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.

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

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.