Cell Cycle Assays-Part I

From alleleblog: http://allelebiotech.com/blogs/

This is the first part of a series of blogs about using fluorescent proteins in cell based assays with established examples, a common theme here at the AlleleBlog.

FUCCI Cell Cycle Sensor

The FUCCI Cell Cycle Sensor is composed of a red (RFP) and a green (GFP) fluorescent protein fused to different regulators of the cell cycle: cdt1 and geminin.

During the cell cycle, these two proteins are ubiquitinated at different time points by specific ubiquitin E3 ligases, which tag them for degradation in the proteasome. The E3 ligases’ activities are regulated temporally and result in the biphasic cycling of GERMINI and CDT1 levels during the cell cycle. In the G1 phase of the cell cycle, GERMINI is degraded; therefore, only CDT1 tagged with RFP is present and appears as red fluorescence within the nuclei. In the S, G2, and M phases, CDT1 is degraded; only GERMINI tagged with GFP is present, resulting in cells with green fluorescent nuclei.

During the G1/S transition, when CDT1 levels are decreasing and GERMINI levels increasing, both proteins are present, so are the tagged fluorescent proteins. When the green and red images are overlaid, nuclei fluoresce yellow. This dynamic color change, from red-to-yellow-to-green, represents the entire cell cycle. This representation can be used to study the effects of elements that may influence cell cycles.

Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, Imamura T, Ogawa M, Masai H, Miyawaki A.Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008 Feb 8;132(3):487-98.

CCNB1-CyclinB(NT)-GFP

In late S phage, CCNB1 promoter will be switched on to drive the expression of Cyclin B N-terminus-GFP expression; thereafter the fluorescent signal will be switched off at the destruction box in Cyclin B N-terminus at the end of Mitosis phase. During the intervening phase the fusion reporter protein will translocate from cytoplasm to nucleus by the cytoplasmic retention signal in the Cyclin B N-terminus.

Thomas N. Lighting the circle of life: fluorescent sensors for covert surveillance of the cell cycle. Cell Cycle. 2003 Nov-Dec;2(6):545-9.

GFP-PCNA/YFP-PCNA
GFP-PCNA, a fusion of GFP and PCNA, has been widely used as a convenient tool to monitor the progress of S phase. At the onset of S phase, GFP-PCNA translocates into the nucleus; at mitosis the nuclear envelope breaks down and the nuclear accumulation of PCNA-GFP dissipates.

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The chairman of the Norwegian Nobel Committee: Why We Gave Liu Xiaobo a Nobel

New York Times, Oct. 22, 2010

我们为什么颁奖给刘晓波

(Thorbjorn Jagland is the chairman of the Norwegian Nobel Committee)

在中国当局对诺 贝尔委员会选择刘晓波(被监禁的政治活动家)作为2010年和平奖得主的谴责无意中说明了为什么值得捍卫人权。

当局声称，任何人都无权干 涉中国的内部事务。但他们错了：国际人权法和标准高于民族国家，而且国际社会有责任确保它们得到尊重。

现代国家制度是国家主权观念演变而 来的，其又是由1648年的威斯特伐利亚和平协议建立的。当时，主权被认为是在一个专制统治者中体现。

但有关主权的想法已经随时间改变 了。美国独立宣言和法国的人权和公民权宣言取代了独裁者控制下的人民的主权作为国家权力以及合法性的来源。

在上个世纪，主权的概念再次改 变了，随着世界从民族主义转移到国际主义。在两个灾难性的世界大战后成立的联合国，让会员国承诺通过和平手段解决争端，并在世界人权宣言中确定全体人民的 基本权利。宣言中说，民族国家将不再有最终的、无限的权力。

今天，普遍人权对世界各地的任意多数提供了一种限制，无论是民主与否。在议会 中的一个多数并不能决定伤害一个少数群体的权利，也不能投票给损害人权的法律。即使中国不是一个宪政民主政体，它是联合国的会员国，而且它已经修改了宪法 以符合世界人权宣言。

但是，刘先生的监禁是清楚地证明，中国的刑法是不符合其宪法的。他被判定犯有“散布谣言，诽谤或者其他手段，颠覆国 家政权，推翻社会主义制度。”但在普遍人权为基础的国际社会，杜绝意见和谣言不是一个政府的工作。各国政府有义务确保自由表达意见的权利——即使说话者主 张不同的社会制度。

这些权利都是诺贝尔委员会捍卫已久的，通过授予那些挣扎着保护它们的人以和平奖，包括安德烈萨哈罗夫为他坚持反对苏联 的人权侵犯，和马丁路德金牧师博士为他争取在美国的公民权利。

毫不奇怪，中国政府已经严厉批评该奖，声称诺贝尔委员会非法干涉其内部事务 和在国际公众的眼睛中羞辱了它。相反，中国应该感到自豪，它已变得强大到足以成为辩论和批评的主体。

有趣的是，中国政府并不是唯一一个批 评诺贝尔委员会的。有些人说，颁奖给刘先生实际上可能恶化中国人权倡导者的境况。

但是，这种说法是不合逻辑的：它导致的结论是我们最好通 过保持沉默来促进人权。如果我们对于中国保持沉默，谁将会是下一个国家要求它保持沉默和不干涉的权利？这种做法将把我们放在一个走向破坏世界人权宣言和人 权的基本原则的道路上。我们绝不能保持沉默。任何国家都没有权无视其国际义务。

中国有充分的理由为它在过去20年来的成就感到自豪。我们 希望看到这一进步继续下去，这就是为什么我们颁发和平奖给刘先生。如果中国是要推进与其他国家的和谐，成为维护国际社会的价值观的一个重要伙伴，它必须首 先给予其所有公民言论的自由。

一个人仅仅因为他表达了他的意见而正在被监禁11年，这是一个悲剧。如果我们要走向阿尔弗雷德诺贝尔所说的 国家的博爱，那么普遍人权必须成为我们的试金石。

Why We Gave Liu Xiaobo a Nobel
By THORBJORN JAGLAND
Published: October 22, 2010

THE Chinese authorities’ condemnation of the Nobel committee’s selection of Liu Xiaobo, the jailed political activist, as the winner of the 2010 Peace Prize inadvertently illustrates why human rights are worth defending.

The authorities assert that no one has the right to interfere in China’s internal affairs. But they are wrong: international human rights law and standards are above the nation-state, and the world community has a duty to ensure they are respected.

The modern state system evolved from the idea of national sovereignty established by the Peace of Westphalia in 1648. At the time, sovereignty was assumed to be embodied in an autocratic ruler.

But ideas about sovereignty have changed over time. The American Declaration of Independence and the French Declaration of the Rights of Man and of the Citizen replaced the control of the autocrat with the sovereignty of the people as the source of national power and legitimacy.

The idea of sovereignty changed again during the last century, as the world moved from nationalism to internationalism. The United Nations, founded in the wake of two disastrous world wars, committed member states to resolve disputes by peaceful means and defined the fundamental rights of all people in the Universal Declaration of Human Rights. The nation-state, the declaration said, would no longer have ultimate, unlimited power.

Today, universal human rights provide a check on arbitrary majorities around the world, whether they are democracies or not. A majority in a parliament cannot decide to harm the rights of a minority, nor vote for laws that undermine human rights. And even though China is not a constitutional democracy, it is a member of the United Nations, and it has amended its Constitution to comply with the Declaration of Human Rights.

However, Mr. Liu’s imprisonment is clear proof that China’s criminal law is not in line with its Constitution. He was convicted of “spreading rumors or slander or any other means to subvert the state power or overthrow the socialist system.” But in a world community based on universal human rights, it is not a government’s task to stamp out opinions and rumors. Governments are obliged to ensure the right to free expression — even if the speaker advocates a different social system.

These are rights that the Nobel committee has long upheld by honoring those who struggle to protect them with the Peace Prize, including Andrei Sakharov for his struggle against human rights abuses in the Soviet Union, and the Rev. Dr. Martin Luther King Jr. for his fight for civil rights in the United States.

Not surprisingly, the Chinese government has harshly criticized the award, claiming that the Nobel committee unlawfully interfered with its internal affairs and humiliated it in the eyes of the international public. On the contrary, China should be proud that it has become powerful enough to be the subject of debate and criticism.

Interestingly, the Chinese government is not the only one to criticize the Nobel committee. Some people have said that giving the prize to Mr. Liu may actually worsen conditions for human-rights advocates in China.

But this argument is illogical: it leads to the conclusion that we best promote human rights by keeping quiet. If we keep quiet about China, who will be the next country to claim its right to silence and non-interference? This approach would put us on a path toward undermining the Universal Declaration and the basic tenets of human rights. We must not and cannot keep quiet. No country has a right to ignore its international obligations.

China has every reason to be proud of what it has achieved in the last 20 years. We want to see that progress continue, and that is why we awarded the Peace Prize to Mr. Liu. If China is to advance in harmony with other countries and become a key partner in upholding the values of the world community, it must first grant freedom of expression to all its citizens.

It is a tragedy that a man is being imprisoned for 11 years merely because he expressed his opinion. If we are to move toward the fraternity of nations of which Alfred Nobel spoke, then universal human rights must be our touchstone.

Thorbjorn Jagland is the chairman of the Norwegian Nobel Committee.

DNA Repair Pathway Factors in Cell-Based Screening for Restoring Patients’ Sensitivity to Cancer Therapies

Cancers undergoing therapies may develop resistance to treatment. Many current cancer treatments, such as cisplatin, function by creating DNA damage, particularly to fast-dividing cells, i.e., most cancer cells. These treatments may be rendered ineffective by DNA-damage response pathways. Cancer resistance to therapies may come from increased activity in nonhomologous end joining, decreased functions of mismatch repair, or reactivation of the Fanconi anemia (FA)/BRCA DNA-damage response pathway, etc. Ironically the loss of function of some of these DNA-damage repair factors may have partially caused the cancer formation in the first place. Regaining their functions in cancer cells possibly contribute to drug resistance. Molecules that disrupt FA/BRCA pathway or other DNA-damage responses could be used to help restore therapy sensitivity.

Like many proteins that function in DNA-damage repair complexes, FANCD2, a member of the FA pathway factor group, is targeted towards chromatin following damage to DNA in a process called foci formation. There have been recent studies that monitored the foci formation of GFP-FANCD2 in small molecule library screening and identified inhibitors to FANCD2 as candidates for a cancer therapy sensitizer. The assays can be improved in a number of ways. There are fluorescent proteins (FPs) that are much brighter than EGFP for increased sensitivity. For instance, the monomeric green FP mWasabi is about 2-3 fold brighter than EGFP, with narrower emission peak, and is more stable under acidic environment. The newly developed lancelet YFP (LanYFP, developed/introduced by Allele Biotech) is astonishingly 10 times brighter than EGFP. Since it has a longer excitation and emission wavelength, it should inherently have a better signal to noise/background ratio compared to EGFP because cells autofluoresce less in long wavelengths. The improved brightness would also help in this respect. The fold difference between foci and LanYFP background will be the same as EGFP, but the contrast will still probably be better because of less autofluorescent background and significantly higher fluorescence reading in foci.

Other factors that may be used as a screening target when fused to effective FPs may probably include:

1) Homologous recombination (HR)
a. End Resection
MRN complex (MRE11, RAD50, NBS1)
CtIP, RPA, ATM, ATR, Exo1, BLM, RMI1, TopIIIa, DNA2, BRCA1
b. Synapsis
RAD51, BRCA2, PALB2, RAD51B, RAD51C, RAD51D, RAD51AP1, XRCC2, XRCC3, RAD54, RAD54B
c. DNA synthesis
DNA polymerase delta, PCNA
2) Nonhomologous End Joining (NHEJ)
Ku70/Ku80, DNA-PK, Ligase IV, XRCC4, XLF
3) Fanconi Anemia Pathway
FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FAAP100, FANCM, MHF, FAAP24, FANCD2, FANCI, FAN1, FANCN, FANCJ, FANCM

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Original post: http://allelebiotech.com/blogs/2010/10/dna-repair-pathway-factors-in-cell-based-screening-for-restoring-patients%E2%80%99-sensitivity-to-cancer-therapies/

Dr. Liu Xiaobo Wins 2010 Nobel Peace Prize

For "his long and non-violent struggle for fundamental human rights in China".

"From my personal angle, I feel in a dictatory society if you want to be a person with dignity, if you want to be a honest person, fight for human-rights improvement, fight for free speech, being ... [in prison] is part of what you are undertaking, and there is nothing to complain," Liu Xiaobo told CNN in 2007, while he was between a series of house arrests."

"Since you chose to do this, you must have a preparation for being in prison," he said. "Entering the prison you must face these things peacefully, not complain [about] others. I even don't complain [about those] ... who arrested me, because this is their inevitable action. I can also not let them arrest me if I chose other way." said Liu before the current prison sentence.  He served one previously for the 89 student movement.

He lost his freedom, willingly, twice, for the freedom of many others.  He is a living example of honor, dignity, bravery, and idealism.  Proud to have shared battle field with the hero 21 years ago.

Congress may let SBIR authority lapse this week

From AlleleBlog: http://allelebiotech.com/blogs/2010/09/congress-may-let-sbir-authority-lapse-this-week/

The SBIR/STTR/CPP now appears likely to expire on Thursday night, September 30.
Some will deny it but here’s what’s happening.

Allegedly the Senate and House were close to a compromise complete with an 8 year
reauthorization of SBIR/STTR/CPP but each time it goes back to the House (Nydia &
Day), they change the VC language to masquerade 100% VC involvement as a compromise.

Because time is so short, the Senate passed a bill (S.3839) to simply extend
SBIR/STTR/CPP through January 31, 2010. The House was going to pass it on Wednesday
with the President signing Thursday. However, the word on the street is that Nydia
Velazquez, chair of the House Small Business Committee, and her illustrious second,
Michael Day, are rejecting the bill and are poised to let SBIR expire if necessary,
at least in the short term.

It seems that Velazquez’s hope is to move the SBIR reauthorization into the lame
duck session and incorporate all her Wall Street investors’ 100% non-compromise VC
ownership and jumbo award support into a must pass, end of the year omnibus bill
that can’t be touched by her detractors.

This sounds like a script for TV, but several years ago we had a similar year end
omnibus situation involving Nydia (as ranking member) and Sam Graves (subcommittee
chair) and BIO/NVCA, but the main difference was that the small business committee
chair was Donald Manzullo who nipped it in the bud. In our scenario today we have
to look to the House leadership to do it, but it will take your involvement.

Many senior people in the democratic party called for the House to support the
Senate compromise bill H.R. 2965, but Nydia ignored those calls, as did Jason
Altmire, the creator of this infamous Altmire Quagmire. Now Nydia’s really “miffed”
because last week she tried to “scrub” H.R.5297, the Small Business Jobs Act of
2010, but the Obama administration and Speaker Pelosi rolled her over and passed it.

CALL TO ACTION

If SBIR is important to you and your company, it’s time to get serious and realize
that this program can, and will go away unless you make a big noise to let your
politician’s know how you feel. All of us are sick of this, and we’re now facing a
lapse. Eight times this program has been deemed important enough to keep going (via
a CR) but will Nydia be successful in blocking this ninth attempt?

Voting will occur in the House on Wednesday and this may be the last time until
after the election that the SBIR extension bill could voted on. That means we must
act on Tuesday, September 28.

Here are some suggestions and rationale behind them.

CALL CALL CALL the House Tuesday September 21! Call Nancy Pelosi’s office at (202)
225-4965, Steny Hoyer (majority leader) at (202) 225-4131, Nydia Velazquez (202)
225-2361, also the House Small Business Committee line (202) 225-4038

Those of you who are good democrats, call the remaining House Democratic caucus
leaders: John Larson 202- 225-2265, Xavier Becerra 202-225-6235, Jim Clyburn
(202)225-3315

Those of you who are good republicans, call John Boehner (202) 225-6205, Eric Cantor
202-225-2815

Tell them in your own words that SBIR is about to expire and is being held hostage
by Nydia Velazquez. Let them know how important continuation of SBIR is to your
business and the country. Ask them to please support S.3839 (additional temporary
extension of programs under the Small Business Act and the Small Business Investment
Act of 1958) to keep the program from lapsing this week.

I realize that I’m asking you to do something that requires a good chunk of your
time. However, at the risk of losing you as a reader I must tell you that I donate
a large share of my time to try and keep you informed about this program, and I’m
not asking you to do anything for me, only for you and others like you. We do have
some good representatives from both parties BUT they need to hear from you and
quickly.

If you’re bold ask, “I would like to know how a party can let itself get hijacked by
a few people (like Nydia) on a vitally important, highly regarded and accepted
program. This action is to the detriment of your constituents, the country, and
yes, even your own party!”

Here’s what’s going on in the back rooms (formerly smoke filled) The Senate agreed
on a 4 month extension for SBIR because they (Senate) largely (including many on the
Republican side) did not feel a reasonable bill could be passed in the lame duck
session. The Senate has offered up some huge compromises that some believe even
James Greenwood from BIO could live with. The very long shot is that with enough
pressure we might get a compromise bill passed by Thursday.

WHAT HAPPENS IF SBIR LAPSES, EVEN FOR A SHORT TIME

This is an interesting question. Theoretically those projects (grants and
contracts) that are already in place should be okay, but some not. All new unsigned
agreements would stop. Agency comptrollers may start adjusting their budgets to put
the overall 2.8% SBIR/STTR back into their own research pools. Administrative
funding for SBIR could be severely cut back. Remember, all of your grants and
contracts are “subject to the availability of funding.”

On the other hand, SBIR can be voluntary, so some agencies may choose to keep their
SBIR doors open, hoping for, or expecting the reinstatement of the program.

In any event, this is bad for you and the agencies.

The Insider will be on the Hill Wednesday and Thursday, so we’ll do a follow up
report to you asap.

Rick Shindell
SBIR Gateway
Zyn Systems
40 Alderwood Dr.
Sequim, WA 98382
360-681-4123
rick@zyn.com
www.zyn.com/sbir

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Dealing with Interferon Response When Doing RNAi

From AlleleBlog: http://allelebiotech.com/blogs/2010/09/dealing-with-interferon-response-when-doing-rnai/

Off-target effects are a major problem when using RNA interference (RNAi) to silence genes in mammalian systems. One potential source of off-target effects, by either transfected siRNA duplexes or transcriptionally expressed shRNAs, is the inadvertent activation of the interferon response. There are several steps that can be taken to deal with this problem.

Delivery
Interferon response is more likely when high levels of siRNA are used; it is important to transfect the minimum amount of the siRNA duplex that gives rise to a specific RNAi response, as assessed by the level of expression of the target mRNA and/or protein. The level of stable shRNA expression achieved by using lentiviral or retroviral vectors is comparatively modest. Unless very high levels of shRNA expression are achieved, for example, by using highly transfectable cells and a very efficient shRNA expression plasmid, nonspecific activation of the innate immune response are less likely to be induced.

Design
Previous work has shown that the interferon response is induced by dsRNAs of ?30 bp in length and that perfect dsRNAs of as little as 11 bp in length can produce a weak induction. One possible approach to solving the problem of nonspecific activation of the cellular interferon response is to design the siRNA duplex or shRNA precursor so that it does not contain any stretches of perfect dsRNA of ?11 bp.

Detection
If activation of the interferon response remains a concern, it is possible to routinely check for this effect during the course of an RNAi experiment. Analyzing the level of expression of an interferon-response gene, such as oligoadenylate synthase-1 (OAS1), interferon-stimulated gene-54 (ISG54), and guanylate-binding protein (GBP), in the transfected or transduced cells by northern blot or RT- PCR assays are commonly used.

Can there be any more convenient alternative method for checking interferon response? One potentially useful product could be HiTiter™ pre-packaged lentiviruses that would have a fluorescent protein (mTFP1, mWasabi, or the brightest FP in lanYFP) under the control of an ISRE (IFN-stimulated response element) or GAS (IFN gamma-activating sequence)*. This could be another group of Product-on-Demand type of reagents, meaning that we will have the design ready, but only to produce them upon ordering. This way the cost to us and the price to customers can be kept at minimum.

To read the wholr blog, click here.

韩寒: 游行的意义

在jiu月18日这个敏感的时刻，我有的朋友开始研究要不要you行。当然，游的主体可以是反ri保diao救船长。终于，在一个很多论坛里连“you 行”两个字都打不出来的国家里，我们有行可以游了。那么，要不要参加这次命题一日游呢？

首先，我认为在现代中国社会中，分为三个阶级，那就是主子，奴才和狗，而我们往往一人饰两角，至于饰演哪两个角色，我想不会有人觉得他在演主子吧。前一阵子，主子需要奴才去附和和伺候，但是现如今，主子需要狗去吼两声，因为在狗的逻辑里，无论主子怎么对待它，只要有外人来犯，狗总是该看家护院的。

当弄明白了这个以后，回头想想就容易多了。但是，在这三个阶级以内，好在我还有选择做花花草草的权力。我的选择依据是，对于相关部门，小事和大事他们的区别就是抗议一次和抗议十一次，有特权有能力的地方尚未出力，除了把人家日本大使变成了应召男郎以外，我们相关部门情绪稳定，并不见什么实际决心，别说武力上，连经济上都不敢有所动作。他们韬光养晦，所以我也韬光养晦。毕竟，我等做狗也罢，但要做一条戏狗，情以何堪。

纵观事态发展，领dao的内心似乎并不愤怒，领dao只是觉得窝囊，那自然，我们也只能跟着觉得窝囊，你哪有上街去表达窝囊的，那岂不是更窝囊。领dao没面子的时候，我们给他们长脸，但领dao有面子的时候，我们被他们掌嘴。我被欺负，我不能游，你被欺负，你让我游，我又情以何堪。你也别说这种民族国土大事应该是我们一起被欺负了，就算政fu不作为，你活的一塌糊涂，也应该挺身而出。我自然可以挺身而出，但我的第一主题就是要求政fu去作为，第二主题才是控诉来犯者，因为领土问题从来都不是老百姓能解决的和该去解决的，尤其是在我国，老百姓自己都没有一寸土地，，所有的一切，都是问政fu租的，所以，理论上，这事对我来说，就是我的房东在和别人就一块在地上的瓦而争执，这块瓦的确是风大的时候从房东的房顶上掉下来的，但房东也不敢去捡，因为可能要和隔壁人家打架。那我等租客在里面搅和什么呢。无土地者要去为他人争取土地，无尊严者要去为他人捍卫尊严，这样的人多少钱一斤？一斤多少个？

但毕竟，这样的you行安全，好玩，显得很酷，关键是游完以后还能正常工作学习，甚至还有助于未来发展，毕竟也算不容易，所以大学生和老百姓抱着尝鲜唱黑脸的角度去游一游无妨。到时候政fu唱一个白脸，说不定能有所见效。况且现在去you行玩的人相比起以前you行玩的人也有着些许不同，以前是彻底的国政不分，被卖数钱，现如今很多青年终于能够将所谓爱国这件事情想的更明白，他们虽然依然愤怒，但开始反思自己为何每次都是那么窝囊和被动，回头也能更客观的看待国家和政fu的关系，这也算是一个进步。对于任何国家来说，国家就是一个女人，zhi zheng者就是占有她的男人，有幸福美满的，有相处和睦的，有家庭暴力的，有关系紧张的，有离婚再嫁的，有不能改嫁的，但无论如何，你爱一个女人总不能连她的男人也一起爱了去。

最后，这些都不重要，最重要的是，我，如果今天能为唐fu珍,谢chao平而you行，那么明天我就一定会为钓 yu岛和奥运火炬而you行。但这又是一个悖论，往往你能够为唐fu珍,谢chao平you行的时候，你往往就不会有钓yu岛奥运火炬之类的事，而且更不会有唐fu珍谢chao平之类的事出现。一个对内不能和平you行的民族，他的对外任何you行是完全没有价值的，那只是一场集体舞。