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1. What is a " ZTL"- Limited Traffic Zone? The centre of Pisa is a
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1. What is a " ZTL"- Limited Traffic Zone? The centre of Pisa is a
1. What is a " ZTL"- Limited Traffic Zone? The centre of Pisa is a limited area (ZTL), in order to access this area you must have a special authorization. All the cars that enter without this authorization are fined because many streets of the centre have got a camera that takes a photo of the plate of the car. These streets are marked by international road signs (see the example below) Remember that the signs are the same in all the countries of Europe. 2. How can I view the photographic evidence of the infringement? You can view the photographic evidence of the offence using the following link: https://secure.comune.pisa.it/tzi/info.jsp Accessing the web page you have to write some information about the infringement: numero verbale- you must write the number of form anno del verbale- you must write the year of the infringement giorno e mese del verbale– you must write the date/ the month of the offence targa del veicolo– you must write the plate number of the vehicle then you must click on "accesso" If you don't view the photo online you can send us an e-mail writing on the subject "copy of photo". Please remember that the electronic device that takes pictures of the plate of the car has been approved by Italian Ministry Of Transport. We're sorry but we're not able to send you further documentation in addition to the photo. 3. How can I pay this kind of fine? It's possible to pay it in the following ways: a) On-line: It should be noted that at the time, this method of payment provides only the utilization of PayPal. 1 – CLICKON www.sepi-pisa.it/sanzioni 2 – CLICK ON THE LINK "Pagamento multe on-line/On-line fines payment" 3 – CLICK ON THE LINK "Pagamento multe on-line/On-line Payment" 4 – In order to pay it's necessary to fill: Targa del Veicolo – Plate number of the vehicle (only alphanumeric characters) Numero del verbale – Number of form fill with the number of form that might be followed by the letter on the basis of the following examples: - if the letter of the infringement is P(1234567/P), fill with 1234567; - if the letter of the infringement is PT (12345678/PT), fill with 12345678T - if the letter of the infringement is VI (121/VI) fill with 123I; - if the letter of the infringement is VC (12345/VC), fill with 12345C Anno del Verbale – Year of the infringement b) Wire transfer, referring to the IBAN number indicated in the notification letter that, in particular, for the infringements with a later date to 28/10/2011 is as follows: SEPI SPA ESTERO PIAZZA DEI FACCHINI 16, 56125 PISA BANCA DI CREDITO COOPERATIVO DI FORNACETTE VIA T.ROMAGNOLA, 101/A 56012 FORNACETTE (PISA) IBAN: IT20R0856270910000010645224 BIC(or swift code): BCCFIT33 Please include the number of the form in the reason for the payment, so we will be able to identify the payment. • If you are in Italy, you can pay the fine in a post office; asking for a "bollettino" (deposit slip) and you have to fill it using the following coordinates: C/C: 59900076 Name of the recipient: SEPI SPA COMUNE DI PISA SANZIONI AMMINISTRATIVE Please write on "Causale versamento" (reason for the payment) the number of the form. Important: after the payment you MUST send us a copy of it by fax or by e-mail using the addresses provided on the fine ATTENTION: PAYMENTS BY CHEQUE ARE NOT ALLOWED 4. It's possible to receive a confirmation of the payment? Yes, it is possible to receive it. You can send us an e-mail writing on the subject "Payment Confirmation Nr. of form ____" The requests for payment confirmation must be sent after the 15th day of the month following that of the payment. 5. What is the time limit for the notification of a fine abroad? According to the Italian Road Traffic Code and the sentence of Corte Costituzionale n. 477 2002, we have 360 days (from the date of the offence) to send a fine abroad. If you have rented a car, it's possible to send the fine to the lessee of the car within 360 days from the date on which the Police Office receives your personal details from your Rentals. 6. How can I lodge an appeal? You can lodge an appeal : • Within 60 days from the date on which you have received the fine. • Only if the fine hasn't been paid. The payment of the fine precludes the possibility of an appeal. You can submit an appeal, ONLY IN ITALIAN LANGUAGE, to one of the following authorities: • Prefect of Pisa, Piazza G. Mazzini 7, 56125 Pisa (Italy) Your appeal must be sent with registered mail. Please retain the proof of receipt. • Justice of Peace of Pisa, Via Palestro 39, 56125 Pisa (Italy). Your appeal must be sent in four signed copies and you must attach to one of these: - A revenue stamp to the amount of 33,50 Euros - A revenue stamp to the amount of 8,00 Euros Your appeal must be sent with registered mail The offender (or a person who has been delegated with a signed copy of the proxy) must assist at the hearing. 7. I'm owner/ I carried a person with a parking permit for people with disabilities, what can I do? You must send : a) a signed letter asking for the deletion of the fine; b) copy of the disability Pass c) proof of the presence in Pisa of the Handicap Pass holder ( ex. Hotel reservation, plane ticket, etc ……) 8. I was staying in a hotel in the restricted zone (ZTL). Was I authorised? If you were guest in a hotel in the restricted zone, you are free from the payment of the violation, but you must contact the hotel and request the permission for your vehicle. Please contact directly your Hotel and ask them to clarify the matter with the Local Police. If your hotel wasn't in the ZTL, you can either pay the fine or send a formal appeal. 9. I paid some administrative fees concerning the fine to the Rentals, what does it mean? If you received an invoice concerning the fine from the Rentals and you've paid it by credit-card, it doesn't mean that you paid the fine itself. Usually the Rental Companies charge the client with administrative fees in order to provide to the Police Office the personal details of the offender. However if you would like to have major information about this invoice, we suggest you to contact directly your Rentals. 10. I received more than one fine, what can I do? If you received two or more fines, we must inform you that all the documents are valid and you must consider the fines separately: for each one you can proceed to the payment or to the appeal.

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- You are here: Groceries
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Finish All In 1 Dishwasher Tablets Regular 56S
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All in 1 Regular
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>30% Phosphates, 5-15% Oxygen-Based Bleaching Agents, <5% Polycarboxylates, Non-Ionic Surfactants, Phosphonates, Enzymes (Protease, Amylase), Perfume, Limonene
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|Reviews for In the Quiet
| Paper Pearls chapter 1 . 2/27/2011
This story was absolutely beautiful, and your writing style was wonderful - so graceful. The elegance of your prose was fitting, as it conveyed the innocence and child-like quality of the dolls perfectly:
"Echo laughs as she tugs on Sierra's hand. They run down the flower covered hill, Sierra struggling to keep up."
There was so much beauty in your language. I applaud your word choice, as the atmosphere was wonderful. Echo's innocence made her feelings for Sierra seem absolutely natural. I found myself wanting them to be together, and when the pair of them embraced, it was very sweet:
"Sierra sleeps in Echo's arms that night, and many more."
Quite rightly, there was none of the emotional depth or worries of an adult relationship. It seemed fitting that the pair of them didn't question anything about their relationship, and that they knew instinctively that it was right.
Well done for writing something so beautiful.
| Rain Addict CM chapter 1 . 4/8/2010
D well written, made me feel all fuzzy inside.
| FemaleSpock chapter 1 . 4/5/2010
Aw, this is sweet! I like that Echo leaves her that message.


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Mazarin is a software development firm based in Colombo, possessing world class technological expertise and process excellence. We provide Java, .NET, Android, iPhone and Open Source technology consultancy services to a growing base of overseas clientele. Our work culture is professional yet informal and conducive to learning and creative thinking. Currently we are looking to fill the following positions.
Software Engineer (.NET)
Technologies of Interest
- In depth knowledge and experience in Object Oriented Design and development patterns
- Proficiency in .NET technologies and frameworks (C#.NET, VB.NET, ASP.NET, ADO.NET and LINQ)
- Experience in database design and development with Microsoft SQL Server 2012/2014/2016
- Knowledge in distributed application development (REST, WCF, Web service, Web API).
- Knowledge in scripting language such as JavaScript, jQuery, AJAX, CSS, XHTML, DHTML, XML
- Familiarity with Front-end frameworks Jquery, Angular JS, React JS.
- Knowledge in using Azure/AWS technology would be an added advantage.
- Additional knowledge in other technologies such as PHP and Java would be an added advantage
Experience and Qualifications
- Bachelor's degree in Computer Science, Information Technology or related discipline
- 2 or more years of post-degree industry experience directly working with the relevant technology and as a software engineer
- Experience in Test Driven Development and technologies such as NUnit and MSTest.
- Hands on experience in Agile project environments and have mastered one or more Agile processes including activities such as test driven development, continuous integration and/or pair programming
- Must have an excellent analytical and problem solving skills
- Knowledge in using UML in different phases of the projects
- Professional certification in relevant technologies would be an added advantage
- Good in spoken and written English to communicate with foreign clients directly
Areas of Responsibility
- Work closely with the project manager, IT architect, product owners and other stakeholders and be part of the decision-making process within the software project to achieve project objectives.
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- Establish standards, functional and technical specifications, solve hardware/software interface problems, define input/output parameters and ensures integration of the entire system or subsystem.
- Learn new technologies quickly and willingness to learn and work on multiple technologies including mobile technologies
At Mazarin you will work with clients, not for them. You will be trusted to rely on your own insights, judgments and skills rather than on rigid mandates. Regardless of your position, you will thrive, learn and grow in a supportive environment that values diversity, collaboration and personal-professional balance.
If you think you got what it takes apply through https://mazarin.bamboohr.com/jobs/ before 31st March 2017. For further information, please contact, + 94 (0) 112 68 1107


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9ct Yellow Gold 18" Curb Chain
Product code: 3791114 £79.99
In stock
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For assistance with your purchase, visit our help centre for advice & tips so you can buy confidently with H.Samuel.
This 9ct yellow gold 18" curb chain will make a perfect gift for a loved one, whatever the occasion.
- Brand
- Loved
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- Curb
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- 9ct Yellow Gold
- Necklace style
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- Length
- 46 cm
- Width
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Courtyard @ Heeren Boutique Hotel
91 Jalan Tun Tan Cheng Lock,
75200 Melaka, Malaysia
Courtyard @ Heeren is located in Malacca, situated in the Melaka Old Chinatown area which is now part of the UNESCO World Heritage City of Malacca. The hotel is located at Jalan Tun Tan Cheng Lock (formely known as Heeren Street). It is just a few steps away from Jonker Street (Jalan Hang Jebat) which is famous for shops selling various types of antiques and handicrafts. Jonker Street is also famous for the weekend (fri, sat & sun) night market which starts from 6pm to 11pm.
In addition to being conveniently located, we provide transportation services as per below
Singapore - Melaka / Melaka - Singapore
RM550.00 per trip (Sedan Car/ Max 4 Pax)
RM800.00 per trip (Max 10 Pax)
Melaka - LCCT & KLIA / KLIA & LCCT - Melaka
RM280.00 per trip (Sedan Car / Max 4 Pax)
RM380.00 per trip (Max 10 Pax)
Melaka - Kuala Lumpur City / Kuala Lumpur City - Melaka
RM320.00 per trip (Sedan Car / Max 4 Pax)
RM420.00 per trip (Max 10 Pax)
Prices are subject to change without prior notice


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John Clifford, 7th Baron de Clifford (1388-1421)
222,950pages onAdd New Page
this wiki
this wiki
John Clifford, 7th Baron de Clifford was born circa 1389 to Thomas Clifford, 6th Baron de Clifford (c1363-1391) and Elizabeth de Ros (c1358-1424) and died 13 March 1422 at the Siege of Meaux of unspecified causes. He married Elizabeth Percy (c1395-1436) circa 1404 JL . Notable ancestors include Charlemagne (747-814), Henry II of England (1133-1189), William I of England (1027-1087), Hugh Capet (c940-996), Rurik (c832-879), Alfred the Great (849-899). Ancestors are from England, the United Kingdom, France, Germany, Russia, Ukraine, Belgium, Ireland, the Netherlands, Spain, Portugal, Italy.
|Offspring of John Clifford and Elizabeth Percy (c1395-1436)
|Joined with
|Thomas Clifford, 8th Baron de Clifford (1414-1455)
|26 March 1414
|22 May 1455
| Joan Dacre (c1418-bef1455)
|Mary Clifford (c1416-1478)
|4 October 1478
| Philip Wentworth (1424-1464)
|Henry Clifford (c1418-aft1460)
Footnotes (including sources)
|‡ General


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Israeli High-tech Fundraising Dips in First Quarter
Decline is led by youngest startups, risking deal flow in future years, report says
Decline is led by youngest startups, risking deal flow in future years, report says
Study by two Haifa University professors finds Israel is turning out enough graduates to meet industry demand
Deal between two Israeli companies would give them a leg up against Uber and Lyft in New York market
Super-Sol sells 200m shekels of shares | Cellcom launches low-cost TV, internet, telephone service | Perrigo jumps on Einhorn's long position | Tel Aviv gets lift from higher Wall Street
Sapir Capital is reportedly in talks over stake worth more than $100 million in state owned mining investment arm Zambia Consolidated Copper Mines Investment Holdings
New chief of the TA exchange says reducing tax from 25 to 10% would lure many more to sagging equity market
Port charges extra 40% to load and unload goods on Shabbat - even when the work only starts the next day
Experts warn that tank is unsound, thousands could die if it falls apart, but Netanyahu says he wants to avoid layoff of workers on site
Stocks and the euro have rallied in an unjustified sigh of relief that Emmanuel Macron is all but assured victory. And if he is?
Private equity fund Fortissimo takes stake in Biological Industries; Pelephone gets approval to retire aging 2G phone service


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This mobile 4.4.2 Android kitkat version. Public needs 6.0 or above versions including 4G . This is important.Read More
Panasonic P55 Novo, After Using Approx 1 Month Then i Share My review
# Phone Touch Is Very faster & Smooth
# Nice Battery Backup
# Very Sharp Quality in Both Camera
Best,affordable premium phone...High lights are 13 mp camera with triple led flash,5 mp front cam with flash,IR,octacore processor.....Read More
Good phone. octa core processor and camera was amazing. but only problem is 4g has not there other wise amazing android phoneRead More
Cons: Only issue which I see is that it takes only 5 to 10 minutes to come to 1% of battery level from 20%.
This happens every time. from 100 to 20 % it gives good battery back up... I mean
i m using this phone from one week ago....n feeling very good nice phone nice speed nice battery overall gud....some additional features are in here...super speed on using net...best phone.. thanksRead More
What more to expect from Panasonic??? Hats off to ur engineers for making such an awesome phone at this price. I am not fan of any particular brand but this brand is pulling me.... and i cannot stopRead More
A nice good Mobil..Read More
Phone is very awsm ...just love panasonic ...i think everyone should by this
Its an affordable phone . just loved it guyzz !
Good perpomanance
Looking so nice
Good battery backup
Camera better..
This range... Good mobile... Features... But camera is not 13 poor like 5 mp.. typeRead More
Nice phone at this price range. No heating problem but rear camera is not so good.Read More
The phone is good with its performance. But front of my pone is not working properlyRead More
niceRead More
goodRead More
I want to return this phone
This phone is not 4G
love its camera and lok alsoRead More
Mst hRead More
It's very unfortunate, I believe I might have got the malfunction one among the well reviewed product! Within hours of use it won't work as expected. Switch off by itself, tried to switch on butRead More
I took the phone considering 4g but the same is 3g phone . Phone reviews are showing it is 4g but it is wrong... Pathetic phone.. hangs a lot..Read More


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If you would like something in your life, like your health, to be better, FIRST express sincere gratitude for the perfection of what you already have.
To understand why this helps, and see evidence that it does, please read the short supporting article.
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It's the Idol's getting rid of stuff sale. Plus other shipments.
Discussion in 'Idol Marine' started by Bob the (reef)builder, 8 Mar 2013.
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text: Kareena Kapoor and Salman Khan have worked together in many superhit films. Recently, Kareena revealed about her first meeting with Bollywoods most loved superstar, Salman.In a candid interview to DNA, Kareena Kapoor said, I first met Salman when he was shooting with Karisma for Nishchaiy.I was nine years old at the time and I told Salman, I am going to work with you when I will be a big star! Thats how our first meeting happened. He looked at me, smiled and said, Oh really? I was just a child, so he was humouring me, she said.Beautiful: This Is What Sanjay Said About Deepika Padukone Kareena Kapoor further added, When he started shooting for Jagruti with Lolo again, I was assisting his sister Baby (Alvira Agnihotri) who was an AD on the sets. Baby one day came and told me, You be my assistant. I was 11 and I became her spot girl running with everything - from her water bottle and food to napkins. This family is extremely precious to me and Lolo, who swears by Salman and considers him to be her bestest friend! Kareena Kapoor Kareena Kapoor Overview Biography Fan Speaks Fan-tastic Photos Photos Videos Upcoming Movies Wallpapers


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Health. Inspiration. Friendship.
22 photos Photo gallery:Mascot Design Revised
Latest News
Mascot Design Revised
28 March, 2017 (10:44)
Tags: mascot, wepaly
At the request of the Esteemed President of Turkmenistan, Wepaly, the 5th Asian Indoor and Martial Arts Games' Mascot, has been redesigned by the National Arts Insitute to included more traditional Turkmen elements. In the new design Wepaly portrays a more realistic representation of an Alabai dog, he is wearing a traditional ceremonial dress and a white telpek fur hat while waving the State Flag of Turkmenistan and the symbol of the Olympic Council of Asia.


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rookierags' Profile
Our focus is to create handmade goods that are made exclusively from repurposed items. We often spend more time searching for things to morph than we do in the actual morphing process. Most of what we work with had a previous life, including the multiple machines and other gadgets we've found useful in the design process. Even the shop owner, Christine, redefined her life after breast cancer and found passion in her work.
Our merchandise is always evolving as we find new ways to resurrect hidden treasures. Each item we offer is one of a kind and no doubt holds a rich history from it's previous life.
- Female
- Born on June 20
- Joined March 13, 2008
Favorite materials
Recycled leather and other fabric, hardware from belts and jewelry, and other used items
- rookierags
Atlanta GA,
United States


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Kids Education Logo Design
This project was awarded to crea8ivedesign for $69 USD.Get free quotes for a project like this
Project Budget$30 - $250 USD
Total Bids77
Project Description
I am looking for someone to come up with 5 logos for my business. My company teaches real life skills such as money management, careers, first aid, etiquette to primary school children in Australia. I want my logo to possess the following qualities: simple, fun, educational and professional.
The name of the business is "WIGU" which stands for "When I grow up...". I would like the four letters WIGU to be the main part of the logo design and "When I grow up..." to be included in a discrete yet clear way. "When I grow up..." needs to be able to be removed from the logo design and for WIGU to still look good on its own.
FORMAT: I would like the logo to be submitted in both ai and pdf format.
Further projects available such as other logo designs and business card designs if I am satisfied with the final product submitted to me.
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Tribeca Coverage International Videos Musicals Hollywood Videos Indie Reviews How ScreenAnarchy Works
Watch A Clip From Ben Wheatley's SIGHTSEERS
Optimum will give the film a UK theatrical release November 30th with a US release to follow via IFC.Chris (Steve Oram) wants to show Tina (Alice Lowe) his world and he wants to do it his way - on a journey through the British Isles in his beloved Abbey Oxford Caravan. Tina's led a sheltered life and there are things that Chris needs her to see - the Crich Tramway Museum, the Ribblehead Viaduct, the Keswick Pencil Museum and the rolling countryside that accompanies these wonders in his life. But it doesn't take long for the dream to fade. Litterbugs, noisy teenagers and pre-booked caravan sites, not to mention Tina's meddling mother, soon conspire to shatter Chris's dreams and send him, and anyone who rubs him up the wrong way, over a very jagged edge...
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Sci fi/Fantasy image bySamuel Morrell
Laides and gentlmen meet the chef of the future! He can slice, dice, julian and even fry, stir and mix and still have time to smoke a sigar while doing it all!
Published More than a year ago
Category Fantasy
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The process of buying a new Ford car or truck can seem overwhelming if you don't know where to begin. Edmunds.com can get you started on the right track with a convenient directory of Ford car dealerships in and around Lettsworth, Louisiana. Compare online price quotes on the new or used car, truck, SUV, minivan, or wagon of your choice to locate the best deals. Edmunds.com makes it easy to find trusted Ford car dealers in Lettsworth, saving car buyers time and money on what will be an important purchase.
Non Display Review Name "This was a great experience right from the start. Jack Broussard, our salesman found our desired vehicle quickly, got it prepped and all the paperwork"... Read more Review by: Bdearborn


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The Great Comeback; How Abraham Lincoln beat the odds to win the 1860 republican nomination
ECELBARGER, Gary
Place Published: New York
Publisher: St. Martin's
Date Published: 2008
Description: Illus. 8vo, black cloth, d.w., corners of cloth bumped, some pages creased in upper right corner margins. New York: St. Martin's Press, (2008).Edition: First
Jacket: Y
Binding: hardcover
Condition: very good(-)
Language: English
Type: Books
Book ID: 254502
By This Author: ECELBARGER, Gary
By This Publisher: St. Martin's


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Need a responsive layout designer
This project received 15 bids from talented freelancers with an average bid price of ₹359 INR / hour.Get free quotes for a project like this
Project BudgetN/A
Total Bids15
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The highly conserved 5' untranslated region as an effective target towards the inhibition of Enterovirus 71 replication by unmodified and appropriate 2'-modified siRNAs
- Jun-Xia Deng1,
- Xiao-Jing Nie1,
- Ying-Feng Lei2,
- Chao-Feng Ma3,
- Dong-Liang Xu1,
- Biao Li1,
- Zhi-Kai Xu2Email author and
- Guo-Cheng Zhang1Email author
© Deng et al.; licensee BioMed Central Ltd. 2012
Received: 22 April 2012
Accepted: 8 August 2012
Published: 13 August 2012
Enterovirus 71 (EV71) is a highly infectious agent that plays an etiological role in hand, foot, and mouth disease. It is associated with severe neurological complications and has caused significant mortalities in recent large-scale outbreaks. Currently, no effective vaccine or specific clinical therapy is available against EV71.
Unmodified 21 nucleotide small interfering RNAs (siRNAs) and classic 2′-modified (2′-O-methylation or 2′-fluoro modification) siRNAs were designed to target highly conserved 5′ untranslated region (UTR) of the EV71 genome and employed as anti-EV71 agents. Real-time TaqMan RT-PCR, western blot analysis and plaque assays were carried out to evaluate specific viral inhibition by the siRNAs.
Transfection of rhabdomyosarcoma (RD) cells with siRNAs targeting the EV71 genomic 5′ UTR significantly delayed and alleviated the cytopathic effects of EV71 infection, increased cell viability in EV71-infected RD cells. The inhibitory effect on EV71 replication was sequence-specific and dosage-dependent, with significant corresponding decreases in viral RNA, VP1 protein and viral titer. Appropriate 2′-modified siRNAs exhibited similar RNA interference (RNAi) activity with dramatically increased serum stability in comparison with unmodified counterparts.
Sequences were identified within the highly conserved 5′ UTR that can be targeted to effectively inhibit EV71 replication through RNAi strategies. Appropriate 2′-modified siRNAs provide a promising approach to optimizing siRNAs to overcome barriers on RNAi-based antiviral therapies for broader administration.
KeywordsEnterovirus 71 RNA interference siRNA 5′ UTR Viral inhibition Antiviral agent 2′-O-methylation modification 2′-fluoro modification
Enterovirus 71 (EV71) has been implicated in numerous epidemics of hand, foot, and mouth disease (HFMD), which primarily affects infants and young children, resulting in the appearance of vesicular rashes on hands, feet, and oral mucosa. More importantly, EV71-infected children can develop neurological complications, such as aseptic meningitis, acute flaccid paralysis, encephalitis, acute cerebellar ataxia, brainstem encephalitis, as well as fatal pulmonary edema or cardiopulmonary collapse [1]. Currently, EV71 is recognized as the most clinically important neurotropic virus since poliovirus has been nearly eradicated in the majority of countries around the world [2]. Significant morbidities and mortalities associated with increasingly frequent HFMD outbreaks throughout the Asia-Pacific region have raised public concerns [3–6]. Despite improvements in virological and medical research, treatments for acute EV71 infections are mainly aimed towards alleviating clinical symptoms, and there is still no specific antiviral agent available against EV71.
RNA interference (RNAi) is a biological and specific post-transcriptional gene silencing mechanism that has been employed in the development of numerous promising therapeutic agents [7]. Since the initial report of RNAi-mediated inhibition of the human respiratory syncytial virus in 2001 [8], a variety of other viruses have been successfully targeted using RNAi strategies [9–13]. Several RNAi-based antiviral therapeutics are currently in various phases of clinical trials for the treatment of viral infections, such as the human immunodeficiency virus, respiratory syncytial virus, and hepatitis C virus [14]. As a positive-stranded RNA virus, EV71 is an attractive target for RNAi therapy due to the functions of its genome as both a replication template and messenger RNA [15]. The destruction of EV71 RNA could eliminate not only protein synthesis but also viral replication.
In spite of the immense attractiveness of efficient gene knockdown as a therapeutic strategy, RNAi applications still face several critical hurdles that have not yet been comprehensively addressed. Identifying the appropriate chemical modifications will be essential to resolving these hurdles and bringing RNAi to the clinic [16]. The properties of small interfering RNAs (siRNAs) can be optimized by incorporation of chemically modified RNA analogues to increase nuclease resistance, improve target specificity and maintain gene-silencing activity [16]. Importantly, the high efficacy of chemically modified siRNAs can be accompanied by low cell toxicity [17]. Recent studies have successfully improved siRNA performance by incorporation of chemically modified RNA analogues against virus infection [18–22]. Among these, 2'-O-methylation (2'-OMe) and 2'-fluoro (2'-F) are classic patterns of chemical modification.
The selection of appropriate siRNA targets is a critical initial step in the development of antiviral RNAi therapeutics. Successful siRNA designs must be based on the conserved sequences of the viral genome. EV71 is a small, non-enveloped, positive single-stranded RNA virus belonging to the genus Enterovirus within the family Picornaviridae. Like all picornavirus, EV71 genome possesses a conserved 5′ untranslated region (UTR) of approximately 740 nucleotides (nt) [23, 24]. The 5′ UTR of EV71 is involved in key steps of the viral life cycle, including replication and translation [25, 26]. Due to its high degree of conservation which renders it less likely to mutate and functional importance for viral replication, the 5′ UTR of the EV71 genome is an attractive target region for siRNA therapeutic treatment. In both cell cultures and animal experiments, RNAi in the form of chemically synthesized siRNA or plasmid-derived shRNA has been used to effectively inhibit EV71 infection, revealing several potential target sequences within the 3′ UTR, VP1, 3D, 3C, and 2C regions of the EV71 genome [27–31]. However, the higher order structure of the EV71 genomic 5′ UTR makes targeting of this region especially challenging. To date, no effective siRNAs targeting the conserved 5′ UTR of the EV71 genome have been reported.
The application of unmodified 21 nt siRNAs and 2′-modified (2′-OMe and 2′-F) siRNAs to target highly conserved 5′ UTR of the EV71 genome for specific viral inhibition was investigated in this study. Identification of effective target sequences within the highly conserved 5′ UTR and optimization of siRNA properties by 2′-OMe or 2′-F modification for future in vivo applications were considered primary objectives. Direct in vitro evaluation explored the effectiveness of siRNAs targeting the 5′ UTR of the EV71 genome in inhibiting viral replication. Meanwhile, we observed good compatibility of the introduction of appropriate 2′-OMe or 2′-F modifications into the siRNA duplexes, which maintained high antiviral activities. Enhanced serum stability was confirmed as a major advantage of the 2′-modified siRNAs in comparison with unmodified counterparts. These strategies may ultimately lead to the development of appropriate chemically modified siRNAs against clinical EV71 infection.
Cell culture and virus strain
Rhabdomyosarcoma (RD) cells were routinely grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA). The EV71 strain (Genbank accession no. HM003207.1) was provided by the Department of Viral Diseases Laboratory, Xi′an Center for Disease Control and Prevention (Xi′an Shanxi, People′s Republic of China). Viruses were propagated and titrated in RD cells.
Design of unmodified and 2′-modified 21 nt double-stranded siRNAs
Nucleotide sequences of siRNAs and their target positions in the EV71 genome (Genbank accession no. HM003207.1)
Chemically modified
Nucleotide sequence
Genomic position
5′-C AGC AAACC AC GAUC AAU ATT-3′(Sense)
5′-U AUU GAUC GU GGUUU GCU GTT-3′(Anti-sense)
si-1 F
5′-C AGC AAACC AC GAUC AAU ATT-3′(Sense)
5′-U AUU GAUC GU GGUUU GCU GTT-3′(Anti-sense)
si -2OMe
5′-C AGAGC AAUU GUUU ACCU ATT-3′(Sense)
5′-U AGGU AAAC AAUU GCUCU GTT-3′(Anti-sense)
si-2 F
5′-C AGAGC AAUU GUUU ACCU ATT-3′(Sense)
5′-U AGGU AAAC AAUU GCUCU GTT-3′(Anti-sense)
5′-(FAM) CAGAGCAAUUGUUUACCUATT-3′(Sense)
Transfection and infection
Transfection was carried out under optimal conditions. RD cells were seeded in 24-well plates (8 × 104 cells/well) without antibiotics the day before transfection to allow adherence and to reach 70–80% confluence at the time of transfection. The culture medium was changed to 500 μl per well of Opti-MEM® (Gibco BRL, Grand Island, NY, USA) immediately prior to transfection. For each transfection, the appropriate amount of siRNA was diluted in 50 μl of Opti-MEM®, while 1 μl of LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA) was also diluted in 50 μl of Opti-MEM® in another tube simultaneously. After incubation at room temperature for 5 min, these two solutions were mixed gently and incubated for 20 min at room temperature to allow the siRNA:LipofectamineTM 2000 complexes to form. Subsequently, 100 μl of each complex was added to each well and mixed by gently rocking the plate. The final concentration of siRNAs was 25 nM, 50 nM, or 100 nM. After 5 h, the medium was removed, and transfected RD cells were infected with EV71 at a multiplicity of infection (MOI) of 0.01 for 1 h. The cells were washed twice to remove any unabsorbed virus, and 500 μl of fresh maintenance medium was added to each well. Cells were then incubated at 37°C, and the morphological changes of RD cells were observed at various time points. Mock transfections were carried out as described above; however, no siRNA was added as a negative control. Cell supernatants and cell lysates were collected at designated post-infection time points and stored at −80°C for further studies.
Fluorescence and flow cytometric analysis
Fluorescein labeled si-2FAM was used to confirm the localization of siRNA in RD cells and the optimal transfection efficiency. RD cells were seeded onto coverslips and transfected with varying concentrations (25, 50, and 100 nM) of si-2FAM. As a control, one of the wells was treated with 100 nM of si-2FAM without LipofectamineTM 2000. The medium was changed at 5 h post-transfection. At 6 h post-transfection, cells were washed twice with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 min. Cells were then washed again with PBS, and Hoechst 33258 (0.5 ug/ml; Sigma, St. Louis, MO, USA) was used to stain cell nuclei. The cells were observed using the FLUOVIEW FV1000 confocal laser scanning biological microscope (Olympus, Tokyo, Japan) equipped with the FV10-ASW system (Olympus, Tokyo, Japan).
Flow cytometry was used to precisely measure the transfection efficiency. RD cells were seeded in 6-well plates and transfected with varying concentrations (25, 50, and 100 nM) of si-2FAM. The medium was changed at 5 h post-transfection. At 6 h post-transfection, RD cells were trypsinized and resuspended in PBS. Transfection efficiency was determined by detection of fluorescein-labeled RD cells using FACSAria flow cytometry (BD Biosciences, San Jose, CA, USA).
Cytotoxicity assay
RD cells were seeded in 96-well plates (1.6 × 104 cells/well) and transfected with 100 nM of various siRNAs. At 24, 48, and 72 h post-transfection, a 20 μl volume of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (5 mg/ml; Sigma, St. Louis, MO, USA) reagent was added to each cell. After incubation at 37°C for 4 h, the cell supernatant of each well was removed. A 150 μl volume of DMSO (Sigma, St. Louis, MO, USA) was added to each well, followed by 10 min of shaking. Absorbance values were measured at a test wavelength of 570 nm with a reference wavelength of 690 nm.
Cell viability assay
RD cells were seeded in 96-well plates (1.6 × 104 cells/well) and transfected with 50 nM or 100 nM of various siRNAs. At the end of the transfection period (5 h), cells were infected with 0.01 MOI of EV71 for 1 h. At 24, 36, 48, 60, and 72 h post-infection, MTT assays were carried out as described above to assess the viability of EV71-infected RD cells transfected with unmodified and 2′-modified siRNAs.
Real-time TaqMan RT-PCR
Viral RNA extraction was performed as previously reported [13]. Briefly, at 36 h post-infection, RD cells were washed with PBS, and 150 μl of CelLyticTM M Cell Lysis Reagent (Sigma, St. Louis, MO, USA) was added into the individual wells containing the RD cells. Viral RNA extraction was then carried out using the QIAamp® Viral RNA Mini Kit according to the manufacturer's instructions (Qiagen, Valencia, CA, USA). Finally, RNA was eluted in 60 μl of elution buffer. Efficiencies of various siRNAs in inhibition of EV71 replication were analyzed using the real-time TaqMan RT-PCR assay. The Human Enterovirus Type 71 Fluorescence Quantitative Polymerase Chain Reaction Diagnostic Kit (Suoao Biomedtech Co. Ltd., Beijing, China) was used in this study. The kit contains a TaqMan probe (5'-FAM-TGCAAGGATGCTAGTGATATCCTGC-TAMRA-3') and a pair of primers (forward, 5'-GCAGCCCAAAAGAACTTCACT-3'; reverse, 5'-ATCTGCCACCCTATCTCCCT-3') that target the 2366–2455 nt region of the EV71 genome (Genbank accession no. HM003207.1). According to the manufacturer's instructions, a reverse transcription reaction was carried out in a 10 μl reaction mixture containing 2 μl of total RNA, 7 μl of EV71-RT MIX, and 1 μl of reverse transcriptase at 42°C for 30 min. Subsequently, the reverse transcriptase was inactivated at 98°C for 5 min. Following reverse transcription, PCR amplification was performed in a 25 μl reaction mixture containing 3 μl cDNA, 20 μl EV71-PCR MIX, and 2 μl Taq Polymerase. The real-time TaqMan RT-PCR conditions were as follows: 1 cycle at 94°C for 2 min, 40 cycles at 93°C for 30 s, and 55°C for 45 s. EV71 RNA levels were detected using the Mx3000P real-time PCR system (StrataGene, La Jolla, CA, USA). A series of 10-fold dilutions of the standard plasmid contained in the test kit were used to obtain the standard curve used in absolute quantitation of viral RNA.
SDS-PAGE and western blot analysis
In order to obtain sufficient viral protein, RD cells in 6-well plates infected with 0.01 MOI of EV71 were lysed with 150 μl of CelLyticTM M Cell Lysis Reagent at 36 h post-infection. The protein concentration was determined by BCA reagents (Pierce, Rockford, IL, USA) according to the instructions provided by the manufacturer. A 40 μg aliquot of each cell lysate was electrophoresed in a 12% polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Western blot was performed according to a standard protocol. Protein detection was performed using an anti-EV71 VP1 mouse polyclonal antibody (Abnova, Taipei, Taiwan) and an anti-β-actin antibody (Sigma, St. Louis, MO, USA). Anti-mouse and anti-rabbit secondary antibodies labeled with IRDye infrared dyes (LI-COR Biosciences, Lincoln, NE, USA) were used in the infrared fluorescence detection procedure. The fluorescent signals on the PVDF membrane were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Quantification of bands was performed using Image J software (NIH, Bethesda, MD, USA).
Virus titering plaque assay
To ascertain the effect of siRNA-mediated intracellular inhibition of EV71 replication on the progeny virus generation, plaque assays were carried out to determine virus titers of the culture supernatants. Culture supernatants from each well were collected at 48 h post-infection and centrifuged at 1000 × g for 10 min to remove cellular debris. Confluent monolayer of RD cells in 6-well plates was inoculated with 500 μl of supernatant from each test group at a 10-4 dilution for 1 h. The media was aspirated off, and each well was covered with 3 ml of 1% (w/v) carboxymethylcellulose (CMC) in maintenance medium. After incubation at 37°C for 72 h, the plaque media was removed, and RD cells were fixed with 20% formalin in PBS. After fixation, the plaques were stained with 1% crystal violet stain for half an hour at room temperature, and the plaques were counted.
Serum stability
Unmodified si-2 and corresponding 2′-modified si-2OMe and si-2 F were used to examine the ability of ribose chemical modifications to improve the serum stability of siRNAs. Various siRNAs were dissolved in RNase-free water containing 10% FBS, 100% mouse serum (Invitrogen, Camarillo, CA, USA), or 100% human serum (Sigma, St Louis, MO, USA) at a final concentration of 10 μM and incubated at 37°C. Aliquots of 5 μl were withdrawn at different time points (0, 0.5, 1, 3, 6, 24, 48, and 72 h) and immediately frozen in 15 μl TBE-loading buffer [TBE (0.1 M Tris–HCl pH 8.4, 2 mM EDTA, 0.09 M boric acid), 7 M urea, 0.01% xylene cyanol, 0.1% bromphenol blue]. RNAs were separated using 15% polyacrylamide-TBE under non-denaturing conditions and visualized by staining with GelRedTM Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA). Gels were imaged using a 300 nm transilluminator and photographed with an ethidium bromide filter and Polaroid 667 black-and-white print film (Alpha Innotech, Santa Clara, CA, USA).
Statistical analyses
All data were expressed as means ± standard deviation (SD). Statistical analyses were performed using one-way ANOVA followed by Dunnett's post-hoc test. Differences with a P-value less than 0.05 were considered to be statistically significant (P < 0.05).
High transfection efficiency of siRNAs using LipofectamineTM 2000
Unmodified and 2'-modified siRNAs showed no toxicity in RD cells
Unmodified and appropriate 2′-modified siRNAs delayed cytopathic effects (CPE) and increased cell viability in EV71-infected RD cells
The protective effect of the unmodified and 2′-modified siRNAs over a period of time after EV71 infection was also evaluated by cell viability assay. At 24, 36, 48, 60, and 72 h post-infection, viability percentages indicated that an obvious elevation of viability could be achieved in RD cells transfected with unmodified (si-1, si-2) or 2′-modified (si-1 F, si-2OMe, si-2 F) siRNAs compared to mock transfection cells and scrambled siRNA transfected cells (Figure 3B). However, 2′-modified si-1OMe was not as effective as unmodified si-1, as there was only a slight elevation of cell viability in EV71-infected RD cells transfected with si-1OMe (Figure 3B). RD cells transfected with effective siRNAs at the concentration of 100 nM were found to exhibit higher viabilities compared with those observed upon treatment with the concentration of 50 nM (Figure 3B). Prolonged incubation up to 72 h post-infection was shown to lead to increased cell death (Figure 3B).
Reduction of EV71 RNA transcripts by unmodified and appropriate 2′-modified siRNAs
It was of interest to know whether the siRNAs could work after EV71 infection. The RD cells were first infected with 0.01 MOI of EV71 for 1 h, followed by treatment with various siRNAs. Also at 36 h post-infection, viral RNA was extracted from the RD cells. The efficiencies of the various siRNAs in inhibiting EV71 replication were analyzed using the real-time TaqMan RT-PCR assay. Results showed that there were significant reductions in EV71 RNA transcripts in a dosage-dependent manner when the infected RD cells were treated with the unmodified (si-1, si-2) and 2′-modified siRNAs (si-1 F, si-2OMe, or si-2 F) after EV71 infection (Additional file 1: Figure S1). Thus, administration of unmodified and appropriate 2′-modified siRNAs to RD cells after EV71 infection could still effectively inhibit EV71 inhibition.
Reduction of EV71-specific proteins by unmodified and appropriate 2′-modified siRNAs
Reduction of viral titers by unmodified and appropriate 2′-modified siRNAs
Serum stability of siRNAs
For many years, there has been an ongoing search for effective vaccines or antiviral agents for the treatment of EV71 infections. Previous studies have shown that EV71 for its survival and replication developed a defense mechanism that could not only eliminate the innate interferon immune response but also weaken the effect of exogenous interferon treatment. The 3C protein of EV71 inhibits retinoid acid-inducible gene I-mediated interferon regulatory factor 3 activation and type I interferon responses [33]. EV71 suppresses Toll-like receptor 3-mediated type I interferon responses by down-regulation of Toll/interleukin-1 receptor domain-containing adaptor inducing beta interferon (TRIF) [34]. EV71 also antagonizes type I interferon signaling by reducing the level of interferon receptor 1 [35]. Traditional interferon therapy, which has been successfully applied to many viruses, showed only weak anti-EV71 activities and is not suitable for the treatment of EV71 infections [35, 36]. Given the limitations of current therapies for EV71 infections, our attention was drawn to the developing RNAi research field. In this study, unmodified and 2′-modified siRNAs were designed to target the highly conserved 5′ UTR of the EV71 genome to inhibit EV71 infection in vitro. When compared with EV71 China strain genomes available in GenBank in the previous 5 years, siRNAs targeting the 115–133 nt sequence of the EV71 genomic 5′ UTR indicated the rates of sequences with complete nucleotide coincidence reached about 71.30%, while the siRNAs targeting the 648–666 nt sequence of the EV71 genomic 5′ UTR showed the rates of sequences with complete nucleotide coincidence (37.04%) and those with the same single-nucleotide difference (41.66%) together reached about 79.70% (Additional file 2: Table S1). Thus, siRNA nucleotide sequence analyses indicated that a mixture of the siRNAs (including consideration of the same single-nucleotide mismatch of target sequences to design a pair of siRNAs) targeting these two regions above may have much broader antiviral effect on diverse EV71 China strains.
Transfection of RD cells with siRNAs targeting the 5′ UTR of the EV71 genome delayed and alleviated CPE related to EV71 infection and increased cell viability in EV71-infected RD cells. The inhibitory effects against EV71 replication were sequence-specific and dosage-dependent, resulting in significant decreases in viral RNA, VP1 protein and viral titer. The most effective inhibition was observed for the siRNAs (si-2, si-2OMe, and si-2 F) targeting the 648–666 nt sequence of the 5′ UTR of the EV71 genome, followed by the siRNAs (si-1 and si-1 F) targeting the 115–133 nt sequence of the 5′ UTR of the EV71 genome. It is likely that variations in efficiency are primarily due to differences in positional accessibility of respective target sequences. The current research demonstrates that the highly conserved 5′ UTR of the EV71 genome can be targeted for inhibition of EV71 replication by efficient siRNAs. The results were consistent with previous studies of siRNAs targeting the 5′ UTR of other RNA viruses such as hepatitis C virus [37, 38] and coxsackievirus B3 [20]. These studies demonstrated that siRNAs targeting the 5' UTR of the viral genome can exert significant antiviral activity. However, siRNAs targeting the highly conserved 5′ UTR of the poliovirus genome failed to inhibit virus replication [39]. It is possible that some siRNAs cannot efficiently hybridize to target sequences of the viral 5′ UTR due to complex secondary structure or protein interactions with RNA motifs that may occlude the target sequences. Thus, the secondary structure of target RNA is an important factor in selecting a suitable target sequence, as it can have a negative effect on RNAi efficiency [40, 41].
When compared to previously reported siRNAs and short hairpin RNAs (shRNAs) [27–31], the target sequence located within the highly conserved 5′ UTR is a superiority with a reduced tendency to mutate. Nucleotide sequence analyses suggests that these siRNAs may have broader antiviral effects among EV71 China strains than the majority of previously reported siRNAs or shRNAs [27–29, 31], because the target selected in majority of previous studies was not conserved in EV71 China strains. Moreover, to tackle the high mutation rate associated with EV71, the siRNAs in our study can be combined with other efficient siRNAs in previous studies simultaneously targeting multiple regions of the genome to increase the efficiency of inhibiting viral replication.
As EV71 infection is transient in nature [27], it is unnecessary to maintain long-lasting viral knockdown effect, which may result in non-specific adverse effects. Synthesized siRNAs used to mediate RNAi against EV71 may be a good strategy for genetic antiviral therapy as it is straightforward and amenable to chemical modification. The 2′-OMe and 2′-F chemical modifications are classic patterns that can increase binding affinity, nuclease stability and be well-tolerated throughout the duplex [16]. These advantages are important for therapeutic applications. To optimize the properties of siRNAs for further research and clinical applications, 2′-OMe or 2′-F modification at U and C sequences on siRNA complementary strands were employed in this study. We compared the serum stabilities as well as gene silencing and cytotoxic effects of the unmodified siRNAs and 2′-modified siRNAs.
Enhanced serum stability is a major advantage of chemically modified siRNAs that can prolong their activity [16]. Both 2′-OMe and 2′-F modifications dramatically increased the serum stability of the siRNAs in the present study. Significant enhancements in serum stability of 2′-OMe or 2′-F modified siRNAs were observed in 10% FBS, 100% human serum, and 100% mouse serum compared with unmodified 21 nt siRNAs. Our research also demonstrated that 2′-OMe or 2′-F modification could improve siRNA stability and protect against nuclease degradation [42].
Several studies have demonstrated that 2′-OMe or 2′-F modification at specific positions in the siRNA duplexes were tolerated without loss of RNAi activity [43, 44]. In this study, the RNAi activities of 2′-modified siRNAs were evaluated. It was found that 2′-modified si-1 F, si-2OMe and si-2 F manifested similar RNAi activity in comparison with unmodified counterparts. The cells transfected with si-2OMe and si-2 F targeting the sequence of nt 648–666 within the 5′ UTR of the EV71 genome had better RNAi activity than si-1 F targeting the sequence of nt 115–133 within the 5′ UTR of the EV71 genome. The results indicated that the RNAi activity of 2′-modified siRNAs can be determined based on the primary sequence of the siRNAs. It is worth noting that the 2′-modified si-1OMe showed significantly decrease in antivirus activity compared with unmodified counterparts. It is not clear why the patterns of 2′-OMe modifications work for one sequence but not for another. Perhaps the introduction of 2′-OMe modifications into the si-1 alters the thermostability of the duplex that could interfere with the activity of siRNA. The results also indicated that siRNA modifications are typically sequence dependent, requiring optimization of the modification strategy for each different siRNA sequence when screening for efficient chemically modified siRNAs.
The application of chemically modified siRNAs to therapeutics has raised a number of concerns about their safety. In this study, cytotoxicity assays were used as a global indicator for monitoring adverse effects of unmodified and the 2′-modified 21 nt siRNAs. Neither the unmodified nor the 2′-modified 21 nt siRNAs showed any significant cytotoxicity in contrast to the mock transfection control up to 72 h post-transfection, even at a high concentration of 100 nM. The results implied that these siRNAs are safe for therapeutic applications.
Our results clearly showed that the 2′-OMe and 2′-F modifications were substantially compatible with the siRNA machinery. The appropriately 2′-modified siRNAs maintained high antiviral activity, were safe and displayed improved bio-stability compared with unmodified siRNAs. The study showed an improvement in triggering RNAi against virus infection using the more potent 2′-OMe-modified and 2′-F-modified siRNAs. These results also represent an important step in advancing siRNA into a broad range of therapeutic areas.
EV71 will very likely continue to cause serious problems in hundreds of thousands of young children in the future. In a direct approach using infectious EV71 to challenge siRNA-transfected cells, we demonstrated that the highly conserved 5′ UTR of the EV71 genome is an attractive target for inhibition of viral replication by the RNAi strategy. 2′-modified siRNA may be more effective and promising than unmodified siRNA as a therapeutic agent against EV71 infection. This study provides new target sequences and highlights the potential of appropriate 2′-modifications in improving properties of siRNAs to overcome the barriers to RNAi-based antiviral therapies and to bring RNAi technology to the clinic in the future.
= Enterovirus 71
- (siRNA):
= Small interfering RNA
= Untranslated region
- (RNAi):
= RNA interference
= Rhabdomyosarcoma
= Hand foot and mouth disease
- (2′-OMe):
= 2′-O-methylation
= 2′-fluoro
- (nt):
= Nucleotide.
This study was supported by the National Ministry of Science "Eleventh Five-Year" Science and Technology Support Project sub-topics (No.077BAI07A12). Many thanks to the Department of Viral Diseases Laboratory, Xi′an Center for Disease Control and Prevention (Xi′an Shanxi, People's Republic of China) for providing the EV71 strain and RD cells lines. We would also like to thank the members of the Department of Microbiology, Fourth Military Medical University for their useful discussion and technical assistance.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Copyright 2016, J. David Goldin.
These are all the programs in the radioGOLDINdex database which credit this artist. These listings are accurate as of February 01, 2017. The programs are listed chronologically, partial dates appear first, unknown dates appear last.
90108. The Fleischmann's Yeast Hour. February 23, 1933. Red net. The first tune is, "Across The Daisy Fields." Conway Tearle is featured in a scene from, "Doctor Jekyl and Mr. Hyde." Rudy sings melodies from, "The Tales Of Hoffman," along with Lois Bennett and Fifi Sherman. Rudy Vallee, The Connecticut Yankees, Jimmy Wallington (announcer), Lou Holtz, Conway Tearle, Rose Hobart, Harold Vermilyea, Louis McHenry Howe (confidential secretary and advisor to president-elect Franklin Roosevelt), Walter Trumble (interviewer), Lois Bennett, Fifi Sherman. 50:56. Audio condition: Excellent. Incomplete.
Report contains 1 program.


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Camp-Kinser Gifts For Men
13 items found
You can send Gifts For Men to Japan APO FPO in the following cities.
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Facebook corporate office in Palo Alto before they move to their new office complex in Menlo Park. I have wanted to visit Facebook since my sister-in-law started working there over a year ago, but today was my first chance. After finding a place to park (not an easy feat for the employees, a little easier for visitors), I took the obligatory photo of the address sign and then headed into the lobby to sign in and take more pictures.
The lobby offered a few more photo opps, including a framed Facebook logo that had signatures of various executives and celebrities, a lego sculpture of the Facebook logo, a "HACK" sign (hack is for all intensive purposes Facebook's motto) and a TV screen that shows all of the connections people are making around the world with Facebook.
Once inside the offices I saw the famous wall (and took a few pictures, I practically blend in). After the wall I looked around the office, saw Mark Zuckerberg, Sheryl Sandberg and what I believe was the rest of the executive team in conference room surrounded by glass and then headed to get some snacks. Free food is a staple in the office and that was great ... I decided to help myself to cookies, Polar Bear Coca-Cola, Starbursts, etc at one of the snack stations. They also have the new Coca-Cola choose your own adventure machine that can make every flavor of Coke and then some.
While eating and taking pictures I got to see more of the office heading over to the employee cafeteria where I got to speak with Facebook's head chef. He was great to talk with and all I can say is the new Facebook campus will have amazing food options for all of the employees (and occasional guests). Lunch was great and reminded me of the tasty buffets on a Royal Caribbean cruise without the ocean view and without having to pay for a cruise.
Did You Know ... that instead of "Casual Friday" a few Facebook staff believe in "Corporate Friday"?
If your still curious about what the inside of the office looks like, search Google Images for "Facebook + Palo Alto" and you can see pictures that other people took.


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Discuss this help topic in SecureBlackbox Forum
TElPKCS11CryptoProvider class
TElPKCS11CryptoProvider is a descendant of TElCustomCryptoProvider.
This class represents a PKCS#11 crypto provider. When this provider is chosen, the implementation of cryptographic functions is delegated to a crypto token..NET:
To use this class in the development and distribution of your software projects, you need to purchase one of the following licenses:
PKIBlackbox OR AdESBlackbox OR SecureBlackbox Standard OR SecureBlackbox Professional OR SecureBlackbox Transports OR SecureBlackbox Data Security


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Privacy Policy
Explanation of contents
In some cases we will ask you to supply personal information (name, telephone number, credit card number etc) so that you can use the service that we offer on our website. This service includes options to receive newsletters and to register interest in our projects.
When you enter your particulars in the various fields, you make it possible for us to give you the service that you want. Every time that you provide us with information Oscar Properties will handle it in accordance with the appropriate legislation.
All information that you supply to us will be used solely by Oscar Properties. We will never pass it on to any third party without your permission unless there is a legal obligation for us. We will store the information in our system for as long as you are using our services, and when we no longer need the information in order to give you the service you want, the information will be removed from our system.
When you visit a website your computer will automatically receive a cookie. Cookies are text files which identify your computer in our server. Cookies do not identify the individual user but only the computer. Many websites use cookies every time anyone visits the website in order to be able to collect information about the number of visitors.
Cookies collect information about which pages are visited by the computer and for how long. As the user of your computer, you can choose, via your computer's settings, either to accept cookies, to receive a message when your computer receives a cookie, or not to accept cookies.
Each time you visit our website, a cookie is downloaded to your computer together with the page. We, and most other websites, do this because cookies enable us to see whether the computer has visited our website previously. All information that we obtain via cookies helps us to give you better service. For example, if you looked for information about projects on your first visit to our website, we are informed about this via your computer's cookie and can take you direct to new projects etc this time [*1].
We may make changes to our policy in the future. The most recently updated version will always be available on our website so that you always know what types of information we are collecting and why.


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Wiz Khalifa – See You Again feat. Charlie Puth
File Size : [ 648 KB ]
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Susan Wilkinson Exhibit Entitled "All Things Bright and Beautiful" at the Prizery starting April 2nd
Parsons-Bruce Art Association is pleased to feature the beautiful paintings of Halifax County resident, Susan Wilkinson. The exhibit, entitled "All Things Bright and Beautiful" is set to open in the Robert F. Cage Gallery at the Prizery Sunday, April 2nd with a reception at 3:00 pm.
Susan studied first at North Georgia College and earned her BFA years later at St. Andrew's Presbyterian College in Laurenburg, NC. After graduation, she settled back in Georgia, combining family life and work as a freelance artist while providing art for several area churches, painting murals, as well as graphite and acrylics portraits.
She was privileged to represent the state of Georgia by decorating an egg for the annual White House Easter Egg Display in 2001. She joined the other 50 artists (from each state and the District of Columbia) to see their eggs displayed in the White House and to attend a reception hosted by the First Lady. Susan used an ancient technique called pysanky, or Ukrainian egg decoration for the Georgia egg; a technique she learned while on vacation.
Susan and her husband, Tommy moved to Halifax County several years ago, and in 2015 she was asked by Parsons-Bruce Art Association to design and contribute an ornament for the Christmas tree in Virginia's Executive Mansion on behalf of Halifax County. That ornament, another egg done with pysanky, will remain in Richmond for use during future holidays.
Susan's work has also been displayed at her senior show at St. Andrew's, the Hudgens in Lawrenceville, the Quinlan and Art in the Square in Gainesville, a solo show in Dalton, GA's Creative Arts Guild gallery, and the Hoschton Heritage Arts Council. She also currently serves on the Parsons-Bruce Art Association Board of Directors.
Susan Wilkinson's art exhibit, sponsored by Parsons-Bruce Art Association, will be on display for viewing and purchasing in the Robert F. Cage Art Gallery at the Prizery from April 2 until May 10. The public is invited to attend the opening reception April 2nd from 3:00 pm until 5:00 pm.
Ansel Adams & Glory of Venice Exhibit at NC Museum or Art
Join the Parsons-Bruce Art Association on a bus trip to Raleigh on March 16, 2017, to the North Carolina Museum of Arts for Ansel Adams Masterworks photography exhibit. Over 40 photographs selected by Adams as a representation of his best works will be on display. Included are majestic American landscapes, such as El Capitan and Half Dome in Yosemite National Park; The Golden Gate Bridge in San Francisco; Monument Valley in Arizona; the Snake River in Grand Teton National Park; and others.
Also on display is Glory of Venice: Renaissance Paintings 1470-1520. The exhibit illustrates a crucial period in the history of Venetian art and culure. Regarded as one of the most exciting periods in Western art history, the exhibit features masterworks from Galleria dell' Accademia and paintings by Giovanni Bellini, Cima da Conegliano, Carpaccio, Titian, and others.
The bus will leave from the parking lot at Main Street United Methodist Church in South Boston at 8:30am and return by 5:00pm on Thursday, March 16th. The trip costs $55 for Parsons-Bruce members and $60 for non-members. The registration fee includes transportation, tickets to both exhibits, docent tours of the exhibits, an exclusive lunch for the participants, and snacks on the return trip.
Parsons-Bruce Art Association is sponsoring a bus trip March 16th to the North Carolina Museum of Art for the exhibit of Ansel Adams Masterworks. The exhibit includes over 40 of Adams' iconic images of majestic American landscapes.


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APPE Special Interest Sections
APPE sponsors a many opportunities for advancing research and professional practice through Special Interest Sections. These interest sections put you in touch with international leaders in your field that are developing and implementing practical ethics programs in all areas of business, education and the professions. Special Interest Sections also organize program sessions at the Annual Meeting, and engage members in ongoing communication and collaboration throughout the year.
Currently, Association facilitates and supports special interest sections for those with particular interests in the following areas:
- Biomedical Ethics
- Business Ethics
- Engineering, Science and Technology Ethics
- Journalism and Media Ethics
- Research Ethics
New Special Interest Sections are being developed in the following areas:
- Action Research Ethics
- Public Health Ethics
- Social Work Ethics
To be added to any of the Special Interest Sections, please complete the online form located on our ecommerce portal. The Special Interest Sections are a benefit of Membership in the Association and are free to join. The portal simply processes your information to send to the APPE Office. Please, contact the APPE Office for additional information at: 812-855-6450 or firstname.lastname@example.com.
Comming soon:
APPE is in the process of creating online forums for discussions on various topics and specifically for the Special Interest Sections. As these forums become available, we will send information about them to our APPE Listserv. To be added to our Listserv, please send a request to the APPE office at: firstname.lastname@example.com.
Please, contact the APPE offices if:
- you are interested in being a part of any Special Interest Sections
- you are interested in being on any Planning Committees for the upcoming Annual Meeting
- you have a suggested topic for consideration at the upcoming Annual Meeting
Current Special Program Topic Sessions for the Annual Meeting
We are currently pursing the following topic session for the Annual Meeting, facilitated by the Special Interest Sections listed above. Follow the links below for additional information regarding these topic sessions.:
- Biomedical Ethics
- Business Ethics
- Empirical Ethics
- Liberty and Security
- Responsible Conduct of Research
Please, contact the APPE offices if you have any questions or would like additional information: email@example.com or 812-855-6450.


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|2,300 sq. ft.
|Property type
|Minimum Stay
|3 - 7 nights
Regency Cabanas Townhomes are gated for your complete protection. A $300,000 infinity edge pool was added, and our townhouse,looks out over this pool. (But not too close, so it's not noisy.) There is also another pool in the complex of about 58 townhomes so there is plenty of room for all swimmers. The garage of our unit will hold 3 cars. The townhouse has over 2300 sq.ft. living space.
The master bedroom is spacious, has a king bed, TV and huge master bath. Each of the bedrooms is beautifully done. Two bedrooms have queen beds and one bedroom has two twin beds. One of the bedrooms also has a twin sofa bed. The dining room has a long table seating 10 comfortably. The open concept family space and 10 ft. ceilings makes the townhome seem bigger than 2300+ sq ft.
Pensacola Beach is known for its beautiful white crystal sand beaches and it is only steps away from Regency Cabanas. No public roads to cross to get to the beach.
Keywords: Beautiful Townhouse - 4 Bedrooms sleeps 9 - You will love. Gated.
I have been coming to Regency Cabanas for many years. My parents used to rent a unit, then they bought one, and then my husband and I bought it from them. This year my husband and I were lucky enough to have an opportunity to purchase one of the newer and bigger ones in our complex, so we can have more room for family visits to the beach. The picture above was taken by my Mom on the beach at Regency Cabanas back in the 90's when our children were young. We look forward to coming to our new place for years to come and hope that you enjoy using it also. If we can do anything to help you please call us at (713)594-8118. Enjoy your stay on Pensacola Beach!!!
Lisa purchased this townhome in 2015
Pensacola Beach has the most beautiful sand beaches in the United States. Additionally, Pensacola Beach is close enough to the many attractions of Pensacola including the Navy Aviation Museum at NAS Pensacola. Also, Pensacola was the original settlement in the United States and two ships from that settlement have been found.
Regency Cabanas is a gated community. We have two pools, one is a $300,000 infinity edge pool which you overlook from the balcony of our unit. Additionally, you are only a short distance from the white sands of the Gulf of Mexico.
A visit to Pensacola Beach is always great, but staying in the "Emerson House" was the icing on the cake. And where else will you get a visit from the owner when a little problem arose? We can't thank Evon and Ralph enough for a fantastic week. All of Our family had a wonderful time. We can't wait to visit again.
We were fortunate to stay in this unit for a week with two other families. The unit is well appointed with tasteful furniture and a well stocked kitchen. The balcony overlooks the beautiful salt water pool and the walk to the beach was only two minutes away. What a gorgeous beach!!! The garage had lots of pool/beach items that we used. I communicated with Lisa, the owner, before our trip. She was very prompt in returning emails to me and went out of her way to accommodate us and make sure that we were well taken care of and comfortable. We were a group of 25 from Texas, Tennessee and Virgina. It was a celebratory trip for our great nephew (6yrs old), who had just finished a three year chemotherapy regimen for leukemia. The week was wonderful. I highly recommend this unit to you. You will not be disappointed!
Stayed in this gorgeous condo while visiting friends and had the best time. Very clean, spacious, and in a great location! We've stayed in this area a couple of times before and each time is more amazing. We will definitely be staying here again soon!
We have been staying in Pensacola the past 40 plus years . We began staying in Regency Cabanas the past 20. I have stayed in probably 15 different units there over the years. This unit is set up perfect for us and our four kids. With ease to beach and pool it is in a perfect location. If you follow the rules and only have the alloted amount of people you will have no problem parking with the huge three car garage. The owner will be putting in new furniture in the fall to make this even a better unit. We have already secured the unit for two weeks next summer. This will be our place of retirement in the years to come and I hope I can find a unit in the Refency Cabanas when the time comes. It is so much better with "low density" compared to high rise units where you have to fight for a place on the beach or the pool.
I've stayed in Regency Cabanas several times over the years; this unit is in a newer section and is a short walk to the white-sand beach. This home has new owners as of April, and they have updated the interior beautifully. This condo is really 3 levels. The first is a garage with storage that has room to easily fit 3 cars and also has an indoor staircase to the condo. The second level has a bedroom and bath, huge open area with living, dining, and kitchen, and a large outdoor patio. Top floor is 3 bedrooms/2 baths with very large master suite. It is clean, tastefully decorated, very inviting, and comfortable! I really enjoyed my stay, and hope to return soon! Thank you!
I wish I could give a better review, unfortunately we were very disappointed! The cleanliness of the condo was an issue my 1 year old granddaughter found a large bottle of Vodka in the cushions of the furniture outside, left by the previous tenant. The condition of the furniture in the living room was dis-questing, we dropped a cell phone and when reaching under the chair to get it found a lot of garbage. One of the ottomans was smashed in. From the pictures we felt a bit deceived we thought we had a water view, we had no view at all other than the other condos. The beach is a 5 min walk from the condo, which would have been fine, if we could have seen any water at all. The bed rooms and baths were nice and well kept up. I would highly recommend finding a different cleaning service the state of the condo when we arrived was extremely dirty and to have a one year old uncover a very large glass bottle of Vodka is not acceptable at all!
My decision to stay here this spring break was an excellent decision for the family. This townhouse is exactly as described and even more beautiful in person. The location is awesome! We have vacationed at different locations included the Portofino and I must say that this place is definitely on my favorite list. We are looking forward to booking again next year and possibly sooner. I am a very picky traveler and I can assure you that you will not be disappointed with this location!
|Rental Period
|Minimum Price
|Additional Nights
|Weekend Night
|Monthly *
|Event Price
Late Spring
16 Apr 2017 - 26 May 2017
4 night minimum stay
for 4 nights
27 May 2017 - 10 Jun 2017
7 night minimum stay
for 7 nights
11 Jun 2017 - 22 Jul 2017
7 night minimum stay
for 7 nights
Late Summer
23 Jul 2017 - 26 Aug 2017
7 night minimum stay
for 7 nights
My Standard Rate
3 night minimum stay
for 3 nights
|Pet Fee
|£231.91 + Tax
|Damage Waiver
|£38.65 + Tax
|Cleaning/Linen Fee
|£170.07 + Tax
|£50.25 + Tax
|Tax Rate
Travel Insurance (optional) is offered by our management company. Your choice.
50% due at time of booking. 50% due 30 days before.
* Approximate monthly price. Actual pricing will depend on the days of the month you stay.


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Definition of Auvergnat
: a native or inhabitant of Auvergne, France
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Origin and Etymology of auvergnat
French, from Auvergne, region of south central France
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