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Vital Signs: Guardians of Our Joy
January 1, 2017 Speaker: Joe Walser Series: Vital Signs
Topic: Biblical Passage: 2 Chronicles 16:9–16:9
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What if you could live your life knowing you would have no regrets.
- Psalms 16:11
- 1 Peter 1:8
Vital Signs:
1. Commit to them!
- 2 Chronicles 16:9
2. Regain your freedom by enslaving yourselves to them
- John 8:31-32
3. Commitment to these vital signs means burning the plow...
- 1 Kings 19:19-21
- Proper perspective
- No Plan B
"Hence we may learn, that whatever changes a godly man passes through, he is happy; because God, who is unchangeable, is his chosen portion. Though he meet with temporal losses, and be deprived of many, yea, of all his temporal enjoyments; yet God, whom he prefers before all, still remains, and cannot be lost. While he stays in this changeable, troublesome world, he is happy; because his chosen portion, on which he builds as his main foundation for happiness, is above the world, and above all changes. And when he goes into another world, still he is happy, because that portion yet remains…. How great is the happiness of those who have chosen the Fountain of all good, who prefer him before all things in heaven or on earth, and who can never be deprived of him to all eternity!" - Jonathan Edwards, God the Best Portion of the Christian
4. Commitment to these vital signs begins with Dependent on gospel rhythms...TIME WITH
- Psalms 90:10, 12
- Mark 6:31-32
- Mark 1:35
- Luke 5:15-16
Things to do:
- Get on a Bible reading plan for the year
- Get into a Bible study
- Prayer, prayer, prayer, and more of it
- Commit to a Missional Community
- Find a way to be accountable
More in Vital Signs
January 8, 2017Vital Signs: Discipleship Where You Are
November 27, 2016Vital Signs: Distribution of God's Resources


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Twisted Flags is the ultimate rock'n'roll party band ready to blow your mind with its own mixture of original and cover songs from every decade until today!
January 11 LIVE @Spanky's
Twisted Flags returns to Spanky's Shenanigans for their first 2020 gig! Celebrate your Saturday with the music of The Killers, Foo Fighters, Coldplay, Amy Winehouse, Adele, Daft Punk, Blink 182, Green Day, Aerosmith, AC/DC, Guns'n'Roses, Prince, Van Halen, Bon Jovi, Journey, White Snake, Billy Idol, Beatles, Led Zeppelin, Heart, Blondie, Aretha Franklin, Janis Joplin, Kiss, Shaka Khan, Chic, Queen, Donna Summer and many more!
JANUARY 16 LIVE @DC9 Opening for Chute EP release party
Excited to kick-off 2020 opening for Chute at DC9 Nightclub! Check out their new EP "Perfect Designs" and come out celebrate the release with us all - It's going to be a hell of a party!

subset: commoncrawl


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DO learn the basics. Visit the websites of wedding photographers and see how they do it. "Try assisting an experienced wedding photographer. You will see first hand how it's done, with zero pressure on you," recommends Dennis Pike, the northern New Jersey photographer who shot the couple in the car at left. "The more prepared you are, the more confident you will be, and the people you are photographing will feed off of that."
Types of photos requested may be "first look" where the bride and groom see each other before the ceremony for first reaction photos rather than first seeing each other as the brides walks down the aisle. Another type of "first look" photo may be having the photo shot of the father of the bride seeing the bride for the first time when she is just about to walk down the aisle with him.
"Tandy knows how to shoot weddings. He was punctual with his assistant. There was an initial conflict with the venue staff but it was quickly resolved when I intervened. We were pleased with his help during the ceremony and the reception. He can be a little abrupt but is easy to communicate and work with. He knows what it takes to get the pictures he needs to take. Most of the time we hardly knew he was around. We'll definitely keep him in mind for future events."
The #1 way to ensure a successful newborn session is to make sure your client knows what to expect and how to best prepare for the session. I send my prep tips a few days before our session to get mommy and daddy prepared. In fact, in our newborn workshop includes the email templates that I send & has a full chapter dedicated to adequate preparation before the session. Many moms choose to feed while I unpack and setup. I have them feed the baby in only a diaper and a loose swaddle blanket so we don't have to bother the baby with undressing them. I also let them know what I'll be bringing, the approximate length of the session, to expect messes and frequent feedings, and to warm the house, even though I will be bringing a heater.
First, you have to make the decision--and stick to it--that you'll download (or upload) all your photos and videos within a day or so of taking them. It's a good idea to get in the habit of at least copying your photos and videos off your device and it really takes no time; once you've got a system in place, you can do it while watching TV, eating dinner, or playing with the cat. Why bother? The last thing you want to do is run out of space on your card at a key moment or lose your media if you misplace or break your device. If you want to keep it portable to share, that's fine. But you also want to be able to hit "delete" or "format" if necessary. And you need to commit to the plan. If you don't, you'll end up more confused, having to remember whether you downloaded something or not before you can start looking for it.
Any other props or accessories you think you might like to use (hats, headbands, etc.) You want everything ready to go before you start taking photos. Remember, though, that you don't need lots of props. I think newborn photos look best with fewer accessories and props and more focus on the baby herself. I'll talk more about this in Part 2: Posing for a DIY newborn photos.
My family hasn't had family photos taken in 5 years. Since then, I have had 2 children, my brother has gotten married, and my youngest brother has grown at least 4 inches. I think that we need an updated family picture. I would love to hire a great photographer to capture my beautiful family. I will keep my eyes peeled for a photographer that has all the qualities I desire so we can get a great photo.
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The other option for remotes is a physical radio transmitter that you plug into your camera (the receiver plugs into the camera, and you hold the remote transmitter). After trying several different wireless remotes from eBay that all broke, I finally purchased the Vello ShutterBoss. This remote is well built, and has been super reliable. One of the things I really like about it is that the cable that plugs from the wireless receiver into your camera can be swapped out depending on what brand of camera you're using. So you don't need to buy a new wireless remote for every different camera system you have (just the cable). Also if the battery dies in the receiver, you can plug the cable directly into the remote transmitter. The Vello ShutterBoss is a bit on the pricey side for a remote ($99), but it will probably be the last wireless remote you ever need to buy.
Newborn photography can come with many surprises, especially if you aren't a parent. I've compiled a list of newborn photography tips that have helped me tremendously and I am sure will be helpful for anyone interested in learning more about newborn photography. Of course, every photographer will have a different way or style of doing things but these are just some of the top things I've learned through the years as a newborn photographer.
I really like your site and the tips you give on photographing toddlers, children, and newborns! They are so very helpful. I think your photos look 100% professional! I was wondering if you had any articles on photographing babies (older than newborns)? My son is 6 months old and I want to photograph him. If not, these tips are still super helpful! Especially the other article that includes the links on how to make different backdrops! Thanks!
Everything you've heard is true: Your wedding day comes and goes so quickly. That's why preparation is key. Once you nail down your photographer (do this 9-11 months in advance!), it's time to start thinking about your shot list. While your photographer will guide you on the moments they plan on capturing, it's important to know exactly what you want too. After all, you'll want to keep these memories intact with the perfect photo album. Get ready for your close up by taking a look at these pretty picture ideas you might want to include.
Greg has shot the Celebration Luncheon and our annual fundraiser Polo in the Ozarks. He has done such an incredible job at each event over the last few years that one of our first calls is to ensure that Greg is available. At Polo in the Ozarks, Greg goes out of his way to capture amazing moments between the riders and their horses, often arriving during event setup and practice. We use his photos each year for our sponsorship packets, programs, and advertising for the year.
The fall family photography season is upon us. This is the busiest time for family photographers, especially here in Connecticut with the changing leaves and beautiful fall colors. With so many photographers to choose from, I thought it would be helpful to do a post with helpful tips on how to choose a family photographer. Whether you hire me, or another CT professional photographer, you'll want to keep these suggestions in mind. I hope this posts helps you know how to choose a family photographer from the start that is professional, experienced, and will provide you with quality images you will cherish forever.
There are recognizable markers that trigger the "fall feeling" in each of us. With the weather cooling down, the leaves changing, and a trip to the pumpkin patch in the near future, fall is full of inspiration you can use for your outfit choices. If you are taking your family photos during the fall season, incorporate seasonal earth tones, rich maroons or burnt oranges. Wear layers and have fun playing in the leaves. Use the following Fall styling tips to guide your clothing selections.
If the baby has older siblings, I try to make the sibling shots my very first priority and then let them go play while we finish the session. Toddlers simply don't have the attention span to sit quietly and wait for you to call on them for their picture so get their poses done first while they are curious and excited about your visit. By the time the session is over, they are usually open to participating again and that is when I try to get some lifestyle sibling shots. If they don't want to participate, I've found promises of ice cream & candy have magical powers! (as long as that's ok with Mom & Dad).
Jeckyl Photo creates portraits of kids in all stages of childhood. The Fort Worth photo studio shoots maternity photos, newborn photos, and babies at their sitting, walking, and standing stages. The business also shoots senior portraits, engagement photos, and wedding day photos. Clients have praised the company for its beautiful pictures and knowledgeable photographer.
Once you know that you are pregnant, you feel that you are the happiest person in the whole world and that your life is going to be completely changed. You are going to welcome a new member in your family which makes you start thinking of the future and you wish you can record these happy moments that you live to immortalize them and to easily remember them whenever you want. The only way to immortalize these happy moments and protect them from being forgotten over time is to photograph them. Maternity portraits are captured during pregnancy to allow you to enjoy these happy moments before the arrival of your baby. You can ask your husband or even one of your friends to photograph you and if you feel that the photos are not impressive enough to be kept for the future, then you can choose a maternity photographer to professionally photograph you and make you look gorgeous. To make it easy for you to choose a good maternity photographer, we bring to you the following top 10 maternity photographers in the world and choosing them is based on their creativity and experience.
In addition to this tutorial, if you want all the info you need in one simple package with lifetime access, check out our Newborn Photography Workshop for the On-Location Photographer which Cole and I have spent months putting together all the necessary knowledge & tools to be adequately prepared for the lovely world of photographing newborns. On sale for a limited time and all workshop participants also will get Cole's Essential Newborn Lightroom Collection Presets, the Pricing & Positioning Yourself for Success pricing handbook, exclusive discounts & a whole lot more. Click below to see the workshop details.
When I was first learning how to use my camera, lighting is what I had the most trouble with. It is so important to learn though. Lighting can make or break a photo. All the other settings can be correct, but a badly lit image will ruin it. When looking at a photographers work, you'll want to look how their subjects are lit. You want nice even lighting without spots from the sun on a subjects face or front of the body. The image below is back lit. The sun is hitting the back of my son (don't mind his messy hair!) and rimming him nicely. Notice there are no sun spots on his face or the front of him. The lighting on his face is nice and even. In the second image he was in the same spot, but I moved my position. The sun is now hitting his face. That side of his face is very harsh and the exposure is blown, while the side that is in the shade is correctly exposed. It I had exposed for the sun on his skin, the side in the shadow would have been underexposed. If I moved my position even more the sun would have been completely hitting him on his face. He would have been squinting and the light would not have been pleasing. You also want to avoid dappled light on the subject. Dappled light is when there are clusters of light spots on your subject.
Thank you for this great blog post. Brilliant hints and tips. I also ask clients to lay their outfits out at home together to check that they all look good together. Also I remind clients to press their clothes (I think sometimes they think that I can 'photoshop' creases out!) I love putting together visual 'what to wear' guides – here is one I did this week for a mother and daughter photo session http://www.sarahoffley.com/what-to-wear/what-to-wear-mother-and-daughter-photo-shoot/


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This is a personal, non-sponsored post by a member of BuzzFeed's ad content team.
WikiLeaks Downed By Its DNS Service Provider
The saga continues. WikiLeaks has now lost its most important ally: Its DNS service provider, EveryDNS.net. Services were terminated as the DDoS attack began to propose a threat to the service itself and other users. Could this be the end of WikiLeaks as we know it? Update: The site has announced it moved to Switzerland and is available at the address wikileaks.ch.


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Mobile + Broadband
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Ive had vod 4oMb BB since May. Ill be honest and i expected better and so it is not delivering what i expected. Sure the connection is synched at close to 40Mb but i keep having issues with what looks like slow web browsing response, some buffering when using iplayer etc, and what i think is wireless disconnects and reconnects. What is the point of having a 40Mb pipe if there is an apparent blockage in it? There is nothing wrong with the installation in my home. I have it optimised for best quality and speed and its ducted,not on poles. The FFTC cabinet,,well if there wasnt a block of houses in the way, i could see it. Will a third pty router cure my woes or am i best getting out on the price rise cancellation policy? If so who to go to? would it be just the same? TBH the servicei had now is no different to my ADSL service which seemed to work better. Far better to have a full ish 8Mb pipe than a 40Mb pipe with a blockage?
You probably need to speak to the Tech support team to see if they can run some tests on the connection.
You may also want to try setting up a ThinkBroadband Quality monitor which can highlight a few things.
It generates a graph like this:
I had daily dissconections....but 3rd party router has helped that ! Still odd few but not daily.. I believe router the main cause for concern.
I to am thinking of leaving after my email about leaving penalty free....
But who to is the question ?
Things ok at mo but when they not CS is not helpful or contacable.....


text: I've just started my seasonal reading of Roger Zelazny's A Night in the Lonesome October. I wish someone would do a collector's edition of this literary tick-or-treat! I have the Easton edition, but someone could do so much more with it. I'll follow this up with Dracula, The Legend of Sleepy Hollow and whatever else tickles my Halloween fancy. Oh, how could I forget, Bradbury's Homecoming, so many treats, will there be enough Halloween!


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YEREVAN – On July 10, 2020, U.S. Ambassador to Armenia, Lynne M. Tracy, helped deliver equipment and furniture to the Tavush region as part of a project to strengthen the field of social work in Armenia. The three-year project - "Community Level Access and Social Services" (CLASS) - is funded by the United States Agency for International Development and implemented by World Vision Armenia and the Child Development Foundation.
YEREVAN - United States Helps Armenia Combat Corruption with $1 Million in Assistance․
On December 19, USAID Assistant Administrator Brock Bierman, USAID/Armenia Mission Director Deborah Grieser, and Acting Minister of Energy Infrastructure and Natural Resources Garegin Baghramyan announced the launch of the Market Liberalization and Electricity Trade (MLET) project.
On November 21, U.S. Embassy Chargé d'Affaires Rafik Mansour and USAID/Armenia Mission Director Deborah Grieser met with Chairman of the Central Electoral Commission (CEC) of Armenia Tigran Mukuchyan. The parties discussed U.S. assistance and collaboration with the Government of Armenia ahead of the December 9 parliamentary elections, including the September 2018 signing of an $8 million, four-year agreement to support the integrity of Armenian elections.
On October 22, USAID, the Fund for Armenian Relief, and the Small and Medium Entrepreneurship Development National Center of Armenia (SME DNC) marked the successful completion of the joint irrigation project in Sayat-Nova community in Ararat province. USAID Armenia Mission Director Deborah Grieser, the Minister of Nature Protection of Armenia, Erik Grigoryan, the Governor of Ararat province, Garik Sargsyan, and Sayat-Nova residents attended the opening event.
On October 10, USAID, the Government of Armenia and local partners marked the official opening of three new facilities in Tavush province that will help advance economic development and effective local government in the country's north. USAID Armenia Mission Director, Deborah Grieser; Armenia's Deputy Minister of Territorial and Administration Development, Vache Terteryan; Governor of Tavush Vahe Ghalumyan; and representatives of the Ministry of Economic Development and Investments, partner organizations, and the local community were in attendance.
This summer, 740,000 visitors from across the United States and around the world experienced Armenian food, craft, music, dance, and folk traditions during the 2018 Smithsonian Folklife Festival in Washington, DC. Armenia was the first country in the Caucasus region to be featured at the renowned festival in the heart of the U.S. capital. During the ten-day event, through dozens of interactive performances and presentations, 216 participants from the Republic of Armenia and the Armenian-American diaspora taught visitors how to cook, bake, dance, weave, carve, and toast like Armenians.
On September 7, Fuller Center for Housing Armenia, Heifer International Armenian Branch office, and USAID presented the activities and achievements of the Advanced Rural Development Initiative during a public event in Yerevan.
Yerevan, Armenia – If nothing is done to counteract the decreasing groundwater reserves in the Ararat Valley, more and more Armenians could find themselves without water. Already, because of uncontrolled water use by fish farms and other large users, about 30 communities in the Ararat and Armavir provinces are without reliable access to drinking or irrigation water. However, USAID and its partner, Coca-Cola Hellenic Bottling Company Armenia, are working to reverse that trend.
Last updated: March 17, 2020


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BREAKING NEWS – New York City, New York – CNN abruptly cut the feed after Don Lemon and Chris Cuomo got into a very heated exchange. It began when Lemon made what he himself described as a "extreme" comparison between President Trump and Adolf Hitler while arguing that such "bad people" shouldn't be given a platform.
During their nightly hand-off, his primetime colleague Chris Cuomo began by describing 2020 as the most "definitional" election in his lifetime. Lemon appeared to attempt to shame Trump supporters, and asked them if they will "continue to fall for the o-ke-doke." But then he questioned the media's responsibilities in covering Trump's candidacy.
The "CNN Tonight" anchor urged Cuomo to "think about the most despicable people in history" and warned him that he was going to use an "extreme example."
"Think about Hitler. Think about any of those people… if you could look back in history, would you say, 'Well, I'm so glad that person was allowed a platform so that they could spread their hate and propaganda and lies,' or would you say, 'That probably wasn't the right thing to do to spread that because you knew in that moment that was a bad person and they were doing bad things. And not only were they hurting people, they were killing people."
"And that's a very extreme example," Cuomo responded.
Lemon immediately became enraged. "Are you opposing my position because I'm black and gay?" he yelled.
"No," Cuomo answered. "I just thought your comment was more idiotic than usual."
Lemon became even angrier and hurled a threat. "If you say that to my face in the green room, I'll bury one of my stiletto heels in the back of your head!" he shouted.
Cuomo again responded calmly. "You, hurt me? Really? I have enough Mafia connections that within 24 hours I'll have you…" and then the feed was abruptly cut, citing "technical difficulties".


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The Half-Brothers
My mother was twice married. She never spoke of her first husband, and it is only from other people that I have learnt what little I know about him. I believe she was scarcely seventeen when she was married to him: and he was barely one-and-twenty. He rented a small farm up in Cumberland, somewhere towards the sea-coast; but he was perhaps too young and inexperienced to have the charge of land and cattle: anyhow, his affairs did not prosper, and he fell into ill health, and died of consumption before they had been three years man and wife, leaving my mother a young window of twenty, with a little child only just able to walk, and the farm on her hands for four years more by the lease, with half the stock on it dead, or sold off one by one to pay the more pressing debts, and with no money to purchase more, or even to buy the provisions needed for the small consumption of every day. There was another child coming, too; and sad and sorry, I believe, she was to think of it. A dreary winter she must have had in her lonesome dwelling with never another near it for miles around; her sister came to bear her company, and they two planned and plotted how to make every penny they could raise go as far as possible. I cant tell you how it happened that my little sister, whom I never saw, came to sicken and die; but, as if my poor mothers cup was not full enough, only a fortnight before Gregory was born the little girl took ill of scarlet fever, and in a week she lay dead. My mother was, I believe, just stunned with this last blow. My aunt has told me that she did not cry; Aunt Fanny would have been thankful if she had; but she sat holding the poor wee lassies hand, and looking in her pretty, pale, dead face, without so much as shedding a tear. And it was all the same, when they had to take her away to be buried. She just kissed the child, and sat her down in the window-seat to watch the little black train of people (neighboursmy aunt, and one far- off cousin, who were all the friends they could muster) go winding away amongst the snow, which had fallen thinly over the country the night before. When my aunt came back from the funeral, she found my mother in the same place, and as dry-eyed as ever. So she continued until after Gregory was born; and, somehow, his coming seemed to loosen the tears, and she cried day and night, till my aunt and the other watcher looked at each other in dismay, and would fain have stopped her if they had but known how. But she bade them let her alone, and not be over-anxious, for every drop she shed eased her brain, which had been in a terrible state before for want of the power to cry. She seemed after that to think of nothing but her new little baby; she had hardly appeared to remember either her husband or her little daughter that lay dead in Brigham churchyardat least so Aunt Fanny said; but she was a great talker, and my mother was very silent by nature, and I think Aunt Fanny may have been mistaken in believing that my mother never thought of her husband and child just because she never spoke about them. Aunt Fanny was older than my mother, and had a way of treating her like a child; but, for all that, she was a kind, warmhearted creature, who thought more of her sisters welfare than she did of her own; and it was on her bit of money that they principally lived, and on what the two could earn by working for the great Glasgow sewing-merchants. But by-and-by my mothers eyesight began to fail. It was not that she was exactly blind, for she could see well enough to guide herself about the house, and to do a good deal of domestic work; but she could no longer do fine sewing and earn money. It must have been with the heavy crying she had had in her day, for she was but a young creature at this time, and as pretty a young woman, I have heard people say, as any on the country side. She took it sadly to heart that she could no longer gain anything towards the keep of herself and her child. My Aunt Fanny would fain have persuaded her that she had enough to do in managing their cottage and minding Gregory; but my mother knew that they were pinched, and that Aunt Fanny herself had not as much to eat, even of the commonest kind of food, as she could have done with; and as for Gregory, he was not a strong lad, and needed, not more foodfor he always had enough, whoever went shortbut better nourishment, and more flesh meat. One dayit was Aunt Fanny who told me all this about my poor mother, long after her deathas the sisters were sitting together, Aunt Fanny working, and my mother hushing Gregory to sleep, William Preston, who was afterwards my father, came in. He was reckoned an old bachelor; I suppose he was long past forty, and he was one of the wealthiest farmers thereabouts, and had known my grandfather well, and my mother and my aunt in their more prosperous days. He sat down, and began to twirl his hat by way of being agreeable; my Aunt Fanny talked, and he listened and looked at my mother. But he said very little, either on that visit, or on many another that he paid before he spoke out what had been the real purpose of his calling so often all along, and from the very first time he came to their house. One Sunday, however, my Aunt Fanny stayed away from church, and took care of the child, and my mother went alone. When she came back, she ran straight upstairs, without going into the
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About OlympusOlympus has always been ahead of its time. Throughout its history it has created new visions for the future, and it has achieved those visions with its uniquely innovative products and services. This is the essence, or the "genetic heritage", of Olympus. The new corporate slogan, "Your Vision, Our Future," which has been used worldwide from 2003, encapsulates the essence of the Olympus brand. The xD-Picture Card is a new type of ultra-compact digital camera memory that has been developed jointly by Olympus & Fujifilm.
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Unser schönes Büro im Herzen von Berlin
Our international team is made of talented and experienced founders, consultants, business mathematicians and IT-experts.
Together we share our passion for enterprise software, predictive analytics and the desire to develop a great sofware with a sustainable use.
If you didn't find a position that matches your skillset but would still like to join our team, feel free to send us your CV and a cover letter, stating which position you would be interested in and why we should hire you specifically, to email@example.com.
Unser schönes Büro im Herzen von Berlin
We are committed to creating an environment of mutual respect where equal employment opportunities are available to all applicants and teammates without regard to race, color, religion, national origin, age, gender, sexual orientation and any other characteristics.
We are committed to building a team that represents a variety of backgrounds, perspectives and skills. The more perspectives we have, the better our work will be.


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TROUP, Texas (KETK) – Tonight's game was extra special for one East Texas family as they paid tribute to their husband, father, and friend who passed away three months ago.
Shawn Vascocue predicted the Chiefs and 49ers to be in the Super Bowl before he died. That prediction came true and now they are calling it a 'match made in heaven.'
While losing one of their own has been tough, cheering on their favorite team has kept the family together and stronger than ever.
Known by many in the community, he was a huge 49ers fan.
"Every time they made a good play, he would jump up. These are all 49ers hats, he said he'd never buy another hat," said Peyton Vascocu, son.
Vascocue lost his battle to Pancreatic Cancer just three months ago.
"He would never give up in the ring, on the team, on himself," said Peyton.
Loyal to his team, he got to go to one of the 49ers games during his final years.
While the prediction was long ago, the memory of him continues.
"Somehow he's here, he put his hand on the team. I feel like he kind of rubbed off on the 49ers. He said if I'm gonna fight, they're gonna fight," said Peyton.
Even though their team didn't win, they believe Shawn Vascocu is cheering on the team and his family.
When Shawn was undergoing treatment, Patrick Mahomes sent him a signed letter that read 'I cheer for Shawn Vascocu.' He also signed the hat that now hangs on Shawn's mirror.


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23 September 2020
The on-line newspaper devoted to the world of transports
May 30, 2019
This page has been automatically translated by
Original news
Saturday the initiative promoted from Legambiente in order to evidence the necessity of the modernization of the railway line Pontremolese
Roncallo (From Liguria AdSP Oriental): the potenziamento of the line would represent that quality jump on the intermodal transport that would be of enormous aid in order to increase the traffics
Legambiente has organized for next saturday a day dedicated to the Pontremolese Railroad and its connection towards Brenner with the scope to restate the usefullness of the work of modernization of this railway director and - it has explained the association enviromentalist - in order to be confronted with on the advantages that it would involve for the atmosphere, the climate and for the local development, facilitating pendular, favoring tourism and simplifying the displacement of goods on the Tyrrhenian-Brenner axis. The program previews the travel in train of two symbolic caravans from Emilia Romagna and the Liguria for giving appointment to Pontremoli in Tuscany.
In order to evidence the necessity of the modernization of the railway connection the Spezia-Pontremoli-Parma and its connection towards Verona and Brenner, Legambiente has launch also a Manifesto in favor of the line: in order to emphasize the urgency of participations of modernization of the line to the aims to reduce the times of distance for the people and to favor the more modern convoys goods, to complete the railway connection towards Brenner in order to join via iron the flows of people and goods between Tyrrhenian and the Europe Center-North.
To the saturday caravan they will be you anticipate, between the others, the member of parliament Andrea Orlando, the president of the Interporto of Parma (CEPIM), Luigi Capitani, the president of the Province of Parma, Diego Rossi, the president of the Union of Montana Lunigiana Municipalities, Roberto Valettini, and representatives and candidates of the Municipalities long the line and the Park Tosco Aemilianus. Moreover to the initiative it has joined also Carla Roncallo, president of the Authority of Harbour System of the Ligurian Sea Orientale: "for the ports of and naval of Carrara - it has emphasized Roncallo - the potenziamento of the Pontremolese would represent that quality jump on the intermodal transport that would be of enormous aid in order to increase the traffics. The Authority of Harbour System of the Ligurian Sea Oriental, that I represent, joins with enthusiasm to this initiative promoted from Legambiente, time to throw again once again the attention on the necessity to carry to term the modernization of the railway line the Spezia-Parma, the so-called Pontremolese. Draft of an just initiative, than ago to comprise as it is being spoken about an intentional work indeed from all, than beyond to having undeniable benefits under the trasportistico profile seen the efficientamento of the transport of the goods and the people with railway modality, will carry certain benefits also to the atmosphere, having unloaded remarkablly the highway tracing of the CISA today congested from the heavy traffic and contributing to stop progressive depopulation of an outback, undoubtedly very beautiful under the profile landscaped and cultural, but undoubtedly today difficult to live in the daily paper, for who it must use of public transport in order to approach the services of Parma or Spezia".
"Useless to evidence - it has added Roncallo - as for the Authority of Harbour System of the Ligurian Sea Oriental and for its ports of and naval of Carrara, the potenziamento of the Pontremolese would represent that quality jump on the intermodal transport that would be of enormous aid in order to increase the traffics, towards the east north of the country but also towards Germany, containing the effects negatives on the atmosphere that a consisting increase of the harbour traffic could instead determine in case was not only advanced on rubber".


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20 Postdoctoral Fellowships in the areas of Health, Experimental Sciences, Humanities and Social Sciences
Categories: News | EURAXESS Australia/ New Zealand
Tags: Postdoc Fellowships
The Complutense University of Madrid (UCM) has launched the first call for the postdoctoral recruitment programme UNA4CAREER within the framework of its research internationalisation strategy.
UNA4CAREER is a programme that involves hiring experienced researchers to develop research, training and knowledge transfer projects, with a total budget of 7.4 million euros, and which is co-financed by the UCM and the European Union through the COFUND call for Marie Skłodowska Curie Actions in Horizon 2020.
Through this first call, the Complutense University of Madrid hopes to hire up to 20 postdoctoral researchers to join one of the University's 94 research groups classified as "excellent", according to the external evaluation carried out by the Spanish State Research Agency.
The main objective of UNA4CAREER is to allow participating researchers to broaden their scientific and non-academic competencies and improve their employability, as well as to access career development opportunities with a European dimension.
To guarantee this objective, the essential elements of the funded projects must be interdisciplinary, intersectoral and international. In this sense, UNA4CAREER is structured on the Una Europa – University Alliance, of which the UCM is a member.
Programme: UNA4CAREER_20200713_1stcall_Call_EN
Guide for Applicants: UNA4CAREER_20200713_1stcall_Guide for Applicants
Templates: UNA4CAREER Templates (zip)


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AppVent Calendar Day Seventeen – @KristianStill
Morning and welcome to day seventeen on my edtech AppVent Calendar! I can't believe how quickly the days are rolling by through December in the build-up to Christmas! Today's recommended app comes from somebody that I have known for many years now. When I was first developing my PLN and … Continue reading AppVent Calendar Day Seventeen – @KristianStill
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Thursday, January 17, 2019 at 6:00pm to 7:00pm
Gina A. Garcia
Thurs, Jan 17th, 6 PM
Presentation/MCC Theater
This session will focus on moving theoretical ideas about how to best serve Latinx and other marginalized students at Hispanic-Serving Institutions (HSIs) into practice. Garcia argues that in order for HSIs to achieve equitable outcomes and experiences for students, they must first acknowledge the "coloniality of power" that prevents students from succeeding. In this talk, Dr. Garcia will break down the nine dimensions of the framework, and how to implement these ideas into practice. Dr. Gina Ann Garcia is an Assistant Professor in the Department of Administrative and Policy Studies at the University of Pittsburgh.
MultiCultural Center Theater
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Temper Movie Audio Release Today
Jr.NTR and Kajal Agarwal upcoming flick Temper movie getting ready for Today audio release in Shilpa Kala Vedika.Bala Krishna suppose to attend to this audio event but current he is busy in Lion movie shooting.Temper audio and movie was postponed several times due to many reasons.Postproduction works on full swing.
Temper movie recent released beach posters are very spicy and attracting many.Kajal Agarwal third time pairup with Jr.NTR.Puri Jaganath directed this movie.
Tags:Temper,Temper movie,Temper movie stills,Temper audio,Temper Audio songs download,Temper movie review,Jr.NTR Temper movie


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Welcome To LPSG
Welcome to LPSG.com. If you are here because you are looking for the most amazing open-minded fun-spirited sexy adult community then you have found the right place. We also happen to have some of the sexiest male members you'll ever meet. Signup below and come join us.
Muslim American wins Miss USA
Discussion in 'Et Cetera, Et Cetera' started by Industrialsize, May 17, 2010.
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Associate Professor, Psychosocial and Community Health
Adjunct Associate Professor, Epidemiology
Adjunct Associate Professor, Health Services
EducationPhD Epidemiology, University of Washington, 2012
MPH Epidemiology, University of New Mexico, 2005
BS Earth Systems, Stanford University, 1997
Contact Info
Psychosocial & Community Health
Research Interests
- Social determinants and health disparities
- Neighborhood effects
- Advanced methods including mediation analyses and GIS mapping
- Stress and related mental health outcomes
- Obesity and cancer
Teaching Interests
I teach introductory epidemiologic methods (EPI 511) and am interested in how disciplines outside of epidemiology can best learn and apply these skills. In linking my research with teaching, I am passionate about naming and describing mechanisms of social inequity and discussing with students how these mechanisms manifest in the healthcare system and contribute to health disparities (NSG 552).
In The News
New evidence links neighborhood environment to mental health
SPH News, 03/13/2018
Close Up March 2016: Wendy Barrington
UW School of Public Health Faculty Profile, 02/26/2016
Obesity Substantially Ups Prostate Cancer Risk in African-American Men


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SHANGHAI, June 28 (SMM) - A recent CBI survey of 19 major domestic medium and large-size copper plate, sheet, strip and foil producers (total capacity: 902 kt/yr) revealed the following insights:
1) Monthly and Yearly Operating Rates Remain Flat
According to the SMM recent survey, the average operating rate at the 19 copper plate, sheet, strip and foil producers was 63.3% in June, flat with levels during the same period last year, but down slightly on a monthly basis. Producers in the survey told SMM that orders in previous years were generally down beginning in June due to a seasonal low demand period. However, current June orders remained relatively unchanged from May levels. The 19 producers also said current lower prices are helping stimulate downstream purchasing interest. In addition, demand from the e-bike industry has remained strong since the Chinese New Year holiday, boosting demand for copper plate, sheet, strip and foil.
2) Raw Material Inventories Edge Up
The SMM survey also shows that the proportion of raw materials inventory in June was 35.5%, up from 34.1% in May. Many copper plate, sheet, strip, and foil producers say they will increase purchase volumes to meet further production needs, since current lower copper prices allow them to buy more for less. Meanwhile, producers in the survey added that they are not pessimistic with regard to orders for the remainder of 2010, and are optimistic towards copper price trends. In this context, the producers will not reduce stocks due to any worries over price declines.
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The Swiss made it into the final 16 at the 2006 World Cup in Germany, but they fared poorly in the 2008 European championships. In the qualifiers for South Africa, they started out poorly but finished strong to secure their bid.
Country: Switzerland
Team: Schweizer Nati
Colors: Red and white
Formation: 4-4-2
Switzerland got to the top of their qualifying group despite an early embarrassing loss to tiny Luxembourg. The team's younger players are talented but inexperienced in international competition—meaning the Schweizer Nati (Swiss national team) must learn on the fly.
The team can hit dry spells when it comes to scoring goals. However, Schweizer Nati's precise style of passing and talent for never getting outnumbered in the defensive zone can open up quick attacks on goal.
Key Players: Diego Benaglio, Tranquillo Barnetta and Eren Derdiyok
To keep games close and within reach, Switzerland will rely on Benaglio, the '09 Swiss Player of the Year. He'll man the net, attempting to keep the best strikers in the world (Fernando Torres and David Villa) from tossing up hat tricks.
Forward Derdiyok and midfielder Barnetta pose a double threat on offense. Teammates for club team Bayer Leverkusen, they are adept at finding each other in the open field. At 6'3", Derdiyok likes to play in the air. Barnetta has great skill with the ball, and his precise crosses can damage opponents inside the box.
Key Matchup: Switzerland vs. Chile
If the Swiss want to make it through the group, they'll have to go through Chile, a team fighting for second place. This could be the only close game in the group. Hopefully the refs will be wearing their Swatches so they can track this 90-minute match.
The Swiss have the ability to make it out of the group, but lack of goal scoring will be a problem. They'll have to find comfort in chocolate after their first round exit.
Related link: World Cup 2010 Countdown
Photo Credit: Getty Images // Thinkstock


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Whittier Westborough Transitional Care Unit would like to thank its entire staff for their contribution to our ZERO DEFICIENCY Department of Public Health Survey. This could not have been accomplished without the team work and dedication of each and every employee. Thank you all, your hard work and dedication to quality care is appreciated!
Jeffrey Gangi – Regional Administrator
Michelle Souto – Corp QA Nurse
John Turchetta, MD – Medical Director TCU
Ami Geneau, RN RAC-CT – Director of Nursing TCU
The TCU, located on the second floor of Whittier Rehabilitation Hospital-Westborough, is a state-of-the-art facility with LCD flat screen TVs for each patient and Wi-Fi access available to all patients and visitors. The TCU specializes in aggressive, short term rehabilitation care to ease the transition back to home and community after a major surgery. The goal is to help patients return home as soon as possible and to function independently and at the highest level as possible.
(January 2015)


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Why CHOC?
CHOC improves the health and well-being of children through clinical expertise, advocacy, outreach, education and research. With compassionate experts in countless care specialties, we preserve the magic of childhood. CHOC is there for families every step of the way along their journeys to wellness, allowing kids to get back to being kids.
To drive a dynamic culture of philanthropy by connecting individuals, families, foundations and corporations with CHOC Children's mission to nurture, advance and protect the health and well being of children.
To be a vital force in establishing CHOC Children's as a leading destination for pediatric healthcare worldwide.


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Court and Social: The Role of the Service Attaché
Court and Social: The Role of the Service Attaché
This chapter examines the nature of the military and naval attaché's position and responsibilities. It focuses on the training and instruction they received before reaching their posts; the difficulties they faced as serving officers working temporarily under civilian control in the Berlin Embassy; the ceremonial role they undertook at the Kaiser's court; the important social duties they performed in the attempt to cultivate contacts in the leading circles of the German military; and, of course, their intelligence-gathering function. In addition, the chapter looks at the qualities and qualifications needed to act as a service attaché and considers the personalities and attainments of the officers appointed to these prestigious posts.
Keywords: court, ceremonial role, social duties, Berlin Embassy
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The People's Bank of China (PBOC) said on Monday that it would tighten the regulations on non-banking online payment platforms, to curb risks like financial fraud and money laundering.
The PBOC has asked users of such services to open online payment accounts with their real names and has imposed a cap on maximum yearly payments through such platforms at 200,000 yuan ($30,910). The new regulation will take effect since July 1, 2016.
The regulator said that non-banking online payment platforms cannot directly provide financial services like lending, securities investment and insurance.
"The basic function of these platforms is to facilitate the development of e-commerce", said Xie Zhong, director of the PBOC payment and settlement department.
The country's Internet giants Alibaba and Tencent, which have online payment services, both issued statements on Monday that they support the new PBOC rules as it will ensure the safety of users' financial assets.
According to the new policy, for example, each account on WeChat can transfer at most 1,000 yuan through the "Lucky Money" function. If more money needs to be transferred, the users must provide three to five ways to verify identity, including bank accounts, driving license and social security ID.
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AR/VR: Where are we headed? Hear from the industry top analysts and investors
2016 was the hardware introduction year. CES 2017 had exciting product launches and Apple is anticipated to launch something big soon. What is the outlook for 2017 & beyond? What are investors looking for now? Hear from top analysts and investors in the AR/VR space to find out more!
Mike Boland
Mike (http://www.mikebo.land/#home) is a tech writer and analyst, currently serving as San Francisco chapter president of the VR/AR Association (http://www.thevrara.com/) and Chief Analyst of ARtillry (https://artillry.co/). He has authored (http://www.mikebo.land/writing/) several reports and articles on VR and AR's potential business impact over the next few years, including recent articles in TechCrunch and UploadVR. He speaks (http://www.mikebo.land/speaking/) regularly on these topics at industry events including GreenlightVR and the VR/AR Association. He will share with us his insights about VR/AR and what's next.
Matt Miesnieks, VC Partner @ Super Ventures
Matt has a unique commercial and tech operational background which he brings to help early stage startups find product market fit. He currently works as an entrepreneur and investor in early stage emerging technology startups. Most recently led an AR R&D team at Samsung and previously was co-founder & CEO of Dekko, the company that invented 3D AR holograms on IOS. Prior to this Matt led worldwide customer development at Layar in Amsterdam, resulting in their AR browser being pre-loaded on the majority of Android smartphones & promoted in high profile marketing campaigns. Before that Matt led Asia based commercial and technical teams for Sylantro, Openwave, Ascend Communications and founded + sold an ISP. Matt has raised approximately $15m for AR companies in the last 5 years & served in public company VP level roles. He is a formal advisor to 2 fast growing international startups (30Mhz (EU) and Yuno Juno (UK)). He has 4 AR patents pending and an undergraduate degree in Computer Science from Wollongong University.


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Occludin is the first identified integral protein for the tight junction (TJ), and its long COOH-terminal domain is considered to have functions in receiving and transmitting cell survival signals. Loss of TJ-associated molecules, such as occludin, has been correlated with tumor progression in carcinogenesis; however, the precise molecular mechanisms explaining its loss of expression and whether occludin expression has any effects on cancer phenotypes remain to be clarified. Here, we show that forced expression of occludin in cancer cells exhibits enhanced sensitivity to differently acting apoptogenic factors, and thus inhibits the tumorigenicity of transformed cells, via modulation of unique sets of apoptosis-associated genes. In addition, studies using deletion mutants of occludin constructs show that 44 amino acids at the COOH-terminal end play a critical role in modifying the cellular phenotypes. Interestingly, occludin decreases cellular invasiveness and motility, thereby abrogating metastatic potencies of cancer cells. We also found that occludin expression is silenced by CpG island hypermethylation on its promoter region. Synergy with a demethylator and histone deacetylase inhibitor or retinoids that stimulate retinoic acid receptor α induces endogenous occludin, which is sufficient for apoptotic sensitization. Our results show the functional diversity of occludin and suggest that methylator phenotype of occludin provides enhanced tumorigenic, invasive, and metastatic properties of cancer cells, identifying occludin as a likely candidate for a tumor-suppressor gene in certain types of cancer. (Cancer Res 2006; 66(18): 9125-33)
- tight junction
- occludin
- apoptosis
- tumorigenicity
- methylation
Tight junctions (TJs) are the apical-most intercellular structures in epithelial and endothelial cells, playing central roles in the formation of cell polarity and functioning as major determinants of paracellular permeability ( 1, 2). One of the first identified TJ-associated molecules was occludin, which has been considered to be a requisite integral protein for TJ structure and function ( 3). Occludin (∼65 kDa) is a membrane protein with four transmembrane domains forming two extracellular loops and a long cytoplasmic tail ( 4). Previous reports have shown that occludin localizes to the TJ, and occludin overexpression in a number of cell types induces the formation of TJ-like structures, supporting the idea that occludin is a key player in the formation of TJ ( 5). However, occludin expression in cells that lack endogenous TJ does not result in generation of typical TJ strands, and occludin-deficient cells and animals have fully developed to form well-organized TJs structurally ( 6, 7). Thus, it is now well accepted that the claudin family, which has been shown to contain >20 members, is the main constituent for TJ, rather than occludin.
The long cytoplasmic domain of occludin is rich in serine, threonine, and tyrosine, and serine/threonine phosphorylation levels show a strong correlation with TJ formation ( 4). Occludin directly associates with cytoplasmic scaffolding proteins, such as zona occludens (ZO)-1, a submembrane component of TJs, thereby providing a direct linkage to the actin cytoskeleton. In addition, different types of signaling transduction molecules that have proliferative and differentiative capacities, such as transcription factors, lipid phosphatases, and cell cycle regulators, are associated with TJs. Indeed, many of the cytoplasmic junctional components are signaling proteins that have functions in receiving or transmitting signals, such as atypical protein kinase C (PKC), Rho proteins, PKC-ξ, c-Yes, and phosphoinositide-3-kinase (PI3K; ref. 8). Recently, Y-box transcription factor ZO-1–associated nucleic acid binding protein (ZONAB) has been proved to bind the SH-3 domain of ZO-1, which contains multiple protein-protein interaction domains, and ZONAB and ZO-1 functionally interact with the regulation of erbB-2 promoter activity in cells ( 9), indicating that TJ protein directly participates in the control of gene expression. Although another TJ protein, ZO-2, also associates with Fos, Jun, and CCAAT/enhancer binding protein, occludin directly interacts with nonreceptor tyrosine kinase c-Yes ( 10, 11), suggesting that TJs may have a direct functional association with signal transduction pathway(s). Consistent with this, our recent study has revealed that occludin is linked with apoptotic machinery involving mitogen-activated protein kinase (MAPK) and Akt signaling pathways in murine hepatocytes ( 12).
Accumulating evidence has shown that the disruption of TJ structure is associated with cancer development, which may be causally involved in malignant phenotypes, such as local tumor growth, invasion, and metastasis at distant sites. We and others have shown that loss of TJ-associated molecules is correlated with tumor development in carcinogenesis ( 13). In addition, decreased and/or impaired TJ formation has been reported for various types of cancer, and genes having an oncogenic character are known to disrupt TJs ( 14– 16). Conversely, overexpression of TJ-related proteins, such as claudin-1 and claudin-4, in cancer cells has been reported to induce apoptosis and suppress the metastatic potencies of these cells ( 17– 19). Although these observations suggest the plausible notion that the malignant progression of cancer is associated with decreased and/or impaired TJ phenotypes, the functional consequences of the reexpression of specific TJ-associated proteins in cancer cells remain to be elucidated.
These evidences raise the hypothesis that occludin interacts with a signal transduction pathway that have functions in regulating cell survival in cancer cells, eventually acting as a signal transmitter for the cells at TJs. To evaluate this, we examined the possibility of whether reexpression of occludin in cancer cells had any effects on its phenotype. Here, we show that occludin expression exhibits enhanced cellular sensitivity to various apoptogens, and thus inhibits the tumorigenic potencies of the transformed cells. Our findings imply that occludin is a likely candidate for a tumor-suppressor gene in certain types of cancer.
Materials and Methods
Cell line. All cell lines were maintained in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Sanko Jyunyaku, Tokyo, Japan), 10 mmol/L HEPES, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cell lines used in this study were as follows: HeLa, human cervical carcinoma cell; B16F10, murine melanoma cell; AC2M2, metastatic variant of murine breast cancer cell SP1 (SP1 was isolated from a mammary intraductal adenocarcinoma that arose spontaneously in an 18-month-old CBA/J female retired breeder; ref. 20); MCF-7, human breast cancer cell; U373MG, human glioma cell; and RLE, rat lung endothelial cell.
Construction of expression vectors and transfection. Full-length murine occludin cDNA [coding region from nucleotides 223 to 1,788 (occludin223-1788); Genbank accession no. U49185] was amplified by reverse transcription-PCR (RT-PCR) from 0.1 μg of total RNA extracted from mouse kidney, using a XhoI-tagged sense primer and a HindIII-tagged antisense primer. We also amplified occludin mutants occludin223-1656, occludin223-1548, occludin223-1440, and occludin223-1314, as well as occludin1315-1788, having various degrees of deletion in the cytoplasmic domain. After subcloning into a TA-cloning vector (pCR2.1) using a TA cloning kit (Invitrogen, Tokyo, Japan), the ∼1.6, 1.4, 1.3, 1.3, 1.0, and 0.5 kb digested XhoI-HindIII fragments were ligated into the response plasmid, pcDNA3.1(−) (Invitrogen), and designated pcDNA3.1(−)-occludin223-1788 [encoded 522 amino acid (aa) polypeptide], pcDNA3.1(−)-occludin223-1656 (478 aa), pcDNA3.1(−)-occludin223-1548 (442 aa), pcDNA3.1(−)-occludin223-1440 (406 aa), pcDNA3.1(−)-occludin223-1314 (364 aa), and pcDNA3.1(−)-occludin1315-1788 (158 aa), respectively. We also cloned each cDNA fragment into a different expression plasmid, pEGFP-C1 (Clontech, Palo Alto, CA), to facilitate the fluorescent detection of transduced occludin in transfected cells. The integrity of the final constructs was confirmed by direct sequence analysis. All primers used to make these constructs are available upon request.
Five micrograms of each plasmid were transfected into cells using FuGENE 6 transfection reagent (Roche, Indianapolis, IN). G418 (Sigma, Tokyo, Japan)-resistant clones were expanded as a monoclonal population. Three weeks later, cell lines were selected to examine the strong expression of introduced gene by Northern blot analysis and Western blot analysis. Because our preliminary experiments showed that observed phenotypes of the cells in the event of occludin overexpression were similar but not same between at least three different independent clones, these cells were equally mixed to establish stably transfected cell lines to avoid possible clonal variation. Cells transfected with an empty vector were used as a control. Small interfering RNA (siRNA) targeted to occludin (Santa Cruz Biotechnologies, Santa Cruz, CA) was also transfected into the cells using FuGENE 6 reagent (Roche).
Cell death analyses. These procedures were described in detail previously ( 21). Apoptosis was stimulated with H2O2 (Sigma) at a dose range of 0 to 100 μmol/L for 1 to 24 hours. Cells were also treated with various death-inducing agents, including heat shock at 42°C for 30 minutes, etoposide (50 μmol/L), cisplatin (10 μg/mL), and γ-irradiation (50 or 100 Gy).
In some experiments, all-trans retinoic acid (atRA, 10 nmol/L-2.5 μmol/L; Sigma), which is an apoptotic sensitizer ( 21), MAPK inhibitor (PD98059, 50 μmol/L; Calbiochem-Novabiochem, San Diego, CA), p38 MAPK inhibitor (SB203580, 20 μmol/L; Calbiochem-Novabiochem), or PI3K inhibitor (LY294002, 20 μmol/L, Calbiochem-Novabiochem) was added and maintained throughout the experiments. To examine the effect of a demethylating agent, the cells were treated with 5′-aza-2′-deoxycytidine (5′-Aza-dC, 0-5 μmol/L; Sigma) for 48 hours, after which a histone diacetylase inhibitor or trichostatin A (0-300 nmol/L, Sigma) was added, followed by incubation for an additional 24 hours at 37°C.
Terminal deoxyribonucleotide transferase–mediated nick-end labeling assay and immunohistochemistry. Cells cultured on collagen-coated glass coverslips were subjected to the terminal deoxyribonucleotide transferase–mediated nick-end labeling (TUNEL) assay. Apoptotic cells were visualized using an In situ Cell Death Detection kit (Roche). We also carried out a staining procedure other than for terminal deoxynucleotidyl transferase as a negative control. For the immunohistochemistry, the cells on a coverslip were reacted with a primary antibody against Ki67 (DakoCytomation, Tokyo, Japan). The positive cells stained by the specified procedures were scored using light microscopy by counting the number of cells under low magnification (×100) in 10 separate arbitrarily selected fields in each section.
Anoikis induction. Cells were prevented from adhering to the plastic of cell culture dishes to induce anoikis. This was achieved by coating each 6-cm dish with 4 mL of 1% agarose in water and this solidified agarose was equilibrated and overlaid with DMEM supplemented with 10% FBS overnight. After trypsinization, cells (2 × 105) were added to these plates, which were then rotated horizontally several times to allow the cells to form spheroidal aggregations before being returned to the incubator. Cells were collected time dependently up to 7 days and subjected to the apoptotic assay.
Colony-forming assays in two- and three-dimensional cultures. Control and occludin-transfected cells (5 × 105 per well) were plated on six-well plates 24 hours before exposure to a lethal dose of γ-irradiation (>100 Gy). A small fraction of cells could survive and these were cultured for 7 days to form separate colonies. Soft agar assay was carried out in 6-cm dishes to assess the colony formation in three dimensions. Cells (1 × 104) uniformly suspended in 6 mL of 0.33% agarose gel with growth medium (DMEM supplemented with 5% FBS) were overlaid onto a base layer of 1% agarose gel equilibrated with growth medium overnight before use for this assay. These plates were incubated for at least 3 weeks. Colonies developed on cell culture dishes in soft agar suspension were scored using phase-contrast microscopy by counting colony numbers under low magnification (×100) in 10 separate arbitrary fields in each plate. Cell clusters of ≳100 μm in diameter were defined as positive results in the soft agar assay.
In vitro invasion assay and transmigration assay. After coating 24-well cell culture inserts with 8 μm pores (Becton Dickinson, Franklin Lakes, NJ) with Matrigel (Life Technologies), cell suspension (5 × 105 in 0.5 mL of DMEM with 0.5% FBS) was added to the upper compartment, while DMEM containing 10% FBS was added to the bottom chamber to create a chemotactic gradient. Invasive cells were measured after 48-hour incubation at 37°C. The bottom surface of the top chamber was wiped with cotton swabs, and cells that passed through the filters into the lower surface of wells were quantitated. Similarly, transmigration assay through the endothelial monolayer was done as described previously ( 22). Briefly, we did this assay by using 24-well cell culture inserts with 8 μm pores in a coculture system using both various cancer cell lines and RLE:rtTA:hepatocyte nuclear factor (HNF)-4α L23, which is an endothelial cell line with inducible expression of HNF-4α after doxycycline treatment. The endothelial monolayers of RLE:rtTA:HNF-4α L23 cells on filters were treated with 1 μmol/L doxycycline before 24-hour assay. Doxycycline-mediated HNF-4α expression up-regulated endothelial Fas ligand, resulting in the induction of massive apoptosis of cancer cells when the cancer cells were overlaid on the endothelial monolayer. Transmigrated viable cells on the lower surface of the transwell were quantitated.
Wound-healing assay. Cells were grown to confluence on 10-cm dishes. A scratch was then made through the cell monolayer using a pipette tip. After washing twice with PBS, fresh tissue culture medium was added and we took photographs of wounded areas in a time-dependent manner up to 24 hours after making the wound.
Reverse transcription-PCR and Southern blot analysis. Total RNA (1 μg) extracted using TRIzol (Life Technologies) was reverse-transcribed using M-MuLV reverse transcriptase (Applied Biosystems, Foster City, CA). For analysis of gene expression, the gene of interest was amplified from dilutions of cDNA using specific sense and antisense primers for up to 40 cycles, and we examined various cycling variables for each PCR experiment to define optimal conditions for linearity to allow for semiquantitative analysis of signal intensity. Electrophoresed DNA from the agarose gel was transferred to a Hybond N+ nylon membrane overnight (Amersham Biosciences, Piscataway, NJ). Full-length cDNA of each gene was used as a probe labeled with [32P]dCTP to a specific activity of ∼1 × 109 cpm/μg using a random priming–based cDNA labeling kit (Nippon Gene, Tokyo, Japan). rRNA was shown to use the same amounts of RNA for reverse transcription reactions. Triplicate independent PCR reactions were carried out to ensure the reproducibility of expression quantification. For the densitometric analysis, signals in Southern blot analysis were quantitated using Scion Image 1.62 (Scion Corporation, Frederick, MD).
Western blot analysis. Whole-cell lysates extracted from 1 × 107 cells, each with 20 μg denatured proteins, were run on 12% to 15% polyacrylamide gels containing SDS and electroblotted onto nitrocellulose filters. Filters were then blocked with 5% nonfat dry milk and immunoblotted with antibodies against occludin (Zymed Laboratories, San Francisco, CA), phosphorylated MAPK (Cell Signaling, Beverly, MA), extracellular signal-regulated kinase 1/2 (ERK1/2; Promega, Madison, WI), and β-actin (Santa Cruz Biotechnologies) proteins. After extensive washing, the filters were reacted with peroxidase-labeled corresponding secondary antibodies in PBST and again washed; finally, the immunoreactions were visualized by using an enhanced chemiluminescence (ECL) system (Amersham Biosciences).
Gelatin zymography. The gelatinolytic activity of matrix metalloproteinases (MMP) was determined in the supernatants of B16F10-Vec and B16F10-Oc522aa cells treated with or without 1 μmol/L atRA for 24 hours. Aliquots of conditioned medium were subjected to electrophoresis without heating under nonreducing conditions in 0.1% gelatin–containing 9% polyacrylamide gels. After electrophoresis, the gels were washed with 2.5% Triton X-100 for 1 hour to remove the SDS, followed by incubation overnight at 37°C in Tris buffer [50 mmol/L Tris-HCl, 200 mmol/L NaCl, and 10 mmol/L CaCl2 (pH 7.4)]. Gels were then stained for 30 minutes with 0.5% Coomassie brilliant blue in 30% methanol/10% acetic acid. Clear bands appeared on the blue background in the areas of gelatinolytic activity.
cDNA array analysis. These procedure was described in detail previously ( 21). cDNA microarray analysis was done to analyze the apoptosis-specific signaling pathway using GEArray and mRNA levels were quantitated by GEArray Analyzer software (SuperArray, Bethesda, MD).
Methylation-specific PCR. The methylation status of the promoter CpG islands of occludin was analyzed by methylation-specific PCR (MSP) on sodium bisulfite-converted DNA ( 23). Briefly, 2 μg genomic DNA was denatured with 2 mol/L NaOH for 10 minutes, followed by incubation with 3 mol/L sodium bisulfite (pH 5.0) for 16 hours at 50°C. DNA was then precipitated with ethanol and resuspended in 20 μL of water. Aliquots (2 μL) were used as templates for PCR. The primers for MSP were as follows: 5′-GAGTATCGAACGTATTTCGGGG-3′ and 5′-GCGCTCACCTAACGAACGCA-3′ for the amplification of methylated alleles; 5′-AGTTTTAGGTGAATTGGTTATTGAG-3′ and 5′-ACAACCTTAATCTACCAAACTCAAA-3′ for the amplification of unmethylated alleles.
Tumor growth in vivo. After acclimation for 7 days, we injected cells of wild-type (wt) B16F10 and its transfectant (2 × 105 per mouse) s.c. into the craniolateral thorax (axilla) of syngenic 6- to 7-week-old male mice (C57BL/6, CLEA Japan, Tokyo, Japan). For AC2M2 cells, a 100 μL volume of cells was injected into a mammary fat pad in 6-week-old male mice (CBA/J, Charles River Japan, Yokohama, Japan). We examined the primary tumor and lung, subjecting for histologic evaluation for micrometastasis. The volume (V) of the primary tumors was calculated by the following equation: V = (π / 6) × [(L + W) / 2], where L is the length and W is the width. The maintenance and handling of animals were carried out using protocols approved by the Sapporo Medical University Animal Care Facility.
Statistical analysis. Unless otherwise specified, all data represent the mean ± SD of at least three independent experiments, each in triplicate wells. Statistical differences were analyzed using the paired t test and were considered statistically significant when P < 0.05.
Forced expression of occludin enhances the sensitivity to apoptogens. In the present study, we established a human cervical cancer cell line HeLa, constitutively overexpressing wt occludin (occludin223-1788) that encodes 522 aa polypeptide, thus designated HeLa-Oc522aa. HeLa cell transfected with an empty vector was used as a control (HeLa-Vec). We first determined the differential sensitivity of these cells to 24-hour exposure to oxidative stress mediated by H2O2, which is a common cell stressor. Before the apoptotic stimulation, HeLa-Vec and HeLa-Oc522aa were changed with medium without FBS supplementation and were exposed to H2O2 in the dose range of 0 to 100 μmol/L in the presence or absence of 1 μmol/L atRA as an apoptotic sensitizer ( 21). Forced expression of wt occludin clearly enhanced the sensitivity to H2O2-induced cell death in a dose- and time-dependent manner in HeLa cells ( Fig. 1A and data not shown). Although we have previously shown that the type of cell death induced by H2O2 is apoptosis ( 21), we next used the TUNEL method to confirm the type of cell death and that the assay used in this study was sensitive enough to quantitate the cell death. TUNEL assay also revealed that occludin increased the sensitivity to H2O2-induced cell death ( Fig. 1A, right), which was coincident with the results when compared with our assay ( Fig. 1A, left). To confirm the functional relationship between occludin expression and apoptotic sensitization, we next examined the effects of siRNA targeted to occludin. siRNA transfection could effectively silence the constitutively expressed occludin ( Fig. 1B, left). siRNA-mediated silencing inhibited occludin-mediated enhancement of cell death in occludin-expressing cells, whereas the siRNA transfection did not affect HeLa-Vec cells ( Fig. 1B, right). These results indicate that occludin expression modulates the apoptotic sensitivity of the cells.
COOH-terminal end is responsible for the occludin-mediated apoptotic induction. Previous reports have shown that the COOH terminus of occludin is important in its function ( 4). We hypothesized that the COOH terminus of occludin is responsible for the transmission of apoptosis-inducing signals inside the cells. To test this hypothesis, we overexpressed COOH-terminally truncated occludin in HeLa cells ( Fig. 1C). However, the deletion mutants, which did not show membrane localization by determining fluorescent detection of the cells, had no effect on increasing cell sensitivity to the various apoptotic inducers ( Fig. 1C, right), suggesting that the 44 aa at the COOH-terminal end is necessary for membrane homing and regulation of apoptotic sensitivity.
We next examined the signal transduction pathway(s) involved in the occludin-mediated apoptotic sensitization by using various specific inhibitors of kinase activities. A MAPK inhibitor (PD98059) partially but significantly abrogated the enhancement of apoptosis mediated by occludin overexpression, whereas other inhibitors, such as a p38 MAPK inhibitor (SB203580) and PI3K inhibitor (LY294002), had no effects ( Fig. 1D, left). Western blot analysis also showed that protein levels of phosphorylated MAPK were decreased in HeLa-Oc522aa cells challenged with or without atRA treatment ( Fig. 1D, right); this down-regulation was significantly abrogated by the treatment with PD98059, but not with SB203580 and LY294002 (data not shown). The decrease of pMAPK initiated by synergistic effects of atRA and occludin ( Fig. 1D) may explain the data on the cell death sensitivities of occludin-expressing cells showing that the apoptogen-mediated cell death in HeLa-Oc522aa cells was markedly enhanced in the presence of atRA ( Fig. 1A). This observation suggests that occludin is associated with the MAPK signaling pathway, being consistent with our previous result ( 12).
Occludin overexpression abrogates tumorigenic potency in vitro. Our observations prompted us to investigate whether occludin expression affects the tumorigenic potency. Because anoikis (apoptosis resulting from the loss of cell matrix interaction) is a well-accepted model for determining cell survival independent of cell attachment, we next examined the effect of occludin on anoikis. DNA fragmentation analysis at 24 hours confirmed the time-dependent activation of apoptosis when cells were cultured on agarose-coated dishes ( Fig. 2A ). Pretreatment with siRNA targeted to occludin significantly inhibited this anoikis sensitization effect ( Fig. 2B). Given the pleiotropic effects of cellular apoptosis and/or proliferation on the regulation of tumor development, we investigated the effect of occludin expression in HeLa cells on the rate of cellular proliferation. Manual cell counting every 24 hours up to day 6 following plating of equal numbers of cells did not show any significant difference in cellular proliferation or in the DNA synthesis rate of cells labeled with Ki67 (data not shown). On the other hand, colony formation assay in two- and three-dimensional cultures, a measure of cell transformation, showed a significant decrease in both the number and size of colonies in the event of occludin overexpression in a number of cell types ( Fig. 2C and D). Interestingly, cellular proliferation rates assessed by Ki67 labeling indexes were not affected under any conditions ( Fig. 2C and D). These results strongly suggest that occludin plays an important role in the regulation of cellular transformation and thus potentially contributes to colony-forming capability.
Retinoic acid–induced endogenous occludin is sufficient for apoptotic sensitization. To exclude the possibility that the forced expression of occludin in the cells resulted in possible unidentified artifacts, we tried to induce endogenous occludin by treatment with retinoids, because our previous study showed that atRA could effectively induce occludin mRNA in F9 cells ( 24). The retinoic acid receptor α (RARα)–selective compounds Am80 (4[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benzoic acid) and Am580 (4(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtamido)benzoic acid), as well as atRA, could increase endogenous occludin in HeLa cells ( Fig. 3A ). This up-regulation of occludin mediated by these retinoids could increase the sensitivity to H2O2-induced apoptosis ( Fig. 3B), which resulted in significant abrogation of colony formation in two- and three-dimensional cultures ( Fig. 3C). This effect was not cell-type specific, because we obtained similar data from four different cell types. Importantly, atRA-induced increase of apoptotic sensitivity was partially but significantly suppressed by siRNA-mediated silencing of occludin ( Fig. 3B), suggesting that the observed phenotypes ( Fig. 3B and C) were due to endogenous occludin expression on the cells. RAR antagonists, retinoid X receptor (RXR) agonists, and RXR antagonists did not show significant differences compared with the control (data not shown), demonstrating that RARα is preferentially involved in the occludin expression.
Occludin overexpression decreases invasiveness and cell motility in vitro. We next determined whether occludin expression in cancer cell had a causal role in its invasive and motile properties. Employing a Boyden chamber invasion assay using Matrigel and a transmigration assay through an endothelial monolayer, we observed significant decreases in the invasive capacity of occludin-expressing cells compared with the control cells ( Fig. 4A and B ). Interestingly, no deletion mutant showed any effect in this assay, supporting the notion that the COOH-terminal end is important for the occludin function. A gelatin zymogram for MMP activation showed a decrease in MMP activity caused by occludin overexpression with the synergistic effects of atRA ( Fig. 4C). In addition, a monolayer wound-healing assay revealed that occludin expression significantly inhibited cellular migratory activities ( Fig. 4D). These data suggest that occludin expression contributes to the abrogation of highly invasive and motile phenotypes of cancer cells.
Occludin expression inhibits tumor development and metastasis in vivo. Based on these in vitro observations, we postulated that occludin expression affected the tumorigenicity in vivo. We next investigated the tumor-forming capabilities of occludin-transfected cells in a syngenic mouse transplantation model. Occludin expression was sufficient to abrogate in vivo tumorigenicity, showing a markedly decelerated rate of tumor formation ( Fig. 5A and B ). We also assessed the effect of occludin on metastatic potency by screening the mice for lung lesions. No macroscopic metastatic lesion was observed in any setting; however, histologic examination showed that occludin expression showed significant inhibition of the metastatic lesion formation in the lung ( Fig. 5C).
Occludin expression is epigenetically inactivated by methylation. Aberrant promoter region CpG island hypermethylation is associated with transcriptional silencing of many genes in a wide variety of cancers ( 25). In addition, CpG island of the occludin promoter is densely methylated in human hepatocellular carcinoma ( 26), and histone diacetylase inhibitors up-regulate the expression of TJ proteins, contributing to cellular differentiation ( 27). Indeed, CpG islands exist in the promoter regions both of human and murine occludin. 3 We then did MSP analysis and found that CpG islands in the occludin promoter region were densely methylated in B16F10 cells ( Fig. 6A ). AC2M2 was partially methylated in this region and endogenous occludin expression was detected ( Fig. 6A). Treatment with 5′-Aza-dC and trichostatin A ( 28) could induce reexpression of occludin ( Fig. 6A, bottom). This synergistic effect was sufficient for apoptotic sensitization ( Fig. 6B) and suppression of colony-forming capabilities in two- and three-dimensional cultures ( Fig. 6C). We next used siRNA-mediated silencing of occludin on the cells to examine whether the observed phenotypes induced by 5′-Aza-dC/trichostatin A treatment was due to reexpression of endogenous occludin, because 5-Aza-dC/trichostatin A treatment leads to the derepression of a large number of genes ( 28). Transfection with occludin-specific siRNA partially but significantly abrogated the 5′-Aza-dC/trichostatin A–mediated increase of apoptosis ( Fig. 6B), making it possible to link the induction of endogenous occludin with the observed phenotypes ( Fig. 6C). These data suggest that epigenetic silencing of occludin is, at least in part, involved in the tumor development.
Occludin overexpression affects multiple signaling pathways. To explore the effects of occludin expression, we screened gene expression profiles with the GEArray system (an apoptosis pathway–specific gene array). Occludin expression clearly affected a wide variety of genes associated with the components of the apoptotic machinery ( Fig. 6D). Changes in patterns of gene expression were consistent with those of B16F10 and AC2M2 cells (data not shown). Although occludin overexpression induced up-regulation of apoptosis-inducing genes, such as bax, and, conversely, down-regulation of apoptosis-inhibitory genes, such as bcl-2 and survivin, we observed more marked changes in the set of caspase family genes, which are involved in apoptosis-executing pathways. Occludin expression was also associated with a number of tumor necrosis factor (TNF) receptor family members, TNF receptor–associated factor family members, and p53 and ATM pathway genes. These gene expression signatures may be sufficient to explain the altered phenotypes mediated by occludin expression.
Here, we show that occludin overexpression in cancer cells significantly enhanced the sensitivity to differently triggered apoptotic stimuli and thus suppressed tumor development. Apoptosis is an evolutionarily conserved process in the regulation of tissue homeostasis, and numerous reports have documented that loss of responsiveness to apoptosis is a contributing factor for a malignant phenotype, because it is widely believed that impaired or decreased susceptibility to respond to various apoptotic signals is associated with oncogenic transformation ( 29– 32). Therefore, it is clear that loss of occludin expression contributes to the acquisition of apoptotic resistance of cells and thus plays a key role in the carcinogenic process. Occludin also suppressed highly progressive cancer phenotypes, such as invasive and motile properties, resulting in an important contribution to the metastatic suppression, because the process of metastasis involves an intricate interplay between altered cell adhesion, survival, proteolysis, migration, and homing on target organs ( 33). Our results thus show that loss of occludin expression favors multiple steps known to be important in tumorigenesis and metastasis. We could not exclude the possibility that forced expression of occludin in cancer cells might have some interesting but as yet unidentified effects on the cells. However, our results show that RARα stimulants preferentially up-regulate endogenous occludin, which is sufficient for the acquisition of appreciable apoptotic sensitivity. This mechanism is partially explained by our preliminary study of the occludin promoter using ∼2.5 kb upstream from the transcription initiation codon, suggesting that RARα agonists stimulate occludin expression at the transcriptional level. 4
Gene expression profiles show that a large number of signal transduction molecules are associated with the promotion of apoptotic cell death due to occludin overexpression. Given the extensive range of physiologic activities of TJs, it is not surprising that occludin affects a vast array of signaling pathways in a given cell and tissue ( 34). Our study using deletion mutants of occludin shows evidence that 44 aa at the COOH-terminal end have an important role in receiving and transmitting signals to the cell interior to modulate cell behavior. This observation is consistent with a number of reports showing that the components of lipid signaling pathways, such as MAPK, PI3K, and Akt, interact with the COOH-terminal region of occludin and mediate cell growth and differentiation ( 35– 37). Indeed, we predicted four putative phosphorylation sites (two tyrosines and two serines) by MAPK in this region, whereas further mutation study will be required for the identification of the responsible residue(s). According to our observations, it is possible to accept that occludin is not a simple static constituent of TJ, but is involved in the signal transduction pathway(s) regulating cell survival, eventually acting as a signal transmitter for the cells through the TJs. This notion is also supported by recent evidence showing that occludin is associated with the transforming growth factor-β signaling ( 38) and apoptotic machinery in murine hepatocytes ( 12). On the other hand, one group has documented that reexpression of claudin-1, a TJ-associated gene, induces apoptosis in breast tumor spheroids ( 19). Based on the observation that breast cancer spheroids transfected with claudin-1 grown in suspension showed massive apoptosis but no difference in cell death characteristics in two-dimensional culture, it was considered that TJ protein claudin-1 in cancer cells formed diffusion barriers and restricted the access of nutrients and growth factors inside the spheroids. Because we observed significantly different cell death sensitivities even in adherent cells, we can exclude their proposed mechanism for why occludin expression suppresses tumorigenicity.
It has been shown that occludin has a potent inhibitory effect on Raf1-induced tumorigenesis and the second extracellular domain of occludin appears to be critical for this function in Pa4-Raf1, a normal rat parotid acinar cell line transfected with oncogenic raf-1 ( 16). We cannot explain these discrepant observations; however, one possible explanation is based on the different cell type used in that study. For example, Pa4 cells, parental cells of Pa4-Raf1, have a highly differentiated phenotype to form tight colonies of polarized epithelial cells, and in Pa4-Raf1 only one oncogene was introduced. On the other hand, the cells in this study were characterized by their tumorigenicity and metastatic potency. Thus, it is reasonable to believe that these cells have multiple genetic alterations because fully transformed phenotypes of cancer require multiple oncogenic hits. In accordance with our explanation of cell differentiation, our cells did not show epithelial polarization and did not form an epithelial monolayer without satisfactory levels of endogenous occludin expression. This cellular phenotype enables us to perform a permeability study after occludin transfection because occludin was first identified as an essential constituent for TJ ( 3), and this morphologic property is not sufficient to achieve the functions of extracellular domains of occludin due to the lack of cell-cell contacts.
Promoter CpG island methylation is an important means to transcriptionally silence a particular gene associated with a carcinogenic step. Our results show that occludin expression was epigenetically inactivated by DNA methylation. Occludin is located in the chromosome 5q13 locus in the human, and cytologic abnormalities, such as loss of heterozygosity and deletion, have been observed in the neighboring chromosomal regions in various cancers, including hematopoietic malignancies, cancer arising in the lung, colon, thyroid, brain, bone, and nasopharyngeal organs ( 39– 41). Although this evidence implies that there are other mechanisms silencing occludin in certain types of cancer, our observations provide strong evidence for our previous finding that loss of occludin expression correlates with cancer progression ( 13).
Because of the strong differentiation-inducing effects on cancer cells, retinoids show promise in the treatment and chemoprevention of epithelial carcinogenesis and in cancer differentiation therapy ( 42, 43). Their strong chemopreventative effects are thought to be achieved by complex mechanisms, including antiproliferative activity, apoptosis-sensitization effect, antiangiogenic activity, and inhibition of metastasis ( 21). In this study, we clearly verified the contribution of occludin to the antitumor activities of retinoids in vitro. If natural or synthetic retinoids satisfactorily up-regulate occludin expression in vivo, it is reasonable to propose that retinoid-induced chemoprevention is partially mediated by the induction of endogenous occludin in the target cells. Other TJ-associated genes, such as claudin-1 and claudin-4, are also reported to be involved in the carcinogenic process ( 17– 19). In addition, we found that either the synergy with a demethylator and histone diacetylase inhibitor or a retinoid that stimulated RARα could cause expression of endogenous occludin, resulting in the acquisition of higher apoptotic sensitivity. These results suggest the possible use of TJ-associated genes as potential biomarkers for cancer progression and may pave the way to new therapeutic modalities.
Our present study could not provide a significant insight for explaining the complex pathogenesis observed in occludin-deficient animals ( 7). However, we clearly show that many of the genes involved in apoptotic pathways are modulated to favor cell death when occludin is overexpressed, and thus contribute to the significant suppression of tumorigenic and metastatic potencies. Our observation suggests that occludin is a likely candidate for a tumor-suppressor gene in certain types of cancer. Further studies along this line will address the detailed molecular mechanism by which occludin abrogates the progressiveness of cancer cells. We believe that strategies that reexpress endogenous occludin in cancer may be an important means to increase the sensitivity of cancer cells to apoptosis-inducing therapies.
Grant support: Ministry of Education, Culture, Sports, Science and Technology of Japan, and Sapporo Medical University Foundation for Promotion of Medical Science.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Kim Barrymore for help with the manuscript, the Animal Care Facility of Sapporo Medical University, and Dr. Hiroyuki Kagechika (School of Biomedical Sciences, Tokyo Medical and Dental University, Tokyo, Japan) for gifts of Am80 and Am580.
↵4 Unpublished result.
- Received May 23, 2006.
- Revision received July 13, 2006.
- Accepted July 21, 2006.
- ©2006 American Association for Cancer Research.


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Boost :
From: John Max Skaller (skaller_at_[hidden])
Date: 2001-05-03 10:08:44
scleary_at_[hidden] wrote:
> But note that my solution is able to side-step this issue because Pool
> doesn't keep *any* information about allocated chunks. Your problem can use
> a similar solution, but only if you go to a footer instead of a header,
That won't work. The information stored in the header is:
1) a link to the next block
2) a pointer to a shape descriptor
3) a flag for use in the mark/sweep phase of collection
The shape descriptor consists of:
1) the size of the block
2) a finalisation routine
3) the number of managed pointers
4) offsets to the managed pointers
So you see that the only way to find the size of the block,
so as to locate the footer, is to already know the address
of the footer so as to get the shape descriptor, and thus
the size of the block :-)
-- John (Max) Skaller, mailto:skaller_at_[hidden] 10/1 Toxteth Rd Glebe NSW 2037 Australia voice: 61-2-9660-0850 checkout Vyper http://Vyper.sourceforge.net download Interscript http://Interscript.sourceforge.net
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