. In the present study, which follows this suggestion as much as possible, the two droplet digital PCR methods (waxy-D1 and ssII-D) have been evaluated for such specific performance criteria. Indeed, the percentage of rain and the peak resolution were calculated at approximately 160 ng of wheat DNA, corresponding to the higher concentration level tested in this study, with a λ value ranging from 0.3 to 0.4. Both methods proved to be compliant with the proposed requirements, with a percentage of rain in the range of 0.403–1.154% for waxy-D1 and in the range of 0.651–1.427% for ssII-D and R s being between 9.21 and 10.10 for waxy-D1 and between 8.11 and 9.95 for ssII-D (see Fig. S1 and Table S5 in supplementary material).
Evaluation of Within-Taxon Allelic Variation of the Target Sequences
In the field of GMO analysis, the adoption of a reference gene target with a stable, known and low copy number, preferably 1 copy per haploid genome equivalent (HGE), and with no allelic variation amongst the cultivars of the same species, is recommended.
In order to evaluate the aforementioned requirements, 16 common wheat cultivars, acquired with the help of CREA (Italian seed testing laboratory and project partner) as varieties cultivated in Italy, were tested with both waxy-D1 and ssII-D endogenous reference PCR systems.
Furthermore, in order to verify the absence of single-nucleotide polymorphisms (SNPs) in the PCR target sequences, the amplicons generated for each cultivar by both waxy-D1 and ssII-D PCR systems were sequenced. The analysed DNA sequences were always identical to the corresponding reference sequences all across the cultivars tested (data not shown).
In the context of GMO quantification, the taxon-specific PCR system should target a single DNA copy per allele within the taxon of interest. The absence of copy number variation within the taxon is generally verified on a representative collection of cultivars, by using real-time PCR (Rønning et al. 2006; Vautrin and Zhang 2007; Chaouachi et al. 2008; Jacchia et al. 2015). The range of variability of Cq values in amplification should not exceed 1 Cq within the taxon (European Network of GMO Laboratories (ENGL) 2015). Moreover, in order to establish gene copy number, Southern blot is considered the most reliable technique, although the process is labour intensive and not automatable. A recent paper (Głowacka et al. 2016) compared Southern blot and PCR-based methods (qPCR and droplet digital PCR) for their ability to determine T-DNA copy number and homozygosity in transgenic plants. Setting Southern blotting as the gold standard, data obtained by ddPCR resulted more accurate than those generated by qPCR. On the basis of these findings, ddPCR technique seems to be, at present, the most convenient tool for routinely assess T-DNA copy number and homozygosity. The validity of ddPCR technique for the determination of zygosity in transgenic plants is also confirmed by Xu et al. (2016). Therefore, the experimental design of the present study is mainly based on ddPCR-generated data set.
Performance Data of waxy-D1 and ssII-D Real-Time PCR Methods in Comparison
Amplification Efficiency, R 2 and Dynamic Range
Slope and R 2 of the 16 regression curves one per cultivar (waxy-D1 method and ssII-D method)
Cultivar A
Cultivar B
Cultivar C
Cultivar D
Cultivar E
Cultivar F
Cultivar G
Cultivar H
Cultivar I
Cultivar L
Cultivar M
Cultivar N
Cultivar O
Cultivar P
Cultivar Q
Cultivar R
Precision—Relative Repeatability Standard Deviation
Precision is defined as the relative standard deviation of test results obtained under repeatability conditions. Five dilutions (1:4) of the two cultivars Aquilante (A) and Palesio (B) were set and analysed as described above, with the lower concentration level (S5) slightly above the limit of quantification of the two PCR methods. Cultivar A (Aquilante) was used as calibrator and cultivar B (Palesio) as unknown sample. According to the MPR guidelines, RSDr% should be ≤25% over the whole dynamic range of the PCR modules. In our results, RSDr% ranged between 3.75 and 20.98 for ssII-D, and between 5.68 and 15.56 for waxy-D1 (Tables S8 and S10 in supplementary material).
Comparison of quantitative data obtained for both waxy-D1 and ssII-D in real-time PCR and ddPCR in parallel
Limit of Detection and LOQ
Absolute limit of detection and absolute limit of quantification of the two real-time PCR methods, waxy-D1 and ssII-D, were estimated on DNA extracted from five cultivars (Aquilante, Palesio, Bologna, Blasco, Albachiara). In order to match each specific Cq value to the corresponding absolute target copy number estimated in ddPCR, data obtained by the evaluation of within-taxon allelic variation of the target sequences were exploited (Tables S11 and S12 in supplementary material). In both cases, limit of detection (LOD) is below 7 copies. Limit of quantification was set to be below the lowest tested amount showing a RSDr% ≤25%. In both cases, the LOQ is below 63 copies. This value is slightly outside the MPR guideline acceptance criteria (i.e. 50 copies), so it was necessary to refine our results by carrying out an additional test for improving the LOQ resolution below 62 copies. A new dilution set (i.e. 1400, 700, 350, 175, 90, 45, 23 and 11 theoretical copy numbers) from Aquilante cultivar DNA was tested in 20 replicates per level. The actual LOQ was then set at 23 theoretical copy number for both methods (see Tables S13, S14, S15 and S16 in supplementary material).
On the basis of our data obtained, both taxon-specific real-time PCR methods show to be promising for being adopted in quantitative GMO analysis. With regard to the choice, as already stated in a previous study (Paternò et al. 2009), from both a theoretical and technical point of view, there is no reason for changing the PCR reference system according to the GM event to be quantified (except for very peculiar situations related to food matrix and amplicon length). Therefore, the use of a single taxon-specific PCR reference system for enforcement purposes is not only advisable for practical reasons but also sensible from a scientific perspective. Nevertheless, the present study could not find any element to prefer ssII-D to waxy-D1 or vice versa, even though waxy-D1 is more supported by previous literature (Huang et al. 2013).
The robustness of a method is defined as the "measure of its capacity to remain unaffected by small but deliberate deviations from the experimental conditions described in the procedure". Currently, MPR guidelines provide some indications on how to evaluate robustness of qualitative and quantitative methods. Nevertheless, no specific suggestions are provided for assessing a taxon reference module to be used in a quantitative method. In our study, standard conditions were compared to deliberately altered conditions by testing ssII-D to waxy-D1 at 50 target copies (higher acceptance limit for LOQ according to the MPR guidelines) and reporting ΔCq values. ΔCq is the difference between the Cq mean obtained in standard condition and the Cq means obtained by each deviating condition. The acceptance criterion adopted was ΔCq ≤ 1. Deliberate changes to the initial protocol did not affect waxy-D1 and ssII-D targets; all the differences tested were ≤0.5 Cq (Tables S17 and S18 in supplementary material).
In the present study, two real-time PCR wheat taxon-specific methods were selected from the relevant literature available as potential candidates for playing the role of a taxon-specific reference system for GM wheat quantification. The first aim of our work was to provide in-house validation data according to MPR and the comparison of the two best-performing reference systems so far available in literature for GM wheat quantification. We hereby also propose the use of digital PCR (and its absolute quantification power) in support of the verification and the validation of taxon-specific reference systems, where intervarietal stability of the target sequence is a priority. Previous performance data were evaluated and, where necessary, integrated according to the MPR guideline requirements for single laboratory validation. The alignment of test conditions permitted the direct comparison of the two candidate methods in the same testing sessions. According to the data obtained, both methods have demonstrated to be in line with the MPR acceptance criteria. In order to support the evaluations and comparisons of these real-time PCR systems, the corresponding droplet digital PCR versions were used, therefore contributing to the bridging process from a consolidated to an emerging quantitative PCR methodology. The results suggest that the two real-time PCR methods are comparable and both eligible as wheat taxon-specific reference systems in GMO quantification assays.
With a view to having additional confirmation of this potential, it will be very interesting to test both methods in combination with a GM-specific ddPCR module (e.g. MON71800-specific or MON71700-specific) as soon as the corresponding CRMs are available.
We are very grateful to our project partners Dr. Elena Perri and Dr. Daniela Villa from Consiglio per la Ricerca in agricoltura e l'analisi dell'Economia Agraria–Centro di sperimentazione e certificazione delle sementi–Sede di Tavazzano (CREA-SCS) for providingthe wheat materials tested in this study.
Financial support was provided by the Italian Ministry of Health through the National Current Research Program (Ref. IZS LT 14/11 RC).
Compliance with Ethical Standards
Conflict of Interest
Annalisa Paternò declares that she has no conflict of interest. Daniela Verginelli declares that she has no conflict of interest. Pamela Bonini declares that she has no conflict of interest. Marisa Misto declares that she has no conflict of interest. Cinzia Quarchioni declares that she has no conflict of interest. Enrico Dainese declares that he has no conflict of interest. Stefania Peddis declares that she has no conflict of interests. Cristiana Fusco declares that she has no conflict of interests. Daniela Vinciguerra declares that she has no conflict of interests. Ugo Marchesi declares that he has no conflict of interests.
Ethical Approval
This article does not contain any studies with human or animal subjects performed by any of the authors.
Informed Consent
Informed consent is not applicable for the nature of this study.
Supplementary material
- Abouseadaa HH, Osman GH, Ramadan AM, Hassanein SE, Abdelsattar MT, Morsy YB, Alameldin HF, El-Ghareeb DK, Nour-Eldin HA, Salem R, Gad AA, Elkhodary SE, Shehata MM, Mahfouz HM, Eissa HF, Bahieldin A. (2015) Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects. BMC Plant Biol. 22;15:183. doi: https://doi.org/10.1186/s12870-015-0570-x
- Anand A, Zhou T, Trick HN, Gill BS, Bockus WW, Muthukrishnan S (2003) Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum. J Exp Bot 54(384):1101–1111. https://doi.org/10.1093/jxb/erg110 CrossRefGoogle Scholar
- Bennett MD, Smith JB. (1976) Nuclear DNA amounts in angiosperms. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 274: 227–274. Kew Royal Botanic Garden. http://data.kew.org/cvalues/
- Burns MJ, Burrell AM, Foy CA (2010) The applicability of digital PCR for the assessment of detection limits in GMO analysis. Eur Food Res Technol 231(3):353–362. https://doi.org/10.1007/s00217-010-1277-8 CrossRefGoogle Scholar
- Chaouachi M, El Malki R, Berard A, Romaniuk M, Laval V, Brunel D, Bertheau Y (2008) Development of a real-time PCR method for the differential detection and quantification of four Solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annum). J Agric Food Chem 56(6):1818–1828. https://doi.org/10.1021/jf073313n CrossRefGoogle Scholar
- Corbisier P, Bhat S, Partis L, Xie VR, Emslie KR (2010) Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction. Anal Bioanal Chem 396(6):2143–2150. https://doi.org/10.1007/s00216-009-3200-3 CrossRefGoogle Scholar
- Demeke T, Gräfenhan T, Holigroski M, Fernando U, Bamforth J, Lee SJ (2014) Assessment of droplet digital PCR for absolute quantification of genetically engineered OXY235 canola and DP305423 soybean samples. Food Control 46:470–474. https://doi.org/10.1016/j.foodcont.2014.06.018 CrossRefGoogle Scholar
- Dobnik D, Spilsberg B, Bogožalec Košir A, Holst-Jensen A, Žel J (2015) Multiplex quantification of 12 European Union authorised genetically modified maize lines with droplet digital polymerase chain reaction. Anal Chem 87(16):8218–8226. https://doi.org/10.1021/acs.analchem.5b01208 CrossRefGoogle Scholar
- Dobnik D, Štebih D, Blejec A, Morisset D, Žel J (2016) Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection. Sci Rep 6(1):35451. https://doi.org/10.1038/srep35451 CrossRefGoogle Scholar
- European Network of GMO Laboratories-ENGL (2015). Definition of minimum performance requirements for analytical methods of GMO testing. Guidance document http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm
- European Parliament and Council of the European Union (2001) Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. Official Journal of European Communities. 1742001 L106(/1)Google Scholar
- European Parliament and Council of the European Union (2003). Regulation (EC) no 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feedGoogle Scholar
- European Union Reference Laboratory for GM Food and Feed (2013a). EU-RL GMFF guidance on testing for GM glyphosate-resistant wheat (MON71800) in wheat grain or in food/feed products containing wheat flour originating or consigned from the US. http://gmo-crl.jrc.ec.europa.eu/doc/Wheat_EURL_testingstrategy_V2.pdf
- European Union Reference Laboratory for GM Food and Feed (2013b). Literature and bioinformatics analysis of wheat-specific detection methods. http://publications.jrc.ec.europa.eu/repository /bitstream/JRC86656/wheat_bioinformatics%20technical%20report_with%20registration.pdf
- European Union Reference Laboratory for GM Food and Feed (2016) - Report on the verification of the performance of a simplex endpoint event-specific method for the detection of event MON71800 in wheat using real-time PCRGoogle Scholar
- Félix-Urquídez D, Pérez-Urquiza M, Valdez Torres JB, León-Félix J, García-Estrada R, Acatzi-Silva A (2015) Development, optimization, and evaluation of a duplex droplet digital PCR assay to quantify the T-nos/hmg copy number ratio in genetically modified maize. Anal Chem 88(1):812–819. https://doi.org/10.1021/acs.analchem.5b03238 Google Scholar
- Gerdes L, Iwobi A, Busch U, Pecoraro S (2016) Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms. Biomol Detect Quantif 7:9–20. https://doi.org/10.1016/j.bdq.2015.12.003 CrossRefGoogle Scholar
- Głowacka K, Kromdijk J, Leonelli L, Niyogi KK, Clemente TE, Long SP (2016) An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants. Plant Cell Environ 39(4):908–917. https://doi.org/10.1111/pce.12693 CrossRefGoogle Scholar
- Hu T, Metz S, Chay C, Zhou HP, Biest N, Chen G, Cheng M, Feng X, Radionenko M, Lu F (2003) Fry J. Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep 21(10):1010–1019. https://doi.org/10.1007/s00299-003-0617-6 CrossRefGoogle Scholar
- Huang H, Cheng F, Wang R, Zhang D, Yang L (2013) Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection. PLoS One 8(9):e75850. https://doi.org/10.1371/journal.pone.0075850 CrossRefGoogle Scholar
- Iida M, Yamashiro S, Yamakawa H, Hayakawa K, Kuribara H, Kodama T, Furui S, Akiyama H, Maitani T, Hino A (2005) Development of taxon-specific sequences of common wheat for the detection of genetically modified wheat. J Agric Food Chem 53(16):6294–6300. https://doi.org/10.1021/jf0505731 CrossRefGoogle Scholar
- Imai S, Tanaka K, Nishitsuji Y, Kikuchi Y, Matsuoka Y, Arami S, Sato M, Haraguchi H, Kurimoto Y, Mano J, Furui S, Kitta K. (2012) An endogenous reference gene of common and durum wheat for detection of genetically modified wheat. Food Hyg Saf Sci. 204 Vol. 53 203–210Google Scholar
- Jacchia S, Nardini E, Savini C, Petrillo M, Angers-Loustau A, Shim JH, Trijatmiko K, Kreysa J, Mazzara M (2015) Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay. J Agric Food Chem 63(6):1711–1721. https://doi.org/10.1021/jf505516y CrossRefGoogle Scholar
- Koppel R, Bucher T (2015) Rapid establishment of droplet digital PCR for quantitative GMO analysis. Eur Food Res Technol 241(3):427–439. https://doi.org/10.1007/s00217-015-2475-1 CrossRefGoogle Scholar
- Leckband G, Lörz H (1998) Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance. Theor Appl Genet 96(8):1004–1012. https://doi.org/10.1007/s001220050832 CrossRefGoogle Scholar
- Lievens A, Jacchia S, Kagkli D, Savini C, Querci M (2016) Measuring digital PCR quality: performance parameters and their optimization. PLoS One 11(5):e0153317. https://doi.org/10.1371/journal.pone.0153317 CrossRefGoogle Scholar
- Matsuoka Y, Arami S, Sato M, Haraguchi H, Kurimoto Y, Imai S, Tanaka K, Mano J, Furui S, Kitta K (2012) Development of methods to distinguish between durum/common wheat and common wheat in blended flour using PCR. Food Hyg Saf Sci 53(5):195–202CrossRefGoogle Scholar
- Morisset D, Štebih D, Milavec M, Gruden K, Žel J (2013) Quantitative analysis of food and feed samples with droplet digital PCR. PLoS One 8(5):e62583. https://doi.org/10.1007/s00216-009-3200-3 2143, 2150CrossRefGoogle Scholar
- Paternò A, Marchesi U, Gatto F, Verginelli D, Quarchioni C, Fusco C, Zepparoni A, Amaddeo D, Ciabatti I (2009) Finding the joker among the maize endogenous reference genes for genetically modified organism (GMO) detection. J Agric Food Chem 57(23):11086–11091. https://doi.org/10.1021/jf902560x CrossRefGoogle Scholar
- Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, Emslie KR (2012) Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 84(2):1003–1011. https://doi.org/10.1021/ac202578x CrossRefGoogle Scholar
- Rønning SB, Berdal KG, Andersen CB, Holst-Jensen A (2006) Novel reference gene, PKABA1, used in a duplex real-time polymerase chain reaction for detection and quantitation of wheat- and barley-derived DNA. J Agric Food Chem 54(3):682–687. https://doi.org/10.1021/jf052328n CrossRefGoogle Scholar
- Shin S, Mackintosh CA, Lewis J, Heinen SJ, Radmer L, Dill-Macky R, Baldridge GD, Zeyen RJ, Muehlbauer GJ (2008) Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum. J Exp Bot 59(9):2371–2378. https://doi.org/10.1093/jxb/ern103 CrossRefGoogle Scholar
- United States Department of Agriculture (USDA). (2015). https://www.aphis.usda.gov/brs/aphisdocs/mt140008_froi_red_20160801.pdf
- United States Department of Agriculture–USDA news. (2016) https://www.aphis.usda.gov/aphis/ourfocus/biotechnology/brs-news-and-information/ge+wheat+washington+state
- Vautrin S, Zhang D (2007) Real-time polymerase chain reaction assay for endogenous reference gene for specific detection and quantification of common wheat-derived DNA (Triticum aestivum L.) J AOAC Int 90(3):794–801Google Scholar
- Xu X, Peng C, Wang X, Chen X, Wang Q, Xu J (2016) Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize. Transgenic Res 25(6):855–864. https://doi.org/10.1007/s11248-016-9982-0 CrossRefGoogle Scholar
- Zhu P, Fu W, Wang C, Du Z, Huang K, Zhu S, Xu W (2016) Development and application of absolute quantitative detection by duplex chamber-based digital PCR of genetically modified maize events without pretreatment steps. Anal Chim Acta 916:60–66. https://doi.org/10.1016/j.aca.2016.02.020 CrossRefGoogle Scholar
Copyright information
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.


text:
In the event your not trapped on getting nearer to CBD then have a look at say Montrose, Bayswater and Kilsyth you might be pleasantly astonished in These parts and price and nonetheless have an honest teach into the CBD…Its OK getting automobile to metropolis but use an online pre booking for parking in any other case its expensive…
Any extra ideas you've got in terms of parking (while I'd probably go ahead and take prepare if we stay in Williamstown) or very good retailers to acquire appliances could be much appreciated. We've chose to lessen delivery (additional the sentimental stuff like little ones's favorite toys and the like) and just Ikea the home with regards to furniture for the start out.
Wild winds and thunderstorms can descend from obvious blue skies and winters is usually rather chilly and really boring, with almost certainly the fewest hours of sunshine located anyplace in Australia.
.He'd love to Stay exactly where I do nevertheless the commute would he says complete him and his spouse as they both work Docklands …So that apart take a look at Williamstown its 25 minutes to Deer Parlk perhaps thirty rush hour however you will be towards the targeted traffic each strategies..Altona is likewise pretty good and it has two shorelines however somewhat missing in browsing ….
Like most stations in this article, Ringwood is pretty gross but they have law enforcement there in the evening so that you (hopefully) received't get stabbed.
I liked Melbourne when I was there, town has an extremely superior sense about this. I'd be tempted to Reside there if I felt I could cope with the winters, but I'm not sure which i can. Most effective I remain in Brisbane then.
Many thanks Mark. I've experienced a very good Go searching (just about on Google Earth and several Web-sites) throughout the last number of times and happen to be seeking closer to Melbourne. Areas like Williamstown, Point Cook, Sandringham, seem to be extra designed up and also have over Clifton Springs and Drysdale as there doesn't appear to be Substantially occurring there. I suppose we gained't truly know till we get there and have a very good Go searching.
Hi Kirsty Wales may be in elements really peaceful some not so, Id liken residing in Point Cook to living in Newtown or Llardrindod Wells, its Alright as alone but travelling out is miles to any where else it feels . The commute is more difficult whether by motor vehicle or train, they did introduce a ferry from Wyndham last year. I'm not so certain its continue to functioning even which was 45 minutes and that Reduce the journey time by 15 minutes. The location just hasn't received likely nonetheless. thats why you gte so considerably more residence for income. You might have read through in my posts in regards to the thought of house loan free of charge and cash in hand at £650 home in Position Prepare dinner I quickly gave up on The thought as they say Location Site Locale, Another destinations you've got picked are all respectable areas. I have a friend who commutes from Frankston within the teach in forty five minutes That's in the event the Convey trains are functioning They are really re carrying out every one of the crossings to really make it quicker but it can have a yr or so, so not constantly an express.
I have a vital problem in advance of answering, Is Mother a giant aspect of this equation E.g kid minder,fall pick up from college etcetera etcetera… I talk to because in on the lookout all over Geelong and Colac you happen to be restricting yourselves with prospects The Seaside little bit is simple anyplace in close proximity to Torquay and about say Ocean grove through I am confident Mom may help there… Certainly where ever There is certainly men and women this is check over here educational institutions and healthcare services but its considerably proportionate to population….Possess a read through of the publish with Matt and myself on This web site .
I would increase to my reviews about Simon Earles really- if anyone is hesitant or Doubtful about completing the assessment they definitely don't have to be.
no just authorized… Then they extra. We are famous throughout the world was the comment, they carried on t say You will have heard of us in the UK…We are certainly renowned there…My bemused respond to was No Sorry your Potentially not that popular there as I haven't heard about you…Maybe I just did nit strike t off with them so go and seem but think about the quantities on job and sit in your automobile at recess and look at …
Some issues to suit your needs The place in United kingdom have you been I'm assuming British isles I check with due to the fact I understand it nicely and When your happy in XYZ area I am able to advise equivalent here…Will your spouse generate when here that more query of when than if actually…Have you ever ruled out private educational facilities? they don't seem to be as lousy as you might Assume £££ and very good. I dint Consider another university move must be ruled out you may decide on a click here now single here and obtain superior, hes is just six It will be an experience for him and he has time with mom when he comes Id be shocked if you have that bit appropriate initial off, I absolutely realize sense of belonging and all Universities close below late December until February… Your wife will almost certainly fly with 3 small children…OUCH..Unwell visit acquire her a espresso with extra photographs of caffeine as being a reward… Sorry just me contemplating out loud again!!! Okay white items no frequently you dont get any, oven excepted 'black gray merchandise' …There's stuff you are able to lend purchase off ebay and many others etc a washer and fridge freezer and so on Offer on once more.
Which are the furniture outlet delahey most effective places to rent a property ? We would not have a car driving possibility do we will count on public transportation and faculty buses. Any info can be enormously useful. Many thanks in your support.
Hello Pam, thank you for the overview! We are glad to hear you will be getting your machien easy and pleasing to use. For those my response who have any queries with regards to your equipment in the slightest degree, remember to Be happy to Call us on 1800 802 924. Kind regards, VibroSlim
Not known Details About home furniture delahey
In the event your not trapped on getting nearer to CBD then have a look at say Montrose, Bayswater and Kilsyth you might be pleasantly astonished in These parts and price and nonetheless have an honest teach into the CBD…Its OK getting automobile to metropolis but use an online pre booking for parking in any other case its expensive…


text:
My desktop computer will not boot into Windows.
I can make it boot into Windows, by first re-entering the BIOS and resetting it.
I can make this problem occur if I unplug the computer for 60 seconds. The computer remembers the current date and time so I'm guessing the BIOS battery is working.
Any ideas?


text:
"Global ISO Shipping Container Market Research Report to 2023" Purchase this Report by calling ResearchnReports.com.
"An intermodal container is a large standardized shipping container, designed and built for intermodal freight transport, meaning these containers can be used across different modes of transport – from ship to rail to truck – without unloading and reloading their cargo."
The Report "Global ISO Shipping Container Market" presents a thorough analytical overview of the past and present state of the market and, with the help of present data, trends, competition, and regulatory framework gives a forward-looking perspective of the way the market will develop over the course of the next few years. Along with qualitative and qualitative details pertaining to the market's key elements, an overview of the chief macro and micro economic factors that will enable companies to the market to surmount competition are also examined in great details.
The Study presents an evaluation of the factors that are expected to inhibit or boost the progress of the global market. The global ISO Shipping Container market has been examined thoroughly on the basis of key criteria such as end user, application, product, technology, and region. An analysis has been provided in the report of the key geographical segments and their share and position in the market. The estimated revenue and volume growth of the market has also been offered in the report.
Download PDF Sample Copy:
Company Profiled: The Top Key Players operating in the ISO Shipping Container Market includes Fortune Container Trading Ltd, Qingdao Double-Friend Container Co., Ltd, J. U. H. Kohrs Elektrotechnik GmbH, Container Produtcs Ltd, CCB Scaffoding Supplies Ltd, CIMC, SINGAMAS, CXIC Group, Shanghai Universal Logistics Equipment, Maersk Container Industry, Charleston Marine Containers, Sea Box, Hoover Container Solutions.
The global ISO Shipping Container market is also presented to the readers as a holistic snapshot of the competitive landscape within the given forecast period. It presents a comparative detailed analysis of the all regional and player segments, offering readers a better knowledge of where areas in which they can place their existing resources and gauging the priority of a particular region in order to boost their standing in the global ISO Shipping Container market.
The key factors that are enhancing the growth of the key segments have been offered in the study. A thorough study of the competitive landscape of the global ISO Shipping Container market have been given, presenting insights into the company profiles, financial status, recent developments, mergers and acquisitions, and the SWOT analysis. This research will give a clear idea to the readers about the overall market scenario to further decide on this market project.
To Get Reasonable Discount:
The research report also mentions the innovations, new developments, marketing strategies, branding techniques, and products of the key participants present in the global ISO Shipping Container market. The reports offer the opportunities and restraint that may hit the market players in the future. Through this report, consumers can easily get the notion for their growth of global ISO Shipping Container products in the market.
Table of Contents
Global ISO Shipping Container Market Research Report to 2023
Chapter 1 ISO Shipping Container Market Overview
Chapter 2 Global Economic Impact on Industry
Chapter 3 Global Market Competition by Manufacturers
Chapter 4 Global Production, Revenue (Value) by Region
Chapter 5 Global Supply (Production), Consumption, Export, Import by Regions
Chapter 6 Global Production, Revenue (Value), Price Trend by Type
Chapter 7 Global Market Analysis by Application
Chapter 8 Manufacturing Cost Analysis
Chapter 9 Industrial Chain, Sourcing Strategy and Downstream Buyers
Chapter 10 Marketing Strategy Analysis, Distributors/Traders
Chapter 11 Market Effect Factors Analysis
Chapter 12 Global ISO Shipping Container Market Forecast
For More Information:
Media Contact
Company Name: Research N Reports
Contact Person: Sunny Denis
Email: Send Email
Phone: +1 888-631-6977
Address:10916, Gold Point, Dr.
City: Houston
State: TX
Country: United States
Website: https://www.researchnreports.com/


text:
In the value Comparison Resource you may occasionally see this icon indicating a car has skilled for your Qualified Pre-Owned plan. The CPO badge distinguishes utilized vehicles that were Licensed by their OEM (i.e Ford). A CPO car or truck need to first fulfill minimum amount age and mileage needs, and afterwards move an intensive inspection.
The Variable Speed Compressor tester might help establish the working affliction of common variable pace compressor control boards by examining the position of your
Since a jigsaw cuts with an up-and-down movement and its blade is comparatively slim, it excels at reducing curves in wood, metal, and plastic. Highly developed models have an orbital location that also moves the blade within a swinging motion for intense woodcutting.
Additionally, this pan is super affordable. Decide one particular up now and also you'll very likely hardly ever utilize it sufficient that you choose to'll have to replace it.
thirteen/ This Bottle Lock slots into most common-sized wine and liquor bottles, and secures by using a 4 amount mix. Following inserting the lock, adjust the combination and twist into place to secure.
We Specially love this salad spinner because the strainer basket can be employed for draining pasta as well as bowl for serving. It's a great multi-function Resource!
We're content to offer you top rated-excellent key appliances from makes you trust at prices you'll adore with our delivers. We've also acquired free shipping and delivery for appliance buys of $396 or even more and The house Depot Safety Approach on most key appliances on the internet and in-store. To learn more about our cost savings check out The House Depot's Appliance Provides For added financial savings.
Whether you happen to be an newbie gourmet or make use of the sink strictly to mix drinks, these things will get any guy's kitchen to another stage.
The companies are rather optimistic when publishing their batch dimensions recommendations for mixers!
Know very well what unit selling price is. The device cost of a product is essentially what the expense of Just about every pound, ounce or gram, square foot or meter, and many others.
When considering virtual machines, consider the tech specs for each to understand what structure the virtual equipment is in, the method prerequisites, just what the opinions have already been like for that Digital device, and the basis/administrative username and password.
With the earlier quarter-century, Eddie Hibbert has patiently restored old mantels, doorways together with other brownstone artifacts, getting a residing legend inside the Ny city salvage Group. Despite the helpful, casual environment all-around his Brooklyn store, the perform is created harmful by Wooden chips, traveling debris and lung-choking dust—some of which emanates from parts that predate the 1978 U.
This prolonged warranty are going to be mirrored in the upper rate these vehicles have a tendency to hold. CPO autos also often feature a absolutely free vehicle background report, roadside assistance systems along with other benefits. To get very clear, just a manufacturer's franchised vendor can sell that company's CPO cars. Which means if a dealership can sell new Hondas, then it could also market Honda CPO utilised autos.
Keep an eye on and regulate well being, location general performance bottlenecks and capability shortfalls and rebalance workloads in advance of demand from customers spikes to enable software functionality, although leveraging self-Studying algorithms and predictive analytics that adapt to the atmosphere.
- 202 Visitors


text: Children's Certificate Template – We are going to serve a lot of examples concerning children's certificate template which you are able to obtain for your recommendation. Actually in my opinion it is not difficult to catch children's certificate template in this website, because we explain many children's certificate template that the writers have provided. You can also refer this this website to your family. But if you aren't able to find what you are searching for then we will recommend you to type other keywords. Well, Children's Certificate Template which you are searching for is really eye-catching for your recommendation.


text:
tá Angelina je veľmi pekne nakreslená.... a ja kreslím a tiež niektoré sa podobajú kresby a niektoré nikto tak si môj človek... :)
Hi my friend! I want to say that this post is amazing, great written and include almost all significant infos. I'd like to see more posts like this. kdafkeckdcecfaeg
Very nice site! cheap goods
Very nice site! cheap goods http://opeyixa2.com/qvovqaa/4.html
I'm seeking weblogs which all have fantastic guidance on what's popular and specifically what the top rated makeup is.. afefcdckegdafbde
But a smiling visitor here to share the love , btw outstanding layout.
Write more, thats all I have to say. Literally, it seems as though you relied on the video to make your point. You clearly know what youre talking about, why throw away your intelligence on just posting videos to your weblog when you could be giving us something enlightening to read?


text:
- Future Mrs.Schug
- 7 years ago
- Wedding: October 2012
Ok Bees I just need to share what is going on… so Fiance and I decided that we were going to stop having sex until the wedding for various reasons. So in august I stopped taking my pill because why pay for a prescription I dont need. Well in sepetmber I had a period… and that was it! So last month (january) I went to the doctors and they mentioned various possible reasons and did some blood tests, but everything came out normal. So they gave me progestrone to see if that would provoke a period and it did! But now the Doctors is just like okay, well contact us if after this you stop having periods again. They have no explanation as to why I did not have a period and they are acting like its no big deal!!! My biggest fear is that I have some problem that will make having children difficult, which is the most important thing to me!! I just needed to get this off my chest. Any advice is appreciated!


text:
Canon PIXMA iP7250 Printer Drivers Download – The market for single functionality, personal computer photo printers may be shrinking, but Canon still has a good range including of course the one we're looking at here, the PIXMA iP7250. In a number of ways it's only a redesign but nevertheless, it comes down to a low wondering price and might generate high-quality prints from an assortment of resources.
How to Install Canon Printer Drivers
Knowing how to install Canon printer drivers is pretty handy, especially if you no longer have the CD. Let's say that you need to re-install your printer, but you have lost your CD. Instead of wasting time trying to find the CD, know that you can install the driver without the CD. For this method, however, you need to be connected to the internet so make sure that your connection is okay.
How to Manage the Installation
Do you know the model of your printer? Do you know the details of it? Consult the manufacturer by visiting the website. It is a guarantee that you should be able to find the needed driver. So, how to install Canon printer drivers? Find the following steps;
- Go to the manufacturer's official website. In this case, you should go to canon.com.
- When you see the site, check the left side. There should be an option for 'Support.' Click on it and click on the current location.
- Go to 'Consumer and Home Office.'
- You should see the dialog for 'Product Family.' Choose 'Printers and Multifunction'
- When you come to the scroll bar section of 'Product Series,' choose the series.
- You should see the dialog 'Drivers and Software.' Click on the model that you want to have.
- From the scroll bar section of 'Drivers and Software,' choose the OS that you have on your computer.
- You should see the download link now. If you click it, it will download straight up to your computer.
- Click on the provided download file and follow the given directions to install the driver. The process isn't complicated as long as you follow the guidelines.
As you can see, it is still possible to install the printer even without using the CD. You should have an active internet connection to make everything okay.
Supported Operating System (OS)
Windows 10 (32-bit),Windows 10 (64-bit),Windows 8.1 (32-bit),Windows 8.1 (64-bit),Windows 8 (32-bit),Windows 8 (64-bit),Windows 7 (32-bit),Windows 7 (64-bit),Windows Vista (32-bit),Windows Vista (64-bit),Windows XP (32-bit)
Download Canon PIXMA iP7250 Printer Drivers
Download for Windows


text:
Memberships Available
Weekdays, excluding Statutory Holidays
There is currently NO running water at the pool. This means that there are NO working washrooms.
We are also offering members the ability to pay for their annual dues in 4 monthly payments. Payments would be due January 15, February 15, March 15 & April 15th. If you choose to take advantage of this payment method, you will be charged the 2018 annual due rate which will be announced by February 14th.
For those members that wish to sell their membership this year, your deadline to request your sale and not be responsible for 2018 fees, is March 15th, 2018. All requests to sell must be received in writing to firstname.lastname@example.com
or by mail to:
Fairfields Swimming Club
P.O. Box 52649, Turtle Creek Postal Outlet
1801 Lakeshore Rd. W.
Mississauga, ON L5J 4S9
Memberships are sold on a first-come, first-serve basis - so get your request in early. The share price of $375.00 will be returned to the seller only after the membership has been resold.
If you have any questions, please feel free to contact us.
2018 Board of Directors
Dear Fairfields Members,
Happy New Year to you all. I hope you have been able to stay warm during this recent deep freeze. I'd like to introduce you to the current 2018 Board of Directors & Support positions.
President - Jenn Tondino
Vice President - Chris Beer
Secretary - Karin Kormann
Director of Membership - Chris Beer
Director of Communications - Lesley Smith
Director of Water Maintenance - Dan Keenliside
Volunteer Coordinator - Lesley Smith
Treasurer (Interim - Jenn Tondino)
Director of Staffing
Director of Facility Maintenance
Social Coordinator
New Membership Coordinator
Grants Coordinator
By-Law Coordinator
Legal Coordinator
Citi*Swim Coordinator
Marketing & Social Media Coordinator
Staffing Support
As a member owned and operated facility we rely heavily on members to volunteer for a variety of roles including joining the Board of Directors. The pool cannot operate without a Board of Directors who ensure that the pool is operational, that we have lifeguards and an amazing level of programming included as part of Membership!
Sadly, we currently don't have some of our vital roles filled for the 2018 season at this time. At this point, we don't have 3 valuable board roles covered for this upcoming season (Treasurer, Staffing & Facility). We also need people to cover the majority of the Coordinator positions for 2018. At this point we are continuing to plan for the upcoming season and will continue to do so until we are unable to move forward any further without those integral roles filled. Once we reach that place, all business at Fairfields will be suspended until a time when we do have those positions filled. Ideally we will have all Board & Coordinator positions filled to help disperse the workload of running a pool.
This will seriously affect all programming, staffing, current & future grant applications and the operation and potential opening of the pool this spring. We would also have to look at options to hire someone to run the day to day business of the pool. Of course, if this is the route we take the annual dues will increase dramatically.
Participating on the Board of Directors doesn't need to be a full-time job (especially if all of the Board and support positions are filled!). However, often board members do spend a fair bit more time than a member who is not on the board. To account for this the members voted in favour that Annual Dues be refunded to Very Active Board Members (those that contribute over 100 hours of valuable service to Fairfields Swimming Club).
We have provided links that briefly outlined the positions below and we ask that you seriously consider helping out so that we can ensure the pool operates next year! If you have any questions or want more details on specific roles please contact at email@example.com.
Thank you in advance to everyone!
For those members that wish to sell their membership this year, your deadline to request your sale and not be responsible for 2018 fees, is March 15th, 2018. All requests to sell must be received in writing to email@example.com
or by mail to:
Fairfields Swimming Club
P.O. Box 52649, Turtle Creek Postal Outlet
1801 Lakeshore Rd. W.
Mississauga, ON L5J 4S9
Memberships are sold on a first-come, first-serve basis - so get your request in early. The share price of $375.00 will be returned to the seller only after the membership has been resold.
THANK YOU to all of our volunteers this 2017 season! Without all of YOU giving your time and talent our members wouldn't have such a wonderful swimming club to participate in!
Fairfields Swimming Club is pleased to announce our 2017 Annual General Meeting. We encourage all members to attend. Please RSVP your attendance to firstname.lastname@example.com or online.
Date: Thursday November 23, 2017
Time: 7:30pm – 9pm
Location: Christ Church, United
1700 Mazo Crescent, Mississauga, L5J 1Y8
Room: Merigold Room
Proposed Agenda
Outgoing 2017 Board Members and New 2018 Board Members are asked to remain for a brief meeting following the AGM.
Call for Board Members & Seasonal Volunteers
Fairfields Swimming Club is run by community members just like you. But it can't run without your help. It's your turn join the Board. It's easy and you can make a big difference for our neighbourhood pool. The vast majority of the 2017 Board is stepping down this year, so we need your help!
All board positions are open for nominations.
We also have Seasonal/Support Coordinator positions available that do not require your attendance at monthly meetings and are primarily an in-season role only.
If you have any questions about Board roles, Coordinator roles, time commitments, or what's involved please contact us at firstname.lastname@example.com.
To nominate yourself or someone else for a Board or Seasonal position, please send an email to firstname.lastname@example.com and let us know the position you are interested in.
2017 Board of Directors
Please note that the pool is now closing at 8.30pm for the remainder of the summer due to reduced daylight hours.
It is also water polo night at Fairfields. Due to the earlier closing, the Jr. Water Polo will run from 6.30pm-7.30pm and Adult (16+) Water Polo will run from 7.30pm-8.30pm for the remainder of the season. If you haven't come out for a game yet season, why not swing by tonight! Guests are welcome for a fee of $5.00 per person.
The hours on the website will be updated to reflect this change by the week-end.
The baby pool will be closed today due to a chemical imbalance and will be reopened tomorrow after a review by the Health Inspector. Sorry for any inconvenience.
The pool will close at 7:30 tonight so that the staff can properly do bathroom renovations, sorry for any inconvenience.