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subset: commoncrawl


text:
Rick Tasco wrote:
: If we are talking Talls then
: surely Pallida, Variegata, Trojana and Mesopotamica would be here. If
: one had the time(probably lots of it) you could go back and trace
: parentages from the 20/30's checklists. Sharon are you there?
Yes, I'm here. But the "spare" time to run the necessary stats isn't!
I have pedigree analysis software, with a database that includes the pedigree of
every known TB ancestor of my seedlings -- plus a number for which I was
assessing potential. And each of those pedigrees goes back to either species
or unknown parentage. The catch is that the "unknown parentage" branches are so
numerous that the largest percentage of ancestry on any TB variety I've assessed
turns out to be "indeterminate". Arilbreds, of course, are another matter --
and that's where I've concentrated my efforts.
Sharon McAllister


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PMFD is committed to assisting people in need, whatever the emergency may be. Recently the staff turned their attention to a local food bank to assist them with providing food to the less fortunate. In November each staff member was asked to donate one can good item, by bringing that food item to each Monday training night.
Today staff delivered 207 pounds of donated food to Second Harvest Food ... (Full Story & Photos)
Yesterday we took delivery of our new MSA G1's. The order included 35 packs, 86 - 45 minute cylinders and face pieces. Training on the new packs is scheduled for January after which the packs will be placed into service. ... (Full Story & Photos)
Another busy month!
10 Days into the final month of the year the PMFD has responded to 26 calls for service, bringing our total for the year to 664.
On Friday December 9th 2016 the Palmer Municipal Fire Department was dispatched to the 4000 block of Green Pond Road (Palmer Township) for the reported detached garage on fire. Crews arrived to find a 3 car garage heavily involved and partially collapsed. ... (Full Story & Photos)
Over the last 4 days the PMFD has responded to 23 calls for Service. As a volunteer company this increased volume of calls puts a huge demand on our membership and we would like to Thank them for their continued dedication. We would also like to Thank Ashley Mesco who was called at 2:00am this morning as we operated at a prolonged operation on route 22. Ashley ran to the store and arrived on scene ... (Full Story & Photos)
The holiday season is upon us. On Saturday November 12, 2016 the members of the PMFD were asked to escort Santa to the Palmer Mall to kick off the Holiday Season. If was a beautiful day and Santa was greeted by a large crowd waiting to get a glimpse of him. Happy Holidays Everyone. ... (Full Story & Photos)
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Best thing we put in our kitchen was drawers instead of cupboards and soft close hinges - love them! Don't skimp on these details because they really are worth having.
Carpet in the bedrooms - good quality with thick underlay and preferably wool so to avoid static.
Security screens so you can always leave the windows open and wide eaves for this reason too.
Use good quality paint internally like wash and wear, it really does make a difference.
Butlers pantry with room for a chest freezer!!! i hate walking outside to get stuff for dinner lol
Those with a big pantry with a sink - exactly what do you use the sink for?
We are renovating & I'm planning a massive walk in pantry in which to keep the small appliances like food processor, juicer etc all set up and ready to go. But I can't imagine what I would do with another sink?
Our pantry leads out to the garage and then bbq so when we're entertaining all bbq dishes are done or rinsed in the back sink so they're ready to be loaded into the dishwasher later..
My husband also brews and uses that sink..
Other appliances like the kettle and Tefal 2in1 are kept in the back, again using that extra sink for cleaning and filling the kettle..
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text: A Collection Of Recipes For The Health Conscious Consumer - Main Page. By Ginger Redeker Last updated: Jul 14, 2014 Get ready to try out some great healthy recipes. This next round of recipes range from Granola and Low Carb Bread to Meringue. Try them out now and see what you think! Ginger's Recipes Ginger's Recipe Collection: A-B Almond Protein Cake Squares, Banana Nut Protein Bread, ...And A Lot More! Ginger's Recipe Collection: C-F Cauliflower Mash, Fat Free Cheesecake Ginger's Recipe Collection: g-m Grilled Asparagus & Tomato Salsa, Maple Pecan Granola Ginger's Recipe Collection: n-r Nutty Dressing Roasted Zucchini & Squash Ginger's Recipe Collection: s-r Savory Steak Zucchini Casserole About The Author Ginger Redeker Along with weight training and riding horses Ginger loves to cook. She will be sharing a lot of information here and mainly cover healthy recipes. View All Articles By This Author Rate This Article


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Fletcher, NC
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Audi Repair in Fletcher
RepairPal has 0 certified Audi repair shops and 24 other repair shops in Fletcher.
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Find RepairPal Certified Audi repair shops in Greer, Greenville and Spartanburg.
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The following 24 shops in Fletcher are not yet RepairPal Certified - Learn More
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T & J Auto Service
4009 Sweeten Creek RdArden, NC 28704
Audi repair 2.2 mi from Fletcher
Import Auto Performance
2129 Hendersonville RdArden, NC 28704
Audi repair 3.8 mi from Fletcher
All German Performance
1194 Hendersonville RdAsheville, NC 28803
Audi repair 7.1 mi from Fletcher
German Motor Werks
1274 Sweeten Creek RdAsheville, NC 28803
Audi repair 7.5 mi from Fletcher
Paul's Foreign Automotive
1404 7th Avenue EastHendersonville, NC 28792
Audi repair 7.6 mi from Fletcher
Meineke Car Care Center
820 Spartanburg Hwy-rt 176Hendersonville, NC 28739
Audi repair 8.1 mi from Fletcher
Js Miller High Country Builder
75 Quail Hollow DrFairview, NC 28730
Audi repair 9.8 mi from Fletcher
Olen Hensley Automotive Inc
221 Amboy RdAsheville, NC 28806
Audi repair 10.3 mi from Fletcher
Xpertech Car Care
1295 Tunnel RdAsheville, NC 28805
Audi repair 10.8 mi from Fletcher
Jack Smith's Advanced Transmission
248 Biltmore AvenueAsheville, NC 28801
Audi repair 11.1 mi from Fletcher
City Transmission Service
51 Southside AvenueAsheville, NC 28801
Audi repair 11.2 mi from Fletcher
Elff Auto Service
151 Coxe AvenueAsheville, NC 28801
Audi repair 11.4 mi from Fletcher
Info Pronto
33 Wake Robin WayAsheville, NC 28805
Audi repair 11.4 mi from Fletcher
Firestone Complete Auto Care
85 Tunnel RdAsheville, NC 28805
Audi repair 11.5 mi from Fletcher
Davidson Automotive
480 Buckeye Access RoadSwannanoa , NC 28778
Audi repair 12.2 mi from Fletcher
Pate's Automotive Services
284 New Leicester HwyAsheville, NC 28806
Audi repair 13.0 mi from Fletcher
Larry's Performance Werks
815 Merrimon AveAsheville, NC 28804
Audi repair 13.9 mi from Fletcher
Redmons Automotive
2368 Us 70 HwySwannanoa, NC 28778
Audi repair 14.4 mi from Fletcher
Bruce Berry Auto Repair
90 Underwood RdFletcher, NC 28732
Audi repair in Fletcher
Genuine Transmissions & Auto Service
5199 Old Haywood RdMills River, NC 28759
Audi repair 4.2 mi from Fletcher
First Gear Transmissions
920 7th Avenue EastHendersonville, NC 28792
Audi repair 7.9 mi from Fletcher
Precision Tune Auto Care
851 Spartanburg HighwayHendersonville, NC 28792
Audi repair 9.1 mi from Fletcher
Precision Tune Auto Care
6 Bryson StreetAsheville, NC 28803
Audi repair 10.0 mi from Fletcher
OGRE/Offroad Research Group
5750 Ashville HighwayPisgah Forest, NC 28768


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Brazil's Oi Said to Weigh Converting Part of Its Debt to Equityby
Company said to discuss idea with international bondholders
Final proposal including haircut seen completed this month
Oi SA, the most indebted phone operator in Brazil, is in talks with bondholders to convert a portion of its $17 billion in debt into equity, according to two people with knowledge of the matter.
The idea is being considered because the Rio de Janeiro-based carrier sees no other way to avoid default given its high level of leverage, the people said, asking not to be identified because the discussions are private. Bondholders represented by Moelis & Co. are weighing whether to accept the proposal, which could offer gains in the future if Oi succeeds in selling part of its assets to competitors, the people said. A final proposal, including the terms of the transaction, will be hammered out this month, the people said.
Oi's press office declined to comment.
Oi, whose name means "hi" in Portuguese, is the fourth-biggest mobile-phone operator in Brazil. It also operates part of the country's landline phone system, which has proven onerous -- the company has a legal commitment to expand and maintain the obsolete network. It had about 5 billion reais ($1.4 billion) of interest expenses in 2015, far more than the roughly 2.7 billion reais of operating income it had available to pay those costs, according to data compiled by Bloomberg.
The telecommunications carrier said last month it hired PJT Partners Inc. for advice on how to manage its debt, and the company announced an agreement April 25 to begin talks with the group represented by Moelis, aiming to quickly complete a restructuring process. Customers won't be affected by the restructuring process, the phone carrier has said.
Oi's total debt represented more than four times its equity as of the end of last year, compared with 47 percent for rival Tim Participacoes SA and 15 percent at Telefonica Brasil SA, according to data compiled by Bloomberg.


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Good news for anyone who's got an extra $30 million laying around! The last private residence at the former Stanhope Hotel is up for grabs and it features a staggering 14 rooms, 10-foot ceilings, and sweeping views of Central Park.
Originally designed by famed architect Rosario Candela, the Stanhope Hotel has since been remastered with all kinds of amenities including a gym and billiards room. Interior designer Felicia Zwebner headed the redesign and detailed:
My inspiration in designing this space came from the fact The Met is right across the street. In keeping with the building's history and architecture the design is a mix of classical and modern elements. The goal was to create a chic, sophisticated lifestyle that is worthy of large entertaining but still feels like a home.
Check out pics of the breathtaking residence from Sotheby's below:
The Morning Email helps you start your workday with everything you need to know: breaking news, entertainment and a dash of fun. Learn more


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by Chris Loomis
|Appropriate Response: More Questions Than Answers
by Chris Loomis
last updated November 28, 2001
So, just how far should security administrators go to protect their systems? What is an appropriate response to a detected security incident? Ask ten security professionals that question and you will most likely get ten different answers. Ask them more specific questions – such as, how do you handle active intrusions? Denial of service attacks? Probes? - and eventually you will be able to piece together their response set, a collection of reactions tailored to particular attacks or threats.
And once the "response" has been determined, how do we decide what makes a particular response "appropriate" or not? Who decides this? If you ask those same ten security professionals directly, each of them will consider their own responses to be, of course, appropriate. Given this, it should come as no surprise that there is no universally agreed upon set of appropriate responses within the security community. The discussion has been contentious - with two major camps emerging from the crowd - the first I will refer to as the Defenders, and the second, the Digilantes.
Defenders are guided by their organization's written security policy. As the name implies, their primary emphasis is on preventing breaches in the first place. If there is an intrusion, a Defender focuses on containing and eradicating the problem, plugging the security hole and getting back to business. While they may consider notifying the ISPs of attackers or perhaps calling in law enforcement, they will not engage in retaliatory actions directly against attackers for a variety of legal and ethical reasons.
Digilantes, or digital vigilantes, on the other hand, have no qualms about striking back against attackers. While they do everything they can to prevent successful attacks, in the event that one does occur they won't even think about reporting it to the authorities. Instead, they prefer to handle things "in house". To them, virtually all responses can be considered appropriate - as long as they don't get caught. Examples of a digilante response may include a denial of service attack, breaking into the attacking machine and neutralizing it, using a variety of techniques to trace an attack back to its point of origin and, in very extreme cases (which may in most cases just be cyber-folklore), visiting that physical location to have a "chat" with the suspected attacker.
Causes of the Schism
So, why the striking divergence of attitudes and approaches? Throughout history, vigilante behavior has emerged to fill a real or perceived inability of conventional law enforcement agencies to adequately and effectively enforce the law. Today this void is, unfortunately, quite real. The inability of law enforcement to successfully combat cybercrime is well documented. The U.S. General Accounting Office recently released a scathing report on the NIPC (National Infrastructure Protection Center), the government's lead cybercrime-fighting agency. While the GAO found too many deficiencies to mention, the general conclusion of the report was that the NIPC has "been impeded by the lack of a comprehensive, government-wide data collection framework for identifying imminent computer-based attacks. Further, the NIPC faces other barriers in issuing timely warnings, including a shortage of skilled staff, avoiding undue alarm for insignificant incidents, and ensuring that sensitive information is protected" (see "Significant Challenges in Developing National Capabilities".)
Governmental cybercrime-fighters won't find a lot of fans amongst the private sector security community either. A recent CIO Magazine poll of 450 chief information officers reveals that only six percent of them believe that existing law enforcement divisions (local and state police, FBI, Secret Service, etc.) are equipped to manage cybercrime (see "CIOs Say Law Enforcement Can't Hack It When It Comes To Hackers".)
Law enforcement is not entirely to blame for the sad state of cybersecurity. After all, they can only enforce the laws that are on the books. Because of our rapid transformation into an interconnected world, the established body of case law pertaining to cybercrime is still rather sparse. Judges are forced to try to apply outdated precedents to contemporary cases since lawmakers, discouraged by the sheer complexity of the issues, have been hesitant to jump into the fray and pass new laws. Instead, those lawmakers are content to stand back and do little, waiting (and waiting...) until the "dueling experts" are able to achieve something resembling consensus on the key issues.
From the point of view of law enforcement, part of the blame may also lie with the private sector itself. Law enforcement agencies need the cooperation and case referrals from the companies they are meant to protect. However, companies are reluctant to enlist the aid of law enforcement for several reasons: they lose control of the investigation, expose themselves to a potential public relations nightmare and, in the case of publicly traded companies, invoke the wrath of their shareholders. The bottom line is that the private sector needs to be able to trust law enforcement - but law enforcement needs to earn that trust. This may take some time.
Digilantes are tired of waiting. To them, we are years away from a legitimate cybersecurity effort. Law enforcement is simply out of its depth. Agencies are overworked, understaffed and underfunded. In addition, until the government can compete with the private sector in terms of compensation, they will not be able to attract the kind of expertise necessary in order to effectively combat cybercrime. Because of this, Digilantes feel compelled to take matters into their own hands. They contend that you can't reason with attackers and you can't coddle them - the only language they understand is force. Attackers search for weak victims. Digilantes feel that if you make it clear to them that you are a formidable foe, the attackers will look elsewhere. This approach can backfire, however, if an admin gets into a shooting match with an attacker who is determined to make it clear that he is the formidable foe.
One of the strongest criticisms of the Digilante approach is that innocent bystanders may get caught in the crossfire, stressing that, "when you strike back - you don't really know who you are hitting." However, Digilantes feel that on the Internet there is no such thing as innocent bystanders - just ignorant bystanders. Digilantes feel that they have a right to defend themselves from whoever is attacking them. If you are going to put a system on the Net without properly securing it and that system ends up being compromised and used in an attack, too bad if you get caught in the crossfire. You have a fundamental responsibility to ensure that your system isn't used to attack others.
Perhaps the biggest problem with digilantism is that it is usually illegal. I say usually because the security community has received mixed signals from law enforcement regarding what is and isn't acceptable - and what laws will and won't be enforced. Winn Schwartau, President of Interpact and founder of Web security service Infowar.com, points out an interesting dichotomy. "If you ask (a law enforcement officer) what the official position of their agency is concerning vigilante behavior, their response will be that they will investigate, pursue and prosecute it as they would any other crime". Off the record, however, they don't object to digilantism as long as you "make sure that you have the right guy, have deniability and don't put their agency in a situation where they have to enforce the official position". However, this should not be construed to mean that people who are caught committing illegal acts are somehow above the law: an illegal act is an illegal act, whether perpetrated by a criminal or in the name of vigilante justice.
How prevalent is Digilante behavior? This is difficult to gauge, considering that it's not something that an organization usually wants to discuss. There have been some studies done in an attempt to quantify the attitudes of security professionals (see Schwartau's book - Cybershock). Even so, it is difficult to discern if organizations are actually engaging in digilantism or are just talking tough in an effort to deter attacks. Regardless of what the numbers are, digilantism will continue to exist as a viable option for many in the security community until they can get some legal clarification, some ethical guidance and until law enforcement improves on its dismal record in cyberspace.
It's not that Defenders don't understand or experience the same frustrations that Digilantes do; it's just that they don't think that retaliation is a particularly effective approach - especially if we are looking for long-term solutions. There isn't any evidence that digilantism has any appreciable deterrent effect. Take out an attacker's zombies and he'll get more. Take out an attacker and he'll be back - and more determined. Computer security professionals have an obligation to uphold the highest ethical and legal standards - digilantism is neither ethical nor legal. Also, when you attack an attacker, you are assuming a risk for your organization that you are most likely not authorized to assume. Defenders repeatedly assert that they have yet to see one written security policy that advocates digilantism as a viable incident response option. (Perhaps this is an unfair argument - for it would obviously be foolish for an organization to put such a policy in writing.)
Defenders don't get mired down in ethical or legal grey areas; for them, the emphasis is on securing their systems and educating the users of those systems. This is a sound strategy regardless of where you fall along the appropriate response spectrum. With threats of legal action against those that fail to adequately protect their systems on the horizon, organizations need to commit the resources necessary to get their systems secured before claims of negligence and failure to perform due diligence start flying.
Defenders are not naïve, they are well aware that all is not well in the security field. Stephen Northcutt, Director of the SANS GIAC Training Program, understands the shortcomings of our current approaches. "Clearly we lost a lot of ground to attackers in 2001. One way that I can see for us to stem the tide is to apply deterrence. One possibility is to increase the number of civil suits - when you get attacked, sue the individual". Obviously, the major roadblock with this approach is determining the actual source of the attacks.
Circle Group Internet was able to do just that after discovering that an attacker had sent over four million pornographic spam e-mails from their systems. Their investigative team was able to track the offender back through several layers of ISPs and servers. Once they had successfully identified the culprit, they sued the attacker and were able to collect a monetary settlement. Not every organization is able to muster the resources necessary to pull off this type of investigation and civil action. An attractive alternative may be to contract out the investigation to a private computer security firm. Once all of the evidence is gathered and analyzed by the firm, the victim organization can decide what they want to do next.
Before any attacks occur, it is critical that an organization map out its response strategy. This includes defining the specific policies and procedures it will employ to counter any anticipated types of attack. However, they need to know their limitations. Tom Perrine, Security Manager for the San Diego Supercomputer Center, points out that organizations need to find a balance between going it alone and calling in the cavalry. "It is important to follow your own policies and do, at least, the beginnings of your own investigations. But in many cases, you will need to bring in law enforcement. Anything else is not being a good Net citizen."
Striking the Balance
So where do you stand? If you're like most people, you find yourself somewhere between the extremes of the Defender and the Digilante. There is no definitive answer – organizations will need to evaluate their particular situation and make their own decisions accordingly. For a moment, though, let's get back to basics. Whether you're talking about defensive actions or offensive actions, computer security is, at its heart, about managing risks. Failure to properly manage those risks may have adverse and unintended consequences.
I am optimistic that law enforcement efforts will eventually come around, but the security community shouldn't kid itself into believing that when that happens then our all of cybersecurity problems will be solved. While the Defender vs. Digilante debate is an important one; we must recognize that each approach is borne out of a much greater concern. Inadequate computer security is a systemic, multi-dimensional problem. Therefore, we need systemic, multi-dimensional answers (I know, easy for me to say).
While I admit that this article poses more questions than it delivers answers, its true intent is to generate discussion. But not just amongst security professionals. If we are serious about this, then we will need to get all of the players involved - industry leaders, software vendors, ISPs, law enforcement, government, etc. Then, and only then, will we be able to formulate and implement the kinds of solutions that truly address the root causes of our security problems.
This article originally appeared on SecurityFocus.com -- reproduction in whole or in part is not allowed without expressed written consent.


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Additional Property InformationThis condo listing located at 4859 Doral Drive, Ann Arbor, MI boasts three bedrooms, three bathrooms and one partial bathroom. The property is currently availible at $439,000.
This property is listed by Nancy Bishop of Charles Reinhart Company based in Michigan.


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Princeton Ultra 40 Driver Download
Princeton Ultra 40 Driver Details:
Princeton Ultra 40 File Name: princeton_ultra_40.zip
Princeton Ultra 40 Driver Version: 604Knxhi
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Driver Download Score68/100
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Published bySarah Roche Modified over 2 years ago
Coping in the subprime region Monetary Management in Crisis Daniela Gabor Department of Economics University of the West of England
The messages of this paper Central bank key in the reconstitution of the Romanian economy as neoliberal economy. What does the central bank do when neoliberal commitments to private finance under threat? – A: it depends. IMF presence changed approach to monetary management, re-assigning the National Bank of Romania (NBR) a central role in the reproduction of neoliberal logics.
Structure The subprime region A Romanian account of how Eastern Europe became subprime – Shifting neoliberal rationalities and modes of economic governance – The crisis of neoliberal capitalism : October 2008 The April 2009 IMF agreement: paradigm change? Conclusion
Features of the subprime region
Exchange rates correction? (2004 Jan = 100)
Foreign loans to the banking sector (% of total funds)
Romania before the 2008 crisis Standard narration of macroeconomic developments – 1990 - 1997: politicized economic decisions prevented implementation of stability orientated policies agreed with the IMF (4 failed SBA) Outcomes: high and volatile inflation, repeated balance of payment difficulties, massive transitional recessions – After 1997: gradual institutionalization of separate economic and political domains Outcome: after a necessary contractionary stabilization, Romania growth leader by 2005, when it adopts inflation targeting
Alternative narration: process of neoliberalization Shifting neoliberal rationalities (Peck and Tickell 2002; Hay 2004) – Roll-back neoliberalism - destructive attack on the Keynesian state during years of exceptional politics – Roll-out, normalized neoliberalism: constructive mode, financialized accumulation as economic imperative (Dumenil and Levy, 2001)
Neoliberalizing central banks international policy advice, IMF in the first instance, constructed central bank as essential instrument of institutional change and neoliberal policy making in formerly planned economies: MONETARISM as legitimizing policy narrative.
1990-1996: roll-back neoliberalism Central bank key to the disciplining of state owned enterprises, the antithesis of allocative efficiency – Contractionary monetary policy (eliminate excess liquidity and contain soft budget constraints) – Exchange rate flexibility: return to equilibrium key to competitiveness Policy practice vs. policy discourse – Liquidity crunches (typical outcome of IMF stabilization program) – Prohibitive loan rates affecting capital investment – Payment blockages – Industrial contraction – Exchange rate driven inflation (devaluations?)
1997 – 2008: Normalized neoliberalism 2 essential policy shifts (1997 IMF agreement) – Market financing of budget deficits – Exchange rate appreciation key to disinflation– sterilized forex interventions (reserve targeting) – structural excess of liquidity on the money market driven by capital inflows 1999 banking crisis: IMF private sector initiative + reduced appetite for emerging market + asymetric distribution of liquidity on the money market => reconfiguration of the banking sector.
Neoliberal regimes of monetary management (1)The transformation of the relationship between wholesale banking and the central bank, where management of liquidity tailors to speculative activity, working similarly under different policy regimes (2) An increasing reconfiguration of banking sector activity through neoliberal logics of financialized accumulation. (3) An increasing vulnerability to international sentiment, produced through practices of sterilized interventions, currency appreciations and expanding current account deficits in the context of a liberalized capital account, an outlook familiar to many Latin American countries (Massad, 1997).
(1) Management of liquidity: structural excess of liquidity on money market - sterilization operations as vehicle for speculative returns - policy models legitimating rather than informing policy practice (monetary vs. Inflation targeting) (Mosse, 2005)
(2) Banking sector under neoliberal regimes The changing configuration of credit, financed by loans from mother banks Credit bubbles and carry trade households (the Swiss connection)
(3) Increasing vulnerability: private debt - Strategies of monetary management altered the term composition of debt-creating inflows, shortening maturity and increasing exposure to short-term foreign currency liabilities. -banking sector financing credit expansion through mother bank loans and foreign owned businesses that preferred intra-company indebtedness. Private foreign debt doubled to 50% of GDP, reserve / short-term debt ratio fell from 170% in 2003 to 75% in February 2009. -Capital flows as prime driver of exchange rate movements. Capital account liberalization - heterogeneous trader community - carry trade activity.
The October 2008 speculative attack Standard and Poors downgrade EUR 2bn short positions opened, betting on depreciation – settlement in domestic currency (overnight or swap) NBR drained domestic liquidity: offshore players failed to close positions, swap rates > 500%, overnight rates> 50%. Liquidity provision through discount window, collateral. Policy controversy: adequate management of liquidity during crisis – Stabilizing overnight interest rates vs. feeding speculative pressures
Crisis responses -High policy rates to mitigate exchange rate pressures -Direct forex interventions However…. -Reserve losses yet domestic currency lost 20% in nominal terms btw. Nov. 2008 and February 2009 -Concerns about roll-over of short-term debts. -Potential currency crisis: IMF
Narrating the crisis: the IMF agreement Applauded NBR for attempts to contain imbalances, glossing over its strategy of real appreciations and strerilized interventions contributed to financial fragility 2 pillar approach – A strong policy program : fiscal contraction, improved bank supervision and regulation, continued exchange rate flexibility – Large external financing assistance : into NBR reserves to stimulate foreign currency lending (reducing reserve requirements on long term forex liabilities) New approach to Private Sector Involvement - convincing mother banks to roll over debt
Monetary management before the agreement: What vehicle for speculative returns if no sterilizations?
Monetary management after the agreement : plus ca change -Neoliberal quantitative easing: a guide for keeping banks happy (and deepen a recession)
Conclusions 1. Neoliberalism's strength - the discursive struggle over the definition of the crisis 2. new opportunities: crisis as an opening for further marketization - pension law, public sector law 3. We are not all Keynesians now!
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"I like this vintage style Santa graphic, but I read that the designer used a cutting machine to cut a red vinyl decal to afix to the cream painted wood board. I don't know what kind of cutting machine she is talking about and I wonder how pricey it is? I'd like an alternative to getting that Santa on there."
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I want to speak up on behalf of SS, since it seems to have drawn its
share of "doesn't work here" comments from Tenn. and NC. Here in NW
Iowa, NE Neb, at least in the several gardens where I've seen it,
including my own, SS is a stunner. It grows as it's supposed to,
stands up well despite those huge flowers, and increases moderately
well. The bloom stalks are as big and fat as any I've ever seen,
and they don't fall over. I've had it for at least 5 years, and I
would definitely not want to part with it.
Arnold Koekkoek
NW Iowa Zone 4 An all-day, all night snow storm has dumped close to
a foot of heavy, wet white stuff on us. Am I ever envious when I
read of some of you listening to robins chirping, crocus blooming.
It's going to be a long time before those things are true here.


text: Douglas Rushkoff joins the rank of French economist Thomas Piketty in expressing skepticism about free market capitalism in the digital age. Groundbreaking companies like Google, Amazon, and Uber operate using a "scorched Earth" method of value creation, says Rushkoff, which resembles 13th century colonialism. To make money, they extract value from communities rather than create it, much like conquistadors would extract precious metals from South American nations. If that sounds like a hyperbolic statement to you, you are probably not alone.


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Elf pets
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Elf on the shelf and reindeer pets play pin the nose on Rudolph! View #2 I downloaded a coloring page and then used the paint program on the computer to color it and make it bigger. Then I printed it on card stock and trimmed the edges a little, glued the picture to blue cardstock, and used my circle paper punch to get the noses.Here is the link to the picture I used. You can color it online too but I wanted it bigger that's why I downloaded it…
Elf Pets Reindeer | Like Looking In a Mirror | The Elf on the Shelf Ideas
If You Were a Reindeer, What Would Your Name Be? | The Elf on the Shelf
Have you ever wondered how Santa makes it around the world in one night? Or how he's able to carry all of those presents? He uses Christmas magic, which is made from Christmas cheer! To help him gather all the cheer he needs, Santa has given a special mission to his newest friend, the Elf Pets® Saint Bernard. Read More
New pet for the elf, need pet waste bags.
Elf arrives with pet reindeer. Picked this up after Christmas for next year...
What would your name be if you were an Elf Pets Saint Bernard? | Elf on the Shelf Ideas | Elf on the Shelf Names | Elf Pets | Saint Bernard
#ScoutElfIdeas for Elves with Reindeer Pets | Elf on the Shelf Ideas | Elf Pets Reindeer
Bringing in a new Elf Pet! Tom the elf rode in on a sleigh pulled by his new friend! Left behind some magic snow & got into the oats for the reindeer when they arrived!! Day 2 ~ 2015
Elf on the Shelf Pets: A St. Bernard Tradition Plush The…


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Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder, characterized by normal post-natal development followed by a sudden deceleration in brain growth with progressive loss of acquired motor and language skills, stereotypic hand movements and severe cognitive impairment. Mutations in the methyl-CpG-binding protein 2 (MECP2) cause more than 95% of classic cases. Recently, it has been shown that the loss of Mecp2 from glia negatively influences neurons in a non-cell-autonomous fashion, and that in Mecp2-null mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern and greatly prolonged lifespan compared with globally null mice. We now report that microtubule (MT)-dependent vesicle transport is altered in Mecp2-deficient astrocytes from newborn Mecp2-deficient mice compared with control wild-type littermates. Similar observation has been made in human MECP2 p.Arg294* iPSC-derived astrocytes. Importantly, administration of Epothilone D, a brain-penetrant MT-stabilizing natural product, was found to restore MT dynamics in Mecp2-deficient astrocytes and in MECP2 p.Arg294* iPSC-derived astrocytes in vitro. Finally, we report that relatively low weekly doses of Epothilone D also partially reversed the impaired exploratory behavior in Mecp2308/y male mice. These findings represent a first step toward the validation of an innovative treatment for RTT.
Rett syndrome (RTT, MIM 3127750) is a rare X-linked neurodevelopmental disorder, characterized by seemingly normal post-natal development until ∼6–18 months of age followed by a sudden deceleration in growth with progressive loss of acquired motor and language skills, stereotypic hand movements, major breathing irregularities, electroencephalogram abnormalities, autistic-like behaviors, increased anxiety-like behaviors, sleep disorders and severe cognitive impairment (1,2). Mutations in the methyl CpG binding protein 2 gene (MECP2) cause more than 95% of classic cases (3–5). MECP2 encodes a multifunctional protein that binds to methylated DNA and mainly acts as a key transcriptional regulator (2,4). How mutations in the MECP2 gene lead to the neurobehavioral features of RTT is still unknown and there is no specific cure for this devastating disorder.
Several lines of mice carrying Mecp2 disruption are now available. Shortly after the creation of Mecp2-null mice, a mouse expressing a truncated form of Mecp2 (Mecp2308) has been generated as this type of mutation is commonly found in classic RTT (6). Mecp2308 mice exhibit behavioral symptoms that recapitulate human RTT phenotype. Interestingly, male Mecp2308/y mice present RTT phenotype earlier than females and thus are predominantly used in RTT studies (6–9).
More recently, induced pluripotent stem cells (iPSC) from patient carrying common mutation in MECP2 have been described and characterized (10–19). RTT iPSC clonal lines and their derived cells stably maintained X-chromosome inactivation (XCI); therefore, the RTT iPSC clonal lines express either the wild-type or the mutant allele of MECP2 and can be studied as isogenic pairs (12,20). Methods to efficiently differentiate iPSC into neural progenitor cells, neurons and/or astrocytes have been reported and provide in vitro models of human RTT neural cells (21).
Earlier studies suggested the idea that RTT is due exclusively to the loss of Mecp2 function in neurons, because Mecp2 was exclusively present in neurons, based on immunohistochemical analyses. However, several subsequent studies showed that Mecp2 is clearly detected by immunostaining in all glial cell types including astrocytes and oligodendrocytes, based on co-staining for the cell-specific markers (GFAP, NG2 and myelin) (20,22–24). Using an in vitro co-culture system, it has been shown that mutant astrocytes from the knock-out RTT mouse model, and their conditioned medium, fail to support normal dendritic morphology of either wild-type or mutant hippocampal neurons (20). Although defective neurons clearly underlie the aberrant behaviors, these data suggested that the loss of Mecp2 from astrocytes negatively influences neurons in a non-cell-autonomous fashion (20). In Mecp2-null mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion, anxiety levels and respiratory abnormalities and greatly prolonged lifespan compared with globally null mice (24). These data showed that astrocytes, like neurons, are integral components of the neuropathology of RTT. However, only few reports have studied the pathogenic mechanism of RTT astrocytes.
Microtubules (MTs) play a crucial role in many cellular processes, such as cellular morphology, cellular division, migration and intracellular transportation of vesicles. MTs are highly dynamic tubular structures composed of α- and β-tubulin dimers, and their constant polymerization and depolymerization is tightly regulated in the cell. In assembled MTs, acetylation of the lysine-40 of the α-tubulin subunit is a post-translational modification that is often employed as a marker for stable MTs, although the relationship between tubulin acetylation and MT stability is not completely understood (25,26). Acetylation of tubulin is mediated by acetyltransferase, while deacetylation is mediated by histone deacetylase 6 (HDAC6) and sirtuin 2 (SIRT2). Recently, we have shown that skin fibroblasts from RTT patients with various mutations in MECP2 present an altered MT stability in response to cold-induced depolymerization and recovery (27). The link between MT dynamics impairment and Mecp2 deficiency has been reinforced by the observation of a reduced acetylation of polymerized tubulin and an elevated level of tubulin deacetylase HDAC6 in fibroblasts from RTT patients and in neurons from Mecp2-null mice (27,28). Interestingly, a reduced MT-dependent transport of vesicles containing BDNF in neurons from Mecp2-null animals has been reported, highlighting the potential perturbation of cellular functions by Mecp2 deficiency-related impairment of MT dynamics (29). We previously showed that astrocytes from Mecp2308/y and Mecp2-null mice have a dramatic increase in MT growth rate (30). However, tubulin acetylation, HDAC6 expression and MT-dependent vesicular transport had not been studied yet in astrocytes.
In this report, we show that astrocytes from Mecp2308/y mice and human astrocytes derived from iPSC from an RTT patient with an MECP2 p.Arg294* mutation have a reduced tubulin acetylation, an increase in HDAC6 level, an altered MT dynamics and an MT-dependent cellular dysfunction such as vesicular transport. We show further that MT dynamics impairment in mouse and human Mecp2-deficient astrocytes was counteracted in vitro by a treatment with an MT-stabilizing natural product, Epothilone D (EpoD). Finally, treatment with EpoD improved the exploratory behavior in Mecp2308/y hemizygous male mice. Overall, through this study, we have validated MTs as a target for therapeutic intervention in Rett syndrome, offering a new strategy for treating this disease by modifying stability of MTs by MT-stabilizing natural products.
Astrocytes from Mecp2308/y mice have a reduced level of acetylated-tubulin and an increased level of HDAC6
The levels of acetylated tubulin, total tubulin and HDAC6 proteins in total proteins extract from confluent cultures of Mecp2308/y and wild-type astrocytes were measured by western blot in three independent experiments. A total of six wild-type and six Mecp2308/y biological replicates were included in the analysis. Mecp2-deficient astrocytes showed a decreased level of acetylated tubulin (0.70× ± 0.10; P-value = 0.03) and an increased level of HDAC6 protein (1.73× ± 0.13; P-value = 0.02) when compared with wild-type astrocytes (Fig. 1A and B). Total tubulin level from Mecp2-deficient and wild-type astrocytes was similar (Fig. 1A). To determine whether HDAC6 is differentially expressed at the transcriptional level, we measured the mRNA expression by quantitative RT–PCR. HDAC6 mRNA level was not significantly different between Mecp2-deficient and wild-type astrocytes (Fig. 1C). Our data suggest that the elevated HDAC6 correlated with a decrease in acetylated-tubulin in Mecp2-deficient astrocytes.
MECP2 p.Arg294* iPSC-derived NPC and astrocytes present a reduced α-tubulin acetylation
To determine whether human astrocytes also presented a reduced α-tubulin acetylation, we examined human astrocytes from isogenic RTT iPSC lines. The iPSC isogenic lines were generated in Pr. Q. Chang Lab from a 11-year-old RTT girl carrying an MECP2 p.Arg294* mutation, a mutation found in ∼5–6% of RTT patients (12). Each line expressed exclusively MECP2 p.Arg294* mutant or wild-type MECP2 allele. The quality of iPSC lines was assessed by karyotyping and teratoma-based in vivo differentiation assay. The iPSC lines had normal karyotype and, after transplantation in vivo, gave rise to cells of the three germ layers (Supplementary Material, Fig. S1). From the two isogenic iPSC lines, we generated neuroepithelial cells with dorsal forebrain regional identity as described previously (31). No difference in the ability of the two isogenic lines to form neural tube-like rosettes was observed. The iPSC differentiation into neural subtypes was monitored by immunostaining for several known markers (Supplementary Material, Fig. S2A). As assessed by quantitative RT–PCR, at 8 weeks of differentiation, MECP2 p.Arg294* and wild-type astrocytes were expressing astrocyte markers at significant levels (Supplementary Material, Fig. S2B). Astrocytes used for further phenotypic characterization were then at 6–10 weeks of differentiation from iPSC. The XCI status and MECP2 allelic expression of astrocytes derived from the iPSC lines were tested to validate the maintenance of their isogenic status. After digestion with methylation-sensitive enzymes, a different androgen receptor hypermethylated allele was amplified in the two iPSC lines with a totally skewed X chromosome inactivation (Supplementary Material, Fig. S3). Moreover, sequencing of MECP2 allele-specific expression showed that iPSC and astrocytes of the same line expressed only the expected wild-type or mutated MECP2 p.Arg294* allele (Supplementary Material, Fig. S3). Human isogenic astrocytes expressing either exclusively the MECP2 p.Arg294* or the wild-type MECP2 allele were produced at the same time from iPSC being at the same passage. At least three independent differentiation experiments from separate iPSC passages were performed to provide the astrocytes tested for the following experiment replicates.
Total proteins were extracted from MECP2 p.Arg294*and wild-type NPCs and astrocytes pairs from four separate differentiations (6–10 weeks of differentiation). The levels of acetylated α-tubulin in MECP2 p.Arg294*cells were significantly lower than in the corresponding isogenic wild-type pair (0.70× ± 0.07; P-value = 0.04), whereas no difference in total α-tubulin level was observed (Fig. 1A). As we observed in Mecp2-deficient astrocytes from mice, a decreased level of acetylated-tubulin was then confirmed in the human RTT iPSC-derived astrocytes.
Astrocytes from Mecp2308/y mice present larger EB3 localization at the MT plus-end
To examine the possibility that Mecp2 plays a role in the stability of the cytoskeleton in astrocytes, we performed MT dynamics assay in astrocytes from Mecp2308/y mice and their control wild-type littermates. Cells were transfected by tracking 'comets' of MT-plus-end binding proteins EB3 fused to GFP (32). Our previous report showed a significant increase in MT polymerization speed in Mecp2-deficient astrocytes (30). Previous report showed that inhibition of HDAC6 activity in CHO cells has been associated with a decrease in MT growth velocity and a shorter binding site of proteins accumulating at growing MT tip (33). To determine if the elevated HDAC6 expression and the increased MT growth velocity in Mecp2308/y astrocytes were associated with accumulation of MT-tip proteins on growing MTs, we measured the size of EB3-comet and observed as postulated that EB3-labeled tails size was larger in Mecp2308/y astrocytes than in wild-type astrocytes (0.503 ± 0.018, n = 42; 0.580 ± 0.015, n = 38; P-value = 0.002) (Fig. 2) reinforcing the idea that MT stability is affected in Mecp2 deficiency.
MECP2 p.Arg294* iPSC-derived astrocytes present a high MT growth velocity and large EB3 MT-binding area
Taking account that cell shape is an essential factor governing MT organization and dynamics and to avoid the high variability in human astrocytes morphology, we choose to obtain a population of human astrocytes with homogeneous morphologies by using non-adherent poly-l-lysine-polyethylene glycol (PLL-PEG)-coated coverslips patterned with adherent 40 µm large fibronectin lines. Cell morphology was homogenized into polarized longitudinal cells (Fig. 3). Astrocytes were transfected with EB3-GFP construct for 24 h before EB3 tracking as previously described (32). Similarly to mouse Mecp2-deficient astrocytes, MECP2 p.Arg294* astrocytes showed a significantly higher MT growth velocity compared with isogenic wild-type astrocytes (0.285 ± 0.011, n = 28; 0.339 ± 0.008, n = 46; P-value <0.001) (Fig. 3). Moreover, the mean EB3 comet size was also significantly larger in MECP2 p.Arg294* astrocytes than in the wild-type human astrocytes (0.448 ± 0.022, n = 28; 0.697 ± 0.038, n = 46; P-value <0.0001) (Fig. 3). These data show that MT stability is affected by Mecp2 deficiency both in mouse and human astrocytes.
Astrocytes from mouse Mecp2308/y and human MECP2 p.Arg294* iPSC-derived astrocytes present an altered directionality of vesicle transport.
To determine if MT-dependent vesicle transport was affected by MT dynamics defect in mouse Mecp2308/y and human MECP2 p.Arg294* astrocytes, and as lysosome vesicles are transported along MTs, we measured the fraction of highly directional versus highly non-directional (ND) mobile lysosome vesicles as described previously (34). Astrocytes cultures were incubated with Lysotracker® and Lysotracker-positive lysosome vesicles were tracked during 2 min movies with a picture acquisition every 2 s. As previously described, vesicles were classified as mobile vesicles by the criteria of a track length over 1 µm (35). Lysotracker-positive mobile vesicles displayed complex motion patterns composed of forward and backward movements interrupted by periods of arrested mobility. We distinguished vesicles moving in a highly directional fashion and vesicles moving in a highly ND fashion by taking account of the ratio of the displacement, meaning the distance between the first and the last spot position, over the total track length of each mobile vesicle. Vesicles with a displacement under 20% of the total track length were classified as highly ND. Around 100–200 vesicles were tracked per cell. Interestingly, Mecp2 deficiency in astrocytes from both models is associated with a slight but significant and reproducible increase in vesicles moving in a highly ND fashion when compared with wild-type astrocytes [mouse astrocytes: 46.93% ± 2.18, n = 55; 55.59% ± 3.67, n = 64; P-value 0.04 (Fig. 4A); human astrocytes: 37.96% ± 1.92, n = 57; 46.70% ± 1.86, n = 74; P-value = 0.001 (Fig. 4B)]. Our data showed for the first time that MT-dependent vesicle mobility is altered in Mecp2-deficient astrocytes.
Epothilone D treatment of mouse Mecp2308/y and human MECP2 p.Arg294* iPSC-derived astrocytes rescues MT growth velocity and EB3-comet size
To investigate the ability of Epothilone D (EpoD) (36), an MT-stabilizing natural product, to correct the impaired cellular phenotype observed on RTT astrocytes, mouse and human MECP2-deficient astrocytes and wild-type astrocytes cultures were incubated with EpoD at a concentration of 10 nm or with vehicle before performing EB3-GFP. Mecp2308/y and wild-type astrocytes treated with EpoD presented a reduced EB3-comet speed (0.213 ± 0.006, n = 42; 0.188 ± 0.008, n = 15; P-value = 0.02; 0.244 ± 0.007, n = 38; 0.194 ± 0.008, n = 27; P-value < 0.001) and size (0.580 ± 0.015, n = 38; 0.505 ± 0.017, n = 27; P-value = 0.002) (Fig. 2). Similarly, the effect of EpoD treatment on human wild-type and MECP2 p.Arg294*isogenic astrocytes was a reduction in EB3-GFP velocity (0.285 ± 0.011, n = 28; 0.220 ± 0.005, n = 8; P-value = 0.02; 0.339 ± 0.008, n = 46; 0.279 ± 0.009, n = 36; P-value = 0.0004) and EB3-GFP comet size (0.448 ± 0.022, n = 28; 0.337 ± 0.016, n = 8; P-value = 0.0002; 0.697 ± 0.038, n = 46; 0.502 ± 0.029, n = 36; P-value = 0.0003) (Fig. 3). Moreover, the growth velocity of MTs (mouse astrocytes: 0.213 ± 0.006, n = 42; 0.188 ± 0.008, n = 27; P-value = 0.119; human astrocytes: 0.285 ± 0.011, n = 28; 0.279 ± 0.009, n = 36; P-value = 0.69) and EB3-comet size (mouse astrocytes: 0.502 ± 0.021, n = 42; 0.505 ± 0.017, n = 27; P-value = 0.93; human astrocytes: 0.448 ± 0.022, n = 28; 0.502 ± 0.029, n = 36; P-value = 0.14) were not significantly different between Mecp2-deficient astrocytes treated with EpoD and wild-type astrocytes treated with vehicle (Figs. 2 and 3).
Epothilone D treatment of mouse Mecp2308/y astrocytes rescues vesicle directionality
Mouse Mecp2308/y astrocytes were treated with 10 nm EpoD for 1 h before performing lysosomes tracking. Mecp2308/y and wild-type astrocytes treated with EpoD presented a reduced percentage of vesicles moving in a highly ND fashion (46.93% ± 2.18, n = 55; 39.07% ± 3.57, n = 15; P-value = 0.04; 55.59% ± 3.67, n = 64; 46.04% ± 3.14, n = 17; P-value = 0.04) in comparison with Mecp2308/y and wild-type astrocytes treated with vehicle, respectively. Vesicle directionality (46.93% ± 2.18, n = 55; 46.04% ± 3.14, n = 17; P-value = 0.81) was not significantly different between Mecp2-deficient astrocytes treated with EpoD and wild-type astrocytes treated with vehicle (Fig. 4). Thus, EpoD is able to rescue the vesicle directionality in Mecp2-deficient astrocytes in vitro (Fig. 4). These data also show that acting on MT dynamics results in a modulation of lysosome vesicle directionality, reinforcing the idea that impairment of MT dynamics leads to vesicular trafficking alteration in RTT.
Epothilone D corrects exploratory behavioral symptoms of Mecp2308/y male mice
Prior pharmacokinetic studies revealed that EpoD has significantly longer resistance time in the brain than in plasma of mice (36), the compound being detectable in brain for at least 1 week after a single 3 mg/kg administration. Moreover, a measurable pharmacodynamics effect was apparent 1 week after a single 3 mg/kg dose of EpoD to wild-type mice, as evidenced by increased levels of acetylated-tubulin (37). In a previous behavioral study, it has been clearly demonstrated that Mecp2308/y mice displayed decreased digging and buried a lower number of marbles compared with wild-type littermates in the marble-burying test (9). Here, based on this study and our in vitro data, we investigated EpoD effects on exploratory behavior in an RTT mouse model. Five-weeks-old Mecp2308/y mice were treated by weekly intraperitoneal injection of EpoD (1 mg/kg/week). To evaluate the effects of EpoD on exploratory behavior, the marble-burying test was carried out on a 12-week treatment schedule. As expected, Mecp2308/y mice buried a lower proportion of 2/3 covered marbles compared with wild-type littermates (26.20% ± 4.95, n = 7; 12.49% ± 2.87, n = 9; P-value = 0.02), indicating decreased environment-directed exploratory behavior (Fig. 5). Proportion of 2/3 covered marbles buried by the EpoD-treated Mecp2308/y mice was not significantly different from that of wild-type mice (26.20% ± 4.95, n = 7; 17.93% ± 3.11, n = 10; P-value = 0.16), thus suggesting that treatment of Mecp2308/y mice with EpoD restored the impaired exploratory behavior (Fig. 5).
MTs are highly dynamics structures and the regulation of MTs dynamics is crucial for cellular functions such as migration, division, polarity and differentiation. The MTs plus-ends display what is called MT instability, which is the succession of slow polymerization and fast depolymerization events, essential for MTs to explore the cell. MTs are polymers of basic α/β-tubulin dimer building units. It is not clear how the functional diversity of MTs is created. One growing hypothesis is that post-translational modifications of tubulin regulate MT functions. Among them, the acetylation of α-tubulin at lysine 40 is found on stable MTs in most cell type. Although the relationship between tubulin acetylation and MT stability is not completely understood, acetylated-tubulin is often considered as a marker for stable MTs. However, this post-translational modification has been reported to play a positive role in motor-based trafficking (27,38–41). Two enzymes have been shown to deacetylate α-tubulin in vitro and in vivo, HDAC6 and SIRT2 (42,43). Based on our and other previous works reporting that astrocytes, like neurons, are integral components of the neuropathology of RTT, we focus our study on astrocytes (17,20,22–24). We have shown for the first time that Mecp2308/y astrocytes display a low level of tubulin acetylation and overexpress HDAC6 protein. This HDAC6 overexpression was not associated with an increase in HDAC6 mRNA transcript, suggesting that Mecp2 may modulate HDAC6 synthesis and/or degradation at the post-transcriptional level. It is interesting to note that similar observations were recently obtained in Mecp2-deficient fibroblasts and Mecp2-null neurons (28). Notably, HDAC6 modulation was shown to modulate some aspects of MT dynamics, such as MT growth velocity, but not the frequency of catastrophe (transition from growth/pause to shortening) and rescue (transition from shortening to growth/pause) events, or the time spent in growing, shortening and pausing (33). We thus focused on this parameter of MT dynamics and observed that Mecp2-deficient astrocytes display increased MT growth velocity. Moreover, HDAC6 activity has been shown to modulate the localization of the plus-end-tracking protein EB1 and Arp1 at the MTs tip (33). We have shown that EB3-GFP comet tail was larger in the Mecp2-deficient astrocytes, which is likely associated with HDAC6 protein overexpression.
Potokar et al. (34,44) have demonstrated the contribution of MTs to directional vesicle mobility in astrocytes. In line with those studies, we showed that altered MT dynamics in Mecp2-deficient astrocytes from Mecp2308/y mice affects the directional mobility of vesicles. Indeed, distinct types of vesicle mobility have been described in astrocytes, consistent with other cell types: a slow and disorganized diffusion transport and an MT-dependent fast and straight trafficking (34). Vesicles were characterized as highly directional when vesicle tracks displaying a straight line and highly ND when vesicle tracks displaying a contorted line. Interestingly, we observed that the percentage of highly directional vesicles is reduced in Mecp2308/y astrocytes compared with wild-type astrocytes. An efficient vesicle cellular transport is necessary for cell function and vesicle exocytosis. As exocytosis from astrocytes is essential in astrocyte-to-neuron communication, our results suggest that altered MT dynamics and vesicle trafficking in Mecp2-deficient astrocytes may lead to an impaired astrocyte secretion that may contribute to the neuronal maturation defect observed in Mecp2-deficient brains. Previous reports have also shown that Mecp2 deficiency impairs axonal transport of amyloid precursor protein without affecting its mRNA and protein levels, and that Mecp2 knockout neurons have fewer fast-moving mitochondria than wild-type neurons (29,45). Those data together suggest that Mecp2 deficiency affects the global MT-dependent transport.
Interestingly, we found in the human MECP2 p.Arg294* isogenic iPSC-derived astrocytes a phenotype similar to the one found in Mecp2-deficient mouse primary culture. It will be of interest to study human astrocytes derived from more RTT patients, with different mutations in MECP2 gene. However, our results highlight the value of iPSC-derived cells as a good model to study Rett syndrome physiology and validate that MT dynamics may be affected in RTT patient cerebral cells and could be a putative target for RTT therapy.
Tubulin acetylation and MT dynamics alteration in Mecp2-deficient cells have been highlighted by our study and others in cerebral cells from three different Mecp2-deficient mice models and in human cells from male and female RTT patients with different mutations in MECP2 genes, providing a robust Mecp2 deficiency-linked cellular phenotype. However, the links between Mecp2 deficiency with impaired tubulin acetylation and increased MT instability have to be further studied to better understand the cause–effect relationships between those cellular events. We suggest that altered MT dynamics leads to an impairment of cellular functions in astrocytes, which may participate in the pathogenic state of Mecp2-deficient astrocytes and contribute to an altered neuronal maturation.
Treatments of 3T3 cells with HDAC6 inhibitor and MT stabilizer both increase the MTs polymer content and the acetylation level of the MT network, and slow down the drug-induced depolymerization of MTs (42). We show in this report that EpoD, a well-characterized MT stabilizer, can restore both MT dynamics and mobile vesicle directionality in Mecp2-deficient astrocytes at a very low concentration. This result indicates that alterations in MT dynamics and mobile vesicle directionality in Mecp2-deficient astrocytes are linked and such cellular phenotypes can be corrected with a regulator of MT dynamics. As a result, MTs comprise a potentially promising target for RTT therapy.
De Filippis et al. (9) recently described that Mecp2308/y mice had a decreased marble-burying behavior that may reflect an impairment of the environment-directed exploratory behavior. This phenomenon was accompanied by increased anxiety and by the absence of spontaneous stereotypic behavior. The performance of RTT mice in the marble-burying test was restored in this study by a selective brain-penetrant agonist (LP-211) treatment, suggesting the reversibility of this Mecp2308-associated phenotype (9). Consistent with these observations, when we employed the marble-burying test, we also noted that Mecp2308/y mice harbored an altered exploratory behavior. Taking account our in vitro results, we studied the effect of an MT-stabilizing molecule in vivo in Mecp2308/y mice. Because paclitaxel, a drug acting against MT depolymerization through its binding to β-tubulin, is not suitable for human neurodevelopmental disorders because of poor blood–brain barrier (BBB) permeability, we evaluated the effects of a brain-penetrant MT-stabilizing natural product, EpoD. Moreover, EpoD has been shown to improve behavior in a mouse model for schizophrenia and Alzheimer-like pathology (46,47). Because RTT patients can only be diagnosed after the beginning of the regression period (9–18 months), we started the mouse treatment on early symptomatic Mecp2308/y mice (1 month old). Here, we show that EpoD treatment improved the RTT mice exploratory behavior in the marble-burying test. Our data provide the first evidence that a regulator of MT dynamics might have a beneficial effect, opening new possibilities for the development of novel pharmacological approaches for the treatment of Rett syndrome. However, MT-stabilizing agents are known to induce side effects when used at the doses required for the treatment of cancer. Hence, it is important to note that the aged mice in this study received EpoD doses that were 30- and 100-fold less, on a milligrams per square meter basis, than was previously used in human testing (48). Thus, the substantially lower EpoD doses used here may avoid the adverse complications associated with MT-stabilizing drugs, such as immune cell depletion and the onset of peripheral neuropathy (49). In the present study, the Mecp2308/y mice receiving EpoD had body weights at the end of the dosing period that were indistinguishable from vehicle-treated wild-type mice, indicating an absence of gross toxicities. Therefore, it appears that the low doses of EpoD used in this study, which were sufficient to improve efficacy outcomes, elicited none of the adverse events that are observed when higher doses of MT-stabilizing agents are used for the treatment of cancer. In conclusion, through this study, we have validated MTs as a target for therapeutic intervention in Rett syndrome, offering a new strategy for treating this disease by modifying stability of MTs by BBB-permeable MT dynamics regulators.
Materials and Methods
The experimental subjects were 5 weeks of age hemizygous Mecp2308/y male mice and wild-type littermates (B6.129S-MeCP2<tm1Heto>/J, stock number: 005439; backcrossed to C57BL/6J mice for at least 12 generations from the Jackson Laboratories, USA) (6). All procedures were carried out in accordance with the new European Communities Council Directive (2010/063 EU) and the implementing French decree of application no 2013-118 01 and its supporting annexes entered into legislation 1 February 2013. All procedures were formally approved by local ethical committees from Paris Descartes University and the Com'Eth (no 17) for the laboratories in Illkirch.
Genotyping was performed by routine PCR technique according to The Jackson Laboratory protocols. The gender was determined using primers for the Sry gene on Y chromosome, which were 5′-CTC ATC GGA GGG CTA AAG TG -3′ and 5′-AAG CTT TGC TGG TTT TTG GA-3′. Genotype was determined using primers for the Mecp2 gene, which were 5′-AAC GGG GTA GAA AGC CTG-3′, 5′-TGA TGG GGT CCT CAG AGC-3′ and 5′-ATG CTC CAG ACT GCC TTG-3′ for Mecp2308/y mice.
Preparation of EpoD
EpoD was prepared as previously described (50,51). The spectroscopic properties of EpoD were identical to those reported in the literature. Compound purity was >95% as demonstrated by liquid chromatography-mass spectrometry. The compound was dissolved in vehicle solution, i.e. 1% dimethyl sulfoxide (DMSO) in saline.
Cortical mouse astrocyte cultures
Primary astrocyte cultures were prepared from 1-day-old wild-type and Mecp2308/y mice, male cerebral cortex according to previously described methods (52). Briefly, after removal of the meninges, forebrains were rinsed and gently dissociated by trituration following 30 min incubation with trypsin 0.05% (Invitrogen, Cergy-Pontoise, France). Cells were collected by centrifugation at 300g for 3 min, resuspended in DMEM supplemented with 10% heat-inactivated FBS, plated in micro-dishes designed for live imaging (µ-Dish 35 mm; Ibidi, München, Germany) and incubated at 37°C in a humidified atmosphere of 95% air, 5% CO2. The purity of the cells, as determined by glial fibrillary acidic protein (GFAP) immunohistochemical staining was >99% (Supplementary Material, Fig. S4). Each experiment was performed with astrocytes primary culture from littermate newborn pups and each litter contained at least two Mecp2308/y and two wild-type pups.
Human astrocytes production from iPSC
Maintenance of iPSC cultures
iPSC lines from MECP2 p.Arg294* RTT patient used in this study have been previously described (12). iPSC were amplified with mTeSR1® medium (StemCell Technologies, Grenoble, France) on matrigel (BD Biosciences, Franklin Lakes, NJ, USA) coated dish and passaged with collagenase IV (StemCell Technologies). iPSC were used for astrocyte differentiation at passage ∼P30–40.
Astrocyte differentiation
Astrocytes with a dorsal regionalization were produced by iPSC differentiation as previously described (53). Briefly, embryoid bodies were generated from iPSC and neuroectoderm induced by inhibition of BMP4 signaling pathway. Columnar neuroepithelial cells formed typical neural tube-like rosettes. Rosettes were dissociated into monolayer of neural progenitor cells with Neurobasal A medium supplemented with 1×N2, 1× B27 without vitamin A, 1×GlutaMAX, 1× PenStrep (LifeTechnologies, Cergy-Pontoise, France) and 10 ng/ml of FGF-2 (Miltenyi Biotech, Paris, France) and EGF (LifeTechnologies). At day 15 of differentiation, highly proliferating neural progenitor cells were switched to astrogenesis by medium supplementation by human recombinant CNTF (BioTechne, Abingdon, UK) and deprivation of FGF-2 and EGF for 2 weeks, then CNTF supplementation was stopped and cells were maintained in culture for months. Most of the experiments were performed on astrocytes at 6–10 weeks of differentiation.
Human androgen receptor assay
XCI status was investigated by sizing the polymorphic CAG trinucleotide repeat on the inactive androgen receptor allele as previously described (54).
Standard G-banding chromosome analysis was performed in Laboratory of Cytogenetics, GHU Cochin-Broca-Hôtel Dieu, Paris, France.
Teratoma assay
Teratoma formation was assessed by injecting hiPSCs into immunodeficient mice as previously described (55).
Allele-specific transcription of MECP2
Total RNA from the iPSC lines and human astrocytes cultures were converted to cDNA with Maxima First Strand cDNA synthesis Kit (ThermoScientific, Rochester, NY, USA). Region containing the mutation was amplified by PCR amplification and sequenced with an ABI 3100® (Applied Biosystems, Courtaboeuf, France). Primer sequences were 5′-ACC GGG GAC CCA TGT ATG AT-3′ and 5′-ATG TCT TTG CGC TCT CCC TC-3′.
Quantitative RT-PCR
Total RNA from the human astrocytes cultures were converted to cDNA with Maxima First Strand cDNA synthesis Kit (ThermoScientific) for quantitative real-time PCR with SYBR Green as detection agent with the LightCycler480 detection system (Roche Applied Bioscience, Indianapolis, IN, USA). Primer sequences are described in Supplementary Material, Table S1. After PCR amplification, a dissociation protocol was performed to determine the melting curve of the PCR product. Reactions with melting curves indicating a single amplification product were considered positive for further analysis. The identity and expected size of the single PCR product were also confirmed by agarose gel electrophoresis. Normalization factor was based on housekeeping gene Gapdh.
Western blot
The levels of HDAC6, α-tubulin and acetylated α-tubulin protein expression were determined by western blot analysis. Briefly, proteins were extracted from cell cultures in RIPA buffer supplemented with protease inhibitors (Sigma-Aldrich). The protein samples were separated by a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to Hybond-C nitrocellulose membrane (GE Healthcare, Piscataway, NJ, USA). After blocking non-specific sites by a TBS-Tween 20 0.05%/5% fat milk solution for 1 h at room temperature, the membrane was incubated overnight at 4°C with a mouse anti-acetylated-tubulin monoclonal antibody (clone 6-11B-1, SIGMA T6793), mouse anti α-tubulin monoclonal antibody (clone DM1A, SIGMA T9026) and goat anti-HDAC6 polyclonal antibody (Santa Cruz Biotechnology sc-5258), washed and incubated with horseradish peroxidase-conjugated secondary antibodies (anti-mouse or anti-goat), and developed with the enhanced chemiluminescence method (ECL; GE Healthcare). We also probed membranes for GAPDH (antibody anti-Gapdh Ambion, dilution 1:1000) for loading normalization. Quantification of protein levels was measured by the chemiluminescence intensity.
Cells were washed twice with PBS and fixed with paraformaldehyde 4% during 15 min. Cells were incubated in 0.1% Triton and 3% bovine serum albumin solution for 1 h and then incubated with goat anti-GFAP (Santa Cruz Biotechnology, sc-6170), mouse anti-OCT4 (clone C-10, Santa Cruz Biotechnology, sc-5279), rabbit anti-PAX6 (Covance, PRB-278P-100) or mouse anti-S100β (Sigma Aldrich, s2532) antibody overnight. After primary antibody washing, cells were incubated 1 h, respectively, with appropriate chromophore-conjugated secondary antibodies (FITC or CY3, produced in donkey, Jackson Immuno Research) and then mounted with Fluoromount mounting medium with DAPI (Sigma Aldrich, St Louis, MO, USA) and analyzed with a Leica DMRA2 fluorescence microscope.
Adherence proteins micro-patterning
A 10× stock solution of poly-l-lysine-poly-ethylene-glycol (PLL-PEG) was prepared dissolving 1 mg/ml of PLL-g-PEG (PLL(20)-[3.5]-PEG(2), SuSoS) in 10 mm HEPES buffer, pH = 7.4, stored at −20°C. By diluting aliquots of the stock solution in the same buffer, 1× solution of PLL-PEG (100 µg/ml) was obtained. Adherent proteins solution was composed of 50 µg/ml of fibronectin (F1141, Sigma-Aldrich) and 50 µg/ml of fibrinogen-Alexa647 (F35200, LifeTechnologies) in 10 mm NaHCO3 buffer, pH 8.3.
A 35 mm glass bottom µ-Dish (81158, Ibidi) was treated with oxygen plasma to clean and activate the glass surface, and turn it hydrophilic. Then a 250 µm thick PDMS spacer cut with a plotter-cutter (Craft ROBOProS, Graphtec) was stuck on the glass surface to define four wells of 4 mm diameter. Each well was incubated with 20 µl of a 1× PLL-PEG solution for 1 h to coat the glass surface. After rinsing with PBS 1×, we achieved the micro-patterning of a mix of fibronectin and fibrinogen-Alexa647 on the surface in each well, with a device developed by ALVEOLE coupling an optical system to a chemical product (Primo™ PLPP™, ALVEOLE, Paris, France). We therefore get horizontal lines of 40 µm in width vertically spaced by 220 µm.
Cell seeding
Human astrocytes (6–10 weeks differentiation from iPSC) were seeded on extemporaneously patterned wells at a concentration of 3000 cells into 10 µl of culture medium. After 1 h, wells were rinsed three times with culture medium to remove non-attached cells and dish was filled with 2 ml of culture medium.
Live cells measurement of MT dynamics
The EB3-GFP construct (generously provided by F. Niedergang, Institut Cochin, Paris, France) has been previously described (32). Briefly, gene-specific primers were designed for cloning of EB3 cDNA, with restriction sites for in-frame cloning after PCR amplification into pEGFP-N1 (Clontech, Palo Alto, CA, USA). The expression of this vector is driven by CMV promoter.
Transfection of mouse and human astrocytes
Two weeks in vitro confluent mouse astrocyte cultures and 6–10 weeks iPSC-derived human astrocytes were transfected using Lipofectamine LTX (Invitrogen) with EB3-GFP vector. Transfected cells were cultured for 24 h prior to imaging.
Live cell imaging and analysis
Prior to imaging, cells were transferred to video medium (DMEM: F12 without phenol red supplemented with 0.3% glucose, 1× N2, 1× B27 and 1 mm pyruvic acid). Cells were allowed to adapt to the new medium in ambient air for at least 30 min at 37°C. Image acquisition and image analysis were performed on the Cochin Imaging Facility. Image acquisition was done using a Zeiss Axiovert 200 M microscope with oil immersion objective 63×/NA 1.4 in a temperature-controlled CO2-incubation system. The acquisition sequence was 1 min long, with scans performed every 1 s. Stacks were built using Meta Morph v7.5 (Molecular Devices, Silicon Valley, CA, USA) and analyzed using the IMARIS software (Bitplane, South Windsor, CT, USA). About 100 comets were tracked in each cell, moving straightforward, radially from the centrosome region to the periphery. Instant velocities of comets were measured between each time point and further averaged over their time courses. Independent experiments were repeated at least three times, a total of 8–46 cells were analyzed for each group.
Live cell tracking of lysosome vesicles
Fluorescent staining and confocal microscopy
Acidic vesicles in 2 weeks in vitro confluent mouse astrocytes and 6–10 weeks iPSC-derived human astrocytes culture were loaded with 200 nm Lysotracker Red DND-99 (Invitrogen, Lieden, The Netherlands) in culture medium at 37°C for 1 and 24 h, respectively. Prior to imaging, cells were transferred to video medium. Cells were allowed to adapt to the new medium in ambient air for at least 30 min at 37°C. Image acquisition and image analysis were performed on the Cochin Imaging Facility. Live stained cells were imaged with an inverted confocal microscope (Zeiss Axiovert 200 M) using oil immersion objective 63×/NA 1.4 Lysotracker Red DND-99 (Molecular Probes, Life Technologies). Time-lapse confocal images were acquired every 2 s for 2 min. Stacks were built using Meta Morph v7.5 (Molecular Devices) and analyzed using the IMARIS software (Bitplane). About 100–200 vesicles were tracked in each cell. Independent experiments were repeated at least three times, a total of 15–74 cells were analyzed for each group.
Vesicle tracking and data analysis
Vesicle mobility was analyzed by IMARIS software (Bitplane) to obtain several mobility parameters (34). Briefly, a 2D Gaussian curve was fitted on the selected vesicles in each image within the time series, and the x- and y-coordinates of the peaks of the curves were connected to acquire tracks of vesicle mobility (trajectories). The current time (time from the beginning of single vesicle tracking), displacement length (distance between the first and last spot position) and track length (TL, the total length of the analyzed vesicle pathway) were determined and the ratio of the displacement, meaning the distance between the first and the last spot position, over the total track length of each mobile vesicle were measured to analyze vesicle directionality.
EpoD or vehicle treatment of Mecp2308/y and wild-type mice
Groups (n = 7–10) of 5-week-old male Mecp308/y mice or 5-week-old wild-type littermates were administered weekly intraperitoneal injections of 1 mg/kg EpoD, or vehicle (DMSO), for a total of 12 consecutive weeks. Experiments have been performed according to guidelines concerning randomization and blinding (56). Treatment continued throughout behavioral testing. We chose to focus on male Mecp2308/y mice because of the unpredictability of heterozygous females' phenotype deriving from the XCI phenomena and because male mice develop RTT pathology more consistently than females. Indeed, hemizygote male mice allow eliminating the confounding effects of a variable phenotype while investigating the role of Mecp2 gene mutations on the development of RTT-like symptoms. Animals were monitored for signs of abnormal behavior or distress, and were weighed weekly. After 12 weeks of dosing, the mice underwent behavioral testing as described below. To unravel the effects of EpoD treatment on exploratory behavior, mice were tested in the marble-burying test.
Marble-burying test
The mouse Tecniplast cages were filled with ∼5 cm of sawdust bedding, lightly tamped down to make a flat, even surface over which nine marbles were evenly distributed. At the end of a 10 min session during which mice were allowed to freely explore the cage, the number of buried marbles (at least 2/3 of their depth) was counted. The test was carried out under dim lights.
Data analysis
Parametric analyses of variance (ANOVA) were performed on all mice behavior data. Specifically, ANOVA model included genotype as between-subject factor and one or two repeated measures factors. Post hoc comparisons were performed using Tukey's test, which can be used in the absence of significant ANOVA results. The Mann–Whitney U-test was applied on latency data that had no normal distribution. Molecular data were analyzed by Student's t-test for quantitative data. A P-value of <0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
Supplementary Material
Supplementary Material is available at HMG online.
This work was supported by the Fondation Jerome Lejeune, Labex 'Who I am?', the Université Paris Descartes and the Association Française du Syndrome de Rett (AFSR, 2005-01, RAK15121KKA). This work was supported by the EU-funded project 'Genetic and Epigenetic Networks in Cognitive Dysfunction' (GENCODYS) consortium (http://www.gencodys.eu/) (FP7-COLLABORATION PROJECT-2009-188.8.131.52/241995 to T.B. and Y.H.), and the French state funds through the 'Agence Nationale de la Recherche' under the frame programme Investissements d'Avenir labelled (grant number ANR-10-IDEX-0002-02, ANR-10-LABX-0030-INRT, ANR-10-INBS-07 PHENOMIN to Y.H.) and the National Institute of Health (grant number U01 AG029213-01A2 to A.B.S). The funders had no role in the study design, data collection and analysis, decision to publish or manuscript preparation.
We thank Olivier Feraud, Matthias Groszer, Alfredo Cabrera-Socorro and Emmanuelle Massourides for advice on iPSC cultures and neural differentiation. A.D. is employed by ALVEOLE.
Conflict of Interest statement: None declared.


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Leon Schuster net worth: Leon Schuster is a South African filmmaker, actor, comedian, and singer who has a net worth of $3 million. Leon Schuster was born in Vereeniging, South Africa in 1951. He graduated from the University of the Orange Free State and then worked as a teacher for two years. Schuster started working for the South African Broadcasting Company and created an Afrikaans radio series. Schuster has released many albums over the years including Leon Schuster, Rugby, You Must Be Joking!, and Catchup Song And Every Cricket Hit. He has been featured on screen in You Must Be Joking!, You Must Be Joking! Too!, Oh Schucks…It's Schuster!, Oh Shucks! Here Comes UNTAG, Sweet 'n Short, Yankee Zulu, Panic Mechanic, Millennium Menace, Mr. Bones, Oh Schuks… I'm Gatvol, Mama Jack, Mr. Bones 2: Back from the Past, Schuks Tshabalala's Survival Guide to South Africa, Mad Buddies, Schuks! Your Country Needs You, and Schuks! Pay Back The Money.
Leon Schuster Net Worth
Leon Schuster
Date of Birth
May 21, 1951 (65 years old)
Screenwriter, Actor, Comedian, Singer
South Africa
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