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This week, my family and I said goodbye to a very important influence in our lives. We lost my grandmother, a woman whose strength and independence has been characterized as a family legacy. She had the same confidence that I see mirrored in my own daughter. The fact my little girl so closely resembled her great-grandmother was a huge point of pride, for my Gigi, my mother and myself. This loss was my daughter's first funeral, and while I know she learned a lot, I think I learned more.
My daughter and I had discussed death in an abstract sense. She had heard, both from her Presbyterian pre-school and her family, that when you die, you go up to Heaven with God. But up until this point, I had never thought she was ready to attend a funeral. I assumed the open casket and crowds of people would be too much for a child struggling to understand why someone she loved had to go away.
However, given my daughter's close relationship with her great-grandmother, I felt like it would be unfair to deny her the chance to say goodbye. We knew my grandmother was sick, but we thought she had months. Then my dad called me during the day and told me to come immediately. There was time for the adults, but no opportunity to get the kids from school and let them say goodbye. The funeral would be my daughter's only chance.
From the very beginning, Brenna seemed to approach the situation with a matter-of-fact point of view. When I tried to sit her down to give her the news, she already knew what I was going to tell her. I sat on the couch and said, "Hunnie, can you come sit with me? Momma needs to talk to you about something." She curled up in my lap and before I could speak she said, "Gigi's dead." I was shocked. I sputtered and asked how she knew. "I just know, Mom," she told me.
I tried to prepare her for the funeral and what she would see. I let her know that her great-grandmother's body would be laying there for us to say goodbye, but that she wouldn't be able to talk to us or respond. She would be listening from Heaven. "It's like she's sleeping," my daughter surmised. I had planned on telling her that next.
Once the day came, I brought my daughter in early before any other guests arrived. I wanted her to have a second to herself, a second to do whatever she needed to do. I was prepared for crying, for confusion, for fear. I braced myself and my mom for my little girl's reaction. My daughter simply walked up, looking at the casket. She asked a couple basic questions like where her legs were, since half of the casket was closed. Then she started examining the flowers that were stretched along the front of the church, asking if she could pick a bouquet to take home.
I have to admit, in those moments, I was a little disappointed. I didn't want my daughter to be sad, but I felt like she didn't understand the gravity of the situation. I was upset and crying and I wanted her to get the chance to feel like bad emotions. My instinct was to try to talk to her, to explain to her why this was serious. I was frustrated when she started giggling with her cousin and playing hide-and-seek in the pews before anyone else arrived. (Never mind that I have played hide-and-seek in those pews countless times in my life.)
Throughout the funeral, my daughter stayed relatively cheerful. When I started crying, she tutted and patted my arm. When the preacher made amusing comments about my grandmother's singing or stubbornness, Brenna laughed the hardest. She seemed completely okay with the situation, comfortable in her surroundings, and accepting of her great-grandmother's passing. It was not what I had prepared for at all. And honestly, she was more composed than I was.
Finally, at the end of the funeral, we were given our final chance to say goodbye. The rest of the guests filed out of the church. The immediate family was left, standing in a semi-circle at the front of the casket. My daughter walked up, she touched her grandmother's face and hands. She whispered softly, so none of us could hear what she was telling her. It was a secret between those two, just as my grandmother would've wanted it to be. She always liked secrets. Then, my little girl turned around, buried her face in my lap and broke into sobs. Every person in that Church lost it, weeping and holding each other and having our last moment with our family's matriarch.
Honestly, I'm not sure if my daughter was responding to her own feelings, or doing what she thought she should, based on watching everyone around her. After I held her and got her calmed down, a sense of guilt seemed to wash over me. I felt bad for wanting her to feel like I did, to grieve as I was. We each process loss in our own way, even children.
Parents want their children to handle funerals and loss in a way that they see as appropriate. Normally, that means we just want them to respond like we do. But we do a disservice to our children when we say that they need to be sad. My daughter was sad in the end, but it would've been fine if she never had that moment of sobbing. It would've been fine if that moment came days later, while we were at home and something reminded her of Gigi. And it'll be fine if all of those emotions come flooding back to us in the next months or years.
Taking a child to a funeral is extremely difficult. It adds just a bit more stress to an already stressful situation. But the biggest mistake a parent can make, a mistake that I fought with this week, is trying to direct your child in how they grieve. We cannot make them process death in the same way that we do, and we shouldn't try.


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Blog Star: Jenny Lawson, The Bloggess
This week we're putting out our list of Blog Stars, a feature story on ten of the best and the brightest in Houston. The bloggers in the story write on a wide range of topics, from sports to politics to the law to food and beyond, and many of them have both a personal blog and gigs writing elsewhere. Humor blogger Jenny Lawson, The Bloggess, was our favorite interview -- the woman cracks us up -- and she has a few interesting gigs we thought Hair Balls readers might appreciate.
Lawson does Ask the Bloggess, a satirical column for a site called PNN, where she gives "really horrible advice." She tackles subjects such as whether a reader's penis is haunted, how to stab people, and when you should or shouldn't commit arson. "Every once in a while I think, 'Somebody is going to do this one day, and I'm gonna get sued,'" she says.
With another mom, Lawson does a blog for the Houston Chronicle called Good Mom/Bad Mom. One day, a porn company sent them a really strange pitch -- clearly, without reading the blog. So they decided to mess with the guy. "We wrote a series of completely clueless things about how we're this parenting blog, and it looks like you have swings, and we're looking for new swings for our playground. We posted the whole thing."
That post led to Lawson's sex column for SexIs, a subsidiary of Eden Fantasies. The column, Lawson writes on her personal blog, is "satirical and relatively safe for work if your boss isn't a douche-canoe." In a recent piece on SexIs, Lawson writes about how her bosses there are considering putting a disclaimer on her writing announcing that it's satire. It all culminates in "the most recent time I sent photos of myself in a ball-gag to an employer":
Courtesy of The Bloggess
Courtesy of The Bloggess
Jenny Lawson is a local -- maybe national -- treasure.
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Analysis: Libya mission is clouded by confusion
Thursday, March 24, 2011; 3:06 AM
WASHINGTON -- President Barack Obama said he was setting clear and unmistakable terms for the U.S. role in Libya: It would be limited, lasting days, not weeks, and its purpose was to protect Libyan citizens.
But that's not the way it's turned out. Less than a week later, the mission has been clouded by confusion and questions about who's in charge and who's doing what - all while the killing of civilians is going on.
The Pentagon claims success in establishing an effective no-fly zone over much of Libya that has grounded Col. Moammar Gadhafi's aging air force. But Gadhafi's tanks and troops are still targeting civilians on the ground.
The administration seeks to minimize current disputes over the reins of leadership, suggesting everything will fall in place quickly, ideally by this weekend.
There are some doubters.
"It could still all come around very quickly in our favor. But if that's to happen, we will have to apply much more intensive military power in an effort to make this succeed," said Aaron David Miller, a former top State Department Mideast negotiator in Republican and Democratic administrations.
"But it doesn't appear to me, given the constraints acting upon us and our own reservations, that we're prepared to do that," said Miller, now with the Woodrow Wilson Center, a foreign-policy think tank. "Right now, it appears to be settling into a stalemate which isn't terribly hurting on the Gadhafi side."
Obama also faces a skeptical audience on Capitol Hill. House Speaker John Boehner, R-Ohio, wrote to the president saying he and others "are troubled that U.S. military resources were committed to war without clearly defining for the American people, the Congress and our troops what the mission in Libya is and what America's role is in achieving that mission."
Boehner said Obama so far had made a "limited, sometimes contradictory case" for the action.
There also seems to be a disconnect between Obama and his military commanders. He keeps emphasizing that the U.S. is just one of many players in the coalition. But in their briefings, the generals and admirals sound like the Pentagon is running the show, at least for now.
To date, the air attacks on Libyan targets have been predominantly American. In a 24-hour period as of late Wednesday, 175 sorties were flown, 113 by the United States, U.S. Navy Rear Adm. Gerald P. Hueber told reporters from the U.S. command ship in the Mediterranean Sea.
His portrayal suggested a long slog might lie ahead.


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— After fuss over Grammys tribute, attention has turned to Academy Awards, and whether late singer will be recognized for her acting work.
Will Oscars honor Whitney Houston?
Current Status: Blessed (1)
Tue Feb 21, 2012 8:12 AM
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Roxann Taylor
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Property DetailsCall Roxann Taylor 620.262.7873 or e-mail: firstname.lastname@example.com // www.weigand.com // All rooms sizes are approximate. All information deemed reliable but not guaranteed. // Truly a one of kind home with the premier lot on the golf course! This home was remodeled for entertaining in mind. Large rooms with vaulted ceilings and many windows, a view from every angle. Walk into the large living area with a beautiful gas, vent-less heat fireplace with remote. All blinds in living room are remotely operated. Two master suites on main level. One has lots of built in storage/closets with patio doors, en suite with stained glass, double sinks, large walk in shower with dual shower heads. Other master suite has small fridge and sink plus attached bath. High end kitchen has many amenities: Large commercial stove/oven, warming drawer, beverage refrigerators, sub zero wine cooler, separate ice maker, 2 sinks and large butler's pantry. This kitchen was designed with entertaining in mind and leads out to the covered deck area. Whole house entertainment system (satellite radio and surround sound), and wired for intercom (all electronics for entertainment system to remain with property) Once outside, you have a large, multi-level covered deck and entertainment area (with speakers and plenty of electrical outlets). Outdoor kitchen has everything that you could need, built in grill, hood vent, sink, and plenty of preparation space. Large outdoor wood, with gas starter, fireplace provides a great gathering area. Main level also offers a large office, craft room (complete with sink and storage), bath and separate laundry room. 3 car garage with separate heat/air. Outdoor wash bay with hot and cold water. Basement has a large family room with gas fireplace, kitchen, bedroom and a full bath. Zone heat and air, two hot water heaters, sprinkler system (which was grandfathered in and DOES NOT use public water), and central vac are just a few more attributes of this wonderful property. Call to schedule your private showing today!
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Additional Property InformationThis single family listing located at 27 Braid Hills Dr, Winfield, KS has three bedrooms, three bathrooms and one partial bathroom. A ranch style property, it is presently priced for $399,000.
Highlights include basement, patio, sprinkler system.
This property is listed by Roxann Taylor of J.P. Weigand & Sons headquartered in Kansas.


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Ocean Indy x Premium Position. This girl is ready to go to the track! SHe has been galloping at the farm and jogging on an exerciser 6 days a week since…SOLD
We are running a Summer Saler (20% Off) till the end of Septebmer on this stunning silver dapple & white Yearling Gypsy Sport Horse filly! "Belle" is…SOLD
Nice filly for any performance event. Freckles Playboy, Doc Olena, Doc Quixote, King, Johnny Tivio, Three Chicks, Chickasha Dan, Cee Bars. Ready to go…SOLD
The mini mule filly is about 34 inches high at 11 months old. Leads very good, has had her hooves trimmed 6 times and she is doing good. I have not messed…SOLD
2 year-old filly by Cats and Dogs out of Marie Jilly. Sire, Cats and Dogs, is a son of Storm Cat and is out of a stakes producing Alydar mare. Dam, Marie…SOLD
Renata is a KFPS registered 2nd premie 2013 Mintse filly out of a Teade Ster Mare. This mare, Willoe, was Keuring Champion and class champion 2 years…SOLD
Adorable, friendly little filly should mature around 31". Has a tiny star and I suspect she may have front socks; both parents are pintos but her lower…SOLD
Very sweet and willing filly. Easy to work around and groom. Leads, Loads, Stands for Farrier, Bathes. Easy to catch. Has been saddled and bridled. Ready…SOLD
New Orleans, LA


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1 item from 2010
19 July 2010 7:05 AM, PDT | backstage.com | See recent Backstage news »
Los Angeles -- James Gammon, the gravel-voiced actor who played the manager of the Cleveland Indians in the 1980s comedy "Major League" and had a host of other movie and TV roles, has died. He was 70.His agent, Merritt Blake, said Monday that Gammon had cancer and died Friday at his daughter's home in Costa Mesa.The craggy-faced actor also played the father on the Don Johnson TV show "Nash Bridges."In addition to dozens of TV and movie appearances, Gammon was a noted stage actor who co-founded the Met Theatre in Los Angeles. Gammon starred in several Sam Shepherd plays, receiving a Tony nomination for his work on a 1996 Broadway production of "Buried Child."Gammon is survived by his wife, a brother and sister, two daughters and two grandchildren.Copyright 2010 Associated Press. All rights reserved. This material may not be published, broadcast, rewritten, or redistributed. »
1 item from 2010
IMDb.com, Inc. takes no responsibility for the content or accuracy of the above news articles, Tweets, or blog posts. This content is published for the entertainment of our users only. The news articles, Tweets, and blog posts do not represent IMDb's opinions nor can we guarantee that the reporting therein is completely factual. Please visit the source responsible for the item in question to report any concerns you may have regarding content or accuracy.See our NewsDesk partners


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Contact Us
COVINGTON, Ga. – What does the coroner do? What does the elected position entail? The Covington News posed these questions to Newton County Coroner Tommy Davis ...
COVINGTON Ga. – Dirt is moving on a 55-unit townhome community in Newton County as a local developer noticed a gap in the affordable home market ...
COVINGTON, Ga. – The completion date for the Highway 36 Henderson Mill Road intersection is still set for Aug. 31; however the work will not be ...
COVINGTON, Ga. – A Covington man was killed and a Jackson man was flown from the scene by air ambulance after a two vehicle collision in ...
COVINGTON, Ga. – Georgia Governor Nathan Deal was one of many state and local officials on hand in Newton County Tuesday morning for the ribbon cutting ...
COVINGTON, Ga – Approximately 100 people were on hand at the Newton County Historic Courthouse July 10 to attend an informational session on the Logistics Education ...
Page 1 of 1
Irvine Duplan of Covington reported a theft at her home on Bear Creek Trail to officers on Thursday.
When local artist Andrew Sabori visited Ellis Island to find out more information about his ancestors, his encounter with a small photo of a mural would change his life.
Oxford native William "Buddy" Smith is about to embark on a new journey in the 24th year of a military career that's already seen a multitude of new experiences.
Filming crews dressed Ga. Hwy 11 to resemble 1950s America for the film '42.' The movie tells the story of Jackie Robinsion, who broke the black color barrier in Major League Baseball when he was signed by the Brooklyn Dodgers in 1947. Robinson is being played by Chadwick Boseman, who has mainly had smaller roles on TV shows, while the role of the famous Dodgers manager Branch Rickey is being played by Harrison Ford. Alan Tudyk, Christopher Meloni, Lucas Black and John C. McGinley are also starring in the movie.
Next up in our primary election poll series is the Democratic race for the District 3 board of commissioners seat between incumbent Nancy Schulz and Tony Flanagan. Who will you vote for? Vote here.
The delivery of this morning's Covington News will be delayed due to an overnight mechanical malfunction. Carriers are currently in the process of delivering the paper, and all subscribers will receive their copy of the Covington News today. For questions regarding your subscription, call 678-750-5014. Thank you for your patience.
Hughson E. Arnold, felony habitual violator, nolle pross. Disrupting traffic, 12 months probation. Cannot drive for entire probationary period.
Officers were dispatched last Friday in reference to a possible kidnapping.
The Porterdale Memorial Library hosted a fun event for children and adults alike on Tuesday. Covington native Andy Irwin told stories from his childhood and performed comedic songs for his audience.
The cloud. It's a term that's been floating around (forgive the pun) in the technology arena for a few years now, but it's being talked about more and more now. So, what exactly is "the cloud" and "cloud computing?"
Chances are, if you use a computer and the Internet, you've had some interaction with cloud computing without even realizing it. Let me tell why the cloud is pretty awesome.
This summer, young artist have the opportunity to learn fine art skills that could last them a lifetime. Southern Heartland Art Gallery is offering its last summer art camp on July 16-19 for participants ages 6 through 12.
Ann Wildmon, Camp Director and one of the 18 artists working at the gallery, said these promising young artists will learn different fine art techniques. Campers will not be occupied with simple crafts involving macaroni and glue.
Job growth and recovering from a troubled economy echoed as some of the needs for Newton County from candidates discussing their platforms at a two-hour political forum held Tuesday night.
Old-time baseball is coming to Social Circle Friday, as Legendary Entertainment is filming the final two scenes of the upcoming Jackie Robinson-inspired movie "42."
Crews will be filming at a private home and in downtown Social Circle at the corner of Cherokee Road and East Hightower Trail, according to a production letter to area residents and merchants.
In their hey-day, independently-owned pharmacies filled Covington, offering their customers personalized service, home delivery, charge accounts and a community gathering spot.
Fire officials have been called to Nisshinbo North America off Ga. Highway 142, after several hundred gallons of hydrogen peroxide spilled out of a container that was being loaded onto a truck.
Page 367 of 671


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Search Moongate
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Pablo Neruda Translations by Jodey Bateman
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Children's Page
At A Bridal | Poem by Thomas Hardy
WHEN you paced forth, to wait maternity,
A dream of other offspring held my mind,
Compounded of us twain as Love designed;
Rare forms, that corporate now will never be!
Should I, too, wed as slave to Mode's decree,
And each thus found apart, of false desire,
A stolid line, whom no high aims will fire
As had fired ours could ever have mingled we;
And, grieved that lives so matched should miscompose,
Each mourn the double waste; and question dare
To the Great Dame whence incarnation flows,
Why those high-purposed children never were:
What will she answer? That she does not care
If the race all such sovereign types unknows.
Thomas Hardy - Poet | Academy of American Poets
Thomas Hardy - Wikipedia
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Pop Culture: Wal-Mart's Grapefruit Soda
Normally the tasting team and I go in to these pop culture reviews on essentially equal footing, none of us terribly familiar with the product at hand. This time I threw the tasters -- Jonathan, Jasmine, Peter and Jay -- a bit of a curve ball, serving up one of my all-time favorite sodas, Wal-mart's unbranded "Grapefruit," without telling them what it was.
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Your employee qualifies as a special needs traveller if she's:
Let's take a closer look at how you need to accommodate each of these special needs travellers.
Here's how to accommodate special needs travellers
#1: Disabled traveller
There are different groups of disabled travellers. These can be for example, people with spinal cord injuries, paraplegics, the hearing impaired, amputees and the visually impaired, says the Health & Safety Advisor.
Here are the general measures you can take to accommodate disabled travellers:
#2: Immune compromised
Who is considered immune compromised?
You must ensure these employees get the necessary vaccine. The travel clinic can help you with this.
'Any adverse reaction to the vaccine of HIV positive individuals should be reported to the travel clinic, who will then report it to the Department of Health', says the Health & Safety Advisor.
#3: Pregnant women
General travel isn't discouraged during pregnancy until close to due date (from 34 weeks) in healthy women. But some airlines impose travel restrictions in late pregnancy and the neonatal period.
Although travel is considered safe, certain vaccines can be harmful (for example live vaccines such as yellow fever or rubella) and must be avoided.
Ideally pregnant women mustn't enter malaria or yellow fever endemic areas.
Now that you know who's considered a special needs traveller, make sure you meet your legal obligations.
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Object: Remove the shuttle; the long, narrow, wavy loop piece can be completely separated from the rest of the puzzle.
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Hawthorn Life insurance adopts software from Dublin-based Financial Risk Solutions (FRS) to meet its Solvency II requirements
Financial Risk Solutions (FRS), a provider of investment administration and compliance software solutions, has announced that its Invest|GRC™ (Governance, Risk & Compliance) product has been implemented by Hawthorn Life, a Berkshire Hathaway company. The implementation is 15 months ahead of the Jan 2016 deadline for Solvency II asset-reporting, which dictates that insurance companies must provide more granular and more frequent insight into the risk and return profile of their investments.
The unique software allows for the fully-automated production of Pillar 3 D1 (investments data) and D4 (investment funds) Solvency II reports by consuming and enriching data from various sources. For Hawthorn Life these sources include Morningstar data and a fund-pricing outsourcer for daily holdings data. The combined data can then be used for monitoring and reporting in the prescribed QRT (Quantitative Reporting Templates) format.
Brian Aherne of Hawthorn Life comments "Through our adoption of Invest|GRC™ being able to view and report on our investments and funds not only helps us meet our regulatory requirements but also benefits our overall business – and we have been able to do this quickly, efficiently and at minimal cost to our business. The process of adopting the software from FRS was seamless and efficient, which means that we are now 15 months ahead of our regulatory deadline and can concentrate on our main business of serving our clients."
Frank Carr, Chief Marketing Officer at FRS says "QRT reporting is quite a challenge for life companies – mainly because of the sheer volume of data and frequency of reporting required. So having one system that can seamlessly facilitate that, yet has the ability to work with existing data sources is important. Having Hawthorn Life adopt Invest|GRC™ is real testament to our ability to deliver a software solution that works – and puts Hawthorn at a real advantage in terms of Solvency II readiness."
Frank Carr, Chief Marketing Officers at FRS.
The news was published on
Actuarial Post
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Jagwire's EZ Handle Single End Straddle Wire is a replacement straddle wire (also called a crossover or transverse cable) for cantilever brakes.
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Monday, December 2, 2013
Biodesix Finds $8.3M In Series E Funding
Boulder-based molecular diagnostics provider Biodesix said this morning that it has raised $8.3M in a Series E equity funding round. The company said the funding was split between 44.3M in new capital, plus $4M in conversion of a convertible note. The funding came from the company's existing investors, who were not named by the company. Biodesix is developing diagnostic testing technology, with a first focus on advanced non-small cell lung cancer (NSCLC). The firm is led by David Brunel. More information »


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If Bill uses holdover relief to pass assets to his daughter Jane, can Jane then subsequently utilise holdover relief to pass assets to her son Bob and so on down the generations? I have assumed so, but cannot find an answer or example anywhere.
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There is no general holdover relief
There is holdover only for gifts of business assets or gifts into settlement, so I'm not sure the question is valid. If you were talking about business assets, for example, there is no reason why successive claims may not be made: the donees will just keep acquiring - effectively - at the asset's base cost, so that if the asset increases in value the held-over gain would get bigger and bigger unlessthe donee drops off the perch and the gain is washed out.
I should have referred to gifts on which IHT is chargeable rather than gifts into settlement - there can be an IHT exit charge and so holdover may apply on the way out of settlement too.
Planning with a partnership?
Just a thought, if these are business assets then you might consider the attractions of estate planning using a partnership. It does all rather depend on the assets and the type of business in which the family is involved.
Virtual tax support for accountants: www.rossmartin.co.uk


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I was born in Manchester in 1967. I am an Economist with a Ph.D. degree. I have been working in the financial trading industry as a professional since 2003. I am a fan of Manchester United team. I am married and have a little princess.
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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
how can I print a concise copy of my jotforms?Asked by wyracrew on July 24, 2014 at 02:30 PM
I have a membership form that is maybe 7 screens long even though it's not that difficult of a form. It's your standard info on kids and then parents (name, address, work details, etc). We need to print these off so we have a hard copy as well as an e-copy, however, when we print the form, it's 7 pages long filled with lots of white space. Is it possible to print a condensed version of the form? In other words, can I get the form down to a page or two where it's just the casts and doesn't look pretty?
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There is a Text field height issue on your form, so we will need to fix it first.
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Conceived and designed the experiments: ML AZ. Performed the experiments: ML BBd GMG CJL JCP. Analyzed the data: ML MP AJVP AZ. Contributed reagents/materials/analysis tools: ML GMG CJL MR MP AJVP. Wrote the paper: ML AZ.
There is no evidence to date on whether transcriptional regulators are able to shift the balance between mitochondrial fusion and fission events through selective control of gene expression.
Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1β is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein. This decrease in Mfn2 is specific since expression of the remaining components of mitochondrial fusion and fission machinery were not affected. Furthermore, PGC-1β increases mitochondrial fusion and elongates mitochondrial tubules. This PGC-1β-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells. Finally, we show that PGC-1β increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor α (ERRα).
Taken together, our data reveal a novel mechanism by which mammalian cells control mitochondrial fusion. In addition, we describe a novel role of PGC-1β in mitochondrial physiology, namely the control of mitochondrial fusion mainly through Mfn2.
Mitochondria are dynamic organelles whose morphology is regulated by fusion and fission processes. A growing body of evidence shows the relevance of these shaping processes in the control of mitochondrial activity and cell metabolism [1]–[7]. Several genes encoding mitochondrial fusion and fission proteins have been recently identified. Mammalian proteins involved in mitochondrial fission are Fission 1 homologue protein (Fis1) and Dynamin-related protein 1 (Drp1). Similarly, Mitofusin 1 (Mfn1), Mitofusin 2 (Mfn2) and Optic Atrophy gene 1 (OPA1) are proteins that participate in mitochondrial fusion in mammals [8]. However, there are no evidences to date that demonstrate the ability of an upstream or transcriptional regulator to shift the balance between mitochondrial fusion and fission events by selective regulation of these proteins.
Several reports provide evidence that Mfn2 protein elicits pleiotropic effects which may be involved in pathology. For instance, Mfn2 is mutated in Charcot Marie Tooth type 2A neuropathy [9] and, interestingly, some of these mutants cause selective defects in mitochondrial fusion [10], reduction in mitochondrial axonal transport [11] or defects in mitochondrial coupling leading to inefficient mitochondria [12]. Defective Mfn2 may also contribute to impaired mitochondrial function in the context of obesity and type 2 diabetes. This notion is supported by the observation that muscle Mfn2 expression is reduced in these patients [1], [13]. In addition, we have previously reported that Mfn2 can modulate mitochondrial activity through changes in the electron transport chain (ETC) and this modulation is independent of its role in mitochondrial morphology [1], [6].
Peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) α and β are important positive regulators of mitochondrial activity and biogenesis in mouse skeletal muscle [14]–[17]. Despite these similarities, PGC-1α and PGC-1β display low overall sequence identity, with the highest percentatges found in two particular domains (activation and RNA recognition domains, with identities of 40% and 50% respectively) [15]. Furthermore, key mitochondrial processes, such as organelle biogenesis and uncoupling, are differentially regulated by these homologues. For instance, in C2C12 muscle cells, PGC-1α but not PGC-1β, increases mitochondrial uncoupling, whereas PGC-1β causes a larger increase in mitochondrial volume than PGC-1α under the same conditions [18]. In addition, while PGC-1β expression in distinct tissues is unaffected by physiological processes characterized by increased energy expenditure, such as cold exposure (in brown adipose tissue), fasting (in liver) or exercise (in muscle), PGC-1α is highly regulated at the transcriptional level under similar physiological challenges [15]–[17]. These data suggest that PGC-1β likely controls basal mitochondrial biogenesis, whereas PGC-1α controls stimulated or regulated mitochondrial activity. In keeping with this view, PGC-1β expression is higher than PGC-1α expression in primary muscle cells under basal conditions [19].
The functional independency in mitochondrial physiology of these homologues is further illustrated by the phenotypes of PGC-1α and PGC-1β knockout (KO) mice. In both animal models, a general defect in the electron transport chain (ETC) system has been described, thereby demonstrating that PGC-1α does not fully compensate the effects of PGC-1β on mitochondria or vice versa [20]–[23]. Furthermore, several mitochondrial phenotypes described in the particular case of PGC-1β-ablated mice cannot be completely explained by impairment of the ETC system. For instance, muscle and liver from PGC-1β KO mouse show a reduction of mitochondrial volume without changes in mitochondria number [20], [23]. This decreased mitochondrial volume together with impaired ETC gene expression may explain the mitochondrial respiration defect found only in muscle strips and not in isolated mitochondria [20]. In keeping with these data, this reduction in mitochondrial size is absent in PGC-1α KO mouse under basal conditions, probably due to normal PGC-1β expression [21], [24]. Of note, despite all these differences, both genes show a diminished expression in the context of type 2 diabetes, suggesting an impairment of mitochondrial effects selectively regulated by each homologue in this disease [25], [26].
We previously reported that PGC-1α was able to induce Mfn2 transcription and that mitochondrial activity regulated by PGC-1α partly depends on correct Mfn2 expression. However, effects on mitochondrial fusion were not determined [27]. In the light of these results and the PGC-1β control of basal mitochondrial biogenesis, we aimed to determine whether PGC-1β was able to control Mfn2 transcription and, therefore, if the mitochondrial dynamics balance could be modulated by transcriptional regulation. Here, we address these questions by determining the role of PGC-1β in the control of mitochondrial fusion using a multi-approach strategy that combines Mfn2 KO cells, PGC-1β-overexpressing muscle cells and PGC-1β-ablated mice.
Our initial aim was to determine whether PGC-1β regulates the expression of Mfn2 in C2C12 muscle cells. Differentiated C2C12 cells show low levels of PGC-1β mRNA as assessed by Northern blot [18] and by real-time PCR (data not shown). To this end, C2C12 myotubes were transduced either with a mouse PGC-1β adenovirus or with a control LacZ adenovirus. Mfn2 mRNA levels doubled in PGC-1β-expressing muscle cells compared to control transduced myotubes (Fig. 1A). To demonstrate that PGC-1β directly increases Mfn2 transcription, we transfected 10T1/2 mouse fibroblasts or HeLa cells with a construct containing a 2-kb fragment (−1982/+45) of the Mfn2 promoter fused to a luciferase reporter gene, together with an irrelevant vector (Basal) or mouse PGC-1β expression vector (PGC-1β). PGC-1β markedly enhanced Mfn2 promoter activity (10.3±0.9- and 4.2±0.6-fold values over basal Mfn2 promoter activity in 10T1/2 and HeLa respectively, Fig. 1B). In a previous study, using electrophoretic mobility shift and chromatin immunoprecipitation assays, we showed that ERRα binds to Mfn2 promoter between nucleotides −459/−396 [27]. This DNA region contains three putative boxes with the capacity to bind nuclear receptors, where box 2 is critical for Mfn2 promoter response to PGC-1α coactivation of ERRα [27]. On the basis of these observations, we determined whether PGC-1β coactivated ERRα through box 2 in a similar way as PGC-1α. We transfected 10T1/2 cells with a construct containing a −459/−352 Mfn2 promoter fragment fused to a luciferase reporter gene or with a mutated version of the same fragment that disrupted box 2 [27]. We observed a marked coactivation of ERRα by PGC-1β in the −459/−352 Mfn2 promoter fragment (Fig. 1C). This effect was completely blunted when box 2 was disrupted (11.9±1.09 vs. 2.8±0.6-fold over basal promoter activity, p=0.001, Fig. 1C). Furthermore, cancellation of box 2 markedly reduced the activation driven by PGC-1β (7.0±0.5 vs. 1.9±0.5, p=0.001) or by ERRα (2.2±0.1 vs. 1.6±0.2, p=0.04), although residual activation was still present. Similar results were obtained when the 2- kb Mfn2 promoter was cotransfected with ERRα or PGC-1β (Figure S1).
To determine whether PGC-1β-mediated Mfn2 transcription leads to enhanced Mfn2 protein expression, we transduced C2C12 myotubes with PGC-1β or two distinct control adenoviruses at a multiplicity of infection (MOI) of 1, 10 or 100. Total protein extracts and mitochondrial-enriched fractions were obtained and analyzed by Western blot. PGC-1β induced Mfn2 in muscle cells (Fig. 2A) and a direct relationship between PGC-1β adenoviral dose and Mfn2 protein induction was detected (Fig. 2A). PGC-1β also increased the cellular content of the constitutive mitochondrial protein Porin (Fig. 2A), used as a measure of mitochondrial mass. Densitometric quantification of Porin induction at MOI 100 showed a 1.8±0.2-fold increase in total lysates (p=0.01, Figure S2) and 1.45±0.003-fold in mitochondrial-enriched fractions (p=0.002, Fig. 2C). Porin induction values in total lysates are consistent with the increase in mitochondrial mass volume reported in PGC-1β-overexpressing C2C12 muscle cells [18].
On the basis of the effects of PGC-1β on Mfn2 and Porin expression, we also analyzed whether this coactivator regulates the expression of other proteins involved in mitochondrial dynamics and in the ETC system. PGC-1β-transduced myotubes induced the expression of Mfn1, OPA1, Drp1 and Fis1 (Fig. 2B) in mitochondrial-enriched extracts (1.7-, 2.2-, 1.4-, or 2.1-fold values over basal values, respectively) to a level similar to that detected for Porin (1.45-fold stimulation, Fig. 2C). Mfn2 displayed a significantly higher increase in expression compared to Porin abundance in mitochondrial-enriched extracts (4.3-fold induction, Fig. 2C). When data were expressed as protein levels relative to Porin expression, we only detected significant stimulation of Mfn2 and Fis1 in response to PGC-1β over-expression (Figure S3). Similar results were obtained in total lysates (data not shown). This superior induction of Mfn2 protein was also detected in C2C12 myoblasts (Fig. 3A).
We also analyzed several subunits of the ETC system in mitochondrial-enriched fractions from C2C12 myotubes transduced at MOI 100 (Fig. 2B,C). All the subunits of complexes I, II, III, IV and V studied were induced in response to PGC-1β (Fig. 2B,C), and the extent of induction was similar to that detected in Porin levels (ranging from 1.3- to 2.1-fold increase).
As PGC-1β induces Mfn2 expression, we next determined whether it was linked to changes in mitochondrial morphology. To this end, we immunofluorescently labelled mitochondria from C2C12 myoblasts transduced with PGC-1β or LacZ adenovirus. PGC-1β caused an increase in the length of mitochondrial tubules (Fig. 3B) in most myoblasts (59% vs. 33%, in PGC-1β and in control cells, respectively, p=0.003) (Fig. 3C). This increased mitochondrial size induced by PGC-1β overexpression was also observed by transmission electron microscopy, showing normal cristae morphology (Fig. 3D). To demonstrate that this increase in mitochondrial length was linked to an increase in mitochondrial fusion, we performed a polyethylene glycol-(PEG) mediated cell fusion assay by using two distinct C2C12 lines stably expressing mitochondrial matrix-targeted GFP (mtGFP) or red fluorescent protein (mtRFP) [28]. Four hours after PEG and in the presence of cycloheximide, a significantly higher percentage (73% vs. 55% in PGC-1β and control cells, respectively, p=0.01) (Fig. 3E) of polykaryons from C2C12 transduced with PGC-1β showed a higher level of mitochondrial matrix content mixing than control LacZ polykaryons (that is mtGFP and mtRFP exchange due to mitochondrial fusion and displayed as yellow mitochondria) (Fig. 3F). This higher mtGFP and mtRFP mixing indicated an increase in the rate of mitochondrial fusion induced by overexpression of PGC-1β. In agreement with previous data [18], the overexpression of PGC-1β in C2C12 myoblasts under these conditions also increased a ~50% the mitochondrial membrane potential values measured using the fluorescent probe JC-1 (data not shown).
To study whether the change in mitochondrial morphology induced by PGC-1β was mediated mainly through Mfn2 activity, we used mouse embryonic fibroblasts (MEFs) from wild-type and from Mfn2 or Mfn1 KO mice [29]. Overexpression of PGC-1β led to an increase in mitochondrial length of wild-type (Fig. 4A,B) and Mfn1 KO MEFs (data not shown), similarly as observed in C2C12 myoblasts (Fig. 3B,C). Importantly, PGC-1β gain-of- function was unable to promote mitochondrial elongation in Mfn2 KO MEFs (Fig. 4C,D), in conditions in which ETC subunits Cox4 and Uqcrc2 were increased 1.6- and 1.5-fold respectively (data not shown). These data demonstrate the requirement of Mfn2 expression for PGC-1β-mediated changes in mitochondrial morphology.
We next studied the effects of in vivo ablation of PGC-1β on Mfn2 expression in gastrocnemius muscles. Mfn2 protein levels were reduced by approximately 50% in KO mice (Fig. 5A). Reduction of Mfn2 levels was relatively specific as indicated by the absence of major changes in proteins involved in mitochondrial dynamics, i.e., Mfn1, OPA1, Drp1 and Fis1 (Fig. 5B). Similar results were obtained using mitochondrial fractions (data not shown). Reduced Mfn2 protein expression paralleled lower levels of Mfn2 mRNA (Fig. 5C).
Soleus muscle from PGC-1β KO mice also shows a decrease in complex IV (subunits Cox4 and Cox5b) mRNA levels [20]. This observation led us to study whether the protein levels of Cox4 and other subunits of complexes I, II, III and V were also altered in gastrocnemius muscle of this mouse model. These mice displayed approximately a ~40% reduction in complex V (ATP5a1) and III (Uqcrc2) (Fig. 5D). Complex IV (Cox4) showed a ~25% reduction and complex I (Ndufa9) a ~20% decrease while no changes were observed in complex II protein levels (Sdha) (Fig. 5D).
The heart is one of the organs with the highest expression of PGC-1β [16]. Interestingly, heart lysates from KO mice displayed a clear reduction in Mfn2 protein levels (WT 1.00±0.06, KO 0.57±0.09, p=0.0015; data not shown), suggesting the impairment of this regulatory pathway also in cardiac muscle.
Liver is a crucial metabolic organ and shows high mitochondrial activity. PGC-1β KO mice also show reduced hepatic ETC gene expression [20]. On the basis of these observations, we next examined whether hepatic Mfn2 expression was altered in PGC-1β KO mice. Hepatic lysates from PGC-1β KO mice were obtained and analyzed by Western blot. Mfn2 protein levels were reduced by 65% in KO livers compared to the control group (Fig. 6A). This decrease was specific since among other proteins involved in mitochondrial dynamics only a modest 12% increase in Fis1 levels was detected (Fig. 6B). Similar results were obtained in mitochondrial fractions (data not shown). Mfn2 mRNA levels were also reduced by 25% in livers from PGC-1β KO mice (Fig. 6C).
Hepatic PGC-1β expression is markedly induced in mice fed a high fat diet, which is, to our knowledge, the only situation reported to induce PGC-1β mRNA levels in adult tissues. Therefore, we analyzed hepatic Mfn2 expression in wild-type and PGC-1β KO mice fed a high fat diet during 6 months. We observed a higher reduction in Mfn2 mRNA levels in livers from PGC-1β KO mice fed a high fat diet (by 45%, data not shown) compared to PGC-1β KO mice fed a chow diet (by 25 %). This was specific to Mfn2 and no changes in Mfn1 expression were detected under normal or high fat diet conditions (data not shown).
The expression of some ETC subunits was examined in liver mitochondrial fractions of PGC-1β KO mice (Fig. 6D). Cox4 levels were reduced by 65%. Subunits of complexes I and V displayed moderate reductions (~20% and ~30%, respectively), while those of complexes II and III remained unaltered (Fig. 6D).
In the present study we demonstrate that mitochondrial dynamics balance can be shifted towards fusion by transcriptional regulation. More specifically, we show that PGC-1β is a regulator of mitochondrial fusion through its effects of selectively promoting Mfn2 expression upon coactivation of ERRα. This new role of PGC-1β in mitochondrial physiology has been demonstrated using both in vitro (muscle cells) and in vivo (PGC-1β-ablated mice) approaches. Firstly, we have shown that PGC-1β overexpression in muscle cells regulates mitochondrial dynamics through a mechanism that involves the preferential induction of Mfn2 expression, among other mitochondrial dynamics effectors such as Mfn1, OPA1, Drp1 and Fis1. Furthermore, PGC-1β gain-of-function results in an elongation of mitochondrial tubules, which is linked to an increased mitochondrial fusion. Importantly, the effects of PGC-1β promoting mitochondrial elongation are not observed in Mfn2-ablated cells, thereby demonstrating that Mfn2 activity is essential for PGC-1β-mediated changes in mitochondrial dynamics.
The relevance of these observations is confirmed by our in vivo data showing that PGC-1β KO mice have a specific reduction of Mfn2 expression in skeletal muscle whereas Mfn1, OPA1, Drp1 and Fis1 levels are maintained. In fact, this preferential regulation of Mfn2 protein levels could explain the reduction in mitochondrial volume, without changes in mitochondria number, observed in liver and muscle from PGC-1β loss-of-function mice [20], [23]. We propose that Mfn2-impaired function in PGC-1β KO mice prevents a normal rate of mitochondrial fusion while physiological mitochondrial fission events remain unaffected, thereby resulting in smaller mitochondria as previously reported for Mfn2 KO or antisense cells [1], [30].
Several lines of evidence demonstrate the relevance of maintaining a proper balance between fusion and fission processes for the specific mitochondrial activity required by distinct cell types [1]–[6]. In this regard, PGC-1β expression is higher in tissues with marked mitochondrial activity (such as liver, muscle and heart), a similar pattern to that reported for Mfn2 [1], [16]. In addition, we have found that PGC-1β regulates Mfn2 expression in liver, skeletal muscle and heart, as PGC-1β KO mice show a marked decrease in Mfn2 in those tissues. The correlational data mentioned before together with the observations obtained in skeletal muscle, heart and liver from PGC-1β KO mice indicate that PGC-1β controls basal expression of Mfn2 in these tissues.
On the basis of these observations, we propose that increased mitochondrial fusion caused by PGC-1β-induced Mfn2 contributes to a fully optimal mitochondrial activity in muscle and liver tissues, which may be defective in skeletal muscle of type 2 diabetic patients. Furthermore, we propose that PGC-1β controls basal mitochondrial fusion in skeletal muscle, whereas PGC-1α would probably control mitochondrial fusion in high energy expenditure situations, such as cold-exposure or exercise, conditions which largely induce Mfn2 expression [27]. This is supported by the fact that PGC-1β expression is higher than PGC-1α expression in skeletal muscle under basal conditions and by the lack of a decrease in mitochondrial size in PGC-1α KO mice [19], [24]. In keeping with this proposal, our data permit to explain the observation of a defective mitochondrial respiration found in muscle strips but not in isolated mitochondria from PGC-1β KO mice under basal conditions [20]. In isolated mitochondria, the integrity of the mitochondrial network and dynamics inside the cell is not maintained and therefore defects in mitochondrial activity secondary to abnormal mitochondrial dynamics are not detectable.
Here, we have also examined the mechanism by which PGC-1β induces Mfn2 transcription, and have identified ERRα as the key transcription factor coactivated by PGC-1β on the Mfn2 promoter. The effect of ERRα and PGC-1β occurred at the level of box 2 (located in the −459/−352 promoter region), although PGC-1β also displayed additional stimulatory actions on the Mfn2 promoter. The same box 2 region is the major component required for PGC-1α activation of the Mfn2 promoter [27]. On the basis of these data, we conclude that PGC-1α and PGC-1β display a common mechanism of activation of the Mfn2 promoter, through nuclear receptor ERRα. However, despite using the same response elements, decreased expression of Mfn2 induced by genetic ablation of PGC-1β in mice cannot be counteracted by PGC-1α action, as shown in liver, heart and skeletal muscle of PGC-1β KO mice, in which Mfn2 expression is decreased, probably due to the low expression of PGC-1α under basal conditions.
In summary, we provide evidence that mitochondrial dynamics balance is selectively controlled by a transcriptional regulator, unravelling an upstream mediator of mitochondrial fusion. Furthermore, we also provide evidence of a novel role of PGC-1β in mitochondrial physiology. Given the crosstalk between mitochondrial activity and dynamics, together with reduced Mfn2 and PGC-1β expression in type 2 diabetes, we conclude that the pathway reported here is not only relevant for the thorough explanation of mitochondrial dynamics regulation and the overall mitochondrial effects of PGC-1β, but also may provide the basis for the correct understanding of the alterations of mitochondrial metabolism associated with type 2 diabetes.
All animal protocols were approved as previously published [20]. Mice were cared for according to the Guiding Principles for Research Involving Animals and Human Beings. Tissue collection was performed as previously published [20].
10 T1/2 , HeLa , HEK293A and C2C12 cell lines were grown as reported [27].
10 T1/2 fibroblasts and HeLa cells were transfected in 24-well plates at 40% confluence using Lipofectamine 2000 (Invitrogen) and harvested 36 h after. Transfected DNA plasmids were 1.04 µg/well and ratios/well were 75 ng to 200 ng of PGC-1β, 200 ng of ERRα, 600 ng of reporter, 40 ng of Renilla and up to 1.04 µg/well of pcDNA 3.1 as an irrelevant vector. 10 µl of total lysates from 10 T1/2 or HeLa cells were analysed using Dual-Glo™ Luciferase Assay System from Promega following the manufacturer's instructions.
Mouse embryonic fibroblasts (MEFs) from wild-type, Mfn1 KO and Mfn2 KO mice [29] were seeded in coverslips 24 h before cotransfection with an empty vector or with a plasmid encoding mouse PGC-1β and GFP (empty vector/PGC-1β:GFP ratio 10:1) in 6-well plates at 40% confluence using Transfectin reagent (1 µg DNA : 3 µl reagent) (BioRad). Transfection efficiencies for wild-type MEFs were ~5–15% and for Mfn2 −/− MEFs were ~40–60% (observed by GFP fluorescence). MEFs were fixed 48 h after transfection.
C2C12 differentiated myotubes were obtained 3 days after adding differentiation medium to 90% confluent cells (5% Horse Serum from Gibco, P/S and 25 mM HEPES). Myotubes were transduced with a LacZ- or a mouse PGC-1β-encoding adenovirus at a multiplicity of infection (MOI) of 1, 10 or 100 during 24 h and processed 2 days after transduction. Myoblasts were transduced at a MOI 200, as they are less sensitive to adenoviral transduction compared to myotubes [31]. Myotubes transduced at a MOI 100 and myoblasts at a MOI 200 displayed 99% of transduced cells (data not shown).
Constructs and adenovirus were previously described [27], [32]. Adenovirus titer was determined in HEK293A cells as previously described [6].
Taqman Probes 20X (Applied Biosystems) for Mfn2, PGC-1β and 3B64 were used. β-Actin primers, RNA isolation, cDNA synthesis and PCR conditions were as described in [27], [32] . Relative expression levels were calculated by ΔCt and the standard curve method [27], [32].
Total lysates and mitochondria from C2C12 myoblasts and myotubes, and skeletal muscle, liver and heart from PGC-1β KO mice were isolated by differential centrifugation as previously described [6]. Protein obtained was quantified by the BCA method (Pierce).
SDS-PAGE, loading controls and densitometric analysis were performed as previously described [6]. Images were processed with Adobe Photoshop CS adjusting levels to reduce background.
The monoclonal antibodies against the ETC system subunits and Porin were described in our previous study [6]. OPA1 and Drp1 antibodies were purchased from BD Biosciences Pharmingen (San Diego, California, USA) and used at a 1/1000 dilution. Polyclonal antibodies generated in rabbit: serum raised against Mfn2 [6] was affinity purified by Sulfo-Link Kit (Pierce, Rockford, Illinois, USA), conjugating the immunogenic peptide (Eurogentec, Liege, Belgium) with an N-terminal cysteine (Nter C-LGPKNSRRALMGYNDQVQRP Cter) to a column and used at dilution 1/500. Mfn1 antibody was from Legros et al. [28] and Fis1 antibody from BioVision (Mountain View, California, USA) (1/500). All antibodies were diluted in PBS 5% dry non-fat milk and incubated overnight at 4°C with orbital agitation. Detection of primary antibodies was performed as previously described [6].
Cells were fixed with PFA 2%/Glutatraldehyde 0.125% in the plate and scrapped gently after 60' of fixation at room temperature. The rest of the protocol was performed as previously reported [6]. Images were taken at 40000x magnification in the electron microscopy platform, Serveis Científico-Tècnics (SCT), Universtitat de Barcelona.
Immunocytochemistry of C2C12 myoblasts and MEFs was performed as previously described, using the fluorochrome Texas red (conjugated to an anti-mouse secondary antibody from Molecular probes) [6] and coverslips were mounted in Mowiol (Calbiochem). Images were taken at room temperature with a Leica laser scanning confocal microscope SP2 (laser excitation 594 nm) using a 63X oil immersion objective (1.4). Images were magnified 5 times with Leica Lite ® software.
Images without magnification (with 30–40 cells per field) were used to count 150–200 cells, which were classified in two groups. Cells with a mean mitochondrial length >4 µm were considered to have elongated mitochondria (Long), the rest were considered short (Short). Mean mitochondrial length was determined (in images magnified 5 times) by measuring the length of 40–60 mitochondrial filaments per cell with Leica Lite ® software.
In the particular case of MEFs, transfected cells were counted (GFP-positive, laser 488 nm excitation, from 60 to 200 cells counted per independent experiment and transfection). In Mfn1 and Mfn2 −/−, we observed two additional cell populations, on the basis of their mitochondrial morphology (see Figure S4), which we named "fragmented", given that most mitochondria from these cells were spherical (similar percentages as previously reported, [3]) , and "perinuclear", for which almost all mitochondria located surrounding the nucleus.
Mitochondrial matrix-targeted GFP and RFP [28] were PCR amplified (with 5′ BamHI and 3′ EcoRI primers) and cloned into pGEM-T easy vector (Promega). Inserts, cloned into pGEM-T, were obtained by EcoRI-BamHI (New England Biolabs) digestion and were ligated (New England Biolabs) into BamHI EcoRI sites of pWPXL lentiviral vector (gift from D. Bach and D. Trono from EPH, Lausanne). Lentiviruses were produced and titrated as described [33]. C2C12 myoblasts were also transduced as described [33].
This assay was adapted from Legros et al. [28]. PEG was purchased from BDH and DMEM from Gibco. Twenty-four hours after transduction of C2C12 cells stably expressing mtGFP and mtRFP, cells were washed with sterile PBS, trypsinized and counted; 1.8.105 C2C12 mtGFP and 1.8.105 C2C12 mtDsRed were coseeded on 25-mm coverslips in 6-well plates. Forty-eight hours after transduction, cells were washed three times with DMEM and then incubated during 30"–40" with 500 µl of PEG:DMEM (1:1 weight:volume). Cells were then washed gently three times with growth medium plus cycloheximide (100 µg/ml) and incubated with this medium. Four hours after of 30"–40" incubation with PEG, cells were fixed (PFA 4% in PBS) and mounted at room temperature.
Polykaryons from cell fusion experiments were counted (75–100 polykaryons per independent experiment) using a Nikon E1000 fluorescence microscope (FITC filter 465–490 nm excitation for mtGFP and G2A filter 510–560 nm for mtRFP) and were classified on the basis of their level of colocalization. Polykaryons classified as "High" contained at least 50% of their green and red mitochondrial matrix-targeted proteins mixed due to mitochondrial fusion (yellow mitochondria). Polykaryons with lower percentages were considered "Low". Single stack representative images shown were taken with Leica laser scanning confocal microscope SP2 (laser emission 488 nm for mtGFP and 561 nm for mtRFP) using a 63X oil immersion objective (1.4) at room temperature. Images were magnified 2–3 times with Leica Lite ® software.
Two-tailed t student test was performed in all experiments. *, # show statistical significance at distinct p values under 0.05.
Figure S1 shows gene reporter experiments performed with a 2 Kb fragment of Mfn2 promoter and its mutated version without a functional Box 2. Figure S2 shows the quantification analysis of Porin induction by PGC-1β in total lysates of C2C12 differentiated muscle cells. Figure S3 shows the effect of PGC-1β overexpression on the abundance of different mitochondrial proteins expressed relative to Porin. Figure S4 shows representative images from different mitochondrial morphologies observed in Mfn2 mouse embryonic fibroblasts.
Box 2 mutation in Mfn2 promoter blocks PGC-1β-dependent ERRα coactivation. 10T1/2 cells were transfected with a 2-Kb (−1982/+45) fragment of Mfn2 promoter (white bars) or its mutated version (Mfn2 promoter Box2 Mut, black bars) together with an irrelevant vector (Basal), 75 ng of PGC-1β expression vector, ERRα or with PGC-1β+ERRα. Graphs display mean±SEM luciferase related to renilla activity values of 3 independent transfection experiments performed in triplicate. *Statistical difference compared to basal group, p<0.05. #Statistical difference compared to wild-type Mfn2 promoter, p<0.05.
Porin expression is increased ~78% in C2C12 myotubes transduced with PGC-1β adenovirus. Total lysates were obtained from non-transduced (NT), LacZ- or PGC-1β-transduced C2C12 myotubes at MOI 100 during 48 h and analyzed by Western blot with specific antibodies raised against Porin. Graphs show mean±SEM of Porin densitometric quantification levels related to LacZ Porin values from 4 independent differentiation and transduction experiments. *Statistical difference, p=0.01.
Normalization of mitochondrial dynamics components and ETC subunits protein levels induced by PGC-1β by Porin levels. Quantification analysis of protein levels of mitochondrial dynamics components and the ETC subunits from complexes I to V detected by Western blot and related to porin levels. Mean±SEM of Non- (white bars) , LacZ- (grey bars) or PGC-1β- (black bars) transduced C2C12 myotubes from n=4 independent differentiation and transduction (MOI 100) experiments. *, statistical difference compared to LacZ transduction, p<0.05.
Representative images from distinct transfected Mfn2 −/− MEFs classified on the basis of mitochondrial morphology. Mitochondria were labelled with anti-Cox1 antibody detected with a secondary antibody conjugated to Alexa 594. Scale bar, 10 µm.
We thank Tanya Yates for editorial support and Lídia Bardia for help in confocal microscopy. Competing interest: Christopher J. Lelliott is an employee and stock-holder of Astra Zeneca R&D.
Competing Interests: Christopher J. Lelliott is an employee and stock-holder of Astra Zeneca R&D.
Funding: This study was supported by research grants from the Ministerio de Educación y Cultura (SAF 2005-00445; SAF2008-03803), Grant 2005SGR00947 from the Generalitat de Catalunya, and CIBERDEM (Instituto de Salud Carlos III). A.Z. is the recipient of a Science Intensification Award from the University of Barcelona. M.L. is the recipient of a predoctoral fellowship from the Ministerio de Educación y Cultura, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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How "Hungry" Works
So I've been doing a lot of research lately on nutrition and I learned something really interesting about how our body decided whether it's full or hungry. I figured I'd share it here because it really helped me understand how a baby's (or child's) hunger cues work. Which I think makes feeding them what they need much easier.
The first thing you have to know is that there are two different "sensors" in the stomach that the body uses to determine when it's full. One measures quantity (the physical volume of food in the stomach) and one measures quality (the caloric density, or richness, of the food you ate). When you reach the threshold for either sensor you body signals your brain that you're full and you stop eating. This is the same whether you're a newborn or an adult.
Generally these two sensors are coordinated so if you're eating the right kinds of foods you should trip both sensors at the same time. This means that your stomach will be filled up and you'll have eaten the correct amount of calories/nutrients. However, if you don't eat the right stuff your sensors won't match and that's when you run into problems. For example, if all you're eating is lettuce you'll fill up your stomach long before you reach you calorie needs. The quantity sensor will trip and tell you you're full. BUT because you didn't get enough calories/nutrients it will tell you you're hungry again as soon as any space frees up. Of course you can also have the opposite problem. If you're eating a really rich piece of food, like a cheeseburger, you'll hit your calorie needs long before you hit your volume needs. At first your body will tell you to stop eating because you tripped the quality sensor, but soon it will realize you're not actually full and tell you to eat again - even though you don't need the extra calories. That's how you become overweight. It's all a big balancing act.
For babies and young children the same thing is true, except that they have the added issue of growth spurts. There are times in their lives where their caloric needs suddenly go up and their quality sensors don't trip even if you filled them up with the right stuff. It can take a little while for their quantity sensor to catch up because the stomach needs to physically stretch out or grow. That when we notice them suddenly getting hungry more frequently. You can either feed them richer foods (as long as they're not too rich) at each meal or add another meal into the mix. Whatever works for you.
For those of you who still have babies who are nursing/on bottles there is another interesting twist to the story. Breast milk and formula deal with the issue of growth spurts in different ways. It starts out the same. The baby will get hungry more often and you'll probably be frustrated with extra feedings for a bit.
For formula fed babies you'll probably start offering a bigger bottle, which essentially stretches out the stomach so that they can take in enough food to hit their quality and quantity sensors at the same time. For breastfed babies the opposite happens. The extra feedings stimulate your breasts to produce higher-calorie milk so the baby can reach their quality sensor without having to stretch out the stomach. Of course there is always some stretching, but not as drastic as it would be for a formula-fed baby. That's why you'll generally see older formula fed babies with large bottles while the same age breast-fed baby may only need half as much expressed breast milk.
So now you know :)


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Skype has released its version 3.0 for Android platforms, incorporating Microsoft login feature and an improved user interface. The UI for tablets such as Nexus 4, Asus' the transformer Prime, the Galaxy Tab 2 and others has been optimized by Skype. The extra screen space available has been made use of well by redesigning menus as well as navigation. The home screen is still available on phones, but in case of tablets, contact list will be placed in front centre for easy access.
Skype version 3.0 also supports SILK, the audio codec of the company. The codec improves the sound quality to its optimum level by collaborating with the speed of your internet connection. It helps the speech to become crisp and clear during the Skype experience.
A slight shortcoming was observed in Nexus 7 which has portrait style camera. The new Skype version is in landscape mode. This wastes a lot of valuable screen space. The Skype experience would have been excellent in Nexus 7 if this could have been taken care of. Despite this, the latest Skype update has brought a lot of design tweaks to the table, giving Android tablets an edge.
The application was also found to be a little slow, especially while launching the app when the splash screen persisted for about 8-10 seconds. But with all the new features, the Skype experience on Android is definitely something to look forward to for Android users. The update is now available at Google Play Store.


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Each month, Rated People connects over 50,000 homeowners with quality tradesmen - simply enter your job details in the form above to get started.
We'll match your job to the relevant tradesmen in your area - those interested will pay to see your contact details and call you to quote.
As each tradesman expresses interest in quoting on your job, we'll send you their profile and ratings to help you choose the right one for you.
After the work is complete, return to the site and rate your tradesman based on their quality of work, reliability and value for money.
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Clue: Unit of oil production: Abbr.
We have 1 answer for the clue Unit of oil production: Abbr.. See the results below.
Possible Answers:
Related Clues:
- Oil measure: Abbr.
- Crude meas.
- Tankerload: Abbr.
- Oil shipment: Abbr.
- Crude container?: Abbr.
- Crude amt.
- OPEC unit
- 42 gal.
- Oil meas.
- OPEC measure
Last Seen In:
- New York Times - February 24, 1998
Found an answer for the clue Unit of oil production: Abbr. that we don't have? Then please submit it to us so we can make the clue database even better!


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Author | NULL | Monday, Jan 01,1900 9:52 PM
Come 2001 and get ready for battle between big-budget extravaganzas. Coming up: two more mega shows from Big Daddy STAR TV. That's just for starters. Also on the anvil: Sony's Jeeto Chapper Phar Ke. Shah Rukh Khan pitching for new entrant B4U, an all-new game show from Zee, which is still smarting over the Sawal Dus Crore Ka fiasco. And the trend-setting Kaun Banega Crorepati going junior with a special series for kids to be anchored by the Big B.
KBC itself may take a small break next year. However, on the anvil is junior version of KBC with kids, expected to hit the small screen around middle of next year. The prize money is to be shared between the kids and their school.
Even the venerable Doordarshan has not been left untouched of the game show mania sweeping the nation. The national channel (DD1) will soon have its own version to be hosted by Kabir Bedi early next year.
Regional channels too have jumped into the fray. Sun TV has its own version of KBC in the form of Kodeeswaran, anchored by cine star Sarath Kumar. Vijay TV and Raj TV are also in the fray.
Raj TV is planning an Rs 1-crore prize money show in January. Vijay TV already runs a game show called Vijay Mahalakshadipathi , which was incidentally hit the screen before KBC made its debut.
According to media planners the trend of channels orienting their programming around mega shows is expected to gain ground over the next year. Live shows and Bollywood-oriented masala fares have been on offer by most channels.
"The channels are increasingly going to put a major chunk of their resources on one or two mega programmes," a media planner said. This is a trend widely observed even in mature but fragmented television market such as the United States.
Programming HeadRadio Mango


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Some children are addicted to sugar, while others are obsessed with the latest Snapchat filters and the endless quest for Pikachu in Pokemon Go — so what's the cure? Pick up one of these delectably marvelous reads. From saving magical kingdoms to skiing undercover at spy school, these 11 middle-grade novels are sure to deliver the extraordinary adventure every kid craves this fall — and parents won't get sued for trespassing trying to catch a digital monster, so that's a win.
What books are your kids (or you) reading? Tweet us your picks @BritandCo!
Brit + Co may at times use affiliate links to promote products sold by others, but always offers genuine editorial recommendations.


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Mother's Day Gifts
Our Mother's Day gift ideas have been carefully selected to give you plenty of inspiration when choosing the perfect present for the special woman in your life. If you're looking for something extra special this Mother's Day, you can be sure that you'll pick the best gift yet for your mum from our range of gorgeous Mother's Day gifts.
Mother's Day is a way to celebrate your mum or mother-figure and show her just how much you love and care with a timeless gift that can be treasured for a lifetime. With a selection of unique gifts to suit every style, taste and budget - choose something that's sure to bring a smile to Mum's face.
With pretty toast racks and quirky coffee spoons you can create the perfect breakfast in bed from the kids to start her day in the very best way. If you plan to surprise her with a bunch of beautiful flowers, make them extra special with one of our unique and stylish vases that she can enjoy using time and time again. We've also got great gift ideas for mums who love baking up a storm in the kitchen or enjoying a glass of wine and their favourite cheese at the end of a long day. Or maybe one of our more heart shaped sentimental gifts will be the key to your Mum's heart this Mother's Day?


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To dream of hiking up mountains or hills is a good omen if the top is reached. If you have trouble climbing, you will have to fight against envy.
Search Results for: hiking
Here's what we've found for you
A warning, the details of which depend on other symbols in the dream. If you dream of an accident while traveling by car, this could be a hint that you should either avoid travel at this time, or, if you can't avoid it, make sure that your car is in the best possible condition before […]
(Curse; Omen; Policeman; Warning sign) In a dream, thunder without rain means a scare, a warning or an ultimatum. It also represents good promises, gracious orders, or the sound of drums. Seeing thunder in a dream means repayment of one's debts, and for a sick person, it means recovering from his illness. Thunder, lightening and […]
The element offire in a dream represents might. Fire in a dream also means love. A product that is touched by fire in the process of its manufacturing or cooking in a dream means arguments and disputes. In a dream, fire also signifies glad tidings, a warning, war, chastisement, power, imprisonment, losses, sins, or blessings. […]
(See Naturalist)
Symbolic of life's journey or the path you are currently on in life


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After driving 14 hours from Florida to Virginia non stop I was done. I had planned on staying with family but it just didn't pan out so I booked a room just for one night. It was extremely close to my family and very easy to get to as well. It seemed like the hotel was just recently renovated and still somewhat under construction but not overly noticeable. The room was great, very clean and modern. For some silly reason I checked out not realizing it would probably just be easier to stay another two nights. We I decided to check back in they gave me a similar room on a different floor. Not even 10 minutes of being in the room and there were children running up and down the hall making all kind of noise, banging on doors. So I went downstairs and the gentleman at the front desk apologized and said he forgot there was a large family taking most of the rooms on that floor and he would move me immediately. I got my original room back, and I have to say it was one of the most enjoyable stays I have ever had in a hotel recently. The staff was helpful and very polite. I would have rated this hotel a little higher but the breakfast was pretty terrible. Granted it's complimentary but what good is that if it's not exactly edible? Anytime I go back I definitely plan on staying here again. The price was also great, I booked by calling the hotel and got a cheaper price than what was online through different hotel sites.
- Official Description (provided by the hotel):
- The Holiday Inn Express Washington DC SW-Springfield is a 10 story hotel with newly renovated guest rooms ideally located 13 miles south of Washington, DC at the junction of I-95, I-395, and I-495 Capital Beltway. Our hotel is 11 miles south of Reagan National Airport (DCA) and with our free shuttle servicing the Franconia-Springfield Metro Station the metropolitan area awaits. Local attractions and businesses include: the White House, Smithsonian, Arlington National Cemetery, Mount Vernon, Amtrak Auto Train, Raytheon, Lockheed Martin, General Dynamics, SAIC, General Motors, the Pentagon, Fort Belvoir, and Quantico. Shoppers will enjoy Springfield Mall, Tysons Corner Mall, and Potomac Mills.The Springfield Holiday Inn Express features 194 guest rooms with newly upgraded decor, complimentary high-speed Internet access (wireless throughout hotel, wired in select rooms), complimentary breakfast, free parking, and outdoor pool. Allow the professional sales staff at Holiday Inn Express in Springfield to assist you with your next banquet or social function; we can accommodate up to 200 people. Stay Smart with us and enjoy all of the convenience of Washington, without all of the hassle! ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Hotels.com, Orbitz, Priceline, IHG, Cancelon, Expedia, TripOnline SA, Travelocity, Hotwire, Evoline ltd and HotelQuickly so you can book your Holiday Inn Express Washington DC SW-Springfield reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Holiday Inn Express Washington Dc Sw-Springfield Hotel Springfield
- Holiday Inn Express Springfield
- Holiday Inn Springfield
- Springfield Holiday Inn


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I myself do not think that. Yet, I wonder what people think about it. Have some friends who happen to be strippers, they're kind of nice.
Most Helpful Girl
Obviously, literally no.
Unless they sleep with clients afterwards. Then yes.21|10|0
What she said.
Most Helpful Guy
A not so close friend of mine strips. I can't say I condone it, but she was my friend before she started, and I wouldn't want to lose a friend over this.00|00|0


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"Haven't seen a dentist in years? Searching for a new dentist?"
Welcome to the Greencastle dental practice of Dr. Elizabeth Scheidt.
Using the latest dental technologies, we can provide you with comprehensive dental care including preventative care, orthodontics, crowns and bridges, veneers, implants, and teeth whitening – ensuring your smile lasts a lifetime.
Our goal is simple: we want you to find our atmosphere calming, the staff friendly, and our dental services exceptional. To learn more about how we can help you keep your smile healthy and trouble-free, please call 765-653-5501. We will be happy to set up an appointment for you.
Our Services
See our wide variety of services; Diagnostic, Preventative, Tooth Whitening, Crowns, Mouthguards and much more! More >>
Common Questions
Check out our FAQ page to find answers to a lot of frequently asked questions about everything dental related. More >>
Book An Appointment
Use our online form to request a time for your next visit. We'll get back to asap to setup a time that works for your schedule! More >>


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Practice Statement
**NEW PATIENTS-Please arrive 15 Minutes prior to your appointment time with your drivers license and insurance cards***New patients, please fill out patient
registration forms here online, prior to your appoitment..
McLaren Macomb-Warren Family & Internal Medicine is Macomb area's premier location for multi-specialty care. The office is a designated Patient Centered Medical Home with a team of experienced physicians committed to providing top-rate health care to the community.
19041 E 12 Mile Rd
Roseville, MI 48066
13425 Nineteen Mile Rd.
Suite 100
Sterling Heights, MI 48313
- 1 other location
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- Primary Care Doctor
- Internist
- Family Physician
In-Network Insurances
- Blue Cross Blue Shield of Michigan
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- HAP (Health Alliance Plan)
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Good hike to the summit. Be careful on the way down. The rocks where plaster was used to prevent erosion are very smooth even when dry.
First half was a nice hike with moderate climb. Second half seemed like as much a rock scramble as a hike. Great view. Quick hike.
We used the same entrance to Ron Coleman Trail at McKelligon Canyon Rd. Just a few feet to the left, we took a trail that goes up near the canyon. The trail is not used to much, so in some sections you can not see it, but with care you can walk with no problems. Once at the top of the mountain ,we took the trail to the left. The trail continues going up and there are some sections that we had to climb the rocks, difficult but exciting, nothing that you can not do. You get to the ridge of the Franklin Mountains and continue on the trail. The last part to get to the Mount Franklin is a rocky area but easy to walk. When we returned to the same top mountain we started, we took another trail to go down. This trail is easier and took us to the McKelligon Rd, then turned to the left and arrived to the point we started. In some parts the trail is difficult, but once you are at the top of the Mount Franklin, you feel a great satisfaction, and you greatly enjoy the view. We recommend good hiking boots, no tennis shoes.


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Orchard Plums, Jamdani
Product Code: JA1727
Rs 3,900
Orchard Plums is an attractive jamdani silk-cotton saree. The lovely, purple saree is embossed with gold floral motifs all over, giving it an alluring look. The gold temples along the light purple borders add to its charm. Pair it with a gold or purple silk blouse and gold accessories for a gorgeous ensemble.


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There are really large animals out there. They are even larger than human. But have you seen large animals that is extraordinary in a sense that they are larger than their normal size?
The world is full of wonderful creatures – some were already discovered while others are yet to be discovered. Because of the revolution in biological technology, cross-breeding of some of the animals may result of an extraordinary large one. But most extraordinary animals were not cross-breed. Most of them were on the wild and was discovered with amazing sizes. If you haven't seen any, you're on the right page. We will show you the top animals with extraordinary sizes. some of them are domestic while others are wild.
You may say it's just a hoax. But don't close your mind yet until you see all of the photos. They really exists. In fact, they were recognized by most of the authoritative body in media and world records. Now, let's begin the tour and start our slideshow. Prepare to be amazed.


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"Excellent Service within A&E"
About: Pinderfields General Hospital Pinderfields General Hospital Wakefield WF1 4DG
Posted by Anonymous
My son recently had to attend A&E after he dislocated his shoulder. He was immediately taken to a cubicle and given pain relief.
We felt well informed about the care/treatment he was receiving.
All staff that we came into contact with were extremley professional and caring.
We hear all too often of bad service but never about the great service - for which this was.
Well done Pinderfields - excellent care!


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The heart of design in the new Mafraq Hospital is a dedication to health and community in the United Arab Emirates. The original Mafraq Hospital has been operating for over thirty years—and they were doing well. But they knew that the future of healthcare could be reflected and modeled in a new, upgraded facility. Something good for health and good for the environment.
Our team of architects, designers, engineers, landscapers, and more chipped in on the new Mafraq Hospital. The design is distinguished by four towers—prominent structures filled with floor upon floor of patient rooms. The new facility houses 499 beds, but was designed to expand internally up to 745 beds if needed.
Each floor features nursing stations and patient control centers, and at the top? Rooftop gardens. Rooms have large windows with ample light, and special floors cater to patients who require additional privacy and attention.
This energy-conscious and forward-looking facility offers a new experience for medical staff and patients—a new standard for modern healing environments.


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Cinisi Vacanze, Cinisi
- Free buffet breakfast and free WiFi
Main amenities
- 8 smoke-free guestrooms
- Rooftop terrace
- Free train station pick-up
- Room service
- Airport shuttle
- Childcare
- Air conditioning
- Daily housekeeping
- Car rentals on site
- Computer station
- Front desk safe
- Laundry service
- Free buffet breakfast and free WiFi
Feel at home
- Free cribs/infant beds
- Babysitting or childcare
- Babysitting or childcare (surcharge)
- Cookware, dishware, and utensils
- Stovetop
- Connecting/adjoining rooms available
What's around
- In Palermo Coast
- Casa Memoria Felicia e Peppino Impastato (0.3 mi / 0.5 km)
- Regional Museum of Palazzo D'Aumale (2.2 mi / 3.6 km)
- Carburangeli Cave (5.2 mi / 8.4 km)
- Bioparco di Sicilia (6.3 mi / 10.1 km)
- Carini Castle (7.9 mi / 12.7 km)
Cinisi Vacanze, Cinisi
Hotel Info: 866-539-8117from $51
- Quadruple Room
- Triple Room
- Double Room Single Use
- Apartment for 4 people
- Double Room
More great choices
At a glance
Key facts
Hotel size
- This hotel has 8 rooms
- This hotel is arranged over 3 floors
- Check-in time 3:30 PM-midnight
- Check-out time is 11 AM
- Express check-in/out
Guests should notify this property in advance of their anticipated arrival time. Please contact the office using the information on the reservation confirmation received after booking.
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- One child (3 years old and younger) stays free when occupying the parent or guardian's room, using existing bedding
- Babysitting/childcare *
- Pets stay free (dogs and cats only)
- 1 per room
- Free WiFi and wired Internet access in public areas
- Free WiFi and wired Internet access in rooms
- Airport shuttle (available 24 hours) *
- Free train station pickup *
- Cruise terminal shuttle *
- Ferry terminal shuttle *
- No parking
- Free nearby parking
- Area shuttle *
Other information
- Smoke-free property
- Resort fee included
Nearby Two Star Cinisi hotels
In the hotel
- Free buffet breakfast daily
- Coffee shop/café
- Room service (during limited hours)
- Bicycle rentals on site
- Mountain biking on site
- Computer station
- Front desk (limited hours)
- Tours/ticket assistance
- Dry cleaning/laundry service
- Laundry facilities
- Luggage storage
- Multilingual staff
- Number of buildings/towers - 1
- Year Built 1830
- Safe-deposit box at front desk
- Rooftop terrace
- Accessible bathroom
- In-room accessibility
- Roll-in shower
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Coffee/tea maker
- Bathrobes
- Iron/ironing board (on request)
- Washer/dryer
- Hypo-allergenic bedding available
- Down comforter
- Soundproofed rooms
- Premium bedding
- Memory foam mattress
- Individually decorated
- Individually furnished
- Separate dining area
- Private bathroom
- Rainfall showerhead
- Shower only
- Bidet
- Free toiletries
- Hair dryer
- 22-inch TV
- Digital TV channels
- Desk
- Free WiFi
- Stovetop
- Cookware, dishware, and utensils
- Free bottled water
- Daily housekeeping
- Connecting/adjoining rooms available
Top Cinisi hotels
Recently reviewed hotels in Cinisi
- Artemisia Palace HotelFrom
"Hotel was well situated, walking distance to all major sites. Room was clean and simple, bed comfortable. The staff were excellent, able to assist with recommendations for…"Aug 17, 2017
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"Loved the hotel, concierge at check In was a typical Sicilian (take that as you will) , but when we returned after dinner the next one was nice "Aug 17, 2017
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"Good hotel but hard to find (small sign with hotel name on side of building. Super secure since it's inside a building that requires 3 separate keys. Awesome location and walking…"Aug 14, 2017
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"Great hotel with a beautiful terrace!"Aug 14, 2017
Also popular with travelers from the USA
Cinisi Vacanze, Cinisi's small print
Also known as
- Cinisi Vacanze
- B&B Cinisi Vacanze Sicily - Province Of Palermo
All guests, including children, must be present at check-in and show their government-issued photo ID card or passport.
Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation.
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
Only registered guests are allowed in the guestrooms.
This property does not have elevators.
For transfers, guests must contact the property 48 hours prior to arrival at the number on the booking confirmation.
Mandatory fees
The charges below are included in the total price displayed:
- Resort fee
Optional extras
Babysitting/childcare is available for an extra charge
Airport shuttle service is offered for an extra charge of EUR 25 per room (one way)
Cruise ship terminal shuttle and ferry terminal shuttle service is offered for an extra charge
Area shuttle service is offered for an extra charge
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Is the description of this hotel not correct? Tell us


text: Maintreedescendants Austin Van Ness (1830-1896) Harriet Eliza Van Ness (1863-1955) Caroline R. Jacobus (1831-1909) Retrieved from "http://familypedia.wikia.com/wiki/Harriet_Eliza_Van_Ness_(1863-1955)/tree?oldid=547966" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Hidden category: Tree pages


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About Beach Street Inn & Suites
With an inspired vision in place, the Beach Street Inn and Suites was purchased four years ago by two local families who immediately started renovating the old Santa Cruz Motel. The new renovations have transformed this singular ocean front hotel property into the premier Boutique Inn and has been rated the best hotel in Santa Cruz by Trip Advisor and has been featured in 2012, 2013 and 2015 Sunset Magazine. Perched atop one of the most spectacular scenic vantage points on the Santa Cruz Coastline, Beach Street Inn and Suites offers guests breathtaking views of the Monterey Bay. We are a full service resort with retail shopping, gourmet café, and heated pool, all on site. This past winter we added a complimentary coffee, espresso and cocoa bar in our guest lounge for your enjoyment.
We are delighted to feature The Picnic Basket café at the base of our hotel. The Picnic Basket dramatically expands on the local and seasonal sourcing philosophy of The Penny Ice Creamery, a local landmark. The Basket offers breakfast, lunch, light dinner, full coffee service, local beer and wine, as well as ice cream. The Picnic Basket is the exclusive provider of in-room service for the hotel.
™, Beach Street Inn and Suites is within walking distance of the Boardwalk, the Municipal Wharf, and our eclectic Downtown, where visitors can enjoy a wide variety of restaurants, shops, art galleries, museums, cinemas and pubs. Our newly renovated rooms and suites offer guests a full range of amenities, including sleeping up to 6 persons, full kitchen, iPod docking stations, DVD players, flat screen TV's, free HBO, local and long distance calls and free Wi-Fi.
Take a tour of our Pool Patio Room and Ocean View Deluxe Veranda, then check our availability online or by calling (831) 423-3031 to book direct for the best Santa Cruz hotel on the beach.


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So this is my first strory. Enjoy!
Have you ever had a dream and in a day or two it some-what becomes reality? If you have you'll know what I'm talking about. I live in Varna, Bulgaria and have two friends that are in a relationship whit two girls. Over a year ago I had a dream. I was in a train station somewhere out side of my city (I study in another city over 200 km away and I see this train station every time when I come home). I was facing one of my friends and his GF when I asked them "Where are the others?". They replied in the same time and tone "Gone." When I woke up, I wondered what the hell did I just dream? I quickly forgot the dream and carryed on. 13 weeks later the couple that was "gone" had broke up. When I heared it the dream came up in my mind.
The train station - I wasn't gonna be there when the event happend.
"Gone." - They were going to brake up. (thank God that it's only this.)
The facing couple - They told me about it.
Since then whenever I had a strange dream I take triple coution. 4 months ago I dreamed that I was driving my father's car on the highway. I felt the cold inside, the panic in me ETC. When I woke up I laughted I sayed to my self "Ok man chill, this time nothing is going to happen. You don't even have license and the old man keeps his key with him always!". The next night I dreamed the street fatality from the new MK (the one in witch a car beheads you). I didn't took this dream seriosly too becouse I was plaing MK till I went to bed (every fight was on that map by the way!). The next day me and my family went out on a ride (with his car on the highway). It was really hot outside so we turned on the air condisioner to cold. I had a weird feeling when my past two dreams poped out. And then it all happend. we got envolved in a carcrash while I was sleeping in the back.The next half month I was in the hospital with a broken neck. When I fell asleep I invisioned me staring to a mirror. My image just spoke these words "I warned you!"


text: Set specific returns for both parties: Identifying specific rewards for each partner will help measure the effectiveness of the partnership, and reduce the chance of surprise or disappointment after the partnership is created. Identify the effort's benefits and requirements: I can't stress enough the need for the entrepreneur to ask: "Am I prepared to work hard to make this partnership work?" Being a good partner is hard work. While tending to your firm's daily operations, you must recognize opportunities you could share and try to exceed the parties' expectations. The partner can seem like another top-line customer, with often indirect revenue or value streams. Partnerships can be very valuable to a young business. But the wrong partner or situation can impede growth. Following these steps can save time and help ensure success.