text:
19 County Road 6341, Granby, Colorado
$349,0004 BedsFor Sale Under Contract
$349,0004 BedsFor Sale Under Contract
19 County Road 6341, Granby, Colorado 80446 - Right at Lake Granby, near Grand Lake. Furnished home, views of lake and surrounding peaks in Rocky Mountain National Park! You'll love the privacy, peace, and quiet of this unique mountain home, with 2 decks to take advantage of the unbeatable views. Lower level features 1 bedroom, 1/2 bath, laundry room, and storage area. Main level has 2 more bedrooms, 3/4 bath, an open kitchen with granite counter tops and bar seats, and a separate dining area with large windows and great views. Access to the top deck is from the dining room. The living room has vaulted ceilings, floor to ceiling windows, wood-burning fireplace, and access to the large deck to take in all the views and great weather. Upper level has a loft, 4th bedroom and 3/4 bath with skylights. Attached 1 car carport, with long gravel driveways for additional/RV parking. 2 hours to Denver, 20 minutes to Grand Lake entrance to RMNP, 15 minutes to quaint town of Granby, 30 minutes to Winter Park ski area. You'll love living here!
Listing Courtesy of ASSIST2SELL RE PROFESSIONALS
"Bob Maiocco exceeded our expectations on every level. Not only does Bob have a very solid knowledge of the real estate market, his negotiating skills are second to none. Bob has a calm and even demeanor when dealing with sometimes stressful situations that can arise when buying or selling real estate. We could not have asked for a more strategic partner to help us navigate the process of making our largest financial purchase."


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Only a week before the Autumn Coin Op Show and NSM has already hit a record breaking amount of sales during 2016.
Just 9 months since the new icon:Storm jukebox was launched at the EAG and it has already proven to be an industry favourite, whether this is down to the breath taking looks or the unbelievable price tag, it really has taken the industry by storm.
As well as enjoying the fruits from the sales of jukeboxes NSM has also hit high levels of success with the revolutionary icon back ground music system and Jukebox App.
According to NSM's Sales Manager Alex Kirby:
"We haven't been as busy as this since the CD Jukebox era, we really have felt a massive difference in sales this year.
The icon range of jukeboxes has never been as affordable and reliable and I think it's a combination of this and the older Digital jukeboxes becoming out dated and expensive to maintain why we are so busy.
Because of our great products and deals, there has never been a better time for operators to update their aging equipment"

subset: commoncrawl


text:
Cost per License:
1 to 3 licenses
$1,150 each
4 to 6 licenses
$950 each
7 to 10 licenses
$800 each
11 and up
$650 each
ReSSA (3.0) and ReSlope (4.0) - Limit 3 sets
$1,250 each
Special Discount: Registered owners of program ReSlope versions 3.0 or 4.0 are entitled for a discounted price of $650, one license of ReSSA for each license of ReSlope. No additional discount, such as educational discount, is available for this special price. This option allows for continued use of ReSlope independent of ReSSA (each has its own hardware key).
Upgrade: Licensed owners of ReSSA 2.0 are entitled to purchase an upgrade. Cost of upgrade from version 2.0 is $500. Cost of S & H is extra. One upgrade per license. In order to upgrade, the original hardware key must be returned.
Educational Discount: Universities worldwide are entitled for 25% discount, regardless of the number of programs purchased. This discount is not applied to the cost of S&H.
ADAMA Engineering keeps a record of purchases. Purchase by a customer having the same name, company affiliation and address in each separate order may entitle that customer for the progressive price noted above. The price will be adjusted for such a customer based on the number of copies purchased over time (however, the cost of S&H is applied for each shipment).
INTERNATIONAL CUSTOMERS: Duty and tax charges (e.g., customs, VAT) are the responsibility of the buyer. ADAMA Engineering is not responsible for duties and taxes.
Note: Each program is shipped with a hardware lock device (dongle). The dongle enables you to run the software, thus constituting a license. Lost dongles will not be replaced. It is recommended to follow a good business practice and insure your license in case the dongle is lost. ADAMA Engineering cannot provide such insurance.
Website designed, constructed and maintained by Ben A. Leshchinsky. Any errors? Email comments and suggestions to email@example.com. Copyright of © ADAMA Engineering, Inc. 2015. Contents of this website may not be reproduced without permission of the webmaster.


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Category Archives: Electric Bass
Smitty & the Line 6 Relay G10
Say hellow to our new favorite way to plug into amps — the Line 6 Relay G10! These are awesome little units. $99 for the transmitter, $179 for the transmitter/receiver in a bundle 😎
Smitty loves these units — come into Music 6000 to try one out!
Peavey Dyna Bass w/Case
Ibanez SR300
Ibanez TMB300 (Navy Metallic)
Ibanez SR500
Fender P Bass (US) w/ Case
$999 *sale*
Ibanez SR750 Bass
Beautiful Ibanez SR750 electric bass guitar at Music 6000 — this thing has gorgeous tone and will make you happy for years to come. Price is too good to believe, come into the shop to see!
Squier 5-String Basses
Get deep with the bass with a 5-string from Squier! These are monster 5-string basses, starting at $349. Come into the shop, make some noise with these things! Give us a call at 360-786-6000 with any questions 🐋
Fender Zone 5-String
Fender Dimension Bass
A new bass design from Fender, the Dimension Bass is a fine addition to Fender's proud bass history. These basses carry a spectrum of great tones — come into Music 6000, plug in, and enjoy!
Fender American Standard Jazz Bass in Olympic White
Just in at Music 6000 is a brand new Fender American Standard Jazz Bass in Olympic White with a Rosewood neck. Get this bass today with a Hard Case for $1399!
The Flagship Jazz Bass
The U.S.-made flagship of the Jazz Bass lineup, and a must-have instrument for bassists who prize the elemental voice and versatility of a timeless Fender classic. The American Standard Jazz bass is the one—the modern incarnation of a sleek and stylishly enduring bass hit, with smooth, fast feel and unmistakable Jazz Bass tone that sings, snarls, grooves and growls with more power and purity than ever.
- Dual single-coil pickups
- Comfortable modern neck profile and fingerboard radius
- Posiflex™ graphite neck support rods
- Combination string-through/top-load high-mass bridge
- Lightweight vintage-style tuners


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- Ling-Bing Tang, Ling-Xiao Tang, Huan-Ye Sheng
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One of the challenging problems in forecasting the conditional volatility of stock market returns is that general kernel functions in support vector machine (SVM) cannot capture the cluster feature of volatility accurately. While wavelet function yields features that describe of the volatility time series both at various locations and at varying time… (More)
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Greyhawk Deities
Yesterday in my opinion and a hitch-hike of good friends travelled to Witchfest. This is an almanac affair organised by The Offspring of Artemis, a group of on a par minded genus that I am a advocate of.
The day was a inundate of seminar, workshops, stalls and postpone music all in one place. Discussion and workshops by Tylluan Penry, James Bennett, Martin Wyse, Damh the Writer, Tam Campbell, Prof. Ronald Hutton and Kate West to name but a few!
Introduce were so plentiful seminar and workshops to covet from it was worrying to work out which ones to go to.
The youthful sermon we attended was 'Shamanism ">
As a result on to Witches Barrier Custom Living - Tam Campbell, Kate West, Martin Wyse, Damh the Writer and Tylluan Penry took the period and answers questions from the addressees. It was very informatative and entertaining!
Gone, on to a invective by Prof. Ronald Hutton on the history of the fae. Fighting fit mesmerizing and exert yourself aggressive with some of his private experiences puzzled in for good debit.
The carry sermon we attended was from Damh the Writer on Ogham and Druidic Tree Mystic, so well attended that the room was full to bursting! Damh was, as consistently very imperceptible and gave a very exert yourself aggressive sermon about ogham, truly gave me a different way of learning it.
A very good day, with ultra cake, shopping and fare of course!
Ample of postpone music in the sundown - Damh the Writer, The Dolmen and Inkubbus Sukkubus in the midst of the line up.
The Offspring of Artemis:
Damh the Bard:
Tylluan Penry:
Martin Wyse:
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The Business Group Blog was created to share and discuss information about challenges and solutions to the health care benefits issues that large employers face today and tomorrow — such as controlling health care costs, reforming the health care delivery system, and engaging employees in their health and benefits — and will provide insight into national health policy issues. We hope you find this information useful and will consider subscribing to the blog and sharing any thoughts or ideas with us at firstname.lastname@example.com.
There are 4 item(s) tagged with the keyword "physical health".
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Large employers are increasingly utilizing networks of volunteer employees as an extension of their wellness staff, commonly called "wellness champions". Their role is to help drive employee participation and engagement in wellness activities at the local level. 77% of large employers have been found to have some type of wellness champion network. While the size of champion networks varies greatly, 56% of employers have at least 50 wellness champions in their network.
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Some companies have made significant progress toward integrating occupational health and safety programs. One example is featured in this post.
Simply adding new well-being programs at your organization is often not enough to increase the health and well-being of your employees. Often times, low engagement rates can become a barrier to creating a culture of well-being at your organization.
Leading employers are changing their focus from offering benefits and programs centered on improving employees' physical health to deploying holistic wellbeing strategies whose purpose is to enhance all the various facets of employees' lives. This shift is taking place as evidence mounts that physical health is only one of a number of factors contributing to outcomes valued by employers. These include improved health, greater productivity and higher performance, accompanied by decreased health care costs and a lower turnover rate.
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text: Spending a summer sanding the fenders of his dad's '56 F100 sparked Tibbett's passion for the car hobby. His penchant to scribble cars in every high school notebook culminated in a ten-year career as art director for some of the most well-known automotive aftermarket brands in the industry. He now turns his snarky pen toward the lighter, quirkier side of the automotive lifestyle as editor of Gearheads4Life.


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text:
Shandar: The Shrunken City
Agnes Bruckner
Ted Nicolaou
Starring: Agnes Bruckner Director: Ted Nicolaou
Two young heroes. One small town.
LOW STOCK: Only 1 copy left
|List Price:
|You Save:
|$7.97 (62% Off)
Available: Usually ships in 1-3 business days
Format: DVD
sku: FUL 375D
Brand New
Related products:
Mysterious Museum ~ $6.98
Night at the Magic Museum ~ $4.98
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DVD Features:
- Rated: Not Rated
- Run Time: 1 hours, 25 minutes
- Video: Color
- Released: June 5, 2012
- Originally Released: 1998
- Label: Wizard Entertainment
- Encoding: Region 1 (USA & Canada)
- Note: Trailers
- Aspect Ratio: Full Frame - 1.33
- Audio:
- Dolby Digital 2.0 Stereo - English
Performers, Cast and Crew:
|Agnes Bruckner
|Directed by
|Ted Nicolaou
|Edited by
|Gregory Sanders
|Screenwriting by
|Benjamin Carr
|Composition by
|Richard Kosinski
|Produced by
|Vlad Paunescu
|Director of Photography:
|Vivi Dragan Vasile
|Executive Production by
|Donald Kushner & Peter Locke
Description by OLDIES.com:
Two kids, GEORGE and LORI - have literally discovered a new world. Hidden deep underground for centuries, the tiny bottled city of Shandar is unearthed in an excavation site. Damaged from the dig, the dormant, miniaturized metropolis reawakens - unprotected from an inter-dimensional team of assassins sent to destroy it. Aided by Shandar's caretaker PRIME, George and Lori must find a way to stop the death squad and restore power to the city before it uncontrollably expands to its normal size annihilating itself and everything around it. Director: Ted Nicolaou. Starring Michael Malota, Agnes Bruckner, Steve Valentine, Jules Mandel, and Ray Laska.
Product Description:
Two young explorers discover a miniature city encapsulated in a glass globe, and fight to protect it from otherworldly invaders in this fantasy adventure for the entire family. For hundreds of years, the tiny city of Shandar has sat dormant underground. But when an excavation project brings Shandar to the surface, George and Lori attempt to get a closer look. Suddenly, the dormant city has sprung to life. But a team of mysterious creatures from another dimension seem poised to destroy it forever. Meanwhile, after being magically transported into Shandar, George and Lori strike up a friendship with kindly caretaker Prime. Now, unless Shandar regains power, it will soon expand to its original size. Should that happen, it would mean complete destruction of not only Shandar, but George and Lori's home as well. With their time quickly running out, the two kids embark on a quest to save the tiny city.
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text: 26 photos $449,000 15.45 acres 2 Beds | 2 Baths | 1 ½ Bath | 3254 sqft 2672 Hwy 95, Council, ID 83612 - Adams County Beautiful Log Home, Out Buildings, Timber, Big Game and Views Impressive log & wood work throughout this well care for home on 15 plus acres. Two sides of the property border forest service ground. Great set up for horses. 2 bedrooms on the main floor & a loft that could be turned into bedrooms if desired. Property sets well off the highway & is surrounded by lots of trees. Amazing deck wraps around 2 sides of the house. Partially finished basement consists of a 2 car garage, storage, shop area & an enclosed room with a half bath. Lots of trees & stunning views. 15.45 acres Wendy Ogden Silvercreek Realty Group


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More Information
- Žižek, Slavoj. The revolt of the salaried bourgeoisie. London Review of Books. 2012 Jan 26; 34(2):9–10.
Available from: http://www.lrb.co.uk/v34/n02/slavoj-zizek/the-revolt-of-the-salaried-bourgeoisie. Žižek begins with the thesis that post-industrial capitalism no longer derives surplus value primarily from exploitation of wage labor, but rather from economic rents on privatized public goods, especially natural resources and (above all) intellectual property. Peter Frase, making a similar point in "Four futures", refers to the resulting system not as capitalism but as rentism (although Žižek himself does not use this term). The shift from capitalism to rentism invalidates classical Marxist analyses of the inevitability of capitalist self-destruction, but leaves open the possibility that rentism may collapse for broadly analogous reasons. Žižek argues that since rentism does not require a substantial bourgeoisie, the former bourgeoisie are transformed into an essentially superfluous class of middle managers, who are paid a "surplus wage" not for services rendered but for the maintenance of social stability. Nevertheless, the lower levels of this "salaried bourgeoisie" are continually at risk of being pushed down into the proletariat, since there is a tendency for the very small rentist elite to concentrate wealth and power in their own hands by withdrawing the surplus wage. And since the rentist elite really has little use for the proletariat either, they tend to be reduced in turn to a nonproductive, marginalized Lumpenproletariat or underclass. The fear of losing the surplus wage and being reduced to the underclass can motivate a "revolt of the salaried bourgeoisie", which is Žižek's characterization of at least some of the popular revolts of 2011. But Žižek leaves open the possibility that an alliance among all of the increasingly dispossessed classes (salaried bourgeoisie, proletariat, and underclass) may lead to the final downfall of rentism.
- Frase, Peter. Four futures. Jacobin. 2012 Winter; 5:27–34.
Available from: http://jacobinmag.com/winter-2012/four-futures/. [anchor]
Frase considers the endgame of our current political struggles, locating future scenarios along two axes: scarcity versus abundance, and social hierarchy versus equality. This yields four possibilities: communism (abundance with equality), rentism (abundance with hierarchy), socialism (scarcity with equality), and exterminism (scarcity with hierarchy). The rentist scenario reveals economic rents (return on use of owned property), rather than labor, as the dominant source of surplus value — especially rents on intellectual property. This analysis of rentism dovetails with similar insights in Žižek's "The revolt of the salaried bourgeoisie", and is applicable in the present, since the relevant economic changes are already occurring. In the most dystopian scenario, exterminism, the ruling class kills off the vast majority of the population in order to secure its continued enjoyment of privilege under conditions of global scarcity, a scenario chillingly anticipated by Hannah Arendt near the end of Eichmann in Jerusalem.


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Advice on writing a personal statement ucas
The personal statement is an important part of the UCAS application. It's your chance to describe you ambitions, skills and experience. Anatomy of the UCAS personal statement. What a successful UCAS personal statement should consist of.
Do you have a specific question about UCAS personal statements? Here we give our answers to some of the most frequently asked questions about writing the personal.
Advice on writing a personal statement ucas
Get into uni is a leading provider of editing solutions serving customers throughout the world. Personal statement samples and writing help from the best editors on the planet
Top tips on writing your UCAS personal statement including how to start your statement as well as advice on structure, how to write it and the layout. Personal statement service. Writing a personal statement can be challenging , so our expert writers are here to help you with this difficult task. Apr 20, 2016 · Definition. A personal statement is an autobiographical essay that many colleges, universities, and professional schools require as part of the admissions.
The personal statement you have written for me is excellent. There is no way I could have improved it so much, thank you for your help and patience. Jun 29, 2015 · The personal statement is a crucial part of university applications in the UK. It's your chance to show what makes you unique, besides your birth name.


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Featured property
Featured property
3 Bedrooms Detached Bungalow for sale
Nestled in the corner of a cul de sac is this beautiful 3 bedroom detached bungalow with very generous driveway spacious low maintenance rear garden.rquite a rare opportunity has arisen…
£225,000 Guide Price
4 Bedrooms Detached House for sale
Guide price of 225,000 - 230,000 a family in mind. this four bedroom detached property with ample space throughout is the perfect next step on the property ladder for a family.
3 Bedrooms End Of Terrace House for sale
Elr are pleased to offer to the market this deceptively spacious 3 bedroom end of terrace property situated to a elevated corner position. with lawned gardens to the front side driveway…
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3 Bedrooms Semi Detached House for sale
Guide price 115,000 - 120,000 this three bedroom, semi detached house stands on a corner plot which requires some modernisation, but offers plenty of potential.
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2 Bedrooms Terraced House for sale
Westfield Road, Bramley, Rotherham, South Yorkshire
Guide price 95,000 - 105,000 no onward chain offered with no onward chain is this beautiful mid terrace property, which has been well maintained throughout with a stylish finish .…
Howard Resindence, Howard Road, Rotherham
Homefinders are delighted to present this opportunity to buy to let investors who are looking for a good return on investment. the development is situated in rotherham in a central…
Marketed by:
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149 Bawtry Road Wickersley
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text: Tonight You Belong To Me (Multitrack a capella) by emilylaras published on 2013-02-15T09:27:21Z I know that almost everybody has done this cover of Patience & Prudence "Tonight You Belong To Me", but I just couldn't help it! I LOVE THIS SONG! And I've been hearing this song over and over in my head since I started watching Ryan Murphy's American Horror Story. I was really in the mood of making an a capella cover version (never done it to any song before) so here it is, Tonight You Belong To Me. All recorded and mixed using GarageBand on my iPad. Enjoy:)) x Genre Acapella Comment by Blu Masibay Fornis this is so beautiful 2013/10/25 08:27:37 +0000 Comment by Om -2.169005,113.870689 folback dong kk emily 2013/09/04 18:14:45 +0000 Comment by Yosha Immawan harmoninya makyuss :0 2013/07/25 06:03:50 +0000 Comment by Constantino Stavroulakis super voice 2013/06/06 06:52:40 +0000 Comment by Wicaksana Arif in silvery moonlight.. (seneng bagian itu) 2013/06/04 03:24:32 +0000 Comment by BeaTW!RE this is beautiful, I honestly hope you make it, the game needs more REAL talented singers like yourself... 2013/06/03 03:15:12 +0000 Comment by mingking so awesome, nothing like layering! 2013/05/05 17:09:12 +0000 Comment by Fahmi Razorblade amazing 2013/04/17 12:38:08 +0000 Comment by Gin Seladipura asikkkkk 2013/03/29 09:54:28 +0000 Comment by gabtunes Heavenly! :-) 2013/03/27 20:00:04 +0000 Comment by adamramadhan lovely 2013/02/25 06:46:51 +0000 Comment by nadyaramly This is so dope!! I do luv ur voice, boo thang! 2013/02/20 13:28:53 +0000 Comment by Panji Singg mau berduaaaa :') 2013/02/15 14:54:04 +0000


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Most Helpful Guy
First photo00|00|0
Thanks for mhg
Most Helpful Girl
The first one00|00|0
Have an opinion?
The second girl
So close - They both appeal to me in different ways
I like the first.
1 filll blanks
2nd all the way
Sharadha ! definitely
top pik
2nd girl, she looked like an easy going person and REALLY happy! :) She's beautiful. Both are pretty girls, I am not saying the girl at the top is ugly as they look similar, I just prefer the girl on the bottom.
They look the same amount of pretty.
B I knw her , Shraddha Kapoor ryt
stop putting up women like this, comparing looks is degrading and stupid!
get a life
@blondman actually im secure, its u who is insecure to judge/rate beauty!!! sit yourself down! beautiful people dont post pics up asking for ratings like thirst monsters. you're parched


text:
By Cohost Dan
Okay guys and gals here is the recipe for my version of the Relson banana shake. As mentioned previously, Relson Gracie, who has been eating this for decades, uses about 6 bananas, cream cheese, and less protein powder. Many people have come up to me randomly and asked "cheese?". The answer is YES! put in the cottage or cream cheese. The cheese is what makes the shake especially good. I personally prefer cottage cheese instead of cream cheese as it is almost pure protein and has little fat. So here goes:
Put in a blender:
1) Two large bananas
2) Two heaping tablespoons lowfat cottage cheese
3) One scoop Vanilla protein powder (Syntha-6 is my favorite)
4) One cup cloudy (non filtered) pure apple juice.
Blend until smooth and enjoy! Sometimes I have the shake for breakfast after a hard night of training. Other times I down one prior to training for some good fuel. Either way it tastes great and has some super nutritional content.
Give it a try and let us know if you like it!


text:
On a university open day we had a visit to the pop up store
organised by the students who are doing the course I am applying for this year.
Unfortunately I couldn't get any photos, but the stores were fantastic.
From the Jewellery store I got this cute little Viva Las Vegas Ring!
Top Topshop
Jeans Customised Primark
Boots Primark
Necklace New Look
Bracelets Can't Remember
Ring Kitche In The Sky Pop Up Store
HOPEFULLY! This will be the last week of waiting for my camera!


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Kate Moss in Vogue UK February 2002
35 comments:
I was waiting for this one :) Too bad I'm not in London anytime soon... xoxReplyDelete
Beautiful shots!!! I love Kate!! She's fabulous :DReplyDelete
xx Mira
She looked so beautiul in this fabulous editorial!ReplyDelete
Don't Call Me Fashion Blogger
She is beautifulReplyDelete
Hi Natalia! I want to visit that exhibit now, I am her fan, grew up with her as one of the most famous model as she is still now. That editorial is gorgeous, made me think of summer. Have a great week, kisses! xoReplyDelete
Hi Natalia! I want to visit that exhibit now, I am her fan, grew up with her as one of the most famous model as she is still now. That editorial is gorgeous, made me think of summer. Have a great week, kisses! xoReplyDelete
cool shoot! Wish I could visit London to see more.ReplyDelete
XO, Gina
Wowww I've not words... so beautiful! She's very cool.ReplyDelete
Kisses and Have a sweet day The Indian Savage Diary
This is most definitely one of the best.ReplyDelete
Stunning editorial and amazing photos!!ReplyDelete
Passa a trovarmi VeryFP
kate moss is just so epic isn't she? :)ReplyDelete
xoxo hobovogue ღ
Wow! This photos were published over a decade ago. Time flies so fast and Kate Moss is ageless!ReplyDelete
Amazing! Thanks for comment on my blog. Greetings from Poland, xoxoReplyDelete
Agreed, Kate looks so fresh faced and youthful here, that first dress is a winner! I am always grateful for your kind comments...Have a great week ahead!ReplyDelete
Yes I heard this! Ah so wish I could attend this exhibit, but thanks for posting these Kate is becoming an icon huh.ReplyDelete
Allie of ALLIE NYC
She's such a beauty, love this editorial.ReplyDelete
xo, Yi-chia
Always Maylee
I like Kate ;)ReplyDelete
what's amazing, is I've never seen this photo shoot of her. wow, her hair was short. so stunning though!ReplyDelete
Seriously Kate Moss looks pretty amazing for 40 (20, 0 - always gorgeous) -jees fab woman. Would love to see that exhibition - wonder if I can make a trip just for that :) Happy Monday Natalia xxxReplyDelete
she's making me feel better about being 40 in a few yearsReplyDelete
Amazing. She looks so different in some of the shots! xxxReplyDelete
Beautiful pictures! She is stunning! <3ReplyDelete
New Blog Post: How To Make Someone's Day
Those pics are amazing!ReplyDelete
I think she is really beautiful :)
Svetlana from Lavender Star
Amazing pics!!ReplyDelete
I wish I could see his exhibition in London but unfortunately I'll have to miss it since I live in Israel.ReplyDelete
Anyway, kate moss is iconically beautiful and I love all the photos on this post :)
These photos are amazing! She always looks stunning!ReplyDelete
i'd be so interested to see the kate moss retrospective!ReplyDelete
I love Kate so much! <3 kissesReplyDelete
Wow, yes I agree. Those cheekbones, and those shots, super gorgeous !ReplyDelete
She is so beautiful!!!ReplyDelete
I love her eye makeup in the third photo. The colors and lighting in the first photo are absolutely stunning.ReplyDelete
The perfection !ReplyDelete


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Station spruce-up in memory of Josh, 18
THE family of a much-loved teenager, who took his own life last year, have praised pupils at his former school who have transformed Harlech train station into a memorial.
In March of last year, Joshua Llwyd-Hopcroft, 18, was tragically found dead by his own father in the fields close to his family home in Llanfair, near Harlech.
Josh's father Dion Llwyd, a team leader with Aberglaslyn Mountain Rescue Team, was left devastated by the discovery, as was the whole community.
In the months following Josh's death, the community has banded together to support the bereaved family.
One act has seen Ysgol Ardudwy, Josh's former comprehensive school, adopt Harlech Train station in the teenager's memory.
The unstaffed station, which sits just below the World Heritage site of Harlech Castle, had lost its sponsorship early last year after the station was regularly vandalised and covered with grafitti.
In a bid to pay tribute to Josh, the school took over the station and worked at turning it into a fitting memorial.
They replaced the 'street art' with a 'tribute board', replanted shrubs in the area and planted 'Josh's Tree' with a commemorative pot.
Josh's family were touched by the community's act of kindness. "It's been an unbelievably hard year for us, but the strength and generosity shown by everyone has helped us more than you can imagine," said Ceri, Josh's sister.
See this week's Meirionnydd edition for the full story, in shops and online now


text:
1. Name of the Institute: TERI University, New Delhi
2. Name of the Programme: Ph.D. (Bioresources and Biotechnology)
3. Duration: 5-7 Years
4. No. of Seats: Not Specified. Admission is subject to vacancies available in the relevant specializations.
5. Brief write-up on the Programme: The doctoral research programme in Bioresources and Biotechnology has been devised to provide a highly academic, knowledge-driven environment that will create scientific talent and innovative minds capable of applying knowledge to benefit society and contribute to its welfare. It is designed to create capacities for pursuing careers in industry by imparting a wide variety of skills to students. A collaborative inter-disciplinary effort between industry and academia is envisioned wherein manpower will be trained in accordance with the changing needs of industry.
6. Eligibility:
For Faculty of Applied Sciences:
(a) M.Sc. or M.Phil. in a relevant field or equivalent.
(b) Bachelor's Degree in Engineering or equivalent.
For Faculty of Policy and Planning:
(a) M.Sc./ M.A. / M.Phil. in a relevant field or equivalent.
(b) Bachelor's Degree in Engineering or equivalent.
Candidates, who posses a B.Tech. Degree in the relevant field or equivalent, are required to have a minimum CGPA of 7.5 on a 10-point scale or 70% marks.
7. Selection Criteria: Admissions to the Ph.D. programmes are made on the basis of a Statement of Purpose, Written Test and an Interview.
8. How to Apply: Students can submit the applications online at the university's website. Alternatively, the application forms can be obtained from the University and a hardcopy can be submitted.
9. Programme Fee: Check the following link for complete fee details:
10. Tentative Months related to Admissions: The Ph.D. admissions are open through the year. The pre-Ph.D. course work commences either in July or in January every year. Applications may be made keeping this in mind. All Ph.D. programmes at the University include mandatory pre-Ph.D. course work, in which students are required to attend courses which are scheduled in the semester.
11. Contact Details:
The Registrar,
TERI University,
10, Institutional Area, Vasant Kunj,
New Delhi – 110070 (India).
Phone No.: (011) 2612 2222, 26139110, 26139011
Fax No.: (011) 2612 2847
E-mail: email@example.com
12. URL {for Ph.D. (Bioresources and Biotechnology)}: http://www.teriuniversity.ac.in/index.php?option=com_program&task=tab&id=1885
Website: www.teriuniversity.ac.in


text:
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Conceived and designed the experiments: CL PI CCL EE NZ CM ED PS NK ML. Performed the experiments: CL PI CCL EE NZ CM ED NB MH. Analyzed the data: CL PI CCL EE NZ NB MH PS NK ML. Contributed reagents/materials/analysis tools: CL PI EE EH MZ PS NK ML. Wrote the paper: PI NK ML.
MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia 1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. elegans MIP-T3 ortholog DYF-11 is an intraflagellar transport (IFT) protein that plays a critical role in assembling functional kinesin motor-IFT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the IFT machinery, including Kinesin-II, as well as IFT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that DYF-11 functions as a novel component of IFT subcomplex B. Consistent with an evolutionarily conserved cilia-associated role, mammalian MIP-T3 localizes to basal bodies and cilia, and zebrafish mipt3 functions synergistically with the Bardet-Biedl syndrome protein Bbs4 to ensure proper gastrulation, a key cilium- and basal body-dependent developmental process. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis and further highlight the emerging role of this organelle in vertebrate development.
The transport of protein complexes and associated cargo along microtubule tracks represents an essential eukaryotic process responsible for a multitude of cellular functions, including cell division, vesicle movement to membranes, and trafficking along dendrites, axons, and cilia. The latter organelles are hair-like cellular appendages implicated in cell and fluid motility, sensing and transducing information from their environment, and development. Their biogenesis and maintenance depends on a kinesin- and dynein-mediated motility process termed intraflagellar transport (IFT). In addition to comprising these specialized molecular motors, the IFT machinery consists of large multisubunit complexes whose exact composition and organization has not been fully defined. Here we identify a protein, DYF-11/MIP-T3, that is conserved in all ciliated organisms and is associated with IFT in C. elegans. Disruption of C. elegans DYF-11 results in structurally compromised cilia, likely as a result of IFT motor and subunit misassembly. Animals lacking DYF-11 display chemosensory anomalies, consistent with a role for the protein in cilia-associated sensory processes. In zebrafish, MIP-T3 is essential for gastrulation movements during development, similar to that observed for other ciliary components, including Bardet-Biedl syndrome proteins. In conclusion, we have identified a novel IFT machinery component that is also essential for development in vertebrates.
Cilia are slender subcellular structures that protrude from the surfaces of most eukaryotic cell types, where they carry out functions associated with sensation and/or motility. Motile cilia are used for the locomotion of spermatozoa or organisms such as the unicellular green alga Chlamydomonas reinhardtii, as well as for generating fluid flow, as is the case in respiratory airways [1]. Non-motile (primary) cilia are nearly ubiquitous in multicellular organisms, and perform a wide range of sensory functions, including chemosensation/olfaction, photoreception, and mechanosensation [1]–[5]. Primary cilia are also associated with several signaling processes critical for development, including Hedgehog signaling, PDGFRαα signaling, as well as canonical and non-canonical (planar cell polarity) Wnt signaling pathways [6]–[12]. Hence, defects in ciliary structure or function affect nearly every organ in humans, and are associated with several pleiotropic genetic disorders. For example, Bardet-Biedl syndrome (BBS), Alström syndrome, Meckel syndrome, Senior-Løken syndrome, Joubert syndrome, and several cystic kidney diseases are all believed to involve dysfunction of primary cilia and/or basal bodies, the modified centriolar structures that nucleate ciliary axonemes [13]–[20].
Cilia are organelles that require several hundred proteins to support their motility and/or sensory and signaling functions [21],[22]. Of particular relevance to the present study, cilia possess a specialized microtubule-based transport system, termed intraflagellar transport (IFT), which shuttles IFT complexes bi-directionally along the axoneme and supports the formation and maintenance of the organelles [23]–[25]. The IFT particles, first observed in Chlamydomonas [26] consist of anterograde Kinesin-2 motor(s) that move cargo into the cilia and a retrograde dynein motor involved in recycling components back to the base (basal body). The molecular motors are associated with two biochemically-separable multisubunit assemblies termed IFT particle subcomplexes A and B [27]–[29]. In Chlamydomonas, IFT subcomplexes A and B consist of at least 6 and 11 subunits, respectively.
In recent years, the nematode Caenorhabditis elegans has emerged as a powerful model organism for the study of cilia and ciliogenesis. The cilia of C. elegans are non-motile and restricted to a subset of sensory neuronal cells principally localized in the head and tail of the animal [30]. While structurally similar to the canonical flagella of Chlamydomonas, C. elegans cilia emanate from a potentially more degenerate basal body (termed transition zone) and exhibit, following the doublet microtubule-containing ciliary middle segment, a pronounced extension of singlet axonemal microtubules in their distal segments [30]. Despite these differences, most, if not all, of the core IFT components identified in Chlamydomonas appear to be conserved in nematodes [31]. Additionally, several other proteins associated with and necessary for the function of the IFT machinery and cilium formation have been discovered in C. elegans, namely DYF-1 [32] DYF-2 [33], DYF-3 [34], DYF-13 [35], and IFTA-1 [36]. Like known IFT particle subcomplex A/B components, orthologs of these C. elegans proteins are enriched within the membrane-plus-matrix fraction of the recently identified Chlamydomonas flagellar proteome [37], supporting the notion that they represent conserved IFT components. In addition, research in the worm has shown that Bardet-Biedl Syndrome (BBS) proteins are themselves associated with IFT and are required to maintain the integrity of the IFT particle during transport along the cilium [4],[32]. The discovery of novel C. elegans IFT proteins suggests that the IFT machinery is more complex than suspected originally from biochemical fractionation studies, raising the possibility that additional components critical for IFT have yet to be identified.
On the basis of several bioinformatic, genomic and proteomic studies, we recently surmised [21] that the microtubule-associated MIP-T3/TRAF3IP1 protein likely represents a previously unknown but conserved ciliary protein. Here, we show that the C. elegans dyf-11 mutant harbors a loss of function mutation in the gene encoding the MIP-T3 ortholog. We demonstrate that the dyf-11 gene product, DYF-11, is a novel IFT-associated protein required for the proper assembly and function of the IFT machinery, as well as the sensory abilities of cilia. Consistent with these findings, mammalian MIP-T3 localizes to the basal body in a pre-ciliated cell and to the ciliary axoneme in ciliated cells. Furthermore, suppressing the Danio rerio (zebrafish) mipt3 gene leads to a range of developmental defects recently associated with cilia function, most prominently cell movement anomalies during gastrulation, a process under the control of the non-canonical Wnt signaling pathway [12]. Our findings therefore demonstrate new roles for the evolutionarily conserved DYF-11/MIP-T3 protein in building and maintaining functional cilia, and in developmental signaling pathways.
Several lines of evidence support the notion that MIP-T3 orthologs have a ciliary function [21]. First, MIP-T3 is found exclusively in ciliated organisms [38],[39]. Second, its expression in C. elegans and Drosophila is restricted to ciliated cells and under the control of an X box motif, which regulates genes required for ciliogenesis [35],[38],[40],[41]. Similarly, Chlamydomonas MIP-T3 (C_140070) is upregulated during flagellar regeneration, and proteomic analyses uncovered MIP-T3 in the Chlamydomonas flagellar proteome [37],[42]. MIP-T3 proteins range in size from 484 to 625 amino acids and have no recognizable domains except for a predicted coiled-coil region near the C-terminus (Figure 1A), providing no indication of their specific cellular function(s).
To test the hypothesis that MIP-T3 encodes a ciliary protein and to analyze its in vivo function, we sought to obtain a strain with a disruption in the C. elegans C02H7.1 open reading frame that encodes MIP-T3 (Figure S1). We noticed that a previously identified mutant, dyf-11(mn392), whose compromised fluorescent dye uptake is suggestive of cilia dysfunction [43], maps within a genetic interval that contains C02H7.1. Sequencing of C02H7.1 in the dyf-11(mn392) mutant strain revealed a nonsense mutation predicted to give rise to a null allele (Figure 1B). Importantly, the dye-filling defect of dyf-11(mn392) is fully rescued by introducing a transgene expressing the wild-type copy of the C02H7.1 coding region fused to GFP (Figure 1C). This confirms the cloning of dyf-11 and demonstrates the functional nature of the GFP-tagged C02H7.1 protein, which we use below for in vivo characterization. We henceforth refer to the C. elegans MIP-T3 gene ortholog C02H7.1 as dyf-11, and its gene product as DYF-11.
To determine if the dye-filling anomaly observed in the dyf-11 strain stems from structural defects in cilia that are normally exposed to the environment, we expressed GFP in the ASER amphid (head) or two PHA/B phasmid (tail) neurons using reporter constructs driven by the gcy-5 or srb-6 gene promoters, respectively; in those neurons, GFP diffuses freely to highlight the entire cell, including the cell body, axon, dendrite, transition zone/basal body and cilium, so as to permit cilium length measurements [44]. Although no morphological defects with the dendritic processes or transition zone positioning were observed, the cilia of dyf-11 mutants were truncated substantially (2.4±0.3 µm and 2.6±0.4 µm for amphids and phasmids, respectively) compared to those of wild-type animals (both 5.7±0.4 µm) (Figure 2A). These findings likely explain the dye-filling defect in dyf-11 animals and directly implicate MIP-T3 in ciliogenesis. Additionally, the observed lengths of dyf-11 mutant cilia are similar to those of IFT subcomplex B mutants.
We next used established assays [30], [45]–[47] to ask whether dyf-11 animals exhibit phenotypes consistent with cilia dysfunction, including anomalies in chemotaxis, avoidance of high osmolarity, ability to form stress-resistant dauer larvae, and lifespan.
We first compared the ability of wild-type and dyf-11 mutant animals to detect a volatile odorant, isoamyl-alcohol. Although the dyf-11 mutants are not impaired in their movement (data not shown), they show a pronounced inability to chemotax towards the attractant, indicative of an abnormal Chemotaxis (Che) phenotype (Figure 2B). In addition, compared to their wild-type counterparts, dyf-11 animals show a clear defect in avoiding moving into a solution of high osmolarity (8 M glycerol; Figure 2C), an Osmotic avoidance-abnormal (Osm) phenotype frequently encountered in ciliary mutants [30],[45]. These dyf-11 mutant phenotypes are consistent with the original observations of Starich et al. [43].
We then tested for the ability of dyf-11 mutants to enter the alternative dauer lifestage at two different temperatures (20/25°C). While wild-type larvae become dauers at these temperatures upon starvation, mutants with defects in IFT (e.g., osm-5) or the cilium-dependent insulin signaling pathway (e.g., daf-16 and daf-2) are either dauer formation-defective (Daf-d) or constitutively form dauer larvae (Daf-c) [47] (Figure 2D). We found that dyf-11 mutants are Daf-d at both temperatures, consistent with abrogated ciliary function [30],[43],[47] (Figure 2D). Lastly, we noted severe male mating defects (data not shown), which can probably be ascribed to the improper mechanosensory/chemosensory functions of cilia in the male tail [48]. For all four behavioral phenotypes observed, the defects could be rescued by expression of the GFP-tagged wild-type DYF-11 protein (Figures 2B–D and data not shown for male mating). Finally, although some ciliary mutants are long-lived [49], for example the che-11 mutant, we found no statistical difference in lifespan between wild-type and dyf-11 animals (Figure 2E).
Intriguingly, dyf-11 mutants also show, compared to the wild-type and rescue strains, a pronounced increase in Nile Red staining within intestinal cells, indicative of an increased lipid accumulation phenotype (Figure 2F). This result is remarkable, as past C. elegans screens for lipid accumulation identified only two ciliary proteins, namely the tubby obesity-associated gene ortholog TUB-1 [50] and BBS-1, which is linked to the obesity disorder Bardet-Biedl syndrome [51]. These observations, coupled with our present findings with dyf-11, suggest an evolutionarily-conserved connection between cilia and lipid homeostasis. It should be noted that since the genome-wide lipid accumulation screens were RNAi-based, it is likely that many more ciliary proteins are involved in lipid homeostasis, as RNAi has been found to be considerably less penetrant in ciliated neurons.
Altogether, our cilium length measurements and sensory behavioral analyses (Che, Osm and Daf) demonstrate that the dyf-11 mutant strain possesses prominent structural and functional ciliary defects, confirming our hypothesis that MIP-T3 plays a role in ciliogenesis and cilia function. The role(s) of DYF-11/MIP-T3 in cilia formation and function appear to be highly specific, as we have not noted any gross morphological, developmental or locomotory defects in dyf-11 mutant animals.
To directly observe whether the C. elegans DYF-11 protein associates with ciliary structures (transition zones/basal bodies and/or cilia), we generated transgenic lines bearing a translational fusion construct of the complete dyf-11 gene (with its endogenous promoter) and GFP. Fluorescence microscopy observation of the lines revealed that dyf-11::gfp is expressed specifically in ciliated (e.g., amphid and phasmid) sensory neurons (data not shown), consistent with expression patterns obtained using transcriptional GFP-fusion constructs obtained in two large-scale studies [35],[52]. Importantly, the DYF-11::GFP protein was found to be highly enriched at transition zones and within ciliary axonemes (Figure 3A, Figure 4). This subcellular localization is indistinguishable from that of other C. elegans proteins associated with IFT, including BBS proteins, newly-discovered IFT proteins, and IFT particle subcomplex A and B proteins [32]–[36],[53]. Consistent with an evolutionarily-conserved role for MIP-T3 at basal bodies and cilia, V5 epitope-tagged human MIP-T3 also localizes to the basal body in cells that have not yet ciliated, and to the ciliary axonemes of ciliated IMCD3 kidney cells (Figure 3B).
Time-lapse microscopy in C. elegans revealed that DYF-11::GFP localization is not static; the GFP-tagged protein moves bi-directionally along the length of amphid and phasmid ciliary axonemes (Figure 3A; Movie S1). Kymograph analyses show that DYF-11::GFP anterograde movement is bi-phasic, exhibiting a velocity of 0.74±0.08 µm/sec along doublet microtubules in middle segments and 1.15±0.21 µm/sec along singlet microtubules in distal segments (Figure 3A). These observations show that DYF-11 is transported cooperatively by Kinesin-II and OSM-3-kinesin in middle segments and then OSM-3-kinesin alone in distal segments, exactly as with other C. elegans IFT/BBS proteins [32],[54]. Importantly, DYF-11::GFP motility depends on the IFT machinery itself, as abrogating the IFT particle subcomplex A protein CHE-11/IFT140, which is required for retrograde transport, results in the expected accumulation of DYF-11::GFP at distal tips (Figure 3C).
Interestingly, DYF-11::GFP can be observed in several additional dendritic extensions that are not typically seen with established GFP-tagged IFT protein markers (see hollow arrowheads in Figure 3A and Figure 4). At least some of these unexpected structures appear to overlap with the cilia of the wing neuron AWC in the DYF-11::GFP-containing strain (Figure S2), which might indicate that the other unaccounted-for structures represent the axonemes of the remaining wing neurons (AWA, AWB). This unexpected observation may reflect stronger accumulation of DYF-11::GFP in the wing neuron cilia, or could be indicative of additional roles for DYF-11 in the development of morphologically distinct cilia (wing cilia have elaborate structures) [30]. Another possible explanation for the observed dendritic extensions is that they represent elongated cilia not sufficiently aligned to enter the amphid channel, a phenotype reminiscent of those recently seen in dyf-5 mutants, which have defects in IFT-kinesin-mediated transport [55].
The molecular architecture of the motor-IFT machinery has been studied in some detail, using mainly Chlamydomonas and C. elegans as model systems [25], [28], [54], [56]–[60]. In C. elegans, the motor-IFT machinery consists of at least 32 components organized into three main modules [58]: a motor module with two kinesin-2-like anterograde motors for anterograde transport (the more canonical heterotrimeric Kinesin-II, and homodimeric OSM-3) and a dynein motor for retrograde transport; another module containing two IFT particle multisubunit subcomplexes (A and B) that are separable genetically and biochemically [27]–[29],[32],[54],[58]; finally, a BBS protein complex/module that mediates the association between Kinesin-II/subcomplex A and OSM-3/subcomplex B [4],[32],[61]. Having identified DYF-11 as a novel IFT protein, we sought to characterize its spatial relationship (i.e., localization in one of the aforementioned modules) and function with respect to other components of the IFT machinery.
To test whether DYF-11 may be a component of either the Kinesin-II or OSM-3 anterograde motor modules, similar to the association of DYF-1 with OSM-3 [32], we queried whether GFP-tagged DYF-11 enters the ciliary axonemes of mutants lacking either motor (compared with Figure 4A, which shows DYF-11::GFP in wild-type animals). We found that in the klp-11 mutant, which lacks Kinesin-II motor function, DYF-11::GFP readily entered the full-length cilia produced by the redundant OSM-3-kinesin (Figure 4B). Likewise, DYF-11::GFP could be detected along the entire length of the ciliary middle segments in the osm-3 kinesin mutant, which specifically lacks distal segments (Figure 4C). These observations suggest that the assembly of DYF-11 with the motor-IFT particle machinery is not dependent on either of the kinesin anterograde motors, and leaves the possibility that it is more closely associated with the BBS protein complex or the IFT subcomplexes A or B.
We therefore analyzed the behavior of DYF-11::GFP in the three available bbs mutants (bbs-1, bbs-7/osm-12 and bbs-8). In all bbs mutants, DYF-11::GFP was distributed throughout the middle segment and the residual distal segment (Figures 4D–F). This suggests that DYF-11 is not tightly associated with BBS protein(s), since all examined BBS proteins are unable to enter cilia in animals lacking bbs-1, bbs-7/osm-12 or bbs-8 [4],[32],[58]. This is also consistent with the fact that the dyf-11 mutant cilia are distinctly shorter (Figure 2A) than those of bbs mutant cilia, which possess part of the distal segment [32],[51],[58]. Instead, the ability of DYF-11::GFP to enter the residual distal segment of bbs mutants implies that it is associated with the OSM-3-kinesin/IFT subcomplex B; this conclusion stems from the fact that in bbs mutants, all tested IFT subcomplex B components enter the distal segment in an OSM-3-dependent manner, whereas subcomplex A components are transported by Kinesin-II, and do not enter the distal segment [32] (see also schematics in Figure 4). Furthermore, DYF-11:GFP travels at a velocity of 1.21±0.19 µm/sec throughout the middle and distal segments of the cilia of osm-12/bbs-7 mutant worms (Figure 4I), indicative of an OSM-3-dependent transport process typically seen for IFT subcomplex B components in a bbs mutant background [4],[32],[58].
When combined with our finding that dyf-11 mutant cilia are truncated to the same extent as those mutants with abrogated IFT subcomplex B components such as OSM-5/IFT88, OSM-6/IFT52, and CHE-13/IFT55-57 (Figure 2A) [55],[56], the above results further support the notion that DYF-11 associates either directly or peripherally with the IFT particle subcomplex B. Previous work has shown that OSM-6 likely anchors IFT subcomplex B at the base of the cilium, possibly at transitional fibers in proximity to the ciliary membrane [56],[62]. For example, OSM-6 can enter the cilium in che-13 and osm-5 mutants, but CHE-13 and OSM-5 are excluded from cilia in an osm-6 mutant background [56]. Therefore, to further resolve the positioning of DYF-11 within the hierarchy of IFT subcomplex B, we examined the localization of DYF-11::GFP in che-13 and osm-6 mutant backgrounds. In both cases, DYF-11::GFP enters the truncated cilia, with little or no observable accumulation in the dendrites (Figures 4G,H). OSM-5::GFP, on the other hand, fails to enter the severely truncated cilia of dyf-11 mutants, showing significant leakage into dendrites (Figure 5H). These data suggest that DYF-11 may be more 'centrally' localized (e.g., closer to the OSM-3-kinesin) than OSM-5, OSM-6 and CHE-13 in the IFT particle subcomplex B, although additional studies are needed to confirm this potential protein topology.
To provide additional insight into the function of DYF-11 with regards to the motor-IFT machinery and cilia formation, we analyzed by microscopy the behavior of several GFP-tagged IFT-associated components in the dyf-11 mutant strain. In addition to the IFT subcomplex B protein OSM-5 (see above), we tested a Kinesin-II component (the Kinesin-Associated Protein 1, KAP-1), homodimeric kinesin OSM-3, a component of IFT-dynein (the light-intermediate chain XBX-1), an IFT particle subcomplex A protein (CHE-11/IFT140), and a BBS protein (BBS-7). In wild-type animals, these proteins always localize prominently at transition zones/basal bodies and clearly undergo bidirectional transport along ciliary axonemes (Figures 5A–F, and data not shown for transport rates) [31]. Remarkably, aside from OSM-3-kinesin, we reproducibly observed abnormal localization for all of these proteins in the dyf-11 mutant strain. OSM-3::GFP consistently localizes to transition zones and the truncated amphid and phasmid cilia of dyf-11 mutants (Figure 5J). In contrast, CHE-11, XBX-1, and BBS-7 were anchored to transition zones but their signal intensities were significantly reduced compared to wild-type, and none of the proteins were observed to enter the (truncated) amphid and phasmid cilia (Figures 5G, I, L). Interestingly, the heterotrimeric kinesin KAP-1 subunit mislocalized consistently along the dendrite, with no apparent anchoring to the transition zones and no observable localization to the ciliary axoneme (Figure 5K). All observations are reproducible and are based on the analysis of at least 50-100 animals, with the observer blind to the genotype and GFP-tagged protein under examination. It should be noted that the observed mislocalizations in the dyf-11 mutant animals appear to be more severe than those typically seen in IFT subcomplex B mutants, perhaps hinting at additional roles for the DYF-11 protein in IFT. Overall, our findings are consistent with DYF-11 playing a critical role in the assembly and thus functions of various IFT-associated proteins, including the Kinesin-II motor, into a functional motor-IFT machinery. Whether DYF-11 operates as an integral IFT component, or potentially as an associated 'cargo' protein that affects the core machinery, remains to be determined.
To provide in vivo evidence that DYF-11/MIP-T3 plays a ciliary role in vertebrates, we turned to Danio rerio (zebrafish), whose ortholog (LOC393572) exhibits 51% amino acid identity to its human counterpart (Figure 1A). We designed a morpholino (MO) to suppress the translation of mipt3 in zebrafish embryos, injected it at the 2-cell stage and phenotyped animals during gastrulation and somitogenesis. We and others showed recently that defects in basal body/cilia function(s) perturb non-canonical Wnt signaling, which in zebrafish manifests as a constellation of gastrulation movement defects that include shortened body axis, broad and sometimes undulated notochords, thinning and elongation of the somites, and failure of tail extension [7], [12], [63]–[65]. We found that disrupting mipt3 phenocopied the gastrulation movement phenotypes seen with other basal body/ciliary mutants [63],[65] in a dose-dependent manner (Figures 6A 6B and Figure S3A; n=100–150 embryos per injection) and with high specificity, since co-injection of a mipt3 MO with a mipt3 capped mRNA rescued >95% of the affected embryos for all phenotypes scored (n=120; Figures 6A–E and Figure S3B and Figure S4). mipt3 morphants were shorter than control-injected embryos at the same somitic age (9±1 somites) and had consistently broadened notochords (Class I), while more severely affected embryos (Class II) also had longer somites as observed by live embryo imaging and confirmed by in situ hybridization staining with riboprobes against myod, pax2 and krox20 (Figures 6C, 6D). In addition, Class II embryos also exhibited detachment of cells along the embryonic axis, probably a result of cell death whose etiology is unclear (Figure 6A), while some embryos failed to complete epiboly and/or were necrotic at the mid-somitic stage (data not shown).
To confirm that the observed defects were a result of defective gastrulation movements, we labeled a single blastomere in MO injected embryos at the 16-cell stage and followed the labeled cell populations through epiboly. As compared to control and WT rescue embryos (Figure 6E), cells converged upon the dorsal midline and extended along the axis aberrantly (Figure 6E). The observed phenotypes were quantified in somite stage embryos by a number of methods. Shortened body axes were measured via body gap angles as described previously [12]. Compared to the average control gap angle of 59°, morphant embryos exhibited a statistically significant increase of the mean angle of 86° (p<0.05; Figure S3B). Further, the broadened body and shortened body axis were quantified by an independent, objective method, where we calculated the ratio of the width of the 5th rhombomere, labeled by krox20 in situ staining, over the distance from the 5th rhombomere to the first somite, labeled by myod staining. Once again, while morphants co-injected with zebrafish mipt3 mRNA were indistinguishable from controls, injection of mipt3 MO alone caused a significantly increased ratio consistent with a wider body and shortened body axis (p<0.05; Figure 6D). Finally, quantification of the efficiency of migration of cells to the midline during epiboly also showed significant differences between controls and mipt3 morphants, a phenotype that could be rescued fully by co-injection of mipt3 mRNA (Figure 6E and Figure S4).
Importantly, MO-based knockdown of both mipt3 and bbs4, which encodes a BBS protein that localizes to centriolar satellites (proximal to basal body) and is necessary for gastrulation movements [63], led to morphogenetic phenotypes that were more severe than with the knockdown of either gene alone. Injection of subeffective doses of mipt3 or bbs4 MOs yielded observable phenotypes in <20% of embryos (n=113 and 127, respectively) (Figures 6A, 6B); by contrast, co-injection of both mipt3 and bbs4 MOs resulted in profound defects in >60% of embryos (Figure 6B), which included not only gastrulation movement hallmarks, such as prominently broadened somites, but also gross morphological defects in severely affected embryos (Figure 6A), including decreased intraocular distance as measured by pax2 expression (data not shown), that might hint at broader functions of mipt3 and bbs4. These results were corroborated by a significant increase in both body gap angles and rhombomere width to rhombomere-somite distance ratios in mipt3/bbs4 morphants as compared to embryos injected with either mipt3 or bbs4 alone (Figures 6C, 6D and Figure S3B).
Human MIP-T3 (Microtubule-interacting protein associated with TRAF3), also termed TRAF3IP1 (TNF Receptor-Associated Factor 3 Interacting Protein 1), is a poorly-characterized protein previously implicated in TRAF3 function and shown to bind microtubules [66]. More recently, Morris et al [67] found that MIP-T3 interacts with the DISC1 (Disrupted-in-schizophrenia 1) protein and is required for its localization to microtubules/centrosomes. Yet, given that MIP-T3 protein orthologs are present in organisms lacking both TRAF3 and DISC1, including B. dendrobatidis, Chlamydomonas and C. elegans (Figure 1A), we hypothesized that MIP-T3 likely also performs a more general, evolutionarily conserved function—one related to cilia formation [21]. Indeed, we now show that C. elegans MIP-T3 (DYF-11) localizes to transition zones/basal bodies and cilia, as does its mammalian counterpart (Figures 3A, B). Importantly, C. elegans DYF-11 is an intraflagellar transport protein critical for the formation of full-length, functional sensory cilia (Figures 1– 3).3). Abrogating DYF-11 function produces ciliary defects consistent with a role for the protein in the IFT process itself: (i) cilia are approximately the same length as those of mutants lacking IFT subcomplex B proteins [56],[58],[68]; (ii) four representative classes of IFT-associated components—OSM-5 (IFT subcomplex B), CHE-11 (subcomplex A), XBX-1 (dynein motor subunit) and BBS-7 (part of the BBS protein complex)—fail to enter the short, residual cilium in dyf-11 mutant animals (Figures 5G, H, I and L); (iii) remarkably, the heterotrimeric Kinesin-II motor subunit KAP-1 also mislocalizes (Figure 5K). These observations suggest that DYF-11 is not only involved in maintaining the integrity of the IFT machinery, but also may help with the assembly of Kinesin-II onto the IFT complex. Moreover, it is possible that a proportion of the C. elegans DYF-11/MIP-T3 protein associates directly with microtubules to influence the function of the IFT motors or components.
Another potentially pertinent discovery by Taya et al [69] is that the MIP-T3-interacting protein DISC1 regulates the Kinesin-1-dependent transport of a protein complex composed of NUDEL/LIS1/14-3-3ε. Consistent with a probable role in transport and neurogenesis, overexpression of a dominant-negative variant of DISC1 results in defective (shortened) neurite outgrowths [70]. Hence, one of the apparent functions of DISC1 as a cohesion factor for a multisubunit protein complex (NUDEL/LIS1/14-3-3ε) has interesting parallels to that of its interacting partner MIP-T3, which we show may function as a subunit of the IFT subcomplex B that possibly links, either directly or indirectly, a ciliary kinesin to the multiprotein IFT subcomplexes A/B (Figure 4). Whether C. elegans DYF-11 has dendrite-associated functions whose disruption could affect ciliogenesis represents an interesting question that will need to be addressed.
Several genome- and proteome-wide studies are in accord with our finding that MIP-T3 plays a critical role in IFT. The Chlamydomonas flagellar proteome uncovered by Pazour et al [37] identified the MIP-T3 ortholog (FAP116) as an abundant protein present specifically in the membrane-plus-matrix but not the axonemal fraction of cilia, precisely like other IFT proteins. Chlamydomonas MIP-T3 is upregulated >3.0 fold during reflagellation [39],[42], again similar to other IFT proteins and consistent with a ciliogenic role. Finally, just as in C. elegans, the Drosophila MIP-T3 ortholog (CG3259) is expressed exclusively in ciliated sensory neurons [38].
Our morpholino knockdown studies in the vertebrate Danio rerio (Figure 6) also suggest that MIP-T3 is likely required for basal body/ciliary functions since it is both necessary for gastrulation movements and also demonstrates a potential genetic interaction with at least one gene encoding a basal body protein (BBS4) in the regulation of this process [7],[65]. These data support the notion that vertebrate MIP-T3 plays an important role in development, potentially helping to modulate Wnt and/or other signaling pathways [12]. Intriguingly, the MIP-T3-interacting protein DISC1 binds FEZ1 [71], a protein that is implicated in neurite outgrowth and co-precipitates with BBS4 [72]. One important question raised by these observations that will need to be addressed in future studies is whether vertebrate MIP-T3 performs functions related to trafficking in neurites, possibly in cooperation with DISC1/FEZ1. If so, then such a finding would suggest that MIP-T3 may have been adapted during evolution to perform two distinct roles in protein trafficking, one in cilia as an IFT protein, and the other in neurites (axons and perhaps also dendrites).
All C. elegans strains were maintained at 20°C, and standard genetic crosses were employed to introduce GFP reporter constructs (transcriptional or translational) into wild-type (N2) or mutant animals. PCR or dye-filling assays were used to follow genotypes, as described [36]. The following mutant strains were used in this study: bbs-1(ok1111), bbs-7/osm-12(n1606), bbs-8(nx77), che-3(e1124) che-11(e1810), che-13(e1805), daf-2(e1370), daf-16(mu86), dyf-11(mn392) klp-11(tm324), osm-3(p802), osm-5(p813), and osm-6(p811).
A translational DYF-11::GFP fusion construct was made by fusion PCR as described [53]. The entire genomic coding region of dyf-11 (C02H7.1), along with 528 bp of promoter sequence 5′ of the start codon, was fused upstream of, and in frame with, the GFP coding sequence. Aside from DYF-11::GFP, the following strains were used: dpy-5(e907); Ex[gcy-5p::gfp+dpy-5(+)] dpy-5(e907); nxEx[osm-12::gfp+dpy-5(+)], N2; myEx10[che-11::gfp+rol-6(su1006)]; N2; Ex[kap-1::gfp+rol-6(su1006)], N2; ejEx1[osm-3::gfp+rol-6(su1006)], N2; yhEx2[osm-5::gfp+rol-6(su1006)], N2; Ex[srb-6p::gfp+rol-6(su1006)] and N2; nxEx[xbx-1::gfp+rol-6(su1006)].
A tagged expression construct for MIP-T3 was generated by LR clonase II (Invitrogen) mediated recombination between the pENTR 221-MIP-T3 (Ultimate ORF clone IOH28851; Invitrogen) and pcDNA6.2/nLumio-DEST (Invitrogen), placing the human MIP-T3 ORF under control of a CMV promoter with an N-terminal V5 epitope tag (pcDNA6.2/nLumio-MIP-T3).
IMCD3 cells were plated on glass coverslips and transfected with the pcDNA6.2/nLumio-MIP-T3 vector when cells reached 60% confluency by using FuGENE6 (Roche) transfection reagent. Twenty-four hours post-transfection, cells were fixed in methanol and stained using mouse anti-V5 (1200, Invitrogen), mouse anti- γ-tubulin or mouse anti- acetylated-tubulin (both 11000, Sigma), and secondary detection carried out with goat anti-mouse IgG antibody conjugated to Alexa 488 dye, and goat anti-mouse IgG antibody conjugated to Alexa 594 (both 11000, Invitrogen). Cells were visualized by fluorescence microscopy.
The C. elegans MIP-T3 gene homolog, C02H7.1, is physically situated close to the interval defined for the dyf-11(mn392) mutant allele (X: −18.27±0.244 cM) [43], suggesting that the genetic lesion lies within this gene. We sequenced the C02H7.1 coding region in the dyf-11 mutant and uncovered a nonsense mutation (TCA→TGA) in the third exon at nt 419 of the coding region. Primer sets used to detect the mutation were: OPS0320 TGGTCGCAATTTGACCACC and OPS0322 TGATCATTCTCGGGCTCTC (fragment 1); OPS0321 GACGATCATGAGATTTCTG and OPS0323 CAACATATTGGTGCAACTTC (fragment 2). A second putative dyf-11 allele, ad1303, had no sequence alterations in exons and complemented the dyf-11(mn392) mutation, suggesting that it represents a different gene.
Dye-filling assays using the fluorescent dye DiI were performed as described [53]. Chemotaxis assays were carried out for 1 hour essentially as described [46], using isoamyl-alcohol as a chemoattractant. A chemotaxis index was calculated as the number of worms in attractant zone minus worms in control zone, divided by the total number of worms. Osmo-avoidance assays were performed as described [46]. Briefly, ~5 worms (for each of at least 20 assays) were placed inside a small ring of 8 M glycerol, and animals found within or beyond the ring after 10 minutes were counted as non-avoiders. An osmo-avoidance index was calculated: (total avoiders–non-avoiders)/total worms.
Lifespan assays were based on the protocol of Apfeld and Kenyon [49]. Animals were grown for at least one generation at 20°C before eggs were collected. At the L4 molt, worms were transferred to NGM plates containing 16 µM fluorodeoxyuridine (FUDR) to prevent progeny growth and kept at 20°C throughout the assay. 100 worms were picked for each strain, at 10 worms/plate. Worms were scored every 1–2 days for viability; those no longer responding to prodding with platinum wire were considered dead, and those that exploded or crawled off the plate were censored.
To test for entry into and exit from the dauer stage, we employed an existing strategy [73]. 10 adult worms were allowed to lay eggs on plates with food at 20°C for 4 hours. Adults were then removed and eggs counted. Eggs were allowed to develop for 4 days at 20°C or 3 days at 25°C, after which they were scored as either Daf-c or Daf-d as follows. To identify Daf-c worms, plates were flooded with 1% SDS, where only dauer larvae remained as live thrashing animals after 15 minutes. To identify Daf-d worms, animals were allowed to grow 4 days following complete consumption of food, after which they were exposed to and unable to survive the 1% SDS treatment. daf-16 (mu86) and daf-2 (e1370) were used as Daf-d and Daf-c controls, respectively. All dauer/lifespan assays were carried out in duplicate or triplicate.
Nile Red staining was performed as previously described [50]. Briefly, Nile Red powder (Molecular Probes) was dissolved in acetone as a 1mg/ml stock solution and kept at room temperature. The stock solution was diluted in 1x PBS to 1 ug/ml and 0.5 ml of diluted solution was applied to NGM plates seeded with E.coli OP50. Plates were allowed to dry for 24 hours. Staged eggs were allowed to develop on Nile Red plates at 20°C. Worms were transferred every 2 days to fresh Nile Red plates and were analyzed two days post-L4 by fluorescence microscopy. Images were captured and processed under identical settings using OpenLab software (Improvision, Inc). Nile Red fluorescence intensity was calculated as the mean pixel intensity after background subtraction.
ASER amphid and PHA/PHB phasmid sensory neuron structures were visualized by expressing the cell-specific transcriptional reporters, gcy-5p::gfp and srb-6p::gfp [36], respectively. In these neurons, the GFP diffuses freely throughout the neuron to mark cell bodies, axons, dendrites, transition zones and cilia. Cilium length was measured from the distal end of the transition zone (visible as a 'bulge' of fluorescence) to the distal tip of the cilium.
Transgenic animals expressing GFP-tagged proteins were mounted on agarose pads and immobilized with 20 mM levamisole. Amphid or phasmid cilia were examined with a 100X, 1.35 NA objective and an ORCA AG CCD camera mounted on an Zeiss Axioskop 2 mot plus microscope. Images and movies were obtained in Openlab version 5.02 beta (Improvision). Kymographs were generated using the MultipleKymograph ImageJ plug-in. Rates from middle and distal segments were obtained essentially as described in Snow et al [54].
Images for intraflagellar transport were collected using a Zeiss Axiovert 200 equipped with a Hamamatsu Orca AG CCD camera, spinning disk confocal head, Zeiss Plan-neofluar 63X, 1.3 NA, water-immersion objective and a 1.5X magnification lens Improvision Piezo Focus Drive. Images for MX488 bbs-7(n1606); Ex[dyf-11::GFP+dpy-1(+)] and MX486 N2; Is[dyf-11::GFP+dpy-1(+)] were collected at 7.5 frames/sec and 4.24 frames/sec respectively. Animals were first anaesthetized with 10 mM levamisole, mounted on agar pads and photobleached for 300–1900 ms before images were collected for 2 minutes. The FRAP module is a Photonic Instruments MOSAIC Digital Diaphragm System with a 488 nm 300 mW laser line. Images were collected using Volocity.
We obtained a translational blocking mipt3 morpholino (MO) from Gene Tools Inc (5′- ACCGATTCGTTCATGGCATCAAACC-3′). The MO was diluted to the desired concentrations in deionized, sterile water and injected into 2-cell stage embryos as described [64]. To rescue the morphant phenotypes, we amplified the open reading frame of zebrafish mipt3 and cloned it into the pCS2+ vector, from which we transcribed RNA using the SP6 mMessage mMachine kit (Ambion). Phenotypes and imaging were performed as described [64].
In situ hybridization, monitoring of gastrulation movements, and measuring of embryo body gap angles were carried out using previously described methods [12]. Measurements of rhombomere width and rhombomere-somite distances were taken on flat-mounted embryos photographed at 10X magnification. Intraocular distance was taken as the measurement of the width of the pax2-expressing region, labeled by ribostaining, and was measured on flat-mounted images photographed at 10X. All experiments were performed blinded to the injection cocktail.
Time-lapse microscopy (4 frames/second) of C. elegans DYF 11::GFP seen moving bi-directionally along amphid cilia, as with other IFT proteins.
Amino acid alignment of C. elegans DYF-11 (C02H7.1) with MIP T3 protein orthologs from C. reinhardtii and H. sapiens. Identical and similar residues are highlighted in black and gray, respectively. Note that the major difference in size between the human and C. elegans/Chlamydomonas proteins are due to two large insertions found in the human protein sequence. The number of residues is displayed at the ends of the sequences. Caenorhabditis elegans DYF-11/MIP-T3: C02H7.1; Homo sapiens MIP-T3: AAF76984; Chlamydomonas reinhardtii MIP-T3 accession number: C_140070.
Fluorescence images indicating the possible presence of DYF 11::GFP in the distal segments of AWC neuron cilia. Two of the highlighted extensions (hollow arrowheads) from the DYF-11::GFP protein overlap with the RFP protein that is most highly expressed in AWC cilia. Two 'branches' of the AWC cilium are shown with arrows, and the bundle of amphid channel cilia are pointed to. The schematic shows the ultrastructure of the AWC cilium, as visualised in Perkins et al. (1986). Images were acquired in the strain OE3657 dpy-5(e907) I; dyf-11(mn392) X; nxEx[C02H7.1::gfp; dpy-5 (+)]; ofEx457 [odr-3::rfp; elt-2::cherry].
Gastrulation phenotypes in mipt3 morphant embryos. (A) Injection of a progressively increasing amount of a translation-blocking mipt3 morpholino (MO) gives rise to a spectrum of gastrulation phenotypes, including shortening of the embryonic axis, broadening and kinking of the notochord, lengthening of the somites and detachment of cells along the embryonic axis. The presence of two of these phenotypes is scored as "Class I", whereas three or more phenotypes are categorized as "Class II". (B) Body gap angle measurements for mipt3 and bbs4 morphants. The gap angle of mid-somitic embryos (nine somites +/− one somite) as defined by the angle formed by triangulating three points (tip of head, tip of tail, center of yolk; see also Gerdes et al., 2007) was calculated to capture the mean length of embryo populations (n=50–70 embryos). On the y-axis, the angle is plotted (in degrees) while the x-axis shows the various injection cocktails. The phenotype is rescued efficiently by co-injection of capped mipt3 mRNA. Note the significantly shorter embryos in the mipt3+bbs4 double morphants. Data were calculated blind to injection cocktail; bars depict standard error.
Quantification of gastrulation movement defects during epiboly. The mean width of fluorescein-positive region was measured across each time-point assayed in nine embryos per category (control, mipt3 morphant, and mipt3 rescue). Asterisks indicate statistically significant differences (p<0.05) between morphants and controls or rescued embryos; the latter two were indistinguishable from each other.
We thank Leon Avery and Joseph Dent for providing us with the ad1303 allele, and T. Stiernagle and the C. elegans Genetics Center for providing strains. We also thank Mathieu Bakhoum, Chrystal Inglis and Harald Hutter for assistance with the project.
The authors have declared that no competing interests exist.
This work was supported by the March of Dimes (to MRL), NIH grants HD042601, DK072301 and DK075972 (to NK), F32 DK0799541 (to EED), as well as the Swedish Research Council (VR) and the Swedish Foundation for Strategic Research (SSF) (to PS). MRL holds Michael Smith Foundation for Health Research (MSFHR) and CIHR scholar awards. PNI holds MSFHR and NSERC post-graduate scholar awards, and MPH holds a MSFHR scholarship.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Mercure Florence CenterMid-scale hotel for business trips or leisure
An elegant, stylish 4-star hotel, equipped with all modern comforts, awaits you in the heart of Florence. A short walk from the cathedral and the Santa Maria Novella train station, amidst culture, art and streets full of stores. A contemporary Lounge Bar, Internet zone, complimentary WIFI, a modular meeting room and 4 luxurious Suites complete the services we offer.
The Mercure Firenze Centro hotel is ideal for a romantic stay in Florence. The cathedral with its majestic Baptistery, the Medici Chapels and the romantic Ponte Vecchio bridge are just some of the treasures located just a few minutes' walk from the hotel.
- Paying car park adjacent to the hotel
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This hotel participates to Planet 21 and is acting in favour of Positive Hospitality.
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Online check-in
Online check-in: Your arrival will be carefully prepared by our team
Fast check-out: Simply return your key
WIFI/Internet Access
- WIFI at the hotel
- High-speed Internet
- 2 Internet points
- WIFI in your room
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- Public indoor parking (Nearby)
Accessibility for people with reduced mobility
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- Small pets are allowed for a supplement
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Via Nazionale 21/23 R
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- AMERIGO VESPUCCI At 6.4km / 3.98 miles
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Stylish Room with double bed. Minibar, safe, tea/coffee making facilities, complimentary WIFI, full HD LCD TV with USB port for smartphones and camcorders. Bathrooms with walk-in shower or bathtub, magnifying mirror. Some rooms have a panoramic balcony.
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Stylish Room with queen size bed. Minibar, tea/coffee making facilities, complimentary WIFI, LCD TV with USB port for smartphones and camcorders. Bathrooms with walk-in shower or bathtub and magnifying mirror. Some rooms have a balcony.
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Soundproofed Superior Room with comfortable twin beds and a sofa bed. Minibar, safe, tea/coffee making facilities, complimentary WIFI, full HD LCD TV with USB port. Bathrooms with walk-in shower or bathtub and magnifying mirror. Some rooms have a balcony.
Deluxe Suite with bedroom and zen lounge
Soundproofed Deluxe Suite equipped with all modern comforts: bedroom and lounge with sofa, double full HD LCD TV with USB port, minibar, safe, tea/coffee making facilities, Nespresso machine, free WIFI. Bathrooms with walk-in shower and magnifying mirror.
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Soundproofed Superior Room with comfortable king-size bed and a sofa bed. Minibar, safe, tea and coffee making facilities, free WIFI, full HD LCD TV with USB port. Bathrooms with walk-in shower or bathtub and magnifying mirror. Some rooms have a balcony.
- 85 rooms
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Enjoy our gourmet snacks and relax at our Bar Apollo with our selection of cocktails, aperitifs and carefully chosen wines. WIFI available at all times.
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Our guest reviews for Mercure Florence Center
100% genuine reviews from our guestsFind out more
It's a great hotel in in impeccable location. However, despite informing the hotel ahead of time of my slightly earlier arrival time (noon to 1pm), I was made to wait for almost two hours for my room as other guests who arrived MUCH LATER than I were given their rooms with no waiting. I was only given a room (and still after waiting for quite a while) after I pointed out to the original staff who served me the very discouraging and unpleasant wait as I watched other guests streaming to their rooms. I don't understand how the hotel system worked and what the point was for asking for my arrival time if my response was completely ignored. Other than that, the room was fantastic, and the other staff were efficient and knowledgeable.
Good location, nice superior room, well trained reception staff. There is only one moment for improvement - there is no available ice machine on the floors (during summer time in Florence with +38 outside it would be very logic).
If you do not want old world Italian charm and prefer a new and modern hotel close to the train station, this is the one for you. Rooms are very large and comfortable. Hotel is very modern and clean. Staff is friendly and very helpful. 10 pot of 10. Stayed here several times and I will be back. Plenty of restaurants, Gelato shops and a very nice supermarket is only 50 yards away.
Concierges are very kind. My room is very wide and comfortable.
We have a most lovely time and enjoyed every minute of it..
Very positive impression, central location close to railway station, well trained staff. Superior room was comfortable enough. There is only one thing was missing - there is no ice machine at the floors, which could be very useful in Italy during summer time with +38 temp. outside
Dear Traveller, I am happy to read you appreciated the quality level of service of our staff, the Superior room, and finally you enjoyed your holiday in Florence. The ice machine is not a Brand standard in Italian hotels, for this reason our Bartender is trained to provide for it upon request. Looking forward to welcoming you again in our hotel I remain, Yours faithfully Lucia Padovan _ General Manager
The location of the hotel is perfect for me. It's so close to the central station and all the attractions. You can travel around very easily. Lots of restaurants and stores nearby, easy to grab some food. Nice staffs, very friendly and helpful.
This was my second time to stay in this hotel. The first time was end July to early August 2016. The experiences for my stays in this hotel were very good. The hotel room is big and clean. Services are very good especially the online check in. We arrived at about 1PM and got our room immediately. Actually, many other Accor hotels cannot do this although the return email from online check-in stated that the room keys will be available upon the guests' arrival for the accor hotel Gold Members. We hope that when we arrive at the same time next year, we can have a room with balcony. The only thing that needs improvement is breakfast. The quality is good but not yet reached value-for-money.
We enjoyed our stay at this hotel in Firenze. The staff was very nice and welcoming. The only downside was the occasional honk with the horn outside our room by impatient italian drivers in the morning but that was only a minor nuisance
It was a great experience but as I´m allergic, i had to took medicines because of the carpet. It´s important that the hotel could offer rooms to allergic person
Everything was wonderful and better than we expected EXCEPT ants in the room even if room exchange eventually.
location is perfect, service of staff also perfect, room conditions perfect too
We had nice stay at this hotel where it is located very near main railways station in Florence. It made our travel to other cities also very convenient. Room is clean, nice and big with balcony to enjoy the outside view.
Great service. My daughter left a pair of fairly expensive pants in the closet of our previous hotel in Rome. Since she was continuing on to stay with a family for 3 weeks and had minimal clothing with her, she really needed them. The concierge at the Mercure called the other hotel for us and had them locate pants and call the courier for a quote.
Need more choices for the breakfast. Too limited. The staff was great.
Very nice and helpful staff at front desk, helped us with our luggage and gave information about things to do around Florence. Situated very conveniently for us as it was close to the train station and had many nearby restaurants. The breakfast was however a little limited in options. The carpet was a bit dirty in our room. Overall very nice experience.
I stayed here just a day before my return flight from Firenze air port. I think 3rd time to stay here. The room was clean and big enough.
Overall very nice hotel and lovely stay. Location is very central, room clean and spacious. Receptionist on check-in was very helpful -she recommended places to go and eat. Only minor negative is room faced out to main street so got noise from bins being emptying and once they forgot to replenish our coffee & cups! Would happily return.
Is a hotel with good infrastructure and well located. Good service
When making a booking at this hotel:
- I earn Status & Rewards points
- I do not earn points
- I can easily convert my HuaZhu Rewards points into Le Club AccorHotels points (and vice versa)
- I can use my Rewards points
- I cannot use my points
- I benefit from {{fidelityCard}} advantages
- I benefit from advantages
- I do not benefit from advantages
- I can take advantage of the Members' rate
- I cannot take advantage of the Members' rate


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Lunch with the Ministers
In Conversation With - Steve Melhuish
Latest News
Ministry of Manpower sets up email helpdesk for Australians seeking to work in Singapore
The Ministry of Manpower have now launched their dedicated help desk for Australian businesses. This is part of the updates to the Comprehensive Strategic Partnership announced last year. You can contact the help desk via email firstname.lastname@example.com or call +65 6871 6047 to inquire about the various pass types and for information on the status of work pass applications.
Lunch with the Ministers
Tue 22 Aug 2017 | 11.30am- 2.00pm | John Jacob, The St Regis Singapore
Australia's Minister for Trade Hon Steven Ciobo MP and Minister for Defence Senator the Hon Marise Payne will be visiting Singapore with a delegation of Australian businesses next week. Join us for a special lunch event on Tuesday 22nd August to hear their plans for the future of trade and defence between our two countries and a panel discussion. .
In Conversation With - Co founder of PropertyGuru
Mon 11 Sep 2017 | 5.30- 7.30pm | Club Co
In Conversation With series provides a community forum where members and guests gather to hear engaging and enlightening conversation. Speakers will present on meaningful topics in subject areas like science and technology, leadership, communications and marketing, diversity, futuristic workspace and adventures.
Special thanks to GEMS World Academy ( Singapore ), our presenting sponsor.


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Main section
Sunday 5 February 2006
399BC Socrates speaks to jury at his trial: 'If you offered to let me off this time on condition I am not any longer to speak my mind... I should say to you, "Men of Athens, I shall obey the Gods rather than you."'
Henry Porter: A few bad cartoons are no reason to fall out I thought I knew exactly where I stood on freedom of speech. But the furore over the depiction of Muhammad raises issues even passionate rationalists must reconsider.
Mary Riddell: The traditional notion of victor and vanquished is no more, leaving the army caught in a no man's land between politician and terrorist.
Andrew Rawnsley: When voices on the right shriek and snarl that he is betraying Conservatism, this helps to define him as modern and moderate.