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WHEN: Saturday, August 10, 11:30am-3:30pm (repeats October 26)
WHERE: Tour departs from the Los Angeles Athletic Club, 431 West 7th Street,
Los Angeles, CA 90014.
COST: $58/person, includes complementary noir-themed gelato from Scoops
MORE INFO: visit http://www.esotouric.com or call 213-373-1947
In August, Esotouric, the eclectic bus adventure company whose
tours reveal L.A.'s secret history, offers a bus tour dedicated to the
city's greatest detective novelist, Raymond Chandler, from the downtown
streets he roamed as a young oil executive to his uniquely poisonous
portrayal of the city's hustlers, heavies and suckers. The tour reveals the
secret historic city that still breathes under our modern streets, and
offers unexpected insight into the life and work of a beloved writer and his
immortal white knight detective hero Philip Marlowe.
And just to show that Chandler scholarship continues to evolve five decades
after his death, Esotouric's Raymond Chandler tour features a startling new
theory developed through Esotouric's original research. Get on the bus to
learn all about the idealistic Los Angeles policeman whose one-man crusade
against the evils of his own department is among the most convincing cases
for a real life prototype of the Philip Marlowe character ever proposed. And
you won't hear about him in any of the Chandler biographies, but only on the
Esotouric bus.
For more original Raymond Chandler research uncovered by Esotouric, see the
blog post "Raymond Chandler and the Creamery" about the criminal happenings
the young writer might have witnessed at his first job in Los Angeles, at
the link below.
RAYMOND CHANDLER'S LOS ANGELES: IN A LONELY PLACE delves deep into a lost
city that exists in shadowy parallels to contemporary LA. Bungalows. Crime.
Hollywood. Blondes. Vets. Smog. Death. This was Raymond Chandler¹s city,
which resonated from deft and melancholy fits of his writer¹s bow. Join
Esotouric on an exploration of the city that shaped Chandler's fiction, and
that in turn shaped his hard-boiled times, in a four hour tour of downtown,
Hollywood and surrounding environs: Musso & Frank, Union Station, the Hotel
Van Nuys, Paramount Studio¹s gates, and beyond. Through published work,
private correspondence, screenplays and film adaptations, the tour traces
Chandler¹s search for meaning and his anti-hero Philip Marlowe¹s struggle to
not be pigeonholed or give anything less than all he has, which lead them
both down the rabbit hole of isolation, depression, and drink.
Get on the bus for a revealing time travel journey into the literary history
of Los Angeles, and discover the places and people who inspired Raymond
Chandler's timeless novels, screenplays and stories. And as a special treat,
the tour's complementary snack break features a selection of noir-themed
gelato flavors from the creative kitchen of Tai Kim's Scoops, the most
interesting and acclaimed ice cream shop in Los Angeles. Scoops doesn't
announce the featured flavors in advance, but Chandleresque offerings on
past tours have have included Coffee/Jack Daniels, Bacon/Caramel,
Guinness/Chocolate and even Nicotine (made with Nicorette gum)!
Upcoming Esotouric bus tour and LAVA special event schedule (free events
Sun. August 4 - South L.A. Road Trip: Hot Rods, Adobes, Googie & Early
Sat. August 10 - Raymond Chandler's Los Angeles
Sat. August 17 - Boyle Heights & The San Gabriel Valley: The Hidden
Histories of L.A.'s Melting Pot
Sat. August 24 - The Lowdown on Downtown
* Sun. August 25 - LAVA's Sunday Salon (info at lavatransforms.org)
* Sun. August 25 - Broadway on My Mind walking tour #2 (info at
Sat. September 7 - Hotel Horrors & Main Street Vice crime bus tour
Sat. September 14 - Weird West Adams crime bus tour
Sat. September 21 - Eastside Babylon crime bus tour
Sat. September 28 - Pasadena Confidential crime bus tour
* Sun. September 29 - LAVA's Sunday Salon (info at lavatransforms.org)
* Sun. September 29 - Broadway on My Mind walking tour #3
Sat. October 12 - Blood & Dumplings crime bus tour
Sat. October 19 - The Real Black Dahlia crime bus tour
Sun. Oct 20 - Crime lab seminar: Los Angeles 1920-1947: The Birth of
Forensic Science (info at lavatransforms.org)
Sat. October 26 - Raymond Chandler's Los Angeles
Sat. October 26 LAVA Literary Salon: A Charles Bukowski Evening (info at
* Sun. October 27 - LAVA's Sunday Salon (info at lavatransforms.org)
* Sun. October 27 - Broadway on My Mind walking tour #4
Sat. November 2 - The Birth of Noir: James M. Cain's Southern California
Sat. November 9 - Charles Bukowski's Los Angeles
Sat. November 16 - East Side Babylon crime bus tour
Sat. November 23 -Weird West Adams crime bus tour
* Sun. November 24 - LAVA's Sunday Salon (info at lavatransforms.org)
* Sun. November 24 - Broadway on My Mind walking tour #5
Esotouric's Kim Cooper and Richard Schave are proud members of LAVA - The
Los Angeles Visionaries Association. http://www.lavatransforms.org


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Here's a sneak peek of my project - please go here to see all the details and a step-by-step of how I put it together!
I will post them again, once we decide on the final design when we know boy or girl!
Thanks for stopping by and Happy Memorial Day!!

subset: commoncrawl


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I've been doodling the past few days, but I've produced nothing but rubbish... this isn't too far above that.
Wanted to try random expressions like you often see for character designs, it's Saber because she came up first in a google for "anime". The range isn't as great as I was intending... Took 90 minutes, which is ok for the linework, but I was intending it to be rougher (so I could do more). No point in applying good linework to all those distended heads. Apparently I'm also bad at being consistent in size since they all needed scaled to match. Sigh...
Thursday, March 13, 2008
Wednesday, March 12, 2008
Halo vs Metroid
Posted by Waseem Bashar at 10:51 AM 1 comment:
Labels: ninjha, video game
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Saturday February 2nd, 3:00pm
Dir. Kaige Chen (China: 1993), 171 min.
Adapted from the novel by Lilian Lee, Farewell My Concubine illustrates China's political turmoil during the mid-20th century. The film is based on the over fifty year friendship of two men who met as apprentices at the Peking Opera in 1924 Beijing, and the woman who comes between them. Farewell My Concubine shared the top price at the Cannes International Film Festival in 1993.
Wednesday, January 30, 2013
FREE SATURDAY SCREENING at CAMERA
Posted by Robyn Zolnai at 11:00 AM
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The Pineapple Express has come to town, and the TODAY show took note.
They never mention the 300 beautiful days we have every year.
Friday, November 30, 2012
Quincy on Today
0 comments Labels: environment, news, warning Links to this post Posted by Sierrosmith at 10:26 PM
Saturday, November 24, 2012
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Research suggests that probiotics work by correcting an imbalance of the friendly bacteria in a person's digestive system, according to WebMD. The digestive system contains over 500 different types of bacteria, which are believed to benefit everything from digestion and intestinal health to the immune system.Continue Reading
Some researchers suggest that certain things, such as infection or taking antibiotics, can cause this group of bacteria to deteriorate, explains WebMD. Research indicates this deterioration may lead to many digestive problems. Building up the numbers of friendly bacteria is thought to treat or prevent many of these problems.
Studies at Yale University from 2011 found that probiotics aid in several digestive conditions, including diarrhea, ulcerative colitis and eczema, that result from an allergy to cow's milk, notes WebMD. Research has also found that probiotics can prevent many digestive conditions such as diarrhea associated with the use of antibiotics and pouchitis, inflammation of the intestines that occurs after surgery. There is also a small amount of evidence to suggest probiotics may aid in other diseases, such as irritable bowel syndrome, Crohn's disease, diarrhea caused by C. difficile bacteria and vaginitis. They may even aid in childhood ailments such as colds, ear infections and strep throat.Learn more about Vitamins & Supplements


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It's been an odd weekend. I really wanted to get on and create some more Haiku comics for my final show, but found it really hard to get going! I spent most of Saturday trying to work on some of my existing ideas that I'd already sketched out, but not being able to get them working to my liking. I decided to go back to the source material and sat and read through some of the Haiku anthologies I got out of the library. It seemed to be a good idea, as I discovered a number of Haiku that I had previously overlooked, but which presented some interesting visual ideas...
Monday, 8 June 2009
Knock Knock. Haiku's there?
Posted by Paul Shinn at 00:17
Labels: comic strip, Haiku
3 comments:
- AnonymousJune 08, 2009
hey! I especially like those with the frog and the rain - it's the feeling of lonliness and the use of color!ReplyDelete
Your haiku comics are so beautiful! I've created a comic strip "Old Pond" featuring two frogs discussing haiku. Haiku and comics are a lot of fun!ReplyDelete
Thanks Tessa. I want to get back to doing some more Haiku Comics. It's funny how we both created comic strips involving frogs and haiku. hehe Small world!ReplyDelete
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Beever uses colors to imply that his work has come to life or to at least give viewers that insight or point of view that for a second they could believe that what they are seeing may be real.
Banksy rarely uses colors. He often incorporates a single color i a specific place in order to attract your attention to a specific portion or area of a work.
Beever's works customarily use the public surfaces of busy sidewalks ,parkground and in small cases the confines of particular efficient areas of a house or building.
Banksy uses large scale buildings and small imperfections in urban architecture to create a thought or idea
Beever's works insist that the realities of the regular world could easily become a figment of reality equipped with extreme imagination.
Banksy uses images to cause you to think about the political aspect of life. His works often push you to question how sound and sensible the actions of the government and those in power truly are.


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Hello once again family and friends!
Can you believe that Christmas has come and gone and the New Year is upon us!? It is time to make those New Year resolutions once again. I have definitely learned the value of setting goals while I have been here. We must prayerfully ponder on how we can improve and then make goals that will help us achieve those improvements. I know that if we make the goals, make the plans, and pray that ANYTHING is possible!
Today all the missionaries gathered at the offices to practice our play. Missionaries are so funny and creative! We also planned the grand finale. Once again, when we came to the part of Mary for the finale, everyone automatically started saying "Hermana Lutze! Hermana Lutze!" I don't even know who these people are!? This is what happens when you are the only white blonde girl in the vicinity. You get picked to do everything and stick out like a sore thumb. So now I am officially the Mary of the grand finale. After our practice, a family, whose nephew collected clothes for the missionaries, brought them in for us. Christmas was here early. It was so nice and thoughtful! As missionaries we will always accept new clothes! We need to do that at home.
We tried to teach but Hermana was so sick it wasn't going well. Eventually we called the Bishop. He and his family took us to a doctor. It turns out that she had Strep throat (I totally called it!) The doctor prescribed her a billion pills! I have never seen so many pills! It was ridiculous! I'm pretty sure it was just so she would have to pay more money. She also had to buy an injection and Hna Sanchez (Bishop's wife)gave it to her. (They give injections at home here.) We stayed home for the night and Hermana Rubina slept. I was so grateful to Bishop Sanchez because he drove us, found the doctor, and paid for it. Without him, I don't know what we would have done! He really taught me what it means to consecrate all that we have to the Lord. We need to each have an eternal perspective in life so that when we are called upon to help our brothers and sisters we will be prepared to willingly sacrifice whatever is required.
Wednesday - ACTIVIDAD NAVIDEÑA!
Today we traveled to a church in San Pedro and watched Avengers 2! Awesome!...except it was in Spanish... and I don't yet understand Spanish movies very well. So I just laughed when I remembered it was funny or when everyone else was laughing!;) After the movie we had a piñata! It was so fun to watch the Elders swinging madly at the air! They were so energetic and enthusiastic to break that piñata! Oh and did you know there is a piñata song!? I will have to write it down and teach you all when I return. After the pinata we ate dinner. Then we went to the park-Bosque Urbano and the Planetarium. Ultimately we ended up at the amphitheater to perform our play! It was awesome! Ours was definitely the funniest and produced the most laughter! After the play various people performed talents. They are so gifted! Watching them helped me realize what it means to use it or lose it. We need to use our talents to help, uplift and bless others because if we don't, what was the point of having that talent? There is no point in only using our talents for ourselves. Oh, I ate some strange desserts today. One was a cream mixture with pineapple, pears, and carrots? The other was a chocolate cake with some kind of fruit in the middle? They were not my favorites.
We had previously bought gifts for our investigators and today we wrapped them! Actually, I wrapped them while Hna Rubina rested some more. That evening we went to the house of Hermana Lourdes and ate dinner with them. We had carne asada, cebollas (onions), grilled peppers filled with cheese, tortillas and guacamole! Delicious! Everyone was saying the guacamole was spicy and burning their mouths. Then they asked if I was having the same problem. I told them that I was fine. I have now passed the Native Mexican Guacamole test! (Thanks Grandma for making salsa so hot that it kills small children.) We gave them our gifts and then went home.
Christmas! Well, it didn't really feel like Christmas. It was sunny and 70 or 80 degrees. We had an Actividad Deportiva. We played different sports. Sadly the sisters cannot play contact sports with the elders so all we played was volleyball or kickball. Not quite as fun as soccer. Then in the evening I got to talk to my family! Best part of the day! Being away from them I have really learned how much I truly love them and am so grateful that I can be with them for eternity! I could never ask for a better gift from the Lord!
This was a mostly normal day of teaching with a few weird moments. We were teaching Maria when her drunk brother walked in. We ended up teaching them both, but let me just say it is HARD teaching someone that is drunk! He asked the strangest questions and constantly interrupted us! Don't drink totally not worth it to be that annoying and dumb. We also went to see our progressing investigator Mario. (The one we found smoking last week.) He doesn't know how to read so he is learning with the Book of Mormon. When we came to visit him today, he had all these doubts and questions about Habakkuk in the Bible. These questions came because he had watched a movie with this person's name. I was confused. ;) We had to explain that movies are not doctrine and are for entertainment and do not always have truth in them.
We taught more people today and really saw some miracles. We visited with Pati who is a recent convert and menos activo. We were teaching her and then had the opportunity to teach her husband who is not a member. We were able to address some of his fears and doubts and now he wants to learn more! This will be so good for Pati to have more support in her religion.
We also taught Omar and Marcia again. Today their son had a rocking horse. He got on it, clicked his tongue, put on his cowboy hat and said 'Yee-haaaw!' He's so adorable! We brought up baptism which opened a whole slew of questions and doubts. However, with the help of the spirit we were able to answer their questions and give them the commitment to pray to our Heavenly Father for an answer. They need to feel the spirit and obtain their own answers of whether or not this is the truth. I am praying they get their answers.
Well, that is all for this week!
I did come up with a new acronym this week. I.L.L. Infectious Love of Life! Friends and Family, life is good. There are bad things that happen, but still, life is good! We have so many blessings especially as members of the Church of JESUS CHRIST of Latter-day Saints! We need to take this life and enjoy it! This is our time of probation! We only get one, don't waste it. Also, our love should be infectious! We are members of the church that has the restored gospel and has everything we need to return to our Heavenly Father!! WE need to share this joy with everyone around us! If we have an Infectious Love of Life then everything will seem a little brighter and go a little smoother. Our trials and troubles will not seem so great especially since this ILL comes from our Savior Jesus Christ! When we are yoked with him, everything is possible!! Spread the ILLness! ;)
Love you all! Hope you are all still becoming and failing forward! The only thing that can stop you is yourself!
Hermana Lutze
P.S. It is freezing here! Day and Night! This wind started blowing and now I feel like I am in Estonia with Kyle! Brrr! I thought I was in Mexico!?
P.P.S We were at a house this week that had cockroaches EVERYWHERE. One of them jumped on my coat. It was a miracle I didn't scream and freak out. My companion saved me.


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Wayne Krantz - Signals - 1990 - Enja
"Krantz has versatility, taste and improvisational eloquence. One of the top 5 CDs of the year" - Jazziz
"Fiery passion and stellar technical facility." - Fanfare
"A remarkable complexity of rhythms and melodic shapes." - Audio
"Good musical ideas and guts, too…as exciting as something you'd have heard at a Mahavishnu Orchestra concert in the '70s. 4 and 1/2 stars." - Downbeat
Guitarist Wayne Krantz's hardness and intensity serve him well on Signals - a respectable fusion date uniting him with drummer Dennis Chambers, keyboardist Jim Beard and bassist Hiram Bullock, among others. When Krantz lets loose and digs into such complex yet accessible jazz-rock as "Faith in the Process," "Don't Tell Me" and "Alliance," the listener can focus on the depth of his playing. Guitarist Leni Stern joins Krantz on the reflective "As Is." © Alex Henderson © 2012 Rovi Corporation. All Rights Reserved http://www.allmusic.com/album/signals-r142310
A great album of instrumental jazz rock/fusion from five masters of their instruments. Wayne Krantz's guitar skills are awe inspiring. He is one of the most innovative guitarists playing today. In 1996 Wayne played with Steely Dan on their world tour. He will amaze you with his unique signature rhythmic approach and totally distinctive style. The bass work from the late Hiram Bullock and Anthony Jackson is an education in itself, and of course the drumming from the unbelievably talented Dennis Chambers is fabulous. There are some quiet moments on the album, but overall it's very, very impressive. The guys produce some magical grooves. "Signals" is VHR by A.O.O.F.C. Listen to Wayne Krantz and Leni Stern's "Separate Cages" album. You can also hear some of Wayne's superb guitar work on Donald Fagen's "Morph The Cat" album. For music in a similar vein, listen to the late Emily Remler's "Transitions" album [All tracks @ 320 Kbps: File size = 76.1 Mb]
1. Alliance 3:38
2. Faith In The Process 3:53
3. One Of Two 4:17
4. Don't Tell Me 5:30
5. As Is 3:30
6. Signals 5:35
7. Sossity; You're A Woman 3:33
8. Music Room 4:50
9. Two Of Two 3:17
10. For Susan 3:41
All tracks composed by Wayne Krantz except "As Is" by Leni Stern, and "Sossity, You're a Woman" by Ian Anderson
Wayne Krantz, Leni Stern - Guitar
Hiram Bullock RIP, Anthony Jackson - Bass
Jim Beard - Keyboards
Dennis Chambers - Drums
Don Alias RIP - Percussion
Wayne Krantz (born July 26, 1956 in Corvallis, Oregon) is an innovative American musician, who is widely recognized as a technically advanced jazz fusion guitarist. He has played with top artists such as Steely Dan, John Zorn, Michael Brecker, Billy Cobham, and others, but currently has a solo act. Krantz released his first album, Signals, in 1991, sporting an array of recognized jazz musicians such as Dennis Chambers, Leni Stern, Anthony Jackson, and others. However, in 1992, he formed a trio with bassist Lincoln Goines and drummer Zach Danziger, and recorded two albums with them; Long To Be Loose, in 1993, and a live album, 2 Drink Minimum, in 1995. In doing so, he began to play periodically at the 55 Bar, a diverse and premier jazz club in New York City. In 1996, Krantz released an acoustic album with Leni Stern, dubbed Separate Cages. Wayne formed a new trio in 1997, consisting of his ferocious guitar skills, complemented by Tim Lefebvre on electric bass and Keith Carlock on drums, new sounds that would change his music's style drastically. On June 28, 2007, Krantz played his final regular Thursday night gig at NYC's 55 Bar. In an announcement by Krantz to his mailing list notifying his fans of the change, he stated a desire to move towards a "louder thing" requiring "bigger rooms, with stages and sound systems to pull it off." Krantz's first three solo albums were released on the jazz label, Enja Records, which was at the time also home to frequent collaborator Leni Stern. However, Krantz's last three albums, 1999's Greenwich Mean, 2003's Your Basic Live, and 2007's "Your Basic Live '06" were both released from Wayne's private website. Like 2 Drink Minimum, these two albums both are excerpts of various sets at the 55 Bar. These two albums also include more use of effects pedals, and are more unscripted and improvised than the previous three, implying Wayne's tendency towards nonconformism on and off the stage. He contributed to Steely Dan co-founder Donald Fagen's newest release, Morph the Cat, and participated in touring with Fagen's band in early 2006. In another rare sideman role, Krantz is featured on tenor saxophonist Chris Potter's 2006 release, "Underground." Additionally, he is to return to the studio in 2006 to record a new solo album, to be followed by touring. It is unknown whether this new album will feature Wayne's existing trio. Even with a lack of a record company, and thus an absence of excessive advertisement and sponsorship, Krantz's music still receives acclaim and a worldwide underground fan base, strongest in the New York metropolitan area and Britain. Wayne Krantz signed with record label Abstract Logix to release his first studio record in over fifteen years.Krantz Carlock Lefebvre (2009) features the core trio of Keith Carlock on drums, Tim Lefebvre on bass, and Wayne on guitar. As a guitarist, Krantz is known for being a relentless individualist, which is evidenced in his improvisational style. In his book "An Improviser's Operating System," Krantz outlines his approach to improvisation, which relies not on licks or memorized fretboard patterns but an awareness of musical "formulas" on the instrument. Krantz's improvisation is known for its spontaneity, and he makes a distinction between this form of improvisation and "composition," the latter of which may include any musical idea that is preconceived on the instrument. While many guitarists rely on other music as a model for their own playing, Krantz is highly committed to realizing his own voice on the instrument and claims not to derive a great deal of inspiration from other music. Though Krantz has studied other players in the past, he does not consider himself a stylist (i.e. someone who models his or her playing on a predefined stylistic template, such as pure bebop, rock, blues or funk.) In addition to its concern for melody and harmony, Krantz's music demonstrates a high rhythmic awareness, often incorporating polyrhythms, odd metric groupings and displaced beats. Krantz is most identified with a Stratocaster-type electric guitar. Recently he has played a model manufactured by Tyler Guitars. Earlier releases such as "Signals" feature a chorus effect commonly associated with fusion guitar. Within several years, however, his sound became more organic, featuring analog effects such as overdrive, wah-wah, and a Moogerfooger ring modulator. For amplification, Krantz has used both Fender Deluxe Reverb and, more recently, Marshall amplifiers for a heavier sound. On May 22, 2004, Krantz married smooth jazz vocalist Gabriela Anders. Together they live in New York City with their daughter Marley.


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ISTP Conference in Toronto
A common assumption in much of psychology is that the body is best (if not solely) understood as a physically deterministic entity, even when the existence of a mind/psyche is granted. Here I wish to explore an alternative conception of the body—that of lived embodiment—and make a modest proposal on its ramifications for how we understand addiction. I begin by discussing some of the important assumptions of the physicalistic model, namely linear temporality, causal determinism, and the notion of "habit" implied in those assumptions. Next, I will discuss an alternative conception of embodiment, drawn particularly from Maurice Merleau-Ponty and supplemented by the phenomenology of Martin Heidegger. The lived body, as found in our everyday movement in the world, exhibits a non-linear temporal horizon through which the world is habitable. I do not act from the temporally punctualized t1 to t2, but I act 'during the lecture,' 'as I cook,' or 'during the conference'; the temporal horizon is spanned. Similarly, it does not follow the if-then logic of causal determinism or rationality, but is based on "motivation"—rather than if I do this, then that will be the result, bodily motility is structured as if I am to do this, then I need to or must do that. For the body to act in a motivated way means its being "geared" towards the world according to specific intentions and, thus, it acts meaningfully. Lastly, "habit," rather than being a learned response to stimuli, is a "style" of being in the world, a particular way of dealing with various contexts. With the above in mind, I propose that the embodied aspect of addiction cannot be fully understood in causal terms, but must include the body's circumstantial grasp of the world, motivation-based actions, and stylistic modes of being in the world.Admittedly, I have not written the paper yet, but I'm pretty certain it will work.
There is so much going on I don't know which sessions I will attend. However, the following definitely stick out at me: Relationship Between Language, Mind & Culture, The "Costs" of Being Dialogical: Assuming the Theoretical Consequences of the Dialogicality of Psychological Knowledge, Hermeneutic and Phenomenological Explorations, Constructing the Self and Identity (I and II), History Without Theory is Empty & Theory Without History is Blind, Exploring Ontological Relationality: Ricoeur, Buber, MacMurray, and Girard, and Embodied Subjectivity. It looks like this will be a really interesting conference and a great chance to network with like-minded pyshologists. So, if you just happen to be in Toronto at the time, come on over and see what theoretical psychology is like.
Labels: Maurice Merleau-Ponty, Phenomenology and Psychology
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I don't remember how many times I've been to Luxembourg, but I do recall that every time I went there I got the same feeling: bored! No sparks to it and most of the people who go visit it have the same impression.
But this week-end we went and visited some friends who moved there from Belgium and got some pretty interesting insights. I must say, I never thought I would say this, but Luxembourg is looking pretty good now. Our dear friends are known to be explorers and always into discovering the places they live in, travel to or just pass by. So, I think this help them see Luxembourg from a different perspective and pass on this new discovery.
Besides the fact that taxes are dreamy, in comparison to Belgium where they tax the air you breath, it appears to be opened to internationals, rich in culture, kids friendly, plenty of things to enjoy and of course well geographically positioned. But I let you make up your own mind when you get there.
Another thing I discover was the tartiflette. I know, it's not Luxembourgish, it's French, but never had it until now. Waw, it was an explosion of flavor, carbs using such simple ingredients. Simply surprising! It is a winter dish, but seriously, have it whenever you can. You only get potatoes, loads of cheese, bacon, sour cream and other beautiful secrets to make a fantastic down to earth dish.
It got me thinking how easy it is to label a place, a person or whatever and how difficult it is to change up your mind afterwards. This trip helped me realize that I have misjudged Luxembourg. Additionally, didn't even know how such a simple dish can be so delicious and surprising.
I don't know if we will ever move to Luxembourg, but I am sure I will eat again tartiflette. I will keep it though for special occasions, it's a whole event to digest it 🙂
Thank you Fiona & Rasmus for being so welcoming, day trips to Luxembourg are more tempting now!


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Sunday, January 10, 2010
Big Oil, the Military Industrial Complex, and UFOs
A major question in the whole UFO phenomena is this: Why is the existence of UFOs and intelligent ET life kept secret from the public? There are a number of possible explanations for this. For instance, perhaps the people at the top believe such information would create a mass hysteria. Another reason may be that the information could conflict with some religious beliefs.
But consider this: What if the primary reason the ET presence is kept secret is because of its potential to eliminate the oil-based economy that world politics and the balance of power is based on?
Assume for a moment that UFOs are indeed real (which I guarantee you will conclude if you free your mind from the stigma society has put on UFOs and spend more than a few passing moments looking into the topic) and this knowledge was made public. Following the public's initial excitement/hysteria regarding ET life and other civilizations in the universe, wouldn't the FIRST question asked be - how the hell do you power those UFOs if it isn't with fossil fuels?
What if UFOs were powered by a source of energy that was free, non-polluting, and unlimited? What if this form of energy existed on earth - only our science hasn't yet discovered how to harness it? Couldn't a free, clean and unlimited supply of energy end poverty, reduce disease, save the environment, and allow world civilizations to flourish?
Now, imagine for a minute you are the person at the head of Big Oil. You have an incredible amount of power through political connections throughout the world. Would you not do everything possible to suppress this information from reaching the public? Maybe this is a cynical viewpoint – but I urge you to polish up on your world history. In particular, read "A People's History of the United States" by Howard Zinn (available free, online, here: http://www.historyisaweapon.com/zinnapeopleshistory.html). History is littered with a small number of people who have had an incredibly disproportionate amount of money, political influence and power. And they do everything possible to keep from losing that power. That is, until a majority of people realize what is truly going on.
You may be thinking - "… wouldn't Big Oil want to get their hands on this clean and free energy so that they could package it and profit from it?" Well, if the system isn't broke – why fix it? From Big Oil's perspective, the system is working fine – it is highly profitable, and ensures their place at the top in the global power structure. Why risk all of their power with the development of a new energy source? What if it was so unlimited they couldn't profit from it? If everyone had a free power source, wouldn't the existing world powers have to make room at the table for all large nations? If energy was no longer the limiting factor of power and influence, wouldn't the nation with the largest population rise to the top? Why would they want that?
What's interesting is that there is substantial amount of evidence that new alternative energy technologies have been suppressed many times in the past. This includes hydroxy gas energy systems, thermal exchange motors, and pulsed motor generators. An example is Stanley Meyer's development of a car that was powered by a water fuel cell. His work quickly attracted the Pentagon - but then his work became ridiculed by the media and a few in the scientific community. Stanley Meyer died suddenly in 1998 after dining at a restaurant. Since then, important pieces of his device have been missing.
Anyone interested in this should look into The Orion Project, a not-for-profit company headed by Dr. Steven Greer and Dr. Theodore Loder to identify plausible alternative energy technologies and assemble teams to develop these systems further (http://www.theorionproject.org/en/index.html). I highly encourage everyone to visit their site to check up on the latest in their research – believe it or not, their work could have an enormous impact on the future landscape of energy.
Next, aside from Big Oil – what is the other major US industry whose livelihood is directly linked to world power and politics? The military industrial complex (MIC). War is very, VERY profitable to the MIC. When the Roswell event occurred in 1947, the US and Soviet Union were in the midst of a cold war. World power was still up for grabs and a crash of a UFO provided a unique opportunity to advance technology for military use.
If you haven't already, please read Col. Philip Corso's book, "The Day After Roswell". In this book, he describes in detail how the military provided previously unknown UFO technology to different research institutions for back-engineering purposes. In exchange for a patent in the "new" technology, the research institutions had to develop the technology for military use. The technological advancements that Col. Corso claim came out of this program were night vision, kevlar, laser, fiber optics, and the microchip. But is it constitutional or lawful for technology to be developed solely for the military sector, while keeping everyone else in the dark about new developments that could eliminate poverty, hunger, disease, and environmental catastrophe?
There is substantial evidence the PRIMARY reason UFOs are kept a secret is for the benefits of Big Oil, and the Military Industrial Complex.
Many UFO researchers are convinced that President Eisenhower was the last US President to be fully briefed on the UFO phenomena. At some point during President Eisenhower's term, the people at the top with UFO knowledge decided it was too risky to involve a person elected by the people. Listen to this extremely revealing clip of President Eisenhower discussing the potential threats of a Military Industrial Complex left unchecked in his farewell address to the nation:
The next US President, JFK, was, reportedly, extremely interested in the UFO phenomena. A number of memos he wrote to the CIA regarding the topic are available here: (http://www.majesticdocuments.com/documents/1960-1969.php). What did JFK find out? Was it more than he was supposed to? Listen to this segment of a speech he gave regarding corrupt organizations within the US government - was he trying to tell us something?
On November 22, 1963, JFK was assassinated. If anyone thinks there are no questions left unanswered about his assassination, I greatly urge you actually read about what happened. For starters, the Secret Service's protection of him during the Dallas parade was laughable compared to the standards for such Presidential events. Not to mention assassination threats had increased in the time prior to this parade. Please watch this 3 minute clip of real footage showing the agent in charge calling off JFKs two most important body guards at the last minute before the parade. The second video discusses more of the Secret Service's shortcomings that day.
I am not arguing that any one of these points proves the UFO phenomena is real – I just think it is important for people to think about this information in a historical context. It's amazing how so many people do not question events that have taken place. For instance, what if the Secretary of Defense came forward and said the Military could not account for over two TRILLION dollars of spending during ONE fiscal year. Wouldn't that be a big story? Wouldn't people demand answers? Well watch this:
Does this not echo the messages given by President Eisenhower and President Kennedy? Where did this money go? What was it spent on? 2.3 TRILLION dollars? That's nearly two and half economic stimulus plans in ONE YEAR! Why aren't people demanding answers on this?
Don't get me wrong – I am a proud US citizen. But evidence suggests that the dynamics identified by Howard Zinn in "A People's History of the United States" still exist today. A small number of people have an enormously disproportionate amount of money, power and political influence. I think people should begin to consider whether the vast majority of US and world citizens are being kept in the dark about the UFO phenomena for the profits of an elite few.
I'll end with the following analogy posted on David Wilcock's webapage (http://divinecosmos.com/index.php/start-here/davids-blog/521-disclosure-endgame). I encourage everyone to check out his thought-provoking material.
"The bully didn't want any of the kids in 'his' schoolyard to play with the other kids -- the ones who lived all throughout the neighborhood.
He guarded the gates. He liked being the Big Fish in a Small Pond. He told elaborate stories of how evil, treacherous and deceptive the other kids were.
He wanted everyone to respect him and pay him for protection, while fearing anyone and everyone else around them -- including him most of all.
If the kids found out they had all these other friends in the neighborhood who could have helped them, they might have quickly forgotten about the bully... and laughed at his once-fearsome authority.
Over the years, the bully grew weak and tired. He couldn't beat people up anymore, so he started paying his friends to do it.
Now he has run out of money to pay all his friends. They, too, became tired of all his lies and backstabbing games -- so as soon as his money ran out, they ditched him.
It took a while for all the kids on the schoolyard to realize the bully could not hurt them anymore. His power was so feared, so legendary, that the stories of his beatings continued to awe and inspire them for some time.
But there were cracks in the armor. A series of signs -- irrefutable and undeniable -- started happening that gradually made the kids realize the bully's power to hurt them had evaporated.
Now all the people he shamed, humiliated and attacked over the years are finally standing up -- and they're going to tell us the truth.
They're going to tell their story... of what it was like to live in fear of this bully for so many years, and never be able to share the secret truth of the horrors he was putting them through.
Once the bully is gone, the children can run and sing and dance and play happily -- quickly recovering from the nightmare of abuse they endured for so long. They won't have to hand over all their lunch money to him and his friends any more.
And they will soon find out they have a lot more friends than they ever could have imagined -- regardless of whatever horror stories the bully tried to tell them about how mean and evil all the other kids were."
5 comments:
I completely agree as far as the whole Big Oil and Military Industrial Complex. It's a little curious how fast we have advanced our technology in the last hundred years. I mean it's possible that we achieved this by ourselves but honestly I feel like there was some kind of influence to it. And your completely right about the oil company and nations, who would want to give up all the money and power if UFO's existed and gave us some new form of energy.ReplyDelete
I have read that there are devices that can be used to create near unlimited forms of energy. One new upcoming energy form would be plasma, a professor named Chernetsky created a paper in the 70's saying that a new type of high-frequency plasma generator could produce 5 times the amount of energy that it consumed. Any new form of energy with a high output and a low input to get it started would result in the Oil companies being obsolete.
I defintely think that if the government is keeping UFO secrets from everyone is to insure that the involvment of their technology doesn't mess with the system of power the world has.
- AnonymousJanuary 21, 2010 at 12:37 PM
I just read something interesting recently that lends to the whole if people are going to lose money then they will do everything in their power to stop it idea. Your idea says that if extraterrestrials were able to give us some form of new energy then the big companies would try to stop it.ReplyDelete
Now if your know your history you would know about Nikola Tesla, whom tried to create the first wireless transatlantic signal against his competitor. J.P Morgan was a silent partner in it, and apparently he found out that Tesla had secret to his invention, I don't exactly remember the name of the project but it was something Wynderclyff or something to that effect in Long Island, NY.
His secret was that this device would also be able to power people across the globe (not sure how far the range was), for free. J.P Morgan found out about this and blackballed Nikola leading him to become blacklisted by hundreds of other companies. The reason for this is that companies wanted to be able to regulate and price energy (electricity), and when they found out Tesla was about to power people for free, then they stopped him.
Wonderful points.ReplyDelete
The sad thing I want to point out, which actually has something to do with what kind of Civilization we are on a Universal scale, is that the oil based companies are slowing down/if not halting human progress. Think about this since we are considered a Type 0 Civilization, since we rely on fossil fuels and oil, and there has been not much of a push forward to other types of energy... when are we going to become a Type 1 Civilization (One that harnesses the power of the planet, aka Solar, water, weather, etc.)ReplyDelete
This makes me upset that the human progress as a whole is stuck on the whims of Big Oil Company. People are so scared about losing their jobs and money, that it stops a LARGE amount of progress. We as a race, can benefit from getting rid of obselete and diminishing sources of energy... Will everyone finally learn to accept other forms once we dry up these resources? I hope we don't have to wait that long or else we'd be at a severe Energy Crisis because if we keep downplaying alternate sources it will lead to bad things.
One main thing that could happen is that we might not be able to finance a large enough revamp of our energy systems, once we dry up our old resources. I really hope people get it through their thick heads that fossil fuels don't last forever...
- AnonymousJanuary 29, 2010 at 2:30 AM
Btw did you hear about the new "sighting" in Brooklyn, NY? There some videos from williamsburg showing what looks like a plane's headlight but it was too lowReplyDelete
Look it up on youtube I can't post links to youtube on here for some reason


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8 After Party: Our Guest Bedroom
>> Saturday, May 15, 2010
Over the past week, we've been hard at work primping our guest bedroom. Keeping it green by decorating with items we already had hanging around the house or recycled from a previous owner, not only did we give ourselves a pat on the back for creating an earth-healthy space but also saved a bundle!
Take a gander at our guest bedroom the day we moved in. You know it was in serious need of a little TLC…
..and now our fabulously planet friendly guest bedroom!
That's our guest bedroom in a nutshell! Check back next week to find out where we snagged all of the goods for this space.


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Friday, December 17, 2010
Peering into the Cold
At some point in the building's history, the basement was extended, an interior staircase was added, and a sort of bathroom was installed. The view shown here is from the basement door, looking along the southeast side of the church and toward the front.
Our plan is to enlarge and replace the windows to capitalize on the southern sun exposure. Between the windows we hope to plant a salad garden, using cold frames to extend our growing and harvesting seasons. But unless we can figure out how to prepare and serve cactus, we'll have to figure out the best way to dig it out of the ground; somewhere in there lurks yet another cactus patch.
Posted by The Butlers at 8:10 PM 0 comments
Building the Bathroom
I'll admit: this whole process is taking longer than we'd hoped. My husband is highly skilled, but highly skilled in completely different areas. He can accomplish projects on an amazingly tight deadline, but building a bathroom isn't exactly radio production.
What's unnerving is that at some point he'll have to disconnect the toilet, and at the rate we're going, it's anybody's guess how long it will take before the new toilet is available for use. We do intend to hire a plumber, but Paul also hopes to set the stage by doing as much of the prep work himself.
Well, Christmas is coming, and that means holiday travel for family celebrations as well as freelance work to fill in for vacationing broadcasters. Whatever Paul can get done tonight and tomorrow is what will welcome us when we return in the new year, and it is what we will be thankful for. Our God provides, and we are grateful.
Posted by The Butlers at 7:15 PM 1 comments
Labels: bathroom, demolition, schedule
Monday, December 13, 2010
We looked at the property twice before closing, once in the high heat of summer and once in its waning days. Both times Paul and I alternately fought our way through the chaos of vegetation and tiptoed our way around "dog mines" on our quest to see the fruit trees and berry patches in the backyard.
It wasn't until the ground was frozen that I decided it was safe to explore the grounds on my own, so I can't exactly wonder that I hadn't seen this espalier before the day Robin and I guarded the burn pile.
It's a curious location for this Golden Delicious apple tree, but the fascination of finding an espalier prompted me to discover the benefits of training fruit trees along a fence or against a wall. After a little reading, I'm eager to find other locations around the yard and give it a try.
One obvious benefit is that it's easy to reach the fruit on an espalier, but it also seems that it will produce more fruit as an espalier; instead of the tree spending its energy on branch growth, it concentrates more on fruit production.
An espalier takes up less room in a garden, so more of them may be planted, and even in small spaces and corners a person can reap a bounty of fruit.
Any fruit tree may be trained in an espalier, but stone fruits such as peaches and cherries are better suited for a more natural, informal pruning into a 2-dimensional shape.
It takes a few years for a fruit tree to get established, and we would have to prune off any fruit that it puts out during that time, but harvesting and putting up the fruit would be well worth the wait.
Posted by The Butlers at 7:59 PM 0 comments
Labels: garden
Saturday, December 11, 2010
Keeping Warm
I didn't want to at first, but Paul thought we really should stretch plastic across the windows. There are storms on the outside, but the various cracks and holes in the stained glass pieces weren't doing us any favors. So on Wednesday, while Paul was working to put insulation back up in the bathroom ceiling, I climbed up the extension ladder countless times with the rolls of double sided tape and sheets of shrinkable plastic and managed to seal off four of the six windows.
I'm pretty satisfied with how they look. Time will tell whether or not this will help us keep our 40-year-old furnace heating costs down.
Posted by The Butlers at 8:55 AM 0 comments
Labels: expenses, stained glass windows
Sunday, December 5, 2010
Beauty in Consensus
Many factors, most of them plumbing related, went into creating this bathroom floor plan, but just as many factors went into determining the flooring itself.
After sorting through the options, we decided that linoleum is the right choice for us: it's very inexpensive, it's not quite as cold on the feet as ceramic tile, it's easy to clean, and it lends a certain vintage charm to the space. The question I needed to work through was how we should use the linoleum tiles.
There are a lot of color and layout options to consider. I'd seen a picture online of a really nice border laid in a contrasting color, and I thought maybe something like that would work here. However, the layout of the room doesn't lend itself well to that approach. Besides, laying a border would also add a lot of work to my poor hubby's burden, so the border is out.
The size of the tile was an issue initially. My preference is not for the 12-inch square tiles; for laying an interesting pattern in contrasting colors, the scale seems a bit too large for the room. Additionally, they're much larger than the 9-inch vintage tiles. However, cutting them down would be wasteful, and not just wasting materials: even if each tiles were cut down into four 6-inch tiles, much of Paul's precious time would be wasted, because, as he said, he would have to lay four times the number of tiles than if we used the whole 12-inch tiles. So for Paul's sake I needed to work with the big tiles as they were.
To avoid ending up with a tile pattern that seems too large for the space, then, we'll be choosing two, low-contrast shades of the same color, probably light yellow, and lay them in a checkerboard pattern. I'm hoping that this will give the impression of design without overpowering the space with busyness, especially since I'd like the hooked rugs I intend to make to have a simple canvas as their backdrop.
Even with the subtle design in the tile layout, I still felt like a diagonal layout was almost necessary to visually unify the two parts of the room, to lead the eye from the vanity to the tub. So I was able to secure Paul's blessing to lay the tile on the diagonal. It will require some extra work, but nothing like the other ideas I bounced past him.
So I'm happy, and Paul's happy. It's a beautiful thing. Hopefully the bathroom will be, too.
Posted by The Butlers at 10:58 AM 0 comments
Labels: bathroom
Saturday, December 4, 2010
Northwest Windows
From left to right: The sheaf of wheat symbolizes the bread of the New Covenant, the open Bible exhorts us to make God's Word central to our lives, and the dove with the olive branch speaks of peace and happiness such as Noah would have experienced following the worldwide flood.
Of all the kinds of stained glass windows that could have been here, I consider myself blessed that these windows feature simple, flowing lines and symbolic representations rather than ornate motives and figurative depictions. They're beautiful, and for the most part, I wouldn't change a thing.
Posted by The Butlers at 9:59 AM 1 comments
Labels: architecture, nave, stained glass windows, tour
Southeast Windows
These are the stained glass windows on the southeast side. Clicking on a picture will enlarge it for looking at the detail.
The window on the left, closest to the front of the nave, features a bunch of grapes to communicate the wine of the New Covenant. The central window portrays a crown and a cross to remind us of Christ our King. The window on the right features an anchor, an ancient symbol of hope.
The windows were installed in 1910, and the glass itself is Tiffany-style glass, composed of many layers of color within each piece. Elliot commented that the glass in the main part of the outer windows looks like mother-of-pearl, and indeed, it does.
I'm sure there's quite a bit more to say about the glass and the style and period of the design, but I'd need to do more research first. When we are able to turn our attention to restoring them, we'll write more about what we learn.
Next: the windows on the northwest side.
Posted by The Butlers at 9:32 AM 0 comments
Labels: architecture, kids, nave, stained glass windows, tour
Not Worn Out Its Welcome
On our way out to The Nest last Tuesday we stopped to pick up this entrance door, shown resting upside down, that we found posted on Craigslist.
The door, while perhaps in need of a refinishing, is solid wood, and it came with a sturdy Anderson storm door and two sidelights for just $125.
I will miss the beautiful beveled glass window in the poor, decrepit door that struggles to keep out the cold, but I'm hoping the replacement side lights will allow in more light than the current ones do.
And I like how the paneled crown top is reminiscent of the detail in the lower portion of the stained glass windows. It's not something that most people would notice, but the unifying detail helps the door seem like it belongs.
Posted by The Butlers at 9:02 AM 0 comments
Labels: architecture, bargains
Prepared for Snow
We've braced ourselves to brave the snow this trip. Paul particularly wanted to beat the snow so he could stretch a tarp over the dumpster and keep the snow from weighing it down when the truck hauls it away.
Posted by The Butlers at 8:47 AM 0 comments
Labels: demolition
Name That Bush
One thing we learned after we bought our house in Chicago: don't yank anything out of the ground until you see the flowers! Thinking that dozens of plants with coarse leaves were just weeds, we pulled as many of them out as we could. Happily, we missed some; in early summer we discovered some unexpected, pretty blooms gracing a coarse-leaved plant which turned out to be a generously hearty campanula.
I don't know what these bushes are, but I do know that they have nothing to fear.
Posted by The Butlers at 8:12 AM 2 comments
Labels: garden
Where There's Smoke...
The wind calm, Robin and I took charge of the burn pile. Mounds of leaves and bits of other burnable junk all waited their turn, and to make a fire strong enough to withstand their frosty moisture, I dragged over a couple of the leave-behind beams, once salvaged from a collapsing homestead somewhere, now rotting in rows in the side yard, and added them to the pyre.
We knocked a pretty big chunk out of the collection of yard junk and even used some of the outdoor time to enjoy a cup of hot chocolate together. We would have done more, perhaps, but we've been sternly cautioned about the noxious nature of sumac, so we left it alone. For now.
Posted by The Butlers at 7:24 AM 0 comments
Labels: cleaning, kids, leave-behinds
Wednesday, December 1, 2010
Along the Sides
More (fuzzy) pictures of the nave. Elliot stayed in the shot to give an idea of the scale, but Aelsa joked that with how quickly he's been growing lately, perhaps there were better people for the job. She wasn't volunteering, however.
Regarding the scale of the windows and the space they fill, it's worth mentioning that the window trim is 10" wide. From the floor to the top of the window trim is 12.5'.
Stained glass window detail coming later.
Posted by The Butlers at 12:48 PM 2 comments
Labels: kids, nave, stained glass windows, tour
Setting the Stage for a Repeat Performance
Posted by The Butlers at 12:35 PM 0 comments
View from the Back
It's hard to get the room to fit into one picture, but here's my attempt to capture it from the back. Notice how the stage is set for the sleeping show. And the stairs to the basement are on the right.
That's it! The space in its entirety is about 28 feet wide by 38 feet long, and we have big plans for it. More on that later.
Posted by The Butlers at 12:31 PM 0 comments
View from the Front
So far I've posted several close-up pictures of isolated elements of The Swallow's Nest. But now here's the beginning of a tour of the grounds to give you a better idea of the space we're adapting.
This is the view from the platform in the front of the nave. The doors in the back lead to the front entry. Yes, the front door is in the back of the room. We hope this won't lead to much confusion. =)
You see the carpeting and the linoleum floor have been removed. The guys finished that up last week. It's interesting to see where the pews sat; marks on the floor reveal where they were bolted to the floor, and stripes of varying colors betray the toll that years of foot traffic took on its once-protective finish.
Our plans are to sand the floor and finish it in a dark brown stain, but that won't be until the bathroom is functional and we have bedrooms designated downstairs: the platform is currently where we stage our airbeds, sleeping bags, and pillows. It's quite a show, but it's been known to put everyone to sleep.
Posted by The Butlers at 12:22 PM 0 comments
Winter Has Come
Winter arrived with snow today after several days of cold, windy weather. And today, after two overnights in our country estate, we make our exit.
If, when he calls in to the Cook County courthouse on Thursday, Paul learns that he doesn't need to report for his jury duty summons on Friday, then we'll drive back out Friday morning.
The plan for this weekend, then, is to frame the bathroom walls, but we still need to lift some of the stubborn linoleum tiles from the concrete floor. I think a heat gun will help loosen it up, and we'll bring one out with us when we come back, but perhaps someone else has a better suggestion... ?
Posted by The Butlers at 11:52 AM 2 comments
Faith: a Work in Progress
Faith is looking at a mess like this and believing that it will one day be beautiful. No wonder faith and hope are so closely linked.
Posted by The Butlers at 9:40 AM 0 comments
Labels: bathroom, demolition
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text:
We're taught not to judge a book by its cover, but sometimes you can judge people by their shoes.
In a study published in the Journal of Research in Personality, researchers from the University of Kansas and Wellesley College found that certain shoe characteristics are telling of the wearer's personal traits. The research, published online this month, explored the concept of thin-slicing, wherein you form impressions based on small amounts of information.


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it's really hard to keep up with a blog during the summertime...
i'm currently accumulating many interesting thoughts and things that i most certainly want to share with you, however, i'm finding it difficult to sit down and make it happen.
i blame my drinking and sports addictions.
i should probably seek help.
until then, i'll leave you with this:
jersey shore: season 2 just started, so let's celebrate how shitty the show is with last season's most memorable moment:
i hope the entire cast drives off a cliff.


text:
Over time, your computer accumulates a lot of junks in a lot of ways, and it can eventually slow your machine to a crawl. So start thinking of your PC the way you think of your car. Like it or not, you can't simply run your machine forever without it developing some quirks. It's an analogy the computer industry needs wants to help people understand: Computers need routine maintenance.
Call us today at 330-865-8800 for more detail.
Our friendly staff is ready to help you with all your computer needs.
Apple Dell Acer Asus Compaq Fujitsu Gateway HP Microsoft


text:
Cellular systems are generally robust against fluctuations of intracellular parameters such as gene expression level. However, little is known about expression limits of genes required to halt cellular systems. In this study, using the fission yeast Schizosaccharomyces pombe, we developed a genetic 'tug‐of‐war' (gTOW) method to assess the overexpression limit of certain genes. Using gTOW, we determined copy number limits for 31 cell‐cycle regulators; the limits varied from 1 to >100. Comparison with orthologs of the budding yeast Saccharomyces cerevisiae suggested the presence of a conserved fragile core in the eukaryotic cell cycle. Robustness profiles of networks regulating cytokinesis in both yeasts (septation‐initiation network (SIN) and mitotic exit network (MEN)) were quite different, probably reflecting differences in their physiologic functions. Fragility in the regulation of GTPase spg1 was due to dosage imbalance against GTPase‐activating protein (GAP) byr4. Using the gTOW data, we modified a mathematical model and successfully reproduced the robustness of the S. pombe cell cycle with the model.
A genetic tug‐of‐war system is used to explore the sensitivity of the fission yeast cell cycle to changes in gene dosage, revealing a deeply conserved fragile core. A mathematical model can reproduce the robustness of the cell cycle, and points to currently unknown regulatory mechanisms.
An experimental genetic tug‐of‐war (gTOW) system is developed for fission yeast that allows quantitation of the robustness of the cell to gene overexpression.
The limits of gene overexpression were measured for 31 fission yeast cell‐cycle regulators.
The robustness of the cell cycle seems to be conserved between distantly related eukaryotes (the budding and fission yeasts).
These data are used to build an integrative mathematical model of the fission yeast cell cycle, which can reproduce the robustness of this system.
Intracellular parameters such as gene expression require optimization, such that cellular functions may be performed effectively (Alon et al, 1999; Zaslaver et al, 2004; Dekel and Alon, 2005; Wagner, 2005). Fluctuations in these parameters lead to various cellular defects. Overexpression of genes involved in proliferation of cancer cells due to gene amplification is a prime example (Albertson, 2006). On the other hand, in order to maintain cellular functions despite environmental change, mutation, and noise in intracellular biochemical reactions, these parameters may have certain permissible ranges, a characteristic termed robustness, which is commonly observed in various cellular systems (Barkai and Leibler, 1997; Little et al, 1999; von Dassow et al, 2000).
We previously reported a method designated genetic 'tug‐of‐war (gTOW),' by which we can measure the limit of gene overexpression in the budding yeast Saccharomyces cerevisiae (Moriya et al, 2006). In gTOW, a target gene with its native regulatory regions is cloned into a particular plasmid, and the copy number of the plasmid is increased by genetic selection. Next, the copy number is measured just before the cellular system halts (i.e., the cell dies), such that the overexpression limit of the target gene is evaluated as the gene copy number limit. As the gene copy number increases, relative overexpression of the gene is expected. If we can measure the copy number limit of gene overexpression, we can evaluate the degree to which the cellular system resists overexpression of the target gene (namely, robustness versus gene overexpression).
Using gTOW, we previously measured the copy number limits of 30 cell‐cycle regulators in budding yeast (Moriya et al, 2006). The data were used to reveal the robustness profile of the cell‐cycle regulatory system, and to evaluate and refine the integrative mathematical model of the budding yeast cell cycle (Moriya et al, 2006; Kaizu et al, 2010). The fission yeast Schizosaccharomyces pombe is distantly related to S. cerevisiae (Sipiczki, 2000), and like S. cerevisiae, is an established model eukaryote for the study of the molecular biology of the cell cycle (Egel, 2004). In this study, we developed a gTOW method using S. pombe and determined the copy number limits of 31 cell‐cycle regulators. The data thus obtained were used to compare the robustness profiles of budding and fission yeasts in order to reveal the conserved/non‐conserved properties of the eukaryotic cell cycle. The data were also used to constitute an integrative mathematical model of the fission yeast cell cycle.
Development of plasmid vectors used for S. pombe gTOW
The scheme of S. pombe gTOW is described in Figure 1A. The plasmid for gTOW must have the following properties: (1) the copy number in each cell is multiple; (2) the copy number is diverse in each cell; and (3) the plasmid contains a gene with a selection bias for increasing the plasmid copy number (leu2d in the case of S. cerevisiae gTOW). Because S. pombe possesses no plasmid with the above properties, we first constructed plasmid vectors for use in S. pombe gTOW.
Given below is a brief summary of the construction of the plasmid (details are given in Supplementary information). We used pDblet (Brun et al, 1995) for the backbone of the plasmid because it satisfies properties 1 and 2 above, and built S. cerevisiae LEU2 with promoter deletions into the plasmid to satisfy property 3. We first used S. cerevisiae leu2d (a LEU2 allele used in S. cerevisiae gTOW). In S. cerevisiae, the selection bias of leu2d to increase the plasmid copy number can be controlled by the leucine concentration in the medium (Moriya et al, 2006). However, this proved impossible in S. pombe because the final copy numbers of the leu2d plasmid (pTOWsp‐H) were the same in media containing different leucine concentrations (data not shown). This is probably due to differences in the leucine‐sensing mechanism between the yeasts. We thus optimized lengths of the LEU2 promoters (Supplementary Table S4) and developed three independent gTOW vectors (pTOWsp‐L, pTOWsp‐M, and pTOWsp‐H) with different biases to increase plasmid copy numbers in leu1Δ S. pombe cells (Supplementary Figure S1). Average plasmid copy numbers of the plasmids in the leu1Δ cell in the leucine+ media (uracil− selection) were 8.5±3.3 (all three plasmids showed no difference), and in the leucine− media were ∼41.3±13.0, 55.4±11.3, and 116.8±7.0, respectively (Figure 1B; Supplementary Table S4). Differences between the gTOW vectors in S. cerevisiae and S. pombe are shown in Supplementary Table S5.
In each plasmid, Ura4‐EGFP was expressed, such that distribution of the plasmid copy number within populations could be monitored. Distribution of the cells for each of the plasmids analyzed by flow cytometry is shown in Figure 1C. In the leucine+ condition (orange in Figure 1C), the plasmids tended to become lost because a portion of the cells showed the same fluorescence as the negative control (gray in Figure 1C). The average GFP fluorescence and copy number showed good correlation (Figure 1B), which indicates that GFP fluorescence is also useful in estimating the plasmid copy number within the cell.
Measuring copy number limits of cell division cycle regulator (cdc) genes using gTOW
We first selected 31 cdc genes (except byr4+, Table I) and measured their copy number limits using gTOW. These genes contain orthologs of the budding yeast cdc genes, which we had previously analyzed (Moriya et al, 2006). They also contained genes implemented into an integrative mathematical model described below. In gTOW, one needs to measure by how much the expression of a target gene can be increased from the native expression level; hence, whole regulatory elements on the 5′ and 3′ regions of the target ORF should be cloned into the plasmids. We thus cloned each target gene just before and after the neighboring ORFs (the case of cdc2 is shown in Supplementary Figure S3 as an example). In addition, we introduced frameshift mutations in five genes just after their start codons to test whether proteins expressed by these genes determine copy number limits (the structure of the frameshift mutant of cdc2 is shown in Supplementary Figure S4 as an example).
Each gene was cloned into pTOWsp‐L, ‐M, and ‐H vectors, and at least two independent clones were studied for each gene. S. pombe cells with each plasmid were grown in leucine+ and leucine− (uracil−) media. After 48 h of cultivation for pTOWsp‐L and ‐M plasmids and 72 h for pTOWsp‐H plasmid, plasmid copy numbers within the cells and GFP fluorescence of the cells were measured and cellular morphologies were observed. Cell size distribution was also monitored as forward scattering (FSC) by flow cytometry, and some examples are shown in Figure 2. Using combinations of leucine+ and leucine− conditions with three vectors, the copy number limit of each gene could be determined precisely (second and third columns in Figure 2). Using fluorescent microscopy, we could assess how cells with high target gene copy numbers (brighter GFP fluorescence) died (fifth and sixth columns in Figure 2). For example, in the case of cdc25, the copy number limit could be determined by the pTOWsp‐M vector, and cells became smaller in the leucine− condition. In the case of rum1, both cells and nuclei enlarged, while in the case of spg1, cells showed multiple septation. The pTOWsp‐H vector for these genes showed a very strong bias for cells with the plasmid in the leucine− condition (red graphs in the FACS histogram in the third column of Figure 2).
In addition, when we analyzed the gTOW experiments by time‐lapse microscopy, we could assess how cells with high target gene copy numbers died. Figure 3 shows an example of time‐lapse observation of a gTOW experiment with the target gene mik1+, which encodes a mitotic inhibitory kinase. Daughter cells with unequally distributed plasmid copy numbers had different fates; cells with low plasmid copy numbers barely grew, and after two cellular divisions growth ceased completely (black arrowhead in Figure 3). Cells with high plasmid copy numbers grew faster because they produced leucine but were unable to divide, resulting in elongation and eventual death (white arrowhead in Figure 3). On the other hand, cells with a balanced plasmid copy number maintained normal growth and division and accumulated in the media (yellow arrowhead in Figure 3).
The copy number limits varied from 1 (one extra copy of the endogenous copy number kills the cell) to >100 (Figure 4), indicating that the cell‐cycle regulatory system has a different robustness against overexpression of each gene, as observed in S. cerevisiae cell‐cycle regulation (Moriya et al, 2006). The frameshift mutants generally showed much higher copy number limits than the wild types (orange bars in Figure 4), but the frameshift mutants of wee1 and rum1 had lower limits than the pTOWsp‐H vector alone, probably because their frameshifts do not completely shut down protein expression. The morphology of cells with high copy frameshift mutant genes was similar to that of cells with high copies of their wild types (Supplementary Figure S6).
Comparison of robustness profiles of cell‐cycle regulation in S. cerevisiae and S. pombe
We next compared the copy number limits of the cdc genes determined in this study with that of the S. cerevisiae homologs determined previously (Moriya et al, 2006; Figure 5). S. pombe genes generally had lower copy number limits (average=52) than those of S. cerevisiae (average=87). We previously reported that cdc genes with lower limits constituted a fragile core that directly regulates the activity of B‐type cyclin Cdk (Cyclin‐dependent kinase) complexes (Moriya et al, 2006). The orthologs that constitute the core structure of S. pombe (Cdc13, Rum1, and Wee1) also demonstrated low limits, which may indicate the presence of a conserved fragile core in eukaryotic cell‐cycle regulation. In contrast, the orthologous components of the mitotic exit network (MEN) of S. cerevisiae showed very different limits from those in the septation‐initiation network (SIN) of S. pombe, although both networks have conserved architecture (Bardin and Amon, 2001; Krapp and Simanis, 2008). This might reflect the different physiologic functions in the two yeasts (namely, budding and fission), although these components have the same origin. These results suggest that comparison of robustness profiles is a useful way to reveal the conserved/non‐conserved properties of cellular systems.
Dosage imbalance causes very low limit of spg1
We recently demonstrated that the M‐phase phosphatase gene CDC14 has a very low limit is because of the dosage imbalance between CDC14 and its inhibitor gene, NET1 (Kaizu et al, 2010). Hence, we investigated whether the low limit of spg1, the lowest copy number gene in S. pombe cell‐cycle regulators evaluated in this study, was also due to dosage imbalance. spg1 encodes a small GTPase involved in the initiation of cellular septation; this activity is regulated by the bipartite GTPase‐activating protein (GAP), which is encoded by both cdc16 and byr4 (Furge et al, 1998; Figure 6A). The copy number limit of cdc16 is quite high (Figure 4), and cells with high copy cdc16 did not show any obvious phenotype (data not shown). In contrast, the limit of byr4 was very low (1.8±0.2, measured by pTOWsp‐L in the leucine+ condition). And cellular lethality due to overexpression of spg1 is nullified by the simultaneous overexpression of byr4 (Furge et al, 1998). We thus assume that proper activation of Spg1 is in delicate balance with Byr4, and hence, the limits of both spg1 and byr4 are quite low. To investigate whether spg1 and byr4 were in dosage balance, we performed two‐dimensional (2D) gTOW (Kaizu et al, 2010). As shown in Figure 6B, byr4 limits increased only when multicopy spg1 was supplied by another plasmid. Moreover, their copy numbers were balanced (Figure 6B, dotted line). Microscopic observation also confirmed our assumption that cells can divide normally only when spg1 and byr4 expressions are balanced (Figure 6C). These results strongly support the theory that spg1 has a very low limit because of the sensitive balance required for GTPase activity.
Development of an integrative mathematical model that reproduces the robustness of cell‐cycle regulation in S. pombe
Robustness can be a measure of plausibility in mathematical models of biological networks (Morohashi et al, 2002). We previously used copy number limits obtained for S. cerevisiae gTOW to evaluate and refine an integrative mathematical model of the S. cerevisiae cell cycle (Chen et al, 2004; Moriya et al, 2006; Kaizu et al, 2010). We thus evaluated and refined the integrative mathematical model, such that the model reproduces robustness (copy number limits of genes) of the S. pombe cell cycle obtained from this study. We previously published a mathematical model for S. pombe cell‐cycle regulation (Novak and Tyson, 1997; Sveiczer et al, 2000), and have independently modified the model from the gTOW experiment, such that the model reproduces more published experimental results. Here, we designated the model 'basic model,' the whole structure of which is shown in Figure 7A (green components; the simplified structure is shown in Supplementary Figure S8). We first investigated to what degree the core model reproduced the copy number limits obtained in this study (see Supplementary information for details). Although we did not use the gTOW data for development of the model, the model predicted the experimental data well (Figure 7B, green circles), and appeared to capture the robustness of fission yeast cell‐cycle regulation. To describe the gTOW data more extensively, we modified the basic model by adding some important regulators (M‐phase phosphatase Clp1, cyclins Cig1 and Puc1) and regulations, and the parameters were optimized by‐hand parameter adjustments (we designated the model 'gTOW model,' shown in red in Figure 7A; see also Supplementary Figure S8). The model successfully reproduced the copy number limits obtained in this study (Figure 7B, orange squares). In addition, the model could also reproduce 42 already published mutant behaviors (Supplementary Table S10). The result of time‐course simulation of the model is shown in Figure 7C. As a result, we can claim that the presented 'gTOW' model is the most detailed model of fission yeast cell‐cycle regulation so far.
Increase of each Cdc2/cyclin complex, but not the depletion of Cdc2/Cdc13 through the competition for Cdc2, determines the upper limits of cyclins (cig1, cig2, and puc1)
S. pombe contains four cyclins (Cdc13, Cig1, Cig2, and Puc1), all of which bind to Cdc2 to form a Cdc2/cyclin complex that performs regulatory functions on Cdc2 (Figure 8A). Cdk's (like Cdc2) are thought to be in excess of cyclins, but in S. cerevisiae it is not tremendously so (Cross et al, 2002). It is thus possible that if cyclins compete with each other for Cdc2, overexpression of one of the cyclins would interfere in formation of other Cdc2/cyclin complexes. Especially since deletion of Cdc13 can lead to lethality, there is a chance that overexpression of the other cyclins is deleterious for the cells because Cdc2 is titrated away from Cdc13 (Figure 8A‐2, case2). In other words, the overexpression limit of each cyclin may be determined by the increased Cdc2/cyclin activity itself (Figure 8A‐2, case1) or by the reduction of Cdc2/Cdc13 complex. To answer this question, we first used the mathematical model to investigate which conditions involve cyclins in competitive situations, and the experimental means to test these conditions. In the gTOW model (and in the basic model), each cyclin is considered to form a Cdc2/cyclin complex immediately after its expression; thus, no monomer cyclins or Cdc2 would be available and one cannot simulate competition with these models. Hence, we further modified the gTOW model, such that the behavior of monomer cyclins and Cdc2 could be simulated (green components in Supplementary Figure S8).
We first investigated the parameters of the condition in which cyclin competition occurs using this model, and observed that the condition is determined by a combination of the dissociation constant of Cdc2/cyclin and total Cdc2 level, and that this condition can be experimentally evaluated by measuring the copy number limit of each cyclin in the Cdc2 overexpression condition (Supplementary Table S12; Figure 8A‐3). If the limit is decreased, then the limit should be determined by an increase in the Cdc2/cyclin activity itself; and if the limit is increased, then the limit should be determined by the reduction of Cdc2/Cdc13 (competition). Figure 8B shows the copy number limits of cig1, cig2, and puc1 with multicopy cdc2; in all cases, the limits were decreased. We thus concluded that upper limits of cyclin genes are determined because of the high Cdc2/cyclin (Cig1, Cig2, and Puc1) activity itself, but not the depletion of Cdc2/Cdc13. This conclusion is supported by the phenotypic observation of gTOW‐cig1+, ‐cig2+, and puc1+ cells (Supplementary Figure S7). The cells did not show any elongated cellular morphology, which is a typical cellular phenotype when Cdc13‐Cdc2 activity is depleted.
Comparison of 2D‐gTOW data in silico and in vivo revealed uncovered regulations in the cell cycle
One of the practical values of the existence of the integrative mathematical model, just as we developed in this study, is that we can define the limit of our understanding and find novel biological knowledge using the model predictions as references of experimental results. We thus measured the copy number limits of the cell‐cycle regulators in the deletion strains, and compared the data with the model prediction in Supplementary Table S11. This '2D‐gTOW experiment' was performed with the deletion strain of each of four genes (cig1, clp1, puc1, and srw1). We chose these genes because the deletion of them does not give so much impact for the upper limits of the other cell‐cycle regulators in the model (Figure 9A; Supplementary Table S11). We thus expected that we could reveal uncovered regulations.
In fact, we found some discrepancies between the gTOW experimental data and the gTOW model predictions (Figure 9A). The rum1 limit in cig1Δ, the slp1 limit in clp1Δ, and cdc25 limit in srw1Δ were much lower than the model prediction. The pyp3 limit in srw1Δ was much higher than the model prediction. These results suggested additional regulations and wrong assumptions within the current gTOW model (summarized in Figure 9B).
gTOW in S. pombe
In this study, we developed a gTOW method in the fission yeast S. pombe. gTOW vectors in S. pombe have different biases for increasing the plasmid copy number. These vectors also have GFP, such that distribution of the plasmid copy numbers can be monitored by flow cytometry, or the plasmid copy number in a single cell can be estimated under fluorescent microscopy. In S. pombe, the nmt1 promoter has been often used to overexpress genes, but the upper limit is difficult to determine by this method. Thus, gTOW provides another means for overexpression of genes and for obtaining unique data to investigate the robustness of cellular systems in S. pombe.
Robustness and fragility in cell‐cycle regulation in S. pombe and S. cerevisiae
S. cerevisiae and S. pombe appear to have conserved fragility in the subsystem that directly regulates B‐type cyclin Cdk1 activity (Figure 5). Because these species are distantly related, with an evolutional distance of several hundred million years (Sipiczki, 2000), fragility in B‐type Cdk regulation would be conserved among eukaryotes. In contrast, networks regulating cytokinesis (MEN in S. cerevisiae and SIN in S. pombe) showed different robustness profiles (Figure 5), which might reflect their different systems of cellular division (budding and fission). Hence, comparison of robustness profiles would be useful to reveal the conserved properties of cellular systems.
In the cell‐cycle regulation of S. cerevisiae, the Cdk1‐counteracting phosphatase gene CDC14 had the lowest copy number limit, the reason being dosage imbalance against the inhibitor gene NET1 (Kaizu et al, 2010). The relatively high copy number limit of clp1, the S. pombe ortholog of CDC14, may be explained by the fact that Clp1 does not have a stoichiometric inhibitor (it seems there is an ortholog of Net1, but acts differently; Jin et al, 2007). In this study, we showed that the small GTPase gene spg1, the gene with the lowest copy number limit among the S. pombe cell‐cycle regulators, was also in dosage balance with the GAP gene byr4 (Figure 6). We therefore believe that dosage imbalance is a general mechanism for inducing cellular fragility in case of gene overexpression.
Integrative mathematical model of cell‐cycle regulation in S. pombe
In this study, we evaluated and modified mathematical models constituted from molecular knowledge using gTOW data. Although the initial model (the basic model) was independently developed from gTOW experiments, the model predicted the experimental results well (Figure 7A). This model is a quantitative model in which biochemical parameters had not been experimentally determined, but it already appears to have captured the robustness characteristics of the S. pombe cell cycle.
By addition of certain components to the model and optimization of parameters, we successfully developed a model (the gTOW model) to reproduce the gTOW data obtained in this study (Figure 7A) and the behavior of 42 other mutants (Supplementary Table S10). The model provided us with two types of predictions that should be evaluated by further research. One of these predictions concerns the effect of gene overexpression on phenotype. With simulations using the gTOW model, three different cell‐cycle defects were observed due to overexpression of each gene (Supplementary Figure S11). Interestingly, the model predicted that rum1 overexpression results in three independent phenotypes according to the level of overexpression (Supplementary Figure S12).
The other prediction concerns the copy number limits of each gene in its deletion strains (Supplementary Table S11). Upon comparison with the data in vivo, this type of prediction is quite useful to find additional regulations that are not implemented in the model (Kaizu et al, 2010). In this study, we measured upper limits of cell‐cycle regulators in four deletion mutant strains, which suggest some novel regulations in the network lacking in our model (Figure 9). We thus showed that the prediction of the model was quite useful reference to indicate the existence of additional (unknown) regulations conferring the robustness of the cell cycle. We propose that the following potential regulatory mechanisms provide an explanation for the 2D‐gTOW data in Figure 9.
(1) Low rum1 limit in cig1Δ: Cig1 might be more important for Rum1 degradation than it is assumed in the model.
(2) Low slp1 limit in clp1Δ: Clp1 might negatively regulate Slp1 activity or the level that is absent in clp1Δ background.
(3) Low cdc25 in srw1Δ: Srw1 might be important for degradation of Cdc25.
(4) Low pyp3 limit in srw1Δ in the model but not in the experiment: Pyp3 might be less important as a Cdc25 backup phosphatase than it is assumed in the model.
At this point, we did not modify the gTOW model with these suggested regulatory interactions because they are still hypothetical. These hypothetical regulations should be tested with additional experiments and the model will be refined with the regulations, just as we did in the budding yeast case (Kaizu et al, 2010).
Evaluation and refinement of integrative mathematical model using robustness information
A considerable body of knowledge has been accumulated for the fission yeast S. pombe and for the molecular details of the regulation of its cell cycle (Nurse, 2002), and this knowledge has been integrated into mathematical models before (Novak and Tyson, 1997; Sveiczer et al, 2000), and further developed in this study. These integrative mathematical models are expected to have very important roles in future life sciences because they can be used to predict behaviors of complicated biological systems that are difficult to handle intuitively.
These models are generally constructed according to the accumulated knowledge on the interactions between relevant molecules, but information on biochemical parameters needed to describe the strength and speed of interactions is usually lacking. Although these parameters are important to guarantee the plausibility of these models, it is sometimes technically difficult to obtain these biochemical parameters experimentally.
On the other hand, it is considered that these parameters should have some permissible ranges because biological systems have robustness (Barkai and Leibler, 1997; Little et al, 1999; von Dassow et al, 2000). Because the permissible ranges have emerged from interactions of components within the system, they are useful indicators for evaluation of the network structures of biological models (Morohashi et al, 2002; Moriya et al, 2006; Kaizu et al, 2010). In this study, we performed experiments to measure the permissible range of gene overexpression and used the data obtained to evaluate and modify the mathematical model. We believe this is a useful scheme for development of integrative mathematical models in which direct experimental measurement of biochemical parameters is difficult.
Materials and methods
Saccharomyces cerevisiae strain BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) (Brachmann et al, 1998) was used for plasmid construction by the gap‐repair cloning method (Oldenburg et al, 1997). Schizosaccharomyces pombe strain FY7652 (h− leu1‐32 ura4‐D18) obtained from NBRP yeast was used for gTOW, apart from 2D‐gTOW (byr4 versus spg1 and cyclins versus cdc2). In 2D‐gTOW of S. pombe, Sp286h+ (h+ ade6 ura4‐D18 leu1‐32), a haploid progeny of Sp286 (BG_0000, Bioneer) was used. For clp1Δ, cig1Δ, puc1Δ, and srw1Δ strains, haploid progenies of BG_0465, BG_4780, BG_4172, and BG_0289 (Bioneer) were used, respectively. The Escherichia coli strain XL1‐blue (Stratagene) was used for plasmid amplification.
Media and growth conditions
YPD and SC medium were used for cultivation of S. cerevisiae and LB broth for E. coli. S. pombe cells were cultured in a manner similar to that described by Moreno et al (1991). EMM medium and plates were prepared using EMM broth powder (MP Biomedicals). Yeast transformation was performed using the LiOAc method (Amberg et al, 2005). Yeasts and E. coli were cultivated at 30 and 37°C, respectively.
Plasmids used in this study
Plasmid vectors used in this study are listed in Supplementary Table S1. Construction details of the plasmids used in this study are described in Supplementary Information. The vectors pTOWsp‐L, pTOWsp‐M, pTOWsp‐H, and pA6R are available from the National BioResource Project (http://yeast.lab.nig.ac.jp/nig/index_en.html). The DNA sequences are provided in Supplementary information.
Measurement of plasmid copy number in S. pombe cells
S. pombe cells with pTOWsp containing target genes were grown in EMM without uracil, transferred into EMM without leucine and uracil, and then cultivated for 48 h (for cells with pTOWsp‐L‐ and ‐M‐based plasmids) or 72 h (for cells with pTOWsp‐H‐based plasmids). In 2D‐gTOW shown in Figures 6B and 8B, pTOW and pA6R derivatives were introduced into Sp286h+ cells, and the cells were grown in EMM without uracil and adenine; they were then transferred into EMM without leucine, uracil, and adenine and cultivated for 48 h. EMM was prepared using EMM broth powder (MP Biomedicals). The plasmid dosage in a yeast cell was determined using real‐time PCR, as described previously (Moriya et al, 2006), except that the pombe‐1 primer set (5′‐CCCTCAGTTGCTTCTCCTAA‐3′ and 5′‐GTGACTTCGTGAATCAAGTG‐3′), the ura4‐3 primer set (5′‐TCTTTGGCTACTGGTTCCTA‐3′ and 5′‐TATGTAGTCGCTTTGAAGGT‐3′), and the ade6‐1 primer set (5′‐GCCCATCGCTTAAACATCA‐3′ and 5′‐TGCATCGGGGTCAGTAAAT‐3′) were used for quantification of genomic DNA, pTOW plasmids, and pA6R plasmids, respectively. Data are averages of at least two independent experiments.
The copy number determined by PCR using the ura4‐3 primer is directly associated with the plasmid copy number in the cell because no sequence on the genomes of FY7652 or Sp286h+ is amplified by the ura4‐3 primer set. In contrast, the copy number determined by PCR using the ade6‐1 primer is one copy more than the plasmid copy number in the cell because an ade6 sequence on the genome of Sp286h+ is amplified by the primer set. The plasmid copy number is shown in all figures and tables. The copy number of a target gene in the wild‐type cell is one copy more than the plasmid copy number as a result of one endogenous copy on the genome.
Analysis of S. pombe cells by flow cytometry
Cells expressing EGFP were directly analyzed by flow cytometry (FACScalibur; Becton, Dickinson and Company). EGFP fluorescence was detected as FL‐1, and cell size was monitored as FSC. The data were analyzed using the FlowJo7 software (http://www.flowjo.com).
Microscopic observation of S. pombe cells
Cells were fixed by 70% ethanol for >10 min, pelleted, and resuspended in the VECTASHIELD mounting medium with DAPI (1/2 diluted with water, Vector Laboratories) and Fluorescent Brighter 28 (final concentration 10 μg/ml; Sigma). The cell suspension (2 μl) was spotted onto a glass slide and covered with an 18‐mm coverslip. The coverslip was sealed with nail polish, and the slide was observed using the Leica DMI 6000B microscope (Leica Microsystems). GFP fluorescence, DAPI and Fluorescent Brighter 28 fluorescence, and RFP fluorescence, were observed with GFP, A, and RFP filter cubes (Leica Microsystems), respectively.
Development of mathematical models and their simulations results are described in Supplementary information. The model files for XPP‐AUT and CellDesigner 4.2 are attached in Supplementary information. The SBML file of the gTOW model shown in Figure 7A can be obtained from BioModels database (http://www.ebi.ac.uk/biomodels‐main/) (ID: MODEL1111040000).
We would like to thank Hiroaki Kitano (Sony CSL), Yuki Yoshida (Sony CSL), Kazunari Kaizu (RIKEN), and Koji Makanae (Okayama University) for their valuable discussions with regard to this project, to Akos Sveiczer (BUTE) for his help at initial stages of modeling. We would also like to thank the Yeast Genetic Resource Center (YGRC), Japan, which is supported by the National BioResource Project (NBRP), for providing us with the S. pombe strain FY7652. This work was supported in part by PRESTO program and Strategic International Cooperative Program of Japan Science and Technology Agency; the Special Coordination Fund for Promoting Sciences and Technology and a Grants‐in‐Aid for Scientific Research on Innovative Areas 'The Physicochemical Field for Genetic Activities' of Ministry of Education, Culture, Sports, Science, and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author contributions: HM conceived and designed the experiments. AC and HM performed the experiments and analyzed the data. OK, ACN, and BN performed the computational analysis. HM wrote the paper.
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplementary Information
Supplementary Information
Supplementary Figures S1–13, Supplementary Tables S1–12 [msb201191-sup-0001.pdf]
This compressed zipped archive contains plasmid sequence files described below [msb201191-sup-0002.zip]
This compressed zipped archive contains ode model files described below [msb201191-sup-0003.zip]
CellDesigner4.2 file of the gTOW model [msb201191-sup-0004.zip]
SBML file of the gTOW model [msb201191-sup-0005.zip]
Movie S1
Time‐lapse microscopic observation of gTOW experiment with mik1+ gene [msb201191-sup-0006.mp4]
This is an open‐access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.
- Copyright © 2011 EMBO and Macmillan Publishers Limited


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Welcome to the publications directory for the Climate Impacts Group and the Climate Dynamics Group. Please contact the web administrator for assistance with any of these publications.
View: Abstract
Douglas-fir growth in mountain ecosystems: Water limits tree growth from stand to region
Littell, J.S., D.L. Peterson, and M. Tjoelker. 2008. Douglas-fir growth in mountain ecosystems: Water limits tree growth from stand to region. Ecological Monographs 78(3): 349-368.
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The purpose of this work is to understand the nature of growth-climate relationships for Douglas-fir (Pseudotsuga menziesii) across the climatic dimensions of its niche. We used a combination of biophysically informed sampling (to identify sample sites) and dendroclimatology (to identify growth-climate relationships) along a climate gradient in northwestern United States mountain ecosystems from the western Olympic Peninsula, Washington to the eastern Rocky Mountain Front, Montana. We used a multi-scale sampling strategy that accounted for continentality, physiography, and topography as non-climatic factors that could influence climate and alter tree growth. We developed a network of 124 Douglas-fir tree-ring chronologies and explored growth-climate correlations across the sampled gradients. We considered two different spatial scales of monthly and seasonal climate variables as potential controlling factors on tree growth.
UW Climate Impacts Group


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Reader recommendation: Water for Elephants
Monitor readers share their favorite book picks.
At first it's hard to get into Water for Elephants by Sara Gruen. But then it prevails as a heart-wrenching story that leads you slowly in. I find I cannot put the book down any longer.
Recommended: Could you pass a US citizenship test?


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Milan: A total of 83 countries have signed a declaration for protection of cultural heritage in Italy's Milan city.
Cultural ministry delegations from the countries signed the declaration on Saturday and expressed "their utmost condemnation of the use of violence against the world's cultural heritage", Xinhua reported.
The Milan declaration appealed to the United Nations (UN) and UNESCO to continue to support the international community in fostering positive communication between different cultures.
Italian Culture Minister Dario Franceschini, who organised this first international conference of ministry delegations from all over the world, announced Italy's intend to set up an international task force to protect the world's cultural heritage in case of terrorist attacks or natural disasters.
"Culture can be an instrument to overcome controversies. We must never underestimate our role and capacity for dialogue," Franceschini said.


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This week, I'm away in Rhode Island, on our annual 4th of July vacation. In my absence, I'll regale you with a family vacation story.
When I sent Uncle Joe the pictures that Maureen sent me from the James Mulvaney family, he called me up, and, among other things, the story of the time they met James on their way out to Breezy Point. A bus from the city would drop people off, and those people would catch rides out to Breezy Point with people heading that way.
There are two versions of this story:
According to Uncle Joe, he was in the car with Nana and Papa, and they saw James and pulled over to give him a ride. Because they were generally catching rides with strangers, he didn't give any thought to whose car he was getting in. He didn't notice who else was in the car until Nana said, "Hello, Jimmy." He was surprised. "Oh! Hello Sister."*
According to Aunt Betty, this event happened when she was in the car with Nana and Papa and a friend of hers. They saw Jimmy and pulled over to give him a ride. He was aware of whose car he was getting into, but when he did, he motioned towards Betty and her friend in the back seat and asked, "So which one's my niece?"
*All of her siblings always called Nana "Sister." She was the baby of the family, and it was the vestiges of having called her "Little Sister" when she was a child.


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By the way, a lot of people have been uploading their Halloween makeup recreations and costume pictures to my Facebook page! Please feel free to do the same if you'd like to share.
If you missed any of these looks and would like to watch the full video, check out my Halloween Playlist where you can find links to all the videos shown here and more from previous years.