text: Bundesliga Cordoba on target as Mainz rout Klopp's Liverpool vor 2 Stunden Jhon Cordoba (r.) capped off a fine display with the third goal in 1. FSV Mainz's 4-0 win over Liverpool FC. Summary Mainz open OPEL ARENA with 4-0 win over Liverpool.Brosinski, Cordoba, Malli and Muto with the goals.An emotional return for Mainz legend Klopp. Colombian striker Jhon Cordoba helped 1.FSV Mainz 05 christen their OPEL ARENA in the the perfect fashion, putting former coach Jürgen Klopp and his Liverpool FC side to the sword with a resounding 4-0 win. Klopp, who led Mainz into the Bundesliga and then the UEFA Cup for the first time before leaving for Borussia Dortmund, returned with a strong team to help the 05ers celebrate the rebranding of their 34,034-capacity stadium. It was an emotional homecoming for Jürgen Klopp, who spent 18 years at Mainz as a player and then coach. Former Bundesliga standouts Joel Matip and Emre Can started for the visitors as well as club captain Jordan Henderson, but that failed to stop the hosts dominating throughout, with Daniel Brosinski's early penalty and a pair of second-half goals from Yunus Malli and Yoshinori Muto complementing Cordoba's strike on the stroke of half-time. At the other end, new Mainz goalkeeper Jonas Lössl shone for 73 minutes, and was delighted in his post-match interview. "[It feels] fantastic, really. It's amazing," said the recently capped Denmark shot-stopper. "The atmosphere out there today, the great weather… and to finish it of with a 4-0 [win], we couldn't have asked for more. [The fans] were amazing. It was really great to be there" What a way to christen the #OpelArena. #Mainz05 beat @LFC 4-0!!! #M05LFC pic.twitter.com/WzaeRKzZi3— 1. FSV Mainz 05 (@Mainz05en) August 7, 2016 The result gives Mainz a welcome spring in their step before they kick off their 2016/17 Bundesliga campaign at Dortmund's SIGNAL IDUNA PARK on 27 August. More news Bundesliga Cordoba to stay at Mainz Bundesliga Humble Cordoba staying true to his roots at Mainz Bundesliga #DontCrackUnderPressure: Mainz stun Bayern Free! Get the Bundesliga newsletter delivered straight to your inbox. Sign up today! Subscribe


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It might be time for you to spend a few minutes looking through your bank accounts as new research by GoCompare has found people are spending an average of £30 on goods and services they don't want or need.
The research found 57% of people haven't reviewed their direct debits or standing orders in the last 12 months, but 14% said they knew they could save money if they went through their statements and 7% know there are subscriptions, donations and memberships they should cancel.
GoCompare estimates we could save an average of £360 in a year by freeing ourselves of these 'zombie payments' that are left unchecked.
How to detox your bank accounts
Here are some of the steps you can take to give your current account a detox – and potentially save hundreds.
Set aside time
The first step is to set aside some time to go through your bank and credit card statement to spot subscriptions, memberships or donations that you could cut or things you are paying too much for.
Gather information
Ideally you should check your statements over the last year to be sure you are taking a look at monthly payments as well as one-off annual charges.
Identify payments for the chop
A quick way to spot things to shed is by identifying which payments are Direct Debits (often shown as DD on statements) and Standing Orders (SO).
A Direct Debit requires you to sign a mandate to be set up and allows a company to take a fixed or variable amount of money from your account. You are entitled to contact your bank to cancel a DD instruction.
A Standing Order is an instruction you can set up that tells your bank to pay a fixed amount at regular intervals to another account.
You can use a SO to keep on paying indefinitely or to end on a certain date or after a set number of payments have been made. You can also change the payment amount or cancel an order whenever you want.
Weed out sneaky payments
You'll need to look harder to spot a Continuous Payment Authority (CPA). This type of payment allows companies to take a regular or one-off payment from your debit or credit card.
Insurers, gyms and subscriptions are often set up using ths type of payment.
The problem is CPAs allow them to take money from an account whenever they want and they can change the payment amount. This is a major problem if fees go up and you don't realise.
You will need to cancel a CPA through the company that is taking the payment.
Flag up payments you don't recognise
If you spot a payment you don't recognise then you should contact your bank immediately.
They might be able to provide more information and expand on the abbreviation or acronym iused on your bank statement.
If the payment is still a complete mystery to you it may be a sign of fraud.
Check terms and conditions
Before cancelling payments for goods and services make sure to check the terms and conditions for any exit penalties or notice periods and whether you may be entitled to a refund.
[Read more: Why life insurance should be on your New Year's resolution list]
'I've done it too': loveMONEY.com's Reena Sewraz confesses
I never thought it would happen to me, but I've been paying for a subscription I forgot I signed up for and don't use.
When I first spotted the payment for an Amazon Kindle Unlimited subscription I thought it was fraud, but tracing back my steps through some old emails I realised I had signed up for a 30-day free trial of the service in September last year, which I forgot to cancel.
Failing to spot my error sooner means I've been paying £7.99 since October 2016 for a service I never use.
In my defence it was easy to miss. Moving to a new house, along with the Christmas shopping rush, meant it wasn't unusual for Amazon payments to come up on my statements during the last few months of the year.
The other problem was the payment was only labelled 'AMZ UK Digital Download' which looks more like a one-off purchase for an e-book or music rather than a subscription to a service.
It's only now in January with the move and festive season over that I realise I've been paying for something I had forgotten I even signed up for and have wasted £31.96 in the process.
My silly mistake has been a wakeup call to detox my bank accounts and you should too.

subset: commoncrawl


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- Average
- Main course: £7
- Food spend: £15
- Opening hours
- Monday to Sunday: 09:00 - 23:00.
Very nice evening the meal was lovely will be returning
Gwyneth Harris-john
08 July 2017
from New Inn, Pontypool
From succulent ribs to irresistible tapas-style tweaks from around the world, Harvester Pontypool serves up a crowd-pleasing selection of freshly made delicacies. This inviting British pub and restaurant is popular with families thanks to its children's menus, and special activities offered to little ones for free.
From its succulent grill selection to the celebrated unlimited salad bar, the Harvester menu is packed full of wholesome flavours for the whole family. Rotisserie chicken cooked in a secret blend of spices is available alongside tender smoked ribs marinated in signature BBQ sauce, while smaller tapas-style dishes are perfect for sharing, with options like spicy crackerjack prawns, and grilled chorizo with padron peppers. There are generous combo platters, juicy steaks, well-stacked burgers, and a great salad selection with premium additions.
All kinds of sweet treats are served on the dessert menu at this Harvester in Blaenau Gwent. The sundae selection loads real dairy ice cream with toppings like warm chocolate fudge brownie, raspberries and honeycomb pieces, while classic British options like Bramley apple pie sit alongside international twists like key lime pie. From the bar, there are world beers, spirits, and speciality soft drinks for the kids.
With a welcoming ambience and reasonable prices, Harvester Pontypool sits just 7 minutes' drive from Pontypool & New Inn Rail Station.
Harvester - Pontypool
Ty Felin Fawr , Lower Mill Field, Pontypool, NP4 0RH
See all Harvester Restaurants
See all restaurants in Blaenau Gwent
Customer reviews for Harvester - Pontypool
- Food
- Value
- Ambience
- Location
- Service
- Drinks
((((( 8 July 2017 by Gwyneth Harris-john from New Inn, Pontypool
Very nice evening the meal was lovely will be returning
((((( 19 June 2017 by Debbie Middleton from pontypool
The staff where more than helpful as I wasn't well they moved me to a table that I was able to sit at & eat .
Food was amazing and the price was right I would go back with bell's on it. Staff where lovely as well, having worked there before I enjoyed seeing some old friends
((((( 17 June 2017 by Jayne Topping from Blackwood
Excellent food and service
((*** 6 March 2017 by Anthony Edgell from Pontypool
Not very good meat very chewer
((((* 22 December 2016 by Janet Barwood from Pontypoo;
We had a very pleasant lunch. The meals were well prepared and presented and the staff were friendly and attentive.
(((** 14 December 2016 by Julie Morgan from aber
Service was good, had a hair in my food so had to send it back. Menu has recently changed I love their slow cooked beef usuallly but was disappointed as they had changed flavour of it not for the better. Most of salad in salad bar totally bland.
((((* 9 November 2016 by Christine Benger from Newport
A Surprise visit from friends left us in a bit of a pickle regarding food, so I checked for availability online and booked a table for an hour later. All food was fab and we were told by our table lady that the place had been busting at the seems only an hour earlier due to a bonfire night crowd. All the salad area had been replenished and had plenty of selection on offer, so recovery had been swift. Staff were excellent, chatty and well mannered. No complaints about any of the food except the "Onion petals" which were a side order and very brown looking, soggy and fatty. My favourite dish is the jerk beef, it is always very, very tasty. Unfortunately I can't speak for the rest of my party.
((((( 9 October 2016 by Diane Rutter from Abergavenny
Booking service was quick and easy to use.my husband and myself had the triple chicken and a drink for twenty pounds, the service was excellent the waitress was helpful and polite nothing was to much trouble.
The food was presented nicely on the plate, it was cooked lovely ample food, the salad bar was clean and tidy all the container was full
((((( 5 September 2016 by Theresa Hayter from Portsmouth
Good food , food value
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Talk:Null Object pattern
|WikiProject Computing / Software
|(Rated Start-class, Mid-importance)
Strange "contradictory" intro statements for examples[edit]
- the C++ example "illustrates how the pattern is more complicated with types"
- the C# example shows how it can be properly implemented (this properly implemented seems to imply the example before is not proper, it however is almost identical).
Kasterma (talk) 17:48, 2 October 2014 (UTC)
Missing example: Qt[edit]
This article should mention the Qt framework. AFAIK, It is the only widely-used C++ library that implements null objects consequently. Ever wondered why the Qt framework uses neither exceptions nor C-style error codes? It is solely because of proper use of Null objects. Vog (talk) 16:23, 4 June 2014 (UTC)
Disagree with the article's statement[edit]
This article is completely wrong. Null pointers and Null variables have little or nothing to do with the Null Object design pattern. http://www.cs.oberlin.edu/~jwalker/nullObjPattern/
- Maybe I phrased things badly - my understanding is that one reason for the pattern is to avoid the checking needed to make sure a variable doesn't have a Null pointer or value as these add to the complexity of code, making it harder to maintain. If I'm mistaken, I'd gladly like to learn better. Autarch 12:23, 28 September 2007 (UTC)
- Upon reading the article, I thought that you were saying that we were using 'null' varaibles and checking whether they were 'null' each time we used them. If I understand correctly, the Null Object Pattern is actually about an object which implements default behavior in absence of something else. For instance, an implementation of an interface whose methods bodies are empty. The article is bad, but strangely it has excellent external links to flesh out a fair article. Ruijoel 13:06, 25 October 2007 (UTC)
I think a better example is that of a logger. Often you see code if logger != null then logger.log ( message ). With the null object pattern you then have a NullLogger which just ignores the input and you get logger.log ( message ). Also, often these null subclasses are singletons (since ignoring everything is often stateless). CaptainPinko (talk) 19:29, 10 May 2010 (UTC)
It looks like the article has been updated, as the content is correct now. The examples are still bad, though. The C example misses the concept entirely (the pattern requires the concept of objects, so I don't know that this pattern can be implemented in C) and while an empty C# array may exhibit similar behavior to a null object, it certainly isn't a good example of the practical use of the pattern. Bgano (talk) 22:35, 18 April 2011 (UTC)
Criticism of Criticisms[edit]
I have not read "Fowler, Martin (1999). Refactoring pp. 261", but the "Critisism" section is very weak and lacking of any actual criticisms. There is no value in saying "this may cause bugs, as references this source". 184.108.40.206 (talk) 22:05, 12 March 2013 (UTC)
function tree_size(null_node) { return 0 }[edit]
Can someone direct me to programming languages where exactly this or a very similar implementation is possible?
Thanks. — Preceding unsigned comment added by Brusinsky (talk • contribs) 12:55, 14 May 2016 (UTC)


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Download Hoval 360° apk 1.0.0 free for Android smartphone
Posted by Hoval in Business | Dec. 12, 2014 | 0 Comments
Apk file size: 20.0 MB
The new Hoval 360° app gives you an all-round view of a wide range of Hoval-related content and services.
The Hoval 360° app brings you a step closer into Hoval's world, putting more information, images, additional animations and film clips right at your fingertips. Via the user menu, you can select between the following features:
- Augmented Reality presents additional information, which is not available in the brochure. There is also a scan-icon, to enable you discover even more.
- QR-code reader can read all QR-code and makes it possible to access even more information.
- HovalTV is your access point to Hoval's world of film, offering technical film clips and much more.
- The partner section enables you to quickly find Hoval's installation partners in your area, providing you with both their location and contact information.
- The contact section contains an overview of Hoval's regional offices and allows you to contact Hoval directly and quickly.
Send your feedback to us by emailing [email protected] and let us know which functions you would like to see added to the Hoval app!
Hoval part of our Business and have average installs from 500 to 1000. Last Update Dec. 12, 2014. Google play rating is 77.1429. Current verison is 1.0.0. Actual size 20.0 MB.


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Dahlia Sky – Sexy Blonde Whore is Brutalized in Grueling Bondage
Released: May 20, 2016
Dahlia is a stunningly beautiful girl. Everything on her from the soles of her feet to her beautiful face, that just screams throat fuck me, makes me want to do evil shit to her. We all know how bad she wants to tormented in bondage. The torment ranges from pussy flogging clothes pins off of her body, to brutal bastinado, to predicaments that push her to the very edge. She perseveres through all of the punishment and begs for her reward like a good little pet.
Spare link: (try when other links are dead)


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Timberland 6-Inch Premium Boot In Brown/Dark Rust Available Now
Monday Oct 10 2016BUY
Timberland reveals an all suede rendition to their iconic 6-Inch Premium Boot ($200). This model is seen dressed in an all dark rust colored suede with traditional Timberland laces. A transparent outsole offers a unique looks to a classic silhouette. The timeless silhouette is finished with a waterproof coating to withstand any weather condition. Purchase your pair online and in-store.


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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.
Problems with the widget "Image Radio Buttons".Asked by Blaick on August 14, 2016 at 10:20 PM
I'm trying to configure the widget "Image Radio Buttons", but so far I've noticed two problems:
- The custom css code doesn't seem to work (or maybe I'm not doing it the right way). I need to center the module, specify the margin between the two pictures, change the size and the color of the text, etc.
-Once I select another field in my form, I can't select this field again. So I can't edit it more than once...
I've tried with another template but I have the same issues.
Could you please help me?
JotForm Support
You need the inject the CSS codes to Image radio button widget as you see below:
ul#images_button_container {
margin-left : 80px !important;
color : red !important;
If you have any question or issues, please do not hesitate contacting us. We will be glad to assist you.`
JotForm Support
I've created a separate thread for you, so anyone in the support staff will be able to assist you with your specific requirement. Please follow up there:
Okay I see, thanks a lot for your help =)


text:
Home page
New Product
About Rich Deer
Factory Introduction
Development History
Company Honor
Contact Us
Order Procedure
Sample Order
Regular Order
Domestic Stock Order
Import And Export
Contact Us
Home : Rich deer > About Us
Factory Introduction
About Rich Deer
Development History
Company Honor
Contact Us
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Copyright (c) 2009 Rich Deer (www.Rich-Deer.com) All Rights Reserved.
Website Support:
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Dan Kimball
Licensed Professional Counselor
Counseling for Men and Couples. I provide an innovative method for men who might not otherwise use mental health services. It is for men who are strong and self-sufficient, but may be at risk for addiction, anxiety, depression, or self-sabotaging behaviors, because they are hesitant to ask for help when they need it.
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Today's Featured Artist: Michael Godard
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Fine Art America is one of the largest, most-respected giclee printing companies in the world with over 40 years of experience producing museum-quality prints. All of our prints are produced on state-of-the-art, professional-grade Epson printers. We use acid-free papers and canvases with archival inks to guarantee that your prints last a lifetime without fading or loss of color.
This is a portrait of Red Wings great Steve Yzerman.
I am a Tampa native and have always lived in the Bay area. In 2001, I graduated with a Bachelors of Fine Arts from the Ringling College of Art and Design. I started painting in 2006 with the encouragement and guidance of a few close artistic friends. Most of my work is done in acrylic and has been displayed in several art shows in Tampa. I have always been a sports fan and have made sports the main focal point of my art. I am skilled in various media including graphite, marker, colored pencil, and acrylic. Outside of sports-themed art and caricatures, I create other works that range from the abstract to realistic portraits. Please contact me if you see something you like or want to commission a piece of art.
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Fine Art America sells thousands of pieces of artwork each month - all with a 100% money-back guarantee. We take great pride in the fact that hundreds of thousands of artists have chosen Fine Art America to fulfill their orders, and we look forward to helping you select your next piece!
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I passed by Shoe Gallery yesterday and saw these just chillin' in the cut in between a couple new releases. Even though these are a couple weeks old, there are still a few left at Shoe Gallery. These are part of the Lance Armstrong and Nike collaboration, "LIVESTRONG" project that was set up to "raise awareness and money for the war against cancer." These are definitely dope running shoes and upon first glance and seeing them in person – I could barely tell it was a shoe by Nike. But its ridiculously good-looking. Definitely make sure to go to Shoe Gallery and see it for yourself. Here are a couple words about the shoes courtesy of NiceKicks (where I also got the picture from as well):
"Designed with a Lunar bottom and a Nike Free Everyday upper, The Nike Lunar Everyday combines two of Nike's most innovative technologies. The Nike Lunar Everyday is a well-cushioned everyday running shoe for longer runs, offering a great ride. The breathable upper mesh of the Nike Lunar Everyday is reinforced with synthetic overlays to add maximum support. No-sew tip construction provides less irritation. Internal heel counter and plush foam package deliver maximum comfort. Utilizing the innovation of Lunarlite foam, the shoe helps protects the foot on long distance runs, distributing the pressure patterns more evenly across the foot."
Shoe Gallery
244 NE 1st Ave
Miami, FL 33132
(305) 371-2063


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|Volume 03, Issue 04
|February 18, 2002
| We annually like to devote an issue of QuintZine to self-assessment, getting to know yourself better, and applying new self-knowledge to personal and career development. We call it our ALL ABOUT YOU issue, and this year we look at exploring and identifying work values. A special feature looks at establishing purpose for your life through four "career investment" areas.
In the spirit of "ALL ABOUT YOU," we have a big favor to ask our readers. We'd like to ask you to take a few minutes to complete a totally anonymous survey. As we vow in our privacy statement, we absolutely will not use the information gained in any other way than to make our Quintessential Career site even BETTER for our readers. Please take our survey. Thank you for your help!
Finally, on a personal note, a birthday wish for a vital member of the Quint Careers team: Happy 15th birthday, JRH!
–Katharine Hansen, editor at email@example.com
| Workplace Values Assessment: Do You Know the Work Values You Most Want in a Job and an Employer — and Does Your Current Employment Reflect Those Values?
by Randall S. Hansen, Ph.D.
People expect to achieve certain ideals from their jobs, employers, and careers. These workplace values, concepts, and ideas that you hold dear have a direct impact on your satisfaction with your job, with your career, and even with your life. When you understand the values you cherish most highly, you can make an evaluation about whether your current employer (or a prospective employer) supports those values. And if you are considering a career change, understanding your values is critical to identifying a new career path.
|How well do you know your workplace values? If you're like most people, you may have done some self-assessment years ago when you were first starting out in your career, but have you taken the time recently to stop and see who and where you are now? After several job changes and promotions, are you still doing the kind of work that really suits you? After several ownership changes, mergers, and acquisitions, are you really with the type of company (and upper management) that respects and rewards your values? As you begin thinking about a job or career change, have you really spent the time thinking about the right job and right employer for what you value — and what you need in your life? Perhaps it is time for a work values check-up.
| Building Your Career Portfolio: Four Career Investments for a Purposeful Lifetime
by Carol A. Poore
"When I was laid off, I was shaking so badly that I didn't know whether I could drive home," said Kristin, 32, a mother of three who lost a public relations job. "My heart was pounding and I felt disassociated, like the whole thing was a bad dream."
After working at the same company for five years, David, 36, was escorted out of his building after being informed that the company was heading in a new direction and no longer needed his type of skills.
Shock, rootlessness and anger are just a few normal responses many feel when they lose their jobs to corporate downsizing. Since the national crisis on Sept. 11, the U.S. Dept. of Labor states that jobless claims are approaching 700,000 across the U.S. — the highest in nearly a decade.
How can you mitigate career risk — otherwise known as the pitfalls of marketplace change, economic turndowns, downsizing, personal health and life changes, and distasteful company politics that can wreck havoc with one's life?
The answer lies in building a CareerPortfolio.
| We've updated our reviews of free and inexpensive online career assessments. We've weeded out old assessment sites that are no longer in operation and added some new ones. These tools provide fun and useful ways to learn more about yourself and to gain food for thought about your career direction: Online Career Assessment Review.
Do you know of a free or inexpensive ($20 or less) online assessment that we should feature in our review? Have any comments about the way we've rated the chart's assessments? Let us know at: mailto:email@example.com
| ImproveNow.com
This site offers a number of assessments to guide career planning and especially to help improve one's current job situation. Assessments include Improve Your Boss, Improve Your Company, The PSI Personality Style Indicator, The JSI Job Style Indicator, and Improve Your JobFit.
The site describes one of our favorites, the PSI, this way: "Understanding our own personal styles and traits (and ideally those of the people we work closely with and for), we have an opportunity to be more successful, higher performing and more satisfied with our jobs and work environment. Taking the Personal Style Indicator assessment and asking all those with whom you work closely to take it, as well, will give you a leg up on improving your working communications and relationships and, therefore (and quite naturally), both your individual and group performance."
Some of the site's assessments are free; others offer a free summary report with a fee for a more detailed report; still others are fee-based. The JSI and PSI can be taken together.
See all our featured Quintessential Sites.
CreditJobsToday.com — where job-seekers in the trade credit industry can search for all types of jobs (by position, company size, location, and keywords), post your resume (confidentially), and use a job agent that will email you job opportunities. Apply directly to jobs via email. Free to job-seekers.
JOBFOOD.COM — a South African job, recruitment, and training site, where job-seekers can search for a job (by job type, industry, location, posting time, and keywords), submit your CV, and find valuable career information. A great site. Free to job-seekers.
naturejobs.com — a job site for practicing scientists and Ph.D. students in the sciences, where job-seekers can search for jobs (by keyword, location, company, and area of expertise), post your resume, and use a job alert tool — and where students can search for graduate assistanceships. Free to job-seekers.
Teens4Hire — a national online job-matching site for teens and the employers who hire them. Teens (14+) can create a personal profile, search for job openings from local and national employers, and apply for positions by forwarding your profile to select employers. Job types include full-time, part-time, seasonal, temporary, vocational, apprenticing, interning, and volunteering. Free to job-seekers.
Find even more career and job site additions to Quintessential Careers by visiting our Latest Additions section.
| Got a career question? The Career Doctor is holding office hours!
Kenn writes: "I would like to find a new career or even new location and would like to try and find someone online that can help me out in this endeavor. Do you have any idea of where I can find a person or site that may be of help?"
Career Doctor Randall S. Hansen responds to the question.
Mikaela writes: "I am 21 and having a mid-life crisis!!! I will graduate from the University of Maryland with a degree in marketing this May. One problem — after a few internships I am convinced that I do NOT want to go into marketing; in fact I do not want to go into business at all. The past few months, I have been drawn to medicine — specifically pediatric cancer — but I'm not sure that med school and being a doctor is in my future. So I have been considering law school — possibly to study medical ethics or patient advocacy. Basically I want to help families who are faced with difficult medical decisions.
| What careers do this? What kind of education should I be trying to get now? Does law school make sense?"
See the Career Doctor's response.
Agnes writes: "I need to know how to write a perfect letter after the first interview for the job of flight attendant. Could you please send me some samples as soon as possible?"
See the Career Doctor's opinion.
Dave Mahloy writes: "I was digging around your career site looking for information on how to make best use of references. I am considering asking two former managers and one personal friend to be references for me but want to know how much coaching I should give them. Do you have any articles or advice on this topic?"
See what the Career Doctor has to say.
Read more from the Career Doctor Archives.
Send your questions to: mailto:email@example.com
| Finding career inspiration is a matter of thinking like an 8-year-old, wrote Dave Murphy recently in the San Francisco Examiner. "If that 8-year-o inside you has died, it makes it just about impossible to change careers," Murphy writes of the wide-eyed innocence with which children dream big dreams of what they want to be when they grow up. Murphy suggests as inspiration not traditional career books, but books of poetry and quotes, as well as biographies of heroes to emulate. "Inspiration is all around you," he writes. "But first you have to let that 8-year-old come out and play."
On her Five O'Clock Club Web site, Kate Wendleton offers a free "Seven Stories Exercise" Worksheet" downloadable as a PDF document. The exercise asks you examine the most satisfying experiences of your life with the goal of identifying the skills you most want to use in the future.
Recruiting Trends reports that demand for sales and marketing professionals, while down from the record-high levels of the past two years, is still relatively strong and should remain buoyant for the rest of 2002. "Although projected new hires are down from the dizzying heights of a year ag , the fact that more than 40 percent of the companies we surveyed will be adding to their sales and marketing staffs during the next six months is a very optimistic sign for the economy," says Management Recruiters International president and CEO Allen Salikof. "There is market share out there to be gained, and sales people will be the drivers who bring that business to their companies." All regions of the country are projecting lower levels of hiring for the first half of the year, but no region is seriously lagging behind the national average."
|Quintessential Career Profiles feature readers who have interesting career stories to tell. Did you obtain a job in an unusual way? Has your career path been out of the ordinary? Have you held one or more unusual jobs? Has your job search been especially troublesome, inspirational, or remarkable? We want to hear from you! Tell us a little about your career story, and we may contact you for a full profile. Write to us and let us know about you. (If your e-mail program doesn't let you click on the link above to open a new e-mail message, just write to email@example.com using the subject line Quintessential-Career-Profiles).
| WATCH FOR feature articles on these topics in upcoming issues of QuintZine:
* How to choose a headhunter/recruiter
* How to write a counteroffer letter
* Home-based careers
* Career strategies for women
* How to start a job club
* Interviewing strategies for teens
* Using informational interviews to research companies
* How to use keywords to enhance your resume's effectiveness
* Letters of recommendation and references
* How to create and use a networking card
* How to resign from your job gracefully
* Quintessential Career Profiles of YOU, our readers
* Q&As with well-known career experts
* Book reviews
. . . and much, much more!
To view back issues of QuintZine, check out the QuintZine Archive.
Don't ever want to miss another issue of QuintZine? Get a free subscription to the email version of QuintZine by completing our subscription form.
We create resumes, cover letters, curriculum vitae, thank-you and follow-up letters, and list-formatted documents, such as salary histories and reference lists. We also can provide critiques and makeovers of your resume or cover letter. Credit cards accepted.
Check out Quintessential Resumes and Cover Letters!
Quintessential Careers also offers writing services for those who have been asked to give a speech and need assistance in researching and writing their remarks.
Read more about this exciting new service by going to Quintessential Speechwriting Services.
Need a speaker for your career-oriented conference or event at your college or organization? The Quintessential Careers Speakers Bureau can help! Our quintessential experts can provide presentations/workshops on a variety of career-related topics.
To find out more, visit: Quintessential Careers Speakers Bureau.
A publication of Quintessential Careers
Publisher: Dr. Randall S. Hansen
Editor: Katharine Hansen


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Related quotes
"A man who is aware only of his personal achievements, will never be aware of the collective failures of his community."
"He's a very humble person. When it comes to personal milestones, that's not what he's in it for. He's in it for the kids."
"If a person is incapable of humbling themselves, the Universe will gladly pick up the task for them, and can assure you; it will not be as kind."


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Transcriptome landscape of the human placenta
The placenta is a key component in understanding the physiological processes involved in pregnancy. Characterizing genes critical for placental function can serve as a basis for identifying mechanisms underlying both normal and pathologic pregnancies. Detailing the placental tissue transcriptome could provide a valuable resource for genomic studies related to placental disease.
We have conducted a deep RNA sequencing (RNA-Seq) study on three tissue components (amnion, chorion, and decidua) of 5 human placentas from normal term pregnancies. We compared the placental RNA-Seq data to that of 16 other human tissues and observed a wide spectrum of transcriptome differences both between placenta and other human tissues and between distinct compartments of the placenta. Exon-level analysis of the RNA-Seq data revealed a large number of exons with differential splicing activities between placenta and other tissues, and 79% (27 out of 34) of the events selected for RT-PCR test were validated. The master splicing regulator
Our data demonstrate unique aspects of gene expression and splicing in placental tissues that provide a basis for disease investigation related to disruption of these mechanisms. These data are publicly available providing the community with a rich resource for placental physiology and disease-related studies.
Pregnancy and parturition require an intricate interplay between maternal and fetal factors, orchestrated by the placenta, which lies at the interface between mother and fetus. The placenta performs multiple functions critical for fetal survival, growth, and development, including transport of gases, nutrients, and waste products, hormone production, protection of the fetus from maternal immune attack, and anchorage of the fetus to the uterus
The value of placental examination is well recognized in the setting of PTB, for instance, which complicates over 12% of all pregnancies in the U.S.
The normal function of the placenta depends on its structural integrity, and the proper growth and development of its structural components require the finely tuned regulation of relevant genes. Thus, alterations in gene expression and RNA processing may represent one of the major molecular mechanisms underlying pathological pregnancies. Previously, numerous studies have investigated changes in global human placental gene expression associated with gestational age
Alternative splicing (AS) is a common mechanism of gene regulation in higher eukaryotes, occurring in over 90% of multi-exon genes in the human genome
In this study, we used high-throughput RNA-Seq to conduct a genome-wide analysis of the normal placental transcriptome. RNA-Seq is a powerful technology for transcriptome analysis that allows global characterization of gene expression and AS at the nucleotide resolution
Overview of the RNA-Seq data
We sequenced pooled mRNA of amnion, chorion, and decidua separately taken from five normal term placentas (3 from male infants and 2 from female infants). For each of the placental tissues, we generated 2 lanes of paired-end Illumina RNA-Seq data with 54 bp and 72 bp in read length and 23-33 million reads of each lane, for a total of 50-60 million paired-end reads per tissue. We only used 50 bp of each end for mapping and analysis based on the sequencing error profile. In addition, we also obtained the Illumina Human Body Map 2.0 (HBM2.0) data with 73-83 million 50 bp paired-end reads from 16 normal non-placental human tissues (adipose, adrenal, brain, breast, colon, heart, kidney, liver, lung, lymph node, ovary, prostate, skeletal muscle, testes, thyroid and white blood cells). We mapped the sequence reads of each tissue to the reference human genome sequence (hg19) as well as all possible exon-exon junctions (Ensembl genes, r57). We obtained a high mapping rate with 70-90% and 7-10% of reads mapped to the reference genome and exon-exon junctions, respectively (Table S1 in Additional file
Additional file 1
Tables S1 and S2 and Figures S1-3 Supplemental Table S1. Mapping statistics of RNA-Seq data from placenta and HBM2.0 tissues. Supplemental Table S2. Distribution of gene expression level (FPKM) of RNA-Seq data from placenta and HBM2.0 tissues. Figure S1. Distribution of gene expression values (FPKM) for all tissues examined in the study. Figure S2. qRT-PCR validation of placenta-enriched SFs ESRP1 and MBNL3. Figure S3. Functional interaction network analysis of genes with enriched expression (EE) and differential splicing (DS) that intersect all three placental tissues: module 2. Circular node: a query gene. Diamond-shaped node: a linker gene. Node color was determined based on whether the query gene shows EE (green), DS (pink), or both (red). The most significantly enriched pathways were highlighted in bigger node size: integrin signaling pathway and ECM-receptor interaction pathway.
Click here for file
Global analysis of gene expression in placenta and other human tissues
Using the uniquely mapped read pairs, we estimated the expression levels of 22,523 protein-coding genes (Ensembl genes, r57) in each tissue using the "Fragments Per Kilobase of gene per Million mapped fragments" (FPKM) metric
Figure 1
Hierarchical clustering analysis of differentially expressed genes among placental and other human tissues
Hierarchical clustering analysis of differentially expressed genes among placental and other human tissues. We calculated expression levels of 51,682 Ensembl genes in each tissue and selected those expressed with FPKM > 5 in 8 or more tissues, which were then ranked based on their coefficient of variation (CV). The heat map was generated by average linkage hierarchical clustering of the top 1,000 differentially expressed genes, using 1-Pearson correlation coefficient as the distance metric. Scaled expression values are color-coded according to the legend in the top left corner.
To obtain a more detailed picture of genes potentially important for normal placental function, we compared the RNA-Seq gene expression profiles between the three placental compartments and the 16 HBM2.0 tissues to identify two types of genes with preferential expression in the placenta: (1) placenta-enriched genes, defined as genes with an FPKM value of at least 1 and greater than 4-fold difference in FPKM between any of the three placental tissues and the average of the 16 non-placental tissues as similarly defined in another study
We identified 938, 865, and 944 genes with at least 4-fold enriched expression in amnion, chorion, and decidua, respectively, as compared to non-placental tissues, including 216 genes shared among the three compartments of the placenta. We also used a similar strategy to generate a list of 758 placenta-enriched genes using the GeneAtlas microarray data set covering whole placental and other human tissues
Figure 2
Analysis of placenta-enriched and -specific genes
Analysis of placenta-enriched and -specific genes. (a) Number of tissue-enriched (blue bar) and tissue-specific (red bar) genes. Tissue-enriched genes were defined as genes with more than 4-fold change in expression and minimum FPKM of 1. (b) Proportions of overlapping genes between the placenta-enriched gene list and the MGI or PTB gene list (see text and Methods for details). The lighter shade indicates the proportion of non-placentaenriched genes while the darker shade indicates the proportion of placenta-enriched genes. P-values were determined by Fisher's exact test. (c) Expression profile of the 70 placenta-enriched MGI list genes. Gene expression values were normalized for each gene and color-coded using the same scheme depicted in Figure 1. (d) Expression patterns of placenta-specific genes in amnion, chorion, and decidua. Color scheme is based on log10(FPKM value).
The RNA-Seq data also allowed us to identify genes whose expression was restricted to the placenta (i.e. not a single read detected in any of the 16 non-placental tissues). We identified a total of 170 placenta-specific genes in the three placental compartments combined. We also used the same criteria to identify tissue-specific genes within the 16 HBM2.0 tissues. Consistent with the pattern observed for the tissue-enriched genes, the three placental tissues were among the tissues with the highest number of tissue-specific genes, only after testes and brain. Taken together, these data indicate abundant tissue-specific activation of gene transcription in the placenta.
Genes enriched in or specific to the placenta play important roles in placental function and pregnancy-related diseases
In order to understand the functional significance of the genes with enriched expression (EE) in the placenta, we asked whether these genes have been implicated in placental biology and/or pregnancy disorders. We compiled two lists of human genes using the Mouse Genome Informatics (MGI) database
Many of the genes associated with placental abnormalities in mice (see the heat map of their expression patterns in Figure
To gain more insight into key processes that may possibly explain functional differences among the three placental tissues, we carried out functional annotation analysis of placenta-enriched genes identified in each of the three placental tissues compared with the other 16 human tissues using DAVID
Table 1
Gene Ontology (GO) analysis of placenta-enriched genes
Number of genes
Fold Enrichment
Bonferroni-corrected P-Value
GO:0007398 ~ ectoderm development
GO:0008544 ~ epidermis development
GO:0007155 ~ cell adhesion
GO:0022610 ~ biological adhesion
GO:0030855 ~ epithelial cell differentiation
GO:0009913 ~ epidermal cell differentiation
GO:0030216 ~ keratinocyte differentiation
GO:0060429 ~ epithelium development
GO:0018149 ~ peptide cross-linking
GO:0031424 ~ keratinization
GO:0043062 ~ extracellular structure organization
GO:0030198 ~ extracellular matrix organization
hsa04512:ECM-receptor interaction
hsa04510:Focal adhesion
GO:0007155 ~ cell adhesion
GO:0022610 ~ biological adhesion
GO:0007166 ~ cell surface receptor linked signal transduction
GO:0007398 ~ ectoderm development
GO:0007223 ~ Wnt receptor signaling pathway, calcium modulating pathway
GO:0008544 ~ epidermis development
GO:0007565 ~ female pregnancy
GO:0009611 ~ response to wounding
GO:0051270 ~ regulation of cell motion
GO:0001501 ~ skeletal system development
GO:0060429 ~ epithelium development
GO:0001568 ~ blood vessel development
GO:0030334 ~ regulation of cell migration
GO:0016055 ~ Wnt receptor signaling pathway
GO:0001944 ~ vasculature development
GO:0042127 ~ regulation of cell proliferation
GO:0035295 ~ tube development
GO:0048514 ~ blood vessel morphogenesis
GO:0001525 ~ angiogenesis
hsa04060:Cytokine-cytokine receptor interaction
hsa04512:ECM-receptor interaction
hsa04340:Hedgehog signaling pathway
hsa05217:Basal cell carcinoma
hsa04510:Focal adhesion
hsa05200:Pathways in cancer
P00034:Integrin signalling pathway
GO:0007565 ~ female pregnancy
GO:0042060 ~ wound healing
GO:0048732 ~ gland development
Analysis was performed on genes identified as being enriched in each of the three placental tissues compared to the other 16 human tissues
# GO term Biological Process categories are based on GO FAT definition from the DAVID website; KEGG and PANTHER are the corresponding pathways defined in the DAVID website.
Although there was no GO annotation shared by all three compartments, we identified several biologically relevant enriched categories that overlap between the two membranous compartments amnion and chorion. For example, epithelium development, one of those categories, explains a common compositional feature that exists between the two tissues with both at least partially consisting of a layer of cells that are epithelial in origin (the amniotic epithelium and extravillous cytotrophoblast)
Among the non-overlapping GO terms, it was noted that there was significant overrepresentation of vascular-related GO terms such as blood vessel development, vasculature development, blood vessel morphogenesis, and angiogenesis in the chorion, while these terms were absent from the amnion, an avascular tissue. One of the genes belonging to these categories is
We found that three GO terms are significantly enriched for the decidua with female pregnancy being the most enriched category, consistent with the role of decidua as a principal source of hormones and cytokines pivotal in the maintenance of pregnancy. It was noted that many of the genes associated with female pregnancy have also been implicated in pregnancy-related disorders. These genes include transforming growth factor beta 1 (
For placenta-specific genes, we further removed genes with extremely low FPKM values (< 0.3) in the placental tissues, which could represent genes with universal low expression in all tissues but sampled by RNA-Seq in the placenta by chance. This led to a final set of 24 placenta-specific well-annotated protein-coding genes with FPKM > 0.3 in at least one placental tissue. The placenta-specific genes are highly enriched for genes encoding pregnancy-related hormones, including pregnancy-specific glycoproteins (
Expression profiles of splicing factors (SFs) in placental and other human tissues
The deep RNA-Seq data also allowed us to go beyond whole transcript level changes, to identify transcript isoform changes due to pre-mRNA alternative splicing (AS). Splicing factors (SFs) are RNA binding proteins that play key roles in AS regulation
To identify SFs with a placenta-specific increase or decrease in expression levels, we compiled a list of sixty well-studied SFs
Figure 3
Expression profile of splicing factors in placental and other human tissues
Expression profile of splicing factors in placental and other human tissues. (a) Heat map showing the expression levels of 60 selected splicing factors across all 19 tissues. Scaled expression values are color-coded according to the legend in the top left corner. Clustering of genes and tissues are both generated by average linkage hierarchical clustering using 1-Pearson correlation coefficient as the distance metric. (b) Expression levels of 3 splicing factors differentially expressed between placental and other human tissues. Each bar labeled HBM2.0 (in blue) represents mean expression value of all 16 HBM2.0 tissues.
RNA-Seq and RT-PCR analysis of exon skipping events in placental and other human tissues
To directly identify AS differences between the placenta and other human tissues, we calculated the exon inclusion level (Ψ) of alternatively spliced cassette exons in each tissue using RNA-Seq reads that are uniquely mapped to the upstream, downstream, and skipping exon-exon junctions of alternatively spliced exons as previously described
Figure 4
Summary of differential splicing events identified by RNA-Seq
Summary of differential splicing events identified by RNA-Seq. (a) Number (percentage in parentheses) of exons with differential inclusion levels ((Δ|Ψ| > 0.1, FDR < 0.1) between given tissue pairs. (b) Venn diagrams showing the distribution and overlap of exons (left) and genes (right) in the three placental tissues that are differentially spliced between the placental and non-placental tissues.
In order to boost the power of RNA-Seq splicing analysis and obtain a robust set of splicing differences between the placental and non-placental tissues, we pooled the RNA-Seq data of all HBM2.0 tissues. We then compared the pooled data to that of each placental tissue. We identified 393, 637, and 402 differentially spliced exons (in 275, 464, and 289 genes) when comparing the pooled non-placental tissues to amnion, chorion, and decidua, respectively (Figure
Figure 5
Examples of exons with splicing differences between placental and HBM2.0 tissues
Examples of exons with splicing differences between placental and HBM2.0 tissues. (a) Exon ENSE00000882762 in
To further confirm the RNA-Seq results of exon splicing, we randomly selected 34 exons in total (including the 3 aformentioned exons) for fluorescently labeled RT-PCR. Using an independent set of term placental samples (
Figure 6
Validation of differentially spliced exons between placental and other tissues
Validation of differentially spliced exons between placental and other tissues.(a) Correlation of exon inclusion level differences between placental and HBM2.0 tissues estimated by RNA-Seq (x-axis) and by RT-PCR (y-axis). The dots are color-coded based on the placental compartment to which the values for other tissues were compared. The grey line indicates y = x. Two dashed lines indicate the 0.1 inclusion level difference, which was used to select target exons for validation. (b) Significant enrichment of ESRP1 targets among exons that are differentially spliced between amnion and other tissues. The darker and lighter shades indicate the proportions of exons with and without splicing differences (according to RNA-Seq) between amnion and other tissues, respectively. P-value was determined by Fisher's exact test. (c) An example of ESRP1 target exons differentially spliced in amnion. Shown are a wiggle plot of RNA-Seq read coverage for
The splicing factor ESRP1 regulates tissue-specific splicing in amnion
The placenta-specific increase in the expres levels of certain master splicing regulators such as ESRP1 and MBNL3 raises the possibility that downstream exon targets of these regulators may have altered splicing activities in the placenta over non-placental tissues. To test this, we studied the splicing factor ESRP1, which had 5.4 fold higher expression in amnion over the average of the 16 HBM2.0 tissues (Figure
Given our moderate sequencing depth in the placental tissues, it is possible that additional ESRP1 target exons with differential splicing in amnion were missed by RNA-Seq. We therefore selected additional 21 ESRP1 target exons besides the aforementioned 5 validated exons for RT-PCR analysis, resulting in 26 exons tested in total. Seven of those exons did not have any RNA-Seq reads presumably due to their relatively low expression levels and the limited coverage depth of our sequencing data. We confirmed that 12 of the 26 ESRP1 target exons showed more than 10% changes in splicing in amnion, with known ESRP1-enhanced exons having increased splicing activities, and known ESRP1-silenced exons having decreased splicing activities. One of the validated ESRP1 target exons was in misshapen-like kinase 1 (
Analysis of pathways influenced by tissue-enriched expression and differential splicing in placenta
The differential gene- and exon-level expression patterns observed between the placental and non-placental tissues may underlie gene pathways that have key roles in the normal biology of the placenta. To identify pathways and molecular networks influenced by placenta-specific gene expression and splicing, we constructed functional interaction (FI) networks
The results of our analysis performed on each of the three placental tissues showed significant enrichment of many functional pathways (Table S3 in Additional file
Additional file 2
Table S3 Enriched pathways (FDR < 0.05) in the whole network and submodules (module size > 50) from functional interaction network analysis.
Click here for file
Figure 7
Functional interaction network analysis of genes with enriched expression (EE) and differential splicing (DS) in the placenta
Functional interaction network analysis of genes with enriched expression (EE) and differential splicing (DS) in the placenta. Shown is module 0 of the interaction network constructed for chorion. Circular node: a query gene. Diamond-shaped node: a linker gene. Node color was determined based on whether the query gene shows EE (green), DS (pink), or both (red). Two large circular clusters represent highlighted significantly enriched pathways in chorion: glypican, SMAD2/3, and TGF-beta receptor signaling pathways (left-hand side; red lines) and HIF-1 alpha transcription factor signaling pathway (right-hand side; blue lines). FOS and SERPINE1 are shared by both groups of enriched pathways, but only shown in the right cluster. Several linker hub genes with dense connections with the highlighted pathways are also shown in bigger nodes.
The analysis performed on genes abundantly expressed and/or differentially spliced in all three placental tissues revealed strong overrepresentation of pathways related to integrin signaling and focal adhesion (Figure S3 in Additional file
Novel transcriptional active regions (TARs)
One major advantage of RNA-Seq compared to microarray technology is its capability to detect un-annotated novel transcripts. To identify novel transcriptional active regions (TARs) in placental tissues, we used the software Scripture
Table 2
Novel transcriptional active regions (TARs) and exons discovered in placental tissues
Total transcripts
Multi-exon# transcripts
Perfect match with annotated transcripts (ignoring transcript start and end)
Total TARs*
Novel TARs (not overlapping with the combined annotation of Ensembl, UCSC, RefSeq and Vega genes)
Internal exons in TARs overlapping annotated transcripts
Total exons
Annotated exons
Novel exons with one end (5' or 3') shared with an annotated exon
Novel exons overlapping with an annotated exon but with no shared 5' or 3' end
Novel exons not overlapping with any annotated exons
Analysis was performed using software Scripture for
*TAR: Transcriptional Active Region after merging overlapping transcripts
# Single exon transcripts were not included in all the downstream analysis
Additional file 3
Table S4 FPKM expression levels in all tissues for the novel TARs identified from the placental tissues.
Click here for file
Figure 8
An example of novel transcriptional active regions (TARs) identified in the present study
An example of novel transcriptional active regions (TARs) identified in the present study. Shown is a novel TAR on chromosome 16 found in amnion. A wiggle plot of RNA-Seq read coverage, structures of 3 alternatively spliced transcripts and ESTs were shown from top to bottom. Note that there is no gene annotated in this region in the indicated annotation databases.
We also used RNA-Seq data to identify novel exons in annotated genes. There are a total of between 93 and 103 thousand exons identified in the TARs overlapping with annotated genes. Although more than 80% of these exons were well annotated with the same 5' and 3' ends, we detected between 494 and 585 totally new exons with no sequence overlap with any annotated exons in the placental tissues. These novel TARs and exons provide a valuable resource for novel transcripts with potential functional significance in the placenta.
With the emergence of new high-throughput technologies such as RNA sequencing, we have recently witnessed a remarkable increase in our knowledge of mammalian transcriptome content and diversity. There has been a particular surge in our understanding of the transcriptome diversity between different tissues and cell types. For example, Wang et al. performed an RNA-Seq analysis of 15 human tissues and cell lines and identified over 22,000 tissue-specific AS events
The majority of previous gene expression studies of human placental tissue have only provided gene-level insights
In the present study, we used RNA-Seq to characterize the transcriptome of selected compartments of the human placenta from normal term pregnancies. RNA-Seq allows an unbiased and sensitive interrogation of the full repertoire of placental mRNA transcripts. We took a two-step approach to analyze the RNA-Seq data at both the gene-level and the exon-level. First, we investigated differential gene expression between the placental and other human tissues to identify genes that are specifically or abundantly expressed in the placenta. Second, we carried out exon profiling as well as SF expression profiling to find AS events and their potential regulators that are differentially present in the placental versus non-placental tissues.
We have compared placenta-enriched genes to genes with putative functional significance in the placenta using the mouse phenotype data and human PTB association study data. We observed that genes implicated in placental abnormalities and PTB are enriched among the genes with placenta-enriched expression profiles. We note that the mouse phenotype data from MGI were generated independent of any previously known gene expression pattern in the placenta. Among such genes are
Because the HBM2.0 data all came from adult tissues, it is possible that some placenta-enriched genes identified in our study reflect age-specific expression signatures. Because of the unavailability of RNA-Seq data from other fetal tissues, we assessed this possibility using the GeneAtlas array data
Given the heterogeneous tissue composition of the placenta, we have characterized the transcriptome profiles of the placenta not only at the whole-organ level, but also at the sub-organ level. It should be noted that the placental samples used in our study (amnion, chorion, and decidua) may not be completely pure, containing minor contamination with other placental components. Nonetheless, our study demonstrated that they are highly enriched for the corresponding tissue types, displaying compartment-specific expression profiles and splicing patterns. The amnion is the innermost layer of the fetal membranes lining the amniotic cavity and is composed of an epithelial cell layer on top of a basement membrane and an avascular matrix
We also examined potential interactions among genes highly expressed and differentially spliced in the placenta compared to other human tissues by constructing FI networks composed of sub-network modules enriched for specific gene categories and functional pathways. Analysis performed separately on each of the three placental tissues revealed enrichment of set of pathways commonly enriched in all three compartments, for example, regulation of cytoplasmic and nuclear SMAD2/3 signaling and TGF-beta receptor signaling. These pathways are known to be involved in a wide range of cellular processes
Our study provides the first comprehensive view of the placental transcriptome at exon-level resolution, and reveals that tissue-specific gene regulation in the placenta involves complex changes in both gene transcription and exon splicing. Our data should serve as a valuable resource for future in-depth investigations into what genes contribute to specification of the placenta. All of the RNA-Seq data can be accessed as the raw RNA-Seq reads and as a processed UCSC Genome Browser custom track
Tissue collection
Fresh human placentas were obtained within one hour of normal vaginal delivery at term with signed informed consent under protocols approved by the University of Iowa Institutional Review Board (200506792, 200411759). The placentas were received largely intact when visually inspected. Each placenta was dissected into the fetal (amnion, chorion) and maternal (decidua) portions. The amnion and chorion were taken from the reflected membranes and separated by blunt dissection. Decidual tissue samples were macroscopically isolated from the maternal-facing surface of the placenta. The dissected tissues were cut into small pieces and placed in RNAlater® solution (Applied Biosystems, Foster City, CA). To ensure that our results better reflect the true nature of the normal term placental transcriptome, we used placentas from term (≥ 37 weeks of gestation) deliveries with spontaneous onset of labor.
RNA extraction
Total RNA was extracted from each tissue using the TRIzol® reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instructions and stored at -80°C until used. For RNA-Seq, we prepared pooled amnion, chorion, and decidua samples, using an identical set of RNA from five different individuals. The pooled samples were of high quality with an RNA integrity number (RIN) > 8. For validation of differential splicing events and splicing factor expression, we generated RNA pools, each for amnion, chorion, and decidua, consisting of 4 biological replicates that are independent from those used in the RNA-Seq experiments. For validation experiments, we purchased total RNA representing all HBM2.0 tissues except white blood cells from Applied Biosystems (Foster City, CA) or Clontech (Mountain View, CA).
Library construction and sequencing
Library preparation and paired-end sequencing were performed by Ambry Genetics (Aliso Viejo, CA). Double-stranded cDNA fragments were synthesized from mRNA, ligated with adapters, and size-selected for library construction according to the manufacturer's protocol (Illumina, San Diego, CA). Each of the three libraries generated was loaded onto one lane of the flow cell at 8 pM concentration. Two paired-end runs (72 bp and 54 bp runs) of sequencing were carried out on the Illumina Genome Analyzer IIx. Initial data processing was performed using RTA 18.104.22.168 (SCS version 2.6.26). Sequence quality filtering script was executed in the Illumina CASAVA version 1.6.0 software (Illumina, Hayward, CA).
Sequence alignment
For each end (forward or reverse) of the paired-end reads from placenta, we trimmed the sequence to 50 bp based on the sequencing error profile. The HBM2.0 data consist of the following tissues: adipose, adrenal, brain, breast, colon, heart, kidney, liver, lung, lymph node, ovary, prostate, skeletal muscle, testes, thyroid and white blood cells. Each tissue came from a single adult donor with ages ranging from 19 to 86. The HBM2.0 data are accessible from EBI ArrayExpress track:
Gene expression quantification using RNA-Seq data
We estimated the gene expression level using RNA-Seq by the Fragments Per Kilobase of gene per Million mapped fragments (FPKM). Ensembl release r57 was used for gene annotation. To avoid the ambiguity of assigning reads to different isoforms of the same gene and obtain a robust estimate of the overall gene expression levels, we used an exon union method by counting all reads mapped to any exon in any of the gene's isoforms. This approach is similar to the original RPKM definition
Placenta-enriched genes based on GeneAtlas array data
We selected 16 tissues from the Human GeneAtlas array dataset
Enrichment of GO functional categories and pathways
To identify the overrepresented functional categories among the genes with enriched expression or differential alternative splicing in the placenta compared to the 16 HBM2.0 tissues, we used the online functional annotation tool DAVID
Placental abnormality- and preterm birth-related genes
To obtain genes associated with abnormal placental phenotypes, we searched the MGI database
Alternative splicing analysis
We focused our analysis on the exon-centric analysis. We only used those reads that uniquely mapped to the splicing junctions to estimate the exon inclusion level (Ψ) of alternatively spliced exons. We used the same formula as in
Fluorescently labeled RT-PCR and qRT-PCR
We validated 2 sets of exons using RT-PCR. One set includes 34 exons that showed significant differential splicing (> 10% inclusion level difference with FDR < 0.1 between one of the three placental tissues and the pooled HBM2.0 tissues). Another set includes 21 known ESRP1 target exons that are predicted to have differential splicing due to differential expression of ESRP1 in amnion. Single-strand cDNA was synthesized from total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. Fluorescently labeled RT-PCR was performed as described
Additional file 4
Table S5 Exon inclusion levels and primer sequences for exons selected for RT-PCR validation.
Click here for file
Additional file 5
Figure S4 RT-PCR analysis of 34 exons that showed significant differential splicing (> 10% difference in exon inclusion level, FDR < 0.1) between placental and HBM2.0 tissues. Figure S5. RT-PCR analysis of 21 ESRP1 target exons.
Click here for file
Functional interaction networks of genes with placenta-enriched expression or differential splicing
We combined the genes with placenta-enriched expression or differential splicing into 4 query gene sets: combination of placenta-enriched genes with FPKM > 1 and > 4 fold enrichment (EE) and genes significantly differentially spliced with FDR < 0.1 and |Ψ1-Ψ2| > 10% (DS) compared to the HBM2.0 tissues in each of the three placental tissues individually and the intersection set of all three tissues. We projected each of the query gene set onto the functional interaction network of human genes from the Reactome database
Discovery of novel transcriptional active regions (TARs)
Scripture software
Data accessibility
All data described here can be accessed from:
AS: Alternative splicing; DS: Differential splicing; EE: Enriched expression; FI: Functional interaction; FPKM: Fragments Per Kilobase of gene per Million mapped fragments; GO: Gene Ontology; HBM2.0: Human Body Map 2.0; IUGR: Intrauterine growth restriction; MGI: Mouse Genome Informatics; PE: Preeclampsia; PTB: Preterm birth; TAR: Transcriptional active region.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JK, KZ, JCM and YX designed the study. KZ, PJ, and JW analyzed data. JK isolated the tissues, prepared RNA for library preparation and sequencing. ZL conducted the RT-PCR validation experiments. JK, KZ, JCM and YX wrote the manuscript. All authors read and approved the final manuscript.
We thank Elizabeth Kenkel for assistance with the PCR experiments and Roger Williamson for providing helpful comments on the manuscript. The HBM2.0 RNA-Seq data were kindly provided by the Gene Expression Applications research group at Illumina. The work was supported by Burroughs Wellcome Fund 1008841.01 (to JCM and YX), NIH grant HD52953 and HD57192 and grants from the March of Dimes (to JCM #6-FY08-260 and #21-FY06575; and to YX #5-FY10-60).
We thank Elizabeth Kenkel for assistance with the PCR experiments and Roger Williamson for providing helpful comments on the manuscript. The HBM2.0 RNA-Seq data were kindly provided by the Gene Expression Applications research group at Illumina. The work was supported by Burroughs Wellcome Fund 1008841.01 (to JCM and YX), NIH grant HD52953 and HD57192 and grants from the March of Dimes (to JCM #6-FY08-260 and #21-FY06575; and to YX #5-FY10-60).
The placenta: from development to disease
The placenta in pre-eclampsia and intrauterine growth restriction
The placenta in preterm birth
Epidemiology and causes of preterm birth
Births: final data for 2009
Profiling gene expression in human placentae of different gestational ages: an OPRU Network and UW SCOR Study
Human spontaneous labor without histologic chorioamnionitis is characterized by an acute inflammation gene expression signature
Gene expression profile in labouring and non-labouring human placenta near term
Differential Placental Gene Expression in Severe Preeclampsia
Gene expression patterns in human placenta
Genomic evolution of the placenta using co-option and duplication and divergence
Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing
Alternative isoform regulation in human tissue transcriptomes
Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches
Splicing in disease: disruption of the splicing code and the decoding machinery
Alternative splicing: multiple control mechanisms and involvement in human disease
A novel Mtd splice isoform is responsible for trophoblast cell death in pre-eclampsia
A novel human-specific soluble vascular endothelial growth factor receptor 1: cell-type-specific splicing and implications to vascular endothelial growth factor homeostasis and preeclampsia
Novel splice variants of sFlt1 are upregulated in preeclampsia
RNA-Seq: a revolutionary tool for transcriptomics
Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation
Mapping and quantifying mammalian transcriptomes by RNA-Seq
A gene atlas of the mouse and human protein-encoding transcriptomes
The Mouse Genome Database (MGD): premier model organism resource for mammalian genomics and genetics
The Mouse Genome Informatics database
Synopsis of preterm birth genetic association studies: the preterm birth genetics knowledge base (PTBGene)
The Preterm Birth Genetics Knowledge Base
DAVID: Database for Annotation, Visualization, and Integrated Discovery
The Database for Annotation, Visualization and Integrated Discovery
PANTHER: a library of protein families and subfamilies indexed by function
Applications for protein sequence-function evolution data: mRNA/protein expression analysis and coding SNP scoring tools
Vascular endothelial growth factor is a chemoattractant for trophoblast cells
Elevated serum levels of vascular endothelial growth factor in patients with preeclampsia
The immunohistochemical evaluation of VEGF in placenta biopsies of pregnancies complicated by preeclampsia
Evaluation of VEGF in placental bed biopsies from preeclamptic women by immunohistochemistry
Gene expression in chorionic villous samples at 11 weeks' gestation from women destined to develop preeclampsia
Performance of messenger RNAs circulating in maternal blood in the prediction of preeclampsia at 10-14 weeks
[Maternal serum concentration of placental growth factor (PIGF) and endothelial growth factor (VEGF) in pregnancies complicated by preeclampsia]
Elevated maternal cortisol early in pregnancy predicts third trimester levels of placental corticotropin releasing hormone (CRH): priming the placental clock
Association between serum tumor necrosis factor-alpha and corticotrophin-releasing hormone levels in women with preterm labor
Tissue-specific splicing factor gene expression signatures
An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells
ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing
Splicing factor and exon profiling across human tissues
An ESRP-regulated splicing programme is abrogated during the epithelial-mesenchymal transition
Global regulation of alternative splicing during myogenic differentiation
MATS: a Bayesian framework for flexible detection of differential alternative splicing from RNA-Seq data
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The integrin alpha 6 beta 4 is a laminin receptor
Genomic organization of the gene coding for TIRC7, a novel membrane protein essential for T cell activation
Identification of new alternative splice events in the TCIRG1 gene in different human tissues
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Alternative splicing events identified in human embryonic stem cells and neural progenitors
Thrombin regulation in mother and fetus during pregnancy
Potential roles of decidual prolactin in early pregnancy
The insulin-like growth factor system and fetal growth restrictionn
Disproportional effects of Igf2 knockout on placental morphology and diffusional exchange characteristics in the mouse
Characterization of genomic variants in CSH1 and GH2, two candidate genes for Silver-Russell syndrome in 17q24-q25
Expression of beta-subunit of HCG genes during normal and failed pregnancy
Expression of human chorionic gonadotrophin alpha and beta subunits is depressed in trophoblast from pregnancies with early embryonic failure
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HLA-G and immune tolerance in pregnancy
Alpha 1 antitrypsin activity is decreased in human amnion in premature rupture of the fetal membranes
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Hypoxia, HIF and the placenta
Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development
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Ultrafast and memory-efficient alignment of short DNA sequences to the human genome
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