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I got a reply from Tascam by e-mail they say it ships "sometime this week! "
I hope this is true.
Greetings Resident.
I got a reply from Tascam by e-mail they say it ships "sometime this week! "
I hope this is true.
Greetings Resident.
"Sometime this week" is no better than "sometime in April," "sometime in May," "sometime in June," "sometime in July," or "sometime in August." Why believe anything these liars say?
I only hope, i will only believe them when they actually send the package. But i got an e-mail and it is clearly saying this week! A lot of people here are complaining that they do not even get a reply from Tascam. Now i got a reply from the sales team, and it looks prommising. If no true is in it well i wait for another year! Always look on the bright sight of l...................!@
Here is the message!
GigaStudio 3 will be shipping sometime next week. You can pre-order by calling us at (323) 278-8584.
Thank You
Sales Department
TEAC America
7733 Telegraph Road
Montebello, CA 90640
Phone: 323-726-0303
Fax: 323-727-7621
And yes ofcourse we can pre-order! How nice of them don't you think! I think the people from tascam are not too bad.
Greetings Resident.
I have the greatest respect for the people that have developed the GigaSampler/GigaStudio software. But as for the company that has understaffed, underfunded and failed to accurately access the launch times of that development team, I do not have any reason feel good will towards their contribution to computer software yet. Letting people pre-order their product is in their best interest as well as the customers and should really be expected of the company. I appreciate your enthusiasm and optimism but would personally advise you to temper it by applying some business sense.Originally Posted by Resident
Every company that sells a product or service is dependant on customers to make money. The easier they make it for people to spend money on their product, the higher the likliehood that people will actually buy their product. The better their relationship with the customer, the higher the chance that the customer will contribute to the company in the future (through word of mouth marketing or by buying future products). Thus, when a company does not do basic things to promote a better releationship with their consumers, such as making it possible to pre-order directly or through their retailers when they've heavily promoted their product for months, then they are shooting themselves in the foot and should be recognized for it. If Tascam were doing something revolutionary in terms of customer service, I would step up and herald their contribution. But I don't this qualifies.
Now don't get me wrong. The GS 3 included libraries look great, the software looks robust, etc. but I was reading the PR back in January and saw the product demoed at NAMM and it is now August with the likely arrival time of the software looking like anywhere from next week to next month. It was originally supposed to be April. That is what I was told in person by the staff at NAMM. They didn't give me qualifications and they didn't allude to any challanges that could present problems. Now, I know that people who get excited about their product can be overly optimistic so I padded by a month or two. That would have placed me in May or June with the ability to use the software on anywhere from 2/3 to all of my projects for the summer. The end of January to the end of April is 3 months. The end of January to the end of August is 7 months. That's over twice as long as I originally expected to wait and I don't feel that Tascam deserves any cheers for it.
Once again, like I said, my qualm is not with the development team who, by every indication, have done an excellent job. But the parent company has no one to blame for their users frusterations but themselves. I hope they really learn from this experience and improve the quality of their relationship to their customers in the future.
Given the tendencies and history of the software industry, accusing Tascam of purposely lying to its customers in this situation lacks logical basis, proof, and is slanderous.
It is EXTREMELY LIKELY that these delays were unforeseen - every software company runs into this. And not one of you people who has the audacity to outrightly call these people liars has a solid footing to stand on. There is a remote possibility that you are correct and these individuals are morally less than your equals. But there's a much greater chance that when pressured to give an estimated release date, they complied and just turned out to be wrong because of factors they hadn't anticipated. Remember when you throw words like "liars" around that you are speaking of individuals, not just corporations, that go home and take care of their families, make donations to charities, and open doors for people. This reminds me of the political hate speech we here all the time. I start to wonder if some of you work or support other companies and wish to damage Tascam.
For what sake you need to estimate the release time! Ok you can misjudge some unforseen problems. But no problems for 6 months! I think and i don't blame individuals you have to be more carefull with promising release dates! You can easiliy preorder the product "HALF YEAR AGO" and then waiting waiting waiting. And now again the SALES people say SOMETIME this week, but you can ofcourse preorder! In marketing terms speaking this is selling AIR! And too many people just like me are willing too believe these lies.
I have no purpose of damaging Tascam or the individual people. I only like the product concept of Nemesys Gigastudio. And am curious for the release of GS 3. But i don't like the way of selling air like the way Tascam is doing. No words but only deeds is the slogan of my homecity "Rotterdam Holland". That means if you promise something you make it work. No means by damaging whatsoever.
Greetings Wilfred.
I saw GS3 at NAB. The demo was impressive. In the lower part of the screen during the demo was a little CPU activity monitor showing performance. The monitor indicated a quite high CPU hit. Someone at the Tascam booth stated that this might be a hold up in getting GS3 to market. I think that ANY software company that is getting a much anticipated product to market has to make sure that the product is as good as it can be before release. Any software that is released without this testing and optimizing is shot down in the marketplace. Tascam can not afford to have negative reviews of GS3 after release because the competition is strong. If they delayed to work out all the problems before release, that is a good thing. IMHO.


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Jose Mourinho has always been known to start games in the press conferences. The Portuguese manager is famed for mind games, which he uses to gain advantage over his opposition. In some ways, his mind games reveal the team Mourinho fears the most or the team he considers to be on the verge of cracking. Since becoming the Manchester United manager in the summer, Mourinho has been pretty much occupied by the top four race when it comes to the Premier League.
United has not been able to make the assault for the title, as Mourinho would have hoped to do in his first season at the club. Mourinho knows pretty well that the manager, who wins the league title for the first time in the post-Sir Alex Ferguson era, will be granted legendary status at the club. Yet, Mourinho would be surprised to see his team remain fixed to the sixth spot for what seems like ages.
The Portuguese appears to be quite envious of his former club, Chelsea, who are the favourites to win the league title as projected by CrownBet (click here to see the latest odds).
Mourinho had been hinting throughout the season that Chelsea has been one directional in their approach. His criticism of Chelsea and the style of play seemed weird considering that the blues have been one of the best in terms of defending and they have also scored much more than United this season. Mourinho claimed that his tactics and style of play were not being given the due credit.
The feeling of sour grapes appears to have elevated even further in recent weeks. Mourinho has now gone on to directly state that the blues have managed to win the league title – despite being only eight points ahead of Manchester City with 39 points still at the disposal. Given the intensity with which Mourinho's comments about Chelsea have gone up, it is clear that the 54-year-old is eyeing up an advantage for the upcoming FA Cup game at Stamford Bridge.
Mourinho will be returning to the scene of the 4-0 humiliation suffered back in October. It remains as one of his biggest defeats in the Premier league. His comments have subtly turned the attention away from this loss, while Mourinho will certainly be hoping that the recent praises upon Chelsea may create some lackadaisical approach coming into the match.
Chelsea are involved only in the FA Cup and the Premier league. The club managed to beat Wolverhampton in the fifth round of the competition, while Manchester United overcame Blackburn Rovers. This high-profile game between the two clubs is scheduled on March 13.

subset: commoncrawl


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It Goes to XI proudly presents the first post from contributing editor Gordon Jones, an NBA authority who actually owned a pair of XI's (white low tops with red patent).
Can someone please teach Dwight Howard a post move?
Go ahead and teach him to shoot free throws while you're at it.
Has any other professional team hired two assistant coaches to work specifically with one player? Orlando has hired Patrick Ewing to work with Howard on his post game and Mark Price to help him at the charity stripe. And apparently, neither is earning his salary.
After watching the Celtics, without their best player Rajon Rondo, thrash the Magic 87-56, I was appalled. Howard went just 4-15 from the field against the likes of Jermaine O'Neal and 54-year-old Kevin Garnett. Oh, and let's not forget Greg Stiemsma. None of those players should have a prayer of holding Howard to those kind of numbers. Howard should be steamrolling through the league to the tune 25 and 20 every night.
And he would, too, if he could develop a post game. I am tired of seeing the league's best center fall back on his only move. The "take a dribble, run throughout the lane and throw up a fading hook that occasionally looks like it could take down low-flying military aircraft move." If he can't dunk, that is his go-to move. No up-and-unders, no bank shots or baby hooks. No fade-away jumpers or dream-shakes. Not a single move that makes it apparent that he plays basketball for a living. Pretty stunning for the best center in the league.
Maybe the Magic should have hired Hakeem Olajuwon or Kevin McHale to be the offensive assistant in charge of Dwight Howard. With a few more moves in his repertoire, Howard wouldn't have to rely so heavily on his brute strength and natural athletic ability. Teams would also brutalize him far less if he shot free throws more consistently. These two improvements would help lengthen his career and increase his stature.
Howard is a fantastic player; he is the best rebounder in the NBA and reigning defensive player of the year, but he also has the potential to be even greater. It's time for Dwight Howard to go from being the best center playing now to being the best center ever.


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Freshman Monroe Scholar Research
Most funding available for undergraduate research goes to upper-level students. It is for advanced research conducted to support declared majors. In contrast, the Freshman Monroe scholarships encourage first-year Scholars to explore any discipline.
Freshman Monroe Scholars may apply for funding to undertake two-week research projects. These projects extend or build on topics they studied in their first year classes.
When freshmen become upperclass Monroe Scholars, they receive funding for a seven-week research project.
The Monroe Scholars Program provides stipends of $1,000 to approximately 50 first-year Scholars. The stipend buys us 80 hours (two full weeks) of your time over the summer. If your project is selected for a grant, you will
- Spend a minimum of two full weeks on your research
- Post a brief abstract of the Monroe project to the Freshman Monroe Research blog
- Post at least three additional entries to the blog over the summer. Your last post will be a summary of your project
- Post at least two substantive comments to other Freshman Monroe Researchers' blogs
- Present your work at the Charles Center-sponsored Summer Research Showcase event in the fall.
You will write your project proposal with guidance from a faculty advisor. If your proposal gets a grant, your advisor does not have to supervise your work over the summer. However, different advisors might be more involved. For example, if your research will take place in your advisor's lab, you will work under their supervision.


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Breakdown of an Accepted Conceptual Reality
Mind bending mushrooms might bend your mind enough to question a status quo, which we have accepted from parents, school, uni, work, media etc. If we have bought and accepted all the things these people tell us about the world – depression is one of the body's calls to make an adjustment and to trigger a similar effect of mushies – to force you to see things differently and to question the status quo – your conceptual reality – The Matrix.
Form is Form, Form is Emptiness
If mushies do not take you to the door of the Buddhist Centre, where they welcome any mind inquisitive to know what is meant by
form is form, form is emptiness and emptiness is form
…depression can also take you to this enquiry.
Anybody who has taken the liberty to pop a few caps a few times, will resonate with the above Buddhist aphorism.
Both liberty caps and depression invite you to stop doing this:
An Aside
Of course, the same enquiry exists in your Christian Church, but because us Westerners are trying to break out of the Matrix, anything Christian is probably conceptually associated with a system that try to keep us blind about the way things really are around here. And because of the energy we have invested in maintaining the status quo ( The Matrix) in place, it will also probably be true that the Christian Church only serves to imprison.
You may not have understood the full import of the previous sentence – it's something to meditate on
Depression – The Upside
The upside about depression is that it is your Soul really trying to break through and be heard. It means that you have a Soul connection; that you are not just a cog in a machine, a socio-path, a minion, a wanton slave. To have a Soul connection means your desire for human happiness is really strong and takes priority over material gains in the world. The stronger the Soul connection, the less you are able to function as a lubricating wheel in the Matrix and be happy at the same time.
Creative expression as a Soul Infused Being can take many forms. They are all efforts at peeling away the onion layers to whittle down to a certain Truth. As such, all efforts are flawed but shining bright.
A Journey Into Light – Alignment with Soul Fusion
One way to open up to the reality of what happens in your awareness, which does not damage the physical body like many hallucinogenics, and which does not require you enter into deep depression, is meditation.
Firstly, you may consider simply calming the mind in meditation. You will certainly benefit from meditations of Loving Kindness. You might be drawn to Vipassana Meditation.
The quantum leaps become possible when we learn about using the mind to move and circulate energies in meditation, and not before we are ready for this. There are many terms to describe this – Kundalini Kriyas, the Five Elemental paths of the Qi, and Swami Satchidanand's great work – Energy Enhancement.
Experiences of energy in meditation (Samadhi) and using this energy to remove blockages (Samyama), just as a farmer would remove the rocks from a water course to irrigate his land – is key to becoming a Soul Infused Being.
A man has blockages like a dog has fleas, and we vow to rid them all.
If you are interested in learning meditation or taking your meditation to the next level, please get in touch.
The post Mushrooms, Depression & Meditation appeared first on Journey Into Light.


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Worship Times
Sunday School
Youth (Jr/Sr High)
Activities & Events
Prayer Request
Request for Space
Contact Us
Church Staff
Donate Online
Governance Board
Web Site
Worship Times
Saturday evenings at 6:00pm
Sunday mornings at 8:00 and 9:30am
11am - "Reveal" (Contemporary worship)
See us on Facebook ( www.facebook.com/revealworshiptinleypark )
Holy Communion is celebrated each 1st and 3rd Sunday,
and corresponding Saturday.
Information about Communion
Worship is the life-blood of a Christian. We realize Christ's promise when He said,
Where two or three are gathered together in My Name, there I am in the midst of them." (Matthew 18:20)
The benefits in the common worship services are strengthening from the Sacraments,
hearing the truth of God from His Word and the joy of spiritual togetherness.


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Professional Cable, LLC, a leading manufacturer of cables and adapters announces that it is now an ICBA vendor partner.
Since 1927, The Independent College Bookstore Association (ICBA) has continued to be the only professional organization in the college store industry dedicated exclusively to institutional stores. The stores own and govern ICBA through an owner-elected Corporate Board of Directors.
ICBA is the organization that originated group buying in the college bookstore industry. Today they continue to deliver unsurpassed and innovative buying programs. There are a lot of "buying groups" in this industry. Independent College Bookstore Association is not one of them. ICBA is more than a buying group.
The Independent College Bookstore Association (ICBA) is a rapidly growing retail cooperative of independent and institutional college bookstores (no leased or private stores) that is one of the premier organizations in the bookstore industry. This nonprofit mutual benefit corporation is owned by its member stores and spans from Hawaii to Maine, from Texas to Minnesota, and throughout Canada.
Professional Cable believes that partnering with ICBA will help increase visibility in the college bookstore market and help to leverage already good relations between the stores nationwide.
Professional Cable offers low-cost retail-packaged cables that are ideal for college bookstores and other retail stores not wanting the fancy packaging that the expensive cables come in. Many buyers are concerned that clamshell packaging is bad for the environment and that Professional Cable's retail poly bags are more environmentally friendly.


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Dealing with Nerves
I can hardly believe it's already audition season again! During this time last year, I had been bouncing across the country for over a month. Juilliard was my seventh and final audition, and by that point, I was honestly too tired to be nervous beforehand. Of course, it was a different story once I got to the school on the day of my audition; I don't think I've ever felt so humbled, inspired, and intimidated all at the same time before.
Nerves are, simply put, really annoying. It's hard to play your best when you feel insecure and scared. I used to get so nervous during performances that my fingers and lips would shake uncontrollably...and as a flute player, it makes playing nearly impossible! As performing artists, we're probably going to spend our lives perpetually nervous, so it helps to at least start embracing instead of repressing your tendencies. Here are some tips on dealing with nerves to succeed in your next performance
1. Breathe slowly. You've heard this one over and over again, but it really does help! To bring your heart rate down and give yourself something to focus on, try taking full, deep breaths. Some people like to follow the 4-7-8 pattern (inhale for 4 seconds, hold for 7 seconds, exhale for 8 seconds). Do whatever works for you to calm down.
2. Know your tendencies. Everyone reacts differently to nerves and stress; some people tense up when they play, some people's mouths get really dry, and some people (like me!) can't stop shaking. Being aware of how you react in high-pressure performances helps you strategize to make your next performance go more smoothly. It also helps to talk to other people and find out their nervous tendencies and methods.
3. See performing in a new light! It helps so much to have a positive mindset to lower your nerves. Thinking to yourself, "I can't wait to play this!" instead of "I'm so scared to play this" makes such a huge difference.
If you're reading this and you have an upcoming Juilliard audition, the light at the end of the tunnel is approaching! You've worked so hard to get here, and you're almost there. If this is one of your last auditions, stay strong and don't forget to enjoy yourself. Best of luck--though you won't need it!


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Constitutively active RAS small GTPases promote the genesis of human cancers. An important goal in cancer biology is to identify means of countervailing activated RAS signaling to reverse malignant transformation. Oncogenic K-RAS mutations are found in virtually all pancreatic adenocarcinomas, making the RAS pathway an ideal target for therapeutic intervention. How to best contravene hyperactivated RAS signaling has remained elusive in human pancreatic cancers. Guided by the Drosophila studies, we reasoned that a downstream mediator of RAS signals might be a suitable anti-RAS target. The E3 ubiquitin ligase seven in absentia (SINA) is an essential downstream component of the Drosophila RAS signal transduction pathway. Thus, we determined the roles of the conserved human homologues of SINA, SIAHs, in mammalian RAS signaling and RAS-mediated tumorigenesis. We report that similar to its Drosophila counterpart, human SIAH is also required for oncogenic RAS signaling in pancreatic cancer. Inhibiting SIAH-dependent proteolysis blocked RAS-mediated focus formation in fibroblasts and abolished the tumor growth of human pancreatic cancer cells in soft agar as well as in athymic nude mice. Given the high level of conservation of RAS and SIAH function, our study provides useful insights into altered proteolysis in the RAS pathway in tumor initiation, progression, and oncogenesis. By targeting SIAH, we have found a novel means to contravene oncogenic RAS signaling and block RAS-mediated transformation/tumorigenesis. Thus, SIAH may offer a novel therapeutic target to halt tumor growth and ameliorate RAS-mediated pancreatic cancer. [Cancer Res 2007;67(24):11798–810]
- Pancreatic cancer
- RAS signal transduction
- RAS-mediated transformation and tumorigenesis
- SINA/SIAH family of E3 ubiquitin ligases
RAS proteins are evolutionarily conserved small GTPases that shuffle between a GTP-bound active state and GDP-bound inactive state ( 1, 2). The RAS signaling pathway controls cell proliferation, differentiation, and survival in all multicellular organisms ( 3– 6). Consequently, proper regulation of RAS signaling is critically important for normal development, and dysregulation of the RAS pathway leads to pathologic diseases ( 7– 9). Indeed, constitutively active RAS promotes cell proliferation, neoplastic transformation, and tumorigenesis ( 7, 10, 11). Oncogenic RAS proteins are commonly detected in human cancers including ∼90% of pancreatic cancers, ∼70% of malignant neoplasias and ∼30% of all human cancers ( 10, 12– 14). Despite the central importance of RAS signaling in human cancer, direct inhibition of hyperactivated RAS has not achieved sufficient clinical efficacy ( 8, 11, 15). We reasoned that targeting a highly conserved downstream signaling module of the RAS pathway might provide a more effective strategy for inhibiting oncogenic RAS signaling and RAS-mediated tumorigenesis in human cancer.
Signaling events downstream of RAS are complex, nonlinear, and dynamic ( 16, 17). RAS transmits signals from receptor tyrosine kinases to the nucleus through multiple downstream signaling pathways, chiefly the RAF/MEK/ERK, PI3K/AKT, and RalGEF pathways ( 4, 6, 18– 20). In addition, genome-wide surveys have found numerous putative RAS target genes mediating RAS transformation ( 21, 22). Despite the identification of these diverse RAS targets, it is still difficult to precisely determine which downstream signaling components represent the best therapeutic targets for blocking RAS-mediated neoplastic transformation/tumorigenesis. Thus, identifying the ideal downstream targets in RAS signal transduction to best contravene oncogenic RAS signaling remains a major challenge in cancer biology.
To address this problem, we exploited the wealth of valuable information and insights gained from genetically tractable model organisms. Genetic studies of Drosophila photoreceptor development have identified many downstream components of the RAS pathway as well as described how the major signaling modules are assembled together ( 23). The Drosophila studies reveal that the proper transmission of RAS/RAF/MEK/ERK signals requires an E3 ligase, seven in absentia (SINA) being the most downstream component identified thus far ( 23, 24). Indeed, loss-of-function sina mutations block the ability of activated RAS/RAF/MEK/ERK to specify the R7 neuronal cell fate by preventing the RAS-regulated proteolysis of SINA substrates ( 23, 25). Thus, the SINA-dependent proteolytic pathway seems to serve as a necessary downstream gatekeeper required for proper RAS signal transduction in Drosophila ( 25).
SINA belongs to an evolutionarily conserved family of E3 ubiquitin ligases. The human genome has two seven in absentia homologues (SIAH1/2) which share >83% amino acid identity with the Drosophila SINA (ref. 26; Supplementary Fig. S1A). Whether human SIAH is a bona fide signaling component of the mammalian RAS pathway is unclear. Due to the extraordinary amino acid conservation shared among the SINA/SIAH E3 ligases, it is conceivable that SIAH-dependent proteolysis might be similarly required for mammalian RAS signal transduction as described in flies. SINA/SIAHs have been implicated in numerous cellular processes from neuronal differentiation, apoptosis, stress response, protein folding to tumor suppression as well as signaling mediated by key signaling molecules including RAS, β-catenin, APC, p53, and NUMB. SINA/SIAHs have been found to interact with and/or degrade more than 28 distinct partners/substrates, most likely through a unique substrate-recognition degron motif ( 27– 29). However, none of the SIAH-interacting proteins are known to play a role in the RAS pathway ( 25, 30– 32). Despite the interest in SIAH-mediated proteolysis in mammalian cells, the role of SIAH in RAS signaling transduction or RAS-mediated oncogenesis remains to be defined.
Activation of the RAS/RAF/MEK/ERK signaling pathway is a major driving force in cell proliferation, transformation, and tumorigenesis ( 8, 9). Therefore, determining whether, similar to that in Drosophila, mammalian SIAH is required for proper transmission of RAS signal and, furthermore, whether SIAH-dependent proteolysis is required for RAS-mediated tumorigenesis are important areas of investigation. In this report, we show that SIAH is required for proper RAS signal transduction. Furthermore, inhibiting SIAH function reduces ERK signaling and blocks RAS-dependent cell transformation and human pancreatic tumor growth in vitro and in vivo. Thus, SIAH may serve as a suitable and novel anti-RAS therapeutic target in the treatment of human pancreatic cancer.
Materials and Methods
Cell culture. Human pancreatic cell lines were purchased from American Type Culture Collection and maintained according to standard procedures.
Reverse transcription-PCR. Qiagen RNeasy Kit was used to isolate mRNA from human cells (Qiagen). The First-Strand cDNA was reverse transcribed with AMV-RT (First-Strand cDNA Synthesis Kit for reverse transcription PCR, Roche) before PCR using gene-specific primer with Taq polymerase (Roche). siah1/2− specific primers were used for PCR synthesis by Taq polymerase (Roche). The 5′- and 3′-primers for siah1 PCR were 5′-ATGAGCCGTCAGACTGCTACAG-3′ and 5′-CAGGACTGCATCATCACCCAGT-3′. The 5′- and 3′-primers for siah2 PCR were 5′-GCCATCGTCCTGCTCATTGGCA-3′ and 5′-ACCAATATGGGAAGGCAGGCAGGAAGGGGC-3′.
Generation of human SIAH1/2proteolysis-deficient mutations. The human SIAH1/2proteolysis-deficient (hSIAH1/2PD) mutant proteins were generated using site-directed mutagenesis to introduce two point mutations (2 Cys → 2 Ser) in their RING domains (SIAH1C41S-C44S and SIAH2C80S-C83S). Two-step PCR was used to synthesize the full-length siah1/2PD transgene using primers 5′-TCTCCAGTCTCCTTTGACTATGTGTTACCGCCC-3′ and 5′-GGGCGGTAACACATAGTCAAAGGAGACTGGAGA-3′ for siah-1. 5′-GAGCTGACCTCGCTCTTCGAGTCTC-CGGTCTCC-3′ and 5′-GGAGACCGGAGACTCGAAGAGCGAGGTCAGCTC-3′ for siah-2. The integrity of siah1/2WT/PD transgenes was confirmed by sequencing. The siah1/2 transgenes were subcloned into pcDNA3, pGEX, and pLentiviral expression constructs.
Expression of human SIAH1/2WT/PD proteins. High-fidelity PCR was carried out using the following primers: (a) siah1-5′-POLYOMA-tagged primer with EcoRI site: 5′-GGCGAATTCAGGAGAACGCCACCATGGAATATATGCCAATGGAAATGGAATATATGCCAATGGAACATATGAGCCGTCAGACTGCTACAGCATTACC-3′ and siah1-3′ primer with XhoI site: 5′-GGCCTCGAGTCAACACATGGAAATAGTTACATTG-3′ for the synthesis of hSIAH1WT/PD with a 5′-POLYOMA tag. (b) siah1-5′-FLAG–tagged PCR primer with EcoRI site: 5′-GGCGAATTCAGGAGAACGCCACCATGGATTACAAGGATGACGACGATAAGAGCCGTCAGACTGCTACAGCATTACC-3′ and siah1-3′ primer with XhoI site for the synthesis of hSIAH1WT/PD with a 5′-FLAG tag. (c) siah2-5′-FLAG–tagged primer with EcoRI site: 5′-GGCGAATTCAGGAGAACGCCACCATGGATTACAAGGATGACGACGATAAGAGCCGCCCGTCCTCCACCGGCCCCAGCGC-3′ and siah2-3′ primer with XhoI site: 5′-GGCCTCGAGTCATGGACAACATGTAGAAATAGTAA-3′ for the synthesis of hSIAH2WT/PD with a 5′-FLAG tag.
Rat fibroblast focus formation assays. Rat-1 fibroblasts were transfected independently with 10 μg of plasmids (1:5 ratio of H-rasV12 with siah1/2WT, siah1/2PD, or pcDNA3) using LipofectAMINE (Invitrogen) in triplicate. After transfection, the cells were split 1:3, resulting in nine 100-mm plates per experimental condition. After 14 to 21 days when the foci became visible, the cells were fixed in 10% methanol and 10% acetic acid and foci were stained with 0.2% Crystal Violet in 10% ethanol. As controls, Rat-1 fibroblasts expressing H-RASV12 alone (positive control) or SIAH1/2WT, SIAH1/2PD, or pcDNA3 alone (negative controls) were assayed for foci formation.
Soft agar growth assays. Soft agar growth assays were performed as described ( 33). Two hundred cells were plated in 24-well plates or 2,000 cells in six-well plates. Triplicates of pLentiviral-infected cells or stable lines were resuspended in 0.4% low-melting agarose and plated on top of a layer of 0.8% agarose in complete DMEM ( 33). The colonies that formed at 2 weeks were imaged and scored. Three separate experiments were done in triplicate, and standard statistical analyses were performed. All data were evaluated by Student's t test with P < 0.001 considered significant.
Generation of stable MiaPaCa cell lines expressing SIAH1/2PD. The pcDNA3 or pcDNA3-siah1/2PD plasmid was used to transfect MiaPaCa cells via electroporation (Bio-Rad GenePulser Xcell). Stable MiaPaCa-pcDNA3 and MiaPaCa-SIAH1/2PD cell lines were established over a 4-week period in 2 mg/mL of G418 selection medium (Invitrogen). Multiple MiaPaCa stable lines expressing SIAH1/2PD at various levels (low, medium, and high) were generated and used in the tumorigenesis assays in athymic nude mice.
Production of pLenti-siah1/2-shRNA and pLenti-SIAH1/2PD viruses. Multiple attenuated Lentiviral vector was used to deliver siah1/2-shRNA constructs and SIAH1/2PD into human cancer cells ( 34). The three-component system was used to generate the Lentiviruses following standard procedures ( 35, 36).
For shRNA knockdown, we employed the MISSION pLentiviral mediated gene-specific short hairpin RNA (shRNA) system ( 37). Control and siah1/2-specific shRNA knockdown constructs were purchased from Sigma. High-titer Lentiviruses were produced using FuGENE (Roche) transfection kit as described ( 34, 36).
hS1-no. 1 CCGGGTCGCCCAAAGCTCACATGTTCTCGAGAACATGTGAGCTTTGGGCGACTTTTT.
hS1-no. 2 CCGGTCACCAGCAGTTCTTCGCAATCTCGAGATTGCGAAGAACTGCTGGTGATTTTT.
hS1-no. 3 CCGGCACACCTTTGAGCTTAATCTTCTCGAGAAGATTAAGCTCAAAGGTGTGTTTTT.
hS1-no. 4 CCGGCCCTGTAAATATGCGTCTTCTCTCGAGAGAAGACGCATATTTACAGGGTTTTT.
hS1-no. 5 CCGGCTGATAGGAACACGCAAGCAACTCGAGTTGCTTGCGTGTTCCTATCAGTTTTT.
hS2-no. 5: CCGGGCTGGCTAATAGACACTGAATCTCGAGATTCAGTGTCTATTAGCCAGCTTTTT.
hS2-no. 6: CCGGGCCTACAGACTGGAGTTGAATCTCGAGATTCAACTCCAGTCTGTAGGCTTTTT.
hS2-no. 7: CCGGCATACGGAGAAACCAGAACATCTCGAGATGTTCTGGTTTCTCCGTATGTTTTT.
hS2-no. 8: CCGGACACAGCCATAGCACATCTTTCTCGAGAAAGATGTGCTATGGCTGTGTTTTTT.
hS2-no. 9: CCGGCGCTAATAAACCCTGCAGCAACTCGAGTTGCTGCAGGGTTTATTAGCGTTTTT.
The pHR-SIN-BX-IRES-Em and pHR-SIN-BX-IRES-SIAH1/2PD bicistronic vector was used to express enhanced green fluorescent protein (eGFP) and SIAH1/2PD, respectively. The viral titer was determined and the Lentiviruses were used to infect cancer cells using an infection ratio of 1:20 (cells to viruses). The biosafety protocols were approved by the Mayo Institutional Biosafety Committee and the BL2+ procedures were followed vigorously.
Athymic nude mice tumor formation assays. One million MiaPaCa or Panc-1 cells were injected s.c. and bilaterally into the dorsal left and right scapular areas of 5-week-old male athymic nude mice NCr nu/nu (NCI/NIH) using 19-gauge needles. Each mouse received two injections of a 200 μL mixture of 1 × 106 cells in 100 μL of 1× PBS and 100 μL of growth factor–reduced MATRIGEL (BD Biosciences). Liquid bandage (NewSkin, MedTech) was used to seal off the injection sites. Six MiaPaCa and two Panc-1 stable lines expressing SIAH1/2PD at different levels along with the control cells were injected into groups of 10 to 15 nude mice. Tumor growth was monitored for 7 to 10 weeks for MiaPaCa and 10 to 15 weeks for Panc-1 cells. Our tumor take rate was 85% to 100%. The tumors were measured with a caliper every 3 days, and tumor volume was calculated as 0.5 × L × H × W. Mice were sacrificed when control tumors reached 15% body weight. The tumors were surgically removed, measured, weighed, and processed. All data were evaluated by Student's t test with P < 0.001 considered significant. All of the animal experiments and surgical procedures were approved by the Mayo Institutional Animal Care and Use Committee.
Histology and immunohistochemistry. Immunohistochemical staining was performed on formalin-fixed and paraffin-embedded tumor tissues as described ( 38). Five-micrometer-cut sections were deparaffinized, rehydrated in graded ethanol, and stained with H&E, von Willebrand factor (vWF), or anti-SIAH 24E6 antibodies.
Western blot and antibodies. Western blot and immunoprecipitation were performed as described ( 25). Mouse anti–SIAH-2, rabbit α-ACTIN, mouse α-FLAG M2 monoclonal antibody (mAb) and EZview Red anti–FLAG-M2 affinity gel were purchased from Sigma and used as described. Mouse-α POLYOMA mAb was used at a 1:10 dilution. Rabbit-α-phospho-ERK, -ERK, -phospho-MEK, -MEK were purchased from Cell Signaling and used at 1:1,000 dilution. Rabbit-α-p21 was purchased from Santa Cruz Biotechnology and used at 1:1,000 dilution. Mouse-α-HIF1α was purchased from BD Transduction Laboratories and used at 1:500 dilution. HRP- and FITC-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories and used at 1:2,000. Enhanced chemiluminescence was performed as described (Pierce).
SIAH is expressed in proliferating human cells. Oncogenic K-RAS is detected in the vast majority of pancreatic cancers and is thought to be an early event in pancreatic tumorigenesis ( 13, 39). However, the roles of mammalian SIAH in the RAS pathway are not well defined. We first developed four mAbs against Drosophila SINA, and tested for cross-reactivity to human SIAHs. Indeed, these anti-SINA mAbs were specific in recognizing the SINA/SIAH family of E3 ligases, as confirmed by Western blot and immunohistochemistry (Supplementary Table S1; Supplementary Fig. S1). Using the anti-SINA/SIAH mAbs, we examined the SIAH expression and localization in normal tissues, human pancreatic cancer cells, and tumor specimens. SIAH expression was detected in all human cancer cell lines and tumor tissues as well as in normal proliferating cells such as skin follicles, kidney, gut, and the germinal center ( Fig. 1 ; Supplementary Fig. S2). The SIAH expression pattern was predominantly nuclear in proliferating cells regardless of the transformation status ( Fig. 1B). In contrast, SIAH expression was undetectable in normal nonproliferating human tissues ( Fig. 1A), indicating a strong correlation between increased SIAH-dependent proteolysis and active cell proliferation, consistent with its roles in modulating RAS-mediated cell proliferation in normal development and tumorigenesis ( Fig. 1; Supplementary Fig. S2).
Because the anti-SINA mAbs recognize both human SIAH-1 and SIAH-2 (Supplementary Fig. S1), we could not differentiate whether one or both SIAH orthologues were up-regulated in proliferating cells. To address this question, we measured the siah1/2 transcript levels in human pancreatic cells. Semiquantitative RT-PCR was performed using siah1/2-specific primers to amplify mRNA isolated from either normal epithelial cells (HPDE6), tumor cells with wild-type RAS (BxPC-3), or cancer cells with oncogenic RAS (Panc-1 and MiaPaCa). Despite their drastic differences in tumorigenic potential, both siah1 and siah2 mRNA transcripts were expressed at similar levels in these proliferating pancreatic cells ( Fig. 1D). Thus, it is likely that SIAH-1 and SIAH-2 are both expressed in normal and neoplastic proliferating cells.
Expression of SIAHPD blocks H-RASV12–mediated focus formation in Rat-1 fibroblasts. Because SINA is a known downstream component of the Drosophila RAS signaling pathway and virtually all human pancreatic cancers contain hyperactivating K-RAS mutations, we next asked whether modulating SIAH activity might provide a means for suppressing RAS-mediated neoplastic transformation/tumorigenesis. It is known that SINA/SIAH functions as a dimer ( 25, 40). The use of dominant-negative mutants can be an effective strategy to inhibit the endogenous protein that are known to dimerize ( 41). Toward this end, we constructed mutant SIAH1/2 proteins by introducing two point mutations (2 Cys → 2 Ser) in the conserved RING domains. These SIAH mutations inactivate the E3 ligase activity and block self-ubiquitination and substrate degradation (Supplementary Fig. S3A). Because these mutant SIAHs bind substrates but cannot degrade them, we termed the mutant forms of SIAHproteolysis-deficient (SIAHPD). Mutant SIAH2PD can bind to wild-type SIAH-2 (SIAH2WT), and the binding of mutant SIAH2PD to SIAH2WT adversely interferes with SIAH2 activity and substrate ubiquitination (Supplementary Fig. S3). Thus, the SIAH1/2PD mutants function as dominant-negatives and are useful tools to dissect the roles of SIAH1/2 in RAS signal transduction.
We first sought to determine whether mammalian SIAH functions downstream of RAS and participates in RAS-dependent transformation. Toward this end, SIAH1/2WT or SIAH1/2PD was expressed in Rat-1 fibroblasts under the control of the cytomegalovirus promoter in combination with oncogenic H-RAS (H-RASV12), and the effects on RAS-mediated focus formation were monitored. Our results showed that SIAH1/2PD expression dramatically decreased the number of foci induced by H-RASV12, as compared with positive controls transfected with H-RASV12 alone ( Fig. 2A and B ). In contrast, SIAH1/2WT expression did not alter the number of foci induced by H-RASv12 under the same conditions, suggesting that SIAHWT expression alone is not sufficient to drive focus formation and cellular transformation ( Fig. 2C). These results indicate that blocking SIAH function inhibits RAS-mediated transformation in fibroblasts.
SIAH is required for anchorage-independent tumor growth of human pancreatic cancer cells in soft agar. We next sought to determine whether SIAH participates in RAS-mediated anchorage-independent growth and malignant transformation of human pancreatic cancer cells. We used either the pcDNA3 or pLentiviral vector to deliver SIAH1/2PD into MiaPaCa and Panc-1 cells, and examined whether SIAH2PD expression could inhibit the anchorage-independent tumor growth of these cancer cells in soft agar. Indeed, the colony formation of both MiaPaCa-SIAH2PD and Panc-1-SIAH2PD cells were significantly less than those of control cells ( Fig. 3b2 and b3 ). Thus, these results support the notion that SIAH2PD expression blocks the RAS-mediated anchorage-independent tumor growth of human pancreatic cancer cells.
To substantiate the above observations, we used an alternative approach to disrupt endogenous SIAH function by using interfering short hairpin shRNA to further delineate SIAH function in the mammalian RAS pathway. Employing the MISSION pLentiviral-shRNA system to silence siah1/2 expression ( 37), we achieved an ∼2- to 3-fold knockdown of siah2 transcripts in human cancer cells using the siah2-specific shRNA constructs (nos. 6 and 8), as shown by semiquantitative RT-PCR, Western blot analyses, and immunochemical staining ( Fig. 3A). Furthermore, to show the knockdown efficacy of these siah2-shRNA constructs, we used a MiaPaCa-SIAH2PD stable line (Supplementary Fig. S4A1, line 7) and showed that the exogenous N-terminal tagged FLAG-SIAH2PD was specifically knocked down by a number of pLenti-shRNA constructs against the siah-2 gene ( Fig. 3a2).
Using these shRNA constructs, we next determined whether a reduction in siah-2 expression affects anchorage-independent cell growth and tumorigenesis of human pancreatic cancer cells. Similar to results obtained using SIAH2PD, the siah-2 transcript knockdown markedly reduced colony formation of these cancer cells in soft agar ( Fig. 3b1 and b2). Interestingly, in analyzing the effects of either SIAH2PD expression or siah2 knockdown on RAS signaling, we observed that the phospho-ERK signal was significantly reduced in these cancer cells with decreased SIAH function ( Fig. 3a3 and a4). The results from these experiments using both shRNA-mediated knockdown and SIAHPD suggest that SIAH is required for RAS-mediated malignant transformation and anchorage-independent tumor growth of human pancreatic cancer cells in soft agar. Furthermore, reduction of ERK signaling upon SIAH2 inhibition suggests that SIAH2 or SIAH2 substrates may be part of a regulatory feedback loop that is involved in modulating RAS/RAF/MEK/MAPK signaling in human pancreatic cancer cells.
Expression of SIAH1/2PD abolishes human pancreatic tumor formation in athymic nude mice. Oncogenic K-RAS mutations are known to drive pancreatic tumor initiation and cancer progression ( 13). We next examined whether SIAH participates in K-RAS–mediated tumorigenesis in vivo. First, we asked whether blocking SIAH function could inhibit K-RAS–mediated pancreatic tumor formation of MiaPaCa and Panc-1 cells (representing highly aggressive human pancreatic adenocarcinomas) in athymic nude mice. SIAH1/2PD or vector control was introduced into these cancer cells using pcDNA3 transfection or pLentiviral infection, and the manipulated cancer cells were implanted in nude mice. Subsequently, the tumor formation of these cancer cells was monitored to determine the efficacy of SIAH1/2PD molecules in blocking RAS-mediated pancreatic tumorigenesis in vivo.
Stable MiaPaCa-SIAH1/2PD cell lines were cloned, amplified, and checked for SIAH1/2PD expression (Supplementary Fig. S4A). One million MiaPaCa cells expressing either pcDNA3 or SIAH1/2PD were then implanted s.c. into athymic nude mice. Tumor formation was monitored over a 7-week period, as shown by the growth curve in Fig. 4B . Fifty-five of 60 injections of 106 MiaPaCa-pcDNA3 cells into 30 nude mice generated large tumors within 6 weeks. In contrast, none of the 110 injections of 106 MiaPaCa-SIAH1/2PD cells into 55 nude mice generated any sizable tumors ( Fig. 4A–C). Thus, the expression of SIAH1/2PD in MiaPaCa cells severely impeded the formation of pancreatic tumors in nude mice ( Fig. 4). These MiaPaCa tumors expressing the mutant forms of SIAH1/2PD did, however, exhibit a slight amount of growth beyond 10 weeks, indicating that these SIAH1/2PD-expressing MiaPaCa cells, although growth-defective, were still viable in vivo (Supplementary Fig. S4C). Because SIAHs form homodimers and heterodimers ( 40), and both SIAH1PD and SIAH2PD have similar antitumor effects for MiaPaCa cells in nude mice, it is conceivable that SIAH1/2PD may interfere with both endogenous SIAH1 and SIAH2 functions. Thus, we focused on characterizing the SIAH2PD phenotypes in subsequent studies.
For analysis of in vivo tumor formation by another independent human pancreatic cancer cell line, an HIV-based pLentivirus that allows for the coexpression of SIAH2PD and eGFP in a bicistronic fashion under the control of the SFFV promotor was used to infect Panc-1 cells ( 34, 36). Almost 100% of the cells were infected as indicated by the eGFP marker ( Fig. 5b2 ). Both heterogeneous populations of Panc-1 cells infected with high-titer pLenti-eGFP control viruses or pLenti-SIAH2PD-eGFP viruses, as well as multiple stable Panc-1-GFP or Panc-1-SIAH2PD-eGFP cell lines were established. The SIAH2PD expression levels in the Panc-1-SIAH2PD stable cell lines are shown ( Fig. 6B , Panc-1-SIAH2PD-eGFP stable line 2 and 3). Tumorigenesis was examined in nude mice using both heterogeneously infected Panc-1 cells as well as the Panc-1 stable lines. One million Panc-1-eGFP and Panc-1-SIAH2PD-eGFP cells were implanted s.c. into athymic nude mice, and tumor formation was monitored over 3 months. The eGFP expression slowed the Panc-1 tumor growth rate due to some GFP toxicity ( Fig. 5b1). Nevertheless, a remarkable blockage in pancreatic tumor growth was observed in Panc-1-SIAH2PD cells, even following extended tumor incubation periods of up to 4 months in nude mice (Supplementary Fig. S4D). Thus, these results clearly show that blocking SIAH function dramatically reduces the tumor formation by two highly aggressive pancreatic cancer cell lines in vivo ( Figs. 4 and 5).
To better understand SIAH1/2PD-mediated tumor suppression at the cellular level, a careful immunohistochemical analysis of the large and small tumors resected from the nude mice was performed to assess changes in cellular morphology, tumor architecture, and angiogenesis. The control and SIAH1/2PD-expressing MiaPaCa and Panc-1 tumors were fixed, sectioned, and stained with H&E, anti–SIAH 24E6 mAb (SIAH), and vWF. The human pancreatic cancer cells could be easily distinguished from the surrounding mouse cells based on their large cell size, large nuclei, and SIAH 24E6 staining ( Figs. 4D and 5C). The anti–SIAH 24E6 mAb specifically stained the proliferating human cancer cells but not the surrounding mouse cells ( Figs. 4d5–d8 and 5c9–c16). Despite the obvious differences in tumor size, the SIAH staining patterns were quite similar in both large and small tumors, suggesting that the implanted human cancer cells were growth-suppressed and tumorigenesis-inhibited in the presence of SIAH1/2PD ( Figs. 4d5–d8 and 5c9–c16). Staining for vWF was also present in all tumors regardless of tumor size, suggesting that the vascularized tissues and blood vessels were not significantly altered by expression of SIAH1/2PD in these tumors ( Figs. 4d13–d16 and 5c17–c24). Thus, the SIAH1/2PD-mediated block in pancreatic tumorigenesis seems to be independent of adverse effects on angiogenesis and tumor vasculature and may rather be a result of altered cancer cell/cell and cancer/stromal cell interactions or some disruptions in tumor microenvironment that compromises tumor growth in vivo.
Because SIAH is expressed in proliferating cells, the reduced growth of pancreatic tumor cells in athymic nude mice could be due to impaired cell cycle, cell proliferation, or apoptosis. To address these possibilities, we monitored the cell cycle and proliferation of MiaPaCa-SIAH1/2PD and control cells in tissue culture. We found that the cell cycle profiles, apoptosis, and bromodeoxyuridine incorporation of MiaPaCa-SIAH1/2PD were indistinguishable from MiaPaCa-pcDNA3 cells (Supplementary Fig. S4B; data not shown). In addition, the MiaPaCa-SIAH1/2PD cells proliferated at the same rate (doubling time, ∼1.5 days) as MiaPaCa and MiaPaCa-pcDNA3 control cells under tissue culture conditions (Supplementary Fig. S4B1–B2). It seemed that blocking SIAH function does not impede cell growth and cell proliferation rate in tissue culture because all of the necessary growth factors and nutrients are provided in the medium. Thus, SIAH may be a necessary licensing factor for tumorigenesis in vivo.
SIAH-2 is known to regulate the hypoxia-inducible factor HIF-1α by targeting prolyl-hydroxylases PHD1 and PHD3 for degradation ( 42). Therefore, we examined whether the lack of pancreatic tumor growth in vivo might be due to a lack of HIF-1α production in response to low oxygen in MiaPaCa-SIAH1/2PD cells. Control and MiaPaCa-SIAH1/2PD cells were incubated under hypoxic conditions, and HIF-1α expression was determined by Western blot. Similar levels of HIF-1α were induced in both control and MiaPaCa-SIAH1/2PD cells under hypoxic conditions, indicating that the SIAH1/2PD-mediated tumor suppression was not due to an aberrant response to hypoxia (Supplementary Fig. S5).
SIAH-deficiency attenuates MAPK signaling. Our results indicate that reduced SIAH function via SIAH1/2PD expression or that siah shRNA knockdown prevents both MiaPaCa and Panc-1 adenocarcinoma formation in nude mice independent of alterations in cell proliferation, apoptosis, angiogenesis, or hypoxia response. Because SIAH is required for RAS-mediated transformation in fibroblasts and tumor growth in soft agar, blocking SIAH function may suppress pancreatic tumor growth by modulating RAS signal transduction. Thus, we examined the alterations of the major signaling pathways that function downstream of RAS in SIAH-deficient cells. The MEK/ERK/p38/JNK/AKT signaling pathways were analyzed by Western blot using phospho-specific antibodies in pancreatic cancer cells untreated or infected with either pLenti-eGFP or pLenti-SIAH2PD-eGFP viruses. Surprisingly, although SIAH is a downstream component of RAS signaling, SIAH-deficiency results in a significant reduction in ERK signaling ( Figs. 3a3 and a4 and 6A and B). This suggests the interesting possibility that the RAS/RAF/MEK/ERK signaling pathway may be modulated by SIAH2 or SIAH2 substrates/partners through a regulatory feedback loop mechanism, thereby controlling RAS-mediated tumorigenesis in human cancer ( Fig. 6C).
Oncogenic RAS mutations promote neoplastic transformation and tumorigenesis ( 10, 13). To date, direct targeting of hyperactivated K-RAS has shown rather limited therapeutic efficacy in the treatment of human pancreatic cancer ( 8). As such, additional targets and therapies are urgently needed. Here, we show that a downstream component of the mammalian RAS signaling pathway, SIAH, plays an essential role in RAS-mediated transformation and tumorigenesis. Thus, as an enzyme, SIAH E3 ligase may serve as an ideal target in the development of novel therapeutics for the treatment of pancreatic cancers in which aberrant RAS signaling is an early signaling event and a major driving force in neoplastic transformation and pancreatic tumorigenesis.
Genetic studies in Drosophila/Caenorhabditis elegans have provided pivotal insights into RAS signal transduction cascades. SINA is the most downstream component identified in the Drosophila RAS pathway ( 23). As a logical extension of our Drosophila studies, we asked whether human SIAH E3 ligases might similarly serve as a downstream gatekeeper for RAS signal transduction in cancer. SIAH is expressed in proliferating cells (both normal and malignant cells) but not in nonproliferating cells ( Fig. 1; Supplementary Fig. S2). Because the RAS pathway is active in proliferating cells, it is conceivable that SIAH expression may be required for transmitting the active RAS signal which drives cell proliferation. Importantly, we observed that blocking SIAH function by SIAHPD suppresses RAS-mediated fibroblast transformation, anchorage-independent tumor growth in soft agar, and pancreatic cancer growth in nude mice ( Figs. 3– 5). Similarly, shRNA-mediated siah mRNA knockdown also reduced the anchorage-independent growth of pancreatic cancer cells in soft agar ( Fig. 3B). Interestingly, SIAH-deficiency in two pancreatic cancer cell lines containing oncogenic K-RAS, MiaPaCa, and Panc-1 resulted in a significant reduction in ERK signaling ( Figs. 3a3 and a4 and 6A and B). Thus, the inhibitory effects of reduced SIAH function on the growth of pancreatic tumors, either through SIAHPD expression or shRNA-mediated siah knockdown, may occur through effects on upstream components of RAS/MEK/ERK signaling as a result of a SIAH- or SIAH substrate–regulated feedback loop mechanism ( Fig. 6C).
Although SIAH is expressed in proliferating cells, human pancreatic cancer cells stably expressing mutant SIAH1/2PD did not exhibit obvious defects in cell cycle progression, proliferation rate, or bromodeoxyuridine incorporation under tissue culture condition. Expression of SIAH2PD or knockdown of siah-2 transcripts in a panel of normal and malignant human pancreatic cells followed by Western blot analysis of components of the RAS/RAF/MEK/MAPK cascade did, however, reveal that ERK signaling in cells with wild-type RAS (HPDE6 and BxPC-3) was less affected than those with oncogenic RAS (Panc-1 and MiaPaCa; Fig. 6A and B). The differential requirement for SIAH in RAS signaling in cells expressing wild-type RAS versus oncogenic RAS could be due to rewiring of cancer signaling pathways induced by hyperactivated K-RAS and a higher dependency on increased SIAH activity for transmitting proper RAS signaling in cancer cells. The roles of SIAH in RAS signaling and RAS-mediated transformation/tumorigenesis are still open areas of investigation. Human pancreatic cancer cells expressing SIAH1/2PD did exhibit a markedly reduced capacity to form tumors in both soft agar and nude mouse tumor assays ( Figs. 3– 5), although there were no dramatic inhibitory effects on cell proliferation and cell cycle progression under tissue culture conditions (Supplementary Fig. S4). Thus, SIAH-dependent proteolysis may play a role in modulating cancer cell behavior and tumor microenvironment in a manner essential for sustaining tumor growth.
Using dominant-negative constructs to block SIAH function may have other unintended side effects. SIAH forms homodimers as well as heterodimers ( 40), and for proteins that form dimers, the dominant-negative approach is a useful strategy for blocking endogenous protein function ( 41). Indeed, the ERK pathway was specifically affected in a similar fashion both when shRNA-mediated siah-2 knockdown and expression of SIAH2PD were used to inhibit endogenous SIAH function, indicating the specificity and efficacy of both agents in blocking RAS signaling in human cancer cells ( Figs. 3a2–a5 and 6A and B). Our results, in which inhibiting SIAH significantly impairs human pancreatic tumorigenesis in nude mice, provides an important step forward in identifying novel therapeutics of blocking RAS signaling by targeting the downstream SIAH-dependent proteolytic machinery. Moreover, by targeting SIAH specifically to inhibit tumor formation, one might avoid complications engendered by other agents that lead to a general inhibition of cell proliferation or proteosome function systemically.
Pancreatic cancer is the fifth leading cause of cancer-related death with a median survival of only 6 months ( 13, 14); however, conventional therapies have little effect on the course of this disease ( 13). As such, there is an urgent need to understand the molecular events underlying the cause and progression of pancreatic cancers and develop novel therapeutic agents to treat this devastating form of cancer. The RAS and ubiquitin-mediated proteolysis pathways have a demonstrated importance in neoplastic transformation and tumorigenesis ( 8, 9, 43– 45). Small molecules that block proteosome activity have shown some clinical promise as novel antineoplastic agents in treating patients with relapsed and refractory multiple myeloma; however, the systemic toxicity due to global inhibition of proteosome function is of significant concern ( 46, 47). Thus, the development of specific inhibitors that selectively target key E3 ligases, such as MDM2 and SIAH, thereby inhibiting important signaling pathways dysregulated in human cancer, would be highly desirable and effective ( 43, 44, 48).
How regulated proteolysis in the RAS pathway contributes to neoplastic transformation/tumorigenesis remains to be elucidated. Here, we show that RAS signal transduction depends on proper SIAH function in cancer cells. (a) SIAH is expressed in dividing cells whereas its expression is absent in nondividing cells. (b) Blocking SIAH function through either the dominant-negative SIAH1/2PD or shRNA-mediated siah-2 knockdown inhibits RAS-mediated foci formation in fibroblasts, anchorage-independent tumor growth in soft agar, and human pancreatic tumor formation in nude mice. (c) Although SIAH is a downstream component of RAS signaling, inhibition of SIAH function results in a reduction in ERK signaling. Thus, SIAH may be a necessary downstream transducer of activated RAS signaling in mammalian cells and that SIAH-mediated proteolysis is required for RAS-dependent neoplastic transformation/tumorigenesis. Furthermore, as the inhibition of SIAH enzymatic activity impaired the ability of highly aggressive human pancreatic cancer cells to form tumors in vivo, this raises an exciting possibility that targeting of SIAH may be a viable anticancer therapeutic option. The next generation of small molecules aimed at targeting SIAH-mediated proteolysis in RAS signaling pathways may yield highly selective therapeutics that could halt the progression of human pancreatic cancer and save the lives of those with this devastating disease.
Grant support: Mayo Clinic Pobanz Family Predoctoral Research Fellowship (R.L. Schmidt), NIH GM 069922 (A.H. Tang), Lustgarten Foundation for Pancreatic Cancer Research (RFA05-046; A.H. Tang), Mayo Clinic Specialized Programs of Research Excellence in Pancreatic Cancer (A.H. Tang), Mayo Prostate Specialized Programs of Research Excellence Career Development Award (A.H. Tang), and a startup fund from the Mayo Foundation (A.H. Tang).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We are grateful to Junjie Chen, Scott Kauffman, Jeffrey Platt, Edward Leof, Peter Harris, Jan van Deursen, Heather Thompson, Adina Bailey, and Nick Zagorski for reading of this manuscript and their valuable comments. We thank Eric Fearon for providing untagged human siah1/2 cDNAs, Thomas Smyrk and Joseph Grande for assistance with immunopathology, Yashuhira Ikeda for pLentiviral vectors and advice for Lentivirus production, Debabrata Mukhopadhyay for assistance with the hypoxia treatment, Karen Lien for assistance with immunohistochemical staining, and James Tarara for assistance with flow cytometry.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received December 6, 2006.
- Revision received September 1, 2007.
- Accepted October 23, 2007.
- ©2007 American Association for Cancer Research.


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U.S. Sends Query to Iran About Ex-FBI Agent's Disappearance
Tuesday, April 3, 2007
The United States sent a formal message to Iran yesterday inquiring into the fate of a former FBI agent who was visiting Iran and has not been heard from since March 8, according to U.S. officials.
The unnamed former agent was on private business, but the United States is now sufficiently concerned that the State Department sent a formal query through Swiss intermediaries asking for information about him, the sources said. The Swiss Embassy in Tehran has represented U.S. interests in Iran since relations were cut off in 1980.
"He is an American private citizen who is in Iran on private business, and we are now pursuing information about his welfare and whereabouts. We don't know where he is. We have no reliable information on him," said State Department spokesman Sean McCormack. "I would not characterize him as a hostage."
The State Department, which has been in constant touch with the man's family and employer, has been trying to track his movements for more than two weeks, U.S. officials said. The missing American was visiting Kish Island, an Iranian resort and free-trade zone off the southwest coast that does not require a visa to visit. He had traveled to Iran from the United Arab Emirates, U.S. officials said.
The FBI and State Department stressed that the man was not working in any capacity for the U.S. government or any U.S. agency. His specialty at the FBI was not Iran, officials added.
The ex-agent, whose name is not being disclosed by the State Department for privacy reasons, had a long career in investigating organized crime in the United States. A middle-aged man who retired from the FBI more than a decade ago, the American worked in private investigations for corporate clients, according to FBI spokesman John Miller. As an FBI agent, he was not assigned to terrorism or Middle East issues, officials said.
The State Department said the missing man was going to meet someone to set up an interview or interviews for a project involving a book and a documentary by an independent producer and author believed to be from Canada, U.S. officials said. He was doing legwork for what a senior U.S. official described as an "innocuous" project that "had no connection with anything political." The official added: "The guy was on a perfectly harmless private activity." But he also may have been doing some work for his own consulting business, U.S. officials said.
The State Department receives information on two or three Americans missing in Iran each year, McCormack said. Although every case is different, some of the missing end up being travelers who do not get in touch with their families. This case does not appear to fit in that mold, however, a U.S. official said.
The case is unrelated to the 15 British sailors and marines seized by Iran in the Persian Gulf on March 23, U.S. officials said. But the United States has its own history on hostages -- most notably the 52 Americans held captive in Iran after the U.S. Embassy takeover from 1979 to 1981.
Kish Island was developed in the mid-1990s in an attempt to attract foreign investment and as a place for Iranians to vacation. The goal was to have a rival for the glitzy sheikdoms such as Dubai across the Persian Gulf. The normally rigid social customs are more relaxed on Kish. More than 1 million people visit every year.


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Nederlandse klantvriendelijkheid in de VS
"Why in the world would you think your (cell) phone would work in your house? The customer has come to expect so much. They want it to work in the elevator; they want it to work in the basement."Hetzelfde Verizon besloot al eerder dat alle mail die niet uit de VS kwam, spam was.
- Verizon baas Ivan Seidenberg in de San Francisco Chronicle


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General Info
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inetnum: 188.8.131.52/22 aut-num: AS262318 abuse-c: HOTEL2 owner: Horizons Telecomunicações e Tecnologia Ltda ownerid: 11.960.585/0001-08 responsible: Noeli Isfer country: BR owner-c: HOTEL2 tech-c: HOTEL2 inetrev: 184.108.40.206/24 nserver: dns1.horizonstelecom.com nsstat: 20170821 AA nslastaa: 20170821 nserver: dns2.horizonstelecom.com nsstat: 20170821 AA nslastaa: 20170821 inetrev: 18.104.22.168/24 nserver: dns1.horizonstelecom.com nsstat: 20170823 AA nslastaa: 20170823 nserver: dns2.horizonstelecom.com nsstat: 20170823 AA nslastaa: 20170823 inetrev: 22.214.171.124/24 nserver: dns1.horizonstelecom.com nsstat: 20170822 AA nslastaa: 20170822 nserver: dns2.horizonstelecom.com nsstat: 20170822 AA nslastaa: 20170822 inetrev: 22.214.171.124/24 nserver: dns1.horizonstelecom.com nsstat: 20170821 AA nslastaa: 20170821 nserver: dns2.horizonstelecom.com nsstat: 20170821 AA nslastaa: 20170821 created: 20121217 changed: 20130121 nic-hdl-br: HOTEL2 person: Horizons Telecom e-mail: email@example.com country: BR created: 20120328 changed: 20151113
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I appreciate ITEAD taking the time to publish 3D models of bezels for the display, however, we're designing a case that will require a much more low profile bezel than the one available.
Is it possible to get a 3D model of the display itself that we can use as a reference to design our own bezel/custom case?
I want to design some bezels / enclosures too. (Actually 7" LCD)
The provided bezel STL file does not fit exactly to the LCD.
STL files are good for 3D printing, but hard to measure.
I could provide STP & IGS CAD files I´ve made of the given dimensions on iTead wiki & a separate email about thickness I´ve got forwarded from Itead.
If someone got the LCD already & could check my 3D file if there are errors?
Most important, to check the "active area" of the LCD, because I found a difference between the provided bezel and the drawings with dimensions.
Hi, maybe this can help?
They are all available on the wiki
As already told,- I´m pretty sure: The first measurements I´ve made on the 3,5" bezel in comparison of the position of active display was NOT correct.
I don´t trust these bezels.
Updated. Both 3D model of the bezel and the display itself.
You can download from the wiki.
Tried to open the STEP files 4,3" & 7.0"Display,- does not work.
File size 5KB only,- please check the STEP files.
Is there any link where I can get the drawing/dimensions of the NX3224T024 for assembly on a box??
STP is not valid, it has errors.
Thank you!
I am now reviewing all of the Feature Requests, this will take some time, patience please.
Rechecking of all the Bezel files will be escalated.
Please also update the models to include all components.
The 3.5in, for example, does not have the coin cell holder ribbon cable connector. Also, all components should be shown so that we can build the design into a product without guessing.
It's very important to supply an accurate model, a non-accurate model as offered is more dangerous than no model at all.
Also, please provide in a valid interchangeable CAD format such as STP.
STL files are not good for anything much other than 3dprinting, which is fine for the bezel however for product integration of the panel its self - no help at all. Most 3DCAD and PCB IDE's will require STEP files.
An erie silience is heard whistling through the air.
Tumbleweed blows past and cobwebs are everywhere.
You begin to notice almost every thread states:
This forum is closed since Nov 15, 2017
Use the new forum at nextion.itead.cc
it has been open since September 2017.


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On July 1, Vermont will become the nation's state first to enact a mandatory GMO-labeling law. Starting tomorrow, all food and drink products made with genetically modified organisms must have warning labels to stay on store shelves in Vermont.
Ahead of the new regulation, The Coca Cola Company said it will likely pull some of its beverages from stores throughout the state.
"To avoid multiple labeling changes, some lower-volume brands and packages we offer within our broad portfolio could be temporarily unavailable in Vermont," Coca-Cola Co. spokesman Ben Sheidler confirmed to Bloomberg.
The world's largest soft-drink company said that its biggest sellers—including Coca-Cola Classic, Diet Coke and Coke Zero—will still be available, complete with new labeling. But for smaller brands, Sheidler says the company is working out how to restructure product packaging to comply with the new law.
Coca-Cola sells more than 100 different beverage brands in the U.S. including Mello Yello, Odwalla, Dasani, Honest Tea, Fuze, Minute Maid and many more.
Earlier this month, several industry groups sued to block Vermont's law, but all were unsuccessful. Some food companies, including Kellogg Co., Campbell Soup Co. and Mars Inc, have previously announced plans to begin shipping products with labels compliant with the new GMO-labeling law.
The Associated Press contributed to this report.


text:
Published By
- Creative Commons Attribution-NoDerivatives 4.0 International License
Document Type
This web page is marked up with Schema.org microdata. Much of the necessary microdata is embedded within the HTML that creates the display you see above. The data that shows below is formatted for machine-reading and rounds out the complete descriptive set for this resource. Want more info about all of this? Go here. You can also view the complete dataset for this resource the way a machine sees it here .Title: Knowledge Management: A Discovery Process
Publication date 2014-03-18
Publication Year 2014
Authors Bernadette Christiansen , Dorothy Wickens , Tim Hanrahan
Copyright holder(s) McKnight Foundation
Keywords knowledge management , DISCOVERY PROCESS , knowledge , workgroups , grantees
Document type CaseStudy
Language English
URL: https://www.issuelab.org/resource/knowledge-management-a-discovery-process.html
Resource provided by IssueLab


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WEBER: Clarinet Concertos Nos. 1 & 2 Concertino, Op. 26 – Paul Meyer, clarinet & cond. l'Orch. De Chambre De Lausanne – Musikproduktion Dabringhaus und Grimm multichannel SACD MDG 940-1922-6 (also 2+2+2), 51:27 (4-1-16) *****:
Classy hi-res recordings of the two Clarinet Concertos by Von Weber.
Just turned 50, French clarinetist Paul Meyer's classy new recordings of Weber's two Clarinet Concertos with the Lausanne Chamber Orchestra eclipse his 1998 versions, for Denon with Günther Herbig conducting the Royal Philharmonic. In fact, this new recording, made in Lausanne's Salle Métropole, eclipses most others and directly competes against Martin Fröst's edgy version with the Tapiola Sinfonietta for BIS.
Meyer, like Fröst, applies his interpretive approach with a splendid, thrilling technique. But where his Swedish colleague's virtuosity is quicksilver and quirky, Meyer's is smoother, more poetic, more mellifluous without any thickening of the sound. And where Fröst offers an unconventional third work, an arrangement for clarinet and strings of Weber's Clarinet Quintet, Op. 34, Meyer takes the conventional route with a charming performance of the charming Op. 26 Concertino.
Balancing the soloist against Weber's rich orchestra is always a challenge for recording these concertos; it was best solved in the analogue days by a 1968 recording made by Gervase de Peyer with the Philharmonia Orchestra conducted by Raphael Frühbeck de Burgos for EMI; it was solved less successfully (technically, not musically) by Karl Leister with the Berlin Philharmonic conducted by Raphael Kubelik on DGG in 1972.
MDG have it set up so that it works better as you increase the volume and realize the orchestra is more present than first realized, while the SACD opens up the space to make things really glorious in surround. And, at all levels and in all modes, as it should be, the sound of Meyer's clarinet is simply gorgeous.
—Laurence Vittes
Copyright © 2016 Audiophile Audition
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Note: Supplemental materials are not guaranteed with Rental or Used book purchases.
Purchase Benefits
What is included with this book?
|Big Ideas: A Framework for Constructivist Curriculum
|Light as an Introduction to Big Ideas
|The Constructivist Teacher: Issues in Implementation
|Balancing Acts
|Zooming In and Out
|Playing with Sound
|Upside Down and Inside Out: The Wacky Curriculum
|Chain Reactions: One Thing Leads to Another
|Table of Contents provided by Publisher. All Rights Reserved.


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For the past year, I've entombed myself in news shows and documentaries, debates and opinion polls, in an attempt to remain well-informed on the latest fiascos of government. Perhaps, I need to give up the continual analyzing by pundits searching for ratings and keep my eye on the road ahead? In my journalistic endeavors, I've attempted to remain somewhat impartial, often failing miserably, and presenting thought food more than absolutes. I doubt if there is such a thing as absolutes in these ongoing political productions other than the absolute of confusion, partisanship, and beating one's head against a wall if one attempts to change the status quo. Depending on where one resides in the economic fabric of things, the changing of the status quo can be a bad, as well as, a good thing.
The rhetoric for or against tax cuts or tax increases, for example, is based wholly on where the speaker resides in the fabric. Those of more wealthy status are against tax cuts that hurt them and help the working folk. What good is a decrease in capital gains taxes going to do for those of us who have no capital gains? What good is a decrease in taxes to small business going to do if they have no customers? Yet, we the consumers tend to be taken in by the carefully structured language of those who are so chagrined at the spending proposals of our new president and the increasing of the Federal debt they will require. Suddenly, borrowing from China is no longer acceptable! Why no mortification at our borrowing practices when it fed their own coffers in the pre-emptive wars and no-bid contracts awarded their buddies? Why no annoyance when the TARP money was dumped into Wall Street with no oversight? Now, that oversight is guaranteed, regulations are to be put in place, and results scrutinized, it's suddenly a bad thing? Why are tax cuts to 95% of the people who buy the products bad, but tax increase to those making millions bad, too? I don't understand that premise.
As most of you, I listened carefully to the "Joint Congressional" message from the president on Tuesday evening. It was a welcome relief to have an intelligent and articulate leader for a change. His confidence in us, the people, is refreshing. His insisting on personal responsibility, a boon. The prioritizing of needed programs that do affect the overall economy like energy independence, healthcare costs, and a strong financial framework, logical. The openness and accessibility promised to the people to the government activities and the White House, unheard of! For this out reach, he does deserve our patience, support, and trust until proven otherwise. I suspect this is why his polls and approval ratings remain high, even with some that didn't cast their vote for him.
I'm personally comforted by the interest shown by the women that come here to Fabulously 40 and Beyond. I've witnessed and read many titillating blogs and discussions among you, taken part in a few myself when time permitted. They were all given in respect and mindfulness of each other and the issues. That is what debate is supposed to be about, not as a whipping post for those that may hold different opinions or understandings. Not as a platform for fear and sensationalism and out of context assumptions. Debate is supposed to be a tool in the learning process, an opening of the mind to consideration of new ideas and thoughts. The women here apply that well. My kudos to all of you.
I'd like to congratulate Yana Berlin on her amazing feat in bringing this website together and connecting so many worthy folks. She's invested a huge amount of time and money and dedication. I've enjoying being a small part of it with a column and private messages. Unfortunately, my writing schedule does not allow time for joining various groups and forums in a Premium Membership capacity, but as long as Yana extends me the column space on the home page, I'll attempt to continue to provide you all with some thought food and topics for your groups to discuss in-depth. Perhaps, some occasional humor.
Years ago, I took part in Spiritual and Philosophical forums on Yahoo and I treasure the experience. I was fortunate enough to make friends that have since turned into real life visits and traveling and closeness. In fact, my son, and now daughter-in-law, met and eventually married as a result of that forum. Yana has provided such a base right here on Fab 40. Life time friendships are about to ensue, my friends. Treasure them.
Susan Haley, Author
**Susan Haley is the published author of two books, several articles on networking, an award-winning poet, a contract copy editor, and book reviewer for AME Marketing. She also contributes a column to "The Florida Writer" the official magazine of the Florida Writers Association, of which she is Facilitator for the Sarasota County Chapter. The audio version of her novel "Rainy Day People" was awarded runner-up Finalist in the 2008 Indie Excellence National Book Awards. She also contributes a variety of editorials and excerpts of her work to various E-zines, newsletters and local papers, and presents editing workshops at Writers Conferences. Her third book, "Songs of the Soul", will be released this fall.
I enjoyed reading your column today....Thanks!
What a delight to read the sentence "His confidence in us, the people, is refreshing. "
I wish all politicians had faith & confidence in the folk who voted in the UK we have no faith or confidence in our Prime Minister - he is with you at the moment can you keep him as a favour to the UK ...
It is very refreshing to see and hear one of our political leaders to have trust in us. Whether we all agree on the politics or not it is nice to see our leader working (right or wrong) for us......
|About this author
|View Blog »


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MESA, Ariz., Sept. 12 (UPI) — A Las Vegas woman who already had four children now has nine with the birth of quintuplets.
Evonne Derrico's babies were born Friday in Banner Desert Medical Center in Mesa, Ariz., a suburb of Phoenix, the Las Vegas Sun reported. The hospital announced the successful delivery Tuesday.
Deon Derrico, the quints' father, told the Sun in a telephone interview from the hospital that the family is doing well: "Excited is an understatement."
But he said he has some work to do to accommodate the five new arrivals.
"We have a vehicle for the family right now, but we don't have a vehicle for the five others," he said. "There's just so much to figure out."
The Derricos moved to the Phoenix area temporarily in preparation for the quintuplets' arrival. Banner Desert has now delivered five sets of quintuplets this year, all born to parents from out of state.
Derrico said the quintuplets were not the result of using fertility drugs.
The two boys were named Deniko and Dariz, and the girls Deonee, Daician and Daiten. The couple already have twin sons, another boy and a girl.
The newborns are in neonatal intensive care at Cardon Children's Medical Center in Mesa while their mother remains in Banner Desert.


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Like many other artists, my passion for music and sound began at young age and is something I could never leave behind. If you are like me, then you can recall the countless times you listened to a particular song and a chill ran up your spine because of the profound effect the music had on you for one reason or another. Maybe you were in love, had just gone through a break-up, got a new job, had an epiphany, experienced something new or rediscovered something old. It doesn't matter what had happened, but in that moment the music you were listening to became a soundtrack for your life. We've all had those moments.
My name is Zamir and I am a musician, songwriter, composer, producer, mix engineer and artist. I started Standard SoundZ in the wake of some down time I had in 2012. During this time I was stuck at home healing from surgeries. As always, I turned to music to help get me through the rough times.
My goal is to assist others who are passionate about their art achieve what they want sonically and to write a variety of music that is relatable on many levels. Whether you are a filmmaker who needs a score or sound design, a band who needs their music mixed, or an artist who needs music and guidance, I want to to help bring your talent to the forefront.
Thank you for visiting my website and I hope we can create something meaningful together!
Zamir is graduate of Musician's Institute of Hollywood and UC Davis and has 15+ years combined experience with all things music and sound.


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Two injured in hawk attacks at Queens university
The injured received lacerations to their heads.
By Cristabelle Tumola
Published : June 02, 2016
Hawks are on the attack at St. John's University in an apparent attempt to protect one of their own.
Two individuals have been attacked since Wednesday, according to media reports. The incidents happened on Wednesday in front of the school's University Center, and Thursday behind St. John's Hall.
In an email alert, reportedly sent to students and staff on Thursday, public safety wrote that "a smaller baby or injured hawk is currently in the bushes near the rear of St. John Hall."
RELATED: 3 charged in armed robbery after shot fired inside St. John's dorm room
"It appears that the larger hawk has become more aggressive in an attempt to protect the smaller or injured hawk."
The email also stated there were "two reported incidents of people being struck in the head by a hawk on the Queens campus," and that "both individuals received lacerations to their head as a result of being struck."
On Thursday DNAinfo also observed "two hawks circling near the baby hawk."
Officers had blocked off the area around St. John Hall and were "keeping their eye on the hawks."
When Hawks are on the prowl at St.Johns... pic.twitter.com/crt5nmR2G2— Tom (@hiitstomm) June 2, 2016


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Princess Kara
Hey loser, I'm going to do some training with you. From now on, you're only allowed to edge. No cumming ever again, lol. And you're only allowed to edge to HumiliationPOV. You're gonna stay here forever. And I'm gonna do that by training you that you don't need any other site or any other brats. You just need us here at HumiliationPOV. Remember no cumming, you're only allowed to edge. You can get so close but you can never feel that sweet release.
Don't you love my sexy outfit? I picked it just for this occasion, because I know how hot I look and I know you're not allowed do cum! LOL! I fucking love teasing you. Just look at my hot young ass, edge to it loser. Get yourself right on the edge and just feel how good that feels. And right when you feel like you can't take it anymore and you're about to explode, take your hand off. And then we're going to start edging all over again. Just so I can see how dedicated you are to following my instructions. Good boy. I know you're getting so weak for me, and my hot young body, and my sweet, bratty voice. I know how to get inside your head, especially when you edge and I deny you, lol.
I know you want to make me happy, and the way to make me happy is to obey me. And you only ever edge to the brats of HumiliationPOV. Keep edging loser, I want you to get so close again to where you think you're going to lose it, and then because you're such a dedicated little loser, I know you're going to stop yourself. I know you're not going to cum because obeying me feels so good. And you're only allowed to edge. This is so much fun, I love fucking with you like this.
But the truth is you love it to, you know that losers should never be allowed to cum. You just need to edge and be brainfucked by the brats of HumiliationPOV. Nothing else in your life feels as good as coming home every night and logging in and edging yourself for hours. You fucking live for that. Edge for me and every other brat on this website. Show me how fucking addicted you are. I know you want to cum so badly by now, but don't. Be a good little obedient loser. You see this is what you need, hot brats telling you exactly what to do. You're too stupid to think for yourself. And you're a chronic masturbator who needs control. Remember loser, condition yourself, you're only allowed to edge to the brats of HumiliationPOV. The more you do it, the more your mind will become conditioned to edge only to this website. We want you so hooked and addicted. I want you edging until your mind breaks.


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Here is a link to the Calfire incident reports on the various California fires.
Overnight fire conditions were very active on several fronts forcing additional evacuations across Northern California. Firefighters are battling 22 large wildfires that have burned nearly 170,000 acres. Firefighters are bracing for the winds to shift this evening and increase in speed.
17 21 people are confirmed to have died in the wildfires this week. 11 people alone died in the Tubbs Fire, making it the 6th deadliest fire in our state's history.
While damage assessment continues on all the fires, it is estimated that at least 3,500 homes and commercial structures have been destroyed by the fast-moving wildfires. Emergency Declarations have been proclaimed by Governor Brown for Napa, Sonoma, and Yuba Butte, Lake, Mendocino, Nevada, Orange and Solano counties.
Red Flag Warnings have been issued for much of Northern California due to gusty winds and low humidity starting today. While these winds may hamper the efforts of firefighters, they will also increase the risk for new wildfires.
Atlas Fire (Southern LNU Complex): Updated: October 11, 2017 10:02 am
County: Napa County,Solano County
Location: off of Altas Peak Rd, south of Lake Berryessa
Acres Burned – Containment: 42,349 acres – 3% contained
Tubbs Fire (Central LNU Complex): Updated: October 11, 2017 10:34 am
County: Napa County
Location: off of Hwy 128 and Bennett Ln, Calistoga
Acres Burned – Containment: 28,000 acres
0% containment
Expected containment date is October 20, 2017
Partrick Fire (Southern LNU Complex): Updated: October 11, 2017 10:18 am
County: Napa County
Location: off Patrick Rd, west of Napa
Acres Burned – Containment: 9,523 acres – 2% contained
Nuns Fire (Southern LNU Complex): Updated: October 11, 2017 10:10 am
County: Sonoma County
Location: Hwy 12, north of Glen Ellen
Acres Burned – Containment: 7,626 acres – 2% containment
The National Weather Service issues Red Flag Warnings and Fire Weather Watches to alert fire departments of the onset, or possible onset, of critical weather and dry conditions that could lead to rapid or dramatic increases in wildfire activity.
4000 firefighters have been deployed


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'CUTS' is a feature length documentary focusing on the hairdressing salon Cuts in London. Cuts was founded by James Lebon during the post-punk, New Romantic scene of the early 80s and is still going to this day.
It began its life in a tiny room in the basement of Kensington Markets where different social strata meshed together to form a movement that included contemporary musicians, photographers, artists, DJs, filmmakers and fashion types.
People such as Leigh Bowery, Christopher Nemeth, Boy George, Tom Dixon and John Maybury all made Kensington Market their home by day.
In the 80s the fashion magazines took their lead from the young people making statements through their DIY clothes and hairstyles. The Face and ID were in their infancy pushing multiculturalism and inclusiveness and at odds with Thatchers declaration 'There is no society'. Thatcher's politics clashed with the flamboyant creativity of the new 'It' kids & Cuts was central to this â?? the first independent hairdessers in London:
The antithesis of Vidal Sasson and the place where the club kids went to get their hair done. Punks, new romantics, rock bands, young stars and the general public all trusted James with creating a 'look' to suit their personality.
This documentary shows the beginning of Cuts through interviews and archival footage. James Lebon & Steve Brooks are interviewed by Paula Yates on her TV show, The Tube on New Years Eve. Cuts is featured in the glossy mags and business couldn't be better. But by the middle of the 80s times were changing; AIDS had become a reality and the DIY attitude of the punk era was being replaced by capitalism & consumer culture.
Change was also inevitable at Cuts and in the mid-80s James left London for a fresh start in New York and Steve Brooks took over the running of the new 'Ladies and Gent's' barber shop in Soho. Steve took on 3 new partners â?? Daniel, Pete and Roy and he referred to the new team at Cuts as â?? 'we were a tribe, we were family.'
Soho is also featured as a character in this film. Footage from the 1950s through to present day shows its ongoing transformation.
The time span of this film allows us to witness change and cultural shifts including the death of the 'people's princess' in 97 and a shift in political regimes in the UK. Tony Blair and New Labour usher in a new era of politics in the UK and with it a nation's optimism. Events of global importance are juxtaposed with events that transpire within the Cuts community
All ideologies are challenged when real life intervenes and the Cuts fairy-tale is no exception. In the beginning we see a carefree utopian workplace within an untarnished vision of trendy London life. In 2010 problems at Cuts come to a head and 'We Are Cuts' is reformed run by Daniel and Pete. As Daniel says ''Cuts is not dependent on one person, it is made up of all the people who have been here over the decades, and hopefully it will continue well after we have gone, taken over by the next generation.â??
Use of funds
We Need Your Help! Your Participation Makes A Difference
Purchases these rewards now will cover the post production costs through to submissions to film festivals. Whatever amount we raise, we can assure 100% will be utilised to fund the completion of this film.
What we need are:
1. Donations
2. Sharing on social media/email
In return for your donations, we have all kinds of rewards, as you'll see to the right, including being the first to see the finished film. Or having you haircut at Cuts, EVERY DONATION MATTERS.
Not just because of the money, but for the support in general and to raise our number of backers. We know that Cuts has a huge outreach of people who have been involved for over 30 years.....and most of these people love Cuts! So we are asking people to please SHARE ON YOUR SOCIAL MEDIA to try reaching as many of these people as possible in the time that this campaign is running.
Help us get the word out there to finish this film so we are able to share it with the world as soon as possible.
Your help empowering this film is critical to the success of this project! You can stay updated beyond the campaign by emailing to sign up to CUTS The Movie mailing list, and please 'like' the project on Facebook.
What We Need, Where The Money Goes
We've pulled many favours already and have onboarded sponsors such as:
Fashion brand –
Hair product – Mojo Hair
MOJO HAIR is the new premium hair styling and grooming range for men that combines pro-salon product styling performance with a modern luxury fragrance. Define your individual style and attitude with MOJO HAIR! Find out more at www.mojo-style.com.
- Final rough-cut edit
- Some post production costs
- We have also taken into account charges via the Crowdfunding platform and the costs of creating, packaging and the shipping of Exclusives.
Any questions? Or not seeing the rewards you'd like? Email us at firstname.lastname@example.com. We'd be happy to answer any questions!
We appreciate anything you can do to help!
Thanks for your support and empowering this project!
Funds at 70%
Reaching 100% of our funding goal will allow us to complete a final 90 minute rough-cut edit, and clear the music and archival rights. Broadcasters are traditionally uncomfortable using their production funds to cover archival costs and the archival footage in this film is essential for the story so this will allow us to fund the clearing of the rights.
Making 70% of our funding goal would be amazing, however we would have to scale back on production costs and festival submissions.
However, Lets dream big and keep our hopes high & CUTS the movie will be a roaring success.
Hurdles & Hopes
In 1996 Jenny Selden and I began filming a documentary about Cuts. It was so different back then. We filmed in an observational way until 1999 on film covering these global events through the eyes of the Cuts community. We captured the daily comings and goings of the colourful characters, the relationships within the Cuts family and the fabulous haircuts.
In 2000, we were unable to get funding to cover the post production of what would have been a very different film than the one that exists today.
It had been stressful, Jen no longer wanted to be involved and I moved back to Australia. But once there, the CUTS film never left my mind and over the next 15 years I continued to visit the UK to film the characters and bring the story up to date.
In 2009, 2011 and 2013, I was back in London filming the Cuts community.
Over this time, filmmaking has completely changed and become far more accessible. This has allowed me to continue working on the edit myself. Using a professional HDV film equipment kit meant I can shoot further footage at Cuts with a minimum of expense.
Little did I know that this venture would take 20 years to be finally finished. When we began filming in 1996 we thought it would probably take 2 years to complete the film. As a result of the length of time that this film has taken, there have been unintended positive consequences.
This film is a fascinating piece of recent British cultural history based in Soho that actually shows the changes in peoples lives and the changing global situation over a time span of 30 years.
It has been a labour of love that now has an end date in sight.
– Sarah Lewis (Director/Producer)
3 years ago
3 years ago
'Hunger TV' supports 'CUTS the movie' crowdfunding efforts!
3 years ago
'Dazed and Confused' are onboard...thank-you!
3 years ago
Excited to share: Weekend Guardian/Observer Article
Press coverage started over the weekend - from Saturday read the Guardian online here and just to prove it happened, see a photo of The Observer in print on Sunday. Enjoy the film's journey and please share with your friends.


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Christmas Outfits
4 comments
Hi, Sarah!ReplyDelete
It's great to see these pictures of you dressed for the holidays. You looked marvelous in your green and orange apparel. Surely you impressed everyone with your authentic period dancing at the 1920s themed Ballroom function.
I hope your year is off to a fine start, dear friend Sarah. Happy 2017 to you!Replies
Thanks Shady, happy new year to you too!Delete
I love your shift dresses - your you-made one is a smasher. (I really need to learn to sew - I'm hoping to knit myself some tops from 60s patterns this year.) Orange and green is a great combination.ReplyDelete
Two fabulous outfits especially that beaded beauty! xxxReplyDelete
Thank you for taking the time to comment, I read and appreciate every one. Please do not comment anonymously - use the Name/URL option instead.
Spam comments will be deleted.


text: 285 Osborne Rd., Adolphus, KY Posted on December 19, 2014 by gregraymer 0 Flares Filament.io 0 Flares × Beautiful 3 bedroom home for sale in Adolphus, KY ! View the Property Details, More Photos, Map & School information here at the updated Single Property Website! Check out this spacious country home that has the feel of an older farm house but was built in 1999.The acreage is on both sides of the road and includes mineral rights.Lots of storage space in the outbuildings and unbeatable quiet, country setting. Lot size and square footage taken from PVA records so buyer(s) should verify for accuracy.To have property disclosures auto-emailed to you dial 270-366-7144, Ext. 154 and wait for text message. Schedule a Showing SOKY Foreclosures View Greg's Listings Videos for Home Buyers Search All Homes For Sale Call 270-282-4005 for more information on this or any other property. Visit Property Website Courtesy: Greg RaymerRaymer Real Estate Services, LLCEmail Me Now270-282-4005Licensed In: KYLicense #: 59997 285 Osborne Rd.Adolphus, KY 42120Bedrooms:3Bathrooms: 3 fullList Price: $ 239,000 Have questions? Ask me.


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DANC 3260, Dance Production (For Majors)
(3.0 Credits)
Course Description
This course is designed to provide dance major students with an understanding of the essential components of dance production. Emphasis is placed on design of technical elements including lighting, costume, sound, and scenic properties as well as the ability to plan and prepare for a dance concert.
Course Objectives
- Understand the technical components of a theater.
- Obtain knowledge in lighting design and lighting instruments.
- Obtain knowledge in costume and sound design and technology.
- Demonstrate the ability to render lighting and costumes designs that support the vision of a dance piece.
- Demonstrate the ability to prepare for and plan a dance production.


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The negroes in negroland; the negroes in America; and negroes generally. Also, the several races of white men, considered as the involuntary and predestined supplanters of the black races. A compilation
Bookwire is a mobile app for your iPhone that gives you the ability to scan a barcode
or enter ISBN, access book records and add titles to wish lists.


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Sizzling Summer Programme: Visitor Golf Tournament
As part of the Bermuda Tourism Authority's Sizzling Summer Programme, a Visitor Golf Tournament is being held at Port...
Copyright © 2018 www.bermudayp.com
All Right Reserved
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Popular soul and R&B band Hindsight will be performing live every Wednesday at Henry VIII's, beginning August 16th. Stop by for drinks, dinner and to enjoy their tunes from 830 to 1130pm each week. There is no entry fee.