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Let me guess, you have been playing New York Times crossword and got stuck on the clue Take another crack at. Well, you have come to the right place to find the answer to this clue.
While some crosswords are much simpler than others, most require that players have a vast knowledge of the language and possibly even various topics. So while crossword puzzles are seen as a more intelligent game, they can be very frustrating for players to complete and even experienced crossword players need some help occasionally.
When finding the answers to the clues becomes too difficult, there's no shame in looking for them online.
Now you have the answer to your clue. Its time to move on to the next clue. You can browse through the list with all the answers to the New York Times crossword of May 1st, 2018. Or you can use the search form below to find the answer (no matter if its New York Times Crossword or any other crossword).
|New York Times crossword of May 1st, 2018 other clues
|Greek letter that represents the golden ratio
|Bishop's headdress
|Pitcher Hershiser
|'Mazel ___!'
|Bit of wear for Colonel Sanders
|Harlem sights
|Play 'monkey see, monkey do'?
|Bride's path
|Hit below the belt
|Poe's middle name
|Busy bees during tax season, for short
|Infamous impaler
|Polynesian land east of Fiji
|Caboose, for a train
|Kovalchuk of the N.H.L
|Prone (to)
|Welcome sight in a desert
|Certain math ratios
|'Good gravy!'
|Large coffee holders
|Push away
|'So ___ me!'
|White House family with the dog Bo
|Corned beef dish
|'Rock-a-bye Baby' setting
|Latin American seafood dish
|Reader of a Fodor's guide
|Winter Olympics equipment
|Corporation named after a mountain
|Long, drawn-out fight
|Word before City or after Fort on Midwest maps
|Cream ___
|'Same here'
|Macaroni or ravioli
|Word stamped on an invoice
|Currency of Tunisia
|Skier's convenience
|'The Simpsons' girl
|Work like a dog
|Dole out
|Slip of paper in a poker pot
|'___ Last Bow' (1917 Sherlock Holmes story)
|Mexican state that touches Texas
|Early means of providing light for a photograph
|Sot's woe, for short
|African game
|More than none but less than all
|Figure in many a sci-fi film
|Stanley who wrote 'The Magic Kingdom'
|Aids for butterfly collectors
|Morrison who wrote 'Beloved'
|___ chart
|Figures on a spreadsheet
|Strong wines
|Airport rental
|Obstacle ... or any one of four black squares in this puzzle?
|Gait faster than a walk
|One of two to four in a standard orchestra
|Go (for)
|Tests the weight of by lifting
|As well
|Part of a cattle roundup
|Going on
|Thin pancakes with sour cream
|Backs of ships
|Perfect diving score
|Two-way, as doors
|Perry of fashion
|Good place to fish from
|Under the weather
We offer support and help with a wide range of crosswords and are guaranteed to make sure you complete everything; the site is also updated regularly to make sure that all of our answers are correct and to add more crosswords to the ever growing list.__IMAGE__
If there is any problem with the answer, please contact us! We will fix the problem.


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If you read the article in Psychology Today, the light-weight puff of pop psychology masquerading as science, David Noose's assertion that the real harm to any society is right wing, ultra-conservative, faith-believing Christians. These peoples as individuals and groups do not believe in "proven" science and reject rational thought as against their religious principles. After all, everyone knows that religious belief is centered on emotional attachment to irrational dogma and requires an unquestioning adherence to religious authority. Well, maybe. Since one can neither prove nor disprove the existence of any god or supreme being or spiritual power, that question is moot. Most "scientific refutation" is not proven theory but the piling up of facts as if the mere presence of these mountains of facts constituted proof. It's sort of like like piling up a large mountain of number on the other side of the equal sign of a quadratic and saying,"See, it works." I do want a better process than what these authors claim is scientific proof.
Anti-Intellectualism is a rather overly broad category and easily bent to almost any meaning any author or speaker may suggest. In its early Greek days the intellect was used to question how one "knew what he knew". This is more the philosophical tradition of questioning one's assumptions about the world in general and specific items. In a sense, we can blame Socrates for this conundrum. But in the world of neuroscience intellect is used in terms of how we understand the world about us. This is a change from the question of what do we know is true to what does it mean? It would seem that meaning is more important that an absolute truth. Of course in medieval times the pursuit for absolute truth was rooted in the sure knowledge of god, who was, after all, the giver of absolute truth. To know god is to know the truth. Not very anti-intellectual, is it? Of course for the peons whose education was stunted by the number of fingers used to count knowing was based on belief. There was the individual belief and the officially proscribed belief. Since the populace did not read and generally relied on priests who could neither read nor write but who could memorize by rote the entire bible, well, everyone was on an equal footing. Belief became knowledge, how could it not? Life for the peons was harsh, work was hard, and death always just around the corner. There wasn't time to speculate as to the truth of the world in which one lived. Work, procreation, and digestion were the principle concerns, the "absolute truth" any peon could know.
So where does the anti-intellectualism come into play? The assumption is that the world is divided between two activities, work and thought. Those who work don't think and those who think don't work. The practical exercise of living in the real world and the impractical exercise of living in a non real world. Working with one's hands and getting them dirty and working in an Ivory tower where one's hands stay clean all day long. There is a certain truth to that divide. I have not met many professors who ever held a job in a factory or worked on a farm. Most of these individuals have, at most, held some part time job where the work was easy, such as in a campus bookstore or even a retail outlet. These are hardly physically taxing jobs and certainly not much strain on the mental processes. It is one thing to go straight from a public or private school system into the university and then on to a position as instructor or professor and quite another to work one's way through college and into a professorship. The latter usually takes longer and gives one a different perspective, even intellectually. Many returning GI.s went to the colleges and universities on the government benefits and a few of them became professors and even administrators within academic organizations. Adele Stevenson was perceived as one of those "Intellectuals" who had always been in politics, never having a "real job" in comparison to Dwight Eisenhower who fought a war (yeah, so he was never on the front line but kicking British butt counts as combat). No one could ever accuse Ike as having the slightest tendency towards abstract thought. He was a man of action, one of us, assuming you had served in the military or had been a war production worker (read factory work). Stevenson was know for great thoughts, not great actions. Yet Ike was hardly the intellectual dullard nor was Stevenson the armchair habitue.
You see, all men and women think. They think when they work in routine occupations. True, only a few may have thoughts about particle physics, but we all think of something. A routine occupation means that one pay minimal attention to the actual work, hence one's minds goes out to play, so to speak. We may day dream, think about tonight's television shows, our favorite sports team, and so on. Some of us may be more concerned with our religion and its tenants. We may have thoughts about the next meeting of the congregation or whether our children have discovered that true belief. In truth, we all think differently and according to our concerns of the day. Just as the liberal progressive is concerned with the perfection of society in the social and political world, the Christians are concerned with the perfection of the individual in the here after. Both individuals have their respective ideals of heaven but the one is worldly and the other is non worldly. For the worldly, science is of greatest concern because they put their faith, oh what a turn of events, into the ability of science to improve conditions in the world, in the lives of individuals, in the progress of mankind towards that perfection they so much adore and believe should be. For the non worldly the concern is about the future after death. For them the world is corrupt and only their god can cure its ills. For this we call them unscientific and anti-intellectual.and yet a great deal of questioning as to what is true goes on in these congregations.
You see, just as in those science classes you took where the teacher used the idea of experimentation to reinforce the validity of the laws of science you were learning, so too, those who undertake to believe in a religious doctrine take the time to study it and experiment in a different manner to ascertain what is true for them. People, science is a religion and has its high priests and its temples as much as any other religion. It may be, perhaps, a little more practical in that it has hands on experiments and observations to "prove" its truth. But science is always subject to revision, it is never static and it is full of contradictions and interpretations. Well, what else is new? Happens in religion as well. New ways of thinking and believing are constantly evolving and tried. Miracles are a little more difficult to prove but belief is what pulls one through this world.
Is Niose the first to notice "anti-intellectualism"? Oh hell no. Individuals have been writing about this perceived problem for centuries. In 1966 Richard Hofstadter wrote Anti-Intellectualism In American Life and did a much better job of explaining the conditions that he believe caused this problem. Well, when one is a lazy "intellectual" and can't be bothered to do his research, what can you expect but another best selling junk book for the choir to read and ooh and ah ha over. And there have been many long before Hofstadter, it is a continuing problem if such a thing really is a problem. After all, isn't a matter of perspective and whether one believes there are correct thoughts and incorrect thoughts? Ah, now there's the rub.

subset: commoncrawl


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The Bank of England has finally dropped the charade and confirmed exactly what we all knew – that inflation is no longer its central target.
The new Governor, Mark Carney, has committed to keeping the Bank Rate at 0.5pc until unemployment falls to 7pc. At 7.8pc today, it is unlikely to reach that level until 2016 or even 2017.
This is a huge departure from the Bank's original aim, set in 1997 by the Labour government, to use interest rates to keep inflation below 2pc and above 1pc.
But a decade later the priorities changed dramatically. By then the state had racked up colossal debts on behalf of taxpayers, which were driven higher by the 2008 banking crisis.
From then on, it was a no-brainer for all those in the ruling elite. With the economy crippled, deflation could set in, and the last thing you want to be holding when prices fall is debt, which grows. Inflation, on the other hand, magics it away.
So since then all policy has been designed to create inflation and keep the economy propped up long enough for those rising prices to work their magic on that debt pile (the Government owes more than £1 trillion, consumers nearly £1.5 trillion).
We knew all this before, to the extent that markets barely fluttered. Interest rate futures point to a first Bank Rate rise in 2017 – the same as they were predicting before Mr Carney's confession.
The economy may be showing some signs of recovery but will ultimately remain weighed down by debt. So nothing has changed. Low rates and high inflation will continue to force Britons to put money into assets, so expect even greater demand for buy-to-let, high-yielding shares, commercial property and any other hard asset. And expect annuity rates to remain artificially depressed.
Savings rates, of course, will remain low, and could sink further if the Government keeps returning to its house price-boosting Funding for Lending toy. So consider a rate fixed for three years – the best deals this week were a 2.55pc rate with the Indian bank ICICI or the over-fifties specialist Saga.
The pathetic rate on that best buy shows how hard this hunt for income has become – and it will remain with us. During the next desperate phase, Your Money will do what it can to help. We have launched an online round-up this week, which will be updated regularly, explaining what next for savings rates and the best ways to make the most of today's miserly deals.
To keep ahead of the pack, go to telegraph.co.uk/savingsrates and bookmark the link.


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Tarascosaurus was a carnivore. It lived in the Cretaceous period and inhabited Europe. Its fossils have been found in places such as Metropolitan France (France).
All these Tarascosaurus pictures were collected from the internet. Enjoy and explore:
- View Tarascosaurus on ancient earth globe
- Random dinosaur
- More from the Cretaceous period
- More in Europe
- All dinosaurs
- Search:
Tarascosaurus was described by the following scientific paper(s):
- J. Le Loeuff and E. Buffetaut. 1991. Tarascosaurus salluvicus nov. gen., nov. sp., dinosaure théropode du Crétacé supérieur du sud de la France [Tarascosaurus salluvicus nov. gen., nov. sp., a theropod dinosaur from the Upper Cretaceous of southern France]. Géobios 25(5):585-594


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Oni (basics)
Salty Runback :: Characters :: Oni
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Oni (basics)
learned alot from this site
Re: Oni (basics)
he still looks like a cross between blanka and a smurf to me
Re: Oni (basics)
lol yeah, except Oni, By finally mastering the Satsui no Hado, the man has become consumed by it. Both his mind and soul are gone, and his body has been transformed into something that is incapable of being called human. What was once an honorable code of fighting only the strong is now a crazed desire to destroy everyone and everything in the universe. The Supreme Master of the Fist, Akuma, is dead. Now, there is only Oni, a demon in the truest sense of the word.
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There's been a lot of news coverage about Samsung becoming the first smartphone manufacturer to be NextRadio®-compatible across all carriers. While radio trade publications are widely touting the news, we've learned that other industries are also examining the impact of Samsung's recent announcement. … Continue reading »
NewBay Media recognized Dial Report with its 2017 Best of Show Award, presented by Radio World magazine. The product stood out among a panel of industry experts who evaluated entries for "design, features, cost-efficiency and performance in service professional users."


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As I mentioned last week, my January organization project is to update my Food Storage Organization. I didn't get as much done this weekend as I hoped I would, but I did manage to straighten up the shelves in the storage room. Here is a picture of how one set of the shelves looks now.
This weekend we also did a Costco run which means that I now have more supplies to put away. While we were there we also picked up another set of shelves. Hubby hasn't put them together yet, but as soon as he does I will start filling them up.
We had debated whether it was worth buying another set of shelves or not. There is space available on the shelves we currently have, but hubby wants me to stock up on as much as I can before the baby gets here. So we finally decided to get the extra shelves. It will be worth it so that I can keep everything organized.
Here is what I hope to accomplish on this project this weekend:
- Have hubby put the new shelves together
- Finishing putting away all the Costco purchases from last weekend
- Buy or order food storage containers for the pantry
Come back next Thursday to see how the project is going. I only have two more weekends to work on this project and I'm beginning to wonder if I'm going to be able to finish it. Obviously I need to get moving and get it done.
Are you currently working on any household organization projects? How are they going?


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"Drag Racer" is a Rock instrumental song by the Doug Wood Band. It was written and performed by the little-known Doug Wood Band in , but since then it became better known as the theme song to the BBC's snooker coverage. It was released as part of the "BBC Snooker Themes" EP, released on BBC. 26 Apr The Doug Wood Band's original snooker theme music boasted the most evocative guitar riff of all time, writes Scott Murray. So why has the BBC. Meet Doug Wood, the man who wrote BBC Snooker's iconic theme tune 'Drag Racer.'.
17 Jul Once voted the most recognised theme tune on British TV, ahead of EastEnders and Watch the Match of the Day theme tune SNOOKER. Misc Television - Bbc World Championship Snooker Theme (Tab) tab by Misc Television with free online tab player, speed control and loop. Correct version. BBC Snooker Theme Song. Tabbed by Bill Runham [email protected] Tabbed By Bill Runham [email protected] This is the BBC snooker.
User image. Bbc Snooker. By: trickydicky. Chords for drag racer cover (snooker theme tune). Play along with guitar, ukulele, or piano with interactive chords and diagrams. Includes transpose, capo hints. 19 Apr Here's a list of walk-on music for all players at the Betfred World Championship: Mark Selby: eez-eh by Kasabian Stephen Maguire: Here I go.


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Debt Consolidation Loans in Oglesby, Illinois
Have you wondered about the most effective way you could consolidate credit debt in Oglesby, Illinois? You'll want to find the very best strategy for you specific financial circumstance and capacity to make payments. The objective of consolidating debts is to help make your life less difficult by restructuring your debt and your monthly payment conditions to something which more effectively fits your needs.
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Disclaimer: Some of the links on this site are affiliate links for which we get compensated.
Things to be aware of with personal credit card debt consolidation in Oglesby, Illinois
A balance transfer card, for instance, can give the advantages of a 0% rate of interest (or very little interest rate) if you understand how to make the most of it with the best payment schedule. For this particular method to succeed, you likely won't find a more affordable payment, and you will need to have a dependable income to handle it successfully. You'll want to have the capacity to pay as much as you can as your payments will go to your principal amount rather than the interest. If you do this right, you can manage to pay back a significant percentage of your total balance in a brief period of time.Read More
When you've got a great credit rating, then deciding on a personal loan for debt consolidation is a great strategy for saving money on high interest credit card debt. A faster term is going to improve your cost savings. The goal should be to minimize how much interest you have to pay during the period of the loan, which is accomplished by merging a low interest rate with a quick payment period. That should add to the benefit that you'll get by using a personal unsecured loan to consolidate your consumer credit card debt.
In case your house has sufficient equity, then you may also consider a home equity loan. As much as possible, the equity should be able to pay for all your high-interest rate credit card debt. Also imperative is the necessity of a effectively monitored repayment plan for the loan. If you don't complete the monthly payments, than the method puts your property at risk.
Know the benefits and drawbacks in Oglesby, Illinois
Nobody should enter into consolidating debts without thinking about the pros and cons of this solution.
To begin with, we can discuss the rewards.
It de-stresses your debt installments. This credit card debt solution combines your numerous financial obligations in to just one. You'll no longer have several accounts to worry about. It can make the monthly payment strategy easier because it calls for significantly less effort to manage.
You get much better conditions on your arrangement. The process of debt consolidation will involve many credit card accounts merged into a completely new arrangement. This adjustment is the perfect time to improve terms on your loan. You can get more affordable interest rates, more versatile plans, or faster terms, providing numerous solutions to lower your expenses.
It provides you with the chance to lessen your rates of interest. Finding a cheaper rate of interest is a crucial part of consolidating debts. You should never opt for consolidation if a more affordable interest rate can't be found. It does not make sense to move your present debts into a newer one with a larger interest.
Now, we'll have a look at some of the cons.
Many incorrectly sense that they're debt free. It's true that you're going to free up your credit lines with consolidation. Yet, it doesn't mean that you've repaid your credit debt. You've merely shifted the debt to a whole new loan balance. Your debt is merged, but the amount of money that you owe remains the very same.
You may end up deeper in debt. When you cave in to the unrealistic sense of credit card debt relief, you may be drawn to make use of your charge card. It will only lead you further into debt.
It isn't going to get rid of the main reason behind the problem. Whatever caused you get so deeply into financial debt will not go away through consolidation. You still have to handle the cause. It simply treats the outward symptoms.
Dependant upon the strategy for debt consolidation you choose, your property may be in jeopardy. Many people decide to place their houses up as collateral, putting their properties in jeopardy if they're unable to pay. The cheaper interest rate of a home equity loan makes this a popular solution, but choosing this method can ultimately result in having your home repossessed.
Request for debt consolidation loan in Oglesby, Illinois
The loan application procedure for debt consolidation loans is dependent entirely on the way you want to consolidate.
If you wish to consolidate by a personal unsecured loan, that will abide by the approach of any sort of unsecured loan. You'll submit a loan application form (or at least the standard loan information) and in most cases, the lending company will carry out a short appraisal of creditworthiness to inform you if you are accepted for the loan. If you are declined, you'll then need to look into other available choices. When you are approved, you will finish the application by providing the appropriate data. If you utilize the web to apply, you might be scanning and giving electronic versions of your data. You can do this on the lender's web site or through electronic mail.
After concluding the criteria, the lender will carry out a more in depth underwriting procedure. Once things are all completed and accepted, that is when you will get the amount you borrow. Some loan companies would require you to open up an account with their organization - this is where they are going to deliver the funds you borrowed.
Home equity loan application forms are the same as mortgage loan application forms. The actual procedure concerned is the same as the approach involved with unsecured loan applications. But, the loan provider will perform a estimation of the home that you'll set up as collateral. In comparison to an unsecured loan, this will be more expensive.
Many people don't realize that they'll save time and money by consolidating their credit card debt with the same lender or financial institution that they are already working with.
Can people with poor credit obtain consolidation loans in Oglesby, Illinois ?
There's a couple different ways to consolidate your credit card debt for those who have weak credit.
One option will be to request a secured or unsecured loan. As a high-risk borrower, you will have to invest higher interest charges. Your low credit score shows the loan provider that you don't demonstrate good behavior with your prior credit balances. Of course, the interest rate of your unsecured loans will be higher than a secured financial loan. The collateral that you'll lay at stake with a secured personal loan will allow you to receive a reduced interest rate.
In case you are not authorized for a personal unsecured loan or you do not have collateral for a secured financial loan, you may have another choice to receive debt consolidation loans. This approach is debt settlement. People who have bad credit will notice that this can be a fantastic alternative to managing debt.
That is an approach to bargaining for the chance to pay for only a part of the debt which you owe. While you could negotiate all on your own, it is much easier to manage this method with a credit card debt settlement business. When you have arrived at an agreement to settle your debt for less than your balance, the lender or collector pardons the remainder of the credit card debt.
Is consolidation a possibility for people who have poor credit in Oglesby, Illinois ?
There are 2 methods for getting low interest consolidation loans. The first option is to have a very good credit score reputation. The financial institution wishes to see that you're not a high-risk client because you have a history of managing your consumer debt reliably. If you want a reduced interest rate loan, your good credit rating could make all the difference in getting authorized.
Trying to find a secured financial loan is your second method for a lower monthly interest. Most of the time, consumers make use of the equity of their home. By putting your property as collateral, the loan provider will feel more reassured that they can get something just in case you decide to default on your loan payments.
Are any personal credit card debt consolidation solutions free of charge?
In case you are searching for a totally free debt consolidation company, we have bad news: There won't be any. All consolidation loans will need to be paid back with interest. Certain services may demand a low origination charge yet it's typically difficult to entirely waive this service charge. Naturally, the business pays the credit agencies to check on your credit report and additional professionals necessary to finalize the personal loan application form. They'll make you shoulder those charges through the application prices.
With a lot of debt consolidation lenders in Oglesby, Illinois, you'll receive a free estimate. The rest of the undertaking will definitely cost money.
Selecting a certified debt consolidation business
The company's accreditations can show you if they are reputable. The lender should be associated with other businesses and organizations. Generally, these kinds of agencies would research representatives before they accept their accreditation. By knowing the requirements and specifications of these organizations, you will know exactly what the debt consolidation company will deliver.
A few typical certifications that consolidation services seek are the below:
Better Business Bureau. The Better Business Bureau Business Partner Code of Conduct is used to rank Business Partners associated with the BBB. You're able to check out their site to learn about the debt consolidation service, any complications that clients experienced with them, and the way they have addressed those concerns.
American Fair Credit Council. This is the top association of Consumer Credit Advocates to help debt overwhelmed clients, encourage the industry's guidelines, and increase decent care of borrowers.
National Foundation for Credit Counseling. The main objective of this institution is on assisting consumers and debt relief agencies to elevate economic circumstances. The Council on Accreditation authorizes consolidation agencies for the NFCC.
International Association of Professional Debt Arbitrators. This organization makes sure that the debt arbitrators doing work for debt consolidation services are qualified and authorized to do business on behalf of their customers.
Other international and national accrediting organizations also exist, and you could find out more by looking into them. If you select a debt consolidation service by looking at credentials, then you can certainly be confident that you're dealing with a legitimate lender.
Examine testimonials from debt consolidation clients
You could also find out much about debt consolidation companies by reading consumer review articles. You'll find client review articles everywhere, which includes internet sites that are specifically created for debt consolidation business reviews. Many are personal financing sites or brand new associations that highlight specific businesses.
You're able to discover the price, the value, the offered solutions, and a lot more from all of these customer reviews. Even though the service fees are a significant component to it, they should not be your primary consideration. A great organization takes more than merely minimal fees. Instead, you'll want to place more importance on the level of satisfaction reported by clients of the organization.
You also want to think of your own debt consolidation requirements. Try to avoid being fooled by the promise of a lower interest. They could demand substantial fees and rigid payment plans. If the process and repayment terms are more critical than a reduced-interest, then be sure the business you'll transact with will help you fulfill your debt relief requirements.
Seeking non profit agencies to consolidate credit card debt
Though you'll find non profit credit counseling services, there aren't any debt consolidation loan companies who aren't involved for the profit. If a debt consolidation business promises to be non profit, you should be suspicious. Any company that provides a loan would require you to pay beyond what you borrowed. The provider usually obtains their gains from management costs and interest charges.
Don't seek out non-profit debt consolidation services that are not available. Rather, look for companies that provide you with minimal service fees and rates of interest. To succeed with a debt consolidation loan, you must incorporate low rates of interest, a good credit reputation, and cost-effective prices.
If you do ever identify a service that promises to be non profit for debt consolidation, you need to be very careful and diligent with your investigation. It's remarkably probable that this kind of service is looking to con you into having to pay excessive service fees.
How to find the most effective consolidation loan companies
Finding the very best consolidation loan companies may not be as difficult as before - owing to the data you'll find over the internet. In reality, you don't need to waste a lot of time investigating companies before deciding on one. Provided that you understand exactly what you need and have an understanding of your particular financial situation, you'll be able to come up with a solid decision. The info on this page ought to be sufficient to ensure you get going in the right path.
Lightstream is a wonderful solution for people with very good credit scores. This business gives unsecured loans to consolidate credit card debt. You won't come across cheaper rates of interest anywhere else, although you have to pass strict credit requirements. To do business with this provider, you need no less than five years of credit with multiple types of consumer debt.
Those trying to find rapid approval for a debt consolidation loan will be smart to investigate Lending Club. This internet based lending company has a fast turn-around time. It could take just a few days to have the loan you want. Your credit score and the sum of the personal loan will ascertain how much time it takes to get approval. Don't worry with regards to transacting with an internet based organization - they have got the most advanced safety precautions so all of your personal financial info are protected.National Debt Relief is a better option for people with bad credit and for those who have already defaulted on their debts. National Debt Relief settles your credit card debt by negotiating with your collectors. With the settlement approach, you could reach an agreement to pay for a portion of the debt balance. After that, the rest of the debt is pardoned. You're able to lower your financial debt and consolidate your credit accounts with this approach.
You should always research any business you consider working with for debt consolidation loans in Oglesby, Illinois; though these three are a good place to begin.
Information on debt consolidation loans in zip code(s): 61348


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On tonight's episode of "The Crypto Show," Reuben Yap of ZCoin joins us to discuss the advantages that ZCoin has over other zero-knowledge proof cryptocurrencies, not to mention just other currencies. We also touch upon the generous contributions that ZCoin continues to make to freeross.org.
In the second hour we talk to Mike Nimitz, an American expat in Acapulco, about his life there and about the lives of other Americans who have moved down there since the first Anarchapulco. We talk about the conference itself as well, among other things.
Source: letstalkbitcoin.com


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Max Polyakov's Firefly Aerospace is back on track, testing its new engine, Firefly Lightning, at their Brigg's facility outside Menlo Park, Texas. CEO, Dr. Tom Markusic, and Max Polyakov plan to make space affordable for the little guy by 2020.
Ironically enough, the rocket industry is not known for doing anything quickly, but Firefly Aerospace is bucking the trend. Just a few years ago, Firefly Aerospace was just a twinkle in Max Polyakov's eye. Now it is regularly testing its flagship engine and is on track to launch its first fully-assembled rocket in late 2019. Even though they have yet to get their first prototype off the ground, they have already started construction on their production facility to pump out two units per month by 2021.
No rest for Tom Markusic
It isn't uncommon for companies to take a breather after a new company takes over ownership, but the last thing on Max Polyakov's mind was giving the fledgling orbital launch company some space. After being purchased out of bankruptcy in 2017, the company immediately began hiring back key personnel, including CEO Tom Markusic.
In less than two years, Max Polyakov (Maxym Polyakov) has helped the company finalized plans for their orbital launcher, Firefly Alpha, tested their new Lightning engine and released a detailed Payload User's Guide to help potential clients plan their first missions.
Image credit: seradata.com
The employees have no time for rest and follow a daily testing schedule of hot burns on their test pad at the Briggs test facility. They have both horizontal and vertical testing facilities that can handle up to 450,000lbs of thrust, although the Lightning is still currently running at a fraction of that. At its peak performance, Lightning plans to provide about one tenth the thrust of the Reaver engines that will eventually be used on the Firefly Alpha's first stage.
Even though Max Polyakov (Maxym Polyakov) is not always present, Firefly staff, engineers and designers often watch or take part in the daily test, which is integral to the success that they have enjoyed. Firefly design, manufacturing, and testing teams enjoy close proximity with each other on the Brigg's compound.
The 200-acre facility is home to more than just the testing facilities. Production buildings, workshops, offices and a control center have all grown out of the farmland surrounding Menlo Park, Texas and are still surrounded by grazing cattle. These rural roots are also slated to be the home of the future full-scale launch vehicle assembly factory within just a few more short years.
Max Polyakov's Firefly plans to turn their relatively small rocket into the most cost effective orbital launch vehicle on the market for small to medium sized satellites. The two stage Firefly Alpha will deliver a modest 1000kg payload to any low-Earth orbit (LEO) within the next two years.
A new, smaller Space Race
More rockets were launched last year worldwide than any year before. In the early 1980's satellites began a rapid miniaturization trend, but rockets have gotten larger and larger. The trend toward larger rockets is an attempt to lower launch costs by making the vehicles large enough that they can be reusable. Companies like Blue Origin and SpaceX are leading the way in this area, but Max Polyakov (Maxim Polyakov) saw that this trend left the majority of future clients forced to leave their smaller satellites on the ground.
Many engineers today are designing CubeSats and small satellites that have no way of getting into space other than begging for a position as a secondary payload on a larger launch. This can mean waiting for years sometimes until a primary payload is going to be launched into a compatible orbit, then applying for a secondary position. According to Max Polyakov (Maxim Polyakov) from Firefly, rideshare launch services, when they can be found, often involve compromise orbits and other undesirable launch parameters. Heavy lift launchers, while offering lower overall cost per kg, often place secondary payloads in orbits that require additional fuel and time to transfer to their proper mission orbits.
That is not to say that there are no small launcher on the market. China, Russia and India each have smaller rockets that carry payloads under 1000kgs, but these options cost an average of $30,000 - $40,000 per kilogram. Max Polyakov, Firefly Aerospace founder, plans to rewrite the price book on small orbital launches by providing a bespoke launch a desired LEO trajectory for a little as $10,000 per kilogram, a fraction of the competition today.
Image credit: newatlas.com
The bigger they are ...
Once the Firefly Alpha is ready to launch, Max Polyakov's Firefly plans to mass produce the vehicle, producing one every two weeks, with the ability to scale up from there.
Image credit: latribune.fr
The company has already signed contracts with the US government, and research and development contracts with DARPA, the Defense Advanced Research Project Agency. They are also currently pursuing certification with the US Air Force, even though it is not required for orbital launches.
Firefly Aerospace does not intend to remain a government contractor though. Max Polyakov (Maxim Polyakov) confirmed that they eventually intend to market their services primarily to the private sector. When asked about the rationale for the extra expenses associated with the US Air Force certification, Max Polyakov cited the fact that the primary cost of a space mission is typically not the launch cost. Very often the overall launch service and resulting impact to mission success is the best measure of value to the customer, not the lowest possible launch cost.
This credibility is key in an industry where most of the players have been at the game for over 50 years. Boeing and Lockheed Martin have been giants in the space industry for decades. Until recently, many people thought it would be impossible for startups to come into the field and innovate their way into a profit position.
The space industry has largely been a government game with little innovation compared to other industries. Rapid prototyping, 3D printing, the availability of computer models and the miniaturization of satellites has lead to a turning point where new companies have been able to enter the field and compete.
Despite being a high tech company, Firefly is incredibly lean and efficient. Max Polyakov (Maxym Polyakov) confirmed that the startup would be able to keep its doors open with just 4 launches each year. With a capacity many times that, and several dozen launches already prebooked, it doesn't seem likely that they will have any troubles financially once they are off the ground.
Along with easing the backlog of launches already in the pipe, Max Polyakov's Firefly also hopes that they will be able to attract new customers. But, since this literally is rocket science, it can be daunting to know where to even start to have your satellite launched to space. To help introduce new clients to the ins and outs of planning a launch, Max Polyakov's Firefly announced the release of its Payload User's Guide for the Firefly Alpha.
Image credit: fireflyspace.com
The guide provides customers with all the information they need on the capabilities, operation and specifications for each possible payload for the Firefly Alpha. With all the recent excitement surrounding the potential of low-cost CubeSats and small satellite constellations, Firefly Alpha hopes to be the rocket that makes actually getting these new tools into space affordable.
In a recent interview, Firefly CEO, Tom Markusic, explained that when they looked at what people wanted to put into space today, the oversized Battlestar Galatica-style satellites were going by the wayside.
Size Matters
The Space Age itself began with a small satellite, Sputnik 1, that weighed in at just 83kg (184lbs). At the time, all it could do was beep out a radio signal, confirming to the world that there actually was an object in space. Sputnik 1 was followed shortly by larger and larger satellites that were filled with heavy remote sensing equipment, communication equipment, GPS stations and various other devices for private and military uses.
In the last 60 years, over 4,250 satellites have made their way off of the surface of Earth, with 93% of them being large satellites weighing more than 1000kg (2200lbs). But, as Max Polyakov pointed out, the percentage is rapidly changing in favor of the little guys. Hundreds of small satellites are being launched every year.
Part of the appeal is that computing and communication equipment has become so much smaller, that even with shielding and guidance equipment, a lot of functionality can be packed into a relatively small package. Founder of Firefly Aerospace Max Polyakov has even sponsored a school that helps engineering students design and build their own satellites. CubeSats are still very popular among college and university students, and microsatellites weighing between 100kg and 1000kg are expected to make up nearly 50% of launched by 2021.
Show me the money
Max Polyakov says that a driving force in the growth of constellation missions is the plummeting cost of building spacecraft. In just the last 10 years costs have decreased by a factor of 5-10 for spacecraft design and development. Many space based services are attracting broad financial investments based on these new business cases such as space based imagery and internet access.
Image credit: communityimpact.com
Max Polyakov, Firefly Aerospace founder, knows. He has put considerable investment into his own remote sensing company, EOS Data Analytics. Just a few years ago, only 2 high-resolution, commercial satellites were available to the public. Now, dozens of agencies and private companies have constellations of hundreds of small satellites that offer the same service for a fraction of the cost. Max Polyakov' Firefly plans to launch a private constellation of near-Earth imaging satellites.
Other startups, such as Millennium Space Systems, specialize in building low-cost small satellites, creating a supply of cargo for Firefly Alpha to deliver. When CubeSats were initially introduced, they were intended to be used for education, training and proof of concept missions. They were made with cheap, off-the-shelf components. But, the quality of these components have proven themselves in space to the point that CubeSats are now regularly used for production commercial missions.
NASA has also ramped up production of small satellites, building a dedicated CubeSat assembly and testing clean room in Pasadena a few years ago. When they were first introduced, CubeSats were difficult to fit in as secondary payloads. Now nearly every mission includes at least a few CubeSats of picosatellites tucked into the corners.
Max Polyakov points out that currently launch opportunities for small class missions are in high demand and are not being met by the launch services market. Launch services tailored to the class of spacecraft and mission requirements will dramatically improve the business cases and success of these new missions. Firefly's Alpha launch vehicle and launch services will have a very positive impact on mission and business case success of constellation and other missions.
Image credit: fireflyspace.com
Like any startup, funding is an issue. Firefly Aerospace's predecessor, Firefly Space Systems, wasn't able to keep up the funding to get it to their first launch. But Max Polyakov has assured Firefly Staff and its CEO that they will have the funding they need to get their Alpha dream off the ground. Their minimalist approach to operations means that once they are selling launches, they will be able to survive even if things start slowly for them.
They are able to keep costs low by focusing on outcomes and making use of the vast amount of experience in the rocket technology in both Ukraine and in Texas. Ukrainian scientists and engineers in Max Polyakov's home country were key in keeping the Soviet Union in the Space Race during the Cold War. Now, specialists from these two worlds are working together to find solutions to the world's problems. Also, being in Texas, Firefly was able to partner with the University of Texas to make use of Stampede, one of the world's top 10 fastest supercomputers.
Image credit: planetary.org
Max Polyakov from Firefly Aerospace is confident that the historically slow pace that the Space Race has taken lately is about to return to a sprint, and he intends to take Firefly to the lead. They have come incredibly far in just 4 years and are back on track, both technically and financially. Markusic is equally positive about the future of the company. They have taken leadership roles and hopefully, if they continue to progress as they have, the sky will no longer be the limit.


text: PLAYER: Rasmus T. S. Petersen LOCATION: Copenhagen, Denmark (soon if all go well Amsterdam, Holland) CHARACTER NAME:Thorolf Oddvarson CHARACTER TITLE: Thorolf The Auroch RACE: Norse (Svea) BIRTHPLACE: Middle Sweden, deep inland OCCUPATION: Warrior/Berserker RELIGION: Odin PHYSICAL DESCRIPTION:A tale and wide young man with shoulder lenght fair white hair, his face i dominated by a hawkish nose and the first traces of what might become a beard (in years), on both his arms he have fresh dragon tatoos ending in a "Uruz" Rune DISTINGUISHING FEATURES: He always carry the horn of a Auroch and his Poleax ADDITIONAL INFORMATION: Thorolf was born, the youngest son of a Old Jarl and one of his female thralls, he was from the day he was born at odds with his 5 day younger half brother Trygved, they are still bitter rivals, Trygveds two brothers normaly have taken his side against Thorolf, who had a very lonely childhood where his only friend was his horse and his halfsister Ravnhild. When his father did die Thorolf did have a run in with Trygved and some of his friends, that did cost Trygved 2 fingers and two friends, and Thorolf his right ear. Trygveds older brothers Thorfin the now jarl Gisli, did settle the case between them, but they did also let Thorolf know that it would be best if he did not show him self near Trygved again. that was the start of Thorolf's wandering, but he have given his word to return and take Ragnhild as his wife. When the Berserkergang come over Thorolf he bellow like a Auroch, since he belive the abillity come from drinking the hot heart blood of a freshly killed Auroch, he did this when huning with his father at the age of 13, and have carryed the horn of that creature ever since FIVE ADJECTIVE THAT CAPTURE YOUR CHARACTER: Reckless, Restless, Young, Lonely, Moody FIVE BEST SKILLS: Hunting, Poleaxe, Rideing, Brawling, Long Bow PRIVELIGED WEAPON: Poleaxe SECONDARY WEAPON: Long Bow NEXT OF KIN:Half Brothers Trygved, Thorfin and Gisli, Half-Sister Ravnhild


text:
Gervase Markham writes about a New Class of Vulnerability that shows the unbridled ego that leads to someone blaming the language and ignoring their own lack of competence.
He writes about a new class vulnerability for what he labels a bug. It's neither new nor a bug. Miyagawa wrote about this in 2009 in Perl: Why parameters() sucks and what we can do, and that's still not the origin of it. I know about in the 90s when I was making these mistakes. If you think you've found something new, you're probably wrong. Don't get too excited too soon.
Gervase is writing about the
param method from CGI.pm. In particular, in list context it returns a list of all the form fields with that name:
my @params = $cgi->param( $field );
If there are no form fields with that name, it returns the empty list. That means when Bugzilla used it in list context to create a hash, it was courting disaster by not properly checking its return value:
my $otheruser = Bugzilla::User->create({ login_name => $login_name, realname => $cgi->param('realname'), cryptpassword => $password});
If there's no
param returns an empty list and
cryptpassword becomes the value for
This is a problem with the competence of the Bugzilla developers. Hey, it happens. But, it's how you react when you mess up that matters. Blaming the language is the wrong move. It's not even a language issue. It's a misuse of a designed and documented API.
But, that's not the real problem here. The syntax and API misuse is the problem. The Bugzilla developers are taking unfiltered and invalidated input directly from the user and doing things with it. Even if the
param method did what they expected, they are still wrong.
This sensationalist posts then make the rounds of the internet, amplified by people who never bothered to learn the tool. It fits their uninformed mental models of the world. Other ignorant people are quick to jump onto that bandwagon because they mirror the behavior of the group they want to belong to. And, most everyone ends up worse for it.
If you mess up, take the blame for it and move on. It makes life so much easier.


text:
Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF‐induced colony dynamics governed by Rac1.
The paper explained
Human epidermal keratinocyte stem cells are critical for regenerative medicine for burns and genetic disorders. In serial culture, the keratinocyte stem cells progressively lose their proliferative capacity and become cells with limited growth (transient amplifying cells), a phenomenon termed clonal conversion that negatively impacts the culture lifespan and the success of cell transplantation. Although, clonal conversion is dramatically increased by stress, an inadequate microenvironment (niche), serial cultivation and age of donor, little is known on the underlying molecular mechanisms.
Here we show that colonies of keratinocyte stems cells differ from those of transient amplifying cells in their organization of actin filament organization, and in their response to epidermal growth factor (EGF). Moreover, inhibition of PI3K or Rac1 in keratinocyte stem cells induces the reorganization of actin filaments and an EGF response that are similar to those of transient amplifying cells. Importantly, continuous Rac1 inhibition in keratinocyte stem cells results in clonal conversion and reduction of growth potential. Together, out data demonstrate that human epidermal keratinocyte stem cells are maintained in culture by EGF signalling and actin filament dynamics in a cooperative manner and we propose the existence of a molecular circuitry that governs clonal conversion.
Modulation of signalling pathways that impact clonal conversion will improve stem cell self‐renewal and engraftment, together with the long‐term maintenance of the regenerated epidermis in transplanted patients.
Actin filament organization and actomyosin contractility are critical for cell shape changes and movements during many developmental processes including gastrulation, tissue morphogenesis and remodelling (Gorfinkiel & Blanchard, 2011; Mason & Martin, 2011). The dynamics of actin filaments and bipolar assemblies of myosin II has been nicely characterized during the morphogenesis of Caenorhabditis elegans and Drosophila melanogaster embryos (Levayer & Lecuit, 2012), and in epidermal keratocyte locomotion in fish (Keren et al, 2009; Schaub et al, 2007; Small et al, 1995). In mammals, the epidermis is a superb model system to study the role of actin filament dynamics in tissue homeostasis because it constantly renews thanks to keratinocyte stem/progenitor cells located in the epithelial basal layer, and in epidermal appendages. Dividing keratinocyte stem cells generate cells with more restricted growth potential that, in turn, generate suprabasal cells that will terminally differentiate to contribute to the barrier function of the skin (Blanpain & Fuchs, 2009; Clayton et al, 2007; Jones et al, 2007; Rochat et al, 1994; Sotiropopulou & Blanpain, 2012). Moreover, actin filaments are reorganized during terminal differentiation of epidermal keratinocytes (Connelly et al, 2010; Lewis et al, 1987; Vaezi et al, 2002), through a molecular mechanism mediated by RhoA and Rac1 (Benitah et al, 2005; Vaezi et al, 2002), the small Rho GTPases that function downstream of epidermal growth factor receptor (EGFR) signalling, and other tyrosine kinase receptor pathways (Raftopoulou & Hall, 2004). However, the impact of actin filament reorganization in epidermal keratinocyte stem cells remains unknown.
Human keratinocyte stem cells are clonogenic and can be extensively cultured (Rheinwald & Green, 1975). Under appropriate conditions, these stem cells, known as holoclones (Barrandon & Green, 1987a), can undergo at least 180 doublings, generating enough progeny to entirely reconstitute the epidermis of an adult human for a lifetime (Mathor et al, 1996; Rochat et al, 1994, 2012). Moreover, clonal analysis has demonstrated that besides stem cells, there are other clonogenic keratinocytes with restricted growth capabilities (Barrandon & Green, 1987a). First, there are progenitors (meroclones) that can only generate an epidermis for a short term when transplanted. Second, there are transient amplifying (TA) cells (paraclones), which growth capacity is limited to a maximum of 15 doublings; obviously paraclones cannot regenerate an epidermis. Termination of a culture of human keratinocytes often results from a phenomenon termed clonal conversion (Fig 1A), the switch of a holoclone into a meroclone or paraclone (Barrandon et al, 2012; Rochat et al, 2012). Clonal conversion thus results in progressive and irreversible restriction in growth potential. It is accelerated by stress, suboptimal culture conditions (inadequate niche), serial cultivation and age of donor. However, reversion of a paraclone to a stem cell‐like phenotype can be obtained by immortalization or oncogenic transformation (Barrandon et al, 1989; Dellambra et al, 2000; Dürst et al, 1987). Recent results also indicate that continuous inhibition of Rho signalling (Chapman et al, 2010; McMullan et al, 2003; Terunuma et al, 2010), and continuous inhibition of mTOR signalling by rapamycin (Brouard et al., in preparation) favour the formation of progressively growing colonies while decreasing the formation of paraclones. Together, these observations suggest that clonal conversion can be reduced or even stopped. Moreover, it is essential to comprehend the molecular mechanisms that govern clonal conversion because cultured human epidermal stem cells can be transplanted onto patients with extensive burns and genetic disorders to regenerate a functional epidermis (De Luca et al, 2006; Gallico et al, 1984; Mavilio et al, 2006; Pellegrini et al, 1999; Rochat et al, 2012; Ronfard et al, 2000). Alleviating clonal conversion will improve stem cell self‐renewal and engraftment, together with the long‐term maintenance of the regenerated epidermis in transplanted patients.
Here, we show that colonies of keratinocyte stem cells differ from those formed by TA keratinocytes in their organization of actin filaments and in their response to EGF and that continuous Rac1 inhibition in holoclones results in clonal conversion.
Clonogenic human keratinocytes respond differently to EGF
Human diploid epidermal keratinocytes form colonies of different shapes when cultured on a feeder layer of lethally irradiated 3T3‐J2 cells, at clonal density (Rheinwald & Green, 1975). After 6 days of culture, some colonies have a nearly circular shape and contain densely packed small basal cells; these colonies grow to a large size if further cultured and can be serially passaged (Supporting Information Fig S1A and S1B). Other colonies have an irregular shape and mostly contain large and flattened basal cells; these colonies soon abort growth and cannot be subcultured (Supporting Information Fig S1A and S1B). The former colonies are initiated by holoclones (stem cells) and meroclones, while the latter are initiated by paraclones (TA cells; Barrandon & Green, 1987a). Because EGFR signalling is important for the successful expansion of diploid human keratinocytes in culture (Barrandon & Green, 1987b), we have further explored how keratinocyte colonies respond to EGF. As expected, progressively growing colonies constantly increased their size during the first 120 min after addition of EGF (30 ng/ml; Barrandon & Green, 1987b), but terminal (aborted) colonies had a strikingly different behaviour. Terminal colonies rapidly shrunk before expanding again in the next 30 min, however, without fully recovering the size they had before addition of EGF (Fig 1B). Time‐lapse imaging showed that the expansion of a growing colony in response to EGF was caused by two mechanisms: a centrifugal migration of the peripheral cells with maintenance of cell–cell contact, and a flattening of the cells located at the center of the colony (Supporting Information Movie S1). In contrast, reduction in size of a terminal colony resulted from the shrinking of each individual cell (Supporting Information Movie S2). This dual effect of EGF could not be explained by an altered expression and/or altered functionality of the EGF receptor, as addition of EGF induced EGFR phosphorylation and internalization in both types of colonies (Fig 1C).
EGF activates extracellular signal‐regulated kinase 1 and 2 (ERK1 and ERK2), through EGFR/RAS signalling. ERK1 and ERK2 (ERK1/2) are involved in migration of various cell types (Huang et al, 2004), and epithelial cell sheets (Matsubayashi et al, 2004). As expected, ERK1/2 were immediately phosphorylated after EGF addition in human keratinocytes, and EGF‐induced ERK1/2 phosphorylation was significantly decreased by U0126, a MAPK/ERK kinase (MEK) 1 and 2 inhibitor (Supporting Information Fig S2A and S2C). Interestingly, addition of U0126 completely inhibited expansion of growing colonies while affecting the dynamics of shrinking of terminal colonies (Fig 1D). Similar results were obtained with another MEK inhibitor (PD98059; Supporting Information Fig S2B). Furthermore, exposed to U0126 for more 2 days increased the expression of involucrin (INV), transglutaminase 1 (TG1), keratin 1 (K1), keratin 10 (K10) and desmoglein 1 (DSG1), and inhibited the growth of growing colonies, even when EGF was added to the culture (Supporting Information Fig S3). These data indicated that colony expansion through EGFR/ERK signalling is essential for sustained growth of keratinocyte colonies. In response to EGFR ligands, cell motility immediately increases through activation of ERK1/2 without de novo transcription (Huang et al, 2004). ERK1/2 then phosphorylates and activates myosin light chain kinase (MLCK; Klemke et al, 1997; Nguyen et al, 1999), which in turn phosphorylates the myosin regulatory light chain (MLC) of myosin II, resulting in actomyosin contraction and cell movement (Totsukawa et al, 2004). In keratinocytes, EGF induced MLC phosphorylation of myosin II, which decreased in presence of MEK inhibitors and a MLCK inhibitor (ML7) (Supporting Information Fig S2C and S2D). ML7 also prevented the expansion of growing colonies and resulted in a delayed shrinking of terminal colonies (Fig 1E), as MEK inhibitors did. These results demonstrated for the first time that growing and terminal colonies of keratinocytes had different dynamics in response to EGF, and indicated that their expansion and shrinking depended on MLC phosphorylation and actomyosin interaction through EGFR/ERK/MLCK signalling.
Actin filaments organize differently in growing and terminal colonies
Phosphorylation of MLC induces bipolar assemblies of myosin II, and enables myosins to interact with actin filaments. Hence, we next visualized the network of actin filaments in growing and terminal colonies by rhodamine‐phalloidin staining. In absence of EGF, short actin bundles were distributed radially in the cells located at the periphery of growing colonies, whereas a well‐developed circumferential actin network was observed in cells of terminal colonies (Fig 2A). Quantitative analysis clearly revealed that orientation of actin filaments in the cells located at the periphery of colonies was significantly different in growing and terminal colonies (Fig 2A and Supporting Information Fig S4). In response to EGF, cytoplasmic protrusions were observed together with radial actin bundles at the leading edge of cells of growing colonies but not in cells of terminal colonies (Fig 2B and C). In both colony types, phosphorylated MLC of myosin II colocalized with actin filaments and was more intense after EGF exposure, whereas it significantly decreased after exposure to MEK and MLCK inhibitors, even in the presence of EGF (Fig 2D). These data further indicated that EGF induced actomyosin interaction through EGFR/ERK/MLCK signalling in growing and terminal colonies of human keratinocytes.
Clonal conversion results in the reorganization of actin filaments
To investigate the relation between the dynamics of a keratinocyte colony and clonal conversion, we isolated two progressively growing clones from a culture of normal human epidermal keratinocytes. These clones were then serially passaged in the presence of EGF. The rate of clonal conversion (switching from growing to terminal) increased with passages as expected; numerous progressive growing colonies were observed in early passages whereas numerous terminal colonies were observed in late passages. Results were identical in clonal and mass cultures, confirming that the switch in the response to EGF and the organization of actin filaments was linked to clonal conversion (Supporting Information Fig S5A and S5B). Next we investigated how colony dynamics is impacted by a ROCK inhibitor (Y27632), a molecule known to favour growing colonies (Chapman et al, 2010; McMullan et al, 2003; Terunuma et al, 2010). ROCK regulates both actin polymerization and MLC phosphorylation (Amano et al, 2010). Addition of Y27632 completely inhibited the response of both growing and terminal colonies to EGF (Fig 3A and Supporting Information Fig S6A). Interestingly, it drastically affected terminal colonies, which expanded similarly to growing colonies (Fig 3A and Supporting Information Fig S6A). Colonies treated with (−)‐blebbistatin, a myosin ATPase inhibitor, also behaved similarly to those treated with Y27632 (Fig 3B). These results further confirmed that actomyosin interaction was essential for colony dynamics in response to EGF. Rhodamine‐phalloidin staining of actin filaments revealed that the organization of the actin network in terminal colonies was now similar to that of a growing colony with Y27632, but not (−)‐blebbistatin (Fig 3C and D). Y27632, but not (−)‐blebbistatin, also diminished pMLC phosphorylation (Fig 3C and Supporting Information Fig S2C). However, Y27632 was not enough to rescue a paraclone from its commitment to terminal differentiation (Supporting Information Fig S6B). Together, these results indicated that inhibiting Rho signalling could remodel the actin filament network of a paraclone, but without consequence for its commitment to terminal differentiation. We further examined the effects of three small molecules, including an actin polymerization inhibitor (cytochalasin D), and two compounds that induce depolymerization of actin filaments (mycalolide B and bistheonelide A) on the growth capacity of human keratinocytes and clonal conversion. However, these molecules could not enhance clonal growth and colony‐forming efficiency (CFE) significantly, as Y27632 did (Supporting Information Fig S7).
We also investigated how colony dynamics is impacted by rapamycin, another molecule that favours the formation of progressively growing colonies while decreasing the formation of paraclones (Brouard et al., in preparation). Addition of rapamycin, an inhibitor of mTORC1 signalling, for 1 h had no significant effect on expansion of growing colonies and shrinking of terminal colonies, regardless of EGF presence (Fig 3E), confirming that mTORC1 signalling had no impact on actin filament reorganization (Benjamin et al, 2011; Laplante & Sabatini, 2012). On the other hand, colonies treated with rapamycin for 3 days behaved similarly to those treated with Y27632 (Fig 3F). Indeed, long‐term treatment of mammalian cells with rapamycin can suppress both mTORC1 and mTORC2 activities (Sarbassov et al, 2006; Supporting Information Fig S2E). Long‐term treatment with rapamycin did not impact the organization of actin filaments, but significantly suppressed the EGF‐induced MLC phosphorylation of myosin II (Fig 3G and H, and Supporting Information Fig S2E). This result further confirmed that EGF‐induced MLC phosphorylation was essential for colony dynamics. These experiments first indicated that rapamycin had a different effect on colony behaviour when present for a short and a long time period; second, that long‐term inhibition of mTOR signalling induced a response similar to the one observed when Rho signalling was inhibited.
PI3K/Rac1 activity regulates organization of actin filaments and the dynamics of keratinocyte colonies
Phosphatidylinositol 3‐kinase (PI3K) signalling is involved in proliferation and differentiation of keratinocytes (Pankow et al, 2006; Sayama et al, 2002), as well as in actin cytoskeletal organization and cell motility in the HaCaT keratinocyte cell line (Pankow et al, 2006). A PI3K inhibitor (LY294002), but not its kinase inactive analogue (LY303511), inhibited PI3K‐dependent Akt phosphorylation in cultured human keratinocytes (Fig 4A and Supporting Information Fig S8E). Without EGF, LY294002 induced the formation of stress fibres and cortical actin bundles parallel to the plasma membrane in both colony types (Fig 4B). Addition of LY294002 for 1 h, followed by EGF exposure, resulted in the shrinking of growing colonies, and in increased shrinking of terminal colonies (Fig 4C). Wortmannin, another PI3K inhibitor, acted similarly to LY294002 (Supporting Information Fig S8). PI3K controls a wide variety of downstream signalling pathways. Rac1, a member of Rho family of small guanosine triphosphatases (GTPases), is a downstream target of PI3K, and coordinates the dynamic organization of the actin cytoskeleton (Ridley et al, 1992). Phosphatidylinositol 3, 4, 5 phosphate (PIP3), a PI3K product, regulates a number of Rac‐specific guanine nucleotide exchange factors (GEFs) that catalyse Rac activation (Cantrell, 2001). As expected, a Rac1 inhibitor (NSC23766) suppressed Rac1 activation in EGF exposed‐cells, similarly to LY294002 (Fig 4A). Rac1 inhibition did not affect phosphorylation of ERK and Akt, the best‐characterized downstream target of PI3K signalling (Fig 4A), but increased rhodamine‐phalloidin staining of actin filaments at the cell periphery and induced actin bundles parallel to the plasma membrane in both growing and terminal colonies (Fig 4B), and resulted in the shrinking of growing colonies, and in increased shrinking of terminal colonies (Fig 4D). An Akt inhibitor (Akt inhibitor VIII) did not alter the organization of actin filaments and the response to EGF of both colony types (Supporting Information Fig S8). Collectively, these results indicated that PI3K/Rac1, but not Akt signalling, regulated actin filament organization and EGF‐induced colony dynamics in human keratinocytes.
Rac1 inhibition promotes clonal conversion
Inhibition of PI3K and Rac1 in holoclones and meroclones resulted in a switch in the organization of actin filaments and EGF‐induced colony dynamics to a pattern similar to that of paraclones. Hence, we further investigated the role of PI3K signalling on clonal conversion in cultured human keratinocytes. Keratinocytes were seeded at clonal density (200 cells), and cultivated for 4 days before being exposed to EGF (10 ng/ml) and various signaling inhibitors (LY294002, NSC23766, Akt inhibitor and rapamycin). After a 3‐day exposure, the growth capacity of keratinocytes was obviously impacted by the presence of the inhibitors, as the colonies were significantly smaller than in control cultures (Fig 5A). Colonies exposed to LY294002, Akt inhibitor and rapamycin mostly contained small cells, while NSC23766‐treated colonies mostly contained large and flat cells. Interestingly, Akt inhibitor and rapamycin decreased the expression of INV, K1, K10, TG1 and DSG1, while NSC23766 increased INV expression (Supporting Information Fig S9A–C). After an 8‐day exposure to various inhibitors, cultures were fixed and stained with rhodamine B. The number of keratinocyte colonies was not affected, but colony growth was impaired with inhibitors (Supporting Information Fig S9D). Rac1 and Akt are distinct downstream targets of PI3K signalling, however, Rac1 is also a substrate of Akt, and its phosphorylation by Akt decreases Rac1‐GTP binding (Kwon et al, 2000). We confirmed that inhibition of PI3K and Akt significantly decreased Rac1 phosphorylation in cultured human keratinocytes (Fig 5B). Exposure to LY294002, Akt inhibitor and rapamycin was sufficient to inhibit Akt phosphorylation at Ser473, a target site of mTORC2 kinase activity (Sarbassov et al, 2005; Fig 5B). Importantly, phosphorylated Akt (Ser473) was only detectable in growth‐arrested cells of terminal colonies, and not in proliferative small cells of growing colonies (Fig 5C). Similar results were also obtained with clones (Supporting Information Fig S5B). Collectively, these data suggested that Rac1 inhibition reduced the growth potential of cultured human keratinocytes. To further address this point, we cultured keratinocytes with different inhibitors for 3 days before cells were passaged at clonal density. The presence of a Rac1 inhibitor markedly reduced CFE from 25 ± 5.3% (mean ± sd) to 7.3 ± 1.3%, while increasing the number of terminal colonies (39 ± 2.6% to 70 ± 7.9%; Fig 5D). Keratinocytes grown in presence of an Akt inhibitor had a decreased CFE (7.8 ± 2.5%), but formed less terminal colonies (28 ± 4.7%) than cells grown without the inhibitor (Fig 5D). No significant changes were observed in cultures treated with a PI3K inhibitor or rapamycin (Fig 5D).
To further confirm these results, we next developed a doxycycline‐inducible target‐specific knockdown system using a single lentiviral vector (Supporting Information Fig S10A). Akt has three isoforms (Akt1/Akt2/Akt3) in mammalian cells, and Akt1 and Akt2 are predominantly expressed in human normal keratinocytes (Thrash et al, 2006). Therefore, we designed transgene cassettes for inducible expression of microRNA(miR)‐typed short hairpin RNA (shRNA) targeted to Rac1, Akt1 or Akt2 gene, and screened and evaluated the knockdown efficiency of these shRNA constructs by Western blotting (Supporting Information Fig S10B–D and Fig 5E). As expected, Rac1 phosphorylation significantly decreased in the Rac1, Akt1 and Akt2 shRNA‐expressing keratinocytes (Fig 5E). Surprisingly, knockdown of Akt1 and Akt2 also resulted in downregulation of Rac1 (Fig 5E). We next seeded keratinocytes at clonal density, and infected them with lentiviral vectors carrying the shRNA transgenes. Keratinocytes were further cultured for 4 days before shRNA expression was induced by treatment with doxycycline. In our system, the miR‐typed shRNA sequence specific to a target gene is transcribed together with the gene of green fluorescent protein (GFP; Supporting Information Fig S10A), and shRNA‐expressing cells are easily traced as GFP‐positive cells. After a 3‐day exposure to doxycycline, keratinocytes transduced with control shRNA gave rise to progressive growing colonies (Fig 5F). However, most of Rac1, Akt1 and Akt2 shRNA‐transduced colonies showed terminal phenotype (Fig 5F). Subsequently, shRNA‐expressing keratinocytes were passaged without doxycycline. The temporal knockdown of Rac1, Akt1 and Akt2 markedly reduced CFE while increasing the number of terminal colonies, and the cultures could not be maintained any more (Fig 5G). Collectively, these results indicated that Rac1 was necessary for maintaining the growing phenotype, and that Akt kinase activity was necessary to promote the terminal phenotype, while Akt expression was required for Rac1 expression.
A colony initiated by a human keratinocyte stem cell grows by a combination of cell migration and multiplication, and requires EGFR ligands (Barrandon & Green, 1987b; Coffey et al, 1987; Rheinwald & Green, 1977). Activation of EGFR signalling immediately induces the lateral expansion of colonies that are actively multiplying (Barrandon & Green, 1987b), and also enhances the helical migration of individual keratinocytes seeded onto a fibrin matrix (Ronfard & Barrandon, 2001). Moreover, EGFR signalling is important for proliferation of keratinocyte stem cells through Lrig1 (Jensen & Watt, 2006; Jensen et al, 2009), for the re‐epithelialization of an epidermal wound (Barrientos et al, 2008; Higashiyama & Nanba, 2005; Gurtner et al, 2008; Pastore et al, 2008), and for the massive ex vivo expansion of human keratinocytes for regenerative medicine (De Luca et al, 2006; Rochat et al, 2012). Our results now demonstrate that the differing organization of the actin filament network in colonies formed by human keratinocyte stem cells (holoclones) and TA cells (paraclones) set up the stage of their response to EGF through EGFR/ERK/MLCK signalling (Fig 6A).
The radial distribution of actin filaments preserved by Rac1 activity in holoclones is required for the expansion of progressively growing colonies in response to EGF. This EGF‐induced colony dynamics results from centrifugal migration of the cells located at the periphery of growing colonies, and is essential for sustained growth of keratinocyte colonies (Barrandon & Green, 1987b). We also demonstrate here that inhibition of EGFR/ERK signalling‐induced colony expansion induces the expression of terminal differentiation markers. Furthermore, EGF increased the clonogenicity, but not multiplication, of human epidermal keratinocytes forming small colonies, which dynamics we examined in this study (Rheinwald & Green, 1977). These data suggest that EGF‐induced colony dynamics defined by actin filament organization is involved in clonal growth and conversion. This study shows that remodelling of actin filaments of paraclones to a holoclones‐type pattern by Y27632 cannot rescue a paraclone from its commitment to terminal phenotype, and that inhibition of actin polymerization and depolymerization of actin filaments are not sufficient to reduce, stop and/or reverse clonal conversion. However, a switch in the organization of actin filaments from holoclone‐ to paraclone‐type by Rac1 inhibition decreases clonal growth, and promotes clonal conversion. These experiments explain that radial distribution of actin filaments is essential for holoclone‐colony dynamics and maintenance of growth capacity of holoclones, but not sufficient for restoration of growth capacity of paraclones.
Continuous inhibition of Rho signalling (Chapman et al, 2010; McMullan et al, 2003; Terunuma et al, 2010), and continuous inhibition of mTOR signalling by rapamycin (Brouard et al., in preparation) favour the formation of progressively growing colonies, while decreasing the formation of paraclones. mTOR signalling plays an important role in balancing cell behaviour (Benjamin et al, 2011; Ma & Blenis, 2009; Laplante & Sabatini, 2012) through mTORC1, which is rapamycin sensitive and acts on translation, and mTORC2, which is rapamycin insensitive and regulates actin filament organization (Jacinto et al, 2004) and actomyosin contractility through Rho/ROCK signalling (Liu et al, 2010). However, little is known on how mTOR signalling impacts human keratinocyte stem cell behaviour (Javier et al, 1997). Our results demonstrate that continuous exposure of a keratinocyte culture to rapamycin inhibits phosphorylation of the MLC and impacts colony dynamics. This indicates that rapamycin can also impact mTORC2, as it has been recently demonstrated (Hosking, 2012; Lamming et al, 2012). Moreover, our results demonstrate that the response of keratinocyte colonies to continuous inhibition of ROCK signalling is strikingly similar to that of colonies exposed to rapamycin, thus suggesting the existence of a functional link between Rho/ROCK and mTORC2 signalling and the significance of phosphorylation of MLC for keratinocyte stem cell maintenance.
PI3K signalling impacts cell motility and actin filament organization, as well as proliferation and differentiation in human keratinocytes (Pankow et al, 2006; Sayama et al, 2002). Moreover, activation of Akt induces keratinocyte terminal differentiation (Calautti et al, 2005; Janes et al, 2009; Thrash et al, 2006), promotes growth arrest (Calautti et al, 2005), and results in clonal conversion (Janes et al, 2009). In contrast, Rac1 is required for clonal growth of keratinocytes (Benitah et al, 2005), and a decreased expression of Rac1 induces keratinocyte differentiation (Nikolova et al, 2008). Our findings indicate that activation of Akt signalling becomes predominant during serial cultivation, with the consequence of decreasing Rac1 activity. This results in remodelling of actin filaments, and in clonal conversion. Hence, we suggest the existence of a molecular circuitry wiring Rac1 to PI3K and Akt (Fig 6B), the modulation of which impacts stem cell behaviour by either enhancing or decreasing clonal conversion. This model presents that PI3K is required for both Rac1 and Akt activity and that inhibition of PI3K causes both Rac1 inactivation and activation, suggesting that the level of Rac1 activity is stable even when a PI3K inhibitor is present and/or that PI3K inhibition does not affect a balance between Rac1 and Akt activity. This can be explained our data why PI3K inhibition does not decrease CFE. Surprisingly, shRNA knockdown of Akt1 and Akt2 results in the decreased expression of Rac1 protein, and accelerates clonal conversion. An Akt inhibitor decreases colony‐forming ability of keratinocytes, but not Rac1 expression. This result suggests the existence of kinase activity‐independent regulation of Rac1 expression by Akt1/2 in keratinocytes. It is likely that Akt1/2 is required for Rac1 signalling and that shRNA knockdown of Akt1/2 interferes with Rac1 signalling and decreases Rac1 protein levels consequently. This point should be considered for regulating keratinocytes stem cells.
In conclusion, our data demonstrate that remodelling of actin filaments and inhibition of actomyosin contractility can impact stem cell behaviour through the modulation of clonal conversion. This is of paramount importance for regenerative medicine.
Cell culture
Human keratinocyte YF29 cells and normal human epidermal keratinocytes (KURABO) were from foreskin of newborns. Frozen cells were thawed and cultivated at clonal density on a feeder layer of irradiated 3T3‐J2 cells at 37°C as described (Rheinwald & Green, 1975; Rochat et al, 1994). The medium was changed every 4 days. Cells were used between passage 4 and 10. For determination of CFE, 100 keratinocytes were cultured as previously described. Cultures were fixed in 3.7% buffered formaldehyde and stained with 1% rhodamine B, and keratinocyte colonies were counted under a binocular microscope.
Inhibitors and antibodies
Pharmacological inhibitors and Primary antibodies used for immunofluorescence and Western blotting were described in Supporting Information Table S1 and S2, respectively.
Measurement of keratinocyte colony area and time‐lapse imaging
Human epidermal keratinocytes were seeded at clonal density in a 35 mm size cell culture dish (Corning) with irradiated 3T3‐J2 cells, and grown for 6 days without EGF. Cells were then cultivated with or without EGF (30 ng/ml) for 3 h. Small molecule inhibitors were added 1 h before starting the EGF treatment. Microscopic colonies were photographed at various times after EGF stimulation and their area was then measured with MetaMorpho (Molecular Devices) or Volocity (PerkinElmer) using a graphic tablet (Wacom). Mean size (±SD) was calculated on measurements obtained from at least five colonies. For time‐lapse imaging, cultures were maintained at 37°C and 10% CO2 in a chamber mounted on an Axiovert 200 M microscope (Zeiss). Images were obtained at 5 min intervals for 180 min.
Measurement of actin filament orientation
An angle of single actin filament against the plasma membrane in a cell localized at the periphery of a keratinocyte colony was defined as an acute crossed‐axes angle (0° ≤ θ ≤ 90°) between a line linking cell–cell contact points at colony edge, and an angle of single actin filament in a cell. See also Supporting Information Fig S4. The angles of three well‐defined actin filaments in individual keratinocytes were measured with Volocity (PerkinElmer) using a graphic tablet (Wacom). The distribution was obtained from at least 75 well‐defined actin filaments in more than 25 cells of growing or terminal colonies in each experimental condition.
Immunofluorescence microscopy
YF29 cells were seeded at clonal density in a 35 mm cell culture dish with irradiated 3T3‐J2 cells, and grown for 6 days without EGF. In some cases, the cells were stimulated with 30 ng/ml EGF for various times in each experiment. After that, the cells were washed twice with cold phosphate‐buffered saline (PBS), and fixed in 4% paraformaldehyde in PBS at 4°C for 10 min. The cells were treated with 0.5% Triton X‐100 in PBS for 10 min and subsequently with 1% BSA. The cells were then incubated with primary antibodies for 1 h at room temperature (RT) or overnight at 4°C, washed in PBS, and incubated with Alexa Flour 488‐conjugated goat anti‐mouse IgG or Alexa Flour 568‐conjugated goat anti‐rabbit IgG (Dako) for 1 h at RT. In some cases, the cells were treated with rhodamine‐phalloidin (Invitrogen) for 30 min at RT. After a wash with PBS, the cells were incubated with Hoechst 33258 (Molecular probes), mounted with a fluorescent mounting medium (Dako), and examined with a Carl Zeiss Axioplan 2 epifluorescence microscopy.
Western blotting
YF29 cells were seeded in a 60 mm cell culture dish (Falcon) with irradiated 3T3‐J2 cells, and grown for 6 days without EGF. In some cases, the cells were stimulated with 30 ng/ml EGF for various times in each experiment. After that, the cells were washed twice with cold‐PBS, and lysed with 1% Triton‐X 100, 5 mM EDTA in PBS containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail 1 (Sigma). For the detection of MLC and phosphorylated MLC, keratinocytes were lysed with SDS sample buffer. Equal amounts of proteins were dissolved in SDS sample buffer and separated by SDS–PAGE. Proteins in the gels were transferred to nitrocellulose membranes (Schleicher & Schuell). After the blocking with 5% skimmilk or 5% BSA in PBS, the membranes were immunoblotted against primary antibodies overnight at 4°C. After a wash with 0.05% Tween‐20 in PBS, the membranes were incubated with horseradish peroxidase (HRP)‐conjugated anti‐rabbit or anti‐mouse antibody (Jackson Immunoresearch) for 1 h at RT. The membranes were washed with 0.05% Tween‐20 in PBS, treated with an enhanced chemiluminescence substrate for detection of HRP (Pierce) for 5 min, and exposed to Kodak BioMAX MR film.
Quantitative RT‐PCR
Total RNA was extracted from cultured keratinocytes with TRIzol reagent (Invitrogen). cDNAs were synthesized from 1 µg of total RNA with SuperScript III (Invitrogen) and hexamer random primers (Invitrogen), according to manufacturers' instructions. cDNAs were adjusted to equal levels by PCR amplification with primers for TBP1. Primers were designed to ensure the uniqueness for each gene, using LightCycler Probe Design 1.0 software and NCBI BLAST. The list of primers used is described in Supporting Information Table S3. Quantitative PCR was performed using LightCycler FastStart DNA Master SYBR Green I reagents, and a LightCycler System piloted by LightCycler 3.5 software (Roche Diagnostics). Differences between experimental samples and controls were calculated on the basis of the method.
Rac1 activity assay
GTP‐bound Rac1 was detected by using Rac1 activation assay kit (Millipore), according to manufacturer's instructions.
Plasmid DNA construction for lentiviral expression vector
The HIV‐based self‐inactivating lentiviral expression vector plasmid CS‐CA‐MCS (Miyoshi et al, 1998) was provided by RIKEN BioResource Center, Japan. Three plasmids, pTet‐On‐Advanced vector, pTRE‐Tight vector (Clontech laboratories), and pcDNA 6.2‐GW/EmGFP‐miR vector (Invitorgen) were obtained, and their components were utilized to construct a transgene cassette consisting of two expression units for inducible gene knockdown in this study. The cDNA fragment of reverse Tet‐controlled transactivator (rtTA) was recovered from the pTet‐On‐Advanced vector and ligated into the pIRES vector (Clontech), upstream the sequence of internal ribosome entry site (IRES). The blasticidin‐resistance gene (Bsd) was ligated into this plasmid, downstream the IRES sequence, resulting in the generation of prtTA‐IRES‐Bsd, containing one transgene unit. On the other hand, a Dra I‐Pvu II fragment of the pcDNA 6.2‐GW/EmGFP‐miR vector containing EmGFP cDNA and pre‐miRNA sequence was recovered and inserted into the pTRE‐Tight vector, downstream the Tet‐responsive promoter. This resulted in the vector pTRE‐EmGFP‐miR, which contained another transgene unit. Finally, the fragment of the rtTA‐IRES‐Bsd was inserted into the lentivirus expression vector plasmid, CS‐CA‐MCS, directly downstream of the CAG promoter sequence, and then another unit, the TRE‐EmGFP‐miR fragment, was inserted into the resulting plasmid, between the Bsd and WPRE sequence. The TRE‐EmGFP‐miR unit was arranged in the opposite direction as the CAG‐driven rtTA‐IRES‐Bsd unit, yielding a new single lentivirus expression plasmid for inducible gene silencing, CS‐CA‐rtTA‐IRBsd‐TRE‐EmGFP‐miR (Supporting Information Fig S10A).
Lentiviral vector for doxycycline‐dependent inducible gene knockdown
In order to construct the lentivial vectors for doxycycline‐dependent inducible silencing of the human Rac1, Akt1 and Akt2 gene expression, a set of synthetic oligonucleotides in each gene designed by BLOCK‐iT miR RNAi technologies of Life Technologies, Inc., was obtained, respectively. The 21nt target sequences were described in Supporting Information Table S4. The double‐strand DNA was inserted between the 5‐miR and the 3′‐miR flanking sequences of the vector plasmid. The miR‐negative control sequence presented by Life Technologies, Inc., was used to construct a negative control vector. Using these plasmid DNAs, lentiviral particles were prepared by standard transfection procedures with packaging plasmid DNAs (pCAG‐HIVgp and pCMV‐VSV‐G‐RSV‐Rev; RIKEN BioResource Center), and used to introduce the transgene into 1 × 105 cells. After transduction of the transgenes at a multiplicity of infection of 5, the cells were treated with 100 nM doxycyline to induce silencing of the gene, and subsequently analysed.
Author contributions
DN, FT and YB designed the experiments; DN and FT performed most of experiments and analysed the data; NM and SM developed the inducible knockdown lentivirus system and produced lentiviruses; YB and SH interpreted the data with DN and supervised the project; All authors discussed the results and commented on the manuscript; DN and YB wrote the manuscript.
Supplementary Information
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
suppMovie_S1 [emmm_201201839_sm_suppMovie_S1.mov]
suppMovie_S2 [emmm_201201839_sm_suppMovie_S2.mov]
suppdata [emmm_201201839_sm_suppdata.pdf]
We thank Dr. K. Hashimoto (Ehime University) for his continuous encouragement and Dr. F. Watt (King's College London) for providing us SY5 and BC1 antibodies. We are also grateful to Dr. A.W. Amici for critical reading of the manuscript. This study was supported by grants to Y.B. from the Eagle Foundation, the Swiss National Science Foundation and the European Economic Community through the 7th EuroSyStem and OptiStem framework program, and to D.N. from the Japan Society for the Promotion of Science (JSPS), Postdoctoral Fellowship for Research Abroad and Grant‐in Aid for Scientific Research No. 22791080 and No. 24791159, and The Nakatomi Foundation.
Supporting Information is available at EMBO Molecular Medicine Online.
The authors declare that they have no conflict of interest.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
- Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO


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|DJI Launches New Osmo Mobile 2 in the Philippines for Php7,900
The DJI Osmo Mobile 2 is "Redisgned for the Storyteller in All of us" according to DJI's branding. As this is a smartphone camera stabilizer, it will enable users to capture smooth videos and high definition panoramas with cinematic movement with ease using their smartphone's camera. DJI has been known to create amazing camera stabilizers due to their amazing three-axis gimbal technology that cancels out unwanted movements in three directions. This will then ensure that your videos stay steady, even with a shaky hand or wobble scenario. It also have DJI's built-in SmoothTrack technology that detects and compensates for the movement of your camera.
The Osmo Mobile 2's design has two things in mind. It has to be lightweight and extremely portable. That is why they chose to use high-strength modified nylon. DJI also used a folding design so that you can bring anywhere you go. There is a two-way mobile clamp that allows you to switch between landscape and portrait orientation. There is also a 1/4" universal screw mount ensures compatibility with your favorite photography accessories.
See Also: Phantom 4 Advanced Vs Phantom 4 Pro: 4 Must-Know Differences
There is also a simplified controls setup that lets you use your smartphone camera like a professional-grade camera. You can control settings such as ISO, shutter speed and more, There is also a new zoom slider that gives you complete control to the point that you can even produce a dolly zoom.
There is also a powerful built-in battery system that gives you 15 hours of recording battery life. This is 3x longer than the Original Osmo Mobile battery. It has a USB port that enables phone charging as well.
The DJI GO mobile app is also compatible that allows you to access intelligent photo and video features to help you create professional grade content. Features such as ActiveTrack, Hyperlapse, Panorama, Lone Exposure and Light Trail mode will be available in the mobile app.
The DJI Osmo Mobile 2 will retail at Php7,900 and will be available through MSI-ECS.


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The Federal Reserve raised rates for the first time this year, a move that's been widely expected for some time. They also signaled that rate increases are likely to continue through 2017, and most observers forecast three more quarter-point hikes by the end of next year.
What does it mean?
To quote from The Hitchhiker's Guide to the Galaxy…
Don't Panic!
Let's look at a the impact on a couple of popular options for investing in real estate.
REITs (Real Estate Investment Trusts) invest in a wide range of income-producing real estate assets, and fall into two general categories: "equity" REITs that own and manage income properties; and "mortgage" REITs that lend (although some REITs engage in both).
- Equity REITs that focus on ground-up development to create their inventory of income properties are heavily dependent on financing. Rising rates will tend to hurt the performance of these REITs, though it probably won't interrupt projects that are underway.
- Equity REITs that focus on acquiring and re-positioning assets can be nearly as capital-intensive, and will also feel some stress as rates rise. Expect new acquisitions to be delayed, and some projects may be canceled if they slip outside of underwriting guidelines.
- Mortgage REITs may be challenged because rising rates tend to decrease borrowing. If their business model is built on lending to equity REITs, don't expect them to outperform.
Other factors weigh on REIT returns. A REIT must distribute at least 90% of its income to shareholders, and without the ability to retain a significant amount of earnings, they'll be less able to ride out challenging markets. And REIT yields over the last 15 years have averaged only 1.28% higher than the 10-Year U.S. Treasury note, which will make some investors wonder if the additional yield is worth the added risk.
As always, the successful REIT investor will look carefully inside each potential investment for weaker holdings that may well drag yield downward.
Residential Housing
Consumer rates were already edging upward before the widely-expected rate hike. A few things to expect:
- HELOCs will begin to reset.
- Stragglers who haven't refinanced will start running for the train as it begins to leave the station.
- Some homeowners may be motivated to buy before interest rates get "too high" in their minds, but with rates still close to historic lows don't expect home prices to surge (from a flood of panicky buyers) or sink (as higher rates start pricing buyers out of the market).
Looking beyond, there are some recent trends whose impact hasn't played out in the economy. Forbes is predicting a "mild rebound" for the 2017-2018 U.S. economic forecast. Delays in household formation, declines in home ownership rates, increases in multi-generational and roommate living arrangements, renewed interest in urban living, and demographic shifts) Some of these may be permanent changes, some may not. But the fact is that personal employment status drives most home buying decisions, not interest rates. And the good news is that further hikes will result from job growth mixed with moderate inflation (and population growth, of course) which will help fuel demand.
But don't forget that all real estate is local, which leads to our third option.
Unlike most other real estate investments, DARE doesn't place multi-year bets on a few large projects. DARE deploys investor capital on short-term residential rehabbing projects—typically 4-8 months—so it can adapt in a way a REIT simply cannot.
DARE also prefers to fund projects that offer multiple exits. By underwriting projects that can be sold to a homeowner or an investor, DARE increases the likelihood of a quick sale, increasing yield. And higher interest rates make other forms of financing even more expensive, driving greater demand for DARE's equity-sharing financing model.
The Bottom Line
Don't Panic! There's no need to quit investing real estate simply because interest rates are rising.
If you'd like to try reading the FOMC's tea leaves in search of deeper meaning, try this article on Bloomberg.