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The History of Wage Inequality in America, 1820 to 1970
In recent decades the United States has experienced a pronounced widening of its wage structure. For the most part, analysis of the recent rise in wage inequality has taken place with the benefits of hindsight--that is, without placing recent changes in the wage structure in historical context. This paper presents such an historical context, by summarizing what is currently known about the evolution of the wage structure from 1820 to 1970. I argue that this evolution was characterized by both episodic change and secular trends. Perhaps the most important secular trend is a long-term rise in the returns to educated labor beginning before the Civil War and continuing until the turn of the 20th century, followed by a decline over the 1900 to 1940 period. In the 1940s substantial further erosion in wage differentials took place, primarily as a consequence of various government policies and increases in the relative demand for less-skilled labor associated with World War II. Although wage inequality today is high by post-World War II standards, it is not particularly high when measured against the pre-World War II experience. As far as government policy is concerned, there is compelling historical evidence that long-term expansion of educational opportunity has been a potent force in narrowing wage differentials.
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|Date of creation:
|Nov 1999
|Contact details of provider:
| Web page: http://www.levyinstitute.org
References listed on IDEAS
Please report citation or reference errors to , or , if you are the registered author of the cited work, log in to your RePEc Author Service profile, click on "citations" and make appropriate adjustments.:
- David G. Blanchflower & Andrew J. Oswald, 1995.
"The Wage Curve,"
MIT Press Books,
The MIT Press,
edition 1, volume 1, number 026202375x, July.
- Blanchflower, David G & Oswald, Andrew J, 1990. " The Wage Curve," Scandinavian Journal of Economics, Wiley Blackwell, vol. 92(2), pages 215-235.
- David G. Blanchflower & Andrew J. Oswald, 1989. "The Wage Curve," NBER Working Papers 3181, National Bureau of Economic Research, Inc.
- Blanchflower, D. & Oswald, A., 1989. "The Wage Curve," Papers 340, London School of Economics - Centre for Labour Economics.
- James, John A. & Skinner, Jonathan S., 1985. "The Resolution of the Labor-Scarcity Paradox," The Journal of Economic History, Cambridge University Press, vol. 45(03), pages 513-540, September.
- John A. James & Jonathan S. Skinner, 1984. "The Resolution of the Labor Scarcity Paradox," NBER Working Papers 1504, National Bureau of Economic Research, Inc.
- Donohue, John J, III & Heckman, James, 1991. "Continuous versus Episodic Change: The Impact of Civil Rights Policy on the Economic Status of Blacks," Journal of Economic Literature, American Economic Association, vol. 29(4), pages 1603-1643, December.
- John J. Donohue III & James Heckman, 1991. "Continuous Versus Episodic Change: The Impact of Civil Rights Policy on the Economic Status of Blacks," NBER Working Papers 3894, National Bureau of Economic Research, Inc.
- Claudia Goldin & Lawrence F. Katz, 1999. "The Returns to Skill in the United States across the Twentieth Century," NBER Working Papers 7126, National Bureau of Economic Research, Inc.
- Costa, Dora L, 1998. "The Unequal Work Day: A Long-Term View," American Economic Review, American Economic Association, vol. 88(2), pages 330-334, May.
- Dora L. Costa, 1998. "The Unequal Work Day: A Long-Term View," NBER Working Papers 6419, National Bureau of Economic Research, Inc.
- Claudia Goldin & Robert A. Margo, 1992. "Wages, Prices, and Labor Markets before the Civil War," NBER Chapters,in: Strategic Factors in Nineteenth Century American Economic History: A Volume to Honor Robert W. Fogel, pages 67-104 National Bureau of Economic Research, Inc.
- Claudia Goldin & Robert A. Margo, 1989. "Wages, Prices, and Labor Markets Before the Civil War," NBER Working Papers 3198, National Bureau of Economic Research, Inc.
- Margo, Robert A. & Villaflor, Georgia C., 1987. "The Growth of Wages in Antebellum America: New Evidence," The Journal of Economic History, Cambridge University Press, vol. 47(04), pages 873-895, December.
- Claudia Goldin & Hugh Rockoff, 1992. "Strategic Factors in Nineteenth Century American Economic History: A Volume to Honor Robert W. Fogel," NBER Books, National Bureau of Economic Research, Inc, number gold92-1, June.
- Thomas J. Weiss, 1992. "U. S. Labor Force Estimates and Economic Growth, 1800-1860," NBER Chapters,in: American Economic Growth and Standards of Living before the Civil War, pages 19-78 National Bureau of Economic Research, Inc.
- Atack, Jeremy, 1985. "Industrial structure and the emergence of the modern industrial corporation," Explorations in Economic History, Elsevier, vol. 22(1), pages 29-52, January.
- Claudia Goldin & Lawrence F. Katz, 1995. "The Decline of Non-Competing Groups: Changes in the Premium to Education, 1890 to 1940," NBER Working Papers 5202, National Bureau of Economic Research, Inc. Full references (including those not matched with items on IDEAS)
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text:
My Cat From Hell shows you a lot of what happens when Jackson rescues cats and their guardians from a hellish situation.
But it can't always show everything.
When Jackson went over to help Roscoe the rambunctious cat and his guardians Danielle and Ruben recently (season 3, episode 5), something happened that spurred Jackson enough to make sure he told you about it.
Here's Jackson:
We didn't show this [on the T.V. episode].
They [Danielle & Ruben] were talking to me about how Roscoe gets out all the time; he's an amazing escape artist.
I said okay tell me about it, and as they were telling me about it – I had been in the house for five minutes – Roscoe reached up, opened the sliding glass door of their patio, and was gone. Like, out onto the patio balcony and was gone; luckily they were on the ground floor.
We got him back pretty uneventfully.
And that's number one by the way:
Cat escapes – don't run after them.
You run and start screaming their name and they will keep running. If you just walk outside, and you breathe and you call their name very gently (even though you're panicking) they won't panic, and you'll be able to swoop them up most of the time. Which is what happened with Roscoe.
But the second thing is this: Roscoe had no collar on. Not only did he have no collar on, this is a chronic escape artist: no collar, no tags, no microchip.
And they didn't have a great picture, a current picture of Roscoe.
These are three major things that have to happen with your cat...
...especially if they're escape artists, but with every cat really.
- First of all, even if your cats are 100% indoors, they should have collars on with tags saying at the very least your phone number.
- Secondly, all cats should be microchipped. Not just for the obvious, which is that if they get picked up, every shelter in the U.S. at this point has a scanner. They will scan, and get your proper information. Also by the way, Ruben and Danielle had just moved. So you have to make sure that the address information that's registered to your microchip is current.
And also this is crazy but true: there are people who drive around and swoop up stray animals. And those animals can be used for laboratory testing. If those animals have microchips, legally they can't be. So that's "kind of important," too.
- Lastly, they didn't have a current picture of Roscoe. If your cat goes missing, you put up a clear, frontal current picture of them. Showing features on their face, the length of their fur, their color...then you make as many flyers as you can saying "This is Roscoe, he's missing, here's my number, call me." If it's a fuzzy picture, if it's one from when he was a kitten – not gonna help you.
So, to sum it up, make sure your cats have a collar and tag, microchip, and clear picture for flyers in case they get lost.
That's what Roscoe was missing.

subset: commoncrawl


text:
There's going to be a set of four Real-D compatible 3D glasses available for purchase when you buy your movie tickets to see Iron Man 3 this May. They're themed as War Machine, Iron Man, Iron Patriot and Tony Stark. It's not just the colors that change on each pair, as the one for Tony is more like a regular pair of glasses while the other three wrap around more like armor. Looks like you're going to want a few extra pennies saved when it comes time to buying those tickets.
(Bad Taste via Bleeding Cool)


text:
I am looking for a spyware solution that keep track of all computers in the network from one client.
We are using Trendmicro and that works fine, but looking for a additional program that could clean up viruses better.
We have the worm WORM_NEERIS.A in the network at moment, we have upgraded all computers so they are imune to it, but everytime installing a new computer, the virus popsup on the new client. Therefore I am looking for a solution that is handled by one client but could reach all clients in the network for scanning spyware and remove it better then Trendmicro.
All ideas of a solution are well appreicated.
Best regards
8 Replies
Jul 16, 2009 at 8:03 UTC
I'm not sure of any central admin spyware/av thatyou will find free, but we use ESET NOD32 and it has worked great for going on 3 years now.
Jul 16, 2009 at 9:41 UTC
Maybe the product: "Spyware Doctor Enterprise" is a solution for your problem.
Website: http://www.pctools.com/spyware-doctor-enterprise/
If that's not doing the job for you, try "Malwarebytes' Anti-Malware".
Website: http:/
Jul 16, 2009 at 1:37 UTC
On a side note.
This might be a really good time to build a honeypot and try to locate where the infection is sourced at. Build an un-patched install with tools to trace port activity and see if you can identify the happy home for this virus.
Jul 16, 2009 at 4:46 UTC
I use Symantec Endpoint Protection managed solution. I can fire off a scan on every node simultaneously if needed. But I think Halcyon has the right idea. stick ethereal on an unpatched system to see where the infection is coming from because you probably have a rogue computer on your network.
Jul 20, 2009 at 8:13 UTC
we've had our best success with PREVX and MalwareBytes. the combination of these two peices saved me enough to justify the costs. well worth it.
Jul 29, 2009 at 8:54 UTC
We use Kaspersky Eterprise (Dumped Trend last year as it seemed to let more spyware and virus's/virii? through than it caught) and apart from a minor outbreak of Kido from a machine still runing Trend so far Kaspersky seems to work. Also the centralised tools for deploying and running removal tools saves a lot of time.
Aug 4, 2009 at 11:56 UTC
Have you tried Super AntiSpyware? Worked well when we needed it.
Aug 5, 2009 at 2:09 UTC
Not sure about a network wide solution, as we actually provide multiple layers of protective solutions.
At the top level, we use Untangle as our Corporate Network. On top of filtering and scanning anything coming to or from our network, it also helps us pinpoint where certain "transmissions" are originating. This has helped us on several occasions to find the "offending" device or devices.
We also are running the Network version of AVG (which covers Malware and Viruses), which gives us full control from an Admin panel installed on one of our servers. We have full control; install, update program, update dictionaries,
Then, we also have Malwarebytes on individual PCs in case we need to clean an infection.
I hope this helps!
This discussion has been inactive for over a year.
You may get a better answer to your question by starting a new discussion.


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Hello all, new to the forums. I picked up this beauty about a year ago at a garage sale in San Francisco. Wondering if anyone could help positively identify it, tell me any sort of value (if any) and age. The bunting is cotton, the flag feels as if it is a wool?/cotton blend or all wool. Bunting has rope at both ends as if it was flown on ship. Only markings on bunting is 4x6. Any help is welcome! Thank you.
Great White Fleet? United Fruit Company?
Need help identifying a flag? This is the place to ask.
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Great White Fleet? United Fruit Company?
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With the aid of google maps and a 3G card, I had no problem finding this hotel. Since I was thirty minutes early for check-in, I dined at a nearby restaurant, ordering four cheese gnocchi that tasted like one-cheese packet dumplings and the entire contents of a salt shaker.
Michel, the owner of the B&B, took me to his dining room for check-in. He commiserated on my chaotic experience at the tourist restaurant, and proceeded to draw on a map, the location of two good restaurants and a gelateria, along with a warning of to be careful about where I ate. I took his advice later, and found the recommendations to be good, and reasonably priced.
My room was fantastic - large, walls decorated with large tiles of Michel's capably taken photographs of nearby beauty spots, a nespresso machine in one corner, and an entire wine rack. I selected one bottle of San Gimignano wine for 10 euro. The room had an attached and private bathroom.
There was no noise from other guests during the entire stay, but some noise from the street in the early morning - if you weren't suffering jetlag, you wouldn't have heard it.
Michel was up at 8 am preparing a great breakfast of cheeses, meats, yoghurts, cereals, chocolate bars, fruits, juices, and individually prepared coffees. The more I dealt with him, the more I became impressed. You only need to see he offers a checkout from 4 am to understand how tirelessly he works. He is the B&B's biggest asset.
Location of the hotel is good - it is a twenty minute walk to the Leaning Tower of Pisa, that takes you through the beautiful town center. It is one street away from the train station - a good way to get to Rome is to take a slow train to Florence, walk around for a few hours, then take the 1hr 40 min non-stop train to Rome; much preferable to flying there from Pisa.
Pisa is small, but located near to some very beautiful places. I didn't have time to see them. The exceptional quality of the B&B will be a large driving force for me to return there and cover the unexplored neighbourhood in the future.
- Reservation Options:
- TripAdvisor is proud to partner with TripOnline SA, Priceline and Booking.com so you can book your B&B Tuscany Gallery 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.


text:
(Original post by h333)
Spoiler:ShowWell, the solution didn't last.Spoiler:ShowOmd love my little bro he found me another charger just nowSpoiler:ShowI am your one and only!! erm namzaat? talk about me being unfaithful....more like I am your aroos/naway.Spoiler:ShowLool. Bless him.
Jk Za sta sara meena laram
You are Here: Home > Forums >< Life and style >< Faith and Spirituality
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- 17-07-2016 22:38
Spoiler:Showza poheegum . za ham mina darsara laram za ma habibi.
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- 17-07-2016 22:44
btw I used to be like that with spiders but now I am like meh.
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- 17-07-2016 23:49
Don't forget to fast tomorrow guys Insha Allah
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- 18-07-2016 00:03
"Buy for yourself (through doing good deeds) while there is still a market and you have the ability to buy."
[Imam Ibn al Qayyim Al-Jawziyyah RahimahuAllah]
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- 18-07-2016 00:04
"The road to Allaah is free from those who have doubt, and those who give in to their lusts. It is full of those having certainty and patience. They are like guideposts along the road."
[Imam Ibn al Qayyim Al-Jawziyyah RahimahuAllah
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- 18-07-2016 00:12
"Everyone will pass through times of sleep and neglect (following his lusts), but do not sink into sleep as the morning of the hereafter is close at hand."
[Imam Ibn al Qayyim Al-Jawziyyah RahimahuAllah]
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2. Reward for fasting for the 6 days of Shawwal
3. Reward for fasting on the white days and tomorrow is the 13th Shawwal.
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(Original post by HAnwar)
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- 18-07-2016 02:49
Alhamdulilah good, just a little sleepy lol
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(Original post by Leukocyte)
Walaykum asalaam
Alhamdulillah nice sunny day, bit of breeze which I like. The problem is I'm at home and I kinda regret not going out with mum to the park.
Alhamdulillah I went out to the park for a bit. Though my friend (who is a grown man, or at least looks like one) was bouncing around trying to catch Pokemon in the park. :?
Jokes I <3 u
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- 18-07-2016 02:51
A tribute to our beloved, the messenger(s.a.w) of Allah (azwj), Muhammed s.a.w, by Ali ibn Abi Talib a.s:
"His place of stay is the best of all places and his origin the noblest of all origins in the mines of honour and the cradles of safety. Hearts of virtuous persons have been inclined towards him and the reins of eyes have been turned towards him. Through him Allah buried mutual rancour and put off the flames of revolt. Through him He gave them affection like brothers and separated those who were together (through unbelief). Through him He gave honour to the low and degraded honour (of unbelief). His speaking is clear and his silence is (indicative) like tongue."
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- 18-07-2016 02:54
Zamestaneh had a couple of ladoo I believe they're called.
Zammy I miss you my little Pakistani gulab jamon. Come back ;(
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- 18-07-2016 02:56
The greatness of the Holy Quran [Ali ibn Abi Talib a.s]:
[Though the chain is not given in nahjul balagha, the full Sanad is contained in Al Kafi and a number of works earlier or at the same time].
"And know that this Qur'an is an adviser who never deceives, a leader who never misleads and a narrator who never speaks a lie. No one will sit beside this Qur'an but that when he rises he will achieve one addition or one diminution - addition in his guidance or elimination in his (spiritual) blindness. You should also know that no one will need anything after (guidance from) the Qur'an and no one will be free from want before (guidance from) the Qur'an.
Therefore, seek cure from it for your ailments and seek its assistance in your distresses. It contains a cure for the biggest diseases, namely unbelief, hypocrisy, revolt and misguidance. Pray to Allah through it and turn to Allah with its love. Do not ask the people through it. There is nothing like it through which the people should turn to Allah, the Sublime.
Know that it is an interceder and its intercession will be accepted. It is a speaker who is testified. For whoever the Qur'an intercedes on the Day of Judgement, its intercession for him would be accepted. He about whom the Qur'an speaks ill on the Day of Judgement shall testify to it. On the Day of Judgement an announcer will announce, "Beware. every sower of a crop is in distress except the sowers of the Qur'an."
Therefore, you should be among the sowers of the Qur'an and its followers. Make it your guide towards Allah. Seek its advice for yourselves, do not trust your views against it. and regard your desires in the matter of the Qur'an as deceitful."
Alternative sources
(1) Al-Zamakhshari, Rabi`, I, 219;
(2) al-Kulayni, Usul al-Kafi, II, 443;
(3) al-Barqi, al-Mahasin, 6;
(4) al-Saduq, al-'Amali, 153;
(5) al-`Ayyashi, Tafsir, II, 262;
(6) al-Harrani, Tuhaf, 71.
(Original post by Inve)
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- 18-07-2016 02:59
Alhamdulillah I'm great! Had a really good day alhamdulillah. Even better when the sun is out. Plus I got my fix of mithai Zamestaneh had a couple of ladoo I believe they're called.
Zammy I miss you my little Pakistani gulab jamon. Come back ;(
Stop making me crave for sweets.
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- 18-07-2016 03:08
SubhanAllah, the following piece of advice from Ali ibn Abi Talib a.s is so profound in meaning and so beautiful, it touched my heart and i have to share it so others may derive benefit as well, inshAllah:
"O people, blessed is the man whose own shortcomings keep him away from (looking into) the shortcomings of others, and also blessed is the man who is confined to his house, eats his meal, buries himself in obeying his Allah. and weeps over his sins, so that he is engaged in himself and people are in safety from him."
يَا أيُّهَا النَّاسُ طُوبى لِمَنْ شَغَلَهُ عَيْبُهُ عَنْ عُيُوبِ النَّاسِ، وَطُوبى لِمَنْ لَزِمَ بَيْتَهُ، وَأَكَلَ قُوتَهُ، وَاشْتَغَلَ بِطَاعَةِ رَبِّهِ، وَبَكَى عَلَى خَطِيئَتِهِ، فَكَانَ مِنْ نَفْسِهِ فِي شُغُل، وَالنَّاسُ مِنْهُ فِي رَاحَة!
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- 18-07-2016 03:11
Tawheed you ok brother?
(Original post by Inve)
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- 18-07-2016 03:29
Feeling sick after eating too much keer.
Oh! The pang of regret when you don't go out and it's sunny and everyone is having a good time. It's bitter
Alhamdulillah I went out to the park for a bit. Though my friend (who is a grown man, or at least looks like one) was bouncing around trying to catch Pokemon in the park. :?
That's Abu Mithai to you, young grasshopper
Jokes I <3 u
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- 18-07-2016 03:31
Apparently 30 somali Muslim were murdered during a prayer ceremony in Baidoa, Somalia by Ethiopian troops.
I can't really understand the somali language so I'm relying on the person who shared the tweets (which contain the news articles) to be truthful.
may Allah grant them Jannah.
Spoiler:ShowNu'man b. Bashir reported that Muslims are like one body of a person; if the eye is sore, the whole body aches, and if the head aches, the whole body aches.
حَدَّثَنِي مُحَمَّدُ بْنُ عَبْدِ اللَّهِ بْنِ نُمَيْرٍ، حَدَّثَنَا حُمَيْدُ بْنُ عَبْدِ الرَّحْمَنِ، عَنِ الأَعْمَشِ، عَنْ خَيْثَمَةَ، عَنِ النُّعْمَانِ بْنِ بَشِيرٍ، قَالَ قَالَ رَسُولُ اللَّهِ صلى الله عليه وسلم " الْمُسْلِمُونَ كَرَجُلٍ وَاحِدٍ إِنِ اشْتَكَى عَيْنُهُ اشْتَكَى كُلُّهُ وَإِنِ اشْتَكَى رَأْسُهُ اشْتَكَى كُلُّهُ " .
Reference : Sahih Muslim 2586 d
In-book reference : Book 45, Hadith 87
USC-MSA web (English) reference : Book 32, Hadith 626
(Original post by IdeasForLife)
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- 18-07-2016 03:36
Apparently 30 somali Muslim were murdered during a prayer ceremony in Baidoa, Somalia by Ethiopian troops.
I can't really understand the somali language so I'm relying on the person who shared the tweets (which contain the news articles) to be truthful.
may Allah grant them Jannah.
Spoiler:ShowAn-Nu'man ibn Basheer reported: The Messenger of Allah, peace and blessings be upon him, said, "The parable of the believers in their affection, mercy, and compassion for each other is that of a body. When any limb aches, the whole body reacts with sleeplessness and fever."
Source: Ṣaḥīḥ al-Bukhārī 5665, Ṣaḥīḥ Muslim 2586
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(Original post by HAnwar)
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Lol I love kheer but it's such a pain to make.
Ignore and report x
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Updated: December 19, 2016
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is back-ordered. We will ship it separately in 10 to 15 days.
At Living Intentions, we strive to produce fun and innovative raw food products that will not only satiate your palette, but nourish your body too. We take great care in each step of our production processes to ensure the best quality of flavor, texture and appearance. We believe that making healthy food choices should be simple and effortless without sacrificing the flavors you love.
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We understand that your property is a big investment and we want our customers to know that we are there for them to help them accomplish the dreams that they may have for there property. Removing a tree sometimes has to be done. And when that time comes, you want to hire a trained professional to complete this task. Sometimes even the smallest jobs come with great causalities in this line of work.
When you hire us to complete your job, we assure a full service so you don't have to worry about anything. We remove everything from the large pieces of wood, to the tiny bits of debris. Our goal is to leave your property with as minimal impact on the landscape as possible. This can always be adjusted according to stand within your budget.
We've done everything from small tree pruning, brush cleanups, medium size trees - To giant removals where cranes are needed to lift out massive pieces of wood. When you call us you can be confident in knowing that you're getting a genuine arborist. A certified tree professional.
Key Benefits of the Service
We take pride in our work and stick to a strict regime of purposeful task completion that keeps all of our customers happy and appreciative of the hard work we do. No shortcuts will be taken and job satisfaction keeps us happy too!
We're an insured company, also with WSIB coverage
Mill your trees trunk into a large wood slab tabletop with us
Have piece of mind knowing your tree is no longer a hazard
Grow grass and get more sunlight for competing growth in your yard
Our arborists take pictures and video of the process for you to keep


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- Knut Alfredsen, Bjørn Sæther
- 1998
This paper presents an object-oriented toolkit for building computer models for water resource planning. The focus is on building a program for flood calculations in river systems with several reservoirs and water transfer structures. The foundation for the flood model is a framework for building hydrologic models. The general framework provides the user… (More)
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- Knut Alfredsen, Bjørn Sæther
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Computer simulation programs have a wide use in hy-drologic and hydraulic applications. We have developed an object-oriented framework that serves as a basis for describing the hydrological system and its processes, and used this as a basis for development of applications. A special consideration is placed on easy reuse and support for further development… (More)
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Swan House Boutique Inn
Ideally situated in the bustling, traditional market town of Beccles , the Swan House offers beautiful and idiosyncratic bed and breakfast accommodation. Our distinctive rooms provide the perfect, cozy base from which to explore the Suffolk heritage coast, the Norfolk Broads and beyond. East Anglia at her most beautiful and varied is literally on the doorstep. After an intrepid day of exploring, come back to a delicious meal in our characterful restaurant, and a good night s sleep in a room designed to provide every comfort. Indulge in fully sprung mattresses, crisp white bedding, flat screen TV s with surround sound and mp3 compatibility as well as delicious little treats and surprises. There s an exquisite three course breakfast which includes home-made tea loaf. Followed by a selection of cured meats and cheeses, fresh fruits with our own original Birchermüesli, croissants, toast and marmalade. All completed with either a classic Egg Benedict egg dipped French toast topped with summer berry compote, natural yoghurt and honey drizzle or a trad English! And all of this is an convivial setting, with open fires to warm you in the depths of winter, and al fresco dining throughout the warmer months.
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Global Start-up Program
Portugal Ventures has teamed up with the Portuguese Foundation For Science and Technology (FCT) to promote the international acceleration of its portfolio companies. FCT established and actively supports the Global Startup Program from The University of Texas at Austin, UT Austin. Portugal Ventures and the Global Startup Program are operating as a cross-functional team to actively develop and expand international revenue opportunities for select companies.
Administered by the IC2 Institute (Innovation, Creativity and Capital) at UT Austin, the Global Startup Program offers a year-long business development and incubation program to assist companies in realizing their international potential more rapidly and economically. Using Austin, Texas, as their launchpad to the United States and the world, companies selected for the program will receive support from dedicated business development specialists and access to a seasoned advisory team exceeding 100 collective years of business development experience leading to more than $300M in realized deal value. These business development specialists will assist Portuguese entrepreneurs and innovators in:
- Refining their product-market fit based on broad feedback from EU, US and other international markets;
- Preparing their product for targeted market readiness;
- Developing a commercialization strategy for international markets;
- Facilitating first customer acquisition in US or other global markets;
- Pursuing sales, partnership, distribution, co-development or in-kind service agreements;
- Extending a Portuguese company financial "runway" through cash conserving strategies;
- Obtaining an advisory network, composed of experts from industry, government and academia.
Company executives, founders, and key leaders must be willing to closely partner and deeply engage with the Global Startup Team to accomplish mutually agreed upon goals tailored to their venture. The CEOs, co-founders, and key leaders of these ventures should have the financial capacity to travel to the United States for short or long term stays to interface with the Global Startup Team in Austin to accelerate business proposition definition, develop market "pitches" and other collateral and meet with potential customers/partners where appropriate in the United States.
The applications are now closed. Applications were accepted from companies in all technology verticals that see an opportunity for international business expansion in their future.
- Global Startup Program 2014 leaflet
- Global Startup Program - Terms of Reference 2014
- Application form
- Pitch template - model pitch presentation
For more information:
UT Austin - Portugal Program,
IC2 Institute The University of Texas at Austin,
2815 San Gabriel St., Austin, Texas 78705 - USA
Phone: -1-512-475-8900
Contact: Marco Bravo


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Spin-off book in the Addicted series
Book 4 in the Calloway Sisters series
With a seven-year age difference, Ryke and Daisy have faced an uphill battle in the eyes of the world and their families. Known as the most adventurous, fast-paced couple, their next step has always been elusive to the rabid media.
Behind the scenes, heartbreaking troubles continue to test Ryke and Daisy's resilience and shape their future together.
They promise: to never slow down. To never compromise who they are. To never abandon their love for each other.
But preserving their happiness also means adding more risks. Ones that even Connor Cobalt wouldn't take. As a professional free-solo climber, Ryke is no stranger to risk, but his next step with Daisy wagers more than just his health.
With their lives on the line, Ryke and Daisy head toward the vast, wild unknown in this epic final conclusion to the Addicted series.
New adult romance: recommended for listeners 18+ for mature content.
©2015 K. B. Ritchie (P)2016 Audible, Inc.
There are no listener reviews for this title yet.
I have been waiting SO impatiently for this audiobook!! Ryke & Daisy are my favorites and this book is just absolute perfection. Krista & Becca Ritchie are superheroes in my eyes.
"A Most Read!"
5 out of 5 Stars
Long Way Down is the 9th/4th book in the Addicted/Calloway Series. I really enjoyed Long Way Down and can't wait to read Some Kind of Perfect. I loved Ryke and Daisy. I am Team Raisy
"They saved the best for last!"
By far my favorite book of the series. It highlighted the multiple dimensions of Daisy and the vulnerabilities of Ryke for the 1st time. We also saw Loren Hale finally come full circle. Excellent work by the Richie sisters!
"love this series."
I enjoyed every book in this series. Great character development and the stories hit every emotion but leave you feeling good at the end.
"Ryke & Daisy"
I love the Addicted Series and this series The Callaway Sisters. Really really enjoyable.
"Great Series!"
This is a fantastic series, with great characters, interesting storyline, and just the right amount of sex/romance. Highly recommend!
"Love this book!"
Narration really brings this story to life! Perfect voices for Ryke & Daisy.
I'm a huge fan of this series - I've invested 8 books into it and I had a LOT of expectations going into this book. I was a bit worried as to whether or not Krista & Becca would meet my expectations.
They totally did.
I LOVE Ryke and Daisy together. I love how they overcame each obstacle that life threw at them. And Krista and Becca made them go through a LOT. WTF ladies??? I was SO mad at the authors a couple of times for testing my heart and tear duct function. Yup, both work. I cried a LOT, I laughed a LOT. I had to put my kindle down to take a break because I was so scared to turn the page. Brace yourself for the angst.
But never fear, Krista & Becca take care of us.
I LOVE Lily and Lo. Especially Loren. He shines in this book and I really appreciated how far his character has grown from the very first book. He is strong. He is dependable. He is there for Ryke the way Ryke was there for him in the past and I was SO proud of him. It's so great to see Lily and Lo as parents too. Moffy = cutest thing.
Connor and Rose are still Connor and Rose. Although I disagreed with a lot of Connor's advice in this book.... He was as arrogant and confident as ever. And I LOVE seeing them as parents too. Jane and Moffy together - so sweet.
Samantha Calloway and Jonathan Hale even have their good moments in this book. Not sure I can really change my opinion of them - I tend to hold on to my resentment a really long time, lol. But it was a pleasant surprise.
This book put me through the ringer. It was unpredictable and full of spectacular life events and full of obstacles. But I loved it all. The writing sucks me in every time and I just don't know what I'm going to do with myself now that I'm done with this book.
And the Epilogue - OMG!!!!!!!!!!!!!!!!!!!!!!!!!! Best. Epilogue. EVER!!!!! I love seeing how everyone ended up. LOVE IT. There were some pleasant surprises in that Epilogue.
Seriously. Book hangover has commenced.
Bottom line: read this book. But start at the beginning!!!!!
I highlighted the majority of this book. LOL. Here are my favorite quotes:
"He Keeps going, he keeps moving , and he picks everyone up when they've fallen behind."
"All I need is you."
"Ryke Meadows endures all things like he was born to last a forever."
"Never give up or back down on the things that fill your soul, Calloway. There is no worse life than a hollow one."
"I'm always kissing you in Paris, Dais. Every fu@king time, I'm kissing you."
"I'm a man of extraordinary talents, but I need you." He swallows. "Do you hear what I'm saying?"
"I love him in every moment. In every smile. In every frown. And I will love him after every long way down."
"Magical things happen in the woods."
"a story and a narration that stole your heart"
* i won this audiobook thanks to the authors
* it took me several weeks to listen to it all because just like reading the book, the story and the words can feel too much at times, it takes and refuses to set you free. i didn't think that the two voices could emphasize this sensation even more but they did, especially ryke's voice and i found myself more than once, tearing up while listening to it (which explains the breaks)
* the quality of the reading is so nice. both the voices go along very well together and they truly give life to the story and to the side characters as well. they didn't do any funny voices but i could very easily dissociate the characters.
* the only flaw for me is that i sometimes find that the speed was too much, i would have prefer it to just be a tiny bit slower.
* truly recommend listening to it, it's a very intense experience, faithful to the book!!
"What A Ride"
A L W A Y S<br/>The Addicted series/Calloways Sisters series hold a special place in my heart and Long Way Down is one of my favorites. As the last solo book for Raisy, this book did nor disappoint. It was filled with ups and downs. I shed so many tears and, as a self proclaimed emotionless robot, it was a bit embarrassing.
I don't know, really? That's like my biggest problem. I WISH there was a book that made me half the things this book alone has. It has no equal in my heart.
Not really, but I really enjoyed their work. I loved Daisy's voice so much and Ryke's just made me swoon <3
I have enjoyed all the books in this series but I have to say this one grabbed me the most. It made laugh and cry. Kept me on the edge of my seat several times. A very good listen!!
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What's wrong with the free versions of both AVG Antivirus and ZoneAlarm Firewall. I have had nothing but the best of results using both of these for several years.
It's your choice but I wonder why spend money for no more protection... just my thoughts!
I recently purchased a new computer and I am looking into getting a new virus scanner and firewall. I am using free versions now. I have ruled out McAfee and Norton because I haven't read many good reviews about their new software. I have narrowed it down to AVG plus Firewall Edition and ZoneAlarm Internet Security Suite. Both products have their advantages, and I have been happy with each companies free versions. I have just one computer not connected to a network. I am sure I can't go wrong with either one. Any help?


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Turner & Gray Smooth Masonry Paint.
Ideal for sound exterior walls including brickwork, concrete, rendering, pebbledash and roughcast.
Drying Time: 2-4 hours (allow 8 hours between each coat).
Covers approximately 10m² per litre depending on the nature and porosity of the substrate.
Two coats will normally achieve the desired look (depending on substrate)
Colour: White
Also available: Magnolia
Size: 10L (Approx.)


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basically do you find it to be a turn off if you find out a guy owns a lot of shoes? I was just wondering because I saw something about this. I'm a sneaker head I buy shoes all the time I own close to 100 pairs mainly jordans and nike sb. I'm just curious because it seems stereotypically women love shoes. but I love sneakers. what's your opinion?
Girls do you find it a turn off if a guy has a bunch of shoes?
Have an opinion?
What Girls Said 4
I reckon it's alright if you love sneakers/whatever and you have many pairs of them. But if it's to the point where you worship them or all you do is flaunt them. Then that's a turn off because it's kinda gay..if you get what I mean. h11|00|0
i understand that I never flaunt them and you would never know I had a ton of shoes unless you kept track of what shoes I wore everyday but what about waiting in line for 15ish hours for a pair of shoes, too far for you?
Hahaha it depends what kinda shoes they were. If they were amazing or if it's that one instance, then it's alright. It's like guys who waited in line for gazillion hours for the Iphone 5 or some videogame..I wouldn't mind, as long as it wasn't every time.
As long he doesn't have more shoes than me its alright00|00|0
It wouldn't be a turn off, but I would be surprised because I've never been out with a guy who had more shoes than me (and I don't even have that many!)00|00|0
no, not at all. why are you so worried? you are not collecting high heels or skirts.00|00|0
What Guys Said 0
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- EXCLUSIVE OFFER: Jim Cramer's Protégé, Dave Peltier, only buys Stocks Under $10 that he thinks could potentially double. See what he's trading today with a 14-day FREE pass.
- The revenue growth greatly exceeded the industry average of 22.9%. Since the same quarter one year prior, revenues rose by 17.5%. Growth in the company's revenue appears to have helped boost the earnings per share.
- CONCEPTUS INC reported significant earnings per share improvement in the most recent quarter compared to the same quarter a year ago. The company has demonstrated a pattern of positive earnings per share growth over the past year. We feel that this trend should continue. This trend suggests that the performance of the business is improving. During the past fiscal year, CONCEPTUS INC turned its bottom line around by earning $0.15 versus -$0.25 in the prior year. This year, the market expects an improvement in earnings ($0.23 versus $0.15).
- The gross profit margin for CONCEPTUS INC is currently very high, coming in at 89.40%. Regardless of CPTS's high profit margin, it has managed to decrease from the same period last year. Despite the mixed results of the gross profit margin, CPTS's net profit margin of 5.44% is significantly lower than the industry average.
- The company's current return on equity greatly increased when compared to its ROE from the same quarter one year prior. This is a signal of significant strength within the corporation. When compared to other companies in the Health Care Equipment & Supplies industry and the overall market, CONCEPTUS INC's return on equity is below that of both the industry average and the S&P 500.
-- Written by a member of TheStreet Ratings Staff
Exclusive Offer: Jim Cramer's 'go-to' small/mid-cap guru Bryan Ashenberg only buys stocks he thinks could return 50-100% See his top picks for 14-days FREE.


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Ashton Eaton
The title of "World's Greatest Athlete" has been bestowed upon the Olympic decathlon champion for decades and Eaton is the greatest of all. Eaton returns to Rio as the reigning Olympic and world champion and is coming off a 2015 outdoor season in which he bested his own world record with a 9,045-point total in the 10-event discipline. Eaton could become the first back-to-back Olympic champion since Daley Thompson's 1980-'84 double.


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Added 29 03 11« Back to A to Z
The good old BMC/Farina theme with twin carburettors, some extra bhp, leather trim, an MG grille, and the odd octagon or two dotted around the scenery. MkIV (1.6-litres/68bhp, automatic option) from August '61. Slightly more 'U' than an Oxford or Cambridge, but better suited to country cabbies than to enthusiasts.
Technical Specifications
- Engine:
- 1489cc/1622cc (S4 OHV)
- Configuration:
- Front engine, rear-wheel drive
- Production dates:
- 1959 to 1968
- Number produced:
- 16,676/14,320


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I used to think I was a beautiful Indian girl.
My crush was being a jerk to me, acting like I was some kind of weirdo or something. I don't know why, I think I hurt his ego.
Now I just feel weird. I have no real incentive to look good. I used to like fashion and clothing etc. but being unemployed I'm not even sure I have a real place to wear all those beautiful things to.
Most Helpful Guy
Totally normal. Everyone feel a little insecure about themselves sometime. Pick yourself up and get back out there!00|00|0
Most Helpful Girl
Honey, we all have our days where we feel terrible. I just went through mine a few days? Weeks ago? The point is, you do not deserve to feel this way and seriously, your crush is a massive egoistical jerk if he treats you like that. Frankly, he just isn't worth your time.
What YOU need is to call up your girlfriends and spend time together. Laugh, cry, eat a tub full of ice cream in front of the television while watching shows like "The Other Woman", "The Duff" or any sitcome, romcom or drama you like that's empowering.
Once that's done, haul yourself out of bed and start pampering yourself and looking good. Dress up and go for a girls' night out. Get manicure and pedicure (if it's allowed I'm not really sure what's acceptable in your culture), go for facial, buy clothes. And then start looking for a job.
When you look good, you feel good. Eat well, exercise, treat yourself well. Who cares about your crush, he's just a complete arse that doesn't know better. YOU ARE BRILLIANT. So put on some attitude and show the world precisely how beautiful you are. You are a strong independent woman in this modern day community. You don't need a man's validation. You just need your OWN self to validate you. <300|00|0


text: Right after the Korean War ended, Korea was just a piece of barren land and here we introduce you two economic heroes who made legends and miracles out of this seemingly impossible land. The lives of these two heroes who were on the evry spot of Korea's economic development called "The Miracle of Han River". lights up the life story of the two legendary heroes, one(namely Jeong, Joo-Young) born and started off as a son of a poor peasant and the other, a drop-out of the renowned Waseda University but who was definitely gifted with native talent in business.


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Call Us Toll Free
Email Us
Customer Services
Call Us Toll Free
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Customer Services
60% off
Was $50.00 Now $20.00
With a high cotton content and just the right amount of stretch, these breathable sports shorts are flattering and supportive. Designed for gentle to mid-intensity work outs, they are finished with stylish marl paneling, with a mid-rise waistband.
- Mid-thigh length
- Soft and comfortable
- 93% cotton, 7% elastane
- Available in black with dark charcoal marl
- Machine wash
Product Code: SECSH
STANDARD NON-TRACKABLE SHIPPING - $9.95 (unless a promotion has been applied) Please allow 6-10 business days for delivery. May require a signature.
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SHIPPING OUTSIDE THE US - Please allow 14 business days for delivery. Click here for International shipping charges
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Returns – FREE (for the US) – Smart label returns are convenient and easy.
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FREE standard delivery on orders over £50 (usually £4.95). For next day delivery, order before midday (£7.95).
Our delivery rates to Europe start from £7 and usually take 5 - 7 working days. If ordering from the US (rates from £15) or Australia (rates from £20), please allow 10 - 14 days for delivery.


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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.
Error: Mozilla firefox has blocked this website from displaying contentAsked by goitom on August 09, 2014 at 03:24 AM
Mozilla firefox has blocked this website from displaying content with security.certificate errors click here for options.
JotForm Support
Please explain further on how you get the reported error. Is it when you view/access a form or when you view/access our website?
But just to add, accessing forms and our website (all domains) using secure URL doesn't give me any issues. The issue might be on your browsers SSL settings. I would advise to clear your SSL Cache, please refer to this guide: http://www.ehow.com/how_6758068_clear-ssl.html. Hope this helps.
Thank you!


text:
60min Squarespace website Intro
In 60 minutes I will:
- Introduce you into the basic Squarespace settings
- Show you how to use the website or blog design tools
- Help you to link an existing domain to it
I will not be able to use custom code like CSS or HTML!
Training & Qualifications
I worked for 2 years in Customer Support at Squarespace.
Availability & Preferences
I'm usually free on weekends and weekdays in the evening (US Eastern Time).
Reviews of Kirsten
Alaina received unit of 60min Squarespace website Intro from KirstenMay 30, 2017
Mauri received unit of Honest, straight forward opinion from KirstenJun 27, 2017
Travis received unit of Honest, straight forward opinion from KirstenJun 23, 2017
Michael received Help with Tell me your secrets from KirstenOct 10, 2016
Simbi received Help with 10-minute Interviews for Simbi from KirstenSep 29, 2016
About Kirsten Penaloza
PROJECT MANAGEMENT Provides organization, time-management and on-site coordination of events, including the following tasks: team leading and coordination of staff, budgeting, billing, making travel ...arrangements, recruiting of volunteers, casting and hiring of personnel, contracting of external providers, full in/outbound correspondence, media outreach, elaborating reports and evaluations, scheduling shifts and leading of meetings (with teams, staff and clients). CUSTOMER CARE Direct communication with customers via email using Salesforce and live chat regarding all levels of customer's websites: Customization of templates, Search Engine Optimization, linking and mapping external domains, addition of third party services through custom code. Troubleshooting with more complex tech issues, as well as billing related aspects, such as refunds, double charges, ownership disputes, inter alia. TOUR GUIDE Leading and coordination of tours of groups with up to 50 people, giving high quality guided tours with detailed explanations of culture, geography, political and economical structures, art and artistic expression of the several traditional regions in Mexico. Coordinates the travel arrangements, solves quickly, efficiently and independently travel issues and emergencies, while offering an excellent time-management. LANGUAGE INSTRUCTOR/ TRANSLATOR Offers private and corporate German lessons one-to-one and small groups, all levels, as well as translations of legal, business and manual documents (from and into German, Spanish and English). show moreOpen to other proposals
Kirsten Penaloza is looking for:
- Experiences
- Fun & Quirky
- Home
- Learning
- Business
- Art & Design


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A mutation in the fifth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel (V317E) resulted in whole cell currents that exhibited marked outward rectification on expression in Xenopus oocytes. However, the single-channel unitary current (i)-voltage (V) relationship failed to account for the rectification of whole cell currents. In excised patches containing one to a few channels, the time-averaged single-channel current (I)-V relationship (I = N ×P o × i, where N is the number of active channels and P o is open probability) of V317E CFTR displayed outward rectification, whereas that of wild-type CFTR was linear, indicating that theP o of V317E CFTR is voltage dependent. The decrease in P o at negative potentials was due to both a decreased burst duration and a decreased opening rate that could not be ameliorated by a 10-fold increase in ATP concentration. This behavior appears to reflect a true voltage dependence of the gating process because the P o-V relationship did not depend on the direction of Cl− movement. The results are consistent with the introduction, by a point mutation, of a novel voltage-dependent gating mode that may provide a useful tool for probing the portions of the protein that move in response to ATP-dependent gating.
- cystic fibrosis transmembrane conductance regulator
- chloride channel
- voltage dependence
the cystic fibrosistransmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette family (24) and functions as a plasma membrane Cl− channel that is expressed in many epithelial tissues (1, 3). Channel activity in CFTR is controlled by protein kinase A (PKA)-dependent phosphorylation of its regulatory domain; subsequent interactions of ATP with the nucleotide binding domains (NBDs) are coupled to channel gating. Neither gating nor conductance of single wild-type (WT) CFTR channels is thought to be voltage dependent. Similarly, macroscopic currents of WT CFTR are time independent.
We studied the impact on channel function of a mutation (V317E) at a site in the extracellular half of the fifth transmembrane domain (TM5). Mutations in this region of TM5 alter the selectivity properties of macroscopic currents (25). Mutations at a roughly homologous position in TM11 (S1118) confer voltage-jump relaxations of macroscopic current, consistent with voltage-dependent gating (31). V317E CFTR macroscopic currents display marked rectification. In principle, this behavior could result from either a change in conduction or a change in gating, but the mutation revealed only a minor effect on single-channel conductance. Instead, we found that the macroscopic rectification of V317E CFTR was due to a voltage dependence of the open probability (P o) of single channels. Hence, these results have implications for understanding the link between ATP-dependent gating and the conformational changes that gate the pore and suggest that V317 and S1118 may be at a key site of conformational change that is associated with channel gating. Portions of these data have been presented in abstract form (25, 30).
Oocyte preparation and cRNA injection.
The methods used were similar to those described previously (19,21). Briefly, stage V-VI oocytes from Xenopus were prepared as previously described (22) and were incubated at 18°C in a modified Leibovitz's L-15 medium with the addition of HEPES (pH 7.5), gentamicin, penicillin, and streptomycin. cRNA was prepared from a construct carrying the full coding region of CFTR in the pALTER vector (Promega, Madison, WI) for WT CFTR or in pBluescript (Stratagene, La Jolla, CA) for V317E and V317Q CFTR. For most experiments, oocytes were injected with ∼5 ng of CFTR cRNA plus 0.6 ng of cRNA for the human β2-adrenergic receptor, which allows activation of PKA-regulated currents by the addition of isoproterenol (Iso) to the bath. Recordings were made 42–96 h after cRNA injection.
Site-directed mutations were engineered in one of two ways:1) with a pSelect vector containing a 1.7-kb fragment of the amino end of CFTR (SacI-SphI) and the Altered Sites protocol (Promega) or 2) with a nested PCR strategy in which the mutation was designed into antiparallel oligomers that were first-round amplified with flanking oligomers (at AflII andSphI, respectively) and second-round amplified with the flanking oligomers alone. In both cases, theAflII-SphI fragment was subcloned back into a pBluescript vector (kindly provided by M. Drumm) containing the human WT CFTR from which the native AflII-SphI fragment had been separated. All mutations were confirmed by direct sequencing of the entire fragment before being subcloned.
Standard two-electrode voltage-clamp (TEVC) techniques were used to study whole cell currents. Borosilicate glass electrodes (Sutter Instrument, Novato, CA) were pulled in four stages and filled with 3 M KCl. Pipette resistances measured 0.4–0.9 MΩ in the bath. Macroscopic data were acquired at room temperature (∼22°C) with a GeneClamp 500 amplifier (Axon Instruments, Foster City, CA) and pCLAMP software (Axon); macroscopic currents were filtered at 500 Hz and acquired by the computer at 2 kHz. Bath solution (ND96) for most TEVC experiments was nominally calcium free and contained (in mM) 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES. The pH was adjusted to 7.5 with NaOH. The oocytes were activated by superfusion of ND96 containing Iso at a final concentration of 0.1–5.0 μM. To limit the activation of endogenous calcium-activated Cl− channels, 1 mM BaCl2 was added to all solutions used in the TEVC experiments. For characterization of permeation and rectification properties, macroscopic currents were elicited by stepping, for 75 ms, from a holding potential of −30 mV to a series of test potentials between −140 and +80 mV in +20-mV increments. Current-voltage plots were constructed from initial data at each potential (average of the first 5 ms) and from data at the end of the 75-ms pulse (average of the final 10 ms) (31). Reversal potentials (E R) were determined by linear regression around the zero-current value. The macroscopic rectification ratio (G +80/G −80) was determined by estimating the currents at E R +80 mV and E R −80 mV, using current-voltage relationships provided by pCLAMP.
All single-channel studies were performed on excised inside-out patches. Oocytes were shrunk in a hypertonic solution of (in mM) 200 monopotassium aspartate, 20 KCl, 1 MgCl2, 10 EGTA, and 10 HEPES-KOH, pH 7.2, and the vitelline membrane was then removed with fine forceps. Pipettes were pulled in four stages from borosilicate glass (Sutter) and had an average resistance of ∼8 MΩ when filled with the standard pipette solution containing (in mM) 150N-methyl-d-glucamine (NMDG)-Cl, 5 MgCl2, and 10 TES, adjusted with Tris to pH 7.4. The low-Cl− pipette solution contained (in mM) 5 NMDG-Cl, 5 MgCl2, 135 mM NMDG-aspartate, and 10 TES, adjusted with Tris to pH 7.4. With the low-Cl− pipette solution, pipette resistances were ∼30 MΩ. For these experiments, data were corrected for junction potentials of ∼9.8 mV, as determined with a free-flowing KCl electrode (n = 5 patches). Seal resistances were in the range of 200 GΩ. In most experiments, the presence of CFTR in the patch was tested by bath application of 1–10 μM Iso before excision. After excision, the patches were perfused with intracellular solution (in mM: 150 NMDG-Cl, 1.1 MgCl2, 2 Tris-EGTA, 1 MgATP, and 10 TES, pH adjusted to 7.4 with Tris). PKA (50 U/ml; Promega) was used to maintain the activity of CFTR. Channel currents were recorded at room temperature (∼22°C) with an AI2120 amplifier (Axon) and were recorded at 10 kHz to DAT tape (model DTC-790; Sony). Data were subsequently played back and filtered with a four-pole Bessel filter (Warner Instruments, Hamden, CT) at 100 Hz and acquired by the computer at 2 ms/point with the Fetchex program of pCLAMP (Axon). Some data were more heavily filtered for display (25 Hz) with the built-in software filter of Igor Pro (version 3.14; Wavemetrics, Lake Oswego, OR).
Kinetic analysis of gating.
Dwell-time analysis of open bursts was performed with Igor software. Open bursts were defined as intervals separated by closings of ≥80 ms, a value previously used in analysis of single-channel data in oocytes (32) and established to discriminate between ATP-dependent gating and intraburst blockade of CFTR in mammalian cells (29). Some of the recordings used in the analysis of burst duration contained more than one simultaneous open level (up to three). For the multiple-channel recordings, the mean burst duration was estimated with the formula tn = Σj · tj /n, wheretj is the time at which the jchannels were open at the same time and n is the total number of transitions from an open burst to an interburst closed state. Thus these multiple-channel open-burst events are transformed ton single-channel open events with burst duration tfor each event. This method was first described by Fenwick and colleagues (7) and has been used in similar types of analysis of muscarinic K+ channels (11) and, more recently, CFTR (26, 32). Given the high degree of filtering used for burst duration analysis, burst duration is equivalent to open time in these experiments.
Variance analysis.
The mean time-averaged currents (I) and their variance (ς2) were calculated from traces at least 15 s in duration. The P o and the number of active channels in the patch (N) were calculated from variance data with the equations ς2 = I ×i(1 − P o) and N= I/(i × P o), wherei is the unitary current. In our patches, we could directly measure i and I; thus these equations yield values for N and P o that are independent of the number of open and closed states.
All reported values are presented as means ± SE unless otherwise noted. Statistical analysis was performed with the t-test for paired or unpaired measurements (SigmaStat; Jandel Scientific, San Rafael, CA) as appropriate for each set of experiments, withP ≤ 0.05 considered indicative of significance.
Whole cell currents exhibit strong rectification.
Exposure of oocytes expressing CFTR and β2-adrenergic receptors to Iso resulted in the development of Cl−current that peaked within 6–7 min. On reaching peak stimulation, oocytes were voltage clamped to a series of potentials (from −140 to +80 mV) to study properties of rectification and the time dependence of macroscopic currents. Figure 1 shows families of whole cell currents from TEVC experiments in WT and V317E CFTR (Fig. 1, A and B, respectively). In this series of experiments, whole cell current-voltage (I-V) relationships were determined by monitoring the current during a 75-ms voltage pulse. Consistent with previous results under identical conditions (32), WT CFTR exhibited time-independent currents over most of the voltage range, aside from a brief relaxation visible at the most hyperpolarizing potentials.I-V relationships for WT CFTR currents (Fig. 1 C) were linear for outward currents but showed significant rectification at strongly hyperpolarizing potentials. As in previous studies, the degree of rectification of WT CFTR macroscopic currents varied between oocytes. Both the rectification and brief relaxation of inward currents may have been due to blockage of the pore by constituents of the oocyte cytoplasm (19, 32). In contrast, V317E CFTR currents exhibited time-dependent behavior at all voltages (Fig. 1 B); inward currents relaxed toward zero, while outward currents increased in conductance through time. This behavior is evident in Fig.1 D, which shows the discrepancy between initial currents (average of the first 5 ms after the voltage jump) and currents recorded after 75 ms (late currents, average of the last 10 ms of the voltage jump). The initial I-V relationship exhibited linear currents at depolarizing potentials. In contrast, the I-Vrelationship constructed from currents at the end of the 75-ms voltage pulse exhibited pronounced outward rectification across the entire voltage range. The outward current amplitude did not approach a constant value during the short voltage pulse, indicating that the underlying time- and voltage-dependent process may be extremely slow. V317E CFTR also exhibited modest tail currents (Fig. 1 B) not seen in WT CFTR with this voltage protocol.
E R in ND96 bath solution did not differ between WT CFTR (−28.1 ± 0.59 mV; n = 12) and V317E CFTR (−27.1 ± 0.58 mV; n = 10). Despite the strong change in rectification during the voltage pulse (Fig. 1 D), the E R in V317E CFTR was not time dependent. Hence, selectivity for Cl− over Na+ did not appear to differ between WT and the mutant and did not change during the time-dependent behavior of V317E CFTR.
To quantify the change in rectification through time, we calculatedG +80/G −80(16) using data from experiments such as those shown in Fig. 1, A and B, as the whole cell chord conductance (G) at E R +80 mV divided by G at E R −80 mV.G +80/G −80 was 1.67 ± 0.13 for WT CFTR initial and late currents and was 1.52 ± 0.08 for V317E CFTR initial currents (P = 0.004 compared with WT CFTR), and 2.24 ± 0.20 for V317E CFTR late currents (P < 0.001 compared with WT CFTR). The smaller degree of rectification of initial currents in V317E CFTR compared with WT CFTR may reflect reduced susceptibility of V317E CFTR currents to blockage by the cytoplasmic constituent(s) or may reflect an impact of holding potential on the process underlying the rectification (seeMechanism of the voltage dependence of V317E CFTR). Hence, the mutant starts out exhibiting less outward rectification than the WT but undergoes a process or processes that both reduces inward current and increases outward current during the 75-ms pulse, leading to pronounced outward rectification in the macroscopic I-Vrelationship. The kinetics of macroscopic currents were not sensitive to a 10-fold increase in bath magnesium concentration (data not shown).
Characterization of unitary currents.
To identify the mechanism responsible for the pronounced outward rectification of V317E CFTR whole cell currents, single-channel studies were performed. Unitary currents (i) were determined from all-points histograms (data not shown) of multiple closed-to-open transitions at a particular potential. In agreement with previous studies that utilized excised inside-out patches (2, 4), we found the i-V relationship of WT CFTR to be linear, yielding a single-channel conductance (g) of 7.26 ± 0.09 pS (n = 13 patches) when measured over the range of membrane potential (V m) of −100 to +100 mV in symmetrical Cl− (Fig. 2,A and B). Single-channel current (i) amplitude for WT CFTR was 0.73 ± 0.01 pA at +100 mV and 0.75 ± 0.01 pA at −100 mV (P > 0.7). In contrast, thei-V relationship for V317E CFTR was found to exhibit slight voltage dependence, with g = 7.65 ± 0.14 pS at negative potentials and g = 6.34 ± 0.14 pS at positive potentials (n = 7 patches; Fig. 2,C and D). i Amplitude for V317E CFTR was 0.64 ± 0.02 pA at +100 mV and 0.77 ± 0.03 pA at −100 mV (P < 0.02). Thus i at positive potentials for V317E CFTR exhibited only 87% of the conductance of WT CFTR. Most importantly, rectification of i was directed inward, opposite to that of the whole cell currents.
To clarify the mechanism leading to reduced g, we studied V317Q. Glutamine and glutamic acid have similar-sized side chains but differ in that glutamine is not charged. The i-Vrelationship of V317Q CFTR was found to have slight voltage dependence, with g = 7.28 ± 0.16 pS at negative potentials and g = 5.91 ± 0.06 pS at positive potentials (n = 5 patches; Fig. 2, E and F).i for V317Q CFTR was 0.59 ± 0.01 pA at +100 mV and 0.73 ± 0.01 pA at −100 mV (P < 0.001). Hence, introduction of a bulky side chain at this position, regardless of charge, appears to result in a slight but significant reduction ofi at positive potentials. Furthermore, the lack of effect of charge at position 317 on the translocation rate of Cl−through the permeation pathway suggests that residue 317 does not directly interact with permeating anions.
The rectification of V317E unitary currents was slight and in the opposite direction of the rectification observed in whole cell experiments. Thus rectification of V317E whole cell currents cannot be explained by voltage-dependent alterations in single-channel conductance.
Characterization of rundown.
To compare the time-averaged, steady-state currents in patches containing either WT or V317E CFTR at multiple holding potentials, it was necessary to control for differences in channel number (N) between patches. We constructed I-Vrelationships by normalizing currents at various potentials to currents at a single potential. This required accounting for time-dependent rundown of channel activity to ensure that all differences inI between WT and the mutant CFTR were due to voltage-dependent, not time-dependent, phenomena.
After excision of a patch from an oocyte expressing WT CFTR, the addition of ATP in the absence of PKA resulted in minimal channel activity, suggesting low phosphorylation (data not shown). Application of PKA (50 U/ml) and ATP (1 mM) resulted in an increase in Ithat reached a steady state of 15.20 ± 2.17 pA in the patch shown in Fig. 3 A. With variance analysis, the P o and N values were estimated to be 0.59 and 34, respectively, for the record shown. Washout of ATP resulted in a dramatic reduction in channel activity that was apparently due to a marked reduction in the opening rate (Fig.3 B). On readdition of 1 mM ATP in the continuing presence of PKA, the steady-state I was 7.59 ± 2.01 pA (Fig.3 C), indicating rundown approaching 50%. Variance analysis of this current yielded P o and Nvalues of 0.29 and 35, consistent with the decrease in current being due to a decline in P o rather than a change inN. The effect of a second washout of ATP was very similar to that of the first (Fig. 3 D). A third application of 1 mM ATP (with PKA) resulted in a steady-state I of 7.72 ± 2.04 pA (Fig. 3 E). Variance analysis of this current yielded aP o of 0.30 and an N of 34. These results suggest that in our system and for a given patch 1) the initial decline in CFTR steady-state activity observed in the presence of 1 mM ATP (Fig. 3, A vs. C) was due to a decline in the P o and not in N, and2) after PKA application, phosphorylation levels were fairly constant within a given patch over several minutes after the initial rundown of activity. To ensure consistent phosphorylation levels in the experiments described in Steady-state I-V relationships, 50 U/ml of PKA were present in all excised-patch experiments. Furthermore, all recordings were performed after the initial rundown in activity. This is important because alteration in phosphorylation levels can affect CFTR gating (5, 12, 17, 23, 27). To account for differences in phosphorylation levels between patches, data at multiple potentials in each patch were referenced to data at −100 mV in the same patch.
Steady-state I-V relationships.
Computed steady-state time-averaged current (I) values were compared in excised inside-out patches containing multiple WT or mutant channels. WT CFTR exhibited a linear I-V relationship, with a E R of ∼0 mV in the presence of symmetrical Cl− (Fig. 4 A). Observation of WT CFTR channel activity at both positive and negative potentials revealed currents of similar magnitude in a given patch (Fig. 4 B). However, this was not the case for V317E CFTR. The I-V relationship for this mutant was markedly rectified in the outward direction (Fig. 4 C) due to the reducedP o at negative membrane potentials (Fig.4 D). Outward currents exhibited a linear I-Vrelationship. Hence the I-V relationship from single channels recapitulated the rectification seen in the true macroscopicI-V relationship from whole cell recording (Fig.1 D).
The behavior of V317Q CFTR channels was also examined at multiple potentials. The single-channel activity of this mutant was found to be voltage independent (Fig. 4 F), the I-Vrelationship being similar to that of WT CFTR (Fig. 4 E). Thus the voltage dependence of gating seems to require introduction of a negative charge.
Mechanism of the voltage dependence of V317E CFTR.
Comparison of V317E and WT CFTR revealed markedly different single-channel kinetics (Fig.5 A). Despite the fact that WT CFTR macroscopic currents showed no voltage dependence, we found that WT CFTR single channels exhibited significantly longer burst durations at negative membrane potentials than at positive membrane potentials (P < 0.05). We suspect that this prolongation of the open-burst duration at negative potentials was due to the voltage-dependent intraburst flicker (not evident in Figs. 2, 4, and 5because of filtering) that has been previously reported (8, 9,17, 19, 28) and likely reflects blockage of the pore by the pH buffer TES in our intracellular solutions (13). If this intraburst flickery behavior is truly indicative of a blocking substrate occluding the pore, then prolongation of the burst duration is to be expected according to the concepts of classic open-channel block (see Ref. 6). However, because the individual dwell time for each blocking event is quite brief (typically <0.25 ms at −100 mV) (19), no significant difference was observed in the magnitude of time-averaged currents at positive and negative potentials (Fig. 4 A). This result is not considered further here. For V317E CFTR, burst durations were significantly reduced compared with WT CFTR over the entire voltage range (Fig.5 B). However, the difference between V317E and WT CFTR burst durations cannot explain the voltage dependence ofP o observed in the mutant, because the burst durations of V317E CFTR at negative potentials were not significantly shorter than the burst durations at positive potentials.
Closed times for either WT or V317E CFTR were fairly consistent in any single experiment, but they varied from patch to patch, probably as a result of variable phosphorylation levels. However, comparison of mean closed times in four different patches containing V317E CFTR revealed significant voltage dependence in the closed duration, with closed times being 4.50 ± 0.93-fold longer (P < 0.05) at V m = −100 mV compared with those atV m = +100 mV in the same patch (n = 4). In WT CFTR, no significant voltage dependence was detected in the closed durations in the same patch, with the closed times at V m = −100 mV being 0.94 ± 0.09-fold of those at V m = +100 mV (n = 5 patches). These data are summarized in Fig. 5,C and D. This result is consistent with the reduced opening rate at V m = −100 mV that is apparent in Fig. 4 D.
One potential explanation for a prolonged closed time is a reduced rate of ATP binding to NBD1 at negative potentials. To test this possibility, we observed the voltage dependence of V317E CFTR activity at 1 and 10 mM ATP. Raising the ATP concentration 10-fold had no effect on i (data not shown) and did not modify the voltage dependence of channel activity observed with 1 mM ATP in the same patch (Fig. 6 A). Furthermore, the multiple of increase in activity that was observed when transitioning from V m = −100 mV toV m = +100 mV was not significantly different (P > 0.5) in paired experiments (2.76 ± 0.37- and 3.04 ± 0.80-fold for 1 and 10 mM ATP, respectively;n = 4 patches; Fig. 6 B). Thus the effect of mutation V317E on channel gating does not appear to change the ATP dependence at concentrations in excess of the reported dissociation constant (29).
Characterization of i and I in asymmetrical Cl−.
To determine whether the voltage dependence ofP o in V317E CFTR was influenced by an anion gradient, we repeated our experiments using a low-Cl− (15 mM) solution in the pipette. Comparing WT CFTR channel behavior at +100 and −100 mV (Fig. 7 A,top and bottom, respectively), one can see a reduced i at +100 mV, due to the decreased driving force for inward Cl− movement, but no drastic difference in the overall P o. Examination of the i-Vfor WT CFTR revealed a 50-mV rightward shift in theE R (59.8 mV after correction for junction potential) in the presence of the low-Cl− pipette solution (Fig. 7 B). As expected for recordings with low Cl− concentration ([Cl−]) in the pipette, the time-averaged I-V for WT CFTR in our asymmetrical Cl− solutions exhibited slight inward rectification.
Comparing V317E CFTR channel behavior at +100 and −100 mV (Fig.7 D, top and bottom, respectively), one can see a reduced i at +100 mV, much like that seen in WT CFTR. However, a large reduction in P o was observed at −100 mV compared with +100 mV. The i-Vrelationship for V317E CFTR was shifted to the right by 49 mV (58.8 mV after corrections) in the presence of our low-Cl−pipette solution (Fig. 7 E). The i-V of V317E CFTR in these conditions showed modestly increased inward rectification compared with that of the WT channel due to the reduced single-channel amplitude at positive potentials (Fig. 2 D), V317E CFTRi amplitude at the most depolarized voltage being only 85% of that seen in WT CFTR. This is very similar to the reduction in outward currents seen in the presence of symmetrical [Cl−] (Fig. 2).
The time-averaged I-V relationship for V317E CFTR in the asymmetrical Cl− solutions showed a pronounced outward rectification (Fig. 7 F), whereas that for WT CFTR remained nearly linear (Fig. 7 C). Neither Goldman-type rectification nor inward rectification of i conferred by the amino acid substitution can account for the rectification observed inI. Hence, as the inhibition of channel opening exhibited at −100 mV was overcome by depolarization, Iincreased. Therefore, similar to the V317E CFTR behavior characterized in symmetrical Cl−, we conclude that outward rectification of I in the presence of a Cl− gradient is likely due to a voltage dependence of P o.
NPo vs. voltage relationship.
We determined the product of N and P o(NP o) in all of our patches, and because bothN and P o varied greatly from patch to patch (but were consistent within a patch after allowing for rundown), we normalized these values to the NP o at −100 mV in each patch. In symmetrical Cl− there is an obvious voltage dependence in NP o for V317E CFTR (Fig.8 A) that was most pronounced at V m values more positive than 0 mV, where net Cl− movement is inward. To determine whether this voltage-dependent increase in NP o could be due to some form of open-channel block at negativeV m, we also performed this analysis on recordings obtained with low-[Cl−] pipette solutions. By shifting the E R for Cl− flow ∼50 mV to the right, we dissociated V m from the direction of anion flow between 0 and approximately +50 mV. With symmetrical Cl− (Fig. 8 A), currents measured between V m = 0 and +50 mV reflected anion entry from the pipette. With reduced bath Cl− (Fig.8 B), currents in the same voltage range reflected anion exit into the pipette. As shown in Fig. 8 B, the increase inNP o was still observed at positive potentials (V m = +10–40 mV), where net Cl− movement is in the same direction (outward) as Cl− movement at −100 mV. This insensitivity (to the direction of ion movement) of the voltage dependence ofNP o for V317E CFTR argues strongly against voltage-dependent blockade of the pore.
Voltage-jump relaxations indicate behavior similar to voltage-gated channels.
The single-channel experiments indicated that the rectification of macroscopic V317E CFTR currents was due to voltage-dependent kinetics of opening and closing. However, the change in macroscopic rectification and the slow relaxations during a voltage step, as shown in Fig. 1, indicate that the voltage-dependent alteration ofP o is a time-dependent process as it is in voltage-gated channels of excitable cells. Because our single-channel recordings were made several seconds after the membrane voltage was changed, they do not reflect the time dependence of this gating process. To determine whether the process evident in V317E CFTR was similar to voltage-dependent gating mechanisms observed in other channels, we asked whether the kinetics of the relaxations at hyperpolarizing potentials were sensitive to V m. Oocytes were held at −30 mV, pulsed to +50 mV for 100 ms, and then stepped for 200 ms to potentials ranging from −120 to −60 mV (data not shown). Relaxations of inward currents were fitted with a second-order exponential in V317E CFTR, whereas only a first-order exponential was required for WT CFTR currents. Time constants for these relaxations in V317E CFTR were strongly dependent on voltage (values for the longer component, τ2, were 157.0 ± 10.7 ms at −120 mV and 294.2 ± 18.2 ms at −60 mV; n = 5 patches; P < 0.001 by paired t-test). The kinetic behavior of V317Q-CFTR macroscopic currents did not differ from WT CFTR (data not shown).
The traces in Fig. 1 show a slow process at depolarizing potentials; as the membrane was held at depolarizing potentials, outward current increased as if the channels were being trapped in a new state with a high opening rate. In contrast, relaxations at hyperpolarizing potentials are consistent with the channels being trapped in a state with a low opening rate. If this interpretation is correct, the kinetics of relaxations to the low opening-rate state (at hyperpolarizing potentials) may depend on the extent to which the pool of channels has previously been trapped in the high-opening-rate state. To determine whether such a state dependency exists, we examined the kinetics of macroscopic currents at −120 mV after depolarizing prepulses that varied in duration from 20 to 250 ms (Fig.9). Oocytes expressing V317E CFTR exhibited an increased conductance at +50 mV as the prepulse duration was lengthened (Fig. 9 A). Concurrently, the peak conductance at −120 mV also increased, as did the magnitude of the relaxation (ranging from ∼25 to ∼40% of the inward current at this potential). This behavior was not evident in WT CFTR studied under identical conditions (Fig. 9 C). The slower component of the relaxation (τ2) in V317E CFTR currents contributed more to the fit as the prepulse duration was lengthened from 20 to 250 ms. Finally, values of τ2 exhibited a clear dependence on prepulse duration (Fig. 9 D). τ2 was significantly increased with prepulse durations >50 ms; this relationship became saturated with prepulses of 200 ms duration. Hence, the process at hyperpolarizing potentials is kinetically linked to the very slow process evident at depolarizing potentials. These results are reminiscent of the behavior of voltage-gated channels of excitable cells (see Ref. 10).
Channel gating in WT CFTR is traditionally thought to be voltage independent. The results presented here demonstrate that it is possible to induce significant voltage dependence in the gating mechanism of the Cl− channel by introducing negative charge at one position in TM5. The voltage-dependent gating of V317E CFTR was evident both in relaxations of macroscopic currents and in the altered gating of single channels. Thus voltage-dependent gating is the mechanism underlying the strong outward rectification of macroscopic currents carried by this channel.
The induction of voltage-dependent gating by substituting a charged residue, but not by substituting a neutral residue, suggests that this site resides in a portion of the protein that is conformationally mobile such that it can move in response to a change in the membrane electric field. The data presented are not sufficient to reveal whether this apparent movement is related to the normal gating cycle of the protein or whether, instead, it reflects a novel motion that would only be seen in this altered protein molecule. In either case, however, the results suggest that it may be possible to use the introduction of voltage-dependent gating as a tool to probe the mobility of segments in the protein and, perhaps, to identify those parts that move in response to events initiated by the hydrolysis of ATP. In the latter case, one might expect a strong interaction between ATP-dependent and voltage-dependent gating events such that, for example, the sensitivity of the channel to activating conditions (PKA + ATP) would become voltage dependent.
Other glutamate substitutions in TM domains 1, 5, 6, and 12 have been investigated [G91E, G314E, and K335E (16); S341E and T1134E (20)], but none of these exhibited voltage-dependent gating (McCarty and Dawson, unpublished observations). Although these mutants have not yet been studied at the single-channel level, no voltage-jump relaxations indicative of voltage-dependent gating were apparent in macroscopic recordings. Hence this phenomenon is thus far specific to the introduction of glutamate at position 317 in TM5. Introduction of a similarly sized but uncharged amino acid side chain at this position (V317Q CFTR) did not affect gating, indicating that it is the charge, not the bulkiness of the side chain, that confers the voltage-dependent behavior.
The voltage dependence of the whole cell I-V relationship is almost purely a gating effect, aside from a minor change in single-channel conductance at positive potentials. The small change in single-channel conductance was found for both V317E and V317Q-CFTR, indicating that it does not result from electrostatic repulsion due to introduction of the negative charge. Hence, this residue apparently has little or no impact on ion permeation, indicating that it does not lie in the permeation pathway. Rather, introduction of a bulky amino acid (glutamic acid or glutamine) at this position may induce rectification in single-channel conductance by altering protein conformation.
The voltage dependence induced by the V317E mutation impacts the CFTR gating scheme at a point distal to the binding and hydrolysis of ATP at the NBDs, as evidenced by the insensitivity of the opening rate to increased [ATP]. Alternatively, it is possible that the mutation resulted in a conformational change that altered exposure of a phosphorylation site in a voltage-dependent manner. However, the kinetic differences were observed immediately upon switching between positive and negative potentials and were observed to be immediately reversible, making it unlikely that rapid changes in phosphorylation had occurred. It also seems unlikely that a mutation in TM5 would alter the structure of the regulatory domain, which includes most of the sites of PKA-dependent phosphorylation in the CFTR. Hence, it appears that the conformational change that is induced by ATP binding and hydrolysis at the NBDs is inhibited at negativeV m in this mutant. It has recently been suggested that the pore of CFTR is not conformationally static in that permeation properties depend on gating status (13, 15, 18,31). Recent work by Kogan and colleagues (14) shows that inhibitors of permeation, such as diphenylamine-2-carboxylate, affect the rate of ATP hydrolysis, suggesting that bidirectional communication between the pore domain and cytosolic gating domains occurs. It is thus possible that the pore of the WT channel undergoes a conformational change at or in the vicinity of position 317, and we have introduced voltage dependence to this process by introduction of a charged amino acid at position 317. To date, the region that experiences this conformational change (i.e., the gate) has not been identified in CFTR. The ability to confer voltage dependence to the gating mechanism, as seen with the V317E mutation, may provide a means to localize the structures that comprise the gate.
We thank Dr. T. C. Hwang and Dr. C. Hartzell for insightful and helpful comments.
↵* Z.-R. Zhang and S. Zeltwanger contributed equally to this work.
This work was supported by American Heart Association Grant 9820032SE and by National Science Foundation Grant MCB-0077575. S. Zeltwanger was supported by National Institute of Diabetes and Digestive and Kidney Diseases 5T32-DK-07656 and by Cystic Fibrosis Foundation Fellowship ZELTWA00F0.
Address for reprint requests and other correspondence: N. A. McCarty, School of Biology, Georgia Inst. of Technology, 310 Ferst Dr., Atlanta, GA 30332 (E-mail: nael.mccarty{at}biology.gatech.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2002 the American Physiological Society


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Web Content, Digital Assets, Form Builder, SEO Tools, Decision Support
Multisite Management, Multilingual Support, Site Synchronization, Deployment Options, Security, Scalability
Personalization, Email Marketing, Mobile, Social Media, Analytics
Integration Platform, Connector for Salesforce, Connector for Microsoft Dynamics CRM, Connector for Marketo, Connector for SharePoint
Product Catalogs, Multiple Storefronts, Targeted Offers, Payment Processing, Order Management
Custom Content Types, UI Framework, ASP.NET MVC Support, Visual Studio Support, Comprehensive API
Behaviour Tracking, Data Integration, 360° Customer Profile
Audience Segmentation, Predictive Analytics, Campaign Recommendations, Campaign Tracking
Marketing Attribution, Conversion Tracking, Persona Profiling, Lead Scoring
Predictive Personalization, Persona-Based Targeting
Your <handler> section is probably invalid.
HTTP Error 500.21 - Internal Server Error
Handler "<HANDLER_NAME>" has a bad module "ManagedPipelineHandler" in its module list
You probably have a handler mapping that does not have the correct precondition.
IIS is not forgiving in regard to typos, and preconditions are case-sensitive.
The text must be preCondition="integratedMode" or preCondition="classicMode".
1) Check this post for more information:
2) Also, set the preCondition mode of your IsapiModule:
<add name="WildCard" path="*" verb="*" modules="IsapiModule" scriptProcessor="%windir%\Microsoft.NET\Framework64\v4.0.30319\aspnet_isapi.dll" resourceType="Unspecified" requireAccess="None" preCondition="classicMode,runtimeVersionv4.0,bitness64" />
<add name="WildCard32" path="*" verb="*" modules="IsapiModule" scriptProcessor="%windir%\Microsoft.NET\Framework\v4.0.30319\aspnet_isapi.dll" resourceType="Unspecified" requireAccess="None" preCondition="classicMode,runtimeVersionv4.0,bitness32" />
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Applicable to:
- Plesk 12.0 for Linux
Release Notes
Odin is pleased to introduce the new Micro-Update #41 for the 12.0.18 version of Plesk.
The 12.0.18 MU#41 update is recommended for all Plesk users and includes general functionality fixes that improve the stability, compatibility, and security of your Plesk server.
To ensure optimal server reliability and security, Odin strongly recommends keeping your operating system and Plesk software up-to-date.
What's Changed
[+] Plesk 12 for Linux now supports multiple PHP versions out of the box. For more details see /213949705
[+] - Added
[-] - Fixed
[*] - Improved
Installation Instructions
#213943585 : Using Micro-Updates in Plesk
A complete list of changes can be found here:
Plesk 12.0 for Windows Systems