Sunday, December 8, 2013

Picture Blog: Naive Human Pluripotent Stem Cells Regrown From Allele’s iPSCs

As we blogged a month ago, the Hanna lab recently published a paper in Nature describing that human ESCs or iPSCs, which typically resemble more of mouse EpiSCs (epiblast stem cells) than ground state mouse stem cells, could be converted to naïve pluripotent stem cells if grown in a stem cell medium that includes hLIF, JNKi, and p38i.  The figure here shows that the reported system did perform well when we at Allele Biotech tested growing our banked iPSCs under similar conditions.  The colonies grown in naive stem cell conditions (B) did become dome-shaped when cultured for longer period of time; when transferred back into regular stem cell medium, the once naive-looking iPSCs formed tighter and “cleaner” colonies than typical “primed” human iPSC colonies.


A, Primed human stem cells: mRNA-iPSC line J-1 grown on CellStar-coated surface and in E8 medium. The cells have human iPSC morphology of being compact in size and in "shiny" colonies. B, Naïve human stem cells: J-1 iPSCs shown 2 days after switching to a medium similar to the Naïve Human Stem cell Medium (NHSM). Compared to primed stem cells in A, the naïve stem cells are more flat and transparent, with no spontaneous differentiation on the edges.

Tuesday, November 19, 2013

Human Pluripotent Stem Cells Are Getting Naïve

 
In the field of stem cell studies there has been a long standing notion that human embryonic stem cells (ESCs) are not equivalent to mouse ESCs isolated from mouse inner cell mass of blastocyst. When induced pluripotent stem cells (iPSCs) were developed by the Yamanaka lab from human adult cells, they were found to be closer to human ESCs but not as “naïve” as the mouse ESCs.
To learn more, see the following key points about naïve stem cell:
The ground state of human iPSCs or ESCs remains the holy grail in stem cell research largely because of its conceptual value, and also because it was difficult to achieve. When mRNA reprogramming was first described by Warren et al. 2010, the hope was that the mRNA-iPSCs could be closer to ground state compared to virus-mediated iPSCs since mRNA-iPSCs had no issue with uncontrolled transgene expression or silencing. However, the human mRNA- iPSCs produced even with our current, much more potent mRNA mix did not grow in dome-shaped colonies like mouse ESCs, making us wonder whether that is achievable. A recent publication by the Hanna group showed that a ground state pluripotency could be achieved by simply growing cells in the presence of a few additional medium factors, mostly controlling signaling pathways. Since it has been shown that the currently available “primed” (not naïve) human iPSCs can already be derived into various tissue types, the practical impact of the new discovery might be more likely found in removing epigenetic memory after reprogramming, or line-to-line variations if a truly naïve state could be achieved.
Technically, any existing human ESCs or iPSCs could be converted to naïve stem cells, according to the new publication. And when the new medium system, termed NHSM for Naïve Human Stem Cell Medium, was applied to iPSCs, it was used 4 days after the start of the reprogramming run.
Key points about naïve stem cells:
1) Stem cells grow in dome-shaped colonies under 2i/LIF conditions.
2) Doubling time is around 14 h compared to 26 h of primed PSCs.
3) Up to 88% single-cell cloning efficiency in the presence of ROCK inhibitor.
4) OCT4 distal enhancer is used more than the proximal enhancer in naïve PSCs.
5) In cells from female donors, naïve iPSCs are at pre-X inactivation state.
6) More E-CADHERIN expression on the surface of naïve stem cells.
7) It is easier to perform gene targeting by homologous recombination in naïve PSCs.
8) Less H3K27me3 in development genes in naïve cells.
9) High efficiency of integration and chimaerism when naïve iPSCs were injected into mouse embryos.
Gafni et al. “Derivation of novel human ground state naive pluripotent stem cellsNature 2013” http://www.nature.com/nature/journal/vaop/ncurrent/full/nature12745.html
New Product of the Month: Nano antibody Line including GFP-nAb and mNeonGreen-nAb for co-IP using fluorescent proteins as precipitation tag, nab your complexes like never before!
Human iPSC Commercial Service and Technology Licensing: mRNA-iPSCs made feeder-free, xeno-free, and footprint-free.

Saturday, November 16, 2013

林昭案卷的来龙去脉

来源:南方周末 作者:彭令范
  
    林昭(1932-1968)遗照。 (南方周末资料图/图)
    林昭案卷的来龙去脉

    编者按:彭令范女士是林昭的妹妹,她这篇文章,详细披露了林昭案历经两次复查,方得平反的过程。读后我们才知道,主持林昭案平反的上海高院院长关子展,原是彭令范在上海第二医学院的老领导,如果不是有此机缘,林昭要脱去“精神病”的罪名,恐怕还要经历很多波折。
  
    彭令范女士现居美国。
  
    林昭难友张元勋去世后,彭令范填了一首词聊表纪念,这里一并发表,以飨读者。

  
    1979年2月28日,我收到北京大学党委寄来的姊姊林昭的右派分子改正通知。这时我想到,应该向有关部门申请复查和平反林昭的反革命罪和死刑判决,同时也必须为我母亲许宪民(1908-1973)的“反革命分子”罪名平反。
  
    母亲受到牵连
  
    “文革”前,我母亲是“民革”苏州市委副主任、市政协委员、中国民主同盟盟员、苏州市汽车公司副董事长、苏州公共汽车公司副经理,她在1949年 后的历次运动中幸存了下来,但在“文革”中却为了林昭的缘故,也成了反革命分子,死于非命。她的平反须通过高一级——江苏省的有关部门甚至民主党派的中央 批准核定。
  
    我同时做这两件事(母亲和姊姊的平反),必须尽一切努力。在那个年代,我用了几乎所有的时间,写了几百封信给我以为能给我们帮助的人,用菲薄的工 资从上海跑南京,跑北京,找寻和访问所有和我母亲、姊姊有关的人,大多数是母亲的同志和朋友,有些是中共党员,在毛泽东去世、“文革”结束后,官复原职, 同时我也作了一些调查研究,找出原始资料和有用证物。没有任何人帮助我做这件事,人们不敢涉及这棘手的问题,包括我的亲弟弟。我完全靠自己做这事,只有上 帝照看着我。
  
    我母亲的追悼会于1980年2月10日在苏州举行,与会者百余人,不无知名人士,如孙中山第八位秘书田桓、台湾裕隆汽车公司老板的长兄严庆祥等,不胜枚举。另外还有来自全国各地的百余份唁电、挽联。《苏州日报》报道了这一新闻,母亲再一次被称为同志。
  
    第一次复查
  
    姊姊的平反过程是非常错综复杂的,并带有一定的神秘色彩。1980年8月22日,我收到上海市高级人民法院寄来的《刑事判决书》(沪高刑复字第435号),判决书虽然宣告林昭无罪,但给出的理由却是:
  
    现经本院复查查明:林昭在1958年被错划为“右派份子”后,因精神上受到刺激,1959年8月开始就患有精神病,嗣后,曾以写长诗、文章等表示 不满,并非犯罪行为。1965年对林昭以反革命罪判徒刑显属不当,应予纠正。林昭被错判服刑后,精神病复发,又曾用写血书、诗歌、日记以及呼喊口号表示不 服,1968年将林昭在病发期间的行为,又以反革命罪处以极刑,显属错杀,应予纠正。据此,本院特重新判决如下:
  
    一、撤销上海市静安区人民法院1962年静刑字第171号和中国人民解放军上海市公检法军事管制委员会1967年度沪中刑(一)字第16号两次判决。
  
    二、对林昭宣告无罪。
  
    所以这里是错划“右派分子”,错判“反革命”服刑,错杀,三错到底,主要归咎于她的“精神病”。我并不认可这平反的“措词”,但当时只能得到这样 的平反判决。因为林昭的“精神病”,法院没有发还家属任何遗物。我曾去信要求发还一些她的遗物(我的目标是她的遗稿),但杳无音讯。
  
    然而奇迹出现了!我所在的上海第二医学院(编者注:1952年院系调整中由圣约翰大学医学院、震旦大学医学院及同德医学院合并而成)的党委书记兼 院长、“党内走资本主义道路的当权派”关子展(1914-1996),“文革”后恢复名誉任上海市高级人民法院院长(编者注:关子展在1979年12月 30日结束的上海市第七届人民代表大会第二次会议上当选为市高级人民法院院长),当我看到他的任命在报上发表后,我立即写信给他。
  
    彭令范赠给胡佛研究所的林昭案卷 (南方周末资料图)
    林昭案卷的来龙去脉

    我与关子展的相识
  
    我与关子展的相识,很有戏剧性。
  
    1973年我母亲去世后,我体内各种生理系统不平衡,经常生病,1976年经急诊住入我们医学院附属的广慈医院(编者注:今瑞金医院前身)。当我 的病情略见好转后,就帮医生护士做些简单的工作。有一天,我到一个小房间给病人发药,一开门,我惊讶地叫道:“关院长!”“我已不再是院长了,”他说。 “不,你是我们的院长,我的院长。”我答道。当时他罹患高血压、心脏病,因为是“党内走资本主义道路的当权派”,在当时是重点打击对象。自此以后,我和关 院长逐渐熟悉和接近。
  
    我们谈医学,他问我:“你是否是好学生好医生?”
  
    我说:“当然。”
  
    他说:“我要问你老师……”
  
    有时他会对护士说:“叫728(我的病床号)来看我。”当时他虽然被允许住进他领导下的医学院附属医院,带有“保护”性质,但被与外界隔离。我是 医生,有些“情报渠道”,所以会告诉他一些敏感消息或道听途说的小道新闻(有时小道亦来自大道)。我尊敬他是我们学院的院长,这本来是人之常情,但这种常 情却超出了“文革”的常情,大概给他留下了良好的印象,中间没有任何政治因素。我从未想到,有一天他会给予我难以想象、不可思议、出乎意料的帮助,使我终 生感激难忘。
  
    林昭的追悼会
  
    我写了好几封信给关子展。第一封是1980年8月26日,然后是同年9月2日和9月26日,提出为林昭开追悼会的要求,同时希望在他的领导下,重新复查林昭案件。我用外交词令暗示,以精神病为由平反似不符实际。
  
    如果不是关院长,其他人大概不会为“精神病”操心,因为林昭已经得到平反了。
  
    结果在1980年12月11日,林昭追悼会在北京中国新闻社召开。与会者有全国政协副秘书长聂真、北大林昭的老师们,还有苏南新闻专科学校、北京 大学、人民大学林昭的同学,以及林昭在人大资料室监督劳动时遇到的刘少奇前夫人王前等,共百余人。我保留了追悼会的录音磁带和所有资料,包括来自全国各地 的唁电、唁信和挽联约八十余份。
  
    参加追悼会大多数人,都希望我能全力去为林昭的“精神病”平反,“只有你能做这件事!”这是人们对我的期待。
  
    《人民日报》报道“子弹费”
  
    1981年1月27日,《人民日报》发表了报道林彪、江青集团案审判情况的长篇通讯《历史的审判》(由新华社总编辑穆青与郭超人、陆拂为合写),其中有一段提及林昭的案件:
  
    她(林昭)就义的详细经过至今无从查考,我们只知道这样一个消息:1968年5月1日清晨,几个“有关方面”的代表找到了她年迈的母亲,宣告林昭 已于4月29日被枪决。由于“反革命分子”耗费了一发子弹,她的家属必须交纳五分钱的子弹费。这真是使人毛骨悚然的天下奇闻!在中世纪被判“火刑”烧死的 犯人无须交付柴火费,在现代资产阶级国家用“电椅”处死的犯人也从未交过电费,唯有在林、江的法西斯统治下,人们竟要为自己的死刑付费,这不能不说是又一 个“史无前例”的创造发明!也许在若干年以后,我们的后代对上述这一切将难以置信,但不幸的是,它确实是发生在我们这一代人生活中的事实。我们每一个活着 的人,都曾经为它感到极度的羞耻。
  
    《人民日报》面向全世界发行,并有各种不同语言的翻译,所以林昭的故事,特别是五分钱子弹费的故事,早在1980年代已在国外流传,我曾看到几个地方(法国等)提及。
  
    这篇通讯发表后,上海公安局很恼怒,“肯定是林昭的那些记者同学干的(写的)!”诚然,陆拂为是林昭北大、苏南新专的两度同学。
  
    再一次复查
  
    1981年2月10日,上海静安区人民法院赵凤岱找我去谈话,他当时是复查组组长,他首先问我林昭的精神状态,“你是医生,你如何看她的精神状 态?”“她去上海精神病总院鉴定过数次,但她仍关在监狱里,没有住精神病院。”我答道。赵先生是一个正派的人,后来我们彼此配合、理解甚佳。有一天,他对 我说:“第一次见到你时,你的眼睛里充满了怒火。”
  
    1981年3月,陈伟斯先生写了最早关于我姊姊的文章《林昭之死》,发表在上海《民主与法制》杂志上。复查组组长赵先生让陈伟斯看了一房间林昭的 档案,只能看,不准抄录,以一天为限。陈伟斯大概是政治系统之外、唯一看到这些档案的人。陈先生是我母亲的同志和朋友(我称他陈叔叔),中共党员、新闻记 者,也是右派分子,曾在劳改营被劳教20年。平反后,重新回到新闻界工作。
  
    与此同时,赵先生曾多次提及“我们的院长对林昭案非常关心并直接参与”。1981年12月30日,我收到上海市高级人民法院的另一份判决书:
  
    本院于1980年8月22日复查……对林昭宣告无罪在案。经本院院长发现,本案改判判决在适用法律上仍属不当,由审判委员会决定再审。
  
    现经本院再审查明:林昭于1958年在北京大学读书期间被错划为“右派份子”,于是以写长诗、文章等表示不满;1968年林昭在被错判服刑期间, 又先后用写血书、诗歌、日记以及呼喊口号表示不服。按林昭以上行为,根本不构成犯罪;而上海市静安区人民法院和中国人民解放军上海市公检法军事管制委员会 均以反革命定罪处刑是错误的。本院于1980年8月22日复查改判时,否定林昭犯有反革命罪,撤销原来两个错误判决,对林昭宣告无罪是正确的。但据以宣告 无罪的理由是说林昭因患有精神病,在病发期间的行为,不应以反革命罪论处。如上所述,林昭的行为既不构成犯罪,故本院(80)沪高刑复字第435号判决在 适用法律上亦属不当,均应与前两个判决一并予以纠正。据此,本院判决如下:
  
    一、撤销本院(80)沪高复刑字第435号、中国人民解放军上海市公检法军事管制委员会1967年度沪中刑(一)字第16号和上海市静安区人民法院1962年静刑字第171号判决。
  
    二、对林昭宣告无罪。
  
    这次复查,不仅去掉了“精神病”的借口,并加以否定。至此,林昭的平反,最终完成了。我没有辜负人们对我的要求。
  
    1982年3月,我收到赵凤岱先生发还我的林昭给人民日报编辑部的信137页。
  
    我从来没有对任何人讲我认识关院长和他在林昭平反中的作用。直到1999年,我写了一篇《我的姊姊林昭》发表在苏南新专的“五七年情缘”专刊上, 约略地提到这事,现在又14年过去了,我应该把这件事讲清楚,在那个年代中,谁帮助使林昭获得了平反,取得了林昭的案卷。
  
    我从来没有机会感谢关院长。他给我的帮助,我铭记终生,感激不尽。但我认为他有一个真正共产党人的良知,捍卫法律的尊严。
  
    神秘人物的馈赠
  
    1982年春末的一天,我们医院党支部书记对我说:“明天下午2点你要到上海公安总局去一次,有人要找你谈话。”“什么主题?”我问道。“你去了就会知道。”“去找谁?”我再问。“没有名字,你准时到,讲你的名字就可以了。”
  
    次日,我到了那里,通报姓名之后,警卫把我领到一个邻近公安总局入口处的办公室。几分钟后,一个四十岁左右、表情阴郁的人走了进来,他注视了我两秒钟,“你是林昭的妹妹?”他问道。“是的。”我说。然后他就开始讲述。我感到很意外,他没有问我任何问题。
  
    “我希望你知道,你姊姊的案例自始至终我们有两种不同的意见,从在监狱里如何对待她(注:林昭被反铐484天),到最后判决死刑,甚至她的平反, 都是非常复杂的过程,可能你也知道一些。因为你姊姊的案子,有些干部丢了工作,甚至坐了牢,下场很悲惨。”我不知道如何应答,也不明白他为什么告诉我这些 情况。我没有准备应该怎么讲,也是怕讲错,“所以有两个司令部?”这是我在这次会见中唯一的几句话。
  
    然后他继续讲:“你姊姊的死刑判决,是中央政治局作出的最后决定。”室内死一样的寂静。我感到空气似乎凝结成冰。我们两人都没有什么要说的了。或者应该说,在任何关键性的时刻,语言都是无力的。
  
    最后,他说:“这里有几本笔记,你姊姊的日记和写作摘要。你大概希望能保留。”“是的,当然!”我答道,“这是不是她的笔迹?”他问道。“是的, 这正是她的笔迹!”然后他问,“你有什么要讲吗?”“没有,感谢你给我她的笔记。”我没有敢问他的名字,我想他也不会告诉我。
  
    这就是林昭三本笔记簿日记和其他资料的来源。
  
    1.《战场日记——留给公众和后世的记录(1967.2.9-2.23)》
  
    2.《血书家信致母亲附血书抗议(1966.10.4;1967.10.14-1968.1.14)》
  
    3.《心灵的战歌,我呼吁人类(1967.11.23-30)》
  
    4.《课卷(1966.1.11-2.27)》
  
    我非常感谢这位神秘的知情人物向我透露林昭案的一点儿内幕,他给我的这些可贵的日记和“课卷”,填补了姊姊狱中生活的空缺,也提供了研究中国近代 史中那些铁腕政治人物的重要线索,同时告诉我们如何研究林昭的案卷。2009年10月,我把这些遗稿和林昭给人民日报编辑部的信,捐赠给了斯坦福大学的胡 佛研究所。
  
    我常常为此人祈祷(虽然或许他并不需要我的祈祷),他大概是一定级别的公安干部,但他仍具有正派、合乎礼仪的人性和善良的道德观念。他给我林昭的 笔记,只因为我是她的妹妹,但或许他要让这世界了解某些内幕,在未来的岁月为历史研究提供线索和暗示。(小标题为编者所加)
  
    小档案
  
    关子展(1914-1996),山西霍县人,1936年参加革命,曾任晋中公安局侦察科长、太原市公安局办公室主任、山西省公安厅调查研究室主 任、华北局社会部办公室副主任等职,1954年调公安部任办公厅副主任、人民防空局局长。1957年调任上海第二医学院党委书记兼院长。1977年任上海 市教育卫生办公室副主任,1978年当选上海市七届人大代表,上海市政协常委,中共上海市市委委员,同年任上海市高级人民法院院长、党组书记。1983年 当选上海市八届人大常委、法制委员会主任。
  
    临江仙·悼张元勋
  
    四十余年弹指去,人间天上茫茫。平生细诉九回肠。营营从楚辞,几度历风霜。劫后风华依旧在,中途不思归航。尘寰错落复流光。麟儿椿萱慰,何必话沧桑。

New Allele Biotech Publication on Stem Cells

Feeder-Free Reprogramming of Human Fibroblasts with Messenger RNA
Current Protocols in Stem Cell Biology • November 13, 2013
DOI: 10.1002/9780470151808.sc04a06s27
Authors: Luigi Warren, Jiwu Wang
This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols.
New Allele Product of the Month: FP-nAb™ products for 100% pull-down

Monday, November 4, 2013

Lab Skills You Stopped Being Proud Of

Molecular biologists who were in graduate school in the 90’s learned how to isolate plasmid DNA from E. coli cultures by a method called “boil-prep” during their first lab rotation. This process involved mixing the bacterial cell pellet in a little bit of detergent, salt and sucrose, dabbed with some fresh lysozyme, and then you are ready to cook, literally! Bacterial cell membranes are disrupted by boiling this soup in a beaker of water over a Bunsen Burner for one minute, and the debris (containing the broken cell membrane and attached chromosomal DNA) is collected by centrifugation in a microfuge at top speed for 10 minutes. Then comes the step that differentiates a true master of lab skills versus a rotation student—if you knew just the right amount of bacterial culture to begin with and handled the E coli pellet by the right techniques, a skillful lab person could collect nearly all the liquid without disturbing the pellet. Pouring out the plasmid-containing supernatant without dislodging the goo on the side/bottom of the tube was such a desirable skill that would not only give you your plasmid but also give you admiration from fellow lab members. That is, of course, if you were doing it before the mid-90’s, because after the introduction of miniprep spin columns by Qiagen, nobody, even the true masters of boil-preps (or its contemporary alkali prep that also involves pelleting by centrifugation and careful removal of tiny volume of liquids surrounding small pellets) would be showing off those skills any longer.
It is actually never easy or fun to collect liquid surrounding small amount of beads or pellets as you always have to struggle to remove as much liquid as possible while trying not to lose any of the beads
Some of the old-timers used to also be very proud of being able to pour a “sequencing gel” (a very thin ~40 cm x 30 cm polyacrylamide gel). I still remember the first time I reported to the second rotation lab at USC. After describing the lab research, the PI showed me around the lab and complained how “Sarah destroyed all my sequencing gel plates”. But consider this, in order to avoid any greasy spot on either plate, you needed to wash both of them fanatically if not religiously. Why? You would have just about a minute’s time to pour non-polymerized acrylamide without leaking from the sides or bubbles forming anywhere in the DNA running lanes, and then inserting a pair of paper-thin combs, all at a speed quicker than TEMED/AP-catalyzed acrylamide polymerization. Good thing that after capillary sequencing was invented, we all happily retired our sequencing-gel pouring skills with a collective sigh of relief.
Technology will always move forward, so will the skills lab researchers will be required to perfect. Using a spin column is very much a “skill-less” technique in contrast to collecting pellets and washing beads after centrifugation, but when there is a choice, people will chose the method that requires “less skills”, such as the spin-column format as the preferred platform for the new FP-nAb™ products.

Sunday, October 27, 2013

When Great is not Good Enough—VHH Antibodies Engineered for 10 Fold Affinity Increase

Single Domain antibodies (VHH fragments, nanobodies, or as we call them, nAbs) have been generated by injecting llamas with ligand-bound GPCR for the purpose of obtaining crystals of active-state structures. Such structural information could be critical in understanding drug functions and screening for new drugs. The unique ability of VHH fragments to fit into protein-protein complex crevices and hold proteins together was demonstrated by two Nature publications from Brian Kobilka’s group at Stanford ([1, 2], also see Allele Newsletter of Sep 4th, 2013). The nano antibody used in those studies, Nb80, showed affinity towards only the active state of the target GPCR.
However, even with an antibody as great as Nb80, the authors were only able to co-crystal GPCR beta2-adrenoceptor (b2AR) with high affinity agonists, not its natural agonists such as adrenaline. In yet another Nature paper published just now, the Kobilka lab showed that Nb80 could be further improved by 10 times in affinity, through in vitro evolution [3]. They presented Nb80 on the surface of yeast using an existing yeast display system, then applied standard limited mutagenesis and magnetic separation technologies for screening. After about 5 rounds of selection, a new version of VHH Nb6B9 was isolated that bound to ligand-loaded GPCR with a kD of 6.4 nM. For the first time, a co-crystal of b2AR-adrenoline was made.

Thursday, September 12, 2013

mRNA Delivery And the Next Wave of Regenerative Medicine

AlleleBlog: Published online by Nature Biotechnology, researchers from Ken Chien’s lab at Harvard and other coauthors showed that modified mRNA of VEGF-A injected intramyocardially resulted in the expansion and directed differentiation of endogenous heart progenitors. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients by directing epicardial progenitor cells toward cardiovascular cell types. This publication appears to be the first example of using mRNA as a delivery platform for cell fate-related therapy. AstraZeneca recently invested $240 million on mRNA-related delivery via Moderna, a company with roots within the Harvard stem cell group.
The drastically increased efficacy of using the mRNA platform was accredited to the pulse-like kinetics of mRNA expression profile. It was explained by the fact that native paracrine signals are often transient and precisely regulated in time and space, therefore the pulse-like expression profile of modRNA might be well suited to delivering paracrine-factor signals. Transfected mRNA molecules do not need to penetrate the nuclear membrane, which greatly enhances the efficiency of protein expression on a per transfected molecule over DNA. mRNAs turn over in a much faster pace than plasmid-mediated transgene expression. This is beneficial to many cell fate decisions as exemplified by this recent publication.
Allele Biotech’s reprogramming technologies, licensed by some of the leading stem cell therapy companies, are built around the mRNA platform. We chose mRNA as our core technology to not only change cell fate, but also direct differentiation. We know this platform is the future for cell fate manipulation because we have seen how robustly mRNA expression made the day-and-night difference in gene expression when compared to plasmid DNA (episomal or not), retrovirus, lentivirus, baculo virus, or even transfected proteins. We could convert human fibroblasts into iPSCs, in bulk, in as short as one week with no more effort than changing mRNA complex-containing medium.
Another recent development in iPSC research is in situ reprogramming. Abad et al. generated mice carrying a Tet-inducible cassette of the four cell-reprogramming factors. They then added feed doxycycline to the animals. After several weeks, teratomas appeared in various tissues, indicating that in situ reprogramming had occurred. The iPSCs created this way did not appear to have much advantage over in vitro produced iPSCs other than they are totipotent (helpful if you are studying placenta). Nevertheless, the concept of changing cell fate in situ as dramatically as complete reprogramming is an important leap of faith. As for the next big step, it is easy to see that mRNAs are well suited for in situ reprogramming, as well as transdifferentiation, and more complex gene delivery than the above mentioned VEGF-A alone in heart treatment.

Monday, September 9, 2013

Picture Blog: How Do You Like Your iPSCs, Clonal or Bulk-Conversion?

Weekly update on an ongoing NIH project for improving mRNA reprogramming at AlleleBlog
Reprogramming of differentiated cells into induced pluripotent stem cells (iPSCs) is commonly considered a stochastic process, i.e. with randomness, which could offer a convenient excuse for the historical inefficiency and inconstancy of making iPSCs. We have demonstrated time and again that by using potent mRNA cocktails, the majority of the fibroblasts seeded in a well can be converted into pluripotent stage in a nearly synchronized manner (Warren et al. 2012, Warren and Wang 2013, and this Allele Picture Blog series). mRNA molecules can function robustly yet transiently while avoiding the need of entering the nucleus, a bottle-neck for all DNA-based vehicles.
Other researchers are used to the idea of clonal expansion partly because isolating iPSCs from “clones” was a common step during reprogramming using viruses or other low efficiency methods, even though those clones were not necessarily from single precursor cells. This week, the Allele iPSC team developed a new way of managing our mRNA reprogramming that allowed us to achieve clonal iPSCs that appear to be a lot purer and more likely true clones compared to previous reports, without compromising any of the main benefits of our protocol, e.g. feeder-free, xeno-free, footprint-free, very fast and highly efficient. This work is currently supported by an NIDA/NIH grant to Dr. Jiwu Wang at Allele Biotech.

Allele scientists lead by Dr. Jiwu Wang have been able to consistently bulk convert
Traditional bulk-conversion by the Allele mRNA reprogramming protocol developed by Warren et al. The picture shows large patches of cells becoming stem cells almost overnight around the 9th day of adding mRNA-cocktail supplement to the media.

With clonal lineage, at bulk-conversion rate, new mRNA reprogramming
Clonal iPSC formation using a modified mRNA reprogramming protocol. The picture shows a typical clone of stem cells that originated from likely single cells.
Warren, Ni, Wang, and Guo, Scientific Reports, 2012
Warren and Wang, Current Protocols, 2013, in press

Monday, August 19, 2013

Human induced pluripotent stem cells provide a great route towards personalized medicine and high accuracy drug screening. Allele Biotech has developed the most efficient method of making human iPSCs by using enhanced mRNAs, which have been adopted by leading pharmaceutical companies for clinical trials. The effects from medium-supplemented mRNAs are robust yet transient, and highly specific compared to both miRNAs (off-targets) and small molecules (unknown targets). To repress cellular immune response to introduced RNA molecules, viral protein B18R was previously used during mRNA reprogramming.
B18R is relatively expensive and inconvenient to use because it requires pre-aliquoting and -80C storage. The protocol has recently been dramatically improved at Allele through an NIDA-funded project. In our latest reprogramming run, all we needed to do was to include mRNA complex in the supplement during medium change for just a week without the need of adding any other type of molecules (such as B18R, miRNA, or chemicals) to help the mRNA mix, unlike all other known mRNA-reprogramming protocols. This advancement can make reprogramming human fibroblasts to footprint-free and xeno-free iPSCs a routine experiment for any lab to perform.
Human R-iPSCs were created without the need of B18R, dramatically reduced the cost and inconvenience. Shown is a newly formed iPSC colony.
mRNA reprogramming used to require B18R to repress cellular immune response from repeated exposure to RNA molecules.  Allele scientists lead by Dr. Jiwu Wang further developed their technology to bypass the use of B18R or any immune repressor.

Sunday, August 4, 2013

Picture Blog: More Efficient Reprogramming for Creating Induced Stem Cells (iPSCs)


Researchers at Allele Biotech achieved reprogramming of human fibroblasts into iPSCs within one week, at “bulk conversion”, and with cells seeded at a much wider density range compared to our previous publications. These significant improvements will further facilitate high throughput, large scale iPSC production using Allele’s feeder-free, xeno-free, footprint-free reprogramming, which was already a preferred method for both clinical applications and stem cell banking. The reprogramming project is currently being funded by the NIDA/NIH.
Dr. Jiwu Wang’s team at Allele Biotech (and his sons) succeeded on turning human skin cells into stem cells in 7 days. They also used a different medium than previously reported, even without B18R. Although the cells in the 15k cell well died, they are still happy of what they accomplished in all other cell densities tested.
Human skin fibroblasts were reprogrammed into stem cells using a proprietary mRNA cocktail in just a week.

Tuesday, July 30, 2013

Visualizing Endogenous Synaptic Proteins in Living Neurons

The recently published method is based on the generation of disulfide-free “intrabodies”, a structure from the 10th fibronectin type III domain known as FingRs. These affinity molecules were fused to GFP for direct fluorescence miscroscopy. The FingRs do not need di-sulfite bonds and are therefore better folders in mammalian cells. Specifically, a library was screened with in vitro display to identify FingRs that bind two synaptic proteins, Gephyrin and PSD95. After the initial selection, the researchers from USC secondarily screened binders using a cellular localization assay to identify potential FingRs that bind at high affinity in an intracellular environment. As it turned out, only 10-20% of the original positive clones bind well inside the cells, suggesting this type of further screening was a critical step.
The expression of intrabody is transcriptionally regulated by the target protein through a ZFN-repressor fusion. This transcriptional control system matches the expression of the intrabody to that of the target protein regardless of the target’s expression level. This design virtually eliminates unbound FingR, resulting in very low background that allows unobstructed visualization of the target proteins. As result, the FingRs presented in this study enabled live cell visualization of excitatory and inhibitory synapses, and apparently without affecting neuronal function.
Technically, the reason to use in vitro mRNA display was required by the need to use a large library (>10exp12, beyond the limit of the more commonly used phase display) to find good binders. A similar visualization system can be established using more potent affinity domains such as the VHH single-domain antibodies that have only one, sometimes dispensable, di-sulfite bond. The VHH domain nanobodies can be more easily isolated from camelid animals. Another improvement to the visualization system can be made by using stronger, superresolution-ready FPs such as mNeonGreen or mMaple to enable single molecule imaging, which is particularly interesting for studying synapses and applied to the BRAIN initiative.

Friday, July 12, 2013

李承鹏: 该不相信历史,还是不相信爱情

1368年,当世第一大屌丝朱重八终于逆袭成功了。

    站在应天城高高的台上,这个开国皇帝、也是帝国最资深的叫化子,不能忘怀当年正是贪官污吏让他流离失所,父母差点死无葬身之地。他心如明镜,官场贪腐让横扫天下的大元瞬间崩溃。他下定决心:绝逼要弄死丫们这些贪官污吏。是的,绝逼!

    那一年,他四十岁。

    他 精力充沛,侦察和分析官员异常财务状况时像一部云计算机。他肃贪苛细,宣布凡贪污60两以上银子的官员将被剥皮揎草,杀得兴起,干脆下令“不足60两也杀 掉”。早年的坎坷使他对贪腐恨之入骨,按现在纪检的说法就是对贪腐“零容忍”。在已知的素材里,可以看到亲任政法委总书记的他抓过的一些案子:“收贿袜子 一双、鞋两双”、“书籍四本、衣服一件”、“围脖一个、网巾一个、圆口衣服一件”……这让他看上去像个收破烂的。

    一方面出于叫化 子护食的本性,另一方面,贫苦出身的他认为乱世就得用重典。杀、杀……他成立了亲军督尉府,就是后来人们在影视剧里很熟悉的锦衣卫。又启用检校,那些军 人、官员、太监甚至和尚的职能很像现在的纪检或者巡视组,四处打探官员的负面。一经查实或不经查实,就会迅速砍掉那些人头。

    他花 了十五年率兄弟们打江山,花了十五年杀掉五万贪官以稳住江山。可贪官越来越多,那时出现这样一种景象,官员正在庭上牛逼哄哄审问犯人时,忽然被一拨更加牛 逼哄哄的锦衣卫冲进来抓走,弄得下面跪着的犯人也莫名其妙。又由于官员已不够用,只好留用一些犯了事的官吏让其戴枷办公。主审的官员和被审的犯人一样戴着 枷锁,官员后面站着监视他的锦衣卫,一俟审毕,再把官员拖出去打板子。这景象,十分壮观。

    多年以后,我们看到同样可爱的景观:早 上在报纸上看到市委的李书记严厉批评官风不正,晚上就在微博得知书记已被纪委带走了;刚刚看到纪委的曾书记抓了贪官,不一会儿就因涉黑被“双规”……新近 的广州白云区肃贪,由于被立案查处的干部多达81名,更由于常务副区长、副区长及原区委书记在内的3名主要领导涉案落马,查处干部太多,导致开会人数凑不 够。

    中国的官场史,一部按了循环播放键的滥剧。

    还是让我们回到明朝。话说贪官屡抓不绝,往往早上抓了三 个,晚上又出现五个。资深叫化子决意祭出群众路线。那时还没有新闻联播,焦点访谈仅限于内阁,他就向全国普遍发行了《大诰三编》,在《民拿害民该吏三十 四》里,他不忿地宣布:那些官员都是傻逼,现在我要动员德高望重的老人和见义勇为的豪侠们来帮我举报官员。后来更规定:任何一个百姓可以直接冲进官府,捉 拿不满意的官员,当官的若敢阻拦,则“夷诛全族”。于是通往首都的路上,常见一群群老百姓押解官员前往南京的盛况,活像黄金周旅游,那些当官的甚至下跪向 百姓求饶……真是大快人心。

    群众路线够彻底,视觉上也有种大革命的波澜壮阔。可是官员们仍然贪污,变花样儿贪污。

    他 郁闷。而且新情况出现了,一方面全国书生们如过江之鲫报考公务员,另一方面,人人自危的京官们每天上朝前要站在家门与妻儿诀别,哪个亲戚欠了钱未还、房契 在哪儿、下一代往什么方向培养、小升初找谁走后门……谁也不敢肯定这天上班之后还能不能再回来。一些官员想辞官。不行,“奸贪小人诽谤朝廷”。

    这个桥段由来已久……多年以后,一个叫刘志军的大官隔着铁栅栏告诉女儿“千万别沾政治”,被官媒批评“中伤政治”。还有一个叫赵光华的四川小官因受不了维稳压力辞去了副镇长的职位,被当成反面教材,被迫上网发表声明。

    压力山大,明朝的一些官员很爱得抑郁症。有的真抑郁,有的装疯。那时并没有太多高楼以供官员跳下来,割静脉自杀的法子也还没有流行开来。但喜欢明史的朋友都知道,有个叫袁凯的监察御史为了保命就装疯,他装疯的办法很有创意:吃屎。

    从南派三叔小说的角度,六百多年后的官员就是六百多年前的转世僵尸。看,龙岩的镇长又上吊了。而之前,福建莆田市市长张国胜在办公楼跳楼自杀,天津市政协主席宋平顺自杀,洛阳公安局纪检书记张广生跳楼自杀,浙江高院副院长童兆洪在卫生间上吊自杀……

    好 吧,还是让我们回到明朝。朱元璋真心郁闷,俸禄虽被抱怨不够养师爷,但你们又为老百姓做了多少事呢?虽说杀掉一些官员是为铲除异己,但很多官员的确贪腐得 不成体统。听听那些刑罚:挖膝盖、抽肠、用开水淋再用铁刷子刷、铁钩把人吊起风干……什么《电锯惊魂》弱爆了。朱元璋奇怪得那张瓦刀脸快形成一个巨大的问 号:“法数行而辄犯,奈何?”。为了几个破钱,丫们不怕死么?

    满朝文武,没人告诉他“渴马守水,饿犬护食”这个道理。一个叫桂彦良的大臣却发表了忠诚却相当二百五的意见:“用德则逸,用法则劳。”陛下该鼓励道德,树立官场道德模范。

    朱 元璋深以为然,开动所有国家机器宣传道德,极品道德文章“八股”也在这时集大成了。他这么推理,把圣人思想像软件一样植入未来官员的脑子里,官员有道德 了,国家自然昌盛了。这个推理影响久远,连后来推翻它的“清”也效仿。甚至几百年后的红朝也号召“流着道德的血液”,公务员统考时圣人、道德、高尚、和谐 等字眼跃然纸上。未来公务员摇篮的大学,则有幸深刻传达了《关于加强和改进高校青年教师思想政治工作的若干意见》……

    朱元璋恨不得在所有官员脑门上纹上“道德”二字,可帝国的吏治仍糟透了,它总共的十六任皇帝中,不乏勤勉之人,可直到末代的崇祯亡国,回天无力。

    帝国只相信两样东西:一是道德,二是酷刑。可从逻辑上,如果道德对约束官员有用,要那些下三滥刑罚干什么,如果酷刑是灵丹妙药,乡试、殿试何不考《论剁去贪官手脚、耳鼻制成人棍置于酱缸对未来吏治的可持续性发展》,至少字面看上去更有震撼力。

    它 从未想到过“法制”、“宪政”这些东西,也不知道差不多在朱重八站在应天城高台上决定玩死贪官的时候,一个叫英国的地方有下议院了。它不知道,锦衣卫、东 厂、检校这些并不是监督,而是监视,而监视只会让贪官更狡猾、更坚定地朋党结私,形成连皇帝也撼不动的利益集团。

    总之,这个精心 设计的帝国亡了。在它亡之前,有一个被人忽略的情节:1583年,万历皇帝在会试时出了一道匪夷所思的题目:朕越励精图治,官场却越腐败、法纪越松懈,到 底是朕缺乏仁爱,还是太优柔寡断呢?在神圣的全国统考时居然出这样的题目,前无古人后无来者。可见万历皇帝对吏治真是愁死了,憋得把本该给贵妃娘娘的私房 话都剧透出来。

    多年以后,红朝拥有了八十一万纪检干部,平均一个纪检干部监视八个官员。这比大明的锦衣卫和检校还要多。外加中央 巡视组和四十五个中央督导组,就是八府巡按的套路。这些日子,有些官员听说巡视组督导组驾临,前列腺都吓得掉裤裆了,每天上班前,说不定也深情回眸一眼平 日爱搭不理的黄脸婆……最近我们常欣闻某某贪官“闪电落马”。可是,现在闪电了,过去的二十多年里干吗去了?亲爱的闪电侠们。

    虽然不再四书五经六艺,但要坚持三个xx八x八x,世界上最神奇的道德就是,刘志军刚跟女孩做了“红楼梦”,就向领导感言“中国梦”。和明朝的贪官别无二致,他们被抓之后总爱玩这感恩呵梦想呵这些调调,仿佛只是误入红尘,内心还是小清新的。

    不 同的是,明朝宁错杀一千,也不放走一个。当今是:诰命夫人被判死缓,银行行长被判死缓,法院院长被判死缓,公安局长被判死缓,刘志军果然也死缓。在争论是 否“废死”时,我们只好调侃“不是取消死刑,而是取消死缓”。但认真你就输了。就算回到大明,抽肠、凌迟、、开水淋再铁刷子刷、钩起来风干……把惩治贪官 基本具体成一个制作腌卤食品的过程,也不会吓跑贪官。

    大明朝不也就出了一个海瑞吗。

    何况海瑞也弄得大家不开心。一个清官无钱葬母,这就不是大时代,也不是小时代,是宵小时代。

    算 了,不说影视,说明朝……《万历十五年》开头,黄仁宇先生专门写到1588年事情,英国大破西班牙无敌舰队。他没有提到的是,整整一百年后,也就是 1688年,英国的光荣革命诞生了,原本也贪腐、朋党、专制、国内形势乱七八糟的英国开始君主立宪,聪明地用分权、宪政、监督等手法治理国家,成为一时世 界霸主。而此时,中国的政权已移交到了“清”,清仍延续覆灭的“明”的道德+酷刑,甚至还采用了一个辅助战术,“文字狱”。帝国从此走下坡路。所谓康乾盛 世是教科书上涂抹的口红,潜伏的危机,均被四库全书那些才子们用修辞手法轻轻抹去。

    然后大家都去看热播电视连续剧《铁齿铜牙纪晓岚》去了。靠,傻逼和绅。哇,牛逼纪晓岚。生生把历史看成了言情。

    几千年来,中国的官场从不缺肃贪,妓院最爱假装打扫内部卫生了,中国官员也是最爱讲道德,婊子最爱述说自己清纯的爱情。很多时候,我们被迫在既有那么多肃贪、又有那么多道德的逻辑矛盾里,相信,丽春院发生过梁山伯与祝英台的爱情故事。

    就像相信人民日报的大标题,死缓彰显了法治精神。其实无论斩立决和还是死缓,都与彰显法治精神一根腿毛关系都没有,只与圣上一拍脑门的力气大还是利益集团拖后腿的力气大,有至深关系。

    公元1644年,崇祯自杀前写下遗诏:“朕凉德藐躬,上干天咎,然皆诸臣误朕。”就是说,你们这些负心人,平时白养你们了,关键时刻一个都不见,是你们搞死我的呀。

    276年前,帝国的第一任皇帝站在城头上发誓要搞死那些奸臣,经过276年卓越的努力,帝国最后一任皇帝终于死在歪脖子树下,哀怨认为自己是被奸臣搞死了。

    看着这枚循环播放键,到底是该不相信历史,还是不相信爱情。

Wednesday, July 10, 2013

Genome Modification—a Practical Approach

http://blog.allelebiotech.com/2013/07/talengenome_editingcrisprcas/

The ability to modify genomes has always been fervidly sought after by molecular, developmental biologists and geneticists as it would provide them with the means for finding out what a particular piece of the genome may do in the biological process they are studying. The discovery of naturally existing P-element helped a generation of Drosophila geneticists and made the fruit fly a prime model system for gene function studies in the 80’s and 90’s. But P-elements inserted at uncontrolled sites, making it essentially a gene transfer vehicle without much control. The introduction of prokaryotic recombination systems, e.g. LoxP and Cre, provided researchers with tools to obtain more control of the inserted genes in a host chromosome during a biological process such as development. Transposons like Sleeping Beauty, Piggybac, or Tol2 made similar experiments possible in mammalian cells.
Still, the randomness of transposon-type elements’ insertion, much like retrovirus or lentivirus, could cause trouble if they land in an undesirable spot. Methods of inserting transgenes only in well-known, harmless, and transcriptionally active regions, so called “safe harbors”, were subjects of interest of researchers and NIH grant topics in the past couple of years under “directed genome editing”. Gene knock-out or knock-in can be achieved through vector-mediated homologous recombination such as the rAAV genome engineering system and the “TARGATT” system, which are commercially available as kits or services.
However, instead of inserting an exogenous gene, it is often highly desirable to modify an endogenous genome sequence, which requires the modification apparatus to first recognize the target sequence. ZFN and TALEN both recognize DNA targets through specific nucleotide binding protein domains, with TALEN having more flexibility if assembled in a “Lego”-like format because each domain can specifically recognize a “C”, “G”, “T”, or “A” base. The description of using CRISPR/cas system in a recent burst of publications opened up new ways of binding to specific DNA sequences and nicking or severing the dsDNA. This system does not require engineeredDNA binding domain assembly; instead, it uses a guide RNA to find the target DNA sequence to direct endonuclease, in a sense quite like RNAi. However, the enthusiasm about CRISPR/cas was somewhat dampened by a report last month in Nature Biotechnology that reported off-target effects of CRISPR/cas was much higher than ZFN and TALEN. Particularly, if mismatches are located in the 5’ portion of the guide RNA targeting sequence, they can be well tolerated up to 3 or 4, even 5 mismatches. Unfortunately this is also similar to the tolerance of the RNAi matching region outside the core 12-base region. The difference is: for RNAi, the off-target damage is temporary and ignorable if the extent is insignificant compared to the effects on the intended target while for CRISPR/cas, an off-target cut on the chromosome is permanent.
On the positive side, in an even more recent publication in Nature Methods, mutant strains of C. elegans were obtained using the CRISPR/cas system and no evidence was found for off-target changes, at least not in an overwhelming fashion. Much value of the estimates of off-target effects relates to the methods used for analysis. Currently, most of the studies looked at potential off-target sides by searching for partial matches. In the future, whole genome sequencing will be increasingly required for submitting such publications.
On a practical note, if you intend to take a dive and try to use any one of these methods, your number one problem will be that none of the methods will result in 100% modification even if you can ignore the off-target problems for now. Therefore, many of our customers ask about a screening strategy. One could use traditional drug selection and fluorescent protein (FP)-based sorting, but these can only help you find cells that are successfully transfected with the ZFN, TALEN, or CRISPR/cas expressing DNA molecules, not necessarily having the genome modification result. We have formulated the idea of inserting the target site into an FP-bearing plasmid as a surrogate target cutting indicator, and use another FP to track transfection of the TALEN plasmid. Nonetheless, in the end, PCR-amplifying the target region of the chromosome and doing either an enzymatic mismatch detection assay (e.g. T7 endonuclease) or sequencing is the only way to know for sure whether genome editing has occurred.

Wednesday, June 19, 2013

李承鹏:投名状

北周的开国者、也是南北朝时期一大猛人,宇文泰,为一统天下曾遍访天下贤才。有天他遇到了号称有诸葛亮之才名的苏绰,向其讨教治国之道。两人一见如故,密谈三日三夜。

  宇文泰问:“国何以立?”
  苏绰答:“具官。”
  宇文泰问:“如何具官?”
  苏绰答:“用贪官,反贪官。”
  宇文泰有些纳闷:“为什么要用贪官?”苏绰答:“无论打江山还是坐江山,都需要手下人为你卖命,可让别人为你卖命就必须有好处,你并没有那么多钱,只好给权,让他用手中的权去搜刮民脂民膏,他不就得到好处了吗?”
  宇文泰问:“贪官得了好处,我有什么好处呢?”苏绰答:“他能得到好处是因为你给的权,为了保住自己的好处,他就拼命维护你的权,有贪官维护你的政权,江山不就巩固了吗。”
  宇文泰又问:“既然用了贪官,为何还要反?”
  苏绰答:“这就是权术的精髓所在,用贪官,就必须反贪官。你看,其一、天下哪有不贪的官?官不怕贪,怕的是不听你的话。以反贪为名,消除不听你话的贪 官,保留听话的贪官。这样可以消除异己、巩固你的权力。其二、官吏只要贪污,把柄就在你的手中。他哪敢背叛你,只会乖乖地听你的话。所以,‘反贪官’驾御 贪官的法宝。如果你所用皆是清官,深得人民拥戴,要是不听话,你哪儿有借口除掉他。假使硬行除掉,也会引来民情骚动。所以必须用贪官,才可以清理官僚队 伍,使其成为清一色的拥护你的人。”
  宇文泰大喜,苏绰忽反问:“如果你用太多贪官而招惹民怨怎么办?”宇文泰一惊,急急请教:“先生有何妙计?”苏绰答:“这就是奥妙所在,加大宣传力 度,祭起反贪大旗,让民众认为你是好的,不好的只是那些贪官,把责任都推到他们的身上,让民众知道社会出现这么多问题,并非你不想搞好,而是下面的官吏不 好好执行你的政策。对那些民怨太大的官吏,宰了他!为民伸冤的同时,再把他搜刮的民财放进你的腰包。这样,不负搜刮民财之名却得民财之实惠。总之,用贪官 来培植死党,除贪官来消除异己,杀贪官来收买人心,没贪财来实己腰包,这才是权谋的最高境界。”

  宇文泰如醍醐灌顶,十年用心用力,终成一时霸业。

  当然,史书上只记载两人有过一次长谈却并无对话内容,并不知上述对话真实作者是谁、是否假托之文。但,这真是一个好的故事。

  这是一个好故事,却不是最早的好故事。差不多在宇文泰与苏绰对话发生的七百多年前,有个叫萧何的人与他的门客也有一次对话。那时萧何已月下追过了韩 信,项羽亦在乌江抹了脖子,刘邦正与异姓王最后一搏。为支持刘邦在前线打仗,萧何在后方大力督办后勤、安抚体恤人心,老百姓很拥戴他。有段时间刘邦特别爱 打听萧何在干什么,使者如实回答“安抚、体恤之事而已”。刘邦听后,沉默不语。

  消息传到后方,门客大惊:“看来萧相国你不久便会满门抄斩了。”
  萧何不解:“我克己奉公,何来满门抄斩之灾?”
  门客:“自入关之后你便兴水利、办实事,深得百姓拥戴,身居相国之位竟从不贪污,还曾把家产拿出来以做军资,这就不合常规。老大屡次打听你在干什么,难道不是怕你借民心、民意图谋不轨吗。”
  萧何深知刘邦性格,黯然:“如何?”
  门客说:“你为什么不干点贱价强买强征农田、掠夺民财之事,以污自己名声,让老百姓都骂你,老大自然就放心了。”
  萧何想了想,依计而行,强买民田及掠人钱财,竟至闹到群众当街举报的地步。刘邦接到探报,不怒反喜,班师回朝时指着萧何取笑:“你这个人,身为相国,跟小老百姓争什么争,啊,哈哈。”

  萧何,遂得善终。

  差不多在宇文泰与苏绰的对话五百多年后,有个叫豹子头林冲的军事干部,因家庭冤屈,一把火烧了草料场跑到了梁山。那天白衣秀士王伦见他来投,说:“若 要入伙,需交投名状”。林冲本来以为就是填个应聘表格之类,却不料这投名状竟是让他下山杀人……纵为八十万禁军教头也知道,不杀不足以表明忠心,不交投名 状就是暗藏贰心。为示忠诚,于是下山与杨志杀得天昏地暗。幸好碰到了晁盖来到,才免得血溅五步。

  当然,老晁后来也被投名状害了命。梁山众兄弟在继任大哥宋江的带领下,为向朝廷表忠心就与方腊激战,整体地交了一个很大很大的投名状,几乎个个死得很难看。

  帝国几千年来的故事基本是这个样子:1、启用大量贪官,2、迫使少许清官变成贪官,3、如果你不想当官想落草,林冲会告诉你——官场是有编制的黑道, 黑道成为官场的预科班。总而言之是交出投名状。中国三千多年文明史,就是三千多年的投名状史。要烂大家一起烂,拆烂污成了入行的敲门砖。你混黑道不杀人, 怎么证明能跟兄弟们同生死;你混官场不贪钱,怎可能与同僚共患难。

  差不多距宇文泰与苏绰对话一千五百年后,这个帝国又破获了很多必将载入史册的案子,经查逾万官员拆了烂污,有枢密使坐拥很多存折、珠宝及很多女人,有 户部侍郎账目异常,有兵部尚书锒铛入狱,最著名的是有江宁织造25年来贪污了很多钱、很多房、以及长达17年卖官……如此波澜壮阔的贪,在检方提出轻刑的 建议后,可真是充分贯彻了当年宇文泰和苏绰的会议精神,他俩从未远去,一直在密室阐述:贪腐,是维护江山的必要手段,反贪,是贪腐一个必要的表演环节。很 多人义愤填膺追问这么多年为什么没发现这些贪官,早年又是谁提拔的贪官?可是读了上面之后你得细想,在一个人治而不是法治、人员是提拔而不是选举、上下级 其实是人身附属关系的国家,擦烂污指数,就是忠诚指数的生动体现。不贪,不能得以提拔。不贿,不仅不得重用。制度性腐败已耳熟能详,也许已是举国投名状。

  还是看故事好玩:明熹宗不杀魏宗贤,而留给崇祯来杀;乾隆不杀和绅,也留给嘉庆来杀。还有不少这样的例子。这说明,先帝并不是从国法和道德来看这些贪 官的,而把他们当工具来使用的。一代枭雄一茬狗,对于崇祯和嘉庆,新一轮的投名状,用不着魏阉和不再细皮嫩肉的和绅来写了。

  谁愿意带一条老狗去打猎呢。

  我的祖国,总这么有深意,我的祖国,从没有一纸官民合同,通篇尽是投名状的禅机。

来源: 作者博客2013-06-18

Allele Biotech Receives $200,000 Grant to Update Its mRNA Reprogramming Commercial Products and Services

On June 10, 2013 Allele received an SBIR award from the National Institute of Drug Abuse (NIDA/NIH) entitled “Revolutionary Technology for Efficient Derivation of Human iPSCs with Messenger RNA”. The goal of the proposed project is to provide to the biomedical research market an advanced reagent kit and services for highly efficient reprogramming of high quality human induced pluripotent stem cells (iPSCs). At the core of this kit is the Allele team’s recent development transcribed messenger RNA (mRNA). Compared to other reprogramming methods, such as lentivirus, Sendai virus, protein, small molecules or any combinations of these reagents, our new generation of the mRNA method often requires less than half the time while sometimes achieving “bulk conversion” efficiency.
While the Allele reprogramming technology was designed for clinical use as the process is feeder-free, xeno-free, chromosome integration-free, as well as without the need for cell splitting, PI, Dr. Jiwu Wang states, “Our purpose of executing the NIH-funded research it to make our method so easy that any researcher can integrate iPSC into his or her projects.” In addition to the extremely high efficiency, mRNA-generated iPSCs should also be more stable because there are no genetic alterations, more uniform among all clones as there is no clonal event, and ultimately suitable for future autologous cell therapy now that creating iPSCs from patient tissue cells should no longer be the rate-limiting steps.
Allele’s business model is to provide cGMP-grade iPSCs to pharmaceutical companies and perform large scale reprogramming by partnering first with university-affiliated hospitals. Great progress has been made in both directions, which has prompted the initiation of a cGMP unit within Allele’s newly acquired building in San Diego.

Saturday, May 4, 2013

齐奥塞斯库

如果极权主义的悲剧不会被忘却,极权主义的喜剧也同样不会被忘却——它们是无法分开的。——诺曼•马内阿

  1789年,法国民众走上街头,将他们的国王路易十六送上了断头台,专制皇权就这样结束了,《人权宣言》确立人权、法治、公民自由和私有财产权等现代政治原则。人类历史上的所谓“现代”就这样拉开序幕。

  在法国大革命200年之后,一场民主浪潮再次席卷世界……在罗马尼亚,民众将一个不戴皇冠的独裁者齐奥塞斯库送上断头台。这一切都发生在短短的7天之内。这7天,或许就是从古代到现代、从独裁到民主的距离。

  从查理一世到路易十六,从墨索里尼到齐奥塞斯库,从萨达姆到卡扎菲,一个又一个暴君走上断头台。每消灭一个独裁者,就有一个民族站立起来,走进现代,这就是人类文明史。中国亚圣孟子曰:“贼仁者谓之贼,贼义者谓之残,残贼之人谓之一夫。闻诛一夫纣矣,未闻弑君也。”

  作为一个反抗法西斯极权的革命者,齐奥塞斯库曾经说过:“任何专制的暴力一旦与人民的正义之师交战,他们必将粉身碎骨。”很多年后,他这句话果然一语成谶。
  一 动物庄园

  历史是吊诡的,人们反对权力,常常是因为自己没有权力。在现代历史中,许多反法西斯者是共产主义者;但在很多情况下,某个极权制度的反对者,有意无意地成为另一个极权制度的支持者,甚至会成为另一个极权制度的制造者。齐奥塞斯库就是这样的。

   1918年,齐奥塞斯库出生在罗马尼亚一个农民家庭,在10位兄弟姐妹中排行第三。因为家庭贫困,他上小学时连课本都买不起,10岁起就不得不进入工厂 做工。当时正值1930年席卷世界的经济大萧条,许多工厂倒闭,大批工人失业,15岁的齐奥塞斯库便加入当时非法的罗马尼亚共产党。因为组织罢工,他曾经 屡次被捕。对齐奥塞斯库来说,身陷囹圄不仅没有什么伤害,反而使他的影响日隆,成为一个知名的“危险共产主义的鼓动者”和“共产主义和反法西斯主义的宣传 活动传播者”。最后一次被捕后,他被判处两年有期徒刑,这场牢狱之灾使他得以结识罗马尼亚共产党领袖格奥尔基•乔治乌-德治,他们共同反抗罗马尼亚的安东 尼斯库法西斯主义政权。这次“铁窗友谊”成为他日后获得政治权力的一个重要转折。

  第二次世界大战时期间,罗马尼亚的安东尼斯库政权加 入德、意、日法西斯同盟。1944年,苏联一方面支持罗马尼亚共产党在首都布加勒斯特发动起义,另一方面苏联红军攻入罗马尼亚,内外夹击下,安东内斯库政 权被推翻,罗马尼亚转而加入了反法西斯的苏联阵营。1945年3月6日,共产党联合其他政党成立联合政府。

  第二次世界大战结束之后, 罗马尼亚被划入苏联势力范围,齐奥塞斯库的职务是罗马尼亚共青团书记。1947年,格奥尔基•乔治乌-德治领导的罗马尼亚工人党夺取政权,宣告正式成立罗 马尼亚人民共和国。齐奥塞斯库成为重要的武装部队副部长。数年之后,齐奥塞斯库已经上升为党内第二把手,仅次于奥尔基•乔治乌-德治。

   1965年,“老大哥”乔治乌-德治去世。齐奥塞斯库顺理成章成为罗马尼亚最高领导人。在当选为罗马尼亚工人党第一书记之后,齐奥塞斯库将工人党改名为 共产党,将国名改为罗马尼亚社会主义共和国,他继续担任罗共中央总书记。1974年,在齐奥塞斯库的策划下,罗马尼亚实行总统制,齐奥塞斯库成为总统,且 拥有了直接颁布法律、任免政府成员的大权。此后齐奥塞斯库一人兼任了罗马尼亚共产党中央总书记、共和国总统、国防委员会主席、武装部队最高统帅、爱国卫队 总司令、经济和社会发展最高委员会主席等党政军最高职务,成为罗马尼亚一切的主宰,甚至连农民在一平方米土地上种几棵玉米也必须要由他来决定。

   齐奥塞斯库一方面批判个人崇拜,另一方面却在罗马尼亚大搞对自己的个人崇拜。党在国之上,而他则在党之上。各种官媒经常说“齐奥塞斯库身上集中了共产党 人最高尚的品质”。上有所好,下必甚焉,这种个人迷信对中国人来说并不新鲜。20多年来,齐奥塞斯库走到哪里,都要组织一场人数众多的盛大欢迎仪式,欢迎 的人群必须提前到场,哪怕风吹雨淋烈日暴晒。齐奥塞斯库一来,一起高呼“万岁”。不仅如此,每逢重要的大会,为了保证理想的效果,“群众演员”之外,官方 还必须组织一批“专业演员”,由思想过硬的警卫部队组成“政治啦啦队”,整齐的坐在前几排。齐奥塞斯库讲话开始,每隔三分钟,这些专业演员就会站起来鼓掌 叫好。后面的群众演员也跟着这些专业演员一起“热烈鼓掌”。齐奥塞斯库每次讲话,都会获得提前设定好次数和时间的鼓掌和站立,非常精确。就这样,一场激动 人心万众拥护紧密团结的共产党大会就顺利制造出来了。

  齐奥塞斯库居住在巴洛克式的豪华宫殿中,过着豪奢的生活。权力在手,乾坤独断, 任人唯亲。齐奥塞斯库自己是党的总书记,夫人是政治局常委和二把手,所有重要部门都被他的子女亲友把持,齐奥塞斯库将国家变成了党天下,最后完全变为家天 下和夫妻政治。他的妻子埃列娜于1973年被选为中央执委会委员(即政治局委员),掌管全党的干部大权;

1980年起,又兼任全国科学和 教育委员会主席。她还俨然以“国母”自居,生日也要全国庆祝;人们在各种大会上的发言,在呼完“敬爱的总书记齐奥塞斯库同志”之后,必呼“尊敬的埃列娜同 志”。在政府里,名义上她是副总理,但实际上她是“超级总理”。罗马尼亚各级官员都习惯地称齐奥塞斯库为“一号”,其办公室为“一号办”,埃列娜为“二 号”,“二号办”。

  齐奥塞斯库有5个兄弟,3个姐妹,全部跟着“老三”齐奥塞斯库鸡犬升天。老大尼库利娜是家庭妇女,老二马林常年担 任罗驻奥地利商务参赞,老四玛丽亚(女)曾在布加勒斯特电磁厂当车间主任,老五弗洛雷亚为前《火花报》记者,老六安德鲁察中将曾任内务部警官学校校长,老 七伊利耶中将曾是罗马尼亚军事科学院历史系教授、国防部副部长兼罗军最高政治委员会书记,老八埃列娜(女)长期在家乡当教师、学监,老九扬曾任农业部长。 齐奥塞斯库的小儿子尼库是罗共中央执行委员会候补委员(即中央政治局候补委员)。据英国《经济学家》杂志统计,齐奥塞斯库家族成员在党政军界担任要职的不 下30人。罗马尼亚完全沦为齐奥塞斯库的家族产业。所谓罗共中央大会,其实就是齐奥塞斯库的家族会议。罗马尼亚人将其嘲讽为“社会主义大家庭”。

   齐奥塞斯库有一爱犬名曰“考布”,罗马尼亚众高官皆尊称其为“考布同志”。齐奥塞斯库认为这种称呼过于平淡,便正式给爱犬授予军衔,“考布同志”摇身一 变成了“考布上校”,由此开创了“狗官”的历史。与很多罗共高干一样,“考布上校”的一切生活用度都享受特供,不仅配有豪华别墅和专车,还有秘密警察充当 保镖,更少不了保健医生日夜照料。据“考布上校”的女助手回忆:“秘密警察告诉我们永远不要喂狗。有专门医生检查狗食(一种从英国进口的肉)。只有医生尝 过后,肉才能喂狗。”在相当一段时期,罗马尼亚驻英国大使最重要的工作就是每星期去一趟圣伯利公司,为考布采购精美的“上校食品”,然后空运回国。而与此 同时,无数罗马尼亚民众正在寒风中排着长队购买可怜的那么一点限供面包。这些垃圾食品对伟大的“考布上校”来说,根本都不屑一顾。“考布上校”陪同齐奥塞 斯库总统视察布兰科温斯克医院时,遭到几只野猫袭击,后果是这家医院马上被夷为平地。

  “考布上校”不仅有助手保镖医生,它还有自己的宠物——一只会说话的鹦鹉。有一段时间,这种鹦鹉每天都说“愚蠢的尼库”。齐奥塞斯库的小儿子尼库已经被指定为未来的接班人,因此这只鹦鹉很快便被秘密警察抓去。结果也审问不出是谁教的这句话,只好把这只鹦鹉杀死了事。

  二 一九八四
  齐奥塞斯库时代的罗马尼亚从不同国家、不同历史阶段和不同意识形态的暴政统治中汲取经验,建立了一个拜占庭式的民族主义—社会主义国家。齐奥塞斯库操纵着公众舆论,完全是纳粹主义与斯大林主义的综合体。1980年发表在《星期》上的社论《理想》中写道:
  我们不需要向任何人解释我们做的事,我们是自由的,我们是大多数,我们是我们自己国家的主人,这个国家数百万忠实的儿子已经做出了历史性的选择——罗马尼亚特色的共产主义。

   齐奥塞斯库以铁腕治国,对内独裁高压,对外独立自主。外交政策中,齐氏奉行的实用主义模糊策略,为自己的独裁统治在包括西方的世界范围内获得了令人吃惊 的友好与支持。从戴高乐毛泽东到福特尼克松,从英国和瑞典的王室到美国和联邦德国的议会,齐奥塞斯库都成为广受欢迎、炙手可热的座上宾。英国女王伊丽莎白 二世授予其荣誉爵士勋位;美国总统尼克松甚至称颂其是“世界上最伟大的领袖之一”。与东欧那些社会主义国家不同,齐奥塞斯库与勃列日涅夫时代的苏联始终敬 而远之,保持着警惕的距离,甚至在“布拉格之春”事件中,他对苏联出兵镇压捷克民众表示强烈谴责。

  1971年,齐奥塞斯库出访了中国 和朝鲜。这次“取经”让这个独裁者被东方式权力美学深深折服,一片红色海洋的文革狂热与同样疯狂的金氏帝国成为齐氏心目中的理想国。回国之后,齐奥塞斯库 就开始了大刀阔斧的极权化运动。所有反对派和异见分子都被关进精神病院,接受“政治偏执狂”或者“老年痴呆症”的治疗,直到最后成为一个模范精神病人,一 个个专心致志地为自己制作棺材……

  在齐奥塞斯库时代,罗马尼亚出台了许多匪夷所思的暴政,最典型的莫如禁止堕胎制度和打字机登记制 度。可以说,没有任何一个欧洲社会主义国家像罗马尼亚这样,受到如此彻底的监管和残酷的压迫。在一个被独裁者和警察奴役的罗马尼亚,所有民众其实都不过是 闷头苦干、愚蠢顺从的奴隶。

  或许是因为出生在孩子众多的大家庭,齐奥塞斯库对罗马尼亚作为小国颇为不满。为了提高人口数量,增强国 力,他废除了关于个人可以自由流产的法律,禁止一切堕胎和节育。这与中国的计划生育正常恰恰相反。后者鼓励人们避孕和流产,前者则将一切避孕和流产都视为 犯罪,这无疑是国家权力对公民身体的占有。堕胎不仅意味着某种错误的、罪恶的道德行为,也是对国家制度的背叛和对抗。齐奥塞斯库认为,胎儿是国家财富,拒 绝生育孩子就是背叛国家。因此,不仅禁止离婚,而且还规定每对罗马尼亚夫妻至少要生4个孩子。同时还规定,不能受孕的女性要交纳惩罚性税金,而违法堕胎者 将受到刑罚和囚禁。为了保证妇女的及时妊娠,罗马尼亚妇女的月经周期都属于国家机密,每个人要随时接受官方的严格检查与盘问。

  斯大林 曾说,权力是没有灵魂的,没有情感的,它只会执行命令,国家机器上的齿轮只会根据指令转动,国家最需要的是没有自己头脑、只会执行命令的工具。在一个机械 化的官僚体制下,所有的机关、工厂、农村和学校等单位,都密布各种严厉的执法者,作为“国家财富”制造者,所有妇女都遭到严密监控和严格检查,避孕工具就 是最可怕的反叛国家的罪证。那些避孕的妇女和默许堕胎的医生一旦败露,等待他们的就是一场牢狱之灾。这些闯入民众卧室的执法者被人们鄙夷地称作“月经警 察”。因为传统的宗教习俗禁忌,很多意外怀孕的少女害怕成为“未婚妈妈”,不是选择不安全的堕胎方式,就是选择自杀。在这种恐怖变态的政治高压下,许多绝 望的妇女铤而走险,试图偷渡多瑙河,到邻国匈牙利寻求庇护。在边境线上被视为叛国者而遭到罗马尼亚军人枪杀的孕妇不在少数。

  在齐奥塞 斯库和他的“月经警察”的共同努力下,罗马尼亚女人果然生出了超过一倍的新生儿。与此同时,非法的地下流产与堕胎屡禁不止,孕妇死亡率也达到一个很高的数 字。正如中国的毛担心大跃进丰收的粮食无处存放,齐奥塞斯库面对迅猛的婴儿潮也措手不及,因为妇产医院的设备、妇产专家、产科医师、儿科医师以及妇幼保健 工作者严重缺乏。仅仅一年中,罗马尼亚的婴儿死亡率就增长了145.6%。这场悲剧令全世界为之哗然,罗马尼亚被人们谴责为“现代社会的滥杀无辜”。齐奥 塞斯库随即命令,只有满月的婴儿才可计入统计数字,然后再核发出生证。如此一来,大量初生即夭折或残疾的婴儿就“不存在”了,婴儿死亡率迅速“下降”,逐 渐恢复正常。

  这项反人性的繁殖国策制造了一场旷日持久的人道灾难,罗马尼亚妇女不仅承受着奇耻大辱,而且被迫沦为国家的生殖机器。另 一方面,孤儿院和收容所里大量被遗弃的孩子“从来没有合法存在过”,这些身体和精神上承受双重伤害的孤儿成为罗马尼亚社会一个永远的伤痛。更为匪夷所思的 是,齐奥塞斯库为了增强新生儿的体质而进行输血,由此导致艾滋病通过血液大量蔓延,这些“艾滋孤儿”成为齐奥塞斯库时代最黑暗的记忆。

   直到1989年,这项罪恶的人口政策才伴随着社会主义政体,和齐奥塞斯库一起在罗马尼亚结束。在此期间,大约有50万的罗马尼亚女性因为秘密堕胎的安全 性而屈辱地死去。很多年后,罗马尼亚导演克里斯蒂安•蒙久用这个题材拍成一部极其压抑的影片《四月三周两天》,在2007年嘎纳电影节上一举获得金棕榈 奖。

  与人口倍增计划类似,齐奥塞斯库对打字机的控制同样极度变态。在极权体制下,文字也是一种对抗权力和保护人性的武器。任何独裁者 都不会轻视文字可怕的力量。如果他掌控文字,他就可以用谎言征服人民;如果民众掌握文字,不仅会危及谎言的存在,甚至会消解权力和暴力的作用。

   在上世纪七八十年代,电脑尚未普及,互联网也还没有诞生。当时,罗马尼亚所有的报纸、杂志、电台、电视台、出版社、印刷厂,统统都是由政府垄断和控制 的;作家也都被圈养在政府管理的“作协”,书籍出版需要经过层层把关和反复审查。在这种密不透风的思想禁锢下,只有个人写作还没有被完全管制。1874 年,美国雷明顿公司制造了世界上第一台打字机,打字机和电话将人类社会的沟通带入机器时代;以至于离开打字机,人们已经无法写字。一个世纪之后的欧美国家 中,打字机已经完全代替笔,成为使用拼音文字的欧洲人的主要写作方式。因此,齐氏政府认为只要控制了打字机,也就等于控制了所有的文字和写作,也就禁止了 言论自由。

  罗马尼亚女作家赫塔米勒说:“我总是警告自己不要接受政府供给人民以‘词’的意义,我也意识到语言本身不能作为抵抗的工 具。语言唯一能做的就是保持自身的纯洁。”作为那个极端社会里的极端因子,被体制编了号的罗马尼亚作家群体彻底成为整个社会所面临绝境的一种象征。齐奥塞 斯库制定了极其严厉的书报审查制度,并设立了一个中央审查机构,罗马尼亚作家不得不“在作品中使用诈术、典故、暗码或粗糙的艺术形象,痛苦而隐晦地和读者 进行沟通,同时又希望能躲开审查者”。赫塔米勒被称为“独裁统治日常生活的女编年史作者”,她长期受到安全部门的监控。其处女作《低地》在出版审查时遭到 严重删改,甚至连“箱子”一词也要删去,免使读者联想到“流亡”。残酷的现实逼迫很多作家不得不走上流亡道路。赫塔米勒后来流亡西德,在《我怕故我写》 中,她这样写道:
  这里不是我的家
  哪里有齐奥塞斯库
  哪里就是异乡

  1980年,齐奥塞斯库正式颁布 了《大罗马尼亚打字机法》。根据该法,每一个罗马尼亚的公民、企业、机关、学校等单位,凡拥有打字机必须要得到警方的许可,领取使用执照;要成为打字员也 必须照此办理,并且要将所打字的样品同时上报。如果打字机需要修理,其使用者及其打字机都需要更新执照。任何继承打字机的罗马尼亚人,都必须将相关证照上 交政府当局,然后再申请取得使用它的资格;如果不把打字机的键盘上交警方,或者私自处理哪怕已经损坏的打字机,都将遭受严厉处罚。

  文字作为思想的载体,控制了文字也就等于控制了思想。对任何统治者来说,一个思想被控制的人才是一个完美的奴隶。在一个彻底消灭自由的国家,权力就获得最大的自由。

   有人说,生活的道路就像一把锋利的刀:一边是地狱,另一边也是地狱,生活的道路从它们中间穿过。罗马尼亚人对这句话应当是深有体会。思想和身体的双重禁 锢使每一个罗马尼亚人都是失去了“自我”;没有“我”,也就没有什么尊严和良心可言。“艺术家用谎言道出真相,政客用谎言掩盖真相。”极权主义的本质是一 个暴力与谎言的游戏,所有人都被卷入共同的阴谋中,每个人都在不同程度上成为罪恶的同谋。不论是官方还是民间,欺骗已经成为蔓延整个罗马尼亚的瘟疫。人们 虽然通过它得以生存,但罪恶感无处不在。对很多人来说,被奴役的生活意味着抵抗、团结及由此而来的折磨和希望。

  三 美丽新世界
   在人类历史上,曾有无数暴君把落后愚昧的国家变得非常强大,如秦始皇、恺撒大帝、奥古斯都大帝、希特勒、斯大林等。事实上,齐奥塞斯库同样做得不错。在 他统治的25年当中,依靠左右逢源的国际关系,全球化经济圈给罗马尼亚带来前所未有的发展机遇,这使罗马尼亚迅速成为欧洲的后起之秀。1965年至 1980年期间,罗马尼亚工业产值持续高速发展,平均每年增长达到11.5%。在高增长高积累的计划经济体制下,罗马尼亚外贸的年平均增长率更是达到 16.5%。这种持续的高速增长使70年代被誉为“齐奥塞斯库时代”。

  中国古语云:“其兴也勃焉,其亡也忽焉。”所有建立在极权之上 的帝国即使再强大,最终也会在倏忽间走向崩溃,这是它的政治结构决定的。一座建立在沙滩上的大厦最怕的就是时间的摧残,一场小小的风暴就可能使其片刻倾 覆。进入80年代后,齐奥塞斯库的“黄金时代”很快就走向终结。受世界经济影响,罗马尼亚经济增速从持续10多年的两位数,急剧下降到2.5%。一个漫长 的下坡路开始了,而路的尽头是一个深渊。

  从1980年开始,罗马尼亚农业连年歉收,粮食短缺使人们连吃面包都成问题。不仅面包店每天 都排着长队,其实买什么都要排队。当时流传着这样的笑话:许多人排长队等候买面包,但能否买到还是未知数。有人骂道:“连面包都吃不上,都是齐奥塞斯库害 的,我去把他干掉!”说完便走了。过了一会儿,这人又回来了。众人问他是否把齐奥塞斯库干掉了。他气呼呼地说:“那里的队排得比这儿还长!”

   进入80年代后,“齐奥塞斯库”在罗马尼亚几乎成为物资匮乏和短缺的代名词。生活必需的农副食品供应极其紧张,几乎快赶上艰苦的二战时期。其它如药品日 用品供应也同样紧缺,煤气、暖气和电力供应严重不足,陷入生活困境的民众怨声载道。1989年10月,在未提前通知的情况下,齐奥塞斯库突然到首都布加勒 斯特的几家大型副食商店视察。看到货架上空空如也,他严厉批评了负责官员。两天后,他再到这几家商店视察,果然看到那里的商品琳琅满目。齐奥塞斯库很满 意,但他走后,这些样品就被从货架全部撤掉了。这就是所谓权力的道场。

  为了解决供应问题,齐奥塞斯库甚至派专家组到中国取经,最后学 到了限量凭证供应的办法。在中国正在退出历史舞台的“粮票”、“布票”和“肉票”等供应票证,在遥远的罗马尼亚竟然咸鱼翻身,再次大行其道。1988年, 齐奥塞斯库访问北京,特意授予邓小平一枚罗马尼亚勋章。然而仅仅一年后,同样的故事却演出不一样的结局。

  从某种程度上,罗马尼亚这种过度“短缺”完全是政治的产物,而不是经济的结果。
   罗马尼亚在1981年的外债为110亿美元,齐奥塞斯库执意要在1990年之前还清全部外债。为了这个目标,当局不得不最大限度地限制进口;与此同时, 又最大限度地增加出口。这种勒紧裤腰带的措施严重影响到民众最基本的生活需求,甚至连食品、煤气、供暖、供电以及日用消费品等都无法得到满足。更加疯狂的 是,本来就民生艰难、经济凋敝,好大喜功的齐奥塞斯库还大兴土木,搞起“形象工程”,这无疑使罗马尼亚人的生活雪上加霜。

  许多城市居 民连日常饮水都发生困难,一个大而无当的多瑙河—黑海运河工程却耗资达数十亿美元。1984年动工的“人民宫”占地面积达33万平方米,建筑空间220万 平方米,是仅次于美国五角大楼的世界第二大宏伟建筑。“人民宫”及其配套的“社会主义广场”和“社会主义大道”等庞大建筑群共花费20多亿美元。颇为讽刺 的是,这项宏伟建筑到齐奥塞斯库死后都还没有完工。

  从某种意义上,经过长期的高速发展之后,国民经济的穷途末路成为对齐奥塞斯库政权的致命打击。涸泽而渔寅吃卯粮的齐奥塞斯库沉迷在权力快感中无法自拔,他完全是在为自己修建一个巨大的坟墓,而且他绝没有想到死神会来得这么早。

  四 沉默的大多数
   历史有一个不变的规律,这就是“变”。长达四分之一世纪的齐奥塞斯库时代在经过一段时间的快速发展后,无论经济还是政治都不可避免地走入了死胡同。这种 弥漫在全国上下的死气沉沉,令许多人感到绝望,家天下的独裁者齐奥塞斯库自然成为众矢之的。罗马尼亚作家诺曼?马内阿在《论小丑:独裁者与艺术家》中写 道:“在1985年到1986年这个期间,政界改变的惟一希望就是一个‘生物方法’,即等待得太久、拖延得太久的领袖之死。”

  当时在布加勒斯特流传着这样一个笑话:许多申请出国的人正在排队领取护照。其中一人回头看到他身后的人不是别人,而是齐奥塞斯库。齐奥塞斯库对他说:“既然大家都要出国,那么我也走。”他无限感激地说:“如果您走的话,我们就不必出国了。”

   马内阿记录了当时罗马尼亚人焦虑的心态,“怀疑主义一直是罗马尼亚人的特征,尤其表现在人们对政治和政治家的态度上,特别是听到政治斗争这种字眼时更是 表示怀疑。当代大小国家许多政治领导人的平庸,以及他们言语中表现出来的愚蠢,只能增加人们的怀疑。最终,这种怀疑就会变成默然和鄙视。”

   “我们都很容易愤怒,但只有很少的人能够表达出来。”这是另一位罗马尼亚作家的话。在极权体制下,大多数民众被排除在政治之外,他们屈辱地忍受着政客们 对他们的利用。很多年以来,整个罗马尼亚社会都缺少公开的讨论,整个国家被简单的煽动性标语操纵着,被安全局这样的特务部门所控制。这种屈辱常常会转化为 对政治冷漠的犬儒状态,但有时候,当这种焦虑积累到一定程度时,怯懦的大众会在突然之间愤怒地爆发。正如马内阿所说:“当一个人的绝望、痛苦和愤怒到了难 以忍受的地步时,他的勇气和尊严就会重新表现出来。”

  人类社会如同一只高压锅,当所有的排气口都被彻底封死后,这只高压锅就变成一枚 危险的炸弹。人作为一种言说动物,一旦失去了言论自由,就会陷入一种沉默的深渊中。在这种沉默中,恐惧逐渐转化为愤怒。各种微不足道的力量慢慢积累,最后 汇集到一起,形成一场可怕的风暴。正如鲁迅先生所说:“不在沉默中爆发,就在沉默中灭亡。”

  1987年,罗马尼亚官方在布拉索夫组织了一场大规模的群众游行。突然之间,这场游行失去了控制,群情激奋的游行者打出了反政府的口号,民众的领袖齐奥塞斯库瞬间就变成了罗马尼亚的国家公敌。
  
反乌托邦电影《V字仇杀队》中有一句经典台词:“人民不应该惧怕政府,政府应该惧怕人民。”电影中有这样一个桥段,总统发表电视讲话,要民众不要相信谣 言,要团结一心维护国家稳定,但电视机前已经无人再听他讲话,人们都已走上街头。当人民不再相信政府和领袖时,接下来会发生什么?很多年后再看,这场失控 的游行其实根本不是一场偶然或意外,子弹挡不住思想,历史就这样已经悄然开始转弯。

  1921年,罗马尼亚共产党诞生;1945年,罗 马尼亚共产党执掌政权,其党员不足1000人。但到了80年代,在这个假面舞会的最后阶段,也就是在共产党当权的40多年后,已经很难在罗马尼亚找到 1000个真正有理想的共产党员了。不幸的是,在这种情况下,齐奥塞斯库领导下的罗马尼亚共产党员人数多达400万。按人口比例算来,这恐怕是世界上最大 的共产党政权了。但没有人否认,党员证只代表一种实现人格依附和财富分赃的证书,人们只想得到一种合法作恶的权力,然后来谋取自己的私利。

   随着戈尔巴乔夫对苏联实行的一系列改革,整个苏联和东欧社会主义阵营发生了多米诺骨牌式的崩溃。1989年10月,匈牙利政局发生剧变,执政的社会主义 工人党改建为社会党,匈开始实行多党制。1989年11月9日,数以万计的民主德国民众走上街头,将这个象征着共产主义极权专制的柏林墙推倒。柏林墙的余 震很快就波及到位于中欧的罗马尼亚。东欧共产党集团的纷纷倒台,使长期以来压抑的罗马尼亚人突然之间看到了远处的光亮。在四处积薪的罗马尼亚,一个小小的 火花都可能引发一场失控的烈火,最后将这座貌似高大的权力大厦烧成灰烬。

  1989年11月29日,著名的罗马尼亚体操明星科马内奇出 逃美国,然后以政治难民的身份寻求政治保护。在1976年蒙特利尔奥运会上,14岁的科马内奇以完美无缺的动作,征服了在场的观众和评委,奇迹般地获得了 世界体操史上第一个满分—— 10分,而且,这样的满分她一下子得了7个,获个人全能、高低杠和平衡木等3项冠军。一时之间轰动了世界体育界,科马内奇被称为“蒙特利尔仙女”、“奥林 匹克公主”。当时齐奥塞斯库的小儿子尼库担任罗马尼亚体育部长和奥委会主席,掌握着科马内奇的生杀大权。有一种广为流传的说法:科马内奇一回国,就被库尼 强奸,之后沦为库尼的性奴。忍无可忍的科马内奇徒步穿越落匈边界,成功逃出罗马尼亚。西方新闻媒体将科马内奇的“恐怖经历”公之于世后,世界大哗,人们无 不为齐奥塞斯库的暴政感到震惊。虽然罗马尼亚当局严密封锁了消息,但无疑构成一场风暴的不祥前兆。

  进入12月,罗马尼亚西部城市蒂米 什瓦拉发生了骚乱。这里距离罗匈边境只有40公里,有很多匈牙利族人居住。刚刚发生的匈牙利政局变革在这里激起强烈的反应。匈牙利电视台多次播放罗马尼亚 匈牙利族牧师特凯什•拉斯洛批评齐奥塞斯库的言论。齐奥塞斯库非常生气,便下令强迫拉斯特迁居。从15日开始,200名保护拉斯特的民众聚集在教堂附近, 阻止政府的强制搬迁。人群中有人喊出“打倒齐奥塞斯库”的口号,抗议活动迅速演变为反政府大游行。人们纷纷加入到游行队伍之中,砸毁齐奥塞斯库画像和雕 像。当军警以暴力阻止时,冲突就在所难免。警方使用了高压消防水龙,很多游行者受伤或者被捕。

  齐奥塞斯库对发生在蒂米什瓦拉的“群体 性事件”非常愤怒,命令国防部长瓦西里•米利亚要毫不留情地使用坦克和装甲车,对示威者进行强力镇压。当时蒂米什瓦拉的游行已经成为全体市民的共同行动, 上万人涌上街头,高喊“要自由”、“要面包”、“要热水”、“要暖气”、“打倒齐奥塞斯库”等口号;部分示威者冲入了市政府,打碎门窗,一些办公设施和汽 车被损毁,整个蒂米什瓦拉如同一座愤怒的火山。

  在掌握罗马尼亚命运的罗共中央执委会,元首齐奥塞斯库再次强烈下达命令,责令国防部使 用坦克部队和摩托化部队,对抗议者采取最严厉的暴力镇压。在巨大的压力之下,内务部部队切断了蒂米什瓦拉与外界的联系,断电、断交通、关闭边境,全副武装 的军队开始对手无寸铁的示威民众实行武力镇压……这场镇压其实就是不折不扣的大屠杀。世界各国媒体纷纷报道了这起由罗马尼亚政府制造的骇人听闻的流血惨 案。有报道称,惨案中有4000多人被杀。后经调查核实,实际死亡人数为147人,受伤335人,失踪25人。

  一场汹涌的社会浪潮化 作鲜血流走了,齐奥塞斯库再一次看到暴力的重要作用。极权只有通过暴力才可以维持稳定,因为权力与民众之间没有任何共识。专制没有妇人之仁,任何统治者都 只有通过暴力镇压才能证明自己的权力,齐奥塞斯库做得极其完美,果断、坚定、冷酷、无情。擦去手上的鲜血,日理万机的元首踏上专机,对伊朗进行为期3天的 国事访问,并与另一个独裁者拉夫桑贾尼谈笑风生。唯一的不同是,他没有像往常一样带上夫人。有作为罗马尼亚政府第一副总理的埃列娜-齐奥塞斯库在家中坐 镇,齐奥塞斯库信心满满地宣称:“我们的形势是稳定的。”

  五 历史的终结
  从伊朗访问归来,齐奥塞斯库发现蒂米索拉的灾难并没有结束,甚至正在向全国蔓延。
   作为一个掌握罗马尼亚达25年的独裁者,齐奥塞斯库身边围满了一层又一层的奴才和献媚者。依靠无法无天无所不为的权力,他不仅仅是一个总统,他其实已经 成为一个呼风唤雨的神。他的妻子埃列娜就称颂道:“罗马尼亚人们不配接受您的统治,因为您太伟大了!”就连齐奥塞斯库自己都相信,他具有神一般的感召力。 “国王不仅要坐在国王的位置上,而且还要坐在上帝的位置上”。文武之道,一张一弛。暴力镇压之后,或许有必要耍一些收买人心的手腕。极其自信的齐奥塞斯库 认为,仅靠自己领袖的魅力和雄辩的口才,可以继续像以往一样,在一场规模宏大的群众大会上一呼百应,让人们相信领袖永远是英明的。如此一来,所有的危机都 将烟消云散……

  12月21日,在布加勒斯特的党中央广场,齐奥塞斯库召开了一场10万人的群众大会。中午12点,齐奥塞斯库和夫人埃 列娜准时出现在党中央大厦的阳台上,他情绪激动地说:蒂米什瓦拉发生的骚乱是流氓、暴徒煽动的,是以破坏国家机关和公共财产为目的的,这是恐怖行动,与反 动势力、帝国主义、沙文主义势力相勾结,试图搞乱罗马尼亚的秩序与稳定……

  “要坚决打退外国的干涉和蒂米索拉流氓集团的动乱!”渐入佳境的齐奥塞斯库提高了嗓音,挥舞着手臂,这预示着马上就要迎来一片群众的欢呼和掌声。权力最幸福最高潮的时刻马上就要来临了。

   突然,从广场的某个角落有人大喊了一声:“打倒齐奥塞斯库!”这孤零零的一声断喝,如同一道闪电划过寂静的夜空。广场上的人们惊呆了,就连齐奥塞斯库举 在半空的右手也僵硬得如同一尊塑像。正在进行现场直播的国家电视台在这一瞬间中断了转播,呆若木鸡的齐奥塞斯库半举起右手的画面,被定格在罗马尼亚每一户 家庭的电视机上。

  等到齐奥塞斯库反应过来,再以提高工资福利进行许愿拉拢时,广场已经被愤怒点燃了,人们高喊着:“蒂米索拉!蒂米索拉!”“打倒杀人犯!”排山倒海的口号声响彻云霄,偌大的广场如同正在喷发的火山口。

  被紧急调来的武装警察头戴钢盔,从四周包围了广场,试图驱散愤怒的人群。
  这种时刻往往也是最能看出人类良心的时候,有的人投靠魔鬼,有的人保卫良心。国防部长米列亚亲临现场,他向警察和军人下令:“不准向人群开枪!”与此同时,有人送来“最高统帅”齐奥塞斯库的命令:“可以开枪,朝天开枪,先警告,如果不成,向腿部开枪!”

  在道义良心的纠结中,米列亚开枪了,但枪口对准的是自己。他无力对抗权力,但在他内心,良心的底线却没有失守,最后选择了自杀。这是改变历史的一粒子弹,它拯救了良心,也因此拯救了罗马尼亚。良心战胜权力,文明战胜暴力,正义在罗马尼亚就这样落地了。

  当齐奥塞斯库愤怒地谴责米列亚是“叛徒”时,他就已经成为良心与正义的敌人,他的所有权力在顷刻之间就丧失了道义上的合法性。在无数正义的人眼中,齐奥塞斯库完全就是一个独夫民贼刽子手。将军的悲愤壮烈与元首的暴虐无情使愤怒之火开始烧向罗马尼亚军人。

   国防部长自杀后,国防部第一副部长斯登古雷斯库自动接任,他拒绝了齐奥塞斯库的命令,下令军队撤回军营。这是对齐奥塞斯库最为致命的一击。在所有的极权 国家,军队都是专制体制最后的堡垒;权力之所以肆无忌惮,就是依靠暴力的强大支撑。一旦暴力抽离,不正义不道德的权力就马上丧失合法性,片刻也无法生存。

   军队宣布中立,大大鼓舞了正义的力量。示威的民众情绪激昂,一起冲向党中央大厦。看到局面彻底失控,齐奥塞斯库夫妇心慌意乱。22日中午,齐奥塞斯库夫 妇乘坐直升飞机逃离党中央大厦。到达博特尼后,他们立即转乘汽车逃跑。这时,他们手下的奴才们都已经树倒猢狲散,四散奔逃。可怜齐氏夫妇惶惶如丧家犬,他 们面前只有一条四面楚歌的逃亡之路。
  
再说布加勒斯特,齐奥塞斯库和他的“贤内助”离开党中央大厦后不久,罗马尼亚民众就在刚成立的政权机构救国阵线的指挥下,形成了一个抓捕齐奥塞斯库的天罗 地网。几乎所有罗马尼亚的广播里都响起了这样的声音:“各位市民请注意,人民公敌齐奥塞斯库和埃列娜正劫持一辆紫色达契亚轿车逃跑,请予以缉拿……”

   可怜的齐奥塞斯库好不容易逃到一个乡下的植物保护局,这时连他的司机都逃跑了。在植物保护局里,人们都围坐在一台电视机前,电视上正在播放关于齐氏夫妇 的通缉令。就在这时,齐奥塞斯库和他的总理妻子埃列娜如同天使下凡一般,突然出现在人们面前,所有在场的人都惊得目瞪口呆……这种尴尬的场面最后被齐奥塞 斯库打破了,他用每个人都很熟悉的腔调问道:“你们局长在哪里……”

  如果极权主义的悲剧不会被忘却,极权主义的喜剧也同样不会被忘却——它们是无法分开的。就这样,一个不可一世的独裁者从高高在上的神,变成了走投无路的阶下囚。

  六 末日的审判
  泰坦尼克没有沉没之前,没有人相信它将会沉没。事实上,泰坦尼克从启航到沉没只用了短短的6天。据说,上帝造人用了7天,而罗马尼亚人消灭他们的“上帝”也用了7天。7天,上帝创造了人类;7天,人类创造了自由。7天,足以改变一个世界,也足以改变一个国家。

这 场突如其来的民主运动后来被称为“十二月事件”,也叫“七日革命”。12月20日,作为总统的齐奥塞斯库结束对伊朗的访问,回到罗马尼亚。7天之后,即 12月26日,齐奥塞斯库已经成为全世界电视新闻上的一具尸体。7天,罗马尼亚从一个依靠谎言、暴力和恐怖维持的专制极权国家,嬗变为一个建立在宪政、法 治和良心上的自由民主国家。

历史告诉人们,民众一旦觉醒,独裁者距离末日只有短短的7天。7天之前,他可以横行霸道,作威作福;7天之后,他必须接受一场良心和正义的末日审判。
齐奥塞斯库夫妇被捕后,为了尽快终止流血冲突,罗马尼亚救国阵线负责人伊利耶斯库授权国防部第一副部长斯登古雷斯库成立特别军事法庭,根据刑事诉讼法规定的特别紧急程序,对齐奥塞斯库夫妇进行审判。

12月25日。特尔戈维什蒂空军中队司令部被改装为临时审判厅,齐奥塞斯库夫妇在这里接受特别军事法庭的审判。审判团由7人组成,齐奥塞斯库夫妇完全否认军事法庭的合法性,并在审判时拒绝回答法官提出的问题。

法 庭指定的辩护律师向齐奥塞斯库夫妇询问,是否要求上诉。根据法律,被告如提出上诉,将由罗马尼亚最高法院进行审理,即使上诉被驳回,也需要一周时间;但被 告如果放弃上诉,这次判决便是终审判决,判决将被立即生效。齐奥塞斯库夫妇对此不予理睬。辩护律师又问齐奥塞斯库夫人,她过去和现在是否有精神疾病。如果 埃列娜说“有”,那么法庭必须将她送到有关医院进行检查,检查过程一般需要好几个月;在此期间,法庭不能对她进行任何判决。但埃列娜的回答是“没有”。

最后,特别军事法庭判处齐奥塞斯库夫妇死刑,他们的罪名包括:“屠杀罪、破坏政权罪、破坏公共财产罪、损坏国民经济罪,以及在外国银行存有10多亿美元,并企图逃往国外……”

在 罗马尼亚南部登博维察县一个兵营的厕所前面,齐奥塞斯库夫妇被军人执行枪决。临刑前,齐奥塞斯库高呼:“自由和独立的罗马尼亚万岁!”他的夫人埃列娜则唱 起《国际歌》:“起来,饥寒交迫的奴隶;起来,全世界受苦的人……”还有一种说法,当时齐奥塞斯库怒气冲天,高喊着:“你们这群叛徒。”然后,4支AK- 47枪同时吐出火舌,齐奥塞斯库夫妇就倒在血泊中。据说两人身上一共有90多个弹孔。

此时正是12月25日,圣诞节之夜。1989年前,耶稣诞生了,人类找到了良心;1989年后,独裁者死去了,人类找到了自由。暴君之后,才是民主。美国总统杰弗逊说:“民主之花是暴君和爱国者的鲜血浇灌出来的。”

审讯和枪决齐奥塞斯库夫妇的过程以神奇的速度被制作成为新闻影片,很快成为罗马尼亚、欧洲乃至全世界的爆炸性新闻。1989年12月26日,全世界几乎所有的电视机上都在反复播放这一历史性的一幕。
后记
1、在处决了齐奥塞斯库夫妇后,罗马尼亚立即宣布废除死刑。
2、齐奥塞斯库政权被推翻后,罗马尼亚共产党被解散,国名由“罗马尼亚社会主义共和国”改为“罗马尼亚”。新宪法出台,新政府实行多党制和自由选举,立法、行政和司法三权分立;取消中央集权的经济制度。
3、在一次纪念“七日革命”的活动中,民选的罗马尼亚总统伊利埃斯库对法国《世界报》记者说:“齐奥塞斯库在党内外建立了强大的镇压系统,扼杀了所有改革意图。一切都恶化了,紧张局势加剧了,直至不可避免地爆发。”
4、 齐奥塞斯库的大儿子瓦伦丁是一个出色的物理学家。齐奥塞斯库死后,他成为一个自食其力的中产阶级。齐奥塞斯库的女儿佐娅是一名数学家,从政府部门提前退 休。经过长期诉讼,她从政府那里拿回了属于自己的财产。其中包括齐奥塞斯库夫妇5万美元的遗产,这是作为齐氏夫妇服务国家应得的报酬。
5、2006年12月14日,经欧盟首脑会议正式批准,罗马尼亚正式加入欧盟。
6、2008年10月14日,通过大量调查取证,罗马尼亚议会宣布,“所有调查证人无一例外地都认为齐奥塞斯库在国外没有存款”。当初普遍认为,齐奥塞斯库有超过10亿美元的海外存款,纯属子虚乌有。
7、2009年,七日革命20周年,一项社会调查显示,尽管仍然有许多人对齐奥塞斯库夫妇没有好感,但越来越多的罗马尼亚民众对在圣诞节那天将国家领导人“像一条野狗似的处死”感到遗憾。当主人齐奥塞斯库像狗一样死去后,像人一样的“考布上校”不知所终。
8、2010年7月21日,齐奥塞斯库及其妻子的遗体被从布加勒斯特的一个墓地中挖出。经过罗马尼亚国家法医学院DNA检测后,确认系前总统齐奥塞斯库夫妇的尸骨。
9、 1989年12月23日,齐奥塞斯库夫妇被罗马尼亚军民逮捕,中国外交部发表声明说:“这是绝对不能容忍的!”1989年12月25日,齐奥塞斯库夫妇被 愤怒的罗马尼亚军民乱枪处决,中国外交部郑重声明:“尊重罗马尼亚人民的选择。”这种中国式“变脸”一时之间成为国际笑柄。从圣诞节到元旦,北京全城狂 欢,大小饭馆无不爆满;借人杯酒浇自己心中块垒,这个遥远的事件竟然一扫很多中国人半年来悲情阴霾……
10、1991年12月25日,最大的极权主义国家苏联帝国成立69周年之际,苏联总统戈尔巴乔夫正式宣布辞职。此前一年,他获得诺贝尔和平奖。飘扬在克里姆林宫顶上空的锤子镰刀旗黯然飘落,苏维埃社会主义共和国联盟彻底终结。
11、 2011年3月24日,俄罗斯《论据周刊》的一篇文章爆料,苏联、美国、匈牙利和罗马尼亚四国情报部门联手,共同策反了齐奥塞斯库的宠臣,通过这场军事政 变推翻齐奥塞斯库,因为美苏都不想让罗马尼亚拥有核武器。很难设想在冷战时期,克格勃和中情局会互相合作,但在戈尔巴乔夫时代,双方却真的走到一起。

来源:《观典》杂志2013年第1卷第2期,有删节

Wednesday, May 1, 2013

The Development of mNeonGreen

http://blog.allelebiotech.com/2013/04/the-development-of-mneongreen/
This week our most recent publication, “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum” will be published in Nature Methods. It has already been viewable online for some time now, here is a link. We believe this new protein possesses a great deal of potential to advance the imaging fields through enhanced fluorescent microscopy. mNeonGreen enables numerous super resolution imaging techniques and allows for greater clarity and insight into one’s research. As a result of this we are taking a new approach at Allele for distribution of this protein, and here we will describe the history of the protein and some of the factors that led us down this path.
mNeonGreen was developed by Dr. Nathan Shaner at Allele Biotechnology and the Scintillon Institute through the directed evolution of a yellow fluorescent protein we offer called LanYFP. LanYFP is a super bright yellow fluorescent protein derived from the Lancelet fish species, characterized by its very high quantum yield, however, in its native state LanYFP is tetrameric. Dr. Shaner was able to monomerize the protein and enhance a number of beneficial properties such as photostability and maturation time. The result is a protein that performs very well in a number of applications, but is also backwards compatible with and equipment for GFP imaging.
Upon publication there was a question of how distribution should be structured. How would we make this protein available to researchers in a simple manner was a very difficult challenge. We also relied heavily on Dr. Shaner’s knowledge and experience in these matters, as he related his experiences to us from his time in Roger Tsien’s lab at UCSD. When the mFruits was published their lab was inundated with requests. The average waiting period was 3 months to receive a protein and they required a dedicated research technician to handle this process. Eventually the mFruits from the Tsien lab were almost exclusively offered through Clontech. Thus we decided that Allele Biotechnology would handle the protein distribution and take a commercial approach to drastically decrease the turnaround time. The next challenge we faced was how to charge for this protein. Due to the cost of developing this protein, which was fully funded by Allele, there is a necessity to recoup our investment and ideally justify further development of research tools, but we also understand the budget constraints every lab now faces. From this line of thinking we conceived our group licensing model; we wanted to limit the charge to $100 per lab. The way this is fiscally justifiable is having every lab in a department or site license the protein at this charge, including access to all related plasmids made by us as well as those generated by other licensed users (Click here for our licensing page). The benefit we see to this is that the protein is licensed for full use at a low cost, and collaboration amongst ones colleagues is not only permissible, it’s encouraged. We saw this as a win-win situation. We would recoup our cost and invest in further fluorescent protein research, and our protein costs would not be a barrier to research and innovation.
The granting of a license to use but not distribute material is not unique to commercial sources. Although academic material transfer agreements typically contain specific language forbidding distribution of received material beyond the recipient laboratory, some researchers choose to disregard these provisions. Unfortunately through this action they are disrespecting the intellectual property rights of the original researchers as well as violating the terms of the legal contract they signed in order to receive the material. We believe most researchers choose to respect the great deal of effort that goes into the creation of research tools for biology and do not distribute any material received from other labs without their express permission. However for a company that funds its own basic research our focus is often on the former example rather than the latter. We believe that this focus artificially drives up the costs of licensing a fluorescent protein and obtaining the plasmid, thus we have chosen to believe researchers will respect our intellectual property as long as we are reasonable in our distribution which is something we have truly striven for.
Additionally we believe the broad-range usage of a superior, new generation FP is an opportunity to advocate newer technologies that can be enabled by mNeonGreen, together with a number of Allele’s other fluorescent proteins (such as the photoconvertible mClavGR2, and mMaple). These new imaging technologies are called super resolution imaging (MRI). They provide researchers with a much finer resolution of cellular structures, protein molecule localizations, and protein-protein interaction information. We have started the construction of a dedicated webpage to provide early adopters with practical and simple guidance, click here to visit our super resolution imaging portal.

Thursday, April 25, 2013

Autologous versus Allogeneic iPSCs in Immune Rejection

http://blog.allelebiotech.com/2013/04/1562/

The enthusiasm of using autologous induced pluripotent stem cells (iPSCs) for cell replacement therapy was dampened by a publication 2 years ago in Nature (Zhao et al, 2011), which suggested that even syngeneic (genetically identical) iPSCs could still invoke strong immune rejection because, as the authors in Yang Xu’s lab at UCSD explained, the iPSCs overexpress a number of tumor antigens possibly linked to genomic mistakes acquired during reprogramming. Embryonic stem cells (ESCs), on the other hand, did not show similar rejection problems in the same studies, indicating that the immune responses were due to somatic reprogramming.
If proven true, the iPSC-specific immune rejection would have been the biggest hurdle for any iPSC-inspired clinical plans. Naturally, a number of labs performed series of experiments that were aimed at addressing the concerns raised by Zhao et al. This month in Cell Stem Cell, researchers from Ashleigh Boyd’s lab at Boston University demonstrated that autologous (self) or syngeneic iPSCs or their derivatives were not rejected (Guha et al. 2013). These iPSCs behaved essentially the same as ESCs in transplantation settings. When immunogenicity was measured in vitro by monitoring T cell responses in co-culture, no immune response was observed either. In contrast, cells and tissues from allogeneic (genetically different) iPSCs were rejected immediately.
In light of this new publication and an earlier Nature paper (Araki et al. 2013), Kaneko and Yamanaka have commented that autologous iPSCs still seem to have a very good chance of being used in cell replacement therapy, pending, of course, additional research and trial results. In their Preview article in Cell Stem Cell (Kaneko and Yamanaka 2013) two points were particularly emphasized: 1) autologous iPSCs are preferred because of the lack of immune rejection; 2) iPSCs generated with footprint-free reprogramming technologies are preferred because the problems reported by Zhao et al 2011 might be correlated with the use of retroviral vectors (even though they also used episomal plasmid-reprogrammed iPSCs). We strongly support both of these points and believe that they point out the direction of future stem cell therapies.
However, we do not agree with the last statement by Kaneko and Yamanaka in that article stating that as a result of the cost and time required to generate iPSC lines from each patient in GMP facilities, iPSC lines from HLA homologous donors will be the choice going forward to clinical applications. First of all, HLA-matched iPSCs should be closer to allogeneic than to autologous iPSCs. From what we just learned in the last round of debates, the field should certainly go with autologous. Second, generating foot-print free iPSCs may already not be the rate-limiting step, even in GMP protocols, compared to downstream differentiations that are required using any pluripotent stem cells. We have shown that human fibroblasts can be reprogrammed in a completely feeder-free, xeno-free, passage-free process, using only mRNAs, in just over a week, achieving sometimes “bulk conversion”—converting nearly all cells within a well into iPSCs (Warren et al. 2012). We have drawn up a plan to establish cGMP protocols and to quickly apply autologous, footprint-free iPSCs to clinical programs through partnerships. The field can move at a faster speed, with all due scientific vigor and caution, if the best technology available is chosen for building the foundation.

Zhao, T., Z.N. Zhang, Z. Rong, and Y. Xu, Immunogenicity of induced pluripotent stem cells. Nature, 2011. 474(7350): p. 212-5.
Guha, P., et al., Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells. Cell Stem Cell, 2013. 12(4): p. 407-1
Kaneko, S. and S. Yamanaka, To Be Immunogenic, or Not to Be: That’s the iPSC Question. Cell Stem Cell, 2013. 12(4): p. 385-6.
Araki, R., et al., Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells. Nature, 2013. 494(7435): p. 100-4.
Warren, L., Y. Ni, J. Wang, and X. Guo, Feeder-free derivation of human induced pluripotent stem cells with messenger RNA. Sci Rep, 2012. 2: p. 657.

Saturday, March 30, 2013

Allele Publishes mNeonGreen as the Brightest Monomeric Fluorescent Protein for Super-resolution Imaging

From AlleleNewsRelease: http://blog.allelebiotech.com/category/ips-and-other-stem-cells/page/2/

This week scientists from Allele Biotechnology and its partner non-profit research institute, the Scintillon Institute, present their latest fluorescent protein, mNeonGreen, in the journal Nature Methods (Nature Publishing Group). In the paper, entitled “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum,” the scientists describe the development of the brightest monomeric fluorescent protein to date.
The scientific efforts to develop this novel fluorescent protein were led by Dr. Nathan Shaner, a leader in the field of fluorescent protein engineering. Fluorescent proteins are highly valuable research tools that allow the labeling and imaging of individual proteins within a living cell, and tracking of their movements and localization in real time through a microscope. However, since the discovery of the original green fluorescent protein in 1993, imaging technology has advanced rapidly beyond the capability of most fluorescent proteins. The newly described fluorescent protein, mNeonGreen, allows researchers to take full advantage of modern super-resolution optical microscopy techniques that enable visualization of structures in living and fixed cells at much smaller scales than are possible using traditional optical microscopy. This improvement will lead to countless new insights into human health and a greater understanding of protein interactions at very small distance scales within living cells. According to Dr. Jiwu Wang, the CEO of Allele Biotechnology, “Super-resolution imaging will become the standard for publication in a short period of time, and mNeonGreen allows researchers to meet this standard while still being compatible with the equipment and methods they already use.”
Prominent researchers within the fluorescent protein field are touting mNeonGreen as a replacement for jellyfish-derived Aequorea GFP, one of the most commonly used fluorescent proteins today. According to lead researcher Dr. Nathan Shaner, “mNeonGreen can be directly substituted for other green fluorescent proteins such as EGFP without the need for any equipment changes,” making the upgrade an attractive prospect for many researchers.
Allele Biotechnology and Pharmaceuticals Inc. is a San Diego-based biotechnology company specializing in the fields of RNAi, stem cells, viral expression, camelid antibodies and fluorescent proteins. The company has co-developed a number of fluorescent proteins and other products for PALM or STORM super-resolution imaging 3D-SIM, and STED imaging. With the arrival of mNeonGreen, Allele plans to collaborate with leading imaging labs, microscope manufacturers, and journals such as Nature Methods to further promote the advantages and capabilities of the latest imaging methods. Additionally, this announcement will coincide with the launch of a new super-resolution imaging web portal and plasmid depository via collaboration with the Scintillon Institute. The Scintillon Institute is a non-profit research institute established in 2012 using seed funding from Allele Biotech. The institute’s researchers are focused on the development of biological tools to improve human health and quality of life, including applications to cancer imaging, regenerative medicine, and sustainable energy and food production.
For details about Allele’s new Superresolution FP distribution method, read our departmental and institutional usage page.

Friday, March 15, 2013

博浪椎:从四大名著看中国之走投无路

看四大名著,不能只看到其中神魔乱舞的有趣,英雄好汉惩恶扬善的痛快淋漓,帝王将相纵横捭阖的权谋秘计,才子佳人荡人心魄的情孽缠绵。我们还可以看到,这里面明明白白写着:中国已经到了走投无路的地步。

孙 悟空大闹天宫,很是让一潭死水的天宫闹腾了一阵子。他提出的革命理论是:皇帝轮流做,明年到我家。这一革命理论,与历来农民起义提出的号召如出一辙。陈胜 吴广的口号——“王侯将相宁有种乎”,不就是孙大圣的轮流坐庄的理论么?大概孙大圣看到玉帝的排场,心向往之,豪言壮语脱口而出:“彼可取而代之。”或者 吃天鹅肉的心理作祟:“嗟乎,大丈夫当如此也。”率领他的猴子猴孙,攻占天庭,把天庭改朝换代成猢狲王朝,这就是孙悟空的革命理想。坐天下的是王,抢天下 的是贼;在天庭里,坐天下的是神仙,抢天下的是妖孽,人间天上,奉行同一道德规条。

玉皇大帝,太上老君斗不过妖猴,于是借师助剿,请来 如来佛,妖猴斗不过佛祖,于是猴子被洗脑,跟随唐僧,充当了镇压各路反叛势力的急先锋,一路降妖除怪,最后官封斗战胜佛。从现有体制的反叛者到忠实维护 者,进而变成现有体制的受益者,孙悟空用了五百多年。天庭也付出了惨重代价,最后双方妥协,互相利用。孙悟空在当初跟他一样身份的妖魔前可以堂而皇之地自 称仙佛,口含天宪,大展神威,实现终极自我价值。天庭也可以利用他充当打手,扫荡其他对现有体制构成挑战的各路反叛势力。孙悟空的七十二变,怎么都比不上 他这第七十三变实在,能得更多实惠。

历代农民起义领袖的嘴脸,在这里被照妖镜照得毫发无隐。斗得过现有体制,就把玉帝的张姓朝廷改姓 孙,斗不过就投降,自己的反叛实力就是跟朝廷谈判的本钱,兄弟队伍的头颅就是投名状,几番含娇弄态之后,即漂白了自己的出身。唐末朱全忠玩的是这一套, 《水浒》里宋江玩的也是这一套。农民起义救中国?痴人说梦而已。

《水浒》里的宋江,玩的也是这一套,不过一在天上,一在地下。世人都怪 宋江,怪他不该投降,他不投降又能怎样呢?梁山事业,实在无路可走。梁山政权,只不过是东京赵家朝廷的山寨版。想当年,赵匡胤在位时,也跟他们的宋大哥一 样英明,一样讲义气,一样替天行道,到如今子孙不肖,宋徽宗赵佶昏庸无比,贪官墨吏扰乱天下,假设梁山事业百年长青,谁能保证宋江的子孙比赵佶一定强,好 汉们的后代一定比高俅雄起?投降了倒好,中国历史省去了一个不必要的循环节。好汉们的理想不过是大碗喝酒大块吃肉大秤分金,贪官们的理想不过是大肆捞权大 把捞钱多睡女人,后者是前者的衍生品,他们只相隔一张纸的距离。梁山与祝家庄,一个由好地主领导,一个由坏地主领导,好地主与坏地主的距离,也不过一张纸 而已。

梁山道路,走下去将无路可走。只可怜了那鲁莽得天真可爱的李逵,为了“替天行道”的杏黄旗永续飘扬,拎着一对大斧排头价砍来去,不知他端的要砍谁?

梁山道路,最终无路可走。

《三 国演义》宣扬的是圣主贤相救国论,翻译成无产阶级口吻,就是只有刘备诸葛亮才能救中国。可惜枭雄斗不过奸雄,贤相斗不过奸相,仁义斗不过厚黑,刘姓天下无 可奈何花落去,圣主贤相的理想一江春水向东流。仁义事业实在让人悲催,刘备诸葛亮只给后人留下一个欲哭无泪的背影。 最终一统天下的司马氏,一点仁义的影子都找不到。

刘备诸葛亮事业的流产,意味着圣贤救国论的破产。

把中国古代的知识 分子分为两拨,《红楼梦》的作者曹雪芹超世独立,一个人站一边,其他所有人站另一边。中国历史上所有读书人的脑袋凑在一起也比不过他思想的深刻。曹雪芹通 过贾宝玉告诉我们,封建社会已是穷途末路。身处康乾盛世而能认识到这一点的,绝无仅有。比他稍前的明末清初的黄宗羲顾炎武王夫之等人,痛心于明朝的灭亡, 沉痛反思的结果无非是皇帝太昏庸,大臣太贪婪顾私利,不去从根本上否定制度,没有从我们固有文化中查找致命之处,反而抱残守缺,对传统儒家沾沾自喜,以为 明的灭亡,不是儒家文化的失败,而是不能遵守儒家戒律的结果。就像《旧约圣经》里的犹太人,每受一次打击,每遭一次失败,都认为是对上帝笃信不坚的后果, 是上帝因为他们在异教间摇摆不定而降罚,因而每一次反思的结果都是更加信仰上帝。鸦片战争后很长一段时间,先进的中国人反思落后挨打原因,还认为我们只是 技不如人,而不是体制不如人,文化已落后。曹雪芹超越了他以前和以后所有的旧式知识分子精英。

他通过贾宝玉的口告诉我们,我们的制度, 我们的文化已是走投无路。贾府里面的男人,从上到下全都灵魂烂透了,封建社会的大厦靠这些人支撑,倾倒是必然的事。不仅如此,贾宝玉还以戏谑的口吻否定了 封建社会的最高道德教条,所谓的“文死谏武死战”。这一条认识是从根基上否定了我们引以为豪的封建道德和封建文化。最崇高的东西都被他看穿看透了,他还能 跟周围的男人沆瀣一气么?他还能从父命读八股考科举光耀门庭为这一腐烂的家族提供继续腐烂下去的养分么?所以贾宝玉清醒坚决地拒绝了合作,不愿为这个注定 要烂掉的社会陪葬。可惜宝玉的心思书中无人理解,就连他深爱的林妹妹也半点不知,他只能“无故寻愁觅恨,有时似傻如狂”了。他的愁恨,他的傻与狂,是出于 绝望,他找不到新的道路。《红楼梦》所深刻揭示的,不是曹雪芹所处的朝代已濒临绝境,此时是大清盛世,正是鲜花着锦烈火烹油无限风光的时代,他揭示给我们 的,是整个封建社会的走投无路。林妹妹的葬花词,在贾宝玉听来,在后世的我们听来,是唱给所有中国人的一首挽歌。

《西游记》《水浒传》告诉我们,农民起义,革命造反不能救中国;《三国演义》告诉我们,伟大领袖不能救中国;《红楼梦》告诉我们,我们真的已走投无路。

我们的文化没有内生性的力量,靠我们的固有文明来救中国,就好像是用中药来救治需要开膛破肚的病人一样无效。

来源:爱思想

Wednesday, March 6, 2013

Conducting Massively Parallel Sequencing

http://blog.allelebiotech.com/2013/03/conducting-massively-parallel-sequencing/

One of the major breakthroughs in modern biology is the development of massively parallel sequencing, also called next generation sequencing (NGS), which enabled the complete delineation of the human genome more than a decade ago. Since then many more species’ genomes have been sequenced, and the cost per genome has dropped from billions to mere thousands of dollars. New discoveries are being made as a result of the capability many research teams now possess to not only sequence chromosomal DNA, but also to identify which regions a protein of interest specifically binds (Chip-seq), analyze a whole transcriptome of a cell population under investigation (RNA-seq), or find out which RNA regions an RNA binding protein resides (CLIC-seq).
While it is inevitable that many PIs will seriously consider the inclusion of deep sequencing in their next grant proposal, it is not necessarily easy to take the first step and get their feet wet, so to speak. Knowing what format (e.g. 454 for longer reads, HighSeq for higher accuracy, or Ion Torren for bench top convenience) to use and how much to pay requires a vast amount of knowledge and experience. Even when you are done with sample prep, amplification and sequencing, to handle such massive amount of data is not trivial—transporting data alone can be a headache. A database server for storage and analysis requires another layer of expertise. There is no easy solution but to get started somehow. However, be prepared to deal with these issues.
Whether the cost on a type of next generation service is justifiable depends on whether it is required for your purposes. For example, when analyzing a person’s propensity of developing a disease by using known, disease-relevant genetic information, often times exome sequencing is sufficient. This costs anywhere between $1,000 to $3,000 with 100X coverage, significantly less than sequencing a complete genome which typically costs ~$5,000 at ~20x coverage.
High coverage sequencing of maternal blood DNA has been developed into clinically approved prenatal diagnosis of trisomy in Down’s syndrome and other chromosomal abnormalities. Transcriptome analysis helped the understanding of how reprogramming works when iPSCs are. Looking forward, with more routine use of deep sequencing we can predict with much more certainty the “off-target” effects of RNAi or cellular toxicity of chromosomal modifications enabled by ZFN, TALEN, or CRISPR. As a matter of fact, we believe that transcriptome sequencing should be required after each RNAi event to prove a specific linkage between knockdown and functions; similarly, whole genome sequencing results need to be provided after making a site directed chromosomal change in the future for high level publications.
*This blog partially resulted from discussions between Jiwu Wang @Allele_Biotech and his colleagues, who are NGS experts at UCSD’s Cellular and Molecular Medicine, Moore Cancer Center, and BGI Americas.

Wednesday, January 16, 2013

Allele Biotechnology Initiates Project On Scaled Manufacturing Of Induced Pluripotent Stem Cells And Differentiation With Chinese Academics

AlleleBlog 01/16/2013 San Diego:
Allele Biotechnology has signed an agreement with Jinan University to develop culturing systems of stem cells and differentiation methods for producing skin tissue cells for wound treatment and stem cell therapy.
San Diego, California (I-Newswire) January 16, 2013 – Allele Biotechnology and Pharmaceuticals, Inc., a San Diego based company with a focus on new technology development, announced today that it has signed an agreement with the Biomedical Institute of Jinan University through a focus group to develop culturing systems of stem cells and differentiation methods for producing skin tissue cells for wound treatment. The joint team will also evaluate using stem cell therapy as potential treatment for arthritis, Lupus, and other autoimmune-related diseases.
Scientists from Allele Biotechnology recently described an important advance in the generation of stem cells capable of producing all the different tissues of the human body. Using messenger RNA molecules and without the need of viral vectors, animal products or feeder cells, this new method can be used to reprogram human fibroblasts into induced pluripotent stem cells (iPSCs). The efficiency is significantly improved over previously reported reprogramming results and the time required to complete reprogramming is slashed in half under optimal conditions.
The Biomedical Institute at Jinan University, a leading comprehensive research university in South China, has focused on translational research in the areas of epidemic diseases and autoimmune diseases. It has broad collaboration with partners and close connections to the biotech industry in China. It was known to have launched (licensed) a number of new biologics in China, and contributed to the understanding and diagnostics of the SARS epidemic in 2003. The institute has also been entitled as the national engineering research center of biopharmaceutics since 2005.
This collaboration will last for at least 2 years, and will go beyond the R&D stage with selected candidates moving into clinical trials, first in China, then in other countries. If the project reaches clinical trials it will be funded jointly by industry and academic partners in the range of $10 million USD.