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Archive for the 'Barack Obama' Category
Is It Time to Get Rid of the Constitution?
By Spartacus Thrace
As 2013 dawns across America, the Progressive Movement appears to be at the zenith of their power within the federal government and throughout major sectors of American society and culture. Buttressed by decades-long control or domination of the academy, the theater, the press, labor, and the Democratic Party, the Progressives have fundamentally changed the state of the nation by creating a secular utopian statist path as an alternative to the religious individualistic minimalist government path set down by the Founders two centuries ago and expressed in the obligations of the government to the governed they enshrined in the Declaration of Independence and the Constitution.
The Declaration has long been treated by many in power as an irrelevant historical relic. The radical notion that the Constitution is a "living document" has gained wide adherence across several parts of the societal spectrum, even among those who hold themselves out as experts on matters of constitutional law. America is now governed by a semi-permanent ruling class, engaged in rampant self-entitlement, seeing itself as separate and apart from — and superior to — the governed. The notions of the American melting pot of disparate peoples and American exceptionalism have been lost to Balkanization of the population along the classic imaginary socialist fault lines of race, ethnicity, gender, and class. The fundamental concept of minimalism — where the power of government is a revocable grant from the governed and the government has only that power and authority necessary to perform the essential duties entrusted to it by the governed — has long been smothered by massive growth in the size, power, and reach of the federal government and the concomitant decrease in individual liberty. Read the rest of this entry »
By Spartacus Thrace
We rightfully expect political candidates and the people around them to be morally upright and of excellent character as a condition precedent to the ability to properly conduct themselves in the affairs of government if and when elected. The fact that a candidate for public office has pornography on his or her computer or subscribes to a pornography feed should be big news in any election, as it says a lot about the morals and character of the candidate. Presidential candidates and their committees are no exception. That is why it is significant and relevant to the election of our next President that the Barack Obama campaign subscribes to the pornography feed, https://twitter.com/ILikeTitsDaily, which features a daily posting of (often gaudy) photographs of the exposed breasts of women. Sometimes the breasts are not completely exposed, often the breasts are framed without showing even the faces of the women, and the sexual organs are exposed in some of the photographs.


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French Customer Service Executive - 3 month FTC
French Customer Service Executive - 3 month FTC
French Customer Service Executive
40 Hours per week. Shifts betwen 7am-10.00pm
1 in 3 weekends
My client is looking to recruit within an already flourishing team, but due to client demands they require experienced French speaking Customer Service Executive to join the team in Manchester
The ideal candidate will have proven customer service experience as well as being able to develop the existing customer relationships.
As a French speaking Customer Service executive, you will demonstrate excellent knowledge of the product and brands; and will identify new target customers to assist the dealer network in this market.
Responsibilities include but not limited to:
- Maintaining and developing relationships with existing customers via telephone calls and emails.
- Demonstrate customer service experience
- Making accurate, knowledgeable responses to customers, and providing dealers with sales opportunities.
- To regularly provide management with appropriate information, analysis and any other input relating to operations involving the above territories.
The ideal candidate will have:
- Proven customer services
- Native or fluent language skills in French and English are essential
- Excellent communication skills both written and verbal
- Excellent Presentation Skills
- Be able to work under pressure
- Can do Attitude is essential
- Creative thinker and persuasive
- The ability to flourish in a competitive environment
- Resilience and the ability to cope with rejection
If you are interested in this role, please apply ASAP. This vacancy is being advertised by Post Recruitment Ltd. The services advertised by Post Recruitment Ltd are those of an Employment Agency, visit our website for more details
Required skills
- Communication Skills
- Customer Services
- French
Reference: 39213292
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About Inter Connection Electric
About Inter Connection Electric
We would like to take this opportunity to introduce you to Inter Connection Electric Inc. Founded in 1987 as Jeff Electric and then incorporated in 1990 under the present name by Jeff Z. Skowronski, for over twenty five years of experience have firmly established us in the electrical, fire alarm and telecommunications industry.
Inter Connection Electric Inc. engages in all phases of electrical installation, from main electrical power entrance service and emergency generators installation to fire alarm systems, data, voice communications, to intercom systems. Our company meets the requirements for both commercial and residential projects. We are fully bonded and insured for both Workers Compensation and General Liability.
Our business operates from its Staten Island facility of 2,200 sq. ft. office and about 5,000 sq. ft. warehouse space, and services all 5 New York City boroughs, as well as Westchester County and parts of New Jersey and Long Island. We have completed jobs ranging from service calls to seven million dollar projects, including a wide range of project types.
Our company is committed to providing fast and professional service to all customers both residential and commercial, and offers free estimates of requested work. We can also provide electrical design and engineering, as well as drawings, plan approvals and Building Department filing for all electrical and fire alarm installations.
We invite you to compare, to talk to our customers, and to visit our jobs in progress! You will see what we have done, what we are doing, and what we can do for you!
Would you like
a no obligation quotation?


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Fabinho: I'll try to convince Mbappe to join Liverpool
Liverpool midfielder Fabinho wants to reunite with Paris Saint-Germain and France sensation Kylian Mbappe at Anfield.
Persuading Kylian Mbappe to leave French champions Paris Saint-Germain and join Liverpool is on the agenda for Reds recruit Fabinho.
Brazilian midfielder Fabinho swapped Ligue 1 outfit Monaco for Liverpool in a deal reportedly worth €50million in the close-season.
Now Fabinho wants former Monaco team-mate and World Cup winner Mbappe – who has committed his future to PSG amid links with Real Madrid – to join him at Anfield.
Mbappe took Russia 2018 by storm, the 19-year-old sensation scoring four goals to claim the best young player of the tournament.
"He [Mbappe] has already said this season he will remain at PSG, but I will try slowly to convince him to come over," Fabinho said.
Fabinho and Mbappe played together in 2016-17 as Monaco upstaged PSG to win Ligue 1.
Mbappe departed Monaco last season and spent the campaign on loan at PSG, where he won the domestic treble.
With both players now plying their trades away from Monaco, Fabinho revealed he was congratulated by Mbappe following his move to Merseyside.
"Kylian Mbappe sent me a text message congratulating me for coming to Liverpool. Unfortunately I have not had a chance to congratulate him for what he did during this World Cup," said Fabinho.
"What France did was fantastic. What Mbappe did in two years of professional football is amazing. He is a phenomenon and needs to be congratulated, and as soon as I have the chance I will."


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Selling Your Home in the Fall & Winter Months
With the time change, the sun goes down around 5 PM, when most folks are on their way home from work. Since most of your guests are visiting you after work, they will now be coming over at night time and your home's outdoor features will be lost in the dark. If you are selling your home, this really will put you at a disadvantage when potential buyers drive by your home and all they see is a dark shape of a home.
Professional Outdoor Lighting System
With a professionally installed outdoor lighting system, your home will stand out in the dark and look much more attractive to potential home buyers. Outdoor lighting will illuminate your property's landscaping, flower beds, and trees.
With outdoor lighting, you can highlight your home's best features. For example, you may wish to showcase your homes front porch and columns. You may want to highlight windows and balustrades. Or you may have recently planted trees along your home that should be illuminated.
Outdoor lighting can be used to highlight walls. Good effects for wall lighting are grazing and washing techniques. Grazing will create beautiful shadows. A washing effect is created when a beam of light is aimed at a wall from two to three feet away.
Lighting Brings Return on Investment
According to many real estate sales experts, adding outdoor and landscape lighting to your home when selling it may bring a 50% return on your outdoor lighting system investment. So what are you waiting for?
Call Creative Nightscapes lighting professionals today for a free estimate to install a lighting system to your property today.


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Abbas, we are waiting for you in Gaza, says Issam Younis at Al Mezan
Diakonia and our partner organizations are deeply concerned about the situation in Gaza and Israel. We call on all parties to protect civilians in order to prevent a humanitarian disaster. Below an open letter from our partner organization Al Mezan's director Issam Younis to President Mahmoud Abbas, written on November 19th 2012:
This is the historic moment that must be taken up. We've waited for you in Gaza for six days. We're still waiting; your people who are being attacked and slaughtered in Gaza. It is not acceptable anymore –no matter what those surrounding you make it look like to you – that you do not come. I do not invite you to shows olidarity with Gaza, but to be in Gaza and with Gaza.
We are waiting in Gaza for you and all the secretary-generals of Palestinian parties. The advocates of divide can stay back in Ramallah. This scandalous schism must end now and here; the schism that made your people bleed. The Palestinian national platform must be restored and Palestinian strive for freedom must be agreed on by all. It is our engagement with the occupation that we must magnify; politically, legally, and diplomatically.
We are waiting for you to come. I am sure you will call the Arab leaders, by whose visits we will be honored; including Morsi and Abdulla, to be part of the historic event that is ending the intra-Palestinian divide from Gaza and in Gaza.
We are waiting to know that you have ordered PLO diplomats in Geneva, New york, Vienna, and Paris; all UN HQ's, to immediately act to convene the UN General Assembly, Human Rights Council, UNESCO and others to condemn the crimes perpetrated against our people in Gaza, and do all they can to secure that these crimes be investigated and punished. It is regrettable we have not so far seen any meaningful diplomatic effort that matches the size of blood and suffering in Gaza.
We are waiting for the orders to our veteran diplomats to get on fire and approach the European Union and other powers to mobilize the most needed pressure on the occupying state.
I will be waiting for you on Rafah Crossing. With me will be hundreds and thousands of your people. No matter how harsh the strikes will be on us, we will be there.


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TC-Helicon VoiceLive 2
What Floor-based harmony and effects processor.
Why Contains the effects necessary to produce vocal sounds from the past, present, and well into the future with more power and voices than ever before.
How Vocal harmonies with up to eight voices that can be controlled via MIDI, guitar, or MP3. Like a great audio engineer, VoiceLive 2 listens to the singer and adjusts EQ, compression, de-essing, and gating to make vocals sound smooth and produced.
Price $945 retail
Web www.tc-helicon.com
Propellerhead Record
What Recording software that puts the musician in focus.
Why Has the feel of a big-league recording studio with the streamlined simplicity of a tape deck.
How Combines recording and a software mixer console with a limitless rack of audio processing gear. For guitar players, the built-in virtual POD from Line 6 brings a wide range of guitar amps and cabinets. The software mixer's sound is faithfully modeled after the renowned SSL 9000K. Efficient coding minimizes concerns about track counts, latency issues, or adding that extra reverb or effect.
Price $299 retail (available September 9th)
Motion Samples Boombonix Drum Samples
What Dirty South 808 drum samples.
Why Samples created with analog drum machines mixed with digital drum synths to give your hip-hop tracks the slap they deserve.
How Included are sounds of sub bass kicks, snappy snares, stacked claps/snaps, crispy clear hats, tom sets, digital percussions, synth effects, and more.
Price $35
Web www.motionsamples.com
Switchcraft SC800
What Instrument direct box.
Why A high quality passive direct box.
How Jensen transformer, dual 1/4" thruputs, all metal Switchcraft 1/4" I/O jacks, 20dB pad, pin 1 ground lift, recessed I/O panel, laser-etched product labeling, and a rugged extruded aluminum enclosure.
Price $270 retail, $199 street
Web www.switchcraft.com/products/sc800.html
Solid State Logic X-Verb Plug-in
What High-end hardware reverb performance in SSL plug-in form.
Why Delivers the density, warmth, depth, and detail usually only associated with high-class hardware reverb units.
How True Reverberation Generator based on unique SSL algorithms. The algorithm-based approach generates stunning sonic results, with lowest possible latencies, and allows dynamic access to all of the fine detail parameters of the Early Reflection and Reverb structure.
Price $349
Web www.solid-state-logic.com
QuikQuak UpStereo 2.00
What Stereo enhancer and expander.
Why Makes your mixes bigger in stereo width and loudness, while being light on your CPU and easy to use.
How Contains a stereo width slider going from mono to wide, bringing the stereo image out and toward the listener. Subtle Air & Bass boosters help the audio breathe. Movable 3D interface, with changeable colors and light positions.
Price Free download
Web www.quikquak.com/Prod UpStereo.html
Akai APC40
What Control surface for Ableton Live.
Why The APC40 is the link between human and computer that balances analytical parameter control with creative expression.
How Communicates bi-directionally with Ableton Live, enabling the controller to display information from the software on its clip matrix of 40 triggers and on LED rings surrounding each knob. Since this product was designed specifically for Ableton Live, users do not need to map its controls for instant performance, while advanced users have the flexibility of mapping controls how they see fit.
Price $599 retail, $399 street
Web www.akaipro.com
Indamixx Netbook Model 2
What Portable music computer.
Why Affordable Linux audio Netbook with preinstalled audio and mobility software.
How Uses custom Linux operating system based on Ubuntu 9.04, 2GB of RAM, 160GB hard drive, 2.0GHz over clocked Intel Atom chip, and 10" widescreen, while being 3G and HSDPA capable, and having up to four hours of battery life.
Price $499
Web www.indamixx.com


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There is a fourth statement that floats in our potion like a lily pod floating in a tranquil pond. It gently attracts our attention. It is worth our while to sit by the pond and ponder the power and worth of words and tales and tellings. Our spirits are calmed as we hearken back to journals and journeys allowing them to guide our lives and our souls.
"I am a stranger among you (23:4)." The Mateh focused on that line. If it were possible for a staff of wood, even a staff formed by G from the Tree of Life/Knowledge to smile a melancholy smile, that is what would have happened. For something as special as the Mateh, time and space bend and flow in many directions and the Mateh can see downstream and upstream with ease. And it saw and heard and felt the words of a Rabbi some four thousand years later, trying to capture the continuous connection to this verse.
He was teaching that this verse was referring to the Jewish soul and hir(1) relationship to this world. A Jewish soul is part of the physical reality, for it is taught to inhabit and elevate it. Virtually all Mitzvot, all Sacred Connections are connections between this world and the higher realms. Virtually all Mitzvot are actions involving elevating physical objects. The challenge for the Jewish soul is to make a dwelling place for the Shehinah (the dwellingness of G) in the material realm, by making sacred everyday actions and mundane materials. And yet the Jewish soul is a 'stranger' in a strange land, the material world. Hir(1) true home, the secret inner home is higher and deeper than the material world. It is in the realm of the spirit. The home of the soul is the world of holiness, the wholeness of harmony. The Jewish soul yearns to return and yet remains through the physical body in the realm of the physical, learning its lessons, teaching its lessons until it is called to separate and return home.
This, the Mateh grokked, it understood. For the Mateh, this piece of staff this piece of wood is the symbol of the residing in, and elevating of the physical. The Mateh was not just a piece or wood. It was a piece of would, a piece of hope. It was the would of elevation. The Mateh is the physical reminder of what the Jewish soul, indeed what all souls could, should and would do and be. The soul would elevate the soporific secular into the stirring sacred. The Mateh felt justifiable pride in its place as the reminder to the two-leggeds of the connection between the material realm and the spirit realm. It felt the heavy weight of the honor of obligation. The Mateh was, is and always will be the symbol of hope. The Mateh of wood: The symbol of would. "Loo Yehi" (That it would be, that it will be)
(1) I use 'hir' to replace the awkward 'his/her'


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Gordon & MacPhail has donated bottle No. 1 of its rare Generations Glenlivet 70 year old to raise funds to aid the relief effort in Japan. "We have decided to donate bottle No. 1 of Generations Glenlivet 70 Years Old to a special charity auction to raise money for the relief effort. All funds will be paid to the Red Cross who will use them to help the Japanese people recover from this devastating natural disaster".
"We all feel helpless in the face of a tragedy so we are looking to do something practical", said David Urqhart from Gordon & MacPhail.
Bonhams Edinburgh, who are giving their services for free, will deliver the special charity auction on Tuesday 29 March 2011 at 7.00pm GMT. The auction will be held at Bonhams 22 Queen Street, Edinburgh and online at http://www.bonhams.com/eur/auction/19503/lot/1
Gordon & MacPhail launched Generations Glenlivet 70 Years Old at a special event at Edinburgh Castle on 8 March 2011. Distilled in 1940 only 100 full sized decanters have been released for sale in 2011. Whisky connoisseur Charles Maclean describes the single malt: "The Glenlivet 1940 opens a door into a different time, another country. To smell and taste this exquisite whisky is to experience the past in a unique way – layer upon layer of flavour, profound and evocative."
The whisky is beautifully presented in a tear-shaped hand-blown crystal decanter with an elegant British Hallmarked silver stopper. The decanter nestles in a sterling silver base and is framed in a handmade box, crafted in Scotland using Scottish Elm.
This is a beautifil whisky and very rare so this is your chance to buy it and support the Japan releif effort at the same time.
Filed under: General Topics, Whisky | Tagged: Glenlivet, Glenlivet 70 year old, glenlivet 70 yr | Leave a comment »


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Dion's Bus Service is available for School Excursions and Private Charter.
We offer you friendly, caring drivers and excellent service.
Let us assist you with your transportation needs, whether it is for a Conference, Special Event, Social outing or Sporting Event. We cater for any group size.
To find out more, select the Bus Hire - Enquiry option, fill in the form and submit it to us. One of our consultants will return a quote to you.


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Climate is not stable. Natural climate variability occurs at all time scales, from year to year and over millions of years. Presently the natural climate variability is superimposed on the human induced climate change. Uni Research investigates how climate varies and change, and the forcing and mechanisms that governs changes in past, present and future climate.
Hidden costs of implementing afforestation as a climate mitigation strategy: A comprehensive assessment of direct and indirect impacts (HiddenCosts)Jan. 1, 2017 - Dec. 31, 2020 | Uni Research Rokkan Centre / Uni Research Climate
OCTEL- Ocean sea-ice atmosphere teleconnections between the Southern Ocean and North Atlantic during the HoloceneApril 1, 2016 - Dec. 31, 2020 | Uni Research Climate
OCEAN-CERTAINUni Research Environment
RISES - Quantifying and understanding rates of ice sheet changeJan. 1, 2018 - Dec. 31, 2022 | Uni Research Climate


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The functional interchangeability of mammalian Notch receptors (Notch1-4) in normal and pathophysiologic contexts such as cancer is unsettled. We used complementary in vivo, cell-based and structural analyses to compare the abilities of activated Notch1-4 to support T cell development, induce T cell acute lymphoblastic leukemia/lymphoma (T-ALL), and maintain T-ALL cell growth and survival.
Principal Findings
We find that the activated intracellular domains of Notch1-4 (ICN1-4) all support T cell development in mice and thymic organ culture. However, unlike ICN1-3, ICN4 fails to induce T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) and is unable to rescue the growth of Notch1-dependent T-ALL cell lines. The ICN4 phenotype is mimicked by weak gain-of-function forms of Notch1, suggesting that it stems from a failure to transactivate one or more critical target genes above a necessary threshold. Experiments with chimeric receptors demonstrate that the Notch ankyrin repeat domains differ in their leukemogenic potential, and that this difference correlates with activation of Myc, a direct Notch target that has an important role in Notch-associated T-ALL.
Citation: Aster JC, Bodnar N, Xu L, Karnell F, Milholland JM, Maillard I, et al. (2011) Notch Ankyrin Repeat Domain Variation Influences Leukemogenesis and Myc Transactivation. PLoS ONE 6(10): e25645. https://doi.org/10.1371/journal.pone.0025645
Editor: Marc Vooijs, University of Maastricht (UM), Netherlands
Received: July 28, 2011; Accepted: September 7, 2011; Published: October 13, 2011
Copyright: © 2011 Aster et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Institutes of Health (WSP, JCA, and SCB) and the Leukemia and Lymphoma Society SCOR Program (WSP, JCA, and SCB). JMM was supported by NIH training grant T32 CA09140. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Notch receptors participate in a highly conserved signaling pathway that regulates cellular differentiation and homeostasis in a dose- and context-dependent fashion (for review, see [1]). Mammals express four different Notch receptors (Notch1-4), large type I transmembrane glycoproteins composed of a series of characteristic structural motifs. Activation of Notch receptors normally depends on two successive types of proteolytic cleavages (for review, see [2]). First, binding of ligands to the extracellular domain of Notch triggers proteolytic cleavage just external to the transmembrane domain by ADAM-type metalloproteases. This creates a truncated membrane-tethered form of Notch that is recognized by the gamma-secretase protease complex. Additional cleavages by gamma-secretase free the intracellular domain of Notch (ICN) from the membrane, allowing it to translocate to the nucleus and form a transcriptional activation complex with the DNA-binding factor CSL and a co-activator protein of the Mastermind-like (MAML) family.
Structural studies have provided a model for the stepwise assembly of the CSL/ICN/MAML transcriptional activation complex [3], [4]. The intracellular portions of Notch1-4 (ICN1-4) contain N-terminal RAM domains, which bind CSL with relatively high affinities [5], [6], [7], and 7 iterated ankyrin repeats. Under physiologic conditions, the RAM domain likely mediates the initial association of ICN and CSL, which enables formation of a CSL∶ANK composite surface that recruits MAMLs, an essential event for transcriptional activation of target genes and subsequent downstream functions [8], [9].
In line with their critical role in assembly of this complex, ANK domains are the most highly conserved part of ICN1-4, followed by the RAM domains (summarized in Figure 1). In contrast, sequences C-terminal of ANK are substantially more varied. At the far C-termini of ICN1-4 are PEST degron domains that stimulate ICN degradation. Between the ANK and the PEST domains lies the most divergent portion of mammalian ICN1-4. In ICN1 this region includes a strong transcriptional activation domain (TAD), whereas the analogous region of Notch2 appears to contain a weaker TAD [10]. The same portion of Notch3 lacks a conventional TAD, but instead is proposed to interact with an as-of-yet unknown zinc-finger transcription factor that contributes to the activation of specific target genes, such as Hes5 [11]. Sequences immediately C-terminal of ANK in ICN1 and ICN2 contain phosphorylation sites that may modulate function in response to cytokines [12], [13], [14]. The function of the region between ANK and PEST in ICN4 is unknown.
The numbers at right are the %identity/%overall identity and similarity for each indicated domain of ICN2, ICN3, and ICN4 relative to ICN1, based on sequence alignments and homology calls performed by Clustal. Similarity is defined as positions with conservative or semi-conservative substitutions. N.S. indicates no significant homology. Domain boundaries were defined based on ICN1, in which the RAM domain and flanking C-terminal sequences span residues 1761 to 1872; the ankyrin repeat domain (ANK) spans residues 1873 to 2123; an ANK flanking region (AFR) spans residues 2124 to 2204; the transcriptional activation domain spans residues 2205 to 2422 [10]; and the C-terminal domain including the PEST degron spans residues 2423 to 2556.
Although ICN1-4 differ in their transactivation of Notch-responsive reporter genes in transient expression assays [11], [15], most direct comparisons performed to date in vivo have not revealed functional differences. Replacement of the last 426 amino acids of Notch2 with the same region of Notch1 (corresponding to the seventh ankyrin repeat, the TAD, and the PEST domain) by gene targeting results in normal mice [16], suggesting that these portions of ICN1 and ICN2 are equivalent functionally. Of relevance to this report, transduced ICN1 or ICN4 both induce human hematopoietic progenitors to undergo T cell development following transplantation into NOD/SCID mice [17].
An important pathophysiologic outcome of ICN overexpression is neoplasia. Retroviral expression of ICN1 in hematolymphoid progenitors is a potent inducer of murine T-ALL [18], and the majority of human and murine T-ALLs harbor gain-of-function mutations in Notch1 (for recent review, see ref. [19]. Feline leukemia viruses that transduce the coding sequences for the RAM and ANK domains of ICN2 accelerate T-ALL development [20], and transgenic LCK-ICN3 mice develop T-ALL with high penetrance and short latency periods [21], indicating that Notch2 and Notch3 also have leukemic potential. Recent deep sequencing studies have identified acquired mutations that result in deletion of the C-terminal PEST domain in 10-15% of human chronic lymphocytic leukemia (CLL) [22], [23], a type of Notch1 mutation originally identified in human T-cell acute lymphoblastic leukemia (T-ALL) [24] that stabilizes ICN1 and enhances the transactivation of target genes in leukemia cells. Conversely, Notch signaling has tumor suppressive effects in the context of squamous epithelium [25], [26], a finding that emphasizes the context-dependent outcome of Notch signaling.
Notch4 was first identified as a proviral insertion site in murine mammary tumors, and enforced expression of ICN4 contributes to development of adenocarcinoma [27]. However, the transforming abilities of ICN1-4 have not been compared directly in vivo in a single cellular context, and other data suggest that ICNs have divergent activities. For example, ICN1 and ICN2 reportedly have opposing effects on the growth of brain tumors [28]. Thus, the physiologic and pathophysiologic interchangeability of ICN1-4 is an open question.
To address this issue, we compared the ability of ICN1-4 to drive T cell development and cause T-ALL in vivo and to rescue T cell progenitors from blockade of endogenous Notch signaling in thymic organ culture assays. We find that while ICN1-4 all support T cell development, only ICN1-3 induce T-ALL efficiently. T cell progenitors expressing ICN4 appear to be actively extinguished and disappear by 6 months post-transplantation, a phenotype resembling that caused by "hypoleukemic" weak gain-of-function forms of Notch1 [29]. Further, studies performed with chimeric receptors allowed us to map the structural basis for this difference in leukemogenicity to repeats 2–7 of the ANK domain, which influence the ability of ICN to activate expression of Myc, a key Notch target gene implicated in leukemogenesis. These studies demonstrate that the transforming activities of Notch receptors in hematolymphoid progenitors are not equivalent, and that this functional divergence is attributable in part to variation in the highly conserved ANK domains.
ICN1-4 Drive T cell Development in vivo and Rescue Developing Thymocytes from the Effects of Gamma-Secretase Inhibitors
When expressed in hematopoietic progenitors, gain-of-function forms of Notch1 cause a CD4+CD8+ double-positive (DP) T cell population to appear in the bone marrow by day 24 post-bone marrow transplant (BMT) [18]. To begin to compare the activities of ICN1-4 in hematopoietic cells, we transduced bone marrow progenitors with MigRI retroviruses of equal titer, and used these cells to reconstitute syngeneic recipient animals. On day 24 post-BMT, the marrow of all ICN1-4 animals contained an abnormal GFP+ DP T cell population, whereas DP T cells were absent from the GFP- bone marrow cell populations of ICN1-4 animals (Figure 2A), as well as MigRI control animals (data not shown). Thus, ICN1-4 all drive ectopic T cell development from bone marrow progenitors.
(A) Bone marrow cells were analyzed by flow cytometry on day 24 post-reconstitution of lethally irradiated recipients with marrow transduced with MigRI-ICN1-4. Representative results are shown. (B) Fetal liver hematopoietic progenitors transduced with MigRI, MigRI-ICN1, or MigRI-ICN4 were used to reconstitute irradiated thymic lobes obtained from the same donor animals. Left hand panels: MigRI control lobes treated with vehicle (DMSO) or the gamma-secretase inhibitor (GSI) compound E (1 microM). Right hand panels: MigRI-ICN1 and MigRI-ICN4 lobes treated with compound E (1microM).
To further study the interchangeability of ICN1-4 in developing T cells, we compared the ability of ICN1-4 to rescue T cell development in thymic organ cultures treated with compound E, a potent gamma-secretase inhibitor (GSI) that blocks T cell development at the CD4−CD8− double negative (DN) 3a stage by inhibiting ICN1 production. In experiments conducted with transduced fetal liver hematopoietic progenitors, ICN1-4 all rescued DP T cell development in the presence of GSI (Figure 2B and data not shown), indicating that each can induce intrathymic T cell development.
ICN4 does not Induce T-ALL or Support the Growth of Notch1-Dependent T-ALL Cells
When activated Notch isoforms are expressed in bone marrow progenitors, the appearance of circulating DP T cells is usually a harbinger of the subsequent lethal T-ALL [18]. Mice receiving ICN1-4 transduced progenitors uniformly developed circulating GFP+ DP T cells by day 21 post-transplant. By day 124 post-transplant, all of the ICN1, ICN2, and ICN3 animals developed T-ALL, but ICN4 animals remained healthy and did not develop leukemia (summarized in Table 1). At necropsy, ICN1-3 animals showed widespread involvement of tissues by leukemic blasts, which had immunophenotypes consistent with immature T cells (data not shown). These leukemias were readily transplantable to secondary recipients (summarized in Table 1).
A clue to the basis for this distinction between ICN1-3 and ICN4 was evident in analyses of bone marrow on day 128 post-transplant. By this time point in ICN4 animals GFP+ cells were markedly decreased in number, whereas the GFP+ DP T cell populations in ICN1-3 animals continued to expand as T-ALL developed (Figure 3A). In contrast to ICN4 animals, GFP+ cells persisted indefinitely in MigRI control animals (data not shown), suggesting that ICN4 suppresses or extinguishes cells with long-term self-renewal capacity. Consistent with this possibility, by 180 days post-transplant ICN4 animals lacked GFP+ Lin−Sca1−c-Kit+ (LSK) bone marrow cells, a population that contains cells with long-term self-renewing capacity [30], whereas a persistent GFP+ LSK cell population was detectable in MigRI control animals (Figure 3B).
(A) GFP+ marrow populations in mice on day 128 post-reconstitution with marrow progenitors transduced with MigRI-ICN1-4. (B) GFP+ Lin-/Sca1-/c-Kit+ (LSK) marrow populations in mice on d180 post-reconstitution with marrow progenitors transduced with MigRI or MigRI-ICN4. (C) T6E transduced with empty MigRI virus or MigRI-ICN1 or -ICN4 viruses were treated with the gamma-secretase inhibitor (GSI) compound E (1 microM) and monitored for growth. (D) DNA content histograms of T6E cells transduced with ICN1 or ICN4 and treated with compound E (1 microM) for the indicated times.
LSK populations in ICN1-3 animals could not be studied because of the development of T-ALL. Of note, however, in prior work we observed that expression of weak gain-of-function non-leukemogenic Notch1 alleles in murine HSCs resulted a burst of T cell development followed by loss of GFP-positivity in the hematolymphoid compartment and no T-ALL development [29], [31], a phenotype closely resembling that produced by ICN4. In addition, expression of ICN1 in Rag1-/- HSCs, which do not develop T-ALL due to defects in lymphoid development, also depletes the LSK population (data not shown). Thus, ICN4 and other forms of activated Notch appear to deplete LSK populations in reconstituted mice, possibly by inducing differentiation at the expense of maintenance of multipotent progenitors.
T-ALL cell lines derived from Notch1-induced T-ALLs require persistent Notch signaling for growth and survival. The murine T-ALL cell line T6E [32] expresses a membrane tethered form of Notch1 that requires gamma-secretase cleavage for activation and can be rescued from GSI-induced apoptosis by retroviral transduction of ICN1, which lies downstream of the GSI blockade. ICN1 rescued T6E cells from GSI induced growth arrest and apoptosis, whereas ICN4 did not (Fig. 3C and 3D). Thus, ICN4 is also incapable of maintaining the growth of established Notch-dependent T-ALL cells.
ANK Domains Determine Notch Receptor Leukemogenicity
The modular nature of Notch intracellular domains and the availability of a high-resolution structure of the ANK domain of ICN1 [3], [33] facilitated construction of chimeric ICNs, which were used to investigate the structural basis for the functional differences between leukemogenic ICNs and ICN4. Because the greatest variation among ICNs is in the region C-terminal of ANK (Figure 1), we anticipated that addition of this region of ICN1 to the RAM-ANK domain of ICN4 would create a leukemogenic chimera. However, the resulting chimeric polypeptide, ICN4/ICN1-CT, created by joining of the RAM-ANK domain of ICN4 to the C-terminus of ICN1 at position J3 (Figure S1), produced an "ICN4" phenotype characterized by the transient appearance of DP GFP+ T cells in the peripheral blood by day 24 post-transplantation and the absence of leukemia (data not shown).
Because the RAM domain of ICN1 is not essential for leukemogenesis [18], we next focused on ANK, which is required for all known physiologic and pathophysiologic Notch functions.
Biophysical differences among the recombinant ANK domains are minimal
Recombinant human ANK1-4 proteins exhibit highly similar circular dichroism spectra (Figure S2A). The programs CONTINLL [34], SELCON3 [35], and K2D [36] on the Dichroweb server [37], yield estimates of 36–40% alpha helix for all four molecules. Moreover, there are minimal differences in the thermal stability of the four ANK domains, with Tm values ranging from 46–50°C for the four proteins: (ANK1 Tm = 50°C; ANK2 Tm = 47°C; ANK3 Tm = 46°C; ANK4 Tm = 50°C; Figure S2B). These data show that the variations in primary sequence observed in the ANK family do not translate to substantial heterogeneity in secondary structure or domain stability. However, a comparison of ANK1-4 surfaces based on the ANK1 crystal structure [3], [33] predicts that while the contacts involved in CSL/ANK/MAML ternary complex formation are highly conserved, surfaces on the convex faces of ANK domains opposite CSL/MAML contacts are more varied (Figure S3), raising the possibility of functional divergence among the four ANK domains.
Guided by these structural insights, we constructed an ICN1(ANK4) chimera by swapping the ANK of NOTCH4 into the ICN1 backbone at positions J1 and J3 (Figure S1). The resulting ICN1(ANK4) polypeptide produced an "ICN4" phenotype in mice (Figure 4A and Table 2), indicating that the ANK domain of ICN4 is limited in its capacity to support leukemia induction. In contrast, ICN chimeras in which the ANK domains of ICN2 or ICN3 were swapped into ICN1 at positions J1 and J3 were fully leukemogenic (Figure 4A; summarized in Table 2).
(A) Kaplan-Meier curves for mice reconstituted with bone marrow progenitors expressing ICN1, ICN1(ANK2), ICN1(ANK4), or ICN1(ANK4r2-7). (B) Western blot analysis of U2OS cells transduced with empty MigRI virus or MigRI viruses carrying cDNAs for the indicated ICN polypeptides. After normalization on GFP expression, lysates were analyzed on Western blots stained with either a rabbit polyclonal antiserum specific for the TAD of Notch1 or a monoclonal antibody specific for GFP. (C) U2OS cells grown in 24-well dishes were co-transfected with 1 ng or 10 ng of pcDNA3 plasmids encoding the indicated forms of ICN, an internal control Renilla luciferase plasmid, and an artificial Notch-responsive firefly luciferase reporter gene plasmid. Luciferase activities were determined in triplicate 44–48 hr post-transfection. Fold stimulations represent the normalized firefly luciferase activity relative to that of the empty vector control, which is arbitrarily given a value of 1. Error bars represent 1 standard deviation. ICN1(ANK2), ICN1 backbone containing the ANK domain of Notch2; ICN1(ANK4), ICN1 backbone containing the ANK domain of Notch4; ICN1(ANK4r2-7), ICN1 backbone containing ANK repeats 2–7 of Notch4.
To assess whether this functional distinction stemmed from differences in protein quantity, we compared the levels of leukemogenic ICN1 and ICN1(ANK2) and non-leukemogenic ICN1(ANK4) chimeric polypeptides in cells transduced with retroviruses of equal titer. Western blots stained with an antibody specific for a common epitope located in the TAD of Notch1 revealed that these forms of ICN were expressed at equal levels (Figure 4B), suggesting that the variation in leukemogenecity stems from qualitative differences. In parallel, we noted that these ICNs activated a sensitive Notch reporter gene to a similar degree (Figure 4C). Together, these findings suggested that the differences in leukemogenic activity stem from qualitative differences in the ability of different ANKs to activate genomic target genes.
Additional experiments were then conducted to further define the structural variation responsible for the observed functional differences. The greatest structural divergence among the ANKs is in the first repeat, which is partially unstructured [3], [33], [38]. We therefore constructed and tested an ICN1(ANK4r2-7) chimera created by swapping ankyrin repeats 2–7 of ICN4 into the backbone of ICN1 at positions J2 and J3 (Figure S1). This chimera also produced an "ICN4" phenotype in mice (Figure 4A; summarized in Table 2).
We also tested the contribution of the ANK domain to the growth maintenance of two Notch1 "addicted" GSI-sensitive murine T-ALL cell lines, T6E and G4A4. These results correlated with leukemia induction, as a chimeric ICN containing the leukemogenic ANK of Notch2 supported T-ALL cell growth and survival, whereas chimeric ICNs containing Notch4 ANK or Notch4 ANK repeats 2–7 did not (Figure 5).
The Notch1-dependent murine T-ALL cell lines T6E and G4A2 were transduced with empty MigRI virus or the indicated forms of ICN and monitored for growth in the presence of the gamma-secretase inhibitor (GSI) compound E (1 microM). ICN1(A2), ICN1 backbone containing the ANK domain of Notch2; ICN1(A4), ICN1 backbone containing the ANK domain of Notch4; ICN1(A4r2-7), ICN1 backbone containing ANK repeats 2–7 of Notch4.
ICN Leukemogenecity Correlates with Transactivation of Myc
Myc is a direct Notch target gene that contributes to leukemogenesis [39], [40], [41], and the failure of certain ICNs to induce T-ALL or maintain T-ALL cell growth might therefore stem from differing abilities to transactivate Myc. This idea was tested using the 8946 T-ALL cell line, which was derived from a murine T-ALL induced with a "doxycycline-off" human MYC transgene. 8946 cells are rescued from doxycycline treatment by transduction of activated Notch1 [39], which induces expression of endogenous murine Myc. We observed that leukemogenic forms of ICN (ICN1 and ICN1(ANK2) rescued the growth of 8946 cells in the presence of doxycycline, whereas non-leukemogenic ICNs (ICN4, ICN1(ANK4), and ICN1(ANK4r2-7)) did not (Figure 6A). None of the ICNs had any effect on the growth of 8946 cells in the absence of doxycycline (Figure S4). Growth rescue correlated with transactivation of the endogenous murine Myc gene, as ICN1 and ICN1(ANK2), but not ICN4 or ICN1(ANK4r2-7), induced Myc expression (Figure 6B). We also noted that upregulation of endogenous Myc by ICN1 and ICN1(ANK2) was blunted by expression of transgenic MYC (Figure 6B). Transgenic or translocation-mediated overexpression of Myc leads to autorepression of intact endogenous Myc loci [42], [43] through a mechanism that appears to involve polycomb family repressors [44]; it may be that autorepression also attenuates ICN transactivation of endogenous Myc in 8946 cells. The inability of non-leukemogenic forms of ICN to upregulate Myc did not reflect a general defect in transactivation of genomic Notch target genes, as all ICNs tested upregulated Deltex1 (Figure 6C), and showed varying abilities to upregulate other Notch1 target genes, such as Ptcra (pre-T cell receptor-alpha), CD25, and Notch3 (Figure S5). Of note, none of the Notch target genes were expressed in the basal state in 8946 cells and, like Myc, showed varying degrees of suppressed responsiveness in the presence transgenic human Myc. Thus, in the MYC-induced 8946 cell line there appears to be no selective pressure for expression of Notch target genes, and indeed enforced Myc expression may be associated with epigenetic changes that negate Notch's ability to upregulate its targets.
(A) Comparison of rescue of 8946 cells by various ICN polypeptides following doxycycline-mediated withdrawal of MYC. 8946 cells were transduced with MigRI viruses, sorted for GFP positivity, and then treated with doxycycline (20 microgram/ml) for up to 8 days. Cell numbers at each time point are shown. (B, C) 8946 cells transduced as in (A) were harvested after 24 hr of treatment with doxycycline (20 microgram/ml) or vehicle (PBS). Levels of Myc (B) and Deltex1 (C) mRNAs were determined by qRT-PCR.
These studies indicate that ICN1-4 share the capacity to drive T cell development, but differ in leukemogenic potential. Specifically, ICN4 has a more limited propensity to cause T-ALL or support T-ALL cell growth, despite its ability to transform cells of other lineages, such as breast epithelial cells. Based on domain swaps, we find that this difference in outcomes stems primarily from variation in the highly conserved ANK domains, which appears to selectively influence the transactivation of various target genes. Our findings (summarized in Figure 7) provide strong evidence for subtle but significant functional divergence in the intracellular domains of mammalian Notch receptors.
The domain organization of ICN chimeras is shown at left, and the ability of each to induce T cell development, induce T-ALL, and transactivate Myc is summarized at right. n.d. = not determined.
The leukemogenicity of various ICN isoforms is correlated with their ability to transactivate Myc, a direct Notch target gene in T-ALL cells [39], [40], [41]. Myc provides important leukemogenic signals, as ectopic Myc expression rescues growth and survival of some, but not all, Notch-dependent human T-ALL cell lines when Notch signaling is inhibited [45], and conditional deletion of Myc impedes Notch1-dependent T-ALL development in mouse models [46]. Notch1 transactivation of Myc has also been implicated in the pathogenesis of other neoplasms, including murine mammary tumors [47].
Although several possible CSL/ICN binding sites have been identified near the proximal Myc promoter [39], [40], [41], additional work is needed to determine how Notch1 transactivates Myc and how ANK sequence variation influences this process. Some Notch target genes are regulated by a sequence-paired-site (SPS), a special "head to head" configuration of CSL binding sites that permits the formation of dimeric CSL/ICN/MAML complexes via interactions involving ANK [48]. Notably, dimerization defective ICN1 mutants induce T cell development, but cannot transactivate Myc, induce T-ALL or maintain T-ALL cell growth [49], a phenotype resembling that produced by ICN4. Thus, one possibility is that ICN4 fails to form functional dimeric transcription complexes on a yet-to-be-discovered SPS that governs Myc expression.
Three key residues involved in dimer contacts in ICN1 are K1946, E1950, and R1985 [48]. These residues vary slightly among the mammalian ICNs; for example, ICN4 has an arginine residue instead of a lysine residue at the position analogous to K1946 in ICN1. While studies in purified systems have not uncovered substantial differences in the ability of ICN1-4 to form complexes with CSL and MAML1 on DNA sequences containing single CSL sites or high affinity SPSs [48], [50], ICN4 might be unable to form dimers on low-affinity, non-consensus, "cryptic" SPSs, which have recently been identified near several direct Notch1 target genes [48]. On the other hand, however, in 8946 cells the ICN1(ANK4) chimeric polypeptide transactivated Ptcra (Figure S5), a dimerization-dependent Notch target that appears to be regulated by a cryptic SPS [49]. A second possibility is that ICN4 is fully competent for dimer formation, but unable to recruit additional unknown factors that are essential for transactivation of a subset of Notch target genes, including Myc. Both ideas are in line with a structural comparison of ANK1-4 (Figure S3), which shows that the most divergent surface residues lie away from CSL/MAML contacts on the backside of the ANK domains. This more divergent ANK surface participates in dimerization and could also be involved in differential recruitment of co-factors to Notch transcription complexes. Testing of these possibilities awaits further characterization of the genetic elements involved in Notch regulation of Myc and the biochemical composition of Notch transcription complexes.
The Notch pathway is an attractive therapeutic target in T-ALL [51], [52], but attempts to treat relapsed/refractory disease have revealed that non-selective GSIs cause "on-Notch" toxicity by skewing the differentiation of epithelial progenitors towards goblet cell fate. The ability of Notch to function as a tumor suppressor raises additional concerns about long-term toxicity associated with Notch pathway blockade. A detailed biochemical comparison of complexes formed by different Notch receptors (e.g., ICN1 and ICN4) may reveal opportunities for development of therapies that selectively interfere with the transactivation of subsets of Notch target genes within particular cellular contexts, thereby obviating toxicities associated with pan-Notch inhibitors.
Materials and Methods
Expression Plasmids
A cartoon depicting ICN1-4 is shown in Figure 1. The constructs encoding ICN1 (amino acids 1759 to 2556) and ICN2 (amino acids 1721 to 2471) have been described [53] The cDNA encoding ICN3 (amino acids 1667 to 2318) was the kind gift of Dr. Urban Lendahl. The ICN4 cDNA was created by PCR amplification of a lambda phage gt11 human umbilical vein endothelial cell cDNA library (the kind gift of Dr. David Ginsburg) with ICN4-specific primers, and encodes amino acids 1472 to 2003 of human Notch4. Each cDNA was engineered to have an ATG start codon within the same Kozak consensus sequence. These cDNAs were cloned into the plasmid pcDNA3 (Invitrogen) and the retroviral vector MigRI, which also contains an internal ribosomal entry sequence and a cDNA encoding GFP. Chimeric ICN cDNAs were created by introducing restriction sites by PCR that permitted the ligation of cDNA fragments of interest followed by site directed mutagenesis (QuikChange Kit, Stratagene). The identities of all expression constructs were confirmed by DNA sequencing. The specific points of joining that were used to create chimeric ICNs and the alignment of the ICN1-4 RAM and ANK domains are shown in Figure S1.
Cell Culture
Cell culture reagents were obtained from Invitrogen. U2OS and 293T cells were obtained from the American Type Culture Collection. U2OS, 293T, and Bosc23 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U of penicillin/ml, and 100 µg of streptomycin/ml. T6E cells and 8946 cells [39] were grown in RPMI 1610 media with the same additions. Cell lines were grown at 37°C under 5% CO2.
Thymic Organ Culture
Harvest of fetal liver hematopoietic progenitor cells and seeding of thymic organ cultures were as described [54]. Briefly, liver cells from day 15 B6 fetal mice were isolated on a Ficoll gradient, washed with PBS, and suspended at 1–2×106 cells/ml in 2 ml of Iscove Modified Dulbecco's medium containing 10% fetal calf serum, 2 mM glutamine, 0.5 mM penicillin and streptomycin, 50 ng/ml stem cell factor, 6 ng/ml interleukin-3, 4 ng/ml interleukin-1beta, and 1 ng/ml interferon-gamma. The following day, the cells were transduced with MigRI retroviruses and used to seed irradiated day 15 fetal thymic lobes (1×105 cells/lobe) harvested from the same fetuses in hanging drops. After 24 hr, reconstituted fetal thymic lobes were placed on 0.8micron polycarbonate membranes (Isopore) in the presence of absence of 1 microM compound E (Tocris), a potent gamma-secretase inhibitor.
Murine Bone Marrow Transplantation
Animal care and experimental protocols were performed according to guidelines from the National Institutes of Health with an approved protocol (801264) from the Institutional Animal Care and Use Committee at the University of Pennsylvania School of Medicine. Mouse experiments were performed in the BRB II/III animal facility at the University of Pennsylvania, which is staffed by animal handlers that are experienced in the care of rodents. Veterinarian services are available at the University of Pennsylvania. A veterinarian reviews all IACUC protocols prior to initiation of the study. The University Laboratory Animal Resources and Department of Laboratory Animal Medicine veterinarian makes regular inspections and provides veterinary care. Every effort was made to reduce the level of pain or discomfort in our studies. Mice were observed daily and when abnormal conditions/behavior was observed, it was reviewed with the attending veterinarian to determine the proper course of action. When euthanasia was required, it was accomplished according to the IACUC guidelines by exposure to carbon dioxide, consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.
Briefly, MigRI plasmids were packaged into ecotropic retroviruses by transient transfection of Bosc23 cells. Retroviral supernatants were titered on NIH 3T3 cells using GFP as a marker of transduction. Bone marrow cells from 5-fluoruracil-treated female 4–8 week old C57BL/6 mice (Taconic Farms) were transduced with GFP-normalized retroviral supernatants in DMEM containing 10% heat-inactivated FBS (Invitrogen), 5% WEHI-conditioned medium, 6 U/ml recombinant mouse IL-3 (Genzyme Corp., Cambridge, MA), 10,000 U/ml recombinant murine IL-6 (Genzyme), 5 U/ml recombinant murine stem cell factor (Genzyme), 4 microgram/ml polybrene (Sigma Chemical Co., St. Louis, MO), 100 U/ml streptomycin (Invitrogen), 100 U/ml penicillin (Invitrogen), and 2 mM L-glutamine (Invitrogen). Transduced bone marrow cells were injected into lethally irradiated (900 rads) 4- to 8-week-old female syngeneic recipients.
Flow Cytometry
Peripheral blood samples and tumor cell suspensions were assessed for GFP+ immature T cells by flow cytometric analysis (Becton Dickinson, FACSCalibur). Cells were incubated with PE-labeled anti-CD8alpha (53–6.7), biotinylated anti-TCRβH57-597), and APC-labeled anti-CD4 (RM4-5) antibodies (Pharmingen). Biotinylated antibodies were revealed with streptavidin-PerCP. Dead cells, identified by forward and side scatter, were excluded from the analysis. FACS results were analyzed using CellQuest software.
Cell Cycle Analyses
Analyses were carried out as described [39]. Live or fixed cells were stained with Hoechst 33342 (4 microM, Sigma B2261) or propidium iodide (40 microgram/ml), respectively, and then analyzed for DNA content and GFP expression by flow cytometry. Data analysis was performed using FlowJo software (Treestar).
Reporter Gene Assays
Notch1 expression plasmids were introduced into U2OS cells by transient transfection with Lipofectamine Plus (Invitrogen) and assessed for their ability to activate an artificial Notch-sensitive reporter gene as described [18]. Briefly, cells in 24-well dishes were co-transfected with 1 ng or 10 ng of various pcDNA3-Notch expression constructs, a Notch-sensitive firefly luciferase reporter gene, and an internal control pRL-TK Renilla luciferase gene (Promega). Total transfected DNA was kept constant by adding empty pcDNA3 plasmid. Normalized firefly luciferase activities were measured in whole cell extracts prepared 44–48 hr after transfection using the Dual Luciferase kit (Promega) and a specially configured luminometer (Turner Systems).
Protein purification
ANK1-4 polypeptides were expressed and purified as described [55]. BL21 (DE3) pLysS Escherichia coli were transformed with the appropriate plasmids and grown at 37°C in LB medium with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol to an OD600 of 0.7–0.9. Protein expression was induced with 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 4 hr, and cells were harvested by centrifugation at 3500 rcf for 30 minutes. Pellets were frozen at −80°C after resuspension in 20 mL lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl) per liter culture. After thawing in the presence of protease inhibitors (Complete, Roche), 5 mM β-mercaptoethanol, and 1 mM EDTA, cells were lysed by sonication and the clarified lysate was applied to glutathione-agarose beads (Sigma-Aldrich). Beads were washed with buffer A (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM DTT) and incubated with approximately 200 ng of tobacco etch virus (TEV) protease per 3 mL pelleted beads for 12–16 hr at room temperature. Following removal of the protease by application of the supernatant to Ni-NTA resin (Qiagen), ANK molecules were further purified by anion-exchange chromatography on a MonoQ 10/100 GL column using a linear gradient from 0.1–0.6 M NaCl in 20 mM Tris, pH 8.0, 5 mM DTT, 1 mM EDTA and size-exclusion chromatography on a Superdex S200 16/60 column in 20 mM Tris pH 7.5, 150 mM NaCl, 5 mM DTT. Proteins were concentrated to 5–10 mg/mL, aliquoted, flash frozen in liquid nitrogen, and stored at −80°C.
Circular Dichroism Spectrocopy
CD spectra were acquired on an AVIV 62DS spectropolarimeter with proteins in 10 mM phosphate buffer at 5 to 10 µM for 1 mm path length cuvettes and 0.5–1 µM for 1 cm path length cuvettes. Concentrations were determined by absorbance at 280 nm [56]. Wavelength scans were performed at 4°C in a 1 mm path length cuvette using a 1 nm step size, a 3-second averaging time, and 5 repeats. Thermal melts were performed by monitoring the CD signal at 222 nm in a 1 cm path length cuvette and varying the temperature from 4°C to 80°C with a thermoelectric temperature controller, using a 2-minute equilibration period, a 1°C step size, and a 30-second acquisition time. Approximate melting temperatures were determined by fitting the thermal denaturation curves with the program IgorPro (Wavemetrics).
Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) Assays
Expression levels of the Notch target genes Myc, Deltex1, Trb2, Notch3, CD25, and Ptcra were determined by qRT-PCR as described [39], [57].
Supporting Information
Figure S1.
Multi-sequence alignment of ICN1-4. The aligned sequences correspond to the RAM and ANK domains of ICN1-4. Hydrophobic residues are colored red, hydrophilic residues are colored green, basic residues are colored magenta, and acidic residues are colored blue. AN and AC designate the N-terminal and C-terminal boundaries of the crystallized ANK domain of Notch1 (residues 1873–2127), while AS brackets the region of ANK that is structured (residues 1909–2123). J1 and J3 designate points of joining in chimeras in which entire ANK domains were swapped; J2 and J3 are points of ANK repeat 2–7 only swaps; while J3 is the point of joining in C-terminal sequences swaps. * = identical residues; : = conservative substitutions; . = semi-conservative substitutions.
Figure S2.
Circiular Dichroism Spectra and Thermal Stability of the Notch1-4 Ankyrin Repeats. The four ANK molecules do not demonstrate heterogeneity in overall secondary structure characteristics. (A) Circular dichroism scans of the four domains at 4°C. (B) Temperature dependence of the CD signal at 222 nm for the four domains, with an apparent Tm between 46°C and 50°C for each ANK.
Figure S3.
Conservation of Surface Residues in the Notch1-4 Ankyrin Repeats. The ANK domain of human Notch1 (pdb ID code 2F8Y) was rendered as a molecular surface using the program PyMol (DeLano Scientific). The inner concave and outer convex surfaces of the ANK domain are shown alone in the top two panels, and in the context of the ternary complex with MAML1 and CSL in the lower panels; "1" and "7" designate the amino- and carboxy-terminal ankyrin repeats 1 and 7, respectively. The ANK surface is colored according to sequence conservation among the ANK domains of Notch1-4 on a sliding scale from dark blue (100% conserved) to red (least conserved).
Figure S4.
ICNs have no Effect on 8946 Cell Growth when Transgenic MYC is "On". 8946 cells were transduced with empty MigRI or the indicated forms of ICN1 and monitored for growth in the presence of vehicle only (DMSO).
Figure S5.
Effect of Various ICNs on Notch Target Gene Expression in 8946 Cells. 8946 cells transduced with the indicated MigRI viruses were sorted for GFP positivity, harvested after 24 hr of treatment with doxycycline (20 microgram/ml) or vehicle (PBS), and analyzed for expression of the various Notch target genes.
We are grateful for the technical help provided by Andrea Carpenter and Hong Sai. We are grateful to the John Morgan and Stemmler ASU (University of Pennsylvania), the Abramson Cancer Center Flow Cytometry Core (University of Pennsylvania), the AFCRI Core (University of Pennsylvania), and the Specialized Histopathology Core of the Dana Farber/Harvard Cancer Center.
Author Contributions
Conceived and designed the experiments: JA WSP SCB . Performed the experiments: NB JM FK LX IM GH YN. Analyzed the data: JCA WSP SCB JM FK LX NB IM GH YN. Wrote the paper: JA SCB WSP. Performed structural modeling: YN NB SCB.
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VIAVI(formerly JDSU) SmartSeries-100A SingleMode OTDR (1310/1550nm, 37/35dB)
Light, compact, hands-free design includes 5" high-visibility outdoor touch screen. Dynamic Range of 37/35dB (1310/1550nm). Dead zones: EDZ (1.35m), ADZ (4m). Integrated CW light source. All-day battery life. Testing of metro & access networks-with tailored OTDR interface & automatic analysis that any technician can understand.
- Description
- Reviews (1)
Sampling PointsUp to 2,56,000 points
|Brand Name
|VIAVI (formerly JDSU)
|Product Title
|VIAVI(formerly JDSU) SmartSeries-100A SingleMode OTDR (1310/1550nm, 37/35dB)
|Part Number
|SmartOTDR 100AS 37dB
|OTDR Type
|Dark Fiber Testing
|Application type
|Last Mile-Access Links and Metro Links (CATV & Telecom)
|Hardware type
|Non-Modular (Dedicated OTDR)
|1310/1550 nm
|Dynamic Range
|37/35 dB
|Firmware Type
|Event Dead Zone
|1.35 meter
|Attenuation Dead Zone
|4 meter
|FTTH/PON Test
|Not Available
|Pulse Width
|5 ns to 20 µs
|Display Size
|5 inch
|Display Type
|Outdoor-Enhanced, 800×480 TFT
|Operation Access
|Touchscreen and Front Panel Buttons
|Internal Data Storage
|10,000 OTDR Traces (typical)
|2x USB2.0 Ports and 1x Mini-USB2.0 Port
|Power Supply
|AC/DC Adapter (O/P: 12V DC 25W)
|Battery Type
|Rechargeable Lithium Polymer
|Battery Backup
|Upto 20 hours (typical, from full charge)
|Other Features
|Light Weight & Compact, Built-in PDF report generation Model for Telecom Contractors.
|175 x 138 x 57 mm
|0.9 kg
|Standard Accessories :
|(1No) SmartOTDR 100AS 37dB
|AC/DC Adapter
|Battery Pack
|(1No) Fixed Internally
|Touch Stylus
|Instruction Guide
|User Manual
|(1No) As Soft Copy in OTDR Base Unit.
|Calibration Certificate
|Carrying Case
1 review for VIAVI(formerly JDSU) SmartSeries-100A SingleMode OTDR (1310/1550nm, 37/35dB)
It is very good product and i bought it at discount rate good compare to other protuct.
Add a review
OTDR's are the testing equipment which is generally employed to test the fiber cables used in communication and networking fields. With the OTDR, we can get a snapshot of the newly installed fiber. This snapshot gives information the technician, about splice loss, measurement length and faults if any available in the fiber. OTDR are most effective in testing long cables approximately 250mts or 800 feet or cable plate with splices.
JDS Uniphase, the company who are pioneer designing and manufacturing products for optical communication network. Having its headquarters situated at Milpitas, California, it is exporting its products all over the world. Currently the OTDR JDSU is available in the name of VIAVI Otdr. With innovative latest technology, this company is now releasing another series of OTDR called Smart Otdr.
Why it is called smart OTDR:
The following features make these series of OTDR as smarter one:
Different combination of essential fiber test capabilities.
Supportive features like fiber end face inspection, optical power measurement, VFL,
Connectivity features like USB, Bluetooth and WIFI.
Smart link mapper application makes it even smarter.
Anywhere accessibility is additional feature of this OTDR.
Enabled with StrataSync.
Application of Smart OTDR:
The smart OTDR series of VIAVI has wide application in testing field. It is widely used in the application of:
Point to point fiber access.
FTTH feeder qualification and drop cables.
FTTA front haul troubleshooting.
Applied in Underbuilding of single mode enterprise networks.
The JDSU powered smart OTDR has tremendous features like automatic macro bend detection; summary result table with pass/fail analysis, Fast report on board report generation, user interface with smartconfig error-free settings is appreciable advantage of this smart otdr. Apart from these features, the price ranges of these series are really appreciable. Based upon the configuration and mode, their price ranges are really appreciable.


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Last Saturday, I was invited to be a guest speaker at a political forum ahead of village council elections in Cavite Province's Barangay Molino V. The forum was spearheaded by a group of citizen volunteers who supported President Noynoy Aquino during this year's general election, and was organized to provide a venue for candidates to come together, present their platforms and visions and to give voters the chance to interact with them directly. In short, it was what Americans would call a town hall meeting.
Three of the four candidates for Punong Barangay, or village chief, attended the forum and each was given ten minutes to present their platforms. Afterwards, there was a short Q and A session to give the villagers a chance to find out a bit more about the candidates' policies.
The three candidates had a lot in common—all called for transparency in the village council and for engaging villagers more in local governance and security. At a glance, some of the concerns covered might seem simple—things like providing street lights for a main road. But at the grassroots level these issues are seen as vital, and candidates need to show that they've got practical solutions to these problems if they want to get elected.
It's a reminder that all politics are, as the old expression goes, local. But chances are that many such issues, even if in this case confined to a village of a few thousand people, are also a concern for the country at large. After all, the bungled hostage rescue last August and the ongoing conflict in Mindanao are both local issues that have had national implications.
On a personal note, one thing that really struck me about Saturday's forum was the positive rapport between the three candidates. Hailing from the same small village, they're practically neighbours and have known each other for decades. It was encouraging to see such positive attitudes, which should help keep people engaged.
Hopefully, as the elections draw nearer, they'll be able to sustain the interest in governance and locals here will be able to add another layer to the Philippines' democratic project.


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Market Watch: The party is surely over for property tycoon Vincent Tchenguiz
News of Vincent Tchenguiz's arrest was greeted with whoops of joy up by pensioners living in retirement developments managed by Peverel
Last week Vincent Tchenguiz was supposed to have been on his yacht in Cannes in the South of France hosting his annual party at the MIPIM property show.
Instead, he was hauled off by police at 5.30am, arrested with six others, including his brother Robert, for questioning about the collapse of the Icelandic Kaupthing bank.
Only the week before, the property tycoon's PR Sean Bellew indignantly disputed my suggestion that the party on board £10million Veni, Vidi, Vici - I came, I saw, I conquered - might not go ahead owing to Tchenguiz losing effective control of the giant Peverel property management company in mid-February.
'Any guest reading this article may think the event was no longer taking place,' he wrote.
The boat is, indeed, moored at the Cannes quayside, but whether any kind of celebration takes place on board is unknown, at the time of going to press.
Meanwhile, news of Tchenguiz's arrest was greeted with whoops of joy up and down the country by pensioners living in retirement developments managed by Peverel, which Tchenguiz bought in 2007.
'Our website has been deluged with posts cheering, "Wey, hey!"' says Melissa Briggs, of CarlEX, the Campaign Against Retirement Leasehold Exploitation.
'I am delighted that the authorities are now investigating thoroughly the financial activities of Mr Vincent Tchenguiz, as I have been investigating them for the past three years.'
A principal concern for Peverel leaseholders is to know that their contingency/sinking funds, which pay for emergency repairs such as new roofs and are held in trust by the company, have been properly protected. They would amount to millions of pounds.
'Both Peverel Group and development bank accounts are entirely separate and unaffected,' said a Peverel spokesman, referring to the arrests.
'Development bank accounts are held in trust with Bank of Scotland. Monies in these accounts are used for individual developments only and are categorically not used as security on loans.'
Since Tchenguiz lost control of Peverel, its fate has been in the hands of Bank of America, which will now make decisions on the company's strategy and management.
As for Vincent Tchenguiz, Peverel now suggests all enquiries are directed to Sean Bellew.
In light of which, one wonders what Kevin Maxwell, son of disgraced tycoon Robert, who was himself acquitted of fraud after the collapse of his father's media empire, is doing working down the corridor from Vincent's office in Mayfair.
'There is no business connection between Kevin Maxwell and Vincent or Robert Tchenguiz,' says Bellew. 'Mr Maxwell simply shares the same office building as Vincent Tchenguiz.'
No comments have so far been submitted. Why not be the first to send us your thoughts, or debate this issue live on our message boards.
We are no longer accepting comments on this article.


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They say it can't be done. But we know Americans can do anything. Even end this epidemic of gun violence. For we are more powerful than any problem when we work as one. Now is the time to unite people, from coast to coast, young and old, liberal and conservative, fed up and fired up, and free our country from what is killing it. It's in our hands.
Brady works tirelessly on Capitol Hill and in state houses across the country to ensure common sense gun laws are enacted and enforced.
We have the strongest legal team in the country, representing victims and survivors among others, to challenge and change laws, state by state and nationwide.
Armed with facts and solutions, every sector of the population can be part of the cultural change needed so all Americans can live free from the fear of gun violence.
There is no one solution to end this epidemic of gun violence and no one side has the answer. So our comprehensive approach fights every aspect with more than 45 years of research and real-world experience. Let's put the Brady plan into action now and get the job done right.The Brady Plan
Jim Brady, a gun owner, Republican and President Reagan's Press Secretary, was shot in the head during the Reagan assassination attempt. Yet he survived, and with his wife Sarah, worked tirelessly to pass the landmark, bipartisan Brady Bill ensuring background checks were conducted on gun sales. But we cannot rest on that victory. American lives continue to be lost every day in our gun violence epidemic. We invite gun owners and non-gun owners to be part of the conversation — and the solution! Working together, there is nothing Americans cannot accomplish.
Brady's rebranding is part of an evolution — not just of an organization, but of two people.
Brady's Team ENOUGH knows that the solution to gun violence is in everyone's hands.
Connect your business to a cause we all share in ways that touch the minds, hearts and lives of every American.


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Event Seven: Dismemberment
Event Seven: Dismemberment[edit]
Located: Central Park[edit]
Dismemberment is another of the event-types which will force Alex to fight droves and droves of enemies using only a specific type of attack (in this case, claws). More specifically, Alex won't have to literally fight everyone with just claw attacks, but claw attacks are the only types of attacks that will count when enemies are killed. Thus, for a kill to count, any said enemy must be killed with the claws. Using other attacks, like kicks and throws, simply will not count, so avoid their use here. Thankfully, there's plenty of time to work with here (a full minute), so scoring the prerequisite amount of kills with your powerful claws to earn a gold medal, which is eighty kills, isn't as hard as it may seem... that is, if you employ the proper tactics.
Here, you're not going to want to run around and seek enemies out. Rather, you're going to want to stay within the confines of the space in which you begin the event, and button mash for the next minute. There are three primary types of enemies that will come at you here. Most of the foes you'll rack up kills by slaying are mindless zombies who can be sliced and diced with ease. Their more obese cousins have a robust health meter that may necessitate a few slices. And then there are the Hunters, pesky foes which will beat you up relentlessly. And while they count towards your kill total, you'll want to avoid them when you can, simply because they can be a bit of a menace when you're trying to do things quickly. As long as you keep button mashing and have a favorable mix up of enemies storming your position, achieving eighty kills (which isn't literal kills but rather points contributed from enemy deaths) within the minute given shouldn't be too difficult of an endeavor for you.


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2017 Update: We still stand by this advice for nail colors for work, but you may also want to check out our latest discussion about work-appropriate nails.
We haven't had a poll on what nail colors are acceptable in a conservative office since 2011 by my count, so here's a fun question for today: what nail colors do you consider appropriate (and inappropriate) for your conservative office? For my $.02, flipping through Elle's recent slideshow on the top nail colors for Fall 2013, I see a lot of dark, muted colors that I probably would try in a conservative office — provided that a) the polish is in impeccable condition, and b) I didn't have any big meetings that day. That said, dark blues and grays are one thing — I can't ever see some of the 3D nail art (pictured) or crazy patterns/colors being appropriate for a conservative office. But I'm curious what you guys think — what colors (beyond pinks and beiges) are appropriate at YOUR office — and when?
Here's what we found on our last poll about which nail colors are acceptable for a conservative office:
And, because there are so many comments below, here's a quick and easy summary of what folks said…
Readers' Favorite Colors for Work:
Essie Ladylike, Mademoiselle, Chinchilly, Sugar Daddy, Ballet Slippers, OPI Lincoln Park After Dark, Little Ondine Cuddle
Some awesome other treatments to look into for healthy, polished nails:
Burts Bees lemon butter cuticle cream, Lush Lemony Flutter, Dior Creme Abricot, Dior Nail Glow, Body Shop Nail Buffer, Bath and Body Works One Minute Manicure, Salley Hansen cuticle massage creme
How about you? What are your favorite nail colors for the office? How has it changed as you've moved from office to office?


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Jackson Brierley Hudson Stoney
Solicitors & Family Mediation Service
We are a friendly and approachable firm of solicitors and have offered legal services to both individuals and businesses in Rochdale and the surrounding areas for well over 300 years.
Today, our solicitors have many years of combined experience and knowledge with differing areas of expertise to enable us to assist clients in a wide range of legal services both for individuals and businesses.
We wish to build and maintain lasting relationships with our clients. We combine our traditional values with up to date legal software and technology and our legal expertise to provide a high quality and efficient professional service that is also attentive and personal for each client.
From 1998 we have also provided a Family Mediation Service through our fully accredited Mediator.
Our costs are discussed at the outset and are transparent and competitive. Fixed fees may be agreed in appropriate cases. Legal Aid may be available in Family Mediation matters.
For You
Divorce & Family
It is an unfortunate fact of life that not all marriages, civil partnerships or cohabitations are successful. ..
Family Mediation
Mediation is a process by which couples seek to reach an agreement about arrangements for separation or divorce…
Managing the Affairs of Elderly People
Advances in medical science mean that people now live well into their eighties and nineties…
Residential Conveyancing
We are members of the Law Society's Conveyancing Quality Scheme (CQS) – the mark of excellence…
Wills, Trusts & Probate
The importance of making a Will cannot be overstressed. Each Will is tailor-made…
For Your Business
Commercial Conveyancing
For our business clients our services extend to the sale and purchase of business premises…
Company & Commercial Matters
For our business clients we can offer advice and services on incorporating a company…
Residential Conveyancing
We are members of the Law Society's Conveyancing Quality Scheme (CQS) – the mark of excellence…
Authorised and Regulated by the Solicitors Regulation Authority. SRA No. 51809
Solicitors Regulation Authority Handbook. (This link will open in another page)


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Security has quickly risen to the top of mind for embedded developers in the past year. Although the Stuxnet worm was a wake-up call for the embedded industry, there have been several other notable incidents since.
For example, as reported on CBS News, attackers were able to gain control of a home insulin pump and change its settings with the ability to seriously harm the patient
In a recent case in South Houston, Texas, CNN reported an attacker gaining control of the human machine interface (HMI) of the SCADA system controlling parts of the water treatment plant. In that case, the HMI security consisted of an easily guessed password.
Security is now a top priority for embedded developers because the systems they build can and will be used in critical infrastructure that is increasingly more automated and connected, in some cases to the outside world.
Some classes of devices are already secure because it was a top requirement from their initial concept, for example, communication devices used by the government and military. A majority of devices, however, are potentially unsecure. What is important to realize is that no device is ever completely secure, but developers need to strive to improve the odds through good design, programming, and configuration.
Security Best Practices
There are some basic rules and principles that help guide design and development decisions when building devices (Table 1 below). First and foremost, it's important to realize that security needs to be built in rather than tacked on. It's important to improve existing and legacy systems the best way possible, and new projects should have security built in from day one. It will pay off considerably down the road.
Table 1: Relationship Between Security Recommendation vs. Best Practices
The following is a list of security best practices (source: Writing Secure Code by Michael Howard and David LeBlanc,, 2004) as applied to embedded development:
* Minimizing the attack surface: Reduce the number of attack vectors into the system. Turn off features, services, and access not necessary for most users.
* Least privilege: Assign just enough privilege to an application, task, or process to achieve the job at hand. Too high a privilege level allows for unwanted access or behavior.
* Defense in depth: Rely on more than one layer of defense and don't count on any one layer as providing complete protection.
* Diversity in defense: Use different types of defense devices, software, or vendors.
* Securing the weakest link: Secure the most unsecure component, interface, or application, the most likely avenue of attack; because any system is only as good as its weakest component.
* Fail-safe stance: Expect vulnerabilities to be found; expect physical and remote attacks on the system.
* Assumptions about external systems: Avoid assumptions about other devices your product will be connected to. You can't assume external devices are secure, and be aware that your device is connected to a wide-open network.
* Security by default: Set the default configuration and behavior of the system to be as secure as possible. Turn off features, services, and access not necessary for most users.
* Simplicity and usability: Use simpler designs that are less likely to have security bugs and vulnerabilities and are easier to understand, audit, and test.
It's important to note that no system is ever completely secure. However, as we shall see later in this article, there are practical steps that can be followed that dramatically improve security for embedded systems. In some cases, these techniques can be applied to existing systems; in other cases, they are proposals for future system designs.


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by Tax Help Expert
The 21% corporate tax rate is definitely historical but nobody is mentioning the losers in this deal; the corporations earning less than $50K.
Until now the first $50,000- was taxed at a rate of 15%, now with the new corporate tax rate everything is being taxed at the new 21% tax rate. So instead of paying $1500- tax on every $10K they will pay $2100-; an increase of 40%.
Join in and write your own page! It's easy to do. How? Simply click here to return to Trump tax calc.
Can't find what you're looking for? You can search this site here.
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How will Trumps Tax Reform Affect YOU?
Check out this new calculator, to see how much you will gain or lose.
Get your exact child tax credit amount with Trump's new Family tax credit.
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Dear Members,
Our term 3 mini kids grading is next week, so make sure you get those forms and payment in to us by or on this Friday. This week we feature Warrior - Three Kings instructor, Mrs Debbie Hart (3rd degree black belt) in our newsletter.
Staff Profile - Mrs Debbie Hart
Mrs Debbie Hart began training in 2003 with her husband and two children and is now a 3rd degree black belt. She has also trained in Balintawak Arnis Cuentada (a Filipino stick fighting martial art) and is a Protect Accredited Trainer (Level 1) through world-renowned self defence experts, Protect Self defence.
In July of this year Mrs Hart competed at the Oceania Taekwon-Do Championships and won back to back silver medals in patterns and power breaking.
One of the many reasons she loves being involved in Taekwon-Do is that she gets to see students gain confidence, achieve their goals and encourage their peers. Mrs Hart became the Head Instructor of the Warrior Taekwon-Do - Three Kings branch in March, where classes are run for children on Tuesday afternoons.
Big Thank You!
Many thanks to the team of helpers who came along to help reset the do-jang mats on Saturday! We really appreciate the help and the mats are looking great!
Class Cancellation Reminder
There are NO CLASSES on Thursday 18 September. However, if you normally attend a class on this day you are welcome to attend a class at the same time on a different day of the same week.
Have a great training week!
Best wishes,
Rachel Bates
Leave a Reply.
November 2019


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PO Box 20729. Charleston, SC 29413, USA
400-498 U.S. 52 Spur Charleston South Carolina 29403 US
(843) 224-8242(843) 224-8242
Daisy's Place Retriever Rescue is dedicated to saving and finding loving homes for Retrievers (and Retriever mixes) six years and older. Occassionally we do take dogs into rescue that are under six years old – some of them come to us from shelters where their correct age was misjudged and sometimes we just can't help but save the younger ones from certain death – we affectionately call these younger honorary Daisy Dogs, 'rosebuds'. We are an all volunteer non-profit organization and all of our dogs are in private foster homes.


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I have been fully swept along with the GBBO magic this year and watching pastry week this Wednesday had us desperate for some freshly baked pain au raisins as soon as it had finished.
So, this weekend, I made laminated dough for the first time in aaaaaages.
It's a faff, and it takes a while, but OMG it was worth it.
I used Paul Hollywood's laminated dough recipe (here), and having started a tad later than I had planned (8pm), I actually ended up squidging a few steps together.
Step 5 calls for 3 envelope folds, chilling the dough for an hour each time so the butter doesn't melt and seep through the dough. In an effort to not stay up 'til 1 am (yes, i'm old an boring now) I completed 2 envelope folds back to back, trying to be as quick as I could. Don't tell Paul! I hoped for the best, popped it back in the fridge to chill overnight and went to bed.
In the morning, I made a creme patisserie using Eric Lanlard's fool proof recipe (here). I just swapped in a teaspoon of vanilla extract rather than steeping the milk with a vanilla pod for a stronger flavour.
Whilst the creme patisserie was cooling, I squeezed half a lemon over two big handfuls of raisins and let them settle.
I have frozen half of the dough for another time. The other half though, I rolled out into a rectangle and smothered it with the creme patisserie and sprinkled on the raisins. This then got rolled up and sliced into rounds.
Turns out, in my infinite ability to be well prepared, I had run out of baking paper. I buttered and floured a flat tray and hoped for the best (for the second time this bake…). The rounds were plopped onto the tray for their last prove.
When we couldn't wait any longer, and the pastries looked like they had puffed up a bit, I popped them in the oven for 20 mins at 200 degrees. I think these are actually a tad over baked, I should have taken them out a couple of minutes earlier – they only take a second to turn – so I will make sure I keep a closer eye on the next batch.
Our craving was sated and we can continue normally again now 🙂
Until next Wednesday that is…
16 Comments on "Pain Au Raisin"
That looks perfect!
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