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A Tasmanian yachtsman has designed what he believes to be the most effective danbuoy system ever invented and is looking for buyers, or even companies to produce his patented design.
Believing the current available danbuoy launch systems to be useless, both as a cruising yacht's emergency aid in a man overboard (MOB) situation and even under the international yacht racing rules where such systems have to be simple and quick; Green believes every yacht racing is, therefore, illegal!
"The present prescribed danbuoy system is too slow to activate with a crewmember having to get to the stern rail, detach and throw," Green states.
"The current requirements state such recovery items must be in easy reach of the helmsperson but, as we know, this is impracticable.
The immediate response by the helm is to stop and turn the boat back towards the MOB.
"In a panic situation and adverse weather, it is very risky trying to get a crew member to the stern rail and even if a crew does succeed, by this time the stern will, most likely, be pointing in the opposite direction!"
So what is Green's solution? To incorporate the acronym KISS: keep it simple stupid.
A danbuoy tethered to a life buoy and self-igniting light that is instantly released from any predetermined location on the vessel within immediate reach of helmsman or other designated postions such as the navigator.
At first call of 'man overboard', a danbuoy system must be fully deployed in seconds. In addition, the danbuoy must be visible from a distance, better than a flag on a stick.
Green's MOBbuoy is a system he designed, built and fitted to his own boat. It is fully deployed within four seconds upon activation, floating a flag two metres above the water and connected to a lifering.
All it takes is the tug on an easily grasped handle that can be led to any predetermined position on the boat. A victim falling from the bow can have a fully-deployed system to hand by the time he clears the stern. This reduces the crew panic to maintain MOB watch, thus enabling the helmsman and crew to take controlled action under less stressful circumstances.
When Cruising Helmsman visited Peter to view the MOBbuoy in action, unfortunately he had sold his yacht so we hand-trialled the deployment off a wharf at Derwent Sailing Squadron.
Being home-built the trial product is roughly made but could easily be tidied up when under production. All the parts of the danbuoy are contained in two differnt sized tubes fixed together. The larger tube contains the ballast and spring mechanism and the thinner tube contains the flagpole on a spring mechanism.
The unit stands about 1.5 metres tall and slots into a bracket fixed to the transom pushpit. A pin at the bottom of the tube stops the MOBbuoy from deploying. Simply by attaching a pull cord to that pin and then routing it to wherever you think it is needed, the MOBbuoy then becomes instantly deployable by anyone.
On his own yacht, Green had his unit deployable from both the helm station and from the navigator's table. Simply clever.
When the pull cord removes the holding pin the weight drops out of the tube and pulls the whole unit out of its bracket. This makes the flagpole extend out of the top of the unit displaying its flag.
Other accessories can be added to the unit: tie a line to a horseshoe ring; include a water-activated light or AIS/GPS unit; rig an unfurling V-sheet. Or even attach other safety equipment for the MOB such as a whistle, radio or torch.The important thing is the MOBbuloy is active in less than five seconds and the boat crew can concentrate on getting the boat back and ready for retrieval.
Peter Green is keen to see his idea installed on boats everywhere, "at this stage I can manufacture these units to order but I would like somebody to pick up my idea on a more professional basis."


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Jakarta: Pakistan captain Mohammad Rizwan pleaded with the Pakistan Hockey Federation not to come hard on his team following their 1-2 loss to India in the bronze medal match of the 18th Asian Games on Saturday.
Japan later rallied to beat Malaysia in a thrilling shoot-out 4-2 to win the gold. Both teams were tied 6-6 at the end of the regulation time.
"They should not be doing any chopping and changing on the basis of this result. It is my humble request to the federation," said a dejected looking captain, adding that his team played badly only in the semi-finals and whatever performance everyone has seen can be credited to the new coach Roelant Oltmans.
"We all want Oltmans to stay so that we can have a strong team for the World Cup. We will be a strong team by the World Cup if we have the same coaching team.
Akashdeep Singh put India in the lead as early as in the third minute. Akashdeep, after receiving the ball from just outside 23 yards, scooped it into the top right corner past a rooted Pakistan goalkeeper Imran Butt.
Chinglensana Singh and Mandeep Singh had chances to widen the lead but a resilient Pakistan defence thwarted their efforts.
Trailing by a goal, Pakistan pressed hard for the equaliser in the second quarter. Forwards Ajaz Ahmad, Muhammad Dilber and Abu Mahmoud all created chances but failed to get the break through. The first penalty corner for Pakistan came in the 21st minute but was neutralised by India. They were awarded two more penalty corners in the same minute but again they failed to make it count.
Indian strikers SV Sunil and Akashdeep had few more scoring opportunities but failed to double the lead. Pakistan upped their tempo further in the third quarter but attempts from Ahmad and the experienced Mohammad Irfan failed to bear fruit.
India's first penalty corner came in the 50th minute, thanks to fine stickwork by Mandeep Singh and the young Harmanpreet Singh made it count with a clinical strike. The 2-0 lead didn't last long, however, with Mohammad Atiq firing a brilliant field goal catching the India defense napping on a counter. Rupinder Pal Singh then missed the target off a penalty corner and Pakistan were still in with a chance to draw level. They mounted tremendous pressure with a series of raids but Indian defence stood tall to defend till the final hooter.
"This wasn't the medal for which we came here. But something is better than nothing and we are happy that we are not going back empty-handed. We fought each and every moment of the game for this," said coach Harendra Singh. "Playing against Pakistan has always been motivational and I am happy that we could get our record straight against them," he added.
Indian women's squash team got a second successive silver medal as they lost to Hong Kong 0-2 in the final. The Indian women's team of Joshana Chinappa, Dipika Pallikal, Sunayna Kuruvilla and Tanvi Khanna, lost their first two singles matches.
Sunayna lost 1-3 (8-11, 6-11, 12-10, 3-11) to Ho Tze-Lok as Hong Kong led 1-0 in the final. Sunayna had defeated Tze-Lok during a Pool B clash but in the final, the 19-year-old was unable to deliver under pressure. Sunayna was fielded ahead of experienced Dipika due to the latter's physical toll during Friday's win over Malaysia in the semi-finals.

subset: commoncrawl


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Before going any further, it's important to begin by saying that the cost of maintaining and running digital signage is much less than you can imagine, on many fronts. For example, just consider all those digital signs in malls, in storefront windows and on the side of buildings in major cities around the world. Can there be a more effective and less expensive form of local advertising than a sign like the Bromley and Co Art Gallery in the storefront windows on Chapel Street in South Yarra?
Featured artists are displayed on huge digital signs in the gallery street-facing windows so that they can entice passers-by into the gallery to peruse the many artists showing there. With that said, that's just one aspect of digital signage that allows you to understand that just a few sales can more than compensate for the cost of the signage, running it and maintaining it. However, that's just the beginning!
Comparison of Hard Copy Signage in Retail
Now let's look at restaurants and stores that traditionally posted sales events on billboard-type signage. It involved a person getting up on an extension ladder to physically change the letters and numbers on those types of signs or posting huge paper signs as often as events or price specials changed. The labour and cost of materials alone are significantly more than the cost of digital signage that can be controlled and changed remotely through proprietary software.
In this light, retail digital signage is so much more cost-effective than ongoing costs to accomplish those tasks mentioned above. If you calculate the printed materials being posted and the cost of labour every time an event or menu changed, it would be far less expensive to purchase digital signage outright. A person sitting at a computer for a few moments to change signs is all the ongoing costs of operating digital signage involves.
Indoor Kiosks in Retail
Not all digital signage is for adverts. Some types are actually functional such as the kiosks in fast food restaurants that allow customers to order from the kiosk and then simply wait until their 'number' is called. As an aside here, although these aren't retail applications, many government and medical offices are using digital signage as a check-in point. It significantly lessons the amount of contact people have with staff and is much more efficient because staff can check in when they are ready to move on to the next person waiting and the process is so much faster.
The same holds true in retail. Digital signage is used to check into Customer Service for questions, tech support or even refunds. Sometimes retail stores take orders online to be picked up at the store. At the appointed time, the customer steps up to the digital signage check-in kiosk, a warehouse worker brings the item or items out, and all is accomplished in moments. This was particularly useful during covid because it limited contact, mitigating the spread of the disease. You can't quantify the savings in terms of health and worker efficiency.
Hidden Costs in Traditional Signage
This brings up a very good point when it comes to all those otherwise hidden costs in traditional retail signage. When it comes to advertising such as the window signage mentioned above, consider how much it would cost to have flyers and mailers printed whenever a new artist is featured or a new sales event is set to begin. Even if you could do your printing at your place of business, bypassing a printing company, the cost of paper, ink, and labour alone would be a tremendous expense.
Then there's the hidden cost to the environment you might not be aware of! All those trees being cut to manufacture paper or cardstock and the carbon footprint of manufacturing paper needs to be calculated as well. Sustainability is a key way in which we, as business owners, can reduce the cost to our planet – the only known inhabited planet in the universe. Digital signage is most often LED signage, which uses light-emitting diodes that utilize a low power consumption. That's a 'cost' you should be aware of as well.
The Final Word
The expected lifespan of digital signage is at least ten years if well maintained. In fact, maintenance is rarely needed unless the display sustains some sort of unforeseen damage from passers-by, customers or even weather events. In cases like that, however, insurance usually covers the loss.
Printed signs last a short time before being obsolete, as in the case of finite events, and outdoor printed signage lasts only as long as it can be protected against UV 'bleaching.' If you want to consider the cost of buying, maintaining, and operating digital signage in retail, it's the most cost-effective alternative available. That's a fact.


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Long-trailed mining claim against Poland gets underway
An Australian mining company has made good on its threat to file a third-party funded treaty claim against Poland, over its alleged obstruction of two coal projects.
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Boxing Day, 2010.
Boxing Day... the day after Christmas. Even when I did celebrate what most (white) Canadians call Christmas I was never sure what Boxing Day was about or how it fit into the religious angle most of the other consumerist debauchery was decorated with to make it somehow ok spiritually. (Like, greed is ok if it's greed in the name of Baby Jesus. Or something.) If Christmas celebrates drinking... I mean, the birth of Christ... Not that it does but way back the church needed a special day (like a sale) for the pagans to party. How else to get those heathens into the "sphere of their control" (Read: God) and make good Christians out of them? It just so happened that Dec 25th was a day the pagans already celebrated with much debauchery and joy so it was a convenient date to make Christ's birthday party.
But where does Boxing Day fit in?
It seems to be all about SHOPPING!!!!
I don't understand that.
I don't understand a lot.
This blog will soon demonstrate that clearly enough.
I'm Gordon Gustav and you are not.


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Metropolitan Baking Co., a Hamtramck-based bakery that supplies fresh and frozen bread products to restaurants, universities, institutions, stadiums, and retail establishments across the U.S., has recently expanded its operations to more than 110,000-square-feet on 20 acres of land.
Metropolitan has been making virtually every kind of bread, bun, and roll product — more than 200 of them — since 1945. The company provides the hot dog and hamburger buns at Comerica Park in Detroit, select buns at Yankee Stadium, as well as dozens of other major league and minor league ballparks from coast to coast.
In addition, Metropolitan services schools from kindergarten to universities, hundreds of independent grocery stores, well-known names like Walmart, Kroger, Sam's Club, and restaurants including some of the largest national chains and popular local spots.
"Hamburger and hot dog buns aren't our only products, not by a long shot," says George Kordas, CEO of Metropolitan Baking Co. "But they pretty much take center stage this time of year when families are in their backyards grilling. We expect to produce millions of buns during the upcoming summer season. In fact, you'll even see our Kordas' brand hotdog buns at Walmart stores across the country this summer."
According to the National Hot Dog and Sausage Council, Americans will consume 7 billion hot dogs between Memorial Day and Labor Day. That's 818 hot dogs consumed by people every second of the traditional summer season.
While the market for coney buns and other bread products continues to grow, Kordas cautions that there has been a perfect storm with a pandemic induced supply chain crunch, labor constraints, bad weather out west, and the war in Ukraine.
Still, Metropolitan is planning to expand its facility in Hamtramck to add 20-30 percent more capacity and 30 employees in the near future to increase efficiencies and continue to remain competitively priced while meeting growing customer demand.
"We're optimistic about the future," says Kordas. "This is not a business where you get ahead by standing still. If you're in Michigan, the chances are that you've recently enjoyed one of our products. We intend to keep it that way."


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Game Vitals:
When: January 18, 7 p.m. EST
Where: Little Caesars Arena, Detroit, MI
Watch: Fox Sports Detroit, NBA League Pass
Odds: Det -2.0
Projected Lineups:
Detroit Pistons (19-24):
Reggie Jackson, Bruce Brown Jr., Reggie Bullock, Blake Griffin, Andre Drummond
Miami Heat (21-21):
Justise Winslow, Tyler Johnson, Rodney McGruder, James Johnson, Hassan Whiteside
Pregame Reading:
No Josh Richardson tonight for Miami:
No Josh Richardson tonight for Heat, due to illness. Wayne Ellington over his illness and available, per @DavidFurones_ Also, Tyler Johnson available after missing practice due to illness Thursday.
— Ira Winderman (@IraHeatBeat) January 18, 2019
DBB Preview, courtesy of yours truly.
Inside the Cylinder goes inside all Dennis Smith Jr.-to-Detroit trade rumors.
Tanking is not the answer to any of the questions the Pistons are asking themselves.
The Pistons defense is trying very hard to give up as few threes as possible - are they trying too hard?
Let's have a good one tonight, y'all.


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Marcus Yallow is no longer a student. California's economy has collapsed, taking his parents' jobs and his university tuition with it. Thanks to his activist past, Marcus lands a job as webmaster for a muckraking politician who promises reform. Things are never simple, though... Soon Marcus finds himself embroiled in lethal political intrigue and the sharp end of class warfare, American style.


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Reality Composer is an easy-to-use tool for creating augmented reality (AR) experiences. With its drag-and-drop interface, you can quickly build interactive 3D scenes that can be exported for use in AR apps. In this article, we'll take a look at how to use Reality Composer to create AR experiences, as well as how to use Sketchfab to quickly create a Web AR experience.
What Capabilities Does Reality Composer Have?
Reality Composer has a wide range of capabilities that make it a powerful tool for creating AR experiences. Some of these capabilities include:
Object Placement
With Reality Composer, developers can place 3D objects in real-world environments using AR technology.
When developers place 3D objects using Reality Composer, the objects can be positioned and oriented in relation to real-world surfaces such as tables, floors, and walls, appearing as if they are actually part of the environment. This creates a powerful illusion of the virtual object's presence in the physical world, enabling users to interact with it and experience it in a way that feels natural and immersive.
Reality Composer allows developers to create animations for their 3D models. These animations can be triggered by user interactions or programmed to play automatically.
Physics Simulation
Reality Composer includes a physics simulation engine that allows developers to create realistic interactions between objects. For example, they can create a ball that bounces off walls and rolls on the floor.
Working with Reality Composer
When you install Xcode 11 or later, Reality Composer is automatically included as one of the developer tools. This means that you don't need to install it separately. Note that you need to participate in the Apple Developer Program in order to download XCode. To open Reality Composer, simply navigate to the menu and select Xcode > Open Developer Tool, then choose Reality Composer.
When you first open Reality Composer, you'll see a blank canvas and a toolbar with a range of objects and behaviours that you can add to your scene. To create a new project, simply drag and drop objects onto the canvas, and use the toolbar to add behaviours like animations and interactions. You can also import 3D models from external sources like Sketchfab or your own 3D modeling software.
One of the best features of Reality Composer is its preview mode, which allows you to test your project in real-time. You can view your AR experience through your device's camera, and interact with objects in the scene to see how they behave.
Using Sketchfab
Sketchfab is a popular online platform for sharing 3D models, and it's also a great resource for finding assets to use in your Reality Composer projects. To use Sketchfab with Reality Composer, simply browse the platform for a model you like, and download it in the appropriate format (such as .usdz for iOS devices). Then, import the model into your Reality Composer project and position it in the scene. You can use the same process to import other assets like textures and animations.
Using Reality Composer with Web AR
Web AR is a powerful technology that allows you to view AR experiences through a web browser, without the need for a dedicated app. Reality Composer is fully compatible with Web AR, and you can easily export your projects in the appropriate format for use on the web. To view your project in Web AR, simply open the link in a Safari on iOS, and point your camera at the marker on the screen.
You can read our article on Native Web AR for more details.
How to Integrate Reality Composer in Apps or Websites
Integrating Reality Composer in apps or websites is easy. Developers can export their AR experiences as USDZ files, which can be easily integrated into iOS apps or websites. They can also use Xcode to integrate their Reality Composer projects into their iOS apps.
In conclusion, Reality Composer is a powerful tool for creating AR experiences. It allows developers to create 3D models and animations, and then place them in real-world environments using AR technology. With its drag-and-drop interface, pre-built objects and animations, and wide range of capabilities, Reality Composer is a great tool for developers who want to create amazing AR experiences without any coding.
Best Practices for Optimizing AR Experiences Created with Reality Composer
Creating an AR experience is one thing, but creating an optimized and delightful AR experience is a whole different ball game. Here are some best practices to follow when creating AR experiences with Reality Composer:
Keep it Simple
One of the most important things to keep in mind when creating an AR experience is to keep it simple. While it's tempting to include a lot of interactive elements and animations, too much complexity can lead to a poor user experience. Instead, focus on creating a few key interactions or animations that enhance the overall experience.
Optimise Your 3D Models
When working with 3D models in Reality Composer, it's important to optimise them for performance. This means reducing the number of polygons and textures wherever possible, as well as minimizing the size of the model itself. By doing this, you can ensure that your AR experiences run smoothly on a variety of devices.
Test on Different Devices
AR experiences can vary greatly depending on the device they're viewed on. To ensure that your experience looks and performs well across all devices, be sure to test your project on multiple devices during development.
Consider Lighting Conditions
Lighting conditions can have a significant impact on how your AR experience looks and feels. When designing your experience, consider different lighting scenarios – both indoor and outdoor – and adjust your design accordingly.
Design for Interactivity
Interactivity is at the heart of any great AR experience. When designing your project in Reality Composer, be sure to consider how users will interact with it and design accordingly. This could mean adding touch or gesture-based interactions or incorporating environmental triggers like location or sound.


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The internet holds the possibility of making you very rich if you know how to use it to your advantage. But to make real money online, you need to own a website.
Some bloggers and website owners earn six-figure incomes through their online ventures. But more than half of web admins rake in less than $5000 a year. The harsh reality is that making money online requires substantial effort, like investing in a good SEO company to build up a well-performing, SEO-optimized site.
Factors that will Help Monetize Your Website Faster.
Before leaping into how you can make money online, it is necessary to understand the factors that distinguish your website from the millions of other websites on the internet.
To make money online, the first thing you need to do is convince search engines like Google and Bing to show your website on the search engine results pages (SERP) against any query related to your website domain or industry. And for this to happen, your website needs to be SEO-optimized. If you are a first-time website owner, investing in a good SEO company is wise to ensure that your website ranks higher on SERPs.
It will help advance your journey a long way if you already have a fan following or an audience for your content. For instance, if you are blogging or writing about travel, having a popular Instagram account on the same subject will drive a significant amount of traffic towards your website from your Instagram account.
Marketing Strategy
Say you don't have an already existing audience. In that case, what you require is a content marketing strategy to grow your audience. A content marketing strategy can show people the value of your content or product and services. Whatever plan you choose to implement, always strive to be authentic.
When you have tuned your website to excel in these three categories, you will be ready to implement the following ways to earn money off it.
Paid Memberships
If you are confident about your content and have a substantial audience, charging for your creations might be the easiest way to earn money. It is especially applicable to you if what you create is of high quality and demand in the market, for instance, studies related to current statistics about a particular location.
You will find this type of revenue system in significant news publishers like The New York Times, which offer only a few free articles per month. Instead, create exclusive content for your audience and put it behind a paywall.
Affiliate Marketing
It is one of the most trouble-free ways to monetize your website. In affiliate marketing, you refer people or audiences visiting your website to other sites that sell products and services. Irrespective of what industry or category your website falls under, there are always products that you can advise your website visitors to buy. E-commerce stores like Amazon, Etsy, etc., offer affiliate marketing opportunities for website owners.
But there is a right way and a wrong way to do this. It is always good practice to be upfront with your viewers and readers about any potential affiliate link in the content. Here are some other points to remember:
- Recommend products that solve problems for your audience by focusing on the value a product or service brings to them.
- Do not spam your content by over-promoting the referral links, as it will surely disrupt your audiences' interest in your content.
- Always try to promote products and services that you have yourself tried first and found helpful.
Sponsored Posts
Sponsored content focuses on promoting a brand and its products and services. The brands approaching you or vice versa will usually be directly related to your domain or industry. For example, if you write about books, you can promote a new book for a publishing house or create something that benefits the publishing house itself.
You can create sponsored content in many forms – videos, blog posts, infographics, podcasts, sales announcements, etc. But before taking on a project, you should consider the following things:
- The degree of your online influence (website visitors and social media followers)
- Number of sponsored posts required per month and the time needed to create them
Display Ads
You must have seen advertisements on almost every website that you had ever visited. These advertisements are called display ads. They are one of the most common ways to make money off your website if you have a steady flow of visitors. For example, the Google Display Network reaches 90 percent of all internet users around the world!
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Ulysses' Gaze
Ulysses' Gaze | |
Directed by | Theo Angelopoulos |
Written by |
Based on | Odyssey by Homer |
Produced by |
Starring | Harvey Keitel Maia Morgenstern Erland Josephson |
Cinematography |
Edited by |
Music by | Eleni Karaindrou |
Distributed by | Roissy Films |
Release date |
Running time | 176 minutes |
Country | Greece |
Languages |
Box office | $42,202 |
Ulysses' Gaze (Greek: Το βλέμμα του Οδυσσέα, translit. To Vlemma tou Odyssea) is a 1995 Greek war drama film directed by Theo Angelopoulos, and starring Harvey Keitel, Maia Morgenstern and Erland Josephson. It is loosely based on Homer's epic poem Odyssey.
The film was selected as the Greek entry for the Best Foreign Language Film at the 68th Academy Awards but it was not nominated.[1][2]
Successful Greek filmmaker, A (Harvey Keitel), returns to Greece. He has come to participate in a screening of one of his earlier films and to begin a personal journey across the Balkans. After the screening is disrupted by local ideological conflict, A takes a taxi from Greece to Albania. Ostensibly A is searching for 3 undeveloped reels of film shot by the Manaki brothers. The mysterious reels could predate the brother's first film, The Weavers, which is believed to be the first film shot in the Balkans.
A's journey fuses his own memories, the experiences of the Manaki brothers, and contemporary images of the Balkans. A drifts from Albania to North Macedonia, Bulgaria, Romania and Serbia. He travels on a train, a barge laden with a statue of Lenin (Polyphemus) and eventually a row boat. Though A makes some acquaintances along the way, he never lingers. His search for the roots of cinema, memory, and the Balkan identity pull him inevitably towards decay and death.
A eventually travels to the besieged Sarajevo. He meets Ivo Levy (Erland Josephson), the curator of an underground cinema archive who had attempted to develop the missing reels before the war. A convinces Levy to continue his work with the reels. The film ends on a rare foggy day in Sarajevo. Ironically the fog protects locals from snipers and gives the city a rare chance to flourish. A explores the city with Levy's family. Near the river the family encounters military personnel and they are executed.
- Harvey Keitel as A
- Maia Morgenstern as Woman In A's Home Town (Penelope) / Kali (Calypso) / Widow (Circe) / Naomi (Nausicaa)
- Erland Josephson as Ivo Levy
- Thanassis Veggos as Taxi Driver
- Yorgos Michalakopoulos as Nikos
- Dora Volanaki as The Old Lady In Albania
- Mania Papadimitriou as A's Mother
The film is part of Angelopoulos's trilogy on borders. It was his first film made outside of Greece. The film screening at the beginning of the film was inspired by a screening of Angelopoulos's earlier film The Suspended Step of the Stork. The dialog played over loudspeakers in the town square was spoken by Marcello Mastroianni[3]
The actor Gian Maria Volonté died during the filming. He was replaced by Erland Josephson and the film was dedicated to Volonté's memory.
The score by Eleni Karaindrou featuring Kim Kashkashian on viola was released on the ECM label in 1995.
- Grand Jury Prize - 1995 Cannes Film Festival[4]
- Critics Award 1995 - European Film Academy
- All-TIME 100 Movies - TIME magazine[5]
- The Top 100 Films of All Time - The Moving Arts Film Journal[6]
See also[edit]
- List of submissions to the 68th Academy Awards for Best Foreign Language Film
- List of Greek submissions for the Academy Award for Best Foreign Language Film
- ^ Margaret Herrick Library, Academy of Motion Picture Arts and Sciences
- ^ "41 to Compete for Foreign Language Oscar Nominations". FilmFestivals.com. Archived from the original on April 7, 2012. Retrieved 4 October 2015.
- ^ Portuges, Catherine (October 1996). "Review of Ulysses' Gaze". The American Historical Review. 101 (4): 1158–1159. doi:10.2307/2169647. JSTOR 2169647.
- ^ "Festival de Cannes: Ulysses' Gaze". festival-cannes.com. Retrieved 2009-09-05.
- ^ "All-TIME 100 Movies". Time. ISSN 0040-781X. Retrieved 2017-12-24.
- ^ "Check Out TMA'S 100 Greatest Movies Of All Time List". GeekTyrant. Retrieved 2017-12-24.
External links[edit]
- Ulysses' Gaze at IMDb
- Ulysses' Gaze at AllMovie
- Ulysses' Gaze at Discogs (list of releases)
- Ulysses' Gaze at Rotten Tomatoes
- Ulysses' Gaze (1995) TIME Magazine All-Time 100 best films
- 1995 films
- 1995 drama films
- English-language Greek films
- 1990s Greek-language films
- Romanian-language films
- Bosnian-language films
- Serbian-language films
- Films directed by Theodoros Angelopoulos
- Films shot in Serbia
- Films based on the Odyssey
- Films set in Greece
- Films set in Albania
- Films set in North Macedonia
- Films set in Bulgaria
- Films set in Bosnia and Herzegovina
- Films set in Romania
- Films set in Serbia
- Films with screenplays by Tonino Guerra
- Films scored by Eleni Karaindrou
- Films shot in Florina
- Greek drama films
- 1990s Greek films
- Modern adaptations of the Odyssey
- Cannes Grand Prix winners
- 1990s English-language films
- 1995 multilingual films
- Greek multilingual films


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Pre-Ballet (Ages 3-4) - Thursdays, February 9 - June 8, 2023, 3:30 - 4:15pm
With Lindsay Shaner
Spring Term: February 9 - June 8, 2023 (16 weeks, no class on February 23 or April 13)
Dance Showcase: Saturday, June 10
Beacon Feeds Dance Studio
This class introduces young dancers to the basic movements of ballet. They will learn the foundational movements of ballet at a fun, easy pace, working toward a performance in June. Special consideration is given to the attention span of children in this age group by incorporating dancing games and imagination.
Girls' attire: black leotard, pink tights, pink ballet shoes. Boys' attire: black shorts or pants, white T-shirt, black ballet shoes.
Scholarship applications available here.
Neon CRM by Neon One |


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There are 2 Comments to "Preach  #fashion #truth #quote #love"
Always warm up before you start https://tutuappx.com/ exercising. Start with static stretches, where you only stretch one muscle at a time.
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text:
patient centered care discussion w4
Question A:
Discuss how patient-centered care is leading to increasingly open models of care. Analyze the benefits and the potential drawbacks of the growing transparency between patients and providers.
Question B:
Discuss the unique features, as well as the advantages and disadvantages, of stand-alone, untethered, tethered, and networked personal health records (PHRs).
Please respond to the initial question by day 5 and be sure to post two additional times to peers and/or instructor by day 7. The initial post by day 5 should be a minimum of 75-150 words. If you use any source outside of your own thoughts, you should reference that source. Include solid grammar, punctuation, sentence structure, and spelling.
Click here to view your discussion rubric.
Note: this is just discussion so it should be very shot


text:
2010 All-Long Island boys track team
Patrick Farmer
Runner of the year
St. Anthony's
300, Senior
Farmer's emergence to the top of the track ladder was quick and smooth. The Virginia-bound sprinter, participating in the sport for the first time after four years of soccer, set Suffolk indoor records in both the 300 and 400. He proved those times were far from a fluke as he won the Nassau-Suffolk CHSAA 300 championship and then the Federation 300 title in 34.86 seconds, edging Bellport's Jaquan Holland (34.32).
Sancho Barrett
55 hurdles, Sr.
Barrett was Long Island's top 55-meter hurdler from start to finish this winter. He capped his great indoor season with a state public schools championship in the event with a career-best time of 7.473. Barrett was beaten out for the Federation title in a photo finish by Sheepshead Bay's Richmond Ahadzi (7.471).
Berfrantz Charles
North Babylon
55, Long Jump, Sr.
Charles was a half-foot away from winning two Federation titles. He won the 55-meter Federation title with a career-best time of 6.391. Charles nearly pulled off the daily double as he set a career mark in the indoor jump with a leap of 23 feet, 8 1/2 inches. Charles was beat out for the Federation by Anthony DeJean of Rome Free Academy, who jumped 23-9.
Joshua Haghighi
Shot put, Sr.
The Maryland-bound Haghighi had two specific goals this season. The first was to win the Federation crown. The second was to throw over 60 feet. It was mission accomplished for Haghighi, who broke 60 feet twice, and won the Federation shot put title with a toss of 59-2 1/2 at Cornell on March 6.
Jaquan Holland
300, Sr.
He had a career indoor season. He was the best in the 300 among public schools in both Nassau and Suffolk and proved it by taking the state public schools championship in a career-best time of 34.62. Holland, who took first in his event at the Suffolk Small Schools championship, has his mind set on resuming his track career at Florida State.


text: Colorful ornaments with a light bulb, socket, and plug make the Electrical Glass Ornaments Card perfect for electricians, electrical companies, and electrical contractors. This unique holiday design features a festive look with a series of round glass ornaments detailed with a Christmas tree, angel, bell, star, tree, and Merry Christmas greeting. Printed on glossy card stock, the Electrical Glass Ornaments Card measures 5" x 7" and comes with many free upgrades, allowing you to add your company name, include a personal message, choose from a variety of different colors and fonts, and order free envelopes printed with your return address.


text:
"Using N-grams to identify edit wars on Wikipedia" in 2019 IEEE Fifth International Conference on Multimedia Big Data (BigMM). pp. 398-403 DOI: 10.1109/BigMM.2019.00021
"Censorship through Copyright: From print to digital media" in Social Scientist vol. 41. nos. 1-2 January-February 2013
"Challenges, Innovations and Commitment: Cultural Intervention and Cultural Resistance" in Economic and Political Weekly VOL 46 No. 24 June 11 – June 17, 2011.
"Performing Change/ Changing Performance: An Exploration of the Life of a Street Play by the Jana Natya Manch" in Asian Theatre Journal vol. 27, no. 1 (2010).
"Theatre for the Ballot: The Use of Street Theatre for Campaigning in the Indian Elections", The Drama Review Winter 2005, Vol. 49, No. 4 (T188).
"'Meanings' of the Revolution: Language in the Street Plays of the Jana Natya Manch" in Language Forum (Jul-Dec 2004).
[Republished in Sudhanva Deshpande ed. Theatre of the Streets New Delhi, Jana Natya Manch, 2007.]
"How Far Radical?: Middle Class Politics, Youth and Women in the Literature of Bengal during National Liberation" in the Journal of the School of Languages (Spring 2005).


text:
Proteins that are localized to the cell surface via glycosylphosphatidylinositol (gpi) anchors have been proposed to regulate cell signaling and cell adhesion events involved in tissue patterning. Conditional deletion of Piga, which encodes the catalytic subunit of an essential enzyme in the gpi-biosynthetic pathway, in the lateral plate mesoderm results in normally patterned limbs that display chondrodysplasia. Analysis of mutant and mosaic Piga cartilage revealed two independent cell autonomous defects. First, loss of Piga function interferes with signal reception by chondrocytes as evidenced by delayed maturation. Second, the proliferative chondrocytes, although present, fail to flatten and arrange into columns. We present evidence that the abnormal organization of mutant proliferative chondrocytes results from errors in cell intercalation. Collectively, our data suggest that the distinct morphological features of the proliferative chondrocytes result from a convergent extension-like process that is regulated independently of chondrocyte maturation.
Growth of endochondral bones derives from regulation of the rate of chondrocyte maturation in the growth plate cartilage (reviewed by Kronenberg, 2003). In the growth plate, resting chondrocytes are a pool of progenitor cells that reside at the ends of embryonic long bones (Abad et al., 2002). Resting chondrocytes mature into proliferative chondrocytes that are characterized by the upregulation of cell proliferation and a change in cell morphology, such that round resting cells give rise to proliferative progeny that are discoid and arranged in columns resembling a stack of coins (Dodds, 1930). As maturation continues, proliferative chondrocytes undergo cell cycle arrest and enlarge, forming prehypertrophic cells. Subsequently, these cells undergo extensive growth to form hypertrophic chondrocytes, which deposit a specialized cartilage matrix that serves as a scaffold for blood vessels and osteoblasts to invade and lay down a true bony matrix(Howlett, 1979; Howlett, 1980; Noonan et al., 1998; Hunziker et al., 1999).
Establishment and maintenance of this cellular architecture is essential for proper function of the growth plate. Previous studies have revealed a complex network of interacting signaling pathways that generate a robust system to regulate the growth plate cartilage. At the heart of this system lie the secreted signaling proteins Indian hedgehog (Ihh) and parathyroid hormone-related peptide (Pthrp; also known as Pthlh) (reviewed by Kronenberg, 2003). Ihh produced by the prehypertrophic chondrocytes acts to induce proliferative chondrocyte formation while simultaneously inducing Pthrp expression in resting chondrocytes (Vortkamp et al.,1996). In turn, Pthrp secreted by resting chondrocytes antagonizes both the action of Ihh and the maturation of proliferative chondrocytes into prehypertrophic chondrocytes. Overlaid on this central interaction are the antagonistic actions of the Wingless/Int-1 factors Wnt5a and Wnt5b that coordinately control the transitions from resting to proliferative and proliferative to prehypertrophic chondrocyte(Yang et al., 2003). Bone morphogenetic proteins (Bmp) and fibroblast growth factors (Fgf) provide additional regulation of chondrocyte maturation(Kronenberg, 2003). Together,these positive and negative interactions help to establish distinct zones of resting, proliferative, and hypertrophic chondrocytes.
Previously, we demonstrated that columns of proliferative chondrocytes form by a process of convergent extension (CE) involving cell intercalation(Li and Dudley, 2009). Thus,cell division displaces daughter cells lateral to the column before cell intercalation movements promote re-integration. Orientation of the division plane and the stacking of chondrocytes depend on non-canonical frizzled (Fzd)signaling and Rho GTPase activity, two components of pathways that additionally regulate CE movements in the neural tube and planar cell polarity(PCP) in epithelial tissues (Klein and Mlodzik, 2005). Proteins displayed on the cell surface via glycosylphosphatidylinositol (gpi) linkages are also important components of the CE pathway (Topoczewski et al., 2001; Shao et al., 2009), and reduction in gpi-anchored proteins affects CE in the axolotl and zebrafish(Drawbridge and Steinberg,2000; Shao et al.,2009). Therefore, we sought to determine whether gpi-anchored proteins are also key regulators of CE and PCP in other tissues.
Gpi-anchored proteins include heparan sulfate proteoglycans (HSPGs) of the glypican family, ephrin A ligands (for Eph receptors), putative adhesion/signaling molecules of the Ly6 family, and enzymes such as alkaline phosphatase (Bernfield et al.,1999; Paulick and Bertozzi,2008). In addition to regulating CE movement, gpi-anchored proteins are thought to play important roles in patterning tissue by regulating cell signaling and cell adhesion. For example, loss of the glypican Dally interferes with Wingless (Wg) signaling and results in defects in patterning of the ventral denticles and wing of Drosophila(Lin and Perrimon, 1999; Tsuda et al., 1999). In vertebrates, manipulation of gpi-anchored ephrin A(Ide et al., 1994; Wada and Ide, 1994; Stadler et al., 2001), a ligand for Eph receptors, or of the ortholog of the gpi-anchored Ly6 family protein CD59 (da Silva et al.,2002; Kumar et al.,2007) in the limb mesenchyme results in defects in cell sorting and skeletal patterning. Therefore, gpi-anchored proteins might mediate the cellular interactions that impart unique identities to subgroups of cells within a field to promote tissue patterning.
Simple genetic tests of these models are confounded by the presence of diverse families of gpi-anchored molecules. As a first approach, we knocked out display of all gpi-anchored proteins on the cell surface using a conditional allele of Piga, which encodes the catalytic subunit of the enzyme complex that generates gpi moieties(Kawagoe et al., 1994; Keller et al., 1999; Keller et al., 2001). Conditional deletion of Piga in the lateral plate mesoderm (LPM) -progenitors of the limb skeleton - surprisingly resulted in a normally patterned limb skeleton. Here, we show that Piga null chondrocytes have normal maturation signatures, but abrogation of gpi-anchors causes cell autonomous defects in maturation rate, in cell morphology, and in chondrocyte organization. Although chondrocytes progress through each stage of the maturation process, mutant growth plates fail to form columns of discoid chondrocytes. Failure of column formation results from a defect in cell intercalation, a coordinated cell movement that depends on cell polarity. In addition, Piga expression is required to properly orient hair cells in the inner ear, a process regulated by the PCP pathway. Collectively, our data firmly establish that gpi-anchored proteins are common components of polarity pathways, including one that regulates CE-like movements in proliferative chondrocytes that in part define the structure and the growth properties of developing long bones.
Mouse strains and animal care
The mouse strains used are Hoxb6::cre(Lowe et al., 2000), lox-Piga-lacZ (Tremml et al.,1999), Prx1-cre(Logan et al., 2002), Ctnnb1tm2Kem (Brault et al., 2001), Foxg1tm1(cre)Skm(Hebert and McConnell, 2000),and TOPGAL [Jackson Laboratories(DasGupta and Fuchs, 1999)]. Animal care and use was in accordance with NIH guidelines and was approved by the Animal Care and Use Committee of Northwestern University. The morning after mating was designated as 0.5 days post coitum (dpc).
Skeletons were prepared as described(McLeod, 1980). Briefly,dissected hindlimbs were dehydrated in 95% ethanol followed by acetone,stained with Alcian Blue/Alizarin Red (Sigma), washed in 95% ethanol, cleared with 1% KOH, followed by a glycerol/decreasing KOH series, and stored in 100%glycerol.
To analyze cell morphology, limbs were dissected, fixed in Bouin's Fixative(Ricca Chemical Company, Texas, USA) at 4°C overnight, dehydrated through an ethanol series followed by xylenes, and embedded in paraffin (Richard-Allan Scientific, MI, USA). Sections were stained with Masson's Trichrome using a standard protocol.
β-galactosidase staining on P0 hindlimbs from TOPGAL mice was performed as described (Kawaguchi et al.,2002). Briefly, limbs were fixed in 1% paraformaldehyde (PFA)/0.5%glutaraldehyde in PBS for 1.5 hours at room temperature, and then stained in X-gal staining solution at 37°C overnight.
Antibody staining
To analyze cell proliferation, cells in S-phase were labeled with bromodeoxyuridine (BrdU) for 2 hours prior to tissue harvest. Tissue was fixed in 4% PFA in PBS at 4°C overnight and prepared for paraffin sectioning as described. Sections of BrdU-labeled tissue were digested in trypsin and depurinated in hydrochloric acid. Incorporated BrdU was detected using a mouse anti-BrdU monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa,USA) and Cy2-labeled donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, Pennsylvania, USA). Nuclei were counterstained with 4′,6-diamidino-2-pheylindole (DAPI, Sigma-Aldrich). The BrdU labeling index was calculated as the percentage of total nuclei that were BrdU positive in each designated box.
P0 cryosections were used for staining with the following antibodies:cleaved caspase-3 (1:500, Cell Signaling); phospho-Smad 1(Ser463/465), 5(Ser 463/465) and 8(Ser 426/428; 1:500, Cell Signaling); phospho-p44/42 (1:100,Cell Signaling); β-galactosidase (1:2000, a gift from R. Holmgren,Northwestern University, IL, USA); acetylated tubulin (1:1000, Sigma). Samples were washed in PBS plus 0.1% Triton X-100 (PBSTx), blocked for 1 hour in 10%FBS/PBSTx at room temperature, then incubated overnight at 4°C with the primary antibody in 2% FBS/PBSTx. Samples were washed and incubated with donkey anti-rabbit Alexa 488 (1:500, Invitrogen) for 4 hours at room temperature, and counterstained with DAPI.
Gene expression analysis
For in situ hybridization (ISH) analysis, RNA probes were produced using T7, T3 or SP6 polymerase in the presence of non-radioactive digoxigenin-labeled rNTPs (Roche) or S35 (PerkinElmer), as described (Murtaugh et al.,1999). For quantification, the length of the expression domain was measured along the longitudinal axis of the cartilage and calculated as a ratio over the total length of the growth plate (articular surface to the mineralized region) using non-radioactive samples.
Chondrocyte isolation and culture
Growth plate cartilage was dissected free of the perichondrium/periosteum,minced with a razor blade, and incubated in 0.5% collagenase in DMEM+10% heat inactivated FBS (Invitrogen) at 37°C for 3 hours. After gentle trituration the resulting cell suspension was passed through a cell strainer (70 μm pore size, Becton-Dickinson), pelleted, and washed twice with serum-free DMEM. For transfection experiments, 2.5×105 chondrocytes were seeded into 12-well tissue culture clusters (Corning) in DMEM+10%heat-inactivated serum with penicillin/streptomycin. Eighteen to 24 hours later chondrocytes were transfected with GFP-gpi or GFP-myr (gifts from A.-K. Hadjantonakis, Sloan-Kettering Institute, NY, USA) using Superfect transfection reagent (Qiagen), according to the manufacturer's instructions. Thirty-six hours later, cultures were washed twice with PBS and imaged by epifluorescence.
Flow cytometry
All incubations were performed on ice in PBS containing 0.1% sodium azide unless otherwise noted. Isolated chondrocytes were washed three times in PBS/azide, blocked in 5% heat-inactivated normal donkey serum (Jackson ImmunoResearch), and incubated for 4 hours in 5% serum containing 1μg/105 chondrocytes of sheep IgG (Jackson ImmunoResearch) or anti-Gpc3 (R&D Systems). After washing three times in PBS/azide, cells were incubated in Alexa-Fluor 488-anti-sheep antibody (Invitrogen) for 1 hour. Cells were washed three times in PBS/azide then fixed in 2% PFA and analyzed using a FACScalibur and Cell Quant Pro software (Becton-Dickinson). For experiments involving removal of gpi-anchored proteins, cells were initially incubated in αMEM (Invitrogen) containing 0.1 units of gpi-specific phospholipase C (Sigma Aldrich) for 1 hour at 37°C.
Alkaline phosphatase activity
P3 limbs were fixed in 4% PFA for 3 hours at room temperature and then prepared for cryosectioning. Alkaline phosphatase staining was performed as described in the development step of the non-radioactive ISH protocol.
Image analysis
Orientation of cell division was determined as described(Li and Dudley, 2009). For analysis of stereocilia, P0 lox-Piga-lacZ/Foxg1tm1(cre)Skmpup inner ears were dissected in PBS/2 mM CaCl2/0.5 mM MgCl2 and fixed in 4% PFA/2 mM CaCl2/0.5 mM MgCl2 at 4°C overnight. The following day, the cochlea were dissected from the temporal bone, and the tissue was permeabalized in PBS plus 1% Triton X-100 (PBSTx) for 1-2 hours, and then blocked overnight in 20%heat-inactivated sheep serum/PBSTx. Cochlea were stained with anti-acetylated tubulin (1:200, Sigma) to visualize the kinocilium or anti-Vangl2 (1:400, gift from M. Montcouquiol, INSERM, Bordeaux, France). All specimens were additionally incubated with Alexa-Fluor 568 phalloidin (Invitrogen) to label the stereocilia and actin cytoskeleton. Images were acquired using a Zeiss apotome deconvolution microscope or Leica Confocal Laser Scanning System and analyzed as described (Qian et al.,2007).
Statistical methods
A two-way ANOVA was used to determine whether the distributions of gene expression domain length were distinct between wild-type and mutant mice. The model contained the main effects of genotype (wild type and mutant) and zone(an interaction effect between the genotype and zone). Different contrasts were constructed to test whether the gene expression of a particular zone was the same between wild-type and mutant mice; if two zones had the same gene expression within the same genotype; and if the gene expression difference between two zones of interest was the same for wild type and mutants. Bonferroni's procedure was used to control the family-wise error rate at significance level 0.05 as multiple tests were performed simultaneously. That is, a test was considered to be statistically significant if the corresponding P-values were below 0.05 divided by the total number of tests. Analysis was based on a minimum of three independent samples per genotype.
Chondrodysplasia in Piga mutant limbs
To determine the role of gpi-anchored proteins in development, we analyzed loss of Piga expression in developing limbs. Piga encodes PIG-A, the catalytic subunit of an essential enzyme for the initial step of gpi-anchor biosynthesis (Fig. 1A) (Miyata et al.,1993; Watanabe et al.,1998; Kostova et al.,2000). Since null Piga alleles are early embryonic lethal(Kawagoe et al., 1994; Rosti et al., 1997), a conditional allele was employed to selectively delete Piga using Cre recombinase (Keller et al.,1999; Tremml et al.,1999). We took advantage of the fact that Piga resides on the X chromosome and mated females homozygous for the conditional allele(XPiga/XPiga) to males heterozygous for the autosomal Hoxb6::cre transgene (cre/+) that expresses Cre recombinase in the posterior LPM(Lowe et al., 2000), which gives rise to the mesenchyme from which the hindlimb skeleton forms. Analysis using reporter alleles shows that Hoxb6::cre transgene function is initiated at 8.5 days post coitum (dpc) and promotes near complete recombination in the hindlimb mesenchyme by 9.5 dpc prior to limb bud outgrowth (Lowe et al., 2000). Our breeding scheme yields equal numbers of genotypically wild-type(XPigaY; +/+) and mutant (XPigaY; cre/+) males (data not shown). By contrast, all females are heterozygous for the conditional allele (XPiga/X). However, due to random X-inactivation, females carrying the Hoxb6::cre transgene (XPiga/X; cre/+)are mosaics composed of both wild-type and null cells. The following studies are based on the analysis of both null mutant (XPigaY; cre/+) and mosaic (XPiga/X; cre/+)limbs.
Given the proposed roles for gpi-anchored proteins in embryogenesis, we were surprised to find normal patterning of the limb skeleton in both mutant and mosaic animals (Fig. 1B). The slight delay in formation of distal cartilage elements that was observed at 12.5 dpc did not translate into patterning defects at later stages. However, Piga mutant hindlimb long bones were significantly shorter(P<0.0001) and wider (P<0.0001) at birth, although heterozygous females presented an intermediate phenotype(Fig. 1C).
A substantial delay in ossification was observed at 14.5 dpc but was absent twelve days after birth (P12). By 15.5 dpc, wild-type cartilage displayed domains of collagen type X alpha 1 (Col10a1)-positive hypertrophic chondrocytes between the collagen type II alpha 1 (Col2a1)-expressing immature chondrocytes and the primary ossification center(Fig. 1D). By contrast, only a single small domain of Col10a1 expression occupies the central diaphysis in Piga mutants. Furthermore, a reduction in mineralization was observed in the periosteum and the primary ossification center at birth,and Piga mutants did not form an extensive trabecular network as observed in wild-type cartilage (see Fig. S1 in the supplementary material). Because matrix mineralization is one function of osteoblasts, we investigated whether gpi-anchored proteins are required for osteoblast formation. Analysis of collagen type I alpha 1 (Col1a1), osteocalcin (OC; also known as Bglap2) and osteopontin (Opn; also known as Spp1) suggested normal or slightly decreased numbers of mature and progenitor osteoblasts (Kratochwil et al.,1993; Sommer et al.,1996). Thus, gpi-anchored proteins are not required for skeletal patterning or osteoblast formation, but might have additional roles in osteoblast cells.
Gpi-anchored proteins are absent from Piga mutant chondrocytes
The absence of major developmental defects could result from a perdurance of gpi-anchored proteins produced prior to recombination or from partial compensation of Piga function by other cellular enzymes. To test these possibilities, we examined whether gpi-anchored proteins were lost in Piga mutant chondrocytes. Alkaline phosphatase (Alp) is a gpi-anchored enzyme displayed on the surface of hypertrophic chondrocytes and osteoblasts. Alp activity was not detectable in Piga null cartilage,but in situ hybridization (ISH) analysis confirmed expression of Akp2(also known as Alpl), the gene encoding bone/liver/kidney alkaline phosphatase (Fig. 2A,B)(Pizauro et al., 1994; Magnusson et al., 1999). Interestingly, in mosaic females, we observed clusters of Alp-positive cells generated from morphologically wild-type columnar chondrocytes beside Alp-negative cells generated from round mutant chondrocytes(Fig. 2A). The phenotypes appeared to remain spatially segregated, as `salt-and-pepper'patterns were never observed with regard to cell shape or Alp activity. We additionally showed by flow cytometry that the gpi-anchored protein glypican 3 (Gpc3) is not displayed on Piga mutant chondrocytes(Fig. 2C; see also Figs S2, S3 in the supplementary material). Moreover, mutant chondrocytes transfected with gpi-anchored green fluorescent protein (gfp) lacked fluorescence in contrast to wild-type chondrocytes (Fig. 2D). By contrast, transfection with a myristolated gfpconstruct resulted in equivalent fluorescence on intracellular membranes of wild-type and mutant chondrocytes (Fig. 2D). Together, these data demonstrate that gpi-anchored proteins are absent from the cell surface of Piga mutant chondrocytes.
Piga mutant chondrocytes proceed through normal stages of maturation
The observation of morphologically abnormal chondrocytes suggested the presence of defects in chondrocyte maturation in mutants. To determine whether loss of Piga expression affects early steps in chondrocyte maturation, we first examined the cellular structure of developing tibiae. In Piga null hindlimbs, resting chondrocytes appeared normal but proliferative chondrocytes failed to flatten or form columns(Fig. 3A,B). Heterozygous females are mosaics with round mutant chondrocytes adjacent to discoid wild-type chondrocytes (Fig. 3C). Bromodeoxyuridine incorporation(Fig. 3B) demonstrated upregulation of the cell cycle in the growth plate cartilage of wild-type(5.7±0.7% versus 12.3±1.6%, resting versus proliferative chondrocytes) and mutant (9.8±0.2% versus 14.7±2.2%, resting versus proliferative chondrocytes) samples. Curiously, we observed a small but significant increase in cell proliferation in mutants compared with littermate controls (e.g. 9.8±0.2% versus 5.7±0.7%, in the resting chondrocytes). In addition, the absence of cleaved caspase-3-positive apoptotic chondrocytes demonstrates that cell viability does not depend on gpi-anchored proteins (Fig. 3B)(Nicholson et al., 1995). Upregulation of the cell cycle suggests that Piga growth plates contain proliferative chondrocytes. To test this possibility, we used ISH analysis to show that mutant growth plate chondrocytes expressed markers appropriate to immature (Col9a1) chondroctyes of the resting(Fgfr1) and proliferative (Bapx1, Fgfr3, Prelp) zones,prehypertrophic chondrocytes (Ihh), and hypertrophic chondrocytes(Col10a1; see Fig. S4 in the supplementary material; see also Fig. 4A). Thus, Piganull growth plates contain a bona fide proliferative zone.
Although each is present, the length of some maturation zones is altered in mutants indicating imbalances in chondrocyte maturation. To quantify this effect, we measured the length of gene expression domains along the epiphyseal-metaphyseal axis and determined the ratio of this measurement to total length of the growth plate (articular surface to the mineralized region). These measurements revealed that total growth plate length is similar at birth (P0) for wild-type (1.05±0.16 mm), mosaic (1.03±0.10 mm) and mutant (1.04±0.16 mm) femurs. The domain of least mature chondrocytes was not significantly shorter in mutants compared with wild type(Fig. 4B; Fgfr1,P=0.033) unlike, the proliferative (Prelp, P<0.0001) and prehypertrophic zones (Ihh; P<0.005; Fig. 4B). By contrast, the region between the Fgfr1 and the Prelp expression domains was expanded (P<0.0001), consistent with the observed upregulation of cell proliferation (Fig. 4B). Collectively, our results demonstrate that morphologically abnormal Piga mutant chondrocytes undergo normal phases of maturation and that the delay in maturation begins at the transition between resting and proliferative chondrocytes.
Additionally, we compared the length of gene expression domains for wild-type and null cells in mosaic cartilage. The Prelp expression domain was similar in size for wild-type chondrocytes in both mosaic and wild-type growth plates (P=0.062; Fig. 5A,B). Similarly, the expression domain of Ihh was the same for wild-type cells in mosaic and wild-type samples (P=0.364; Fig. 5A,B). Likewise, gene expression domains were similar for mutant chondrocytes expressing Prelp (P=0.944) and Ihh (P=0.610; Fig. 5A,B), whether the Piga null cells were in a mutant or a wild-type environment. The delayed maturation of Piga mutant chondrocytes in mosaic cartilage suggests a defect in signal reception. However, we did not detect a decrease in Ihh, Bmp, Fgf, or canonical Wnt signaling in mutant chondrocytes(Fig. 4A,B, Ptc1; see Fig. S5 in the supplementary material; see also Fig. 6C). Moreover, to confirm that canonical Wnt signaling is unaffected, we demonstrated that conditional inactivation of β-catenin (Ctnnb1tm2Kem)(Brault et al., 2001) in the cartilage does not alter morphology or organization of proliferative chondrocytes (Fig. S6 in the supplementary material).
Piga mutant chondrocytes fail to intercalate
One intriguing observation is that although mutant chondrocytes ultimately progress through all stages of maturation, columns of discoid cells never form, suggesting that cell morphology and maturation are regulated by distinct mechanisms. Column formation in chondrocytes occurs in two steps with cell division displacing daughters laterally before intercalation integrates them back into the column (Dodds,1930; Li and Dudley,2009). Absence of columns could result from defects at either step. We first determined the orientation of cell division(Fig. 6A) and showed that wild-type and mutant resting chondrocytes divide in arbitrary planes(Fig. 6B), whereas 74% of mutant proliferative chondrocytes divide at angles nearly perpendicular to the axis of growth similar to wild-type chondrocytes(Fig. 6B). Although 26% of the mutant proliferative chondrocytes divide at non-wild-type angles, these improperly aligned cell divisions cannot account for the 100% of mutant chondrocytes that fail to form columns, which suggests that the primary defect is not in establishing the division plane.
Next, we tested for defects in cell intercalation. Initially, we considered using the lacZ cassette embedded in the conditional Pigaallele to label mutant cells, but we found that the lacZ gene product was not detectable using chromogenic substrates, immunofluorescence or ISH. Therefore, we used a property of TOPGAL transgene expression - patchy across the tissue but uniform within a chondrocyte column - to test whether Piga null cells intercalate into columns. Because cells within an individual column derive from a single progenitor cell, epigenetic regulation of TOPGAL results in a short-term lineage mark(Fig. 6C). We analyzed groups of labeled cells in TOPGAL mice that were additionally wild-type or mutant for Piga (Fig. 6C). Because chondrocytes in wild-type columns undergo limited cell divisions(Dodds, 1930), we determined total cell number and extent of lateral dispersion in small groups of labeled cells (<16 cells/group). Labeled wild-type cells are contained in groups distributed in the longitudinal axis that are generally one or two cell diameters wide (Fig. 6C,D). By contrast, mutant cells were preferentially distributed laterally within the growth plate (P<0.0001, compared with wild type; Fig. 6D). Consistent with this,we found that groups of Piga mutant cells were smaller than wild type(P<0.0001), presumably a result of the lateral spread of progeny into adjacent sections.
Failure of column formation could also result from defects in cytokinesis,as shown for integrin β1 mutant chondrocytes(Aszodi et al., 2003). However,in Piga mutants, chondrocytes showed normal cell surface display of integrin β1, and multinucleate or apoptotic cells normally associated with cytokinesis defects were not observed(Fig. 3B; see also Fig. S7 in the supplementary material; data not shown). Moreover, we found in both wild-type and mutant cartilage that dividing cells in the resting and proliferative zones showed a normal flattened interface that included a well-formed midbody (Fig. 6E),a microtubule-based structure assembled during anaphase that is important for completion of cytokinesis (Raich et al.,1998). These observations suggest that cell division occurs normally in Piga mutants and thus the requirement for gpi-anchored proteins is in the downstream cell intercalation process.
Gpi-anchored proteins regulate cell polarity
The requirement for gpi-anchored proteins for polarized cell movements in cartilage and in axis formation in the axolotol and zebrafish suggests that gpi-anchored proteins are general components of polarity pathways(Drawbridge and Steinberg,2000; Topczewski et al.,2001; Shao et al.,2009). We tested this hypothesis using the sensory epithelium of the vertebrate inner ear. The sensory epithelium contains four rows of hair cells each displaying a `V'-shaped bundle of actin-rich stereocilia with a single microtubule-based kinocilium at the apex (reviewed by Kelly and Chen, 2007). Alignment of stereocilia and kinocilium across all hair cells in wild-type cochlea is regulated by the PCP pathway (reviewed by Klein and Mlodzik, 2005). To test whether gpi-anchored proteins regulate cell polarity, we crossed XPiga/XPiga females to Foxg1tm1(cre)Skm males, which express Cre recombinase in the otic vessel (Hebert and McConnell,2000). Hair cells in Piga mutant ears display disrupted architecture at the base of the hair cells and poorly aligned stereocilia and kinocilia of the outer hair cell layers(Fig. 7A-C), as has been shown for PCP mutants such as Vangl2 and scribble(Montcouquiol et al., 2003). Additionally, Vangl2 is mislocalized in Piga mutant outer hair cells(Fig. 7C). Collectively, our data establish a link between gpi-anchored protein activity and tissue polarity pathways that pattern the sensory epithelium of the inner ear.
Gpi-anchored proteins function cell autonomously
Conditional inactivation of Piga results in the loss of gpi-anchored cell surface proteins, as determined by the absence of Alp activity on mutant chondrocytes and osteoblasts, the lack of Gpc3 on mutant chondrocytes, and the failure of transfected mutant chondrocytes to display gpi-anchored Gfp, findings that are consistent with previous studies in the hematopoietic lineage (Tremml et al.,1999). Interestingly, Alp activity is not observed in the matrix of mutant cartilage, demonstrating that secreted forms are not generated by the absence of anchor addition. This finding is consistent with previous studies demonstrating that failure of anchor addition leads to retention in the endoplasmic reticulum (Field et al.,1994; Doering and Schekman,1996; Doering and Schekman,1997) and protein degradation(Ali et al., 2000). In this context, the presence of secreted epitope-tagged gpi-anchored Knypek in zebrafish embryos injected with morpholinos directed against pigp(Shao et al., 2009) suggests an incomplete knockdown of transamidase activity. However, secreted forms, if they exist, probably do not act dominantly because small patches of wild-type or mutant cells surrounded by cells of the opposite genotype are readily observed in mosaic females. Moreover, in mosaics, transitions in maturation state for cells of one genotype are unaffected by the presence of large numbers of cells of the opposite genotype. Together, these data indicate that the observed phenotype is the result of cell autonomous Pigaexpression and does not reflect global defects in signaling pathways that regulate growth plate function.
Gpi-anchored proteins are not required for tissue patterning
Numerous studies have demonstrated important roles for gpi-anchored cell surface proteins in controlling cell signaling and cell adhesion during embryogenesis. For example, dally and dally-like encode two gpi-anchored HSPGs of the glypican family that contribute to patterning of the Drosophila wing through the regulation of signaling by secreted factors of the Wg/Wnt and Hh families (Lin and Perrimon, 1999; Tsuda et al., 1999; Lum et al.,2003; Kirkpatrick et al.,2004). In the vertebrate limb, gpi-anchored ephrin A ligands are proposed to regulate cell sorting in the autopod through interaction with the EphA4 and/or EphA7 receptor tyrosine kinase(Wada et al., 1998; Stadler et al., 2001), and a gradient of CD59 has been linked to regeneration of the proximodistal axis in amputated newt limbs (da Silva et al.,2002; Kumar et al.,2007). By contrast, elimination of Piga expression in the posterior LPM lineage does not alter patterning of the limb skeleton. Even mosaic limbs do not show patterning defects predicted from errors in cell sorting (Davy et al., 2004). One caveat to this interpretation is that we do not currently have robust assays to verify the absence of gpi-anchored proteins in the early embryonic limb buds. However, given that Hoxb6::cre catalyzes near complete recombination in the LPM by 9.5 dpc (Lowe et al., 2000), it is reasonable to assume that mutant early limb mesenchyme cannot generate new gpi-anchored proteins. Proteins produced before 9.5 dpc could function early but would be rapidly diluted as local cell proliferation drives limb bud growth. Thus, total depletion of gpi-anchored proteins is likely to occur early in limb bud development and certainly before digit formation when the gpi-anchored ephrin A ligands are supposed to act(Wada et al., 1998; Stadler et al., 2001).
The nonessential function of gpi-anchored proteins is not limited to the cartilage, as we show that loss of Piga expression in the otic placode does not alter the gross structure of the inner ear, although hair cells in the cochlea do display altered cell polarity. Similarly, defective gpi-anchoring of proteins does not substantially affect patterning of the skin(Hara-Chikuma et al., 2004),the neural tube (Nozaki et al.,1999), or the zebrafish embryonic germ layers(Shao et al., 2009). Despite potential effects on key growth factor signaling pathways, gpi-anchored proteins are not absolutely required for many processes. This might reflect compensation by, or predominance of, a non-gpi-anchored protein. For example,mosaics for a null mutation in the non-gpi-anchored ephrin B1 ligand show defects in cell sorting and patterning predicted for loss of ephrin A ligands in the vertebrate limb (Davy et al.,2004). Thus, the gpi-anchored class of cell surface proteins might primarily function to fine-tune cell-cell interaction pathways to promote robustness.
Gpi-anchored proteins regulate chondrocyte maturation
Loss of gpi-anchored proteins leads to a reduced rate of chondrocyte maturation that is first observed at the transition from resting to proliferative chondrocytes, as demonstrated by the decreased size of the proliferative zone despite an increase in resting chondrocyte proliferation. The size of and transitions between maturation zones are controlled by the action of a complex network of synergistic and antagonistic pathways regulated by secreted signaling proteins (Karsenty and Wagner, 2002; Kronenberg,2003; Yang et al.,2003). HSPGs, including gpi-anchored glypicans, are important regulators of these signaling pathways (reviewed by Häcker et al., 2005). In cartilage, HSPGs might determine the extracellular distribution of secreted ligands (Koziel et al., 2004). However, in mosaics, in which wild-type cells display normal zones of maturation consistent with normal ligand distribution, Piga null cells show delayed onset and reduced size of maturation zones equivalent to that in mutant cartilage. The failure to initiate transitions in maturation state synchronously with wild-type neighboring cells suggests that Piga null cells either do not correctly detect or fail to respond appropriately to growth plate signals.
Defects in signaling are consistent with a role for gpi-anchored proteins as co-receptors for signals that promote chondrocyte maturation, or as antagonists of factors that repress maturation. Likely candidates for these functions are gpi-anchored HSPGs of the glypican family. Consistent with this interpretation, many similarities exist between mutants in Ext1(Koziel et al., 2004), an enzyme involved in the production of HSPGs, and Piga (see Table S1 in the supplementary material). Interestingly, a hypomorphic allele of Gpc3 displays defects in chondrocyte maturation similar to those of Piga mutants, suggesting that Gpc3 is the primary gpi-anchored regulator of chondrocyte maturation in the growth plate(Viviano et al., 2005). If so,it will be important to determine the functions of the other glypicans expressed in the growth plate (see Fig. S3 in the supplementary material).
Gpi-anchored proteins regulate tissue polarity
The fact that Piga null cells eventually pass through all phases of chondrocyte maturation while defects in cell shape and cell arrangement in proliferative chondrocytes are absolute suggests that distinct mechanisms regulate chondrocyte maturation and proliferative chondrocyte behavior. The arrangement of proliferative chondrocytes into columns of stacked cells is a multi-step process that includes two planar processes -aligned division planes and cell intercalation - and that is regulated by non-canonical Fzd signaling(Dodds, 1930; Li and Dudley, 2009).
In the absence of gpi-anchored proteins, the spindle orienting mechanism is functional, but imprecise, resulting in a percentage of chondrocytes that divide out of plane. A similar percentage of misaligned division planes are observed in kidney tubules lacking the atypical cadherin Fat4, a component of the PCP pathway (Saburi et al.,2008). However, the Fat4 mutant phenotype is more severe than that of Piga mutants in that a substantial number of Fat4 mutant cells align the mitotic spindle orthogonal to wild type. In this respect, the Fat4 phenotype approximates, but is less severe than, the uniform distribution of division planes in chick chondrocytes deficient in non-canonical Fzd signaling(Li and Dudley, 2009). Although gpi-anchored glypicans are components of the non-canonical Fzd pathway (Caneparo et al., 2007; Shao et al., 2009), the weak division plane phenotype of Piga mutants suggests that gpi-anchored proteins either are largely expendable because of redundancy or are not core components of the Fzd pathway that regulates the division plane. In this latter model, one possibility is that gpi-anchored proteins regulate a parallel process that influences the division plane. For example, division of most cells occurs according to Hertwig's rule, which predicts alignment of the mitotic spindles parallel to the long axis of the cell(Wilson, 1900; Wilson, 1925), and might involve interaction between cell-matrix adhesion and the actin cytoskeleton(Toyoshima and Nishida, 2007). Even though tissue polarity pathway function can override Hertwig's rule(Gong et al., 2004), perhaps the system is more robust when components of polarity pathways are aligned with a prominent axis defined by cell shape/cell adhesion that might be lacking in round Piga chondrocytes.
Interestingly, although the plane of cell division is largely normal,mutant cells fail to intercalate and, as a result, clones of Piganull cells expand laterally across the proliferative zone instead of forming columns aligned to the longitudinal axis. It remains to be determined if the small number of stacked chondrocytes in a mutant clone results from occasional, normal cell intercalation events or from aberrant division planes in a non-intercalating clone. Nonetheless, these results refine our previous model (Li and Dudley, 2009) by demonstrating that establishment of the division plane and the process of cell intercalation are differentially regulated, despite both events being sensitive to non-canonical Fzd signaling. Moreover, lateral expansion of the proliferative chondrocytes correlates with the increased width and decreased longitudinal growth of mutant cartilage, providing further evidence that cell intercalation events orient both local and tissue-wide vectors of growth in cartilage through a process of CE.
Towards a mechanism of convergent extension in cartilage
How cell polarity pathways influence cell shape and promote cell intercalation is currently under debate. One possibility is that gpi-anchored proteins regulate non-canonical Fzd signaling. In this regard, two non-exclusive models have been proposed for the role of the gpi-anchored proteins in CE. First, the glypican Knypek has been shown to act in conjunction with the secreted canonical pathway inhibitor Dickkopf (Dkk) to alter the specificity of Fzd signaling from activating canonical to activating non-canonical pathways (Caneparo et al.,2007). Second, gpi-anchored proteins might stabilize Fzd receptors on the cell surface potentially promoting membrane localization of Dishevelled, an event associated with non-canonical PCP signaling(Shao et al., 2009). In this context, the observation of reduced Vangl2 localization in hair cells might reflect defects in Fzd signaling rather than a direct interaction between Vangl2 and gpi-anchored proteins.
The effectors of cell polarity pathways are most probably regulators of cytoskeletal dynamics and cell-matrix interactions. In particular, matrix structure is an important determinant of cell shape and cell intercalation(Davidson et al., 2006). Consistent with this, null mutants in cartilage-specific Col9a1(Blumbach et al., 2008; Dreier et al., 2008) and the cell-matrix adhesion receptor integrin β1(Aszodi et al., 2003) result in round, disorganized proliferative chondrocytes that are grossly similar to those of Piga mutants. However, loss of either integrin β1 or Col9a1 function results in a more severe phenotype than displayed by Piga null cartilage, and expression of Col9a1 and cell surface localization of integrin β1 are unaffected in Pigamutant cartilage (see Figs S4, S6 in the supplementary material). Together,these data suggest that gpi-anchored proteins do not regulate the production of Col9a1 or integrin β1. Thus, as with Fzd receptors,gpi-anchored proteins might be required for the appropriate trafficking or stability of cell adhesion molecules, or to properly organize the extracellular matrix to promote column formation.
CE and PCP are two processes that define the cellular architecture of many tissues. Although once thought to be highly specific to certain tissues, these processes are being discovered in diverse cell types, including chondrocytes. The involvement of non-canonical Fzd signaling, Rho GTPase activity, and now gpi-anchored proteins in both CE and PCP suggests that common mechanisms regulate the architecture of all tissues.
We thank Monica Bessler and Michael Kuehn for mice; Yingzi Yang and Hank Kronenberg for ISH templates; Mireille Montcouquiol for the Vangl2 antibody;Anna-Katerina Hadjantonakis for plasmids; Roxanne Edge for technical advice;Robert Holmgren, Alec Wang, Carole LaBonne and Richard Carthew for comments and discussion. This work was supported by the Cellular Molecular Basis of Disease Training Grant (MJA), the National Institutes of Health/NIAMS(AR054857), the American Cancer Society, the Alumnae of Northwestern University, and by the National Center for Research Resources (NCRR). Deposited in PMC for release after 12 months.


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The riots that are going on across the country and the world have escalated to the point where people are blatantly attacking police vehicles and police officers to get their message across that they will be heard and they are not going to take it anymore.
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2023 State of the City Address
The Greater Idaho Falls Association of Realtors® (GIFAR) hosted the City of Idaho Falls and Mayor Rebecca Casper for the 2023 State of the City Address. The address was given on Jan. 18 at the luncheon, with over 150 people in attendance.
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Repo Reaper Ring
Repo Reaper Ring
The embodiment of death, made from 925 Sterling Silver.
Legend says that the Grim Reaper appears when someone is just moments from death. It is believed that the he uses his sharp scythe to separate the human soul from the body. He then accompanies the soul to its final resting place, which is either Heaven or Hell.
In Roman mythology, the collector of souls was actually portrayed in a positive manner as a sign of good luck.
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