Top 10 List of Most Viewed AlleleBlogs in 2011

The ballot is in—among the “usual suspect” hot topics, iPS takes the top honor and most entries; Camelid antibodies, although not really presented as a typical AlleleBlog in 2011, made it to the top 3. shRNA cloning and RNAi screening are still on a lot of people’s minds, so it seems.

Method: total visits to each blog since our new webpage was launched in July was counted.

1) Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming
http://blog.allelebiotech.com/2011/10/fusion-of-the-transcription-domain-to-ips-factors-radically-enhances-reprogramming/

2) Methods of iPSC Generation Update
http://blog.allelebiotech.com/2011/08/methods-of-ipsc-generation-update/

3) About 50 Papers Cited the Use of GFP-Trap Camelid Antibody So Far in 2011
http://blog.allelebiotech.com/2011/09/about-50-papers-cited-the-use-of-gfp-trap-camelid-antibody-so-far-in-2011/

4) Big Potential in Using Protozoans for Producing Mammalian Proteins
http://blog.allelebiotech.com/2011/09/big-potential-in-using-protozoans-for-producing-mammalian-proteins/

5) How do you make shRNA-expressing viruses for function screening?
http://blog.allelebiotech.com/2011/11/how-do-you-make-shrna-expressing-viruses-for-function-screening/

6) Creating ground-state human iPSCs
http://blog.allelebiotech.com/2011/10/creating-ground-state-human-ipsc/

7) Recombinase-Mediated Cassette Exchange (RMCE) and Integrase Swappable in vivo Targeting Element (InSITE)
http://blog.allelebiotech.com/2011/03/recombinase-mediated-cassette-exchange-rmce-and-integrase-swappable-in-vivo-targeting-element-insite/

8) Development of Cell Lines from iPSCs for Bioassays
http://blog.allelebiotech.com/2011/11/development-of-cell-lines-from-ipscs-for-bioassays/

9) Choosing siRNA, shRNA, and miRNA for Gene Silencing
blog.allelebiotech.com/2010/02/choosing-sirna-shrna-and-mirna-for-gene-silencing/

10) Allele Biotech’s Box Swap Program
http://blog.allelebiotech.com/2009/07/allele-biotechs-box-swap-program/

Have a successful 2012!

From AlleleBlog: http://blog.allelebiotech.com/2011/12/top-10-list-of-most-viewed-alleleblogs-in-2011/

“活着”之后的张艺谋和谈论“革命”、“民主”、及“自由”的韩寒

张艺谋的“活着”可以说是中国当代电影中最深刻、最有影响力的、反应中国近代社会、历史的作品之一。它在中国的禁演减小了其影响力，在时间上也似乎把张导演的电影生涯画了一条界线----之前的张艺谋作品是给大众看的；之后的是为了掌权者或投资人而让大众看的。比如《英雄》的唯一思想就是“秦王不能杀”，即使他施行暴政、残忍肆虐，但他有一个“打下很大的天下”的宏大志向，用现代的话说叫“正在下一盘很大的棋”。其实纵观人类现代史，顶尖的恶魔大都也有这样的宏大志向及信心、耐力、甚至智慧去实现（或几乎实现或暂时实现）其志向，比如希特勒、波尔布特、等等。张艺谋之后的开幕式、《黄金甲》，不用重复评价了，引王朔的话说：“我倒是认为张艺谋同志后来有点像一个装修大师了，他现在到处把人家弄得金碧辉煌，这是为挣钱吗？我不太明白，（他）挺有钱的啊”。也许张艺谋有了钱之后还有“更高追求”？进一步更大胆地猜一下，他还把自己当艺术家？张艺谋重返艺术的努力，姑且认为有这种努力的话，结晶成了《三枪》和《山楂树》，或者是荒诞的娱乐以买笑，或者是虚佞的情节以煽情。而其艺术糟粕之登封之作恐怕要数近作《十三钗》了，在南京大屠杀这样的灾难中选择无处不在的性暗示作为故事的主线，以妓女的命不如学生的命值钱的逻辑，裹上一面民族主义的大旗，加上中国电影史上最高制作费的招摇和国家支持的奥斯卡入围来试图成就艺术。何其荒诞。艺术的没灵魂直接反映的是制作者的无灵魂。

不管你喜欢不喜欢他的艺术，张艺谋还是在人们心目中常被划在艺术家的一群中。韩寒是作家，当然也和艺术家有关联，但显然他最成名的原因是他的时政评论。韩寒以前通过网络文章表现出来的自由自在的思维、无拘无束的态度、和同情弱小的情怀使他成为拥有最多读者群和“时代”杂志风云人物候选人，用他自己的话说是“我候选了时代周刊的两百个影响全球的人物，中国同时入选的还有敏感词，敏感词和敏感词等人” （其中的一个敏感词是刘晓波，剩下大部分是高级官员，在中国也能以和刘晓波敏感相反的原因成为敏感词，比如防止被人骂）。韩寒在今年圣诞节前后以评革命、民主、和自由的三段网文正式直接地进行了政治评论。他的基本观点是中国没有民主自由是百姓素质太差；统治者的党之所以有了执政合法性是因为他们已经大到了人口的很大比例，所以我中有你，你中有我，党即民民即党；革命的结果是可怕的，等着执政者变好吧。这些显然和政府一贯拒绝施行民主所用的原因是很一致的，韩寒甚至因此得到了“环球时报”主编和其他不少著名5毛的公开支持和赞赏。不知道韩寒被这样的，曾经被他无情嘲讽过的人夸是喜、是悲、是无奈，还是尴尬，但这和他失去民众支持的悲哀比起来已经不重要了。正像我想问新近投奔党国怀抱并拒绝谈论6.4的北岛—--还记得你自己写的“高尚是高尚者的墓志铭，卑鄙是卑鄙者的通行证”吗？很多人在问韩寒----还记得你自己写的“你可以不为自由而战，但不能为高墙添砖”吗？

在生物学上目前最难也可能是以后最后需要科学家攻克的难题是脑神经学；在人类社会上最难解释也最难预见的可能是人心之变化。希望这些我很遗憾看到的变化主要局限在艺术家身上吧。这篇感慨既然从评论艺术作品作品开始，就以引用艺术作品结束：还记得《桃花扇》里李香君对侯朝宗撕扇明志时引用的李清照的“乌江”吗？“生当作人杰，死亦为鬼雄。至今思项羽，不肯过江东”。

北大校长对北大教育成功与美国教育失败的评价

昨日，周其凤站在家乡长沙市一中的讲台上，面对长沙四大名校中学生进行演讲。演讲会进行了2个多小时，对于现在很多人否定中国的教育，周其凤持不同态度，“我认为美国的教育一塌糊涂，他们的每一任总统都不懂得尊重人，总是把自己的意愿强加于别人，如此看来，他们的教育是一塌糊涂的。”周其凤认为中国的教育很成功，理由是中国这些年都在飞速发展，“我们的国家在进步，靠的就是我们的教育培养的人才。”

就是这位校长前段时间写了一首歌《化学是你化学是我》，在网上迅速“蹿红”infamous，甚至被封为“神曲”。

就是这所学校有“孔三妈”当教授。

我的北大校长是丁石孙，还记得他6.4站在校门口阻止学生出门，却在人民大会堂什么代表大会上激烈地为学生说话。

周其凤来圣地亚哥还好没去，不然没的掉了价。

方励之、李淑娴：谨借楚辞八句以献林昭八秩冥寿

惜林苑之曾信兮，受命诏以昭时。
奉先功以照下兮，盛气志而过之。
君无度而弗察兮，使芳草为藪幽。
卒没身而绝名兮，不毕辞而赴天。

2011年12月12日Tucson

注：李老师是我的大学物理老师，借她和方教授引的楚辞表达对我心目中最优秀的校友的敬意。也顺祝方李两位金婚之喜。

Localization imaging with standard fluorescent proteins on live cells—Bayesian modeling

The resolution of optical microscopy is limited by the Abbe limit, the diffraction limit roughly half the wavelength of the light used (e.g. green light is around 500 nm, its Abbe limit is 250 nm). Super-imaging fluorescence often involves switching fluorophores between a dark and a bright state, building a high-resolution image from many single, localized fluorophores. These technologies have become relatively well-known in the past years, including stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM), photoactivatable localization microscopy (PALM), and a number of variations.



STED and saturated structured illumination (SSIM) require specialized microscopes to shrink the effective size of the scanning beam or to extract information from hidden patterns. Localization techniques require non-overlapping fluorophore emission by activating a small populations of fluorophores, e.g. those of photoswitchable FPs such as Dendra,mEos, or mClavGR2. Cox et al. recently reported in Nature Methods that by using Bayesian modeling they were able to utilize many overlapping fluorophores to obtain localization from blinking and bleaching. This allows high resolution imaging at 50 nm using wide field microscopy, regular FPs, and on live cells.



The technology’s novelty and focus were mostly on modeling. The authors used podosome (cytoskeletal structures associated with cell adhesion, migration, and disintegration of the extracellular matrix) danymics imaging as the first example to demonstrate the power of the new method. The software for data analysis is provided at http://3bmicroscopy.com. The technology is termed 3B analysis for Bayesian analysis of the Blinking and Bleaching. Bayesian: Bayesian probability as "a degree of plausibility of a proposition (belief in a proposition) based on the given state of knowledge," in contrast to interpreting it as a frequency or a "propensity" of some phenomenon.

Cox et al.: http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.1812.html

How do you make shRNA-expressing viruses for function screening?

Allele Weekly Blog: http://blog.allelebiotech.com/2011/11/how-do-you-make-shrna-expressing-viruses-for-function-screening/

Most people use standard cloning procedures when trying to insert shRNA templates into lentiviral vectors, i.e. anneal a pair of long oligos with sticky ends and ligate the dsDNA into a linearized plasmid with compatible overhangs. However, since typical lentiviral vector plasmids have terminal repeats and are relatively large, when ligated to hairpin sequence-containing shRNA templates, recombination often occurs inside bacteria that results in smaller plasmids. This problem is common for cloning shRNA or other unstable DNA pieces into viral vectors. This cloning issue is further compounded by the fact that it is difficult to sequence any shRNA template region because the hairpin may block the progress of the DNA polymerase used in sequencing, sometimes requiring several repeats under different sequencing conditions, incurring high costs charged by sequencing service providers.

To deal with these aspects of the cloning difficulties, particularly for the purpose of increasing cloning efficiency RNAi-based screening, we compared three different strategies

First, we built a smaller shRNA cloning vector to clone and sequence shRNA templates prior to transferring to lentiviral vectors. This smaller vector does not have a severe recombination problem and is easier to sequence in the hairpin-containing region. After an initial round of cloning with this new vector, we further improved it by inserting an XbaI and a NheI site between the BamHI and SpeI insertion sites, so that any plasmid preparations can be screened for recombinants by a simple XbaI or NheI digest before sequencing. After cloning into this intermediate vector, the shRNA expression cassette can be transferred into the lentivirus vectors with some flanking viral sequences so that the insert size will be around 1kb.

Second, we developed a novel DNA preparation procedure after realizing that DNA damage during miniprep of vector plasmids and gel purification of vector fragment increased recombination of these constructs, which were already less stable than usual due to hairpin structures. This procedure of DNA preparation avoids UV or guanidium exposure, which can cause nicks on double-stranded DNA and facilitate recombination. This new procedure relies on purifying DNA through surface-binding to regular reaction tubes treated with a proprietary reagent (SurfaceBind Purification). The process simply requires adding a proprietary, guanidium-free binding buffer to the DNA, which has been processed in a specially coated tube (eppendorf or thin-wall PCR tube), and purifying directly in the same tube. Vectors prepared this way indeed provide more colony counts and a higher percentage of correct constructs as shown by our test runs. The procedure also requires less time and the purified DNA can be dissolved in volumes as small as a few microliters.

Third, to enable truly high throughput shRNA screening (i.e. looking for effective RNAi reagents), we further tested and adapted a ligationless cloning protocol that can be handled by a liquid handler almost entirely. In order to increase throughput, we designed a drastically different procedure that could bypass ligation and sequencing altogether before functional tests. Briefly, DNA molecules that would provide enhanced recombination were created by one round of PCR, purified directly in the surface bind PCR reaction tubes (any template DNA would be removed with DpnI enzyme that cuts non-PCR DNA), pooled, and transformed in bacteria directly. DNA plasmids from transformed bacteria can be used for lentivirus packaging, bypassing sequencing at the initial screening stage, and choose single colonies for sequencing only after a shRNA sequence shows promise in functional assays. This is based on the fact that such cloning rarely has any background colonies, and that among all oligos (if using the correct grade of oligos from validated suppliers) inserted this way, a good portion encodes the correct sequence.

New Products of the week: 100x 15mm EcoCulture Vented Dishes for better stem cell attachment and less plastic waste to the environment, APB-CS-114TC.

Promotion of the week: Buy 1 Stealth Express IPS Induction PCR Template Set, get 1 SurfaceBind RNA Purification Kit free. Use code FreePureRNA.

Development of Cell Lines from iPSCs for Bioassays

The reprogramming of differentiated somatic cells to pluripotency holds great promise for drug discovery and developmental biology. Using immortalized cell lines for drug screening assays has its limitations, such as questionable relevance; and the use of primary cells is often hindered by supply difficulties. Thanks to pioneering work by the Yamanaka, Thompson, and other groups, the feasibility of creating iPSCs has generated an opportunity to provide cell lines with stem cell properties in a virtually unlimited supply [1, 2]. These cells can be derived into different cell types for specific assays, even with patient- or genotype-specific background. Technologies are being developed to produce re-differentiated cells of a number of lineages.

Take cardiomyocytes as an example. There are a number of conventional methods for inducing stem cells into cardiomyocytes: through embryoid body (EB) formation, co-culturing with visceral endoderm-like cell line (END-2), and monolayer caridomyocyte differentiation with defined growth medium and protein factors [3]. A recent publication showed that using appropriate concentrations of BMP4 and activin-A in BSA-containing medium cardiomyocytes might be achieved from iPSCs or ESCs in about 6 days [4].

Transdifferentiation, or direct reprogramming, by introducing a group of 3 cardiomyocyte-specific factors, investigators could directly program cardiac or dermal fibroblasts into cardiomyocyte-like cells [5]. Although much refinement and characterization of these directly reprogrammed cardiomyocyte-like cells, termed iCMs, will be needed before the process can become widely used, this work raised the possibility of quicker and perhaps more efficient ways of generating cells for assays. Similar transdifferentiation has resulted in induced neuron (iN) cells, also by introducing 3 tissue-specific transcription factors [6]. Therefore, it seems that by using defined combinations of tissue-specific transcription factors it is possible to generate cells of different tissue types. It is also possible that by using different, developmental stage-specific transcription activator sets, transdifferentiation can be conducted in a stepwise way and make sure cells at each step is pure. This strategy may be particularly attractive if its efficiency can be improved by the techniques developed for iPSC creation. After all, reprogramming to pluripotency and transdifferentiation to different tissue types must share certain mechanistic steps in their respective processes.

In addition, it has been reported that by briefly overexpressing the Yamanaka iPS factors and controlling growth conditions, mouse fibroblasts could be transdifferentiated up to 40% in 18 days without reversing back to pluripotency [7]. It would be interesting to see if by transient expression of iPS factors via mRNA then switching to cardiomyocyte-specific transcription factors, we can increase the efficiency for direct reprogramming. Use of chromatin-modifying chemicals that were already shown to directly reverse and alter cell fates might also be used to assist direct reprogramming. We believe that a systematic approach for studying these reprogramming aspects should benefit the iPS fields.

1. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.
2. Yu, J., et al., Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-20.
3. Vidarsson, H., J. Hyllner, and P. Sartipy, Differentiation of human embryonic stem cells to cardiomyocytes for in vitro and in vivo applications. Stem Cell Rev, 2010. 6(1): p. 108-20.
4. Elliott, D.A., et al., NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods, 2011.
5. Ieda, M., et al., Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell, 2010. 142(3): p. 375-86.
6. Pang, Z.P., et al., Induction of human neuronal cells by defined transcription factors. Nature, 2011. 476(7359): p. 220-3.
7. Efe, J.A., et al., Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol, 2011. 13(3): p. 215-22.

New Products of the week: T7 RNA Polymerase, high quality for demanding in vitro transcription requirements.

Promotion of the week: GFP-Trap, buy 2 of any package and get 1 of equal or less value free. Use code FreeTrap, follow deals quickly on Facebook.

李承鹏：一只叫萨克斯风的破鞋

我小时候在新疆，特别喜欢看抓破鞋。因为相比于其他各种类型的坏人，搞破鞋的貌似长得好看些，也不像其他坏人被抓斗时尽说些很艰深的事情，破鞋说的通俗易懂。那时哈密有个露天的“小河沟电影院”，河水从天山化解而下，清凉蜿蜒，两岸稀拉长着些胡杨，破鞋们就从电影院出发，脖子上挂着破鞋，成双成对沿着河岸被游行，边走边交待怎么搞上的破鞋、如何接头、如何亲嘴……剩下的就不许讲了，但仅仅这样，已让我觉得很是有趣。



那时对破鞋的定义不仅是奸夫淫妇，在野地里搞对象也算搞破鞋，因为搞对象就该在屋子里搞，野外搞，当然就是搞破鞋。有个姓安的小伙总是被抓，因为不仅喜欢在野外搞破鞋，还要吹着萨克斯风。虽然当时在新疆，用萨克斯风进行汇报演出不算搞破鞋，可在野外对着女人吹萨克斯风就是十足的搞破鞋。跟其他人不一样，交待到最后，姓安的常被工宣队的要求来一段萨克斯风，他会面带微笑，吹上一段，很好听。让我从小就觉得萨克斯风就等于搞破鞋，而搞破鞋其实是件挺美好的事情。



我只是一直不明白，为什么给领导汇报演出时吹萨克斯风不算搞破鞋，搞对象时吹萨克斯风就算搞破鞋。这个问题，我听那个姓安的小伙也问过，工宣队大概表达了这样的意思：萨克斯风是外国乐器，要是吹锁呐问题就小些（注：当时还没上演红高梁），总之领导听什么都没问题，因为领导更有社会主义道德。



前段时间，国家广电总局决定限制各卫视娱乐节目，我觉得“限娱”没有问题，问题在于为什么只限制19：30-22：00的娱乐节目，不限制19：00-19：30的那档娱乐节目。那是一档看的人没当真、念的人没当真、写的人没当真、下命令的人更不当真，可大家集体假装很当真的样子且一当真就是几十年……的王牌娱乐节目。可现在的状况跟小时候很相像，工宣队与广电总局的逻辑也很一样：屋子里就是搞对象，野外就是搞破鞋；给领导汇报演出时吹萨克斯风不算搞破鞋，给对象吹萨克斯风就算搞破鞋……地面频道的不算娱乐，上了星就是娱乐，19：00-19：30不算娱乐，19：30-22：00就是低俗娱乐。



所以我国的事情一点没变，表面看在限娱，实际上还是在抓破鞋。



我常幻想，多年之后，天安门广场矗立起一座娱乐博物馆，盛况空前地记录着以下的抓破鞋：禁《流星花园》，限穿越剧，限网络音乐，限谍战剧，禁同性恋题材，声讨郭德纲，禁《蜗居》……这个国家太多的破鞋。我不解为什么要怕人民搞破鞋，其实让他们搞破鞋，就没精力搞破坏。竟听说八成群众纷纷支持限娱，表示“是该提升一下道德了”，哪天我一定要会会该名永远叫“八成”的群众，问他是不是“八戒”表亲。又听说文化部为避免暴力提升道德，决定推出“绿色游戏”，我好奇绿色游戏里的悟空是否不可拿金箍棒，只可拿祥云火炬……大家知道最近我国又很爱谈道德了，因为佛山两岁女孩被反复辗压十八路人却漠然置之，让长期在19：00-19：30里群HI的礼仪之邦很难堪。总得找出原因，却不能说是官德倒退让民德倒退，所以找来找去，终于在司机、路人、家长之外找到娱乐这个大破鞋。



抓娱乐这大破鞋符合这里一惯的政治逻辑。早年的《海瑞罢官》掀起文革，前两年中国音协声讨过低俗音乐，有个老同志痛心疾首：我很担心自己的孙子孙女，他们现在十多岁，很容易灵魂就受到网络低俗音乐的污染，很担心等他们成为社会中坚力量时这些恶劣影响还在。可并没见有谁受低俗音乐污染，因为这么轻易就污染，它就不是音乐，是原子弹。



把道德下降归罪于娱乐，这可太娱乐了；说娱乐败坏了道德，这本身就不道德。港台的娱乐就低俗，可没有七十码、地沟油和见死不救，人家买东西好好排队，保钓却也冲到最前头。至于领导说的高雅艺术以提升道德，我对这个可真是很怀疑。我还记得《辛德勒名单》有个情节：屠城的那个晚上，犹太人纷纷躲藏在楼梯间、墙体夹层。纳粹军就用听诊器去听墙体里有没有呼吸声。有个犹太人不小心碰到了钢琴，士兵们发疯般冲上楼去一通扫射，从而掀开了第二次屠杀的序幕。可在机关枪声、惨叫声中，长夜里忽然响起一阵悦耳的钢琴声，很优秀的琴声，流畅而激昂，有一种巴赫式的宗教宁静。两个士兵被琴声吸引，竟在门边讨论，一个说：是巴赫。另一个说：不，是莫扎特……我一直以为这是视死如归的犹太艺术家临终的演奏，可画面摇起，一个表情肃穆的纳粹军官，一个高雅艺术的爱好者。



纳粹军队可谓二战时期音乐素养最高的一支军队，希特勒和戈培尔都曾强力在军队推行高雅艺术。希特勒本人是瓦格纳的粉丝，德国空军轰炸伦敦前大多要听贝多芬《第三交响曲》，奥斯威辛集中营司令官克拉麦杀人时甚至要听舒曼的梦幻曲……可见高雅艺术提升道德是个伪概念，艺术欣赏力跟杀不杀人并没关系。否则以后监狱里不安狱警，安装一水儿的高保真黑胶唱机，罪犯也不越狱了。大街上要碰到绑匪，直接播出《众神的黄昏》，一听感动得化了：哈里路亚，不能杀人了，去唱诗班吧。



当然要提升道德，可不要用抓破鞋的方法去提升道德，也可以抓破鞋，可不要一边抓破鞋一边自己在搞最大一只破鞋。希特勒、戈培尔当初就用抓破鞋手段摒弃一切低俗艺术，甚至一度禁止电台播放爵士乐，因为爵士乐来自美国，这多么低俗。后来虽然允许在舞厅里演奏爵士乐，但已是只能用小提琴和大提琴演奏“洁本”了。想像一下，用小提琴和大提琴演奏的爵士乐，就跟中国小脚老太太跳芭蕾一样古怪了，可希特勒认为，这样的艺术改造才能让帝国的意志更统一、更强大、更能忘记痛苦。



所以现在还呼吁“人民有低俗的权利”的朋友就很不上道了，此时我真切地政治敏感到这次祖国真是要推动限娱——道德——文化的一体化强国工程。表面上是在限娱乐，其实在抓破鞋，表面在提升道德，其实在统一思想，又不好意思给没头脑的刁民明说，绕了好大一个圈子，你看，我们很早就不方便谈政治了，后来也不好谈历史了，谈地理其实也是敏感瓷，现在连风月都开始不许谈了，所以只好谈谈道德。限娱乐是为了抓破鞋、抓破鞋为了促道德、促道德必然结果是，建成一个正确的文化体系……



是该社会主义文艺复兴的时候了，像美国那么没道德的国家都能成文化大国，有道德的我们更是前程远大。虽然我们没有一个好大学，没有一部好电影，没有一个好作家，没有一个好博物馆，没有一档好电视节目，没有一个真实历史……但必须指出，我们有论语心得，有建党伟业，有孔子学院，有大爱无疆，有19：00-19：30，还有西门庆故居。虽然我们报刊杂志不太说真话，但印刷品数量是全球第一。虽然我们出版审查是严了一点，但实在不行，还可以出手抄本。虽然我们有个别无德贪官，但贪污几千万的十大品牌市长李启红“还是有很多好的品质，骨子里无比热爱党”。虽然我们的舆论监督遇到些问题，可监督舆论从来不是问题，你看前面我那篇文章，虽然只有一个标点符号，却能有三十多万点击率，这才叫传媒大国、文化强国，这才叫软实力，名副其实。



最后一个故事，是文章写到这里时发现Richard Overy介绍的：“上世纪三十年代早期，苏联视爵士乐为一种文化颠覆，跳爵士舞，也作为堕落的资产阶级生活方式。可是低俗堕落的资产阶级生活方式实在诱惑太大了，官方不得不让步，成立国营爵士乐团，但只允许演奏旋律柔和的舞厅曲目，或是改编自俄罗斯民歌的音乐。一九四五年以降，爵士乐因为冷战头号敌人美帝国主义，更是罪加一等。到了一九四九年，苏联萨克斯风的生产与销售皆为非法”。



让我们最后一次谈谈风月吧，原来老大哥早就抓获了一只叫萨克斯风的破鞋。一只叫萨克斯风的破鞋，一个叫李启红的道德，一个只剩下标点的文化。

Creating ground-state human iPSCs

AlleleBlog

Murine pluripotent stem cells can exist in two distinct states, blastocyst-derived LIF-dependent embryonic stem cells (ESCs) and epiblast-derived bFGF-dependent stem cells (EpiSCs). Murine ESCs and similar iPSC lines are more of the “ground-state” in terms of developmental status, as reflected by the lack of X chromosome inactivation in female cells and their abilities to pass as single cells. Human iPSCs, like human ES cells, are more similar to mouse EpiSCs. Unfortunately these human pluripotent stem cells are difficult to genetically manipulate, e.g. knockin or knockout. They also grow slowly, with doubling time averaging 36 hours. In order to create ground-state human iPSCs, several approaches have been tested, including reprogramming iPSC-derived fibroblasts, continuously expressing 5 iPS factors (Oct4, Sox2, Nanog, c-Myc, and Klf4), or using chemicals to inhibit chromatin modifying enzyme HDAC. While these approaches succeeded to certain degrees, the resulting cell lines seem to have some limitations, such as limited passage numbers.

Retinoic acid (RA) signaling is involved in many aspects of embryonic development. RA receptor (RAR), together with one of its heterodimerization partners, steroid hormone receptor Lrh-1, was recently found to be able to synergize with the 4 common iPS factors (Oct4, Sox2, Klf4, and c-Myc) to induce mouse and human fibroblasts into ground-state iPSCs. The pluripotent cells created by the so-called F6 factor combination show no X chromosome inactivation if from female origin, can fully activate the endogenous Oct4 promoter, express Rex1 (which is specific to mouse ESCs, not EpiSCs), and grow with a 16 hour doubling time. All these mouse ESC-like features were achieved without detectable expression of the exogenous factors once iPSC colonies formed, indicating transient F6 expression is capable of effectively initiating endogenous stem cell factors. Remarkably, these stem cells can maintain their undifferentiated status in mouse ESC medium for 50 passages or more. This work, published this month in Proceedings of National Academy of Science USA [1], provided the stem cell research and application field with a very desirable choice of human stem cells.

As opposed to ~16 days with F4, it appears that the time required to induce adult fibroblasts into pluripotent stem cells is as short as 4 days if F6 factors are introduced on a murine stem cell virus (MSCV) vector with an integrated piggyback transposon. As the authors noted in their discussion, the speed-up benefit should be particularly advantageous for transient transfection approaches such as mRNA reprogramming. The bottom line from this paper and the engineered factor papers (see the previous AlleleBlog article under “iPS and other Stem Cells”) is that iPSC reprogramming is only going to get faster, which means that hopefully in the near future creating iPSCs will become a routine experiment as easy as a simple transfection.

Wang, W., J. Yang, et al. (2011). “Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1.” Proc Natl Acad Sci U S A.

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Solving the world’s problems with new biotechnology

The ability to isolate, create, synthesize, or artificially evolve living organisms towards desirable phenotypes may be increasingly important for solving many of the problems the world is facing. Such problems may include creating renewable energy using biowaste, finding biocontrol products that kill food-spoiling fungi “organically”, or assaying pathogens in the field using synthetic biological detection systems. With the arrival of synthetic biology, “it is possible to design and assemble chromosomes, genes and gene pathways, and even whole genomes”, according to the J. Craig Venter Institute. That is, if you know which genes or gene pathways you would need to put into the synthetic genome that would lead to the desired traits. So far, most published synthetic biology work involves bringing in transcription factors from a non-host source to set up an artificial network like circadian oscillators, showing that it can be done and it is interesting.

Through the process of evolution biological systems aptly self-engineer favorable traits in order to survive, but these changes require millions of years to manifest. However, there are quicker adaptations to environmental cues, such as developing antibiotic resistance, which can be achieved through a small number of mutations in hundreds or even dozens of generations. The question is how to harness this kind of adaptation for new strains that can be used as products with defined purposes? As a first requirement, you must have an assay for identifying the wanted mutants or method for augmenting their subpopulation, which is not necessarily easy and normally takes some clever designs to establish. Since evolutionary success in nature results from continuous “rounds” of gene mutagenesis, expression and selection, an evolution in the lab should ideally proceed with continuity. Previously, each round of mutation and selection takes a few days to complete. Recently, Esvelt et al. in David Liu’s lab at Harvard demonstrated one way of doing in vitro continuous evolution, by creating a lagoon of mixed E. coli and phages. By continuous dilution of the phage population through outflow, those phages that remain in the pool with properties that help them propagate in the host bacteria will have a better chance to regenerate and accumulate mutations towards the design of the assay [1].

AlleleBlog: http://blog.allelebiotech.com/2011/10/solving-the-world%e2%80%99s-problems-with-new-biotechnology/

Another aspect of natural evolution is that it occurs in a heterogeneous environment separated into niches of subpopulations with uneven stress levels. Although most evolutions with human intervention were conducted in a homologous population under the same stress and selection, a spatially complex environment may speed up evolution. This may not be easy to imagine, but if a mutant acquires some level of resistance to its environmental stress level and has a chance to move to join a population under higher stress, its relative fitness will likely increase. In addition, in a smaller population in the niche under higher stress, the mutant with marginally beneficial properties acquired under lower pressure can take over more quickly. This was demonstrated by Zhang et al. who showed that with a gradient of antibiotics applied to an array of microwells interconnected through tiny channels, new resistant strains can evolve in less than a day. Without the gradient, or separate the interconnected niches into discrete wells, no resistant populations could be obtained [2].

With more understandings like these and equipped with large scale gene synthesis, chromosome assembly, and deep sequencing technologies, we should see increasing numbers of human-made organisms serving special needs for food, health, energy, and the environment. Synthetic biology or artificial evolution won’t solve all the world’s problems, but if applied effectively and diligently, they can certainly help with many critical aspects as the technology “coevolves” with the environment.

[1] Kevin M. Esvelt, Jacob C. Carlson, & David R. Liu. “A system for the continuous directed evolution of biomolecules” Nature 499, 2011.
Qiucen Zhang, Guillaume Lambert, David Liao, Hyunsung Kim, Kristelle Robin, Chih-kuan Tung, Nader Pourmand, Robert H. Austin. “Acceleration of Emergence of Bacterial Antibiotic Resistance in Connected Microenvironments” Science 333, 2011.

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Originally at AlleleBlog at blog.allelebiotech.com

Storm, by 2011 Nobel Prize in Literature winner, Tomas Transtromer.

“突然，漫游者在此遇上年迈
高大的橡树――像一头石化的
长着巨角的麋鹿，面对九月的大海
那墨绿的城堡

北方的风暴。正是楸树的果子
成熟的季节。在黑暗中醒着
能听见橡树上空的星宿
在厩中跺脚”

——托马斯·特郎斯特罗姆《风暴》；李笠译《特郎斯特罗姆诗全集》

By 2011 Nobel Prize in Literature winner, Tomas Transtromer.

Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming

AlleleBlog:

Induced pluripotent stem cells (iPSCs) can be achieved through introduction of a small group of stem cell specific transcription factors. Ever since this was first demonstrated by Takahashi and Yamanaka, there have been relentless efforts for improving the efficiency of this generally inefficient process. There is also a general opinion that iPSCs are different from each other and from embryonic stem cells (ESCs) in various aspects, depending on the method of the induction. As a result, another focus of the reprogramming field has been to find ways for creating iPSCs that are as close to ESCs as possible. One of the parameters for defining stem cell status is their epigenetic characters; epigenetic changes have been demonstrated to occur during reprogramming of subsequent differentiation.

In fact, it seems that reprogramming can be largely described as a process composed of chromatin remodeling and specific transcription activation. Strong transcription activators are known to effectively recruit multiple chromatin remodeling complexes when exerting their functions. A good example is MyoD, a master transcription factor for skeletal myogenesis that can “single-handedly” switch (transdifferentiate) the fate of differentiated cells. Hirai et al. speculated that since MyoD is such a strong transcription factor, it may be able to increase chromatin accessibility to iPS factors if fused together. When transduced on retroviral vectors, Oct-TAD (Transcription Activation Domain) of MyoD, in combination with Sox2 and Klf4, increased the number of iPSC colonies by 40-fold. Additionally, these iPSCs appeared to quickly adopt stem cell gene expression profiles, days faster than when traditional Oct4, Sox2, c-Myc, and Klf4 were used; and sometimes the levels of pluripotency genes even exceeds those seen in ESCs. Amazingly, when using the fusion assisted method some colonies are formed without the help of feeder cells, a requirement of ESCs grown in similar medium. Does this mean that these iPSCs can even be more “stem-like” than embryonic stem cells?

Like MyoD, VP16, also widely known for its strong transcription activation domain, when fused to iPS factors, was shown to exhibit a similar stimulation effect on reprogramming. Although the details of the fusion arrangements and specificity appear to differ between MyoD and VP16, the fact that two research groups could achieve similar results using comparable strategies provides a good argument that other labs should at least consider this method when creating mouse or human iPSCs. Previously in our blog we have discussed using iPS factor mRNAs, a method originally developed by Warren et al., for substantially shortening the time required for reprogramming and making it more robust across cell types and media conditions. If the new TAD-fusion factors are used also in the mRNA format, then the protocol might be further shortened and simplified. If successful, this non-integrating approach could become a dominant method in the field, even making competitive non-integrating method such as Sendai and plasmid-based miRNA irrelevant.

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Big Potential in Using Protozoans for Producing Mammalian Proteins

Recombinant protein expression is critical for functionally studying proteins, preparing antigens, providing tissue culture growth supplement, and producing certain therapeutic compounds. Like many molecular biology labs, we have used several heterologous protein expression systems over the last decade including E. coli, yeasts, insect cells and mammalian cells from various species. It is widely accepted that these systems present increasing functional relevance from bacteria to mammalian cells, with accompanying increase in difficulty and cost. The benefits of using cells from higher species are often reflected in post-translational modifications (PTMs), such as glycosylation, phosphorylation, etc.

There is yet another system that could be easy to handle while maintaining mammalian-like PTMs–parasitic protozoan Leishmania tarentolae. L. tarenolae is a unicellular organism, its host is lizard. Even though it’s a vertebrate parasite, this species poses no risk to humans. Amazingly, L. tarenolae individuals can be grown on agar plates for clonal selection or in simple liquid media like E. coli. Their optimal growth temperature is 27C, and they do not require shaking; thus they are suitable for growth in insect cell incubators or even at room temperature. The most important advantage of this system is that oligosaccharide structures of proteins produced in this organism resemble those of mammalian cells much more closely than even insect cells, i. e. the N-glycosylation profile can be basically identical to a biantennary fully galactosylated Man3GlcNAc2core-a-1,6-fucosylated structure found in mammalian cells.

IFrom our first-hand experience, the handling of this species is extremely convenient. While we heavily promote the baculovirus expression system (BVES) for most of our custom protein production projects (we carried out one NIH project for producing human glycosylated cancer antigen proteins using a modified BVES recently), we now believe that there is a good chance that many of the proteins we have been producing could be produced in the protozoan system with potentially better efficiency.

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About 50 Papers Cited the Use of GFP-Trap Camelid Antibody So Far in 2011

With their ability to quantitatively pulldown GFP-tagged proteins, GFP-Trap (or RFP-Trap for DsRed-derived fluorescent proteins) beads have gained ground in becoming the reagent of choice for immuno-coprecipitation. The complexes isolated from GFP-Trap agarose or magnetic beads can be easily analyzed without interference from light or heavy IgG chains typically present after monoclonal or polyclonal antibody precipitation. Since the market launch of GFP-Trap, in each of the past 3 years, the number of publications citing GFP-Trap more has than doubled and there is no sign of that rate slowing down any time soon.

In 2011 alone, 48 research groups have published their results with data generated through use of GFP-Trap (not including other related products such as GFP-Booster, GFP-MultiTrap). Research topics in these recent publications include identification of domains of the zinc finger protein 638 (ZNF638) that interacts with C/EBP? when promoting adipocyte differentiation [1]; identification of phosphorylation site on Cdc42-associated kinase (Ack) by LC-MS/MS after immunoprecipitation [2]; and analysis of the activities of myosin heavy-chain kinases (MHCKs) in wild-type vs Htt mutant Dictyostelium discoideum, a cellular model for studying the Huntingon disease [3].

The use of GFP-Trap beads is a simple bind-wash-elute procedure that involves just one antibody already immobilized on either agarose or magnetic beads. Camelid antibodies, especially their VHH single domain fragments such as those used in GFP-Trap or RFP-Trap, are very stable (they can be shipped and temporarily stored at room temperature). The consistency of performance is very high; as a matter of fact, this line of products requires the lowest amount of technical support among all of our products. If you are still using tags like FLAG, V5, HA, etc., you should consider trying GFP as both a fluorescence and co-IP tag in your future experiments for obtaining results you previously could not obtain.

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Blog References:
[1] Meruvu, S. et al. “Regulation of Adipocyte Differentiation by the Zinc Finger Protein ZNF638″ JBC 2011
[2] Shen, H. et al. “Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin” Molecular Biology of the Cell 2011
[3] Wang, Y. et al. “Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation” Molecular Biology of the Cell 2011

北岛以一句“卑鄙是卑鄙者的通行证，高尚是高尚者的墓志铭”而著名

"今天他当然更清楚，在一个不要墓志铭的世界，卑鄙者的通行证是抢破头的。所以他也迫不及待地实践自己的名言去了。" 曹长青：北岛回国跟宣传部长唱红诗


 可叹，又一个张艺谋。其实为求名逐利也不一定是这些“精英”表现人格低下的原因，很多时候他们只是看不见或者没时间去看世上的邪恶与不公正。

这两年我有机会与之交谈世事的北大同学中，除一人有共鸣，一人观点相反并激烈辩论外，多数的反应是问我是否CND看多了，或者是不是FLG。与我辩论者的言辞基本符合网上所谓“五毛”的观点，只是更自信一些，让我觉得很多被人在网上贴“五毛”标签的人可能不是拿钱发贴，而确实相信那些别人不相信他们会相信的观点。不管你信不信，反正我相信我的在美国读过博士，现在美国工作，多半是美国公民的大学同学不是“五毛”。人的观点的差距可以是巨大的，即使从表面上看来我们有着相似的背景。

另一同学曾道:"我已经可以退休了，你还在奋斗。。。“ Yeah right! 就象你一辈子的求学、训练、竞争、工作都只是为了尽快走到退休那一步。如果只为自己或自己的孩子们想，要退休当然可以，在自己的庄园上在种些果树养些动物，钓鱼打猎都是乐趣，或者写一些书，文学、历史、科学史亦无不可。但正像Richard Gere  在 Primo Fear 里说的”If you can still play the game (be a trial lawyer), why become a referee (court judge)?" I am still in my prime for science, and fight for what I believe is right.

Large Intergenic Non-Coding RNAs (lincRNAs) Play Large Roles in Pluripotency

AlleleNews

here may be only 21,000 distinct proteins encoded in a human genome but there are also thousands of transcripts that do not encode any proteins in human cells.  These so called lincRNAs have been a subject of speculations in terms of functions and mechanisms underlying the functions.  In a recent Nature Article, Eric Lander’s group at Harvard and their colleagues have shown by RNAi that ES-cell expressed lincRNAs function in keeping pluripotency.  They seem to maintain expression of pluripotency-related factors such as Oct4 and Nanog, while at the same time lincRNA genes appear to be targets of these factors’ transcriptional control.

As shown by immuneprecipitation, lincRNAs bind to chromatin proteins such as those of the polycomb family and influence chromatin structures and gene expression, at least as part of their functional mechanism.  It would be interesting if the RNAi-against-lincRNAs experiments were complemented by transgene expression, something we might see in follow-up mechanism studies on lincRNAs.  Pulldown of lincRNAs instead of the polycomb proteins could be helpful in identifying their unknown partners. 

It will also be interesting to see how lincRNA profiles correlate with iPSCs created by different methods or from different sources.  It may be worth it to even try and use lincRNAs as reprogramming reagents as mi-RNA302, miRNA369, etc.

Guttman et al.  2011, Nature

http://news.allelebiotech.com/index.php?mod=article&cat=iPSStemCells&article=1841 

Methods of iPSC Generation Update

AlleleBlog

Induced pluripotent stem cells can be directly generated from adult cell cultures through the introduction of a group of factors, e.g. Oct4, Sox2, Klf4, and c-Myc (the Yamanaka factors) [Takahashi and Yamanaka, 2006]. Additional factors such as Nanog and Lin28 can either substitute some of the Yamanaka factors or supplement them for higher reprogramming efficiency [Yu et al. 2007].

The original pluripotent stem cells induction methods involved retrovirus or lentivirus that would leave foot-print in the host genome, a concern for clinical use of iPSCs. Several groups have tried to create iPSCs without integrating viruses, such as using small molecules, directly delivering proteins instead of cDNAs, viruses with RNA genomes, episomal systems, or removable elements such as PiggyBac or Sleeping Beauty transposons. From the literature and our first-hand experience in the iPS market, none of these methods has become a widely applicable tool, mostly due to impractically low reprogramming efficiency.

In addition to low efficiency, RNA viruses, such as the sendai virus, are still viruses and have virus-associated risks. Episomal plasmids or removable transposons still involve DNA, so the possibility of genomic integration by recombination remains. In case of some transposons such as PiggyBac, there is an additional question about the degree of removal – whether it is certain that all integrated transposons, often inserted within genes, are deleted; in case of transposons similar to Sleeping Beauty, the small footprints they leave behind may post a concern.

The method of choice for generating zero-footprint iPSCs should clearly be RNA-based without the involvement of virus. Luigi Warren and his former colleagues at Harvard demonstrated that by using in vitro transcribed iPS factor mRNAs with modified CTP and UTP, and 5’-cap can effectively reprogram a number of different human as well as mouse cells. The efficiency even exceeds those by using retrovirus or lentivirus by 10 to 100 fold. Furthermore, the RiPSCs created with mRNAs appear to be closer to hESCs as shown by expression profiling.

Very recently, a few miRNAs that have high expression levels in stem cells were shown to be able to reprogram mouse and human somatic cells when expressed together from a lentivirus [Anokye-Danso et al. 2011]. while that work used lentivirus, thus not directly applicable to the current project, Miyoshi et al. later showed that by using synthesized mature miRNA (overlapping but not the same set of miRNAs as used by Anokye-Danso et al.) reprogramming cold be achieved without viral infection. We believe that this is a promising method and would like to pursue it further and to find out whether these mi-iPSCs relate to hESCs as closely as R-iPSCs. Because transfecting synthetic miRNAs “does operate at considerably lower efficiency” in terms of iPSC creation [Miyoshi et al. 2011],alternatiuve protocols may include transfecting the iPS factor mRNAs together with various miRNAs at different doses and frequencies.

New Product of the Week: EF1a-lacZ lentivirus particles, for expressing nuclear lacZ in virtually any human or mouse cells.

This week save 25% on photoconvertible fluorescent protein mClavGR2 cloning plasmids. Email oligo@allelebiotech.com with code FPBLOG0831.

Blog References: Warren, L. et al. “Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA” 2010 Cell Stem Cell 7(5): 618-30

Anokye-Danso, F. et al. “Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency” 2011 Cell Stem Cell 8(4): 376-88
Miyoshi, N. et al. “Reprogramming of mouse and human cells to pluripotency using mature microRNAs” 2011 Cell Stem Cell 8(6): 633-8
Kim, H. et al. “miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells” 2011 Cell Stem Cell 8(6): 695-706
Takahashi, K. and Yamanaka, S. “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” 2006 Cell 126(4): 663-76
Yu, J. et al. “Induced pluripotent stem cell lines derived from human somatic cells” 2007 Science 318(5858): 1917-20

让中国男人羞愧的南国烈女——渺小(实名梁海怡)

注：本文来自网络全文刊登
  
     （一）：心中的隐痛
  
    渺小失踪六个多月了
    渺小是一位普通的网民。同时，她又是半年多来最令我牵肠挂肚的美丽女子……
    渺小是今年初于哈尔滨人民广场失踪的——二月二十日的上午，她孤身一人前往哈尔滨人民广场向行人派发传单，并发表强烈要求实行宪政民主的激情演说……结果便可想而知了。
    “渺小”真名叫梁海 怡。广州市从化人。多年前，“千里姻缘网线牵”，网恋，让这位来自南国——珠江三角洲的“靓女”，与北国冰城哈尔滨的一位英俊儒雅的大学教师于先生走上红地毯。后来，他们有了一位可爱的小男孩。
渺小并非我的亲戚，本人与她没有发生过浪漫悱恻的爱情故事。半年来，她之所以最令我“牵肠挂肚”，更非咱这位小老头暗恋上这位年仅三十有五的美丽少妇，而是她“有之，请自海怡始”的殉道精神和“我不入地狱谁入地狱”的决绝（注1）。

    渺 小失踪半年多了，每想起她，心中便隐隐作痛和愧疚不安……为何会“隐隐作痛”？原因是：同为“受难的耶稣”，有些人却备受天下舆论关注，广受社会各界的舆 论声援和经济支持，有人甚至因受难而改变了人生命运，成为名利双收的英雄……而更多人则成了饱吃“哑巴亏”的殉道者——蒙难之后，不只个人饱受精神肉体摧 残，而家人也因此饱受精神惊吓的同时，生活因此陷入水深火热之中——我所知道的曾建余、唐荆 陵、姚 立 法……等等等等同胞就是如此。
    ——这，就是现实中国的悲哀：由于严酷现实环境所致，所有追求“人民共和”理想者人人皆是散沙一粒。若遭遇不测，且在社会上没有一定知名度，结果往往是吃尽黄莲苦也不为人所知！
    每想至此，心中岂能不“隐隐作痛”？
    就 比如渺小吧，每想到她目前的苦难和处境，怎不让人有揪心之痛？——她只是一位极小范围内被人所知的小网民，远远没有老艾和老冉的名气。甚至没有唐荆陵和姚 立法的名气。所以，她的壮举只为极少数QQ群中网友所知。她“失踪”后，也只有她QQ群内的网友经常发出“寻人启事”。我曾经打通她爱人的手机，然而，要 么是关机，要么是支支吾吾很快挂掉电话……
    一想起渺小目前的处境，心中怎不“愧疚不安”？——身为七尺男儿，却没有“有之，请自李悔之 始”的殉道精神和“我不入地狱谁入地狱的决绝；身为同志，半年来，除了打过几次电话关注之外，没有对受难的“渺小”施之援手。甚至没有写过一篇文章对其安 危作出呼吁。尽管我屡次奋笔疾书，只是囿于现实环境最终放弃了——写了也极难发表，岂不白费一番心血？
  
    （二）：绝非一时心血来潮之举
  
    在万马齐喑的现实中国，渺小二月二十日之壮举，用“力拨山兮气盖世”来形容也不为过——女中伟丈夫梁海 怡在偌大的人民广场孤身振臂一呼，神州数亿男人瞬间皆成侏儒！
    如此一位年纪年年的弱女子，竟作出如此惊世之举，精神之源、力量之源来自何方？是否一时心血来潮之举？……相信不少人都会对这个问题抱有不小的疑问。
    渺小二月二十日的壮举令人敬佩，但如果没有去年冬哈尔滨一次刻骨铭心的聚会，我亦会怀疑渺小是否“一时心血来潮之举”。
    事 情得从本人去年的东北行说起——应黑龙江友人杜兄之邀，我于去年十一月底到黑龙江大庆访友。家在哈尔滨、从未见过面的老网友渺小得知我到了大庆，在QQ上 对我说：“李老师，您可是咱的‘娘家人’，我们一家人得知您来到黑龙江都十分高兴，无论如何要到哈尔滨一趟啊。……”我当时愉快地答应了渺小的邀请。第三 天，杜兄亲自开车与我一起前往哈尔滨……
    一路走高速公路，大庆到哈尔滨只用了两小时车程。下车后，我与杜兄见到了典型南国女子身材的渺小和她身材高挑、风度儒雅的丈夫于先生……随即，夫妇俩领我和杜兄来到一间装潢考研的韩国餐馆。
    在 韩国餐馆吃的是铁板牛肉。渺小和她丈夫于先生的豪爽热情令人感动。尤其是渺小，在长达四个多小时的用餐过程中，又是举杯，又是切肉、煎烤，热情殷勤让人有 些难为情……然而，随即而来的，却是几个小时的尴尬——虽然事先我与杜兄达成这样的“共识”：席间只叙乡情友情，不谈“政治”。然而，“物以类聚，人以群 分”，客套话没说几句，政治话题便成了整个聚会的主题。
    令我没想到的是：纤弱文静的渺小，竟比网上所接触、了解的渺小要壮怀激烈得多。尤 其是她的许多激烈言论，不但让我和杜兄深感意外，更让一旁的他丈夫于先生深为担忧——他先是陪着笑脸劝阻渺小，后又转为委婉的批评。没想到的是，于先生的 劝阻和批评惹得渺小毫不留情的嘲讽和怒斥，当着我和杜兄的面痛斥丈夫是“犬儒”，是“做惯了奴才的小人”……
    渺小的“河东狮吼”，让我与杜兄尴尬异常
    。她的丈夫于先生却不恼不怒，自始至终脸带微笑。为了不让我们产生误会，他特地解释道：
    “海怡很有抱负和血性，很有正义感。这一点应当肯定。但我们不能成为一对‘革命夫妻’，她没有工作单位还好，如果我也跟她一样，肯定被开除。我可要养家糊口啊。”
    于先生的话让我和杜兄深深点头。然而，渺小却不依不饶厉声痛斥丈夫道：
    “于××，我鄙视你这种瞻前顾后的软骨头。中国有血性的男人都死绝了！不自由，毋宁死！一河之隔的韩国人能做到的，为什么中国人做不到？这就是中国人的无耻——都想让别人去牺牲，自己坐享成果！中国如果要靠你们这帮软骨头男人，再过一千年也会做奴才！”
    渺小这番毫无顾忌，毫不留情面的话，让我和杜兄脸上红一阵白一阵。幸好我俩都是曾经沧海之人，心中再咋的，表面功夫还是到家的——仍然微笑面对渺小和于先生夫妇。
    于先生可能感到渺小太过了，于是对渺小打哈哈、话中带刺回嘲道：“老婆大人，我承认我是奴才，是犬儒，是软骨头…但你还不是只会在网上闹革命？”
    于先生的话让渺小更加怒不可歇，她点着于先生的鼻尖痛斥道：“于××，你走着瞧吧，我要让你领教一下梁海怡究竟是什么人！”……
    午 宴在令人难堪的气氛中结束了。分别时虽然彼此热情握手话别，但我内心却笼罩着一片阴云：渺小的激烈既使我难堪，更令我深深忧虑……不过，我却并未将渺小 “我要让你领教一下梁海 怡究竟是什么人”这番话放在心上。三个多月后渺小在哈尔滨人民广场振臂一呼，我才痛省自己是“以小人之心度君子之腹”了。
    这位“君子”就是南国烈女梁海怡！
    渺小，你绝不“渺小”！在你的壮举面前，数亿中国男人才立马变得渺小不堪！
  
    2011年8月25日于云南大理初稿（作者李悔之）
  
    （注 1）“有之，请自海怡始”： 1898年9月21日，慈禧太后就发动政变，囚禁光绪皇帝并开始大肆搜捕和屠杀维新派人物。谭嗣同当时拒绝了别人请他逃走的劝告（康有为经上海逃往香港， 梁启超经天津逃往日本），决心一死，愿以身殉法来唤醒和警策国人。他说：“各国变法，无不从流血而成，今中国未闻有因变法而流血者，此国之所以不昌也。有 之，请自嗣同始。”

林昭《秋声辞》

冯士彦: 【祭园守园人】今天，辛亥百年的秋瑾就义日。特在这个日子，首发冯士彦先生受托成稿的《林昭<秋声辞>全注释》——以为林昭、秋瑾、辛亥百年共同的纪念。

林昭十四万言书附录之二

       秋声辞 （并序）

    在狱三秋，侘傺（1）长恨；秋心秋绪，郁作秋声。即用鉴湖女侠（2）断句为韵，并作辘轳体（3）以敷陈其意。有愿补石（4），不避续貂（5），回环往复，声气尚应。后生其再来人欤？抑前贤馀烈（6）之荫也！哀时明志，未辨今昔，成仁取义（7），誓继踵武（8）。        

                              一九六三年十月林昭自志

秋风秋雨愁煞人，凭对遥天吊荆榛（9）。狐鼠（10）纵横山岳老，脂膏滴沥（11）稻粱贫。

为悲寂寞求同气，敢避艰难惜一身。夜夜肠回寒蛩（12）泣，丹心未忍逐青磷（13）！

劫里芳华不成春，秋风秋雨愁煞人！青衫泪浥朱颜悴，碧血花催白发新。

决死精卫（14）战浩荡，伤心子规（15）哭沉沦。齐家报国犹虚说，愧负望殷父老亲。

《哀江南赋》（16）墨溶尘，抱恨楚囚（17）志未伸。霾露霾霜（18）瘦生菊，秋风秋雨愁煞人！

宁随兆庶盟朝日，岂戴独夫（19）蹑佞臣（20）。唱彻招魂（21）金铁寂，肝肠百沸湿罗巾！

忧乐苍生夙愿真，壮怀激烈（22）照天陈。吞毡（23）谁复思侯汉，蹈海（24）我终不帝秦！

赤水赤原病体国，秋风秋雨愁煞人！此身定化干城剑（25），贯日横空泣鬼神。

浩歌慷慨夺江津，最是知音第五伦（26）。翰墨请缨（27）彰素志，榛苓（28）补石证前因。

凌霜劲节千钧义，挥刃英谋一念仁。莫笑猖狂乔作态（29），秋风秋雨愁煞人。

注释

（1）、侘傺（chàchì）：失意，抑郁的样子。

（2）、鉴湖女侠：清末民主革命烈士秋瑾（1875～1907），字璇卿，别号竞雄，绍兴人，又称鉴湖女侠。她的《绝命词》：“秋雨秋风愁煞人！”当时报刊所载皆如此，惟灿芝本作“秋风秋雨愁煞人”，后从之。此断句，系清吏逼供，秋瑾不语，写此七字作答。古歌有“秋风萧萧愁杀人，出亦愁，入亦愁”句。秋瑾留下诗词125首，近百分之四十直抒悲愤的愁绪，在愁云中闪露赤诚、挚爱。何谓“愁”？愁，自以为合理而迫切的物质或精神诉求，想实现而实现不了的那种心理状态。愁，可望不可及的忧思，一种观念被现实否定的剧烈痛苦。愁，不是冰隙弥散的冷雾，是骨髓蒸腾的火焰。秋瑾愁欧风美雨难以飘洒故国神州，愁同胞之不觉醒，愁知音之稀少，愁离情别绪之难以释怀，愁壮志之终于不酬！秋瑾的愁，比天大，比一江春水深，无舟车载得起，仿佛集古往今来之愁于她的一身。林昭越发深化了秋瑾的愁，她“但觉寒冷彻骨沉痛欲绝而悲愤无已”！

（3）、辘轳体：杂诗诗名。五言或七言律诗五首，将第一首起韵的第一句全句，分别置于其他四首押韵的四个位置中，在第二首为第二句，第三首为第四句，第四首为第六句，第五首为末句。五首诗的韵节，如辘轳旋转而下，故名。

（4）、补石：补天窟窿的五色石。神话中创造人类的始祖女娲氏，稳定大地，炼五彩石补天塌裂的孔洞，治平洪水，杀死猛兽，使人民得以安居。秋瑾诗“衔泥有愿誓填海，炼石无才莫补天”；“炼石无方乞女娲，白驹过隙感韶华”，足见林昭熟知和参证了秋瑾的诗。

（5）、续貂：狗尾续貂。貂尾不足，用狗尾代替，比喻用差的东西续接在好的东西后面，这里是林昭的自谦。

（6）、余烈：先人留下的功业，见贾谊《过秦论》。（另有解为祸患，非此。）

（7）、成仁取义：成仁，牺牲自己，成就仁德；取义，舍弃生命，取得正义。指为正义献出生命。

（8）、踵武：脚迹，跟随前人的脚迹走，喻继承前人的事业。

（9）、荆榛（zh俄n）：荆棘和榛莽，比喻幽暗僻远，处境险恶。

（10）、狐鼠：城狐社鼠，钻进城墙里的狐狸，躲进社稷台里的老鼠，比喻依势为奸的人，因顾忌而难以消灭他们。

（11）、脂膏滴沥：脂膏，生物体中的油脂，喻人民用血汗挣来的财富。滴沥，水稀疏下漏，也形容水滴声。民脂民膏被无节制地挥霍。

（12）、寒蛩（qióng）：蛩，蟋蟀。天寒蟋蟀噤声，秋寒砭骨，蟋蟀即将不再鸣叫。

（13）、青磷：忽隐忽现的青色野火，俗称“鬼火”。

（14）、精卫：神话中鸟名，又称“冤禽”。相传炎帝之女，游东海淹死，化为精卫，久久衔西山木石填东海。陶渊明诗“精卫衔微木，将以填沧海”，咏其事。

（15）、子规，杜鹃鸟，古人以其鸣声哀凄，以杜鹃啼血形容。杜甫诗句：“两边山木合，终日子规啼。”李白诗句：“又闻子规啼夜月，愁空山。”

（16）、《哀江南赋》：北周庾信赋篇名。《哀江南赋》感慨身世，伤悼梁王朝覆亡，对梁朝政治腐败，倒行逆施，颇多暴露，情调危苦悲哀。

（17）、楚囚：楚人之被俘者，囚徒。泛指处境窘迫的人。林昭自谓“奉着十字架的自由志士”，她这个囚徒，是相信上帝的基督徒。

（18）、霾露霾霜：霾（mái），大气混浊，系悬浮细微烟尘所造成，能见度低，天空微黄、桔红色。霜、露洁白，严重污染，故称之。

（19）、独夫：原指暴君商纣，泛指残暴无道，众叛亲离的独裁者。

（20）、佞（nìng）臣：善以花言巧语谄媚君王的臣子。

（21）、招魂：古丧礼，人初死，招其灵魂回家。又《楚辞》篇名，屈原作品，招回忠魂。

（22）、壮怀激烈：豪壮的胸怀，激烈的情绪，形容昂扬的情志。岳飞词“抬望眼，仰天长啸，壮怀激烈”，表现爱国激情，英雄气概。林昭恪守的信念是：“祖国至上，自由万岁；公义永存，青春必胜！”

（23）、吞毡：苏武出使，被匈奴扣留19年，不降，饥渴时啮雪吞毡（吃雪解渴，嚼毡充饥），维持生命，历尽艰危，终回故土。

（24）、蹈海：战国时，齐人鲁仲连，又名鲁连、鲁仲子，善谋策，周游列国，为人排难解纷，是一个“志愿者”。他宁可跳海而死，也不尊秦为帝，曾说以利害，劝阻赵国平原君向秦国屈服。

（25）、干城剑：干，盾牌；干和城都用以防御。干城剑，喻捍卫人民权益的武器。

（26）、五伦：也称“五常”。封建宗法社会把君臣、父子、夫妇、兄弟、朋友，叫“五伦”。第五伦即朋友关系。人和人之间的正常关系和应当遵守的行为准则，朋友间讲“诚信”。

（27）、翰墨请缨：翰，毛笔；翰墨，笔墨、文辞，也指书法、绘画。缨，绳子、革带；请缨，请求接受自己为国效命。信奉民族意识、基督精神，据此递上请战书，决心抛头颅。

（28）、榛（zhēn）苓：榛，木名；苓，草名，卷耳。《诗经·邶风·简兮》：“山有榛，隰（xi）有苓，云谁之思，西方美人。”（高山有榛树，洼地甘草长，我心里想着谁呀？那西方的美男儿。）谓榛木、苓草，坚贞守位，各得其所；自己身处囚牢，想做山野的榛苓而不得。

（29）、乔作态：乔，做作，假装。作态，作秀，装模作样。乔作态，这里指狱吏、审讯员对林昭的诬蔑和斥责之词。

李承鹏：墙里扔出的一根骨头

前天在微博上看到，把国家和政府当父母，为了不给父母添乱就从不在两会上投反对票的倪萍获得“共和国脊梁”的殊荣，出于对脊梁这根很敏感的骨头理解的不同，大家议论纷纷，我说：倪萍确实是共和国的脊梁，只是患了颈椎病。

我 觉得把国家和政府当父母，是一个很欠的说法。通俗来说国家就是保镳政府就是物管，是纳税人聘来的，可大家显然没跟小区保安和物业喊过父母吧。其次，为不给 保安和物业添乱，哪怕它们做得再差，也深情地不投反对票……这样的逻辑很差劲，除了会导致中国这个小区下水道持续内涝，一定会导致“你到底是为党说话还是 为老百姓说话”这样的励志名言及墓志铭言，教育出更多的软骨头而不是脊梁。所以我反对从不投反对票的倪萍是共和国脊梁，我不认为这么说有什么不对，不为老 百姓说话的代表即使是脊梁也是颈椎病的7，而不是迎风招展的1。

好玩的是出现了很多水军帖子，除了每回都会整编制出现的“大姐好人哪” “谦虚，低调，实在”“大姐像一阵春风化解了人间”知音体（每回都发这些，雇的水军太没创意了），这次增派了很多的“不给国家添乱就是好脊梁，操李全家” “李就是想搞乱中国，操他全家”“我们这个世界，大姐无疑是建设者，搅和者如大眼、奸详、含含之流利用当下的民主宽容无时无刻不在大呼小叫，其实是在捞取 个人的好处!历史上的汉奸大多数都是出自于这种类型的搅和者!”……这时我不笑是很难的，因为大量ID都是刚注册的或只有几篇散文美容帖的，最好玩的是 “大眼明显是故意的，他有私心，想引起领导的注意，他想当人大代表”。

我对水军无所谓，虽然派水军已是笔仗中很土鳖的一招了，可水军多 一点，水涨船高可力保我国航母不至搁浅。操我全家及让我道歉也无所谓，我天天都在向共和国道歉，我家那条柴狗正在发情期也正急着找同类，谢谢舍身帮忙。让 我觉得要写点什么的是，看到倪萍更新了一封给我的信：《李承鹏，你的微博我看了》——

最近我获了两个奖。一个是“全国中青年德艺双馨文 艺工作者”奖，获奖那天组委会安排我接受媒体采访，我婉拒了，我说：“有德有艺是一个文艺工作者应具备的基本条件，不用表扬。”另一个就是昨天你说的那个 奖，我具体获得的是纪念建党90周年•共和国脊梁系列活动“十大杰出艺术成就奖”。我的现场获奖感言是这么说的：和同时获奖的田华老师、刘兰芳老师、张继 刚他们相比，我真的不配拿这个奖，如果能退的话，这个奖我退了吧。我仅是沾了职业的光，又出名又得利的，我知道自己，我会努力的。
其实，无论你怎么说，我都能理解你。圈内我们都叫你李大眼，我还签了本《姥姥语录》托人转给你，并写了这样的话：你的几本书我都看了，散文写得真好，尤其是写张国荣，还有写你的母亲。交换吧，你也看看我的书。
最后我想说，姐姐我从来没觉得自己是脊梁，盼你能理解。

表 面看这是一篇特别谦虚厚道和温暖感动的文章，可透着一种春晚体的假，假装老百姓贴心小棉袄，实是有关部门的铁马甲，假装趴着接地气，其实在琢磨抽掉想翻墙 的梯子。你都不婉拒获德艺双馨奖，却要婉拒采访。都婉拒采访了，可还是要告诉亿万观众“有德有艺是一个文艺工作者应具备的基本条件，不用表扬”。这么裸的 假，我雷鸣般地想起雷锋做好事从来不留名，就是写在日记上。另外一个雷鸣般响起的就是倪萍获奖感言居然是要退掉共和国脊梁奖。多么熟悉的场景，就是这样， 领导每回都说“咳，我真的不想当这个官啊，可群众不答应啊”，倪萍说“哎，我是真不想领这个奖啊，可恐怕退不掉啊”。姐，你退掉它吧，我打赌，真退还是能 退掉的。

我也不认为倪萍提及的那些人配得上共和国脊梁，靠演些红色电影讲些价值观混乱的评书或是导一场耗费民资毫无实用的大型晚会就脊梁了，你说的是蚯蚓的脊梁吗。也别说德艺双馨奖了，我说过我俩唯一的分歧就在德艺双馨的定义，大家都说，苍井空听了，会含泪退役的。

其 实我觉得跟倪萍打笔战没意思，也不想绑架倪萍去反对什么。我只是觉得派水军这做法太不脊梁了，我要告诉倪萍：这个国家，因为种种原因，你我都是戏子，且不 幸是三四流的戏子，举国都被一根看不见的线牵着在演皮影。可蹩脚戏子中还是有高下之分的：知道自己在演戏的，和忘了自己在演戏的。你碰巧就属于那种经典入 戏太深的，你真相信靠煽情的眼泪就清涤了街市上的耻辱，靠略颤的尾音就共鸣出一个幸福大家庭的心声。可我要告诉你，那是你的幻觉。太多的人民上不起学找不 到工作喝着毒牛奶住着高价房，老了火化时却成得道高僧，因为烧成灰后闪现出好多三聚牌舍利子……其实你也不是看不到，开始你是装看不到，后来你就真看不到 了。你盈眶的泪，已自顾自凝化成一副美瞳隐形眼镜，看什么都叫幸福、安康、合家欢乐、举国欢腾。

倪萍是个好人，可这就是好人倪萍现象。 这个国家总有这么一群人，他自己装看不见，后来真看不见，不仅他看不见，还不许别人看得见。他从不投反对票，他唯一投的反对票就是，反对别人投反对票。因 为他凭借这个方法赚到很多，上面有大佬罩着，下面有脑残欢呼。到后来，他不再是当初那个只是有些傻气的人，而成为精明的帮凶，他其实确知自己要什么，怎么 要。动作娴熟，表情纯真，下手狠毒，倘若你发现这些技巧，他会深情地望着你说：呀，我只是一个孩子，想帮父母操点心。

问题是，这么多年，都姥姥了，还装孩子，不烦吗。

花 了一辈子演乖孩子，而且高龄之际竟演出这么清纯的一嗲，所以好人倪萍不是脊梁，其实是是伎俩，是化骨绵掌，因为这么一伎俩，高堂之上本有些心虚的父母就坦 然了：这样一群代表民意的脊梁，油价就不用降了，房价不算太高，税费还是嫌有些低哈，总之一切都是发展中必然遇到的问题嘛。还有一些小棉袄式絮叨的话，比 如教我厚道的。我觉得最厚道的是帮人民说话，而不是成为两会一枚永恒不倒的“同意牌”假肢。教我善良的，这国家的人民被善良害过几千年了，生活中哪个教你 善良的人没害过你。以及倪姐夸我散文写得好张国荣写得好，这只是中文系的基本功，不值一提，且这国家已让我没心情写散文，我写的《李可乐抗拆记》比所有的 抒情散文都好。因此交换彼此的书就不必，我俩不是同一路人，你我的区别，就是《李可乐抗拆记》和《姥姥语录》的区别，这不是两本书，是两个中国。不如你带 我去姥姥的菜市场，我带你去拆迁现场，看我俩谁先崩溃，以后你在两会上反对一次父母，我在文章里表扬一次父母。倪，敢？

最后，我对脊梁是这样看的：人体最重要的中枢神经系统，实现大脑指挥屁股。如果屁股指挥大脑，不叫脊梁，当了一辈子代表连句有担当的话都不敢说，这脊梁，顶多是野夫说的一根墙里面扔出来的骨头。

你敢保证每回墙里扔的都是骨头？我知道的一个真实故事，那人一直在等骨头，等啊深情地等，结果扔出来一砖头。