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PETERSON REVIEWS
Possession May 26, 2016
Andrzej Żulawski
Isabelle Adjani
Heinz Bennent
Margit Carstensen
Michael Hogben
Shaun Lawton
2 Hrs., 3 Mins.
ossession (1981) is a tightrope walk of a movie. One step in the wrong direction and it'd be the most freakishly overwrought psychological thriller to exist within the art-house sphere. It's a divorce movie without expected tenderness or melancholy. Instead it's a filmed nightmare in which all locked up emotional turmoil is amplified and where otherworldly terror supplements erotic angst. The film has been attributed to genres varying from horror to
drama to suspense, but it defies categorization for the most part. This is a movie that emphasizes an unhealthy relationship between its audience and its director — violent, bizarre images are hurled at us at the speed of light, and yet we get off on the cinematic masochism on display.
Such is not so much a result of our own sadism as it is an effect of Possession's operatic mania. Co-written (with Frederic Tuten) and directed by Andrzej Żulawski, an unrelenting auteur, it's a wonderfully insane take on the marital drama, far past the verge of lunacy throughout its unforgettable 123 minutes. It begins conventionally enough. In Possession, we're dropped into the lives of Anna (Isabelle Adjani) and Mark (Sam Neill), a married couple with a young son (Michael Hogben). Little is known about them besides the fact that Mark is a spy returning from a long-in-the-making espionage mission, Anna a housewife. Upon his arrival back home, Anna announces that she wants a divorce. Her reasoning is vague. But the lack of background knowledge hardly matters in the context of the film: it's a study of separation and the mental devastation that oftentimes follows suit.
From there does Possession quickly escalate, remaining to be at high dramatic volume until it reaches its stunning conclusion. Like John Cassavetes's ordinary-by-comparison Faces (1968), its impact does not have much to do with plot; it's all in the acting, and the staging by its maker. And Possession is singular in its presentation, Żulawski and his actors in such overdrive mode that one false move could potentially kill them.
A claim as dramatic as that one raises concerns, I know. But consider the work done by the film's exceptional artists. Adjani and Neill, both remarkable, do more than just live vicariously through the dialogue and emotional components of their characters. They reach beyond what's expected of any individual in their profession, putting their bodies through tremendous strain, their own emotional stability at risk.
As Adjani's character is written to convey increasingly erratic behavior — Anna's reason for desiring dissolution is strange and genuinely unnerving — most scenes put the actress through hell. Coming to mind particularly is the sequence in which Anna has an inexplicable mental/physical breakdown in the underground throes of the area's subway station. In those moments, Adjani is required to throw her body about like a wild psychiatric patient and scream her head off, all with virtuosity. In other time frames, outlandish monologues are pivotal. To deliver such a blisteringly difficult performance is a feat, but Adjani, one of the best of her class, throws herself into the role to great effect. (Though rumors abound that she attempted suicide following production due to the emotional overextension.) By comparison, Neill's character is not required to be as ferociously manic as Adjani's; he, rather, perpetrates and receives violent behavior as if he were a lightweight champion or a punching bag. Clearly, Mark is an embodiment of all faithful husbands within a divorce, conflicted with self-doubt and unfathomable rage toward his significant other. Internal madness, however, is never kept in hiding. Neill is excellent.
And the mastery of these actors is not something to be taken lightly: without them, I'd imagine Possession being a thoroughly misconceived mess. While it is a divorce movie, it is also much more than that, being a surrealistic horror show with touches of the fantastical that push it far past the limits of most in its genre. We're left rattled, but maybe like a roller coaster, it's a thrilling kind of rattling. Just make sure you can take it before you get on the ride. A


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tradition Free Videos
The most beautiful town, where many people dream, is known as "the land of God". X9b
Colds and coughs are always bad. Cook it with honey and drink a bowl of coughs to get rid of it!
China's 200-year-old seaweed house, a roof to use 10,000 Jin of seaweed, so far can prevent wind and rain! Nine hundred and fifty-three
China's "most valuable" four tea trees, insured 100 million yuan, 24 hours of dedicated care! CX2
China's "most tyrant" village: 2000 people live in villas, per capita GDP reached 250 thousand, only because of this ability! Uob
Which girl in our country is the most "beautiful" row second, the first I do not accept! C0u
China's most "precious" trees, once used to be firewood, now several thousand kilograms! KOF
China's most insignificant vegetables, a "foreign name" export to the United States, sell more expensive than meat! TbG
China's most dangerous villages, villagers dare not go out at night, only dare to take the designated route during the day! 2Sw
China's largest nun island, more than 20,000 women retreat, but at night there is a doctrine unacceptable! TZa
The earliest town in China sleeps at 3 p.m. every day. There is only one man in the town! AB9
The most expensive tree in our country has sold half a million fruits! Can't afford it! XJ3
Ranking the most suitable cities in China: Hangzhou ranked only fifth, the first foreigners admire! 5y0
Why are there more and more fish in Chagan Lake in China? That's because of "bathing fish"! PPc
Several provinces that can bear hardships in China are ranked third in Anhui, and the first is unexpected. 8Df
Our country, a unique nation, has a super high face value. In order to ensure the purity of descent, special regulations are adopted for marriage. H19
How to eat these three kinds of food often can enhance immunity, nourish the spleen and strengthen the stomach! VSr
Use it to boil water, gargle once in the morning and once in the evening, killing oral bacteria, alleviating periodontal odontosis 8f3
Adopt a group of dogs, do not live cattle people build their own dog paradise! Cost no money! BW8
The most mysterious door, tens of thousands of tourists queuing for 3 hours is also willing, what dHw inside?
To go to the beauty parlor all day, it is better to eat more of these three kinds of fruits. Beauty products are more effective than skin care products.
How abnormal is the Japanese in detail? The last toilet was a bit useless. Tqn
Bali "Kissing Day" can not refuse to be kissed and cried, which is clearly a hooligan ah! BtS
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redpajama_set_name: RedPajamaCommonCrawl


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Stavovým prostorem se v informatice rozumí konfigurace diskrétních stavů sloužící jako výpočetní model. Formálně může být stavový prostor definován jako čtveřice [N, A, S, G], kde:

 N je množina stavů
 A je množina přechodů mezi stavy
 S je neprázdná podmnožina N obsahující počáteční stavy
 G je neprázdná podmnožina N obsahující cílové stavy

Na procházení stavového prostoru je založena metoda řešení úloh zvaná Prohledávání stavového prostoru. S analýzou stavového prostoru souvisí také Teorie grafů.

Reference

Literatura

Výpočetní modely

redpajama_set_name: RedPajamaWikipedia


I just hate it when my walls are empty. It makes the room so boringgg. A printed tapestry adds life and color to the room by taking up a huge portion of the wall! There are so many to choose from.
Because seriously, what would we do without coffee? It makes grabbing coffee on the go quick and easy so you won't be late for that 8 am class!
Although it's not NECESSARY, having a printer in my dorm room freshman year made my life so much easier. Instead of running to the library every time I wanted to print something, I was able to just do it in my room! Also a big time saver.
String lights make the room so much cozier! When I get stressed out, I like to turn off all the lights except for my string lights and I can relax more and get in a zen mood.
I have a bajillion sticky frames. They are so cute! Everyone needs some photos from home on the wall and these are fun, easy and decorative.
Having a desk lamp with a USB port my freshman year was so useful. Whenever I had to work at my desk and needed to charge my phone, I could do it right there instead of having to walk across the room!
Who's excited to move back in?!


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Fla. Court - Cigarette Firms To Face Punitive Damages
A state appeals court in Florida Wednesday refused to shelter U.S. tobacco companies from punitive damages in a follow-up trial to last July's sweeping liability verdict for as many as 1 million sick smokers.
The ruling knocked down arguments by Philip Morris Cos. Inc. and other defendants in the Engle vs RJ Reynolds class-action that potentially hundreds of billions of dollars in punitive damages be fixed through thousands of individual trials. It also sets the stage for a possibly massive award against beleaguered U.S. tobacco companies by year's end, analysts have said. The follow-up trial, now scheduled for November 1, had been delayed by appeals by tobacco industry lawyers. They argued that the trial judge was improperly allowing punitive damages, usually the biggest portion of cash payments awarded by U.S. juries, to be set for all Florida's sick smokers at once. Analysts have said the prospect of individual trials taking decades was a much milder threat to the financial health of cigarette makers than the possibility of a single massive judgement. The three judges of Florida's Third District Court of Appeal ruled after an hour-long hearing in which Judge David Levy said the court had no intention of "micromanaging" the pioneering class-action suit. Cigarette makers may appeal on other issues later, the judges said in a brief written ruling. Tobacco companies stocks fell after the ruling. Philip Morris, maker of top-selling Marlboro cigarettes, was off 2-1/4 to 28-11/16, after touching a 52-week low of 28-9/16. RJR Tobacco Holdings Inc., maker of Camel cigarettes and the No. 2 U.S. tobacco company after Philip Morris, also touched a new 12-month low of 23-1/2 before recovering slightly to 23-3/4, off 2-3/16.


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Maoism-Third Worldism
Maoist Information Website
Books by Jason Unruhe
Books by Mason Steiner
Books by PrairieFire
Mises the Hypocrite: When Reality Trumps Praxeology
Jason Unruhe / 2011 05 21
A commentator on my last post has alerted me to this fascinating article on the Mises.org blog:
socialdemocracy21stcentury.blogspot.com/ –
Richard M. Ebeling, "The 'Other' Ludwig von Mises: Economic-Policy Advocate in an Interventionist World," Mises Daily, March 26, 2010
This provides a discussion by Ebeling of Mises' policy advice to the old Austro-Hungarian empire and then the Austrian republic which replaced it, on the basis of "lost papers" of Mises recovered in 1996 and the 3 volume Selected Writings of Ludwig von Mises.
One of the more interesting points that emerges is Mises' policy advice for Mexico:
"in a lengthy monograph that he wrote during the Second World War devoted to economic reform in an underdeveloped country like Mexico, he took as 'given' that the politics of Mexican society was not ready to fully privatize, say, the national railway system or the oil industry. So as a 'second best,' Mises proposed transforming the railway system into a government-owned but privately managed corporation with strict rules and procedures to assure it was run in a relatively 'business-like' manner with the least likelihood of political interference. He even supported limited and temporary subsidies to assist poor Mexican farmers to establish themselves as more-successful private enterprisers.
And on tariffs, he did not propose immediate abolition of trade barriers in Mexico. He accepted that there were many industries that had grown up behind the tariff walls, and that they would resist immediate repeal of trade protectionism. So, instead, he advocated 'incrementalism,' i.e., a gradual reduction of the tariff barriers over several years."
Now, in contrast to many other Austrian views on economics, Mises' plans here appear relatively reasonable. You might even say that reality penetrated his ideological mind and overcame his praxeological theory.
But one will have to wonder how a man who proclaimed that all government intervention in the economy leads to socialism or chaos could advocate "temporary subsidies to assist poor Mexican farmers" without devastating self-contradiction. How could Mises possibly believe that his "temporary subsidies" would be abolished after some limited period of time, without sending Mexico on the path to socialism or chaos?
In an attempt to defend Mises from Schuller (1950), Rothbard characterised Mises' position as follows:
"When Mises presents us with the choice between the free market and socialism, he is saying that in-between systems of a hampered market are not coherent, consistent systems. He demonstrates that any measure of government intervention in the market creates problems and consequences which present the people with a further choice: repeal this measure, or effect another measure of governmental intervention …. interventionist measures logically lead to one or the other [sc. free market or socialism]. Since a socialist system cannot exist, the only intelligent choice is the purely free market. … Mises demonstrates that every form of government intervention in the market creates consequences that lead to an economy worse than that of the free market, …. For Mises, all government intervention in the market is irrational and therefore contrary to economic law" (Rothbard 1951: 184).
Perhaps Rothbard exaggerated Mises' position somewhat, but a reading of Human Action mostly confirms his view:
"The maintenance of a government apparatus of courts, police officers, prisons, and of armed forces requires considerable expenditure. To levy taxes for these purposes is fully compatible with the freedom the individual enjoys in a free market economy. To assert this does not, of course, amount to a justification of the confiscatory and discriminatory taxation methods practiced today by the self-styled progressive governments. There is need to stress this fact, because in our age of interventionism and the steady "progress" toward totalitarianism the governments employ the power to tax for the destruction of the market economy. Every step a government takes beyond the fulfillment of its essential functions of protecting the smooth operation of the market economy against aggression, whether on the part of domestic or foreign disturbers, is a step forward on a road that directly leads into the totalitarian system where there is no freedom at all" (Mises 1996: 282–283).
Commenting on his support of the gold standard and attacking opponents of it, Mises makes a bold claim:
"However, the futility of interventionist policies has nothing at all to do with monetary matters. It will be shown later why all isolated measures of government interference with market phenomena must fail to attain the ends sought. If the interventionist government wants to remedy the shortcomings of its first interferences by going further and further, it finally converts its country's economic system into socialism of the German pattern. Then it abolishes the domestic market altogether, and with it money and all monetary problems, even though it may retain some of the terms and labels of the market economy" (Mises 1996: 474).
One sentence in this passage deserves emphasis:
"all isolated measures of government interference with market phenomena must fail to attain the ends sought".
If Mises really believed this, then his support for "temporary subsidies to assist poor Mexican farmers" stands as a bizarre, illogical position for him. According to him, even isolated instances of government intervention with the market must fail.
So how, then, could Mises' market-distorting subsidies possibly achieve the goal he wanted for them? A miracle? Libertarian magic dust?
We are faced with yet another instance of Mises' intellectual hypocrisy and logical contradictions.
A more serious accusation against Mises is his ridiculous view that social democracy leads to fascism. Let's take an example from history which is highly relevant to Mises' theory.
Engelbert Dollfuss became Chancellor of Austria in 1932. In March 1933, Dollfuss effectively abolished democracy, and established an authoritarian regime, but was assassinated in July 25, 1934 and replaced by Kurt Schuschnigg, who was Chancellor from July 1934 to the Anschluss in March 1938.
It is claimed that before 1934 Mises had become an economic adviser to Dollfuss, even a close adviser (see Hans-Hermann Hoppe, "The Meaning of the Mises Papers," Mises.org, April 1997).
And what exactly happened in Austria in this period when Mises may have had some influence on economic policy? Let's look at a few quotes from the specialist literature on the history of Austria in the 1930s:
"In tackling the economic crisis the Dollfuss-Schuschnigg dictatorship pursued harsh deflationary policies designed to balance the budget and stabilize the currency. The government's program featured severe spending cuts, high interest rates, and frozen wages. From an orthodox economic point of view there was considerable success: by 1937, both industrial and agricultural production had surpassed the levels of 1929; trade was more favourably balanced; the National Bank had liquidated most of its foreign debt and even accumulated reserves of gold and foreign exchange. In a sense the Christian Corporative regime demonstrated the viability of the Austrian state, but it did so at the cost of alienating a majority of the Austrian people. On the eve of Anschluss a third of the population was still out of work, while those fortunate enough to have jobs were bringing home paychecks considerably smaller than before the Great War" (Bukey 2000: 17).
"Beginning in in 1931, [Austrian] unemployment grew rapidly, reaching a peak in 1933–6, with between 24 and 26 per cent of the labour force out of work …. When, in 1937 and 1938, there was a modest recovery, unemployment never dropped below the 20 per cent value. This had a devastating effect on the legitimacy of the Austrian system …. As the Austrian government sustained its reluctance to apply Keynesian policies, the economic recovery never entered a serious tale-off phase in the second half of the 1930s. Linked to an exhausted determination of the Austrian government to resist the pressures from Germany, the economic crisis of the 1930s should be seen as an additional reason why the Austrian society was receptive to the annexation by Germany in March 1938" (Gerlich and Campbell 2000: 55).
Austrians are fond of pointing to the US recession of 1920-1921 as (alleged) proof that austerity brings prosperity, but you will not find them using 1930s Austria as proof of that, even though their hero Mises may well have had a hand in the contractionary policies pursued by the Austro-fascists.
In reality, it was the vicious austerity and deflationary economics imposed on Austria that led to some measure of public support for the Nazi takeover (Utgaard 2003: 72).
Let apologists for Mises explain whether he supported or even designed those policies.
A note of thanks to Iain of "An Anarchist FAQ," whose insightful comment on the previous post inspired me to think more seriously about economic policy under Dollfuss and Mises' connection to it.
Bukey, E. B. 2000. Hitler's Austria: Popular Sentiment in the Nazi Era, 1938–1945, University of North Carolina Press, Chapel Hill, North Carolina.
Ebeling, R. M. 2010. "The 'Other' Ludwig von Mises: Economic-Policy Advocate in an Interventionist World," Mises Daily, March 26.
http://mises.org/daily/4189
Gerlich, P. and D. Campbell, 2000. "Austria: From Compromise to Authoritarianism," in D. Berg-Schlosser and J. Mitchell (eds). The Conditions of Democracy in Europe, 1919–39: Systematic Case Studies, Macmillan, Basingstoke. 40–58.
Hans-Hermann Hoppe, "The Meaning of the Mises Papers," Mises.org, April 1997.
http://mises.org/freemarket_detail.aspx?control=137
Mises, L. 1996. Human Action: A Treatise on Economics (4th rev. edn), Fox and Wilkes, San Francisco.
Rothbard. M. N. 1951. "Mises' 'Human Action': Comment," American Economic Review 41.1: 181–185.
Schuller, G. J. 1950. Review of Human Action: A Treatise on Economics by Ludwig von Mises, American Economic Review 40.3: 418–422.
Schuller, G. J. 1951. "Mises' 'Human Action': Rejoinder," American Economic Review 41.1: 185–190.
Utgaard, P. 2003. Remembering and Forgetting Nazism: Education, National Identity, and the Victim Myth in Postwar Austria, Berghahn Books, New York and Oxford.
2011 05 21 in Commentary.
Mises on Nazi Economics Debunked
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3 thoughts on "Mises the Hypocrite: When Reality Trumps Praxeology"
jsn ecri says:
2011 05 22 at 06:21
great!!! p p p
Edniv says:
I don't know if you have seen this video but I thought you would be interested. He dispels some myths surrounding socialism:
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We found 46 records for hard money lenders in Semmes, AL. In this city, the mean loan amount is $283,750. Interest rates on private loans made to Semmes borrowers average 11.1%. 20 months is the mean length for loans offered in this city. The average points charged for loans is 2.5%. The average LTV offered for loans in Semmes is 74%.
Curtis closes on a $160,000 rehab project in the Cody subdivision of Semmes, AL, using a private money loan from Boom Finance Company . The borrower will need to bring 30% of the purchase price in cash to closing based on a 70% loan-to-value stipulated by the lender. This makes the principle amount from Boom Finance Company $112,000. The terms of the note also include a one percent origination fee which is to be paid at closing and a 18 month, interest-only note with a 8% rate of interest.
Curtis will have to fund a total of $32,400 up front to cover the $48,000 down payment in addition to the $1,120 origination fee. he must then pay $747 monthly to Boom Finance Company . If Curtis meets his goal of a $192,000 total sales price at the end of the loan term, he would pocket a total profit of $17,440 after repaying the principle amount and subtracting the money he contributed at closing, the origination fee, and the total monthly interest payments.


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HGG-Advances
Article| Volume 30, ISSUE 3, P474-484, September 12, 2016
Small-Molecule Targeting of E3 Ligase Adaptor SPOP in Kidney Cancer
Zhong-Qiang Guo 12
12 Co-first author
Zhong-Qiang Guo
CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China
Department of Urology, Peking University First Hospital, Beijing 100034, China
Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China
Tong Zheng 12
Tong Zheng
Laboratory of Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
University of Chinese Academy of Sciences, Beijing 100049, China
Baoen Chen 12
Baoen Chen
Cheng Luo 12
Cheng Luo
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Sisheng Ouyang 12
Sisheng Ouyang
Shouzhe Gong
Jiafei Li
Liu-Liang Mao
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China
Fulin Lian
Department of Analytical Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Yong Yang
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Yue Huang
Li Li
Jing Lu
Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Bidong Zhang
Luming Zhou
Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, and College of Life Sciences, Beijing Normal University, Beijing 100875, China
Hong Ding
Zhiwei Gao
Liqun Zhou
Guoqiang Li
Ran Zhou
Ke Chen
Jingqiu Liu
Yi Wen
Likun Gong
Yuwen Ke
Shang-Dong Yang
Xiao-Bo Qiu
Naixia Zhang
Jin Ren
Dafang Zhong
Cai-Guang Yang
Jiang Liu
Hualiang Jiang 13
13 Lead Contact
Hualiang Jiang
Open ArchiveDOI:https://doi.org/10.1016/j.ccell.2016.08.003
Previous ArticleFeedback Activation of Leukemia Inhibitory Factor Receptor Limits Response to Histone Deacetylase Inhibitors in Breast Cancer
Next ArticleAn Integrated Model of RAF Inhibitor Action Predicts Inhibitor Activity against Oncogenic BRAF Signaling
Computational design and synthetic optimization yield SPOP inhibitors
SPOP inhibitors suppress oncogenic SPOP-signaling pathways in ccRCC
SPOP inhibitors induce cell death and prevent tumor progress of ccRCC
Inhibiting SPOP-substrate interactions may be a potential approach against ccRCC
In the cytoplasm of virtually all clear-cell renal cell carcinoma (ccRCC), speckle-type POZ protein (SPOP) is overexpressed and misallocated, which may induce proliferation and promote kidney tumorigenesis. In normal cells, however, SPOP is located in the nucleus and induces apoptosis. Here we show that a structure-based design and subsequent hit optimization yield small molecules that can inhibit the SPOP-substrate protein interaction and can suppress oncogenic SPOP-signaling pathways. These inhibitors kill human ccRCC cells that are dependent on oncogenic cytoplasmic SPOP. Notably, these inhibitors minimally affect the viability of other cells in which SPOP is not accumulated in the cytoplasm. Our findings validate the SPOP-substrate protein interaction as an attractive target specific to ccRCC that may yield novel drug discovery efforts.
Dysregulation of ubiquitin signaling is associated with multiple cancer development; however, to date no therapeutic treatment that targets ubiquitin signaling has succeeded in a clinical setting. The E3 ligase adaptor SPOP is overexpressed in virtually all ccRCC and promotes tumorigenesis by acting as a key regulatory hub. Our work has identified a small-molecule inhibitor that disrupts SPOP-substrate protein interactions, prevents ubiquitination and degradation of the oncogenic SPOP substrates, and kills human ccRCC cells. These results have demonstrated that targeting SPOP might be specific to kidney cancer, and that small-molecule inhibitors would be potential candidates for a targeted therapy of ccRCC.
Ubiquitin signaling regulates many cellular processes depending on ubiquitination and degradation of diverse cellular proteins (
Jung et al., 2009
Jung T.
Catalgol B.
Grune T.
The proteasomal system.
Mol. Aspects Med. 2009; 30: 191-296
Nakayama and Nakayama, 2006
Nakayama K.
Ubiquitin ligases: cell-cycle control and cancer.
Nat. Rev. Cancer. 2006; 6: 369-381
). The process by which protein ubiquitination and degradation is mediated by ubiquitin signaling involves multistep enzymatic reactions catalyzed by a cascade of enzymes, including ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. E3 ubiquitin ligase recognizes the substrate proteins and catalyzes the ubiquitination of those proteins for targeted degradation through the 26S proteasome (
Ciechanover, 1998
Ciechanover A.
The ubiquitin-proteasome pathway: on protein death and cell life.
Sun, 2003
Sun Y.
Targeting E3 ubiquitin ligases for cancer therapy.
Cancer Biol. Ther. 2003; 2: 623-629
). BTB domain-containing speckle-type POZ protein (SPOP) is an adaptor of E3 ligase, and can mediate E3 ubiquitin ligase recognizing the substrate protein (
Pintard et al., 2003
Pintard L.
Willis J.H.
Willems A.
Johnson J.L.F.
Srayko M.
Kurz T.
Glaser S.
Mains P.E.
Tyers M.
Bowerman B.
The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase.
Zhuang et al., 2009
Zhuang M.
Calabrese M.F.
Liu J.
Waddell M.B.
Nourse A.
Hammel M.
Miller D.J.
Walden H.
Duda D.M.
Seyedin S.N.
Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases.
Mol. Cell. 2009; 36: 39-50
). The MATH domain of SPOP is involved in the specific recognition of substrates (
Hernandez-Munoz et al., 2005
Hernandez-Munoz I.
Lund A.H.
van der Stoop P.
Boutsma E.
Muijrers I.
Verhoeven E.
Nusinow D.A.
Panning B.
Marahrens Y.
van Lohuizen M.
Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase.
Proc. Natl. Acad. Sci. USA. 2005; 102: 7635-7640
Li et al., 2011
Li C.
Ao J.
Fu J.
Lee D.F.
Xu J.
Lonard D.
O'Malley B.W.
Tumor-suppressor role for the SPOP ubiquitin ligase in signal-dependent proteolysis of the oncogenic co-activator SRC-3/AIB1.
Oncogene. 2011; 30: 4350-4364
Nagai et al., 1997
Nagai Y.
Kojima T.
Muro Y.
Hachiya T.
Nishizawa Y.
Wakabayashi T.
Hagiwara M.
Identification of a novel nuclear speckle-type protein, SPOP.
FEBS Lett. 1997; 418: 23-26
). SPOP plays important roles in development (
Liu et al., 2009
Ghanim M.
Xue L.
Brown C.D.
Iossifov I.
Angeletti C.
Hua S.
Negre N.
Ludwig M.
Stricker T.
Analysis of Drosophila segmentation network identifies a JNK pathway factor overexpressed in kidney cancer.
Zhang et al., 2006
Zhang Q.
Zhang L.
Wang B.
Ou C.Y.
Chien C.T.
Jiang J.
A hedgehog-induced BTB protein modulates hedgehog signaling by degrading Ci/Gli transcription factor.
Dev. Cell. 2006; 10: 719-729
) and tumorigenesis (
An et al., 2014
An J.
Wang C.
Deng Y.
Yu L.
Huang H.
Destruction of full-length androgen receptor by wild-type SPOP, but not prostate-cancer-associated mutants.
Cell Rep. 2014; 6: 657-669
Ci W.
Karmakar S.
Chen K.
Dhar R.
Fan Z.
Guo Z.
Zhang J.
Ke Y.
SPOP promotes tumorigenesis by acting as a key regulatory hub in kidney cancer.
) by mediating the ubiquitination of multiple substrates. Dysregulation of ubiquitin signaling is associated with multiple cancer development, which makes it an attractive anticancer target (
Anderica-Romero et al., 2013
Anderica-Romero A.C.
Gonzalez-Herrera I.G.
Santamaria A.
Pedraza-Chaverri J.
Cullin 3 as a novel target in diverse pathologies.
Redox Biol. 2013; 1: 366-372
Chene, 2004
Chene P.
Inhibition of the p53-MDM2 interaction: targeting a protein-protein interface.
Mol. Cancer Res. 2004; 2: 20-28
Mani and Gelmann, 2005
Mani A.
Gelmann E.P.
The ubiquitin-proteasome pathway and its role in cancer.
J. Clin. Oncol. 2005; 23: 4776-4789
). Therapeutic treatment that targets ubiquitin signaling has yet to succeed in a clinical setting, however (
Jia and Sun, 2011
Scf E3 ubiquitin ligases as anticancer targets.
Curr. Cancer Drug Targets. 2011; 11: 347-356
Soucy et al., 2009
Soucy T.A.
Smith P.G.
Milhollen M.A.
Berger A.J.
Gavin J.M.
Adhikari S.
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E3 ubiquitin ligases as cancer targets and biomarkers.
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Wu L.
Grigoryan A.V.
Hao B.
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Specific small molecule inhibitors of Skp2-mediated p27 degradation.
Chem. Biol. 2012; 19: 1515-1524
Renal cell carcinoma (RCC), which accounts for about 4% of all cancers, is one of the most common malignancies (
Siegel et al., 2013
Siegel R.
Naishadham D.
Jemal A.
Cancer statistics, 2013.
CA Cancer J. Clin. 2013; 63: 11-30
), notorious for its resistance to radiotherapy and chemotherapy. Clear-cell renal cell carcinoma (ccRCC) is the most common pathological subtype of RCC, which accounts for about 75% of all cases (
Lopez-Beltran et al., 2006
Lopez-Beltran A.
Scarpelli M.
Montironi R.
Kirkali Z.
2004 WHO classification of the renal tumors of the adults.
Eur. Urol. 2006; 49: 798-805
). The drugs approved for the treatment of metastatic ccRCC mainly target vascular endothelial growth factor, the mammalian target of rapamycin, or the phosphatidylinositol 3-kinase pathway; however, these drugs do not cure patients (
Brugarolas, 2007
Brugarolas J.
Renal-cell carcinoma–molecular pathways and therapies.
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Patil et al., 2012
Patil S.
Manola J.
Elson P.
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Escudier B.
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Atkins M.
Bukowski R.
Motzer R.J.
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J. Urol. 2012; 188: 2095-2100
). Therefore, there is an urgent need to identify small-molecule inhibitors that target the more effective tumor target proteins.
We have shown that SPOP is overexpressed in virtually all ccRCCs (
), and overexpressed SPOP accumulates in the cytoplasm of ccRCC cells, which is a nucleoprotein originally (
). Furthermore, SPOP serves as a regulatory hub in order to promote ccRCC tumorigenesis through the ubiquitination and degradation of multiple regulators of cellular proliferation and apoptosis (
). Above all, the ideal characteristics of SPOP, such as overexpression in ccRCC, dislocation in the cytoplasm, its vital role in ccRCC tumorigenesis, and its degradation of substrates by recognizing their SPOP-binding consensus (SBC) motif, make SPOP a potential antitumor target for treating ccRCC. Here we aim to identify small molecules that can inhibit the SPOP-mediated ubiquitination-signaling pathway as well as SPOP inhibitors that suppress the proliferation of ccRCC both in vitro and in vivo.
Development of SPOP Inhibitors
In order to inhibit the tumorigenesis caused by cytoplasmic SPOP, we aimed to identify small molecules that directly target the SPOP protein and disrupt the SPOP-substrate interactions. Structural studies have demonstrated that SPOP selectively binds with its substrates by recognizing their SBC motif (Φ-π-S-S/T-S/T; Φ is nonpolar, π is polar) (
). Based on the structural coordinate of the SPOP-puc_SBC1 complex (PDB: 3HQL) (
), we have performed computational screening through a hierarchical strategy that combines pharmacophore modeling and molecular docking (Figure 1A ). One hundred and nine compounds were selected from the SPECS database, which contains ∼200,000 drug-like structures. We first performed a fluorescence polarization (FP) assay in order to measure the ability of small molecules to competitively inhibit puc_SBC1 peptide binding to SPOP protein in vitro. The saturation binding assay showed that the affinity (KD) for puc_SBC1 peptide binding to SPOP is about 5.93 μM (Figure S1A), which is comparable with the reported affinity, suggesting that the recombinant SPOP protein is in the active form (
). Compound 6a (Figure 1B) was identified as an initial hit capable of competing with puc_SBC1 peptide binding to SPOP with an estimated KD value of 62 μM (Figure 1C). Additional synthetic optimization (Figure S1B) of the chemical core successfully yielded the lead compound 6b (Figure 1B) that is more active, with a KD value of 35 μM on FP measurement (Figure 1C). The 6b-property-like compound 6c (Figure 1B) fails to compete with peptide binding to SPOP protein, however (Figure 1C). In order to further validate if SPOP inhibitor could disrupt SPOP-substrate interactions, we performed an in vitro pull-down assay. The result shows that compound 6b obviously reduces combinative phosphatase and tensin homolog (PTEN) protein binding to SPOP, while the inactive analog compound 6c does not observably disrupt the protein interaction between SPOP and PTEN in the pull-down assay (Figure 1D). These results demonstrate that the small-molecule inhibitor appears to disrupt the SPOP-substrate interactions in vitro. The inhibitory activities of these compounds against the cell proliferation of ccRCC cell line A498 were then investigated (Figure 1E). Consistent with the observed trend in the competition on peptide binding to SPOP protein (Figure 1C), compound 6a displayed a moderate inhibitory activity with an observed half-inhibitory concentration (IC50) of 18.1 μM, whereas the lead compound 6b is more active, with an IC50 of 2.8 μM. Compound 6c serves as a negative control in the following studies, as it neither alters the SPOP-substrate interaction nor inhibits ccRCC cell proliferation (Figures 1C–1E).
Figure 1Development of SPOP Inhibitors
(A) Work flow of the structure-based virtual screening of inhibitors of SPOP-substrate interactions.
(B) Structure of inhibitors as identified through virtual screening and subsequent synthetic optimizations. Compound 6a shows moderate inhibitory activity, 6b is the most active inhibitor, and 6c is the negative control.
(C) Inhibitors competitively inhibit puc_SBC1 peptide binding to SPOP23−337 measured by FP assay.
(D) In a pull-down assay, 6b disrupts protein binding between SPOP and PTEN. In the negative control experiment, protein interaction is intact in the presence of compound 6c.
(E) Proliferation experiment on A498 cells in the presence of inhibitors. IC50 (μM) values of inhibitors were calculated from three independent experiments.
Error bars represent mean ± SD. See also Figure S1.
In the virtual screening protocol, we have applied tests in order to filter the pan-assay interference compounds (PAINS) (Figure 1A). Furthermore, we have performed experimental tests of compound 6b. When compound 6b was added to a concentration of 200 μM, 40-fold excess over SPOP protein, we observed subtle changes in the circular dichroism (CD) spectra (Figure S1C), indicating no gross structural perturbations of the SPOP protein. In addition, high-performance liquid chromatography (HPLC) analysis confirms that compound 6b is in a highly pure form (Figure S1D). Dynamic light scattering was performed in order to test the solubility of the compounds in cellular assay media. Neither particle formation nor compound aggregation occurred at a concentration of 300 μM in the cellular assay media (Figure S1E). Compound 6b is not intrinsically fluorescent itself (Figure S1F). In addition, the intracellular delivery of compound 6b was quantified by HPLC-tandem mass spectrometry (MS/MS) analysis, the intake rate of which is rather high (up to ∼30%; Figures S1G–S1I), indicating that the small-molecule inhibitor shows excellent cellular permeability and quickly accumulates inside cells. All these data demonstrate the quality of the lead compound 6b that does not behave as a pan-assay interfering compound.
The Small-Molecule Inhibitor Binds to and Stabilizes the SPOP Protein
In order to understand the mode of action of the inhibitor, we tested if compound 6b directly targets SPOP in vitro using surface plasmon resonance technology. Compound 6b exhibited moderate binding affinity to either the truncated SPOP23−337 or full-length SPOP protein with an estimated KD constant of ∼19 μM (Figures 2A and S2A), whereas the inactive compound 6c could not observably bind to SPOP under the detection conditions (Figure S2B). In nuclear magnetic resonance (NMR) experiments, dose-dependent attenuation of signals was observed in the T1ρ NMR spectra and positive saturation transfer difference (STD) signals were detected in the STD spectrum, which indicate that the full-length SPOP protein interferes with the state of compound 6b (Figure 2B). Similarly, the SPOP23−337 truncation protein also interferes with compound 6b as observed in the NMR experiments (Figures S2C and S2D). No interference with NMR signals was observed in the negative control experiments conducted on compound 6c (Figures S2E and S2F). In order to assess the plausible binding site of compound 6b, we investigated the binding between 6b and different SPOP mutants. It is of note that the hotspot residues in the MATH domain of SPOP that have been demonstrated as essential for SBC motif binding (
) were also observably seen to contribute in a major way to compound 6b binding, since the affinities were significantly attenuated in different SPOP mutants (Figures S2G and S2H). In addition, none of the SPOP mutants tested in the NMR experiments showed gross structural changes verified by the CD spectra (Figure S2I). With these NMR-based binding studies, we attempted to generate a computational model to illustrate the possible interactions between compound 6b and the SPOP protein (Figures S2J and S2K), which might aid in the design and development of new small-molecule modulators targeting SPOP. However, how the BTB domain or its interaction with the MATH domain is involved in inhibitor binding to SPOP remains to be further explored.
Figure 2SPOP Inhibitor Binds to SPOP
(A) 6b binds to SPOP23−337 protein as shown by surface plasmon resonance measurements. Graphs of equilibrium response unit responses versus compound concentrations are plotted. The estimated KD is 19.1 μM.
(B) NMR measurement of direct binding between 6b and SPOP. T1ρ NMR spectra for 6b (red), 6b in the presence of SPOP at 2.5 μM (blue), 5 μM (cyan), and 10 μM (green). The STD spectrum for 6b is recorded in the presence of 5 μM SPOP.
(C) Representative western blots for the stabilization of the SPOP protein. CETSA was assayed in HEK293 cell lysate and in intact cells in the presence of 20 and 5 μM 6b, respectively.
See also Figure S2.
In order to further verify the interaction between the inhibitor and the SPOP protein, we performed a cellular thermal shift assay (CETSA) that enables us to assay for affinity, cellular uptake, and target engagement in vivo. Compound 6b was analyzed for thermal stabilization of SPOP in HEK293 cell lysates (Figure 2C, top panel). Representative western blots for the stabilization of SPOP are shown. The detected soluble SPOP proteins clearly differ at denaturation temperatures ranging from 60°C to 68°C with and without compound 6b, indicating that the small-molecule inhibitor directly bound to the SPOP protein. The whole-cell CETSA with compound 6b produced a thermal shift for SPOP that did not differ significantly to that observed in the cell lysate CETSA (Figure 2C, down panel). These results demonstrate that compound 6b was able to enter cells and bind to the SPOP protein. In contrast, the inactive analog compound 6c did not observably stabilize the SPOP protein in these CETSA detections (Figure S2L), indicating that compound 6c could not bind to the SPOP protein. Taken together, the CETSA analysis supports that compound 6b directly binds to and thermally stabilizes the SPOP protein in vivo.
SPOP Inhibitors Repress the Proliferation of ccRCC Cells
In normal cells, SPOP localizes in the nucleus (Figure 3A ), which enhances apoptosis (
). In ccRCC cells, however, SPOP is overexpressed and mislocalized to the cytoplasm and therefore enhances proliferation and promotes tumorigenesis (Figure 3B) (
). These differences indicate that targeting cytoplasmic SPOP may provide a novel opportunity to selectively kill ccRCC cancer cells rather than normal cells. To this end, we tested the cell proliferation of six ccRCC cell lines and one non-tumor kidney cell line, HK-2 (human proximal tubule epithelial cell line), in the presence of SPOP inhibitors (Figure 3C). Compound 6a and 6b efficiently inhibit the growth of ccRCC cells with IC50 values ranging from 1.8 to 24 μM for different ccRCC cell lines, whereas compound 6c shows no inhibitory effect on cell proliferation in these ccRCC cells. In addition, compound 6a or 6b have minimal effect on the growth of HK-2 cells. The aqueous soluble compound 6b-HCl also efficiently inhibits the growth of ccRCC cells and displays a similar IC50 for each ccRCC cell line compared with the compound 6b (Figure S3A).
Figure 3SPOP Inhibitors Suppress the Proliferation of ccRCC Cell Lines and the Growth of Primary ccRCC Cancer Cells
(A) SPOP localizes in the nucleus of HK-2 cells.
(B) The relative abundance of SPOP protein minimally varies in six ccRCC cells. Much less SPOP was detectable in HK-2 cells. GAPDH is used as a loading control.
(C) Cell proliferations of six ccRCC cell lines and one non-tumor HK-2 cell line in the presence of 6a-c.
(D) Inhibition of the proliferation of the primary ccRCC cells isolated from seven patients. The proliferation of A498 cells is considered a positive control, and proliferation of HK-2 cells is considered a negative control.
The cell proliferations assays were performed in triplicate, and error bars represent mean ± SD. See also Figure S3.
Overexpression and dislocation in cell cytoplasm make SPOP a regulatory hub for the promotion of ccRCC tumorigenesis (
). However, SPOP is not observably overexpressed in other cancer cells (
) and fails to promote tumorigenesis (
Barbieri et al., 2012
Barbieri C.E.
Baca S.C.
Lawrence M.S.
Demichelis F.
Blattner M.
Theurillat J.P.
White T.A.
Stojanov P.
Van Allen E.
Stransky N.
Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer.
Nam H.J.
Pyo K.E.
Jang M.J.
Kim I.S.
Kim D.
Boo K.
Lee S.H.
Yoon J.B.
Baek S.H.
Kim J.H.
Breast cancer metastasis suppressor 1 (BRMS1) is destabilized by the Cul3-SPOP E3 ubiquitin ligase complex.
Biochem. Biophys. Res. Commun. 2011; 415: 720-726
) in, for example, prostate cancer and breast cancer. In order to identify if the small-molecule inhibitor specifically kills ccRCC cells depending on SPOP overexpression and dislocation, we compared the cell proliferation of the A498 cell line and six other cell lines including five non-SPOP cancer cell lines and one non-cancer cell line in the presence of compound 6b. The IC50 for A498 cell proliferation measured less than that for other tested cell lines by more than 5-fold (Figure S3B).
In order to further investigate the target engagement of the SPOP inhibitor, we generated stable SPOP knockdown A498 cell lines using a small hairpin RNA (shRNA) method and demonstrated how the cellular activity of the SPOP inhibitor could be affected (Figure S3C). The level of SPOP knockdown was verified using western blot. After 72 hr exposure, compound 6b significantly inhibited the proliferation of A498 cells in the scrambled shRNA control group. Our data further show that polyclonal cells with a stable knockdown of SPOP expression in A498 cells become resistant toward 6b treatment. Overexpressed SPOP can enhance cell growth in kidney cancer cells. The growth of polyclonal cells with SPOP knockdown adapted to the situation with low levels of SPOP. SPOP contributes less in the stable knockdown cells compared with the wild-type A498 cells. Therefore, the cells with SPOP knockdown become less sensitive to compound 6b treatment. Similar results were observed in the regulation of the skp1-skp2 signaling pathway with small-molecule inhibitors (
Chan et al., 2013
Chan C.H.
Morrow J.K.
Li C.F.
Gao Y.
Jin G.
Moten A.
Stagg L.J.
Ladbury J.E.
Cai Z.
Xu D.
Pharmacological inactivation of Skp2 SCF ubiquitin ligase restricts cancer stem cell traits and cancer progression.
). These results indicate that the small-molecule inhibitors suppress the proliferation of ccRCC cells by targeting SPOP activity specifically.
SPOP Inhibitors Could Kill Primary Human ccRCC Cells
We next sought to examine the effect of SPOP inhibitors on the growth of primary ccRCC cancer cells. Notably, compound 6a, 6b, and 6b-HCl effectively inhibited the proliferation of all primary ccRCC cells isolated from seven patients (Figures 3D and S3D). The IC50 values for primary cancer cells were slightly higher than those for ccRCC cell lines (Figure 3C), which may be caused by the growing speed of primary cancer cells being slower than ccRCC cell lines. Compounds 6b and 6b-HCl showed more inhibitory behavior than 6a, which is consistent with the results observed on ccRCC cell lines. These data further demonstrate that small-molecule inhibitors targeting SPOP-substrate protein interaction could kill kidney cancer cells.
Inhibitors that Disrupt SPOP-Substrate Interaction Thus Block the Ubiquitination and Degradation of Tumor Suppressors In Vivo
Previously, we showed that SPOP directly binds to PTEN and dual specificity phosphatase 7 (DUSP7) (
). In order to address whether SPOP-substrate protein interactions could be disrupted in the presence of SPOP inhibitors, we conducted a co-immunoprecipitation experiment. Inhibitors 6a, 6b, and 6b-HCl significantly disrupt SPOP binding to PTEN and DUSP7 in a dose-response manner; however, SPOP-substrate protein interactions were intact in the presence of compound 6c (Figures 4A, 4B, and S4A ).
Figure 4SPOP Inhibitors Inhibit the Ubiquitination and Degradation of Tumor Repressors In Vivo
(A) 6a disrupts SPOP-PTEN interaction in HEK293 cells.
(B) SPOP-PTEN and SPOP-DUSP7 protein interactions are inhibited in the presence of 6b in a concentration-dependent manner. These interactions are intact in the presence of 6c.
(C) 6a inhibits PTEN ubiquitination in HEK293 cells.
(D) 6b inhibits the ubiquitination of PTEN and DUSP7 in a dose-dependent manner. 6c is inactive and does not inhibit the ubiquitination of PTEN or DUSP7.
(E) 6b inhibits the degradation of PTEN and DUSP7 in a concentration-dependent manner in A498 cells. The downstream p-AKT and p-ERK1/2 abundances are observed to decrease.
(F) 6a increases the accumulation of PTEN in both a time- and concentration-dependent manner in A498 cells.
(G) 6a increases the accumulation of PTEN, while it decrease the levels of p-AKT in Caki-2 cells.
SPOP promotes the ubiquitination and degradation of tumor suppressors PTEN and DUSP7 in kidney cancer (
). PTEN phosphatase acts as a negative regulator of the phosphoinositide 3-kinase/Akt pathway (
Wu X.Y.
Senechal K.
Neshat M.S.
Whang Y.E.
Sawyers C.L.
The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway.
Proc. Natl. Acad. Sci. USA. 1998; 95: 15587-15591
). DUSP7 is one of the DUSPs and dephosphorylates extracellular signal-regulated kinase (ERK), which mediate cellular proliferation, apoptosis, and differentiation (
Chappell et al., 2013
Chappell J.
Sun Y.H.
Singh A.
Dalton S.
MYC/MAX control ERK signaling and pluripotency by regulation of dual-specificity phosphatases 2 and 7.
Gene Dev. 2013; 27: 725-733
). We wondered if small-molecule inhibitors kill ccRCC cells by increasing the cellular abundance of PTEN and DUSP7 and therefore inactivate downstream signaling pathways. The interference of SPOP-substrate protein interactions by a small molecule could inhibit the ubiquitination of PTEN and DUSP7 in vivo. We observed that PTEN and DUSP7 ubiquitination was completely inhibited when HEK293 cells were treated with compounds 6a, 6b, or 6b-HCl (Figures 4C, 4D, and S4B). As a result of the inhibition of PTEN and DUSP7 ubiquitination, we observed the steady accumulation of cellular PTEN and DUSP7 proteins in a concentration-dependent manner when A498 cells were treated with inhibitors 6b or 6b-HCl for 12 hr (Figures 4E and S4C). The accumulation of PTEN and DUSP7 tumor suppressors should subsequently lead to decreases of phosphorylated Akt and ERK. As expected, a decrease of p-Akt and p-ERK was clearly detected, but a total cellular abundance of Akt and ERK proteins in A498 cells was not detected (Figures 4E and S4C). Similarly, a higher dosage of compound 6a also elevates accumulation of PTEN in both A498 and Caki-2 cells and decreases phosphorylated Akt in Caki-2 cells (Figures 4F and 4G). Notably, compounds 6b and 6b-HCl have a minimal effect on the cellular abundance of PTEN or DUSP7 in HK-2 cells, in which SPOP is mainly located in the nucleus (Figure S4D), and this potentially correlates with the insensitivity of HK-2 cells to SPOP inhibitors. Furthermore, we have performed an RT-qPCR in order to detect the cellular levels of transcription of SPOP, PTEN, and DUSP7 genes (Figure S4E), which indicated that, as we predicted, compounds 6b or 6b-HCl have a minimal effect on the transcriptions of these genes.
In order to clarify that the small-molecule inhibition of ubiquitination of SPOP substrates is not non-specific, we tested the effect of inhibitor 6b on ring finger protein 5 (RNF5) self-polyubiquitination. RNF5 is another E3 ligase that mediates self-polyubiquitination (
Qian et al., 2013
Qian M.X.
Pang Y.
Liu C.H.
Haratake K.
Du B.Y.
Ji D.Y.
Wang G.F.
Zhu Q.Q.
Song W.
Yu Y.
Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.
Cell. 2013; 153: 1012-1024
). Compound 6b could not inhibit the self-ubiquitination of RNF5 in HEK239T cells (Figure S4F). These data indicate that small-molecule inhibitors could specifically disrupt SPOP-substrate protein interactions and inactivate downstream signaling pathways in ccRCC cells.
SPOP Inhibitor Displays Favorable Pharmacokinetics
In order to assess the utility of compound 6b in animal experiments, we investigated its pharmacokinetics. Nude mice were treated with a dose of 80 mg/kg of 6b in 0.5% 2-phenylethanol and 1.5% Tween 80 by intraperitoneal injection. Nude mice were grouped randomly (n = 3 per group) and killed at 0.25, 0.5, 1, 2, 4, 6, 10, and 24 hr after injection. Blood was harvested and plasma was analyzed by LC-MS/MS. Compound 6b was found to have a peak concentration of 1,070 ng/mL at 0.5 hr after injection (Figure S5A and Table S1). Compound 6b might be retained in nude mice with a half-life of 6.52 hr. Compared with the 3,515 hr × ng/mL area under the concentration-time curve (AUC0-∞), the 3,321 hr × ng/mL of AUC0-24 indicates that 6b can be almost entirely eliminated within 24 hr.
SPOP Inhibitors Are Nontoxic to Animals
In order to determine if compounds 6b or 6b-HCl are suitable for in vivo studies, we examined the induced toxic effects of the compounds in BALB/c or ICR mice. The female and male BALB/c mice (n = 3) were treated daily with 80 mg/kg of 6b or vehicle control in 0.5% 2-phenylethanol and 1.5% Tween 80 by intraperitoneal injection for 6 days. No evidence of body weight loss or mice death was observed (Figure S5B), nor was any obvious weight loss or physical damage observed in the organs (Figure S5C). Encouragingly, daily treatment of 6b in elevated dosage (120 mg/kg) for 6 days also showed no evidence of weight loss or mice death on BABL/c male mice (n = 3) (Figure S5B). Blood was harvested in heparin anticoagulant tubes and variant kinds of blood cells were calculated using complete blood count (CBC) analysis. The results show no significant difference in hematopoiesis between the vehicle control group and 6b-treated mice (Table S2). Similarly, daily treatment of 80 mg/kg of 6b-HCl on ICR mice for 7 days had no obvious effect on body weight (Figure S5D). Blood was subjected to CBC analysis and plasma biochemical analysis. The results revealed no obvious difference between the vehicle control and inhibitor 6b-HCl-treated groups (Tables S3 and S4). These results indicate that compounds 6b and 6b-HCl, at a dosage of 80 mg/kg, are safe for antitumor efficacy studies.
SPOP Inhibitors Reduce the Growth of ccRCC Xenograft in Nude Mice
In order to evaluate the antitumor activity of SPOP inhibitors, we administrated them into nude mice bearing subcutaneous A498 tumor cell xenografts and monitored the tumor growth rate with and without treatment with the compounds. Compounds 6b and 6b-HCl were administered by intraperitoneal injection once per day for 21 days. Two dosage regiments (60 and 80 mg/kg/day) were tested. A clear dose-dependent reduction in A498 tumor growth rate could be observed in mice treated with either compound 6b or 6b-HCl compared with the vehicle-injected control (Figures 5A and S5E ). The negative effect of the compounds on A498 tumor growth could be clearly observed in the reduced variance in tumor size or weight with respect to the control as measured at day 21 (Figures 5B and S5F). Compound 6b-HCl has few side effects on body weight compared with compound 6b, as shown in Figure S5G. An initial ∼15% maximal weight loss in mice injected with compound 6b was observed during the first 3 days of treatment, after which this stabilized in all groups (Figure S5H). Histopathological analysis was performed on mice injected intraperitoneally with compound 6b (80 mg/kg/day) for 21 days. H&E staining of multiple organs, except for kidney sections, revealed that no significant differences were observed between the vehicle- and compound-treated groups (Figure S5I), suggesting the low toxicity of small-molecule inhibitors.
Figure 56b Dose-Dependently Impairs the Growth of Xenografted A498 Cells in Nude Mice
(A) Volume measurements of A498 xenograft tumors treated with vehicle or compound 6b at the indicated dosages for 21 days. 6b was administered by intraperitoneal injection once per day at 60 or 80 mg/kg. Changes in mean tumor volume are given relative to untreated tumor volumes. Error bars represent mean ± SD, with n = 6 for vehicle control, and 60 and 80 mg/kg groups. p Values were obtained by unpaired t test.
(B) Volume distribution at day 21 of A498 xenograft tumors treated with vehicle or 6b.
(C) Quantification of PTEN and DUSP7 accumulation and repression of p-AKT and p-ERK at day 21 of A498 xenograft tumors treated with vehicle or 6b.
(D) Schematic overview of the mechanism that the small-molecule inhibitor uses to block SPOP-substrate protein interactions in ccRCC cancer. 6b disrupts the SPOP-mediated protein interactions, inhibits the ubiquitination and degradation of tumor repressors PTEN and DUSP7, and decreases the downstream phosphorylation of AKT and ERK; ultimately, this process leads to the death of the ccRCC cells.
See also Figure S5 and Tables S1–S4.
Lastly, we checked the effect of the inhibitor on oncogenic SPOP signaling in xenograft tumors. As expected, PTEN and DUSP7 proteins are observably elevated in the compound 6b-treated groups, and p-AKT and p-ERK levels are decreased (Figure 5C). The level of SPOP remains relatively steady in different samples. These results are consistent with the observations in the ccRCC cell experiments (Figure 4C).
Although abnormal activation of ubiquitin signaling in multiple tumors indicates that it might be an important tumor target, no therapeutic treatment that targets ubiquitin signaling has clinically succeeded to cure patients. E3 ligase complexes that recognize and degrade substrates directly are highly diverse. Different E3 ligase complexes target specific substrates. Our recent study showed that the ubiquitination-signaling pathways contribute to tumorigenesis of ccRCC through the activation of the hypoxia regulatory network (
). We wondered if the identification of small-molecule inhibitors targeting the E3 ligase Cul3 pathway would achieve favorable therapeutic effects on ccRCC cancer.
SPOP, meeting the following criteria (
), is an attractive target against kidney tumors: (1) SPOP is overexpressed in 85% of kidney cancers and nearly 100% of primary and metastatic ccRCC tumors (
); (2) overexpressed SPOP dislocates in the cytoplasm of ccRCC cells, cyto-SPOP promotes ccRCC tumorigenesis by acting as a key regulatory hub, and a small degree of interfering RNA knockdown of SPOP induces ccRCC cells apoptosis (
); (3) SPOP is expressed at a very low level in adjacent normal kidney tissues and locates in the cell nucleus, inhibition of which has a minimal effect on normal cell growth and function (
); and (4) most importantly, SPOP is an adapter of E3 ligase, and degrades substrates by specifically recognizing their SBC motif (
), making SPOP a classically "undruggable" target in terms of protein-protein interaction. Development of small molecules targeting the E3 ligase adapter SPOP represent a potential approach against kidney cancer.
Although some inhibitors targeting other E3 ligases-substrate interactions have been developed, such as murine double minute 2, von Hippel-Lindau disease tumor suppressor, and Kelch-like ECH-associated protein 1 (
Bulatov and Ciulli, 2015
Bulatov E.
Ciulli A.
Targeting Cullin-RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation.
Biochem. J. 2015; 467: 365-386
Zhao et al., 2015
Zhao Y.
Aguilar A.
Bernard D.
Wang S.
Small-molecule inhibitors of the MDM2-p53 protein-protein interaction (MDM2 Inhibitors) in clinical trials for cancer treatment.
J. Med. Chem. 2015; 58: 1038-1052
), a chemical compound that can disrupt the SPOP-substrate interaction is unknown. Here, we have identified small-molecule inhibitors that target SPOP and inhibit its activity in ccRCC for the first time. In order to find the inhibitors, we utilized a combinative method of virtual screening, pharmacophore modeling, and molecular docking, leading to the discovery of hit compound 6a, and subsequently the lead compound 6b, by synthetic optimization. Distinct from most Cullin-RING ligases, SPOP has a Cullin adaptor BTB domain and substrate receptor MATH domain for integration. We initially aimed to specifically target the MATH domain of SPOP in order to disrupt its interactions with substrates. However, it remains unclear if the compounds really target the MATH domain of SPOP rather than both MATH and BTB domains. Indeed, in cells we have been able to convincingly show biochemically the therapeutic potential of blocking SPOP activity to stabilize PTEN and DUSP7 in order to upregulate their AKT- and ERK-driven cancer-signaling pathways, with phenotypic consequences in both immortalized and primary patient ccRCC cell lines and ultimately in vivo in mice.
Unlike genetic techniques, small-molecule inhibitors are usually thought to be imperfectly specific for inhibiting a single target (
Davies et al., 2000
Davies S.P.
Reddy H.
Caivano M.
Specificity and mechanism of action of some commonly used protein kinase inhibitors.
Biochem. J. 2000; 351: 95-105
). In order to verify that compound 6b is indeed on its target, we performed a CETSA in order to test the compound's ability to stabilize SPOP proteins in vivo. As we expected, the results of CETSA assays demonstrated that compound 6b thermally stabilizes SPOP protein both in cell lysate and intact cells. Furthermore, we observed that cells with the reduced level of SPOP expression become more resistant to compound 6b treatment. In addition, we tested the effect of SPOP inhibitors on HK-2 cells, which is an SPOP-negative normal kidney epithelial cell line. Consistent with our hypothesis, SPOP inhibitors minimally impaired HK-2 cells both in proliferation growth and the substrate signaling pathway. This result may indicate that the small-molecule inhibitor specifically kills ccRCC cells depending on SPOP overexpression and mislocalization. Lastly, a therapeutic dose of SPOP inhibitors has minimal effect on the weight of nude mice or on damage to organs, which would also partially eliminate the off-target effect of SPOP inhibitors. These data have revealed the cellular target engagement of the SPOP inhibitor inside the cells.
In summary, based on the previous observations that SPOP located in the nucleus of the normal cell induces apoptosis (
Byun et al., 2007
Byun B.
Tak H.
Joe C.O.
BTB/POZ domain of speckle-type POZ protein (SPOP) confers proapoptotic function in HeLa cells.
Biofactors. 2007; 31: 165-169
), while the accumulation of SPOP in the cytoplasm of the ccRCC induces proliferation (
), our studies convincingly demonstrate that using small molecules to interfere with the binding of the E3 ligase adapter SPOP to tumor suppressors PTEN and DUSP7, leading to the inactivation of its downstream Akt/ERK signaling pathways, is feasible with regard to the suppression of the proliferation of human ccRCC cancer cells (Figure 5D). To this end, our study implies that SPOP would not classically be viewed as "undruggable," and opens up an avenue that small-molecule-targeting cytoplasmic SPOP signaling might be more specific to ccRCC cancer cells, which would be a promising strategy to combat kidney cancer in future therapies.
Cell Lines and Primary Cells
All cell lines were obtained from the American Type Culture Collection or the National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China) and maintained according to the supplier's specifications. All human tissue samples were obtained from individuals undergoing kidney resection at the Peking University First Hospital (Beijing, China). All subjects provided written informed consent, and approval for the study was obtained from the Ethical Committee of the Beijing Institute of Genomics, Chinese Academy of Sciences.
Compounds and Plasmids
Compounds 6 were derivatives of the core structure obtained from the structure-based screening of the SPECS library, and the synthetic route is described in detail in the Supplemental Experimental Procedures. The hydrochloride form of compound 6b was used in all studies. All compounds were fully characterized by spectrum analysis (Figure S6). Plasmids for Flag-SPOP, myc-PTEN, and myc-DUSP7 have been described previously (
). Human SPOP cDNA was obtained from the German Resource Center for Genome Research (RZPD). For the construction of the 3 × Flag-SPOP, the SPOP fragment open reading frame was subcloned between the HindIII and EcoRV sites of a p3 × Flag-CMV10 vector. To construct myc-PTEN and myc-DUSP7, the fragments containing the coding sequences of PTEN and DUSP7 were inserted in the pcDNA4-myc-His vector (Invitrogen). The pcDNA3-HA-Ub was a kind gift from Dr. Y. Yang at Beijing Institute of Genomics, Chinese Academy of Sciences. All constructs were verified by standard DNA sequencing.
The following antibodies were used for immunoblotting, immunoprecipitation, and in vivo ubiquitination assays: SPOP (clone 6C) (
), Flag (Sigma, F1804), c-Myc (Sigma, M4439), HA (Sigma, H9658), GAPDH (Sigma, G8795), PTEN (Cell Signaling, no. 9559), DUSP7 (ABGENT, AP8450a), AKT (Cell Signaling, no. 9272), p-AKT (Thr308) (Cell signaling, no. 4056), ERK1/2 (Cell Signaling, no. 9102), and p-ERK1/2 (Thr202/Tyr204) (Cell Signaling, no. 4376).
BALB/c and ICR mice were obtained from Shanghai SLAC Laboratory Animals and Shanghai Sippr-BK Laboratory Animal, respectively. Six- to 8-week-old animals were used for the studies and were allowed unrestricted access to food and water. The Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica approved all animal procedures and conformed to all regulatory standards.
Xenograft Tumor Growth
A498 cells (1 × 107) were mixed with Matrigel (BD Biosciences) in a 3:1 volume on ice and subcutaneously injected into the forelimb armpit of nude mice (BALB/c nu, 4–6 weeks old) to generate xenografts. The mice were grouped randomly (n = 6 or 8 per group) and treatment was initiated when the mean tumor volume was approximately 125 mm3. The mice were treated intraperitoneally with 0.9% saline solution containing 0.5% 2-phenylethanol and 1.5% Tween 80 (vehicle), and 60 or 80 mg/kg of 6b or 6b-HCl daily for 21 days. Tumor volume was recorded by the measurement of two perpendicular diameters of the tumors using the formula V = length (mm) × width (mm)2/2, and the body weight of the animal was measured every 3 days. The volumes of tumors treated with the vehicle control and the compounds were compared and p values were determined by a two-tailed Student's t test. At the completion of the study, the mice were killed and necropsied, and tumor tissue was removed for further studies.
The Liu laboratory initiated this project, evaluated compound activity using an MTT assay, and validated the cellular signaling pathway. The Jiang laboratory identified the hit compound in order to disrupt SPOP-substrate interactions. The Yang laboratory optimized the hit structure by synthesizing numerous compounds, conducted the mechanism studies in vitro, and performed animal experiments. C.-G.Y., J.L., and H.J. conceived the project. C.L., S.O., and R.Z. designed and performed the structure-based computational screening; S.G. conducted the synthetic optimization with help from L.-L.M., B.Z., and S.-D.Y.; T.Z. performed the binding studies and characterizations of lead compound with help from F.L., Y.H., L.L., H.D., J.L., Y.W., and N.Z.; B.C. and J.L. carried out the proliferation assays and xenograft experiments; Y.Y., Z.G., and D.Z. performed the pharmacokinetics experiments; J.L., L.G., and J.R. conducted the toxicity studies; Z.-Q.G. performed the coIP detections and ubiquitination-signaling studies with help from L.Z., L.Z., G.L., K.C., Y.K., and X.-B.Q.; Z.-Q.G., T.Z., and C.-G.Y. wrote the manuscript. All authors discussed the results and commented on the manuscript.
We thank Dr. M. Zhuang (ShanghaiTech University) for the kind gift of plasmid pET23a encoding the MBP-tag, Dr. Z. Xu (GE Healthcare) for help with surface plasmon resonance experiments, and S.F. Reichard, MA, for editing the manuscript. This work is supported in part by the National Key Research and Development Projects (2016YFA0501500 to C.-G.Y., 2015CB856200 to J.L., 2015CB910304 to H.J., and SS2014AA020608 to Y.K.), the National Natural Science Foundation of China (91313303 to C.-G.Y., 81171902 to J.L., 21210003 and 81430084 to H.J., 81101940 to K.C., and 81402112 to Y.K.), and the Youth Innovation Promotion Association of the Chinese Academy of Sciences to K.C.
Download .pdf (8.45 MB)
Document S1. Supplemental Experimental Procedures, Figures S1–S6, and Tables S1–S4
Accepted: August 8, 2016
Received in revised form: February 15, 2016
Received: May 24, 2015
DOI: https://doi.org/10.1016/j.ccell.2016.08.003


text:
Q: Jquery: apply class to all text on the webpages I'm using the jquery plugin trocar to make the text of a webapge editable on the fly.
for example

             This text is editable.

In above i have added a class, which uses by following js code to make that editable.
js code:

On webpage i want every pieces of text to editable.
On way is to add text-poi class to every single text element , but would be tiring.
Is there any better way to do that ?

A: A better and safer solution would be to reference only tipical text tags, I would avoid * selector:
$('p, span, h1, h2').trocar({[...]});

A: How about:
$('*').trocar({
    'on': 'focus',
    'off': 'blur',
    'endOnEnter': true,
    'filterPaste': true
});

A: here is what i did and it worked for me

   < all your content here >

and in the script section at the bottom before the  tag ....

hope this helps

redpajama_set_name: RedPajamaStackExchange


text:
Svétloie (en rus: Светлое) és un poble de la província d'Astracan, a Rússia, segons el cens del 2010 tenia 254 habitants.

Referències

Pobles de la província d'Astracan


text:
Bill Haskell
The principle responsibility of the Commodore is to ensure regulatory compliance and to foster the ongoing development and vitality of the Club for current and future members. Bill has respectfully held the position of Commodore for many years and is also the Club's Licensee.
Bill sails regularly on Good Life II and represents the Club in international races such as PacRim.
Vice Commodore:
Rick Pacey
Responsible for the setting, administration and conduct of the club's sailing programme. Rick is an experienced sailor across differing boat types and classes, including offshore, coastal and regular competition in state and national regattas.
Rick is currently a co-owner and skipper of 'Elusive' and also brings an extensive corporate, business and project management background to the club.
Rear Commodore:
David Simm
Responsible for the organising and supervising the maintenance of the Club premises, ensuring its compliance with relevant authorities, liaising with Council on issues affecting the Club premises and is the Club's representative on the Soldiers Point Management Plan Committee.
David is the owner / skipper of 'Chilli'.
Club Captain:
Alan Richards
Acts as the Safety Audit Officer, assists in conducting Equipment Audits, sits on the Race Committee, responsible for the Club moorings and rounding marks, and assisting the Vice Commodore with preparation of the Race Programme, Notices of Race, Sailing Instructions, Race Courses, and other race related documentation.
Alan is an experienced sailor on a wide range of yachts and actively sails his joint owned Etchells 'Broomstick'. Alan is a shipwright by trade but is currently working as a work health and safety consultant, which adds high value to his role on the Board.
Marina Budisavljevic
Responsible for distributing information and notices to all members, editor of the monthly newsletter "The Bilge", manages the website, attends to all correspondence, ensures practices of the Club are in line with the Constitution, coordinates social functions, and prepares the agenda and minutes for Board Meetings. Marina is also assisting with the membership, sponsorship and publicity of the Club.
Marina sails on 'She's The One' and is a keen ambassador for women in sailing. Marina brings value to the Board from her professional experience as an urban/environmental planner.
Honorary Treasurer:
Ann Evans
Ensures regulatory and statutory requirements are adhered to with regard to taxation, ASIC, insurances, is responsible for all banking and payments, undertakes/supervises stock take, prepares all accounting records, financial statements and reports for internal and external review and audits.
Ann is the Club's Honorary Treasurer and brings a wealth of experience both as a Chartered Accountant and as Treasurer of several other not-for-profit organisations.
Ken Peachey
As Race Officer, Ken is responsible for managing 'SAIL100', the Club's race management system including the on going review of handicaps and the production of race results.
Ken is owner / skipper of 'Una Vita' and a member of the Race Committee.
Michael is taking on responsibility for helping to develop growth in Club member numbers. He brings valuable experience to the Board particularly with his extensive knowledge of real estate, property matters and business management.
Micheal is owner / skipper of 'She's The One'.
Steve Plante
Steve is responsible for the maintenance of the Club's rounding marks and moorings alongside the Club Captain.
He and his wife Judy moved up to Port Stephens just in 2016 having retired in Sydney and Steve has been sailing competitively since 2000 and is owner / skipper of 'Twitcher'.


text:
Us Cities Paris Agreement
Author: craftybroads
He said: "Climate change is the catalyst, but these plans go much further, how to create cleaner, more inclusive, more egalitarian cities, with better housing and better jobs." Among the cities best assessed to meet their emissions reduction targets, the NRDC is working to make the Global Climate Action Summit a success, building on more ambitious commitments to the landmark 2015 agreement and enhanced initiatives to reduce pollution. Recognizing that many developing countries and small island developing states that have contributed the least to climate change are most likely to suffer the consequences, the Paris Agreement contains a plan for developed countries – and others that are able to do so – to continue to provide financial resources to help developing countries reduce and increase their capacity to withstand climate change. The agreement builds on the financial commitments of the 2009 Copenhagen Accord, which aimed to increase public and private climate finance to developing countries to $100 billion per year by 2020. (To put it in perspective, in 2017 alone, global military spending amounted to about $1.7 trillion, more than a third of which came from the United States. The Copenhagen Pact also created the Green Climate Fund to mobilize transformation funding with targeted public dollars. The Paris agreement expected the world to set a higher annual target by 2025 to build on the $100 billion target by 2020 and create mechanisms to achieve this. "I was president of the C40 cities when the 2020 deadline was set and I challenged global cities to put in place their own climate plan, which protects people, creates green jobs, ends inequality and builds the future we want," Hidalgo said before the event. In addition to this declaration, 211 climate mayors have adopted the Paris Agreement goals for their cities since President Trump announced his withdrawal, 13 governors have created the bipartisan U.S. climate alliance and 17 governors have issued individual statements that assist Paris. Today`s declaration joins this growing movement of sub-national leaders and civil society by announcing that these leaders are not just moving forward, but moving forward together.
The Secretary-General`s Special Representative for Climate Policy today announced the $70 million American Cities Climate Challenge, a major new effort to accelerate progress in reducing greenhouse gas emissions while pushing the local economy into a period of federal inaction. The Challenge is open to America`s 100 most populous cities.
← Uk Health Agreement With Australia Uwa Enterprise Bargaining Agreement →
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Copyright © 2021 Crafty Broads. All rights reserved.


text:
BENEFIT CONCERT SCHEDULED
A daylong concert to benefit Florida Cystic Fibrosis Inc. begins at 10 a.m. Sunday at C.B. Smith Park at the intersection of Flamingo Road and Pines Boulevard in Pembroke Pines.
Scheduled to appear at the concert are The Fabulons (oldies), The Jetters (country), Tommy Gunn and Full Clip (blues/contemporary), The Roach Thompson Blues Band (rhythm and blues), Michelle Barica (contemporary) and musicians playing Latin music and jazz.
Music Aid, the concert organizer, will donate proceeds to the fight against cystic fibrosis, which causes deterioration of the lungs and digestive tract. The concert ends at 5 p.m. Admission is $7.50. Children under 12 are free. Advance admission is $6, and tickets are available by calling 680-9270.


text:
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#Copyright 2014 LinkedIn Corp.
#
# Licensed to the Apache Software Foundation (ASF) under one
# or more contributor license agreements.  See the NOTICE file
# distributed with this work for additional information
# regarding copyright ownership.  The ASF licenses this file
# to you under the Apache License, Version 2.0 (the
# "License"); you may not use this file except in compliance
# with the License.  You may obtain a copy of the License at
#
#   http://www.apache.org/licenses/LICENSE-2.0
#
# Unless required by applicable law or agreed to in writing,
# software distributed under the License is distributed on an
# "AS IS" BASIS, WITHOUT WARRANTIES OR CONDITIONS OF ANY
# KIND, either express or implied.  See the License for the
# specific language governing permissions and limitations
# under the License.
-->
<!DOCTYPE html>
<html>
  <head>
    {%- if page_title %}
      <title>{{ page_title }}</title>
    {%- endif %}
    {%- if css_includes %}
      {%- for css_include in css_includes %}
        <link rel="stylesheet" href="{{ css_include }}">
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      <!-- Latest compiled and minified CSS -->
      <link rel="stylesheet" href="https://maxcdn.bootstrapcdn.com/bootstrap/3.3.1/css/bootstrap.min.css">
    {%- endif %}
    {%- if js_includes %}
      {%- for js_include in js_includes %}
        <script type="text/javascript" src="{{ js_include }}"></script>
      {%- endfor %}
      <script src="http://code.jquery.com/jquery-1.11.0.min.js"></script>
      <script src="http://code.jquery.com/jquery-migrate-1.2.1.min.js"></script>
      <!-- Latest compiled and minified JavaScript -->
      <script src="https://maxcdn.bootstrapcdn.com/bootstrap/3.3.1/js/bootstrap.min.js"></script>
    {%- endif %}
  </head>
  <body>

redpajama_set_name: RedPajamaGithub


text:
{% extends 'templates/nhs_transaction_layout.html' %}
{% import 'includes/form_macros.html' as form_macros %}

{% block afterHeader %}
  {% include "includes/service-headers/low-income.html" %}
{% endblock %}

{% block content %}

    Do you or your partner have a mortgage or loan secured on your home?

  <form action="" method="get" class="form">

  <fieldset>

        <input type="radio" id="email" name="contact">

          Yes

        <input type="radio" id="mobile" name="contact">

          No

        How much is your mortgage or loan payment each month?

Include any endowment premiums linked to the mortgage.
Don't include any other insurance premiums or any arrears.

      <input type="text" id="email-address" class="form-control">

  </fieldset>

    {{
      form_macros.button({
        href: 'ground-rent',
        label: 'Save and continue'
      })
    }}


President Trump showed little concern for water protectors or Native sovereignty when he fast-tracked the Dakota Access Pipeline in spite of environmental concerns. Now he claims to support Native American sovereignty by promising to relax regulations so Native nations can dig up natural resources on their reservations and create jobs.
To be clear, Indian Country is not monolithic its opposition to extractive industries that hurt Mother Earth. We wrote a piece April 5 titled: Indian Country is Divided on Mining Coal, Drilling for Oil; More Clashes Ahead. Yet Trump's support for tribal sovereignty here seems dubious at best, a tool to support his pro-fossil fuel agenda.
Trump made his comments today while meeting with state and tribal leaders. Rather than do more interpretation, here are his words directly from a White House transcript.
…. I'm proud to have such a large gathering of tribal leaders here at the White House. I look forward to more government-to-government consultations with tribal leaders about the issues important to Indian Country. We love Indian Country, right?
To shed some light on what "energy dominance" means, check out this chart. It shows that the United States is now a net exporter of finished petroleum products, including gasoline. Yes, we still import crude oil, but we are importing more than we need for domestic use. The various pipelines proposals such as Keystone XL and the Enbridge Line 3 expansion through northern Minnesota are not about our own energy security, they are about increasing corporate profits from exporting energy.
Read Trump's full statement here.


text:
New Internships at OneEurope!
Thanks to significant growth and new partnerships, we have been able to create several new exciting positions!!
17 Aug 2013 | tags: Internship, Join Us, Volunteer and Inspire Europe!
Gain experiences, boost your CV and make friends all across the continent, while contributing to a more united and democratic Europe! Here are the new positions we have available!!
- Article Editor - Social Media Manager - Initiatives Reporter - Editor of our new Video section - Infographic Editor - Cartoonist / Caricaturist - Graphic Designer
We will provide training and guidance throughout, while allowing you to develop in the areas YOU are most interested in.
All internships are on-line, from the comfort of your home, and completely flexible in terms of the days and times suitable to you. The typical weekly commitment is 5-7 hours, which can be done at the best time for you.
If none of the above roles are of interest to you, check out our many other opportunities, for Writers, Ambassadors, Fundraisers, Designers and much more: http://one-europe.info/join-us
Apply by sending an e-mail to: info@one-europe.info
Internships, is this the new millennium's slavery?
Exploring the discrepancy between what an internship should be and what it actually is
by Fábio Lopes Paulos on Apr 01, 2016
What it's really like to work for international organisations – the interns speaking (Part II)
Thousands of young people dream about a career in global institutions. But is an internship the best route into employment there?
by Tanya Zhekova on Aug 31, 2015
What it's really like to work for international organisations – the interns speaking (Part I)
Ivan Botoucharov Co-Founder of OneEurope	read more
Nathan Wheeler Following extensive work with Amnesty International researching death penalty cases in the United States and forced evictions in Italy Nathan's work shifted to human rights and its interaction with religious rights. He studies Law in Dublin City University and has an avid interest in the foundati..
Peter Josika Peter Josika is a Swiss-based Austrian author, Historian and Political Scientist specializing in topics related to European politics, federalism, centralism, nationalism, post-War expulsions and minority rights. He can be contacted on europaderregionen.org.