英文写作的一些经典著作

If you’ve been hanging around Scientist Sees Squirrel, you’ve surely notice that I’ve written a guidebook for scientific writers.  I’m biased, of course, but I think The Scientist’s Guide to Writing is pretty good – and if you write at all, I think reading it can help.  (Why not go buy yourself a copy?  I’ll wait.)  But if you’re serious about your writing craft, I hope The Scientist’s Guide won’t be alone on your shelf.  It isn’t alone on mine.

Here are a few books that I think could profitably keep The Scientist’s Guide to Writing company.  (UPDATED: see the Replies thread for reader suggestions!)  These aren’t guidebooks.  There are, of course, plenty of other guidebooks out there; they overlap to some extent with The Scientist’s Guide and hence are competitors of a sort*. Instead, the books I’m sharing with you today are those that are usefully different (from The Scientist’s Guide and from each other).  I’ve found each helpful in my own writing.  Few scientists are likely to read all these books, but any scientist can benefit from reading a few of them.  If you’re nerdy enough to put a writing book on your Christmas list, be assured, Santa will understand.

  • How to Write a Lot (Paul Silvia). (Deals with writing of any sort.) This is not a book about writing (the noun), but about writing (the verb) – that is, it’s concerned with the process of writing and the behaviour that lets someone do a lot of it (and oh boy, do we scientists have to do a lot of it). It’s a very short book that answers its own title with tips on time management and the psychology of productivity.  In my career I may have met two or three people who don’t need to read this book.  It’s hard to recommend it too highly.
  • On Writing: a Memoir of the Craft (Stephen King.) (Writing of any sort, plus some autobiography.) Yes, that Stephen King. King’s writing is enjoyed by many, and reviled by many more. It may not be your cup of tea, but there’s no denying he’s produced a lot of it, and that it’s never unclear – two things we’d all like to emulate in our scientific writing.  King has advice to give about both: behaviour that favours productivity, and composition that favours clarity.  Some of the latter is specific to fiction – but if you have an open mind, not nearly as much as you’d think.  The advice is interwoven with stories from King’s life.  Both are worth hearing**.  The book is conversational and easy to read (much like King’s fiction).  You might think a writer of mass-market horror would have little to say to a scientist writing for our scientific literature.  You would be wrong.

  • The Elements of Style (Strunk and White). (Writing of any sort.) This may be the most famous writing book there is. It has passionate fans and equally passionate despisers (I’m in the former camp). Its writing advice (largely on style) is simple and indispensable, and it’s delivered with wit and occasional cantankerousness.  It’s short enough to read in an hour or two, but insightful enough to read repeatedly.
  • The Sense of Style (Pinker). (Writing of any sort.) This, despite its title, is largely a book about vocabulary and grammar. Don’t panic, though; it’s not an indigestible one. Pinker’s interest is in how writers can harness understanding of vocabulary, grammar, and psychology to write clearly.  You could be forgiven for flipping quickly through Chapter 3, which diagrams sentences and dissects their syntax in more depth than you might want (although there are lessons to reward you if you stick with it).  But the remaining chapters are both useful and entertaining. Yes, entertaining – especially the final chapter, which dissects so-called “rules” of grammar that aren’t really rules, but are counterproductive.
  • Style: Lessons in Clarity and Grace (Williams and Bizup). (Writing of any sort.) Williams’ book is perhaps best thought of as a longer and less crotchety version of Strunk and White. It’s also a lot like Pinker’s book – only Pinker writes like a psychologist who’s discovered composition, while Williams writes like a scholar of composition who’s discovered psychology.  This book aims to help writers produce text that’s coherent and clear, but that also that has the ill-defined property of “grace”  (closely related to what Pinker (and Sword, below) would call “stylishness”.  We’re not used to thinking of scientific writing as potentially graceful, but we should be.  (By the way, the newest edition of this book, linked here, is ludicrously expensive.  Don’t buy it.  Any edition back to the original will be equally useful.)
  • Stylish Academic Writing (Sword). (Writing in all academic disciplines). Sword argues that (much) academic writing is horrible, even though we largely know how it ought to change.  Her goal is to make academic writing more vivid, passionate, and elegant. She gives examples of elegant writing from many disciplines, and breaks down ways that any writer can emulate them.  Some are ordinary (use less jargon); others are more exotic to the average scientific writer (opening “hooks”, or creativity and humour).  This is a short and engaging book that will be of special interest to someone who can write a basic, functional paper, but who aspires to writing one colleagues will admire.
  • Communicating Science (Gross et al.). (Writing in the sciences.) This is not a book about how to write – it’s a book about how we’ve written. Gross et al. trace the history of writing in the sciences – the history of structure, of style, of vocabulary, of grammatical choices, and much more.  It’s addressed more to the field of science studies than to practicing scientific writers, so it’s probably better grazed than devoured.  However, I think it’s very useful to know not just how scientists write, but how we’ve come to write the way we do.  It’s one way we can escape from the trap of simply modeling the last five years’ worth of papers, and thus perpetuating practices without thinking about them.  I wouldn’t dip back into this book often enough to own it, but I’m glad my library has a copy.
  • The Scientific Literature: A Guided Tour (Harmon and Gross). (Writing in the sciences.)  This follow-up to Communicating Science isn’t just more of the same.  The approach is again historical, but this book is more of a field guide crosspollinated by an anthology.  Where Communicating Science constructs figures and tables to analyze how usage has changed, A Guided Tour displays examples of figures and tables (and text) to show that change.  Just as interesting and perhaps even more useful, A Guided Tour examines the modern diversity of writing forms and styles – for instance, illustrating the use of nonstandard structures and nonstandard forms (including the humour and beauty I’ve written about elsewhere).  The examples are taken from a wide range of papers, from classics of early history to famous modern papers to the obscure but fascinating.  There’s a lot one can learn from the tour and from the tour guides.
  • The Little, Brown Handbook (Fowler and Aaron). (Writing of any sort.) This is the only boring book on this list. Boring – but crucial. It’s a reference manual to grammar, composition, and rhetoric. Do your coauthors constantly change your punctuation?  Do you have trouble keeping “which” and “that” straight?  Do you worry about when and how to use the subjunctive mood?  (Or for that matter, do you wonder what the heck the subjunctive mood is?)  All writers have issues like this at some point, and having a reference manual on your shelf is the cure.  There are several; but this one is comprehensive, well organized, and straightforward to use.

来源: https://scientistseessquirrel.wordpress.com/2016/12/08/some-other-good-books-on-scientific-writing/

Excel 函数 VLOOKUP 的使用方法

当我们使用 Excel 统计数据时,往往需要引用另一张表格中的部分数据。我们拿最常见的发工资这件事举个例子。

假设公司原先有一张记录了「工号、姓名、性别、手机号、身份证号」的员工信息表。最近需要为员工发工资了,公司又统计了一张含有「姓名、工资金额、工号、银行卡号」的工资表。我们需要做的是在「员工信息表」中加上银行卡号和工资金额信息,去掉不需要的敏感信息。

需要将两张表格的信息组合起来

然而令人头疼的是,两张表格的顺序不尽相同,无法直接将工资单的数据直接复制进员工信息表中,一一填写未免过于麻烦。遇到这种情况应该怎么办呢?

Excel 内置的 VLOOKUP 功能就是为此而生的,可以用它来实现数据的查找与引用。它就像我们去银行取钱时,银行根据我们给出的「银行卡号」,来告诉我们卡里的「余额」一样,使用起来并不麻烦。VLOOKUP 就可以实现类似的功能。

这里给出一份 📑 演示数据.xlsx(点此下载),文中的所有内容都可以在这张表格中实践。(其中的所有敏感数据均为随机生成的假数据)

VLOOKUP 是什么?

VLOOKUP 其实是一个函数,它作用是「纵向查询」,可以按列查找值,并返回另一列的值。

就像上面讲的「根据银行卡号查余额」一样,我们可以查询任意一列的数据,来得到与它对应的其他数值。所以 VLOOKUP 通常被我们用来查询数据引用数据

在实际使用 VLOOKUP 之前,需要简单了解一下它的基础语法。

许多地方都把 VLOOKUP 语法写的很复杂,包括 微软官网的 VLOOKUP 文档 也使用了比较严谨的描述,需要脑子转个弯才能明白它在讲什么:

=VLOOKUP(查阅值、包含查阅值的区域、区域中包含返回值的列号以及(可选)为近似匹配指定 TRUE 或者为精确匹配指定 FALSE)

其实,VLOOKUP 的语法很简单,翻译成一眼就能看明白的话就是——VLOOKUP 函数需要有 4 个部分:「需要查什么、查哪个区域、第几列、是否返回近似值」

这个式子中的最后一个值是选填的,0(False)是精确,1(True)是近似。

实际使用时只要记住最简单的 =VLOOKUP(查什么,区域,第几列,0)这样一个式子,就能顺利地使用 VLOOKUP 函数了。

111.png

这个式子里还有一个地方需要注意,「需要查的内容」必须是「查哪个区域」第一列。很多时候查不出数据,就是因为没有遵守这个规则。不过这个问题也是有办法可以解决的,文章下面会提到。

我们先来看一看 VLOOKUP 的基础用法。

基础用法:查询单个信息

VLOOKUP 的最基础用法实际上和搜索类似:用「工号」去查询「工号和工资对应信息」,来获得「工资」这个数值。

222.png

在这个例子中,VLOOKUP 函数的作用就是告诉电脑「我的工号,工号和工资信息,工资在第几列,是否模糊查询」 。比如我想查询工号为 13062714 的工资,工号和工资分别在 A 和 C 列,需要查询的数据在第 3 列。

于是只要把内容一一对应,就可以很容易获得一个式子——

=VLOOKUP(13062714,A:C,3,0)
=VLOOKUP(工号,工号和工资在 A 到 C 列,工资在第 3 列,使用精确匹配)

Excel 就能通过这个式子明白你想做的事情,把需要的工资信息显示出来。

常见用法:批量引用数值

有人可能会问:「Excel 里已经内置了搜索功能,为什么不用搜索功能直接查姓名呢?」

因为如果只查一次姓名,用直接搜索并不麻烦。但如果需要一次处理成千上万个姓名,搜索就显得心有余而力不足,此时用 VLOOKUP 的优势就体现出来了。

333.gif

现在我们需要为每个人自动填写工资信息,只要对上面这个式子=VLOOKUP(13062714,A:C,3,0)稍加改动就可以实现。那就是把具体的工号「13062714」改成工号所在的单元格「A2」,得到=VLOOKUP(A2,A:B,2,0)这个式子,然后拖拽单元格右下角的十字标,对下列所有的内容进行自动填充,就可在瞬间完成批量查询工资信息了。

=VLOOKUP(A2,A:B,2,0)
=VLOOKUP(每个人的工号,工号和工资在 A 和 B 两列,工资在第 2 列,精确匹配)

在这个用法中,只是将一个固定的值变成了动态的值,产生的效果也从单一的值变成了动态的值。这种方法与其说是批量查询,不如说是批量引用,这样的做法在后面还会用到许多次。

组合技巧:配合 IF 实现组合判断

VLOOKUP 函数中的每一个部分都可以和其他函数组合使用。当 VLOOKUP 和 IF 组合起来时还可以有更多的用法。

比如通过 IF 来判断工资是否超过 10000 元。这个用法中,VLOOKUP 起到过滤的作用,将需要用到的数据作为过滤条件,实现组合判断。

=IF(VLOOKUP($E2,$A:$C,3,0)>10000,">10000",VLOOKUP($E2,$A:$C,3,0))
=如果(判断查询数值是否大于 10000,是则显示 >10000,否则显示数值)

通过这条式子,得出的结果是「如果超过 10000 元就显示 >10000,如果不足 10000 元就显示具体金额」。

444.png

组合技巧:更友好地显示错误数据

当我们使用 VLOOKUP 时常常会碰到一种情况:找不到符合的数值。这种情况下会默认显示为 #N/A 来表示错误。但这个字符串往往会让其他看表格的人摸不着头脑。而配合 Iserror 函数就可以让这个错误值看上去变得更友好,比如查不到某个工号时显示「查无此人」,我们可以用下面这个式子来实现。

=IF(ISERROR(VLOOKUP($E2,$A:$C,2,0)),"查无此人",VLOOKUP($E2,$A:$C,2,0))
=如果(判断查询数值是否有错,是则显示「查无此人」,否则显示数值)

通过 IF、ISERROR 和 VLOOKUP 一起使用,就可以实现对错误值的处理。

555.png

组合技巧:一次搞定多列信息

上面的技巧大多是围绕着两列函数进行的,我们可以直接使用自动填充来实现一次性填充多列。但是这样会出现一个问题,公式中的所有数值都会向右移动一列,这就可能会导致 VLOOKUP 失效或错误。如果希望一次返回多列内容,是不是只能靠复制粘贴来解决呢?

其实我们可以用 $ 和 COLUMN 来实现一个全表格通用的 VLOOKUP 函数。在式子的列数前加上一个 $ 标记。这样,需要筛选的内容、范围都不会因为向右移动而改变。

=VLOOKUP(A2,A:C,2,0) // 原式
=VLOOKUP($A2,$A:$C,2,0) // 加上 $ 来固定列

但是这样又出现了一个问题,VLOOKUP 式子中的第三个值「第几列」不会自动改变,但我们希望它根据表格内容自动变化。这里可以配合 COLUMN 函数一起使用。

COLUMN 函数的作用是「返回选中的列数」。比如 =COLUMN(D10) 中,不管单元格中的值是什么,这个式子的结果永远都是 4,因为 D 这一列就是第 4 列。如果不输入,直接使用 =COLUMN() 就会返回当前这一列的列数。

了解了 COLUMN 的用法,再对式子做一次调整,加入 COLUMN(C1) 函数。

=VLOOKUP(A2,A:C,2,0) // 原式
=VLOOKUP($A2,$A:$C,2,0) // 加上 $ 来固定列
=VLOOKUP($A2,$A:$C,COLUMN(C1),0) // 用 COLUMN 函数使得列数据自动改变
=VLOOKUP($A2,$A:$C,COLUMN(C1)-1,0) // 如果需要查询第二列数值,则需要改变列的关系,比如 -1 列
=VLOOKUP(固定查询,固定信息列,自动改变需要的列数,精确查询)

现在对右侧的一列进行自动填充,就能获得正确的数值了,有效实现了数据的自动更新。

COLUMN 函数中采用的是单元格而非具体数字,所以内容会随着自动填充而改变。这样就实现了横跨多列拖拽也能完美自动填充的效果

666.png

组合技巧:配合 IF 实现换列查询

有时候我们会碰到一种情况,表格的第一列是「工号」,第二列是「姓名」。那我想用第二列的「姓名」去查「工号」该怎么办呢?

这个问题在于,VLOOKUP 默认在第一列的数据中查询,在工号这列找姓名当然是找不到的。所以我们就要想办法把这两列调换一个位置,让 VLOOKUP 函数得以正常运行。但是公司内部的表格结构通常是统一的,不能随意调换位置。这里就要配合 IF 函数来实现换列查询。

IF 函数有一个用法可以实现调换两列的位置 IF({1,0},B:B,A:A),这个函数的作用就是把 B 列和 A 列互换一个位置。

然后再用 VLOOKUP 函数查询调换位置后的第二列,我们可以得到这样一个公式——

=VLOOKUP(E2,IF({1,0},B:B,A:A),2,0)
=VLOOKUP(查什么,调换两列,查询调换后的第 2 列,精确匹配)

不要看式子变得这么复杂,它和上面那些式子的区别也只有「查哪个区域」变成了 IF({1,0},B:B,A:A),总体的结构仍然没有改变。如果换个表格改为第三列是姓名。其实也是一样的,把 B:B 换成 C:C 就好了。

777.png

高级用法:实现多个条件查询

有时候满足条件的值有许多种,比如演示数据中有两个叫「林晓」的员工,此时我们可以通过工号+姓名的方式来实现准确的匹配。

遇到这种情况,我们可以用 & 把条件组合起来查询,也就是 E2&F2,但是情况比想象的要复杂一些,这个公式会报错,因为 VLOOKUP 函数默认只允许单个条件搜索,如果要用双重条件,就需要给出两个条件的范围。那么将范围 A:A&B:B 组合起来是否就可以了呢?但是仍然报错。

这个问题对很多人造成了困扰,实际上,这里需要用到前面提到的 IF({1,0}, , )将多列的数据组合为数组,才能进行查询。在这里应该用 =VLOOKUP(E14&F14,IF({1,0},A:A&B:B,C:C),2,0)

=VLOOKUP(E2,A:C,3,0) // 单条件查询
=VLOOKUP(E2&F2,A:C,3,0) // 错误示范 1
=VLOOKUP(E2&F2,A:A&B:B,3,0) // 错误示范 2
=VLOOKUP(E2&F2,IF({1,0},A:A&B:B,C:C),2,0) // 将多列的数据组合为数组
=VLOOKUP(两个查询条件,需要查询的内容组成数组,查询的列数,精确查询)

当我们使用到数组进行查询时,完成编辑后需要按下 Ctrl + Shift + Enter 才能使其生效。

888.png

实例讲解:引用另一张的表格信息

有了上面这些技巧,再回到开头提到的问题,是不是可以很快解决了?

当你输入 =VLOOKUP 函数后,只要直接打开另一张表格,就可以选中其中的数据了。Excel 会自动帮我们把引用的数据转换为可识别的信息并加入到公式中。

我们需要查找的信息是「工号」这类具有唯一性的信息,所以利用「常见方法:批量查询信息」中的内容可以轻松搞定。银行卡号的批量填充。

=VLOOKUP(A2,工资单信息!C:D,2,0) // 银行卡号

在示例表格中,「工资单信息」的排序方式是姓名、工资金额、工号、银行卡号。工资金额在工号之前,利用「组合技巧:配合 IF 实现换列查询」中的内容,将两列信息进行调换,即可实现工资金额的自动填充。

=VLOOKUP(A2,IF({1,0},工资单信息!C:C,工资单信息!B:B),2,0) // 工资金额

999.png

注意事项:VLOOKUP 是动态的

VLOOKUP 函数的最基本作用之一是「引用」,所以得到的值是动态改变的。当你在一张表格中使用 VLOOKUP 时,如果源数据发生了变动,VLOOKUP 函数查询到的值也会跟着变动。

VLOOKUP 还支持跨工作表、跨文件引用数值,这个功能方便了使用,但万一数据源文档被删除,引用的数据也会消失。为了防止这种情况出现,可以在使用 VLOOKUP 获取数值之后,再来一步「复制 – 粘贴为值」来格式化数据,这样就不会让引用的数据消失。

10000.png

除了上面这些技巧之外,VLOOKUP 还有一个同胞兄弟:HLOOKUP。与 VLOOKUP 的纵向查询对应,HLOOKUP 可以实现横向的数据查询。

TDP-43相分离与神经退行性疾病

蛋白质内含体,包括一些错误折叠的蛋白质或者是蛋白质的碎片,在多种神经退行性疾病中都存在,例如老年痴呆(AD)、帕金森(PD)、额颞叶痴呆(FTD)、亨廷顿舞蹈症(HD)以及脊髓侧索硬化症(ALS)等神经退行性疾病的重要特征。这些错误聚集的蛋白具有高度无序的结构域(Intrinsically disordered regions, IDRs),IDRs有时候也被称作低复杂度(Low complexity, LC)结构域,IDRs是蛋白质能够进行相分离(Phase separation or phase transition)的重要标志之一。

在神经退行性疾病的病人样本中错误定位、错误折叠的蛋白中包括TAR DNA -binding protein 43 (TDP-43),TDP-43是ALS中运动神经元的神经退行性病变中会异常的蛋白质聚集物,是ALS以及FTD重要病理学标记。TDP-43具有一段IDRs,这种高度无序的结构域的存在给了科学家们一个提示,那就是TDP-43的突变而造成的神经退行性疾病机制是由于相分离。

近日,Neuron上背靠背发表了两篇关于TDP-43的研究从不同方面对TDP-43通过相分离对细胞坏死、细胞核内TDP-43清除、核质转运以及相分离的调控进行了解释。分别是来自于宾夕法尼亚大学Don W. Clevelan研究组的Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, loss of Nuclear TDP-43, and Cell Death 以及来自于匹兹堡大学Christopher J. Donnelly研究组的RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43

Xnip2019-03-07_22-44-33.png

那么什么是相分离呢?相分离是指能够进行自我组装成的无膜细胞器,比如P颗粒、核仁、应激颗粒(Stress granules)、Cajal小体以及一系列具有能够相互融合、具有最小表面张力、与溶液进行动态物质交换并且与液体性质类似的现象(『珍藏版』Cell发布“相分离”研究指南)。

Clevelan研究组发现在不同的细胞系的生理条件下,发现定位在细胞核中的TDP-43无论是内源抗体染色的或者是外源转入表达的会形成明显的相分离(图 1A-1B),并且形成的这种小颗粒能够进行融合和分离,同时也通过荧光淬灭恢复实验证明形成的颗粒具有很好的动态动力学特性。

222.jpeg

图1 生理条件下TDP-43形成的相分离现象。 A)TDP-43在不同细胞系中免疫染色的结果,绿色颗粒即由TDP-43形成的相分离聚集的液滴;B)荧光蛋白融合TDP-43在细胞内形成的相分离的液滴。

随着细胞衰老或者是病理过程中,由于核孔复合体的减少,细胞质内TDP-43积累量变多。为了模拟这种过程,Clevelan研究组构建了删除入核序列(NLS)的TDP-43,通过药物诱导后,存在于细胞质中的TDP-43能够形成明显的相分离的液滴,并且在胞质中形成液滴能能够招募正常转入细胞核内的TDP-43(用与删除NLS的不同的荧光蛋白分别标记),这给了作者一个提示,随着细胞衰老,这种细胞质中逐渐积累的异常的TDP-43的液滴会加速细胞核中TDP-43的清除,最终引起神经退行性疾病的发生。

为了模拟细胞受到胁迫的情况,作者使用亚砷酸盐在对细胞进行诱导,亚砷酸盐诱导会产包含RNA的应激颗粒,诱导后的不同时间后发现,虽然在最初TDP-43在细胞质中形成相分离的现象与应激颗粒的相伴产生,但是诱导之后通过更长时间地观察发现TDP-43产生的液滴与应激颗粒几乎不存在共定位,并且TDP-43形成的液滴也不会被应激颗粒特异性抗体所标记。说明TDP-43在细胞质中形成的液滴不依赖于应激颗粒的产生。但是亚砷酸盐诱导TDP-43与应激颗粒共定位的液滴与不依赖于应激颗粒产生的液滴相比,动态性要差很多,光漂白后几乎不能恢复(图2 )。因此,亚砷酸盐诱导后TDP-43会在细胞质中形成凝胶态的TDP-43液滴333.jpeg

图2 通过亚砷酸盐诱导产生的TDP-43液滴具有凝胶或者是蛋白聚集物的特定,动态型更差,光漂白后几乎不能恢复。

去掉NLS后定位在细胞质中的TDP-43在亚砷酸盐诱导后,会逐渐将正常的TDP-43从细胞核中清除出核,并招募到细胞质中形成的TDP-43的液滴中,并且最终造成神经细胞存活率明显的下降。在TDP-43突变造成的神经退行性疾病中,细胞质中TDP-43的积累是由于核质运输被破坏,从而导致细胞核中的TDP-43被清除出核,并且在此过程中细胞质中形成的液滴中磷酸化的TDP-43也大量积累。

当健康的神经元细胞在经历瞬时胁迫诱导后,会引起细胞质中形成TDP-43相分离的液滴,该液滴中富集磷酸化的TDP-43,并且会影响到核质运输。进一步的胁迫诱导或者是衰老的发生后会引起TDP-43形成胶状或者蛋白质聚集颗粒,造成神经元细胞核找到呢TDP-43被完全清除,最终造成细胞坏死引发神经退行性疾病(图3)。因此,找到目前有可能会引起TDP-43错误累积和错误定位形成的因素以及伴随发生的相关事件,很有可能会降低该基因突变或者是错误积累引发的神经退行性疾病。

444.jpeg
图 3 TDP-43相分离与最终引发神经元细胞坏死模式图

而Donnelly研究组从一个不同的角度对TDP-43的相分离以及与神经退行性疾病进行了研究。首先他们利用Cry2olig,从拟南芥中得到的一个隐花色素蛋白的一个光裂酶同源区域的一个变体,能够在蓝光诱导下发生多聚反应 (图4A)。他们将Cryolig加在TDP-43全长的N端,在没有了蓝光诱导的情况下,TDP-43主要集中在细胞核里,但是当进蓝光诱导的时候会发现,TDP-43会从细胞核中逐渐被清除并在细胞质中形成蛋白的内含体(图4B),在细胞之中形成的这种蛋白聚集小体动态性不佳。对这种聚集的内含体免疫荧光染色体发现其p62(ALS的病理性特征标志物)以及磷酸化TDP-43含量很高(图5),与ALS病人的脊髓组织切片的结果不谋而合。因此作者通过在将Cry2olig与TDP-43进行融合在细胞中建立一个能够良好的拟合神经退行性疾病中TDP-43形成相分离现象的模型。

555.jpeg

图 4 TDP-43与Cry2olig形成融合蛋白的模式图(A)以及蓝光诱导后TDP-43发生错误定位,从细胞核中被清除出来,并且形成蛋白内含体的相分离的现象(B)。

5556.jpeg

图 5 ALS病人脊髓切片的p62染色以及磷酸化TDP-43染色。

在2017年Brangwynne研究组建立的一种基于Cry2WT的检测蛋白LCD或者说是IDRs是否具有驱动相分离的能力光诱导系统:Optodroplet system【1】。Donnelly研究组他们使用的Cry2olig与Cry2WT功能上基本相似,但是CryWT对蓝光更敏感,发挥作用的作用的饱和浓度更低,相对来说更不可控,因此他们只将TDP-43的LCD放入该系统中进行测试,发现该LCD能够明显的产生可逆的相分离的液滴(图 6)。

666.jpeg图6 TDP-43的LCD能够在Optodroplet系统中蓝光诱导产生可逆的相分离的现象。

但是他们将TDP-43的全长放入Optodroplet系统中之后发现,并不能产生相分离的现象,但是原本的TDP-43的全长是有相分离的能力的。因此作者对该现象进行思考,将TDP-43全长放到Optodroplet系统后不能发生相变是否是由于TDP-43全长中存在RNA-binding domain(RNA-recognition motifs,RRMs)存在起的,因为已有实验发现,当包含RRMs的区域删除后,TDP-43形成蛋白聚集小体的能力会增强。因此作者首先将该RRMs区域单独拿出来放入Optodroplet系统中后发现,该区域不能发生相分离现象(图 7)。当把具有相分离能力的LCD区域与RRM区域放在一起时也并不能引起相分离现象,而将RRM中已知的能够显著降低TDP-43的RNA结合能力的五个位点突变后,RRM-LCD能够产生明显的相分离的现象,而这五个位点的突变其实并不是完全消除TDP-43的RNA结合能力而只是降低而已。

777.jpeg

图7 TDP-43的RNA结合能力阻止TDP-43的光诱导产生的相分离能力。

由此作者对于RNA对于TDP-43形成的相分离的调节作用产生了兴趣。为了验证该想法,他们体外纯化了TDP-43的全长以及TDP-43-5FL(包含RRM五个突变),并且合成了TDP-43特异结合的RNA序列,他们发现随着加入的RNA的总量的提升,TDP-43野生型全长形成小液滴的能力明显下降,但是TDP-43-5FL对于RNA的加入没有明显的响应(图 8)

888.jpeg

图8 TDP-43的RNA结合能力阻止TDP-43相分离能力。

总的来说,Donnelly研究组在活细胞中建立了更好的光控的研究TDP-43相分离的体系,并且他们发现RNA能够调控TDP-43的内含体的形成。他们的工作还建立了对于人类神经元细胞中异常的相分离的毒性作用。未来他们将致力于研究TDP-43有神经毒性相分离的其他特性以及造成神经退行性疾病的下游过程。最后他们发现的TDP-43特异结合的RNA的策略能够抑制异常的TDP-43的相分离的形成这一点,可能会为未来研究神经退行性疾病的治疗方案提供可能的参考思路。

以上的两篇文章都发现TDP-43的形成的相分离现象与不可逆的蛋白质聚集而造成的神经退行性疾病病理性特征进行了解释,也给神经退行性疾病的治疗提供了可能的方案。未来关于相分离的研究将给予很多疾病极其致病机理给出可能的阐释,也将有助于人类对于相关疾病的药物研发、治疗途径进行研究。

本文来自:http://www.jintiankansha.me/t/JKnSywp6iR

Lifting the Curtain: a Beginners Guide to iPS Cell Culture

I think it is fair to say that most people who have experience with cell culture know that there is at least some degree of “black magic” that goes into getting a particular protocol to work. In my experience, I’ve found this to be especially the case with iPS/ hES cell culture. In this series of blog posts I hope to shed a little light on this “black magic,” to talk about what I’ve found works, and hopefully to generate a platform for others to share their secrets as well.

Even though iPS cell culture is a relatively new technology, there are already tons of protocols for culturing them—each with its own variations on the amounts of reagents to add to culture media, methods of passaging, ways of freezing down lines, and the list goes on. Clearly, there are countless variables to test if you want to optimize your culture strategy. In addition, however, I have found that not only are there variables in technique, there are also lots of differences in iPS lines, even when they are all reprogrammed from normal patients. These differences may be illuminated in ways like pluripotency tests, where one line may take exactly six weeks to form a clear teratoma while another line may not exhibit tumors until 10 to 12 weeks. This might not sound too surprising on paper, but when you have injected a couple different lines on the same day and six weeks down the road all your lines except for one have teratomas, it is easy to think that that last line just didn’t work. If you wait another couple weeks, you may be surprised to find a cage full of mice with teratomas. Also, differences in lines may become very obvious while trying to differentiate iPS cells down a particular lineage. Currently I have been working on driving cells down the hematpoietic lineage, and I’ve found that the culture conditions for differentiating one line are quite different from differentiating another line. Even variables as small as the line’s growth rate or passaging timing may be different. My point with all this is simply that you should be aware that these differences exist and to be open-minded if your experiences with one line do not translate 100% to those with another line.

So, moving on to the good stuff—how to deal with some of these variables. I’m going to give you the “Dummies” edition of what specifically I have found to work well culturing my cells.

iMEFs vs. Matrigel
There are two main ways to culture iPS cells: you can culture them on a feeder layer using irradiated mouse embryonic fibroblasts (iMEF), or you can culture them feeder free. Depending on your desired application, both methods have their benefits.

Here is the breakdown of what I have found using iMEFs.

iMEFs are really great if you are thawing a line you’ve never worked with before. They are reliable in culture for a good 10 days, which should give you enough time to see a couple small colonies form. Also, the iPS colonies formed on the iMEFs will be nice and uniformly shaped, so you will be able to clearly identify where your colonies are and where differentiation (if any) is occurring. However, there are a couple things that must be taken into account using iMEFs. First, you must use good-quality iMEFs. If they are not high quality, they will not provide the appropriate feeder layer and support that your iPS colonies need, resulting in failure to seed or improper seeding that leads to differentiation. You want your colonies to fit fairly snugly between the iMEFs so that they can stay contained and undifferentiated. However, if they are too snug (the iMEFs are plated too densely), the colonies will grow vertically and risk differentiating on account of not having the space to expand horizontally. I’ve used both homemade and purchased iMEFs and have found for my needs it is more cost effective to buy them. I get them from global stem (cat # CF-1 MEF), and it costs $24 for a vial of 2M cells. I plate them at 200k/well of a six-well plate and do this by splitting one iMEF vial over 10 six-well plate wells (ie: 1 and 2/3 plates). I’ve tried plating anywhere from 100K to 300k, and 300k was definitely way too much, but 100k was a bit too sparse for my iPS cells to seed well. Making sure they are evenly spread out over the plate is also really important, so be sure to do the “T” motion at least three times in the hood and then at least one more time in the incubator. My only comment about the homemade iMEFs is that, unless you are making them to share with many others (and therefore can take turns harvesting, irradiating, and preparing them), it’s a lot of work and may not necessarily save you that much money. The main downside to using iMEFs is that it’s a much more time-consuming process. In order to passage or seed iPS cells onto them, first you would need to gelatin-coat your plates, which will take a minimum of four hours to set. Then you can plate your iMEFs, but those need to sit overnight in order to plate properly. Ultimately, then, this means that preparing your plate needs to start one or two days before you want to plate your iPS cells on it.

Feeder free, on the other hand, is very quick to prepare, taking only one to two hours to set. The main products on the market right now for this are Matrigel (BD), CELLstart (Invitrogen), and Vitronectin XF (Stem Cell Technologies). I have only tired Matrigel, but from the descriptions of CELLstart and Vitronectin, they sound very similar. Another benefit of using one of these feeder-free systems is that they are quite a bit more streamlined and simple. The media usually comes as part of a kit, where you only have to add a couple of things (if anything) in. There is usually some sort of standardization with these systems allowing you to purchase not only your media but also a recommended passaging reagent and freezing reagent, which can be nice as well. My last comment about working feeder free is to make sure you are buying the hES-grade material. The first time I ordered Matrigel, I didn’t realize that there were differences in grade and purchased a non-ES cell-grade one. After about six days in culture, the Matrigel would degrade and my colonies would lift off the plate with the matrix and basically be completely destroyed.

Passaging
There are two main methods for passaging hES and iPS cells: using an enzyme or manually detaching the colonies. Depending on the status of your plate and colonies, one method may be more useful to you than the other. In my experience, when you have fewer than 20 colonies (per well in a six-well plate), it is much better to passage manually. This gives you much more control over what you are detaching from the plate and bringing over to your fresh plate. This should also then be passaged at a 1:1 split unless the colonies you have are pretty large. Even though there are lots of different methods and tools you can use for manual passaging, I’ve found the most effective way to do this is to just use a p200 pipette and tips. This seems to be the perfect size to allow you to score larger colonies into sections while also scraping up the smaller colonies with one scratch. I’ve tried using Pasteur pipettes with the tips curved using a Bunsen burner, but this seems to yield too blunt and irregular tips. I’ve also tried using different-sized needles to break iMEFs off the colonies and score the colonies into smaller pieces, but this often scrapes plastic off the bottom of the plate, getting pieces of plastic mixed into the colony.

If you have over 20 colonies per well, I think it is much easier to go with an enzymatic passage. If you are using a feeder layer, the quality of the colonies may be slightly worse than with manual passaging, because you are picking up the iMEFs in addition to your colonies, which can result in your colonies forming large clumps in the new plate rather than seeding nicely into the new feeder layer. For hES and iPS cell culture, the enzyme used shouldn’t break the cells into a single cell suspension (like Trypsin/EDTA); rather they should be broken into smaller clumps for optimal seeding and growth capabilities. I have used 1mg/ml collagenase type IV (Invitrogen) diluted with DMEM-F12 for passaging with a feeder layer, and Dispase (Stem Cell Technologies) also at 1mg/ml when passaging from Matrigel. Both collagenase and Dispase keep the cells in clumps. Once prepared, collagenase only stores for two weeks, so be sure to not hold on to it for any longer than that; the enzyme becomes weak and ineffective. Even if you are passaging enzymatically, I have found it very helpful to do a little colony cleaning manually beforehand. Getting rid of partially differentiated colonies, breaking up larger colonies into smaller pieces, and teasing away some iMEFS can really make a big difference in the quality of your cells.

When you are removing the cells from the plate after the enzyme treatment, a really effective method for scraping is the “car wash” method. This is done using a 5ml glass serological pipette. While tilting the culture plate slightly forward so that the media forms a pool at the bottom half of the well, you pull up the media, and while releasing the media, scrape in a zigzag pattern from the top of the well towards the bottom. By releasing the media while gently scraping, you help keep the colony-removal process gentle and the cells in bigger clumps. Once you have cleared the top half of the well, flip the plate around so that the other half is on top and repeat the process.

Antibiotic Use
Many people have very different views from mine on the use of antibiotics for hES/iPS culture. I feel very strongly about culturing antibiotic-free, and I will explain why. First of all, it allows you to have more control over the status of your cells. If there is any sort of breach in sterility, without antibiotics, you will immediately know and be able to deal with it by getting rid of the contaminated plates. You never have to live questioning if a plate is infected or not, meanwhile exposing your other plates to the potential infection. Everything is very clear; infections are obvious and therefore can be dealt with swiftly, without jeopardizing the rest of your cells. One of the few times in my iPS culture experience that I was using an antibiotic in my media, after not knowing whether a particular plate was infected, one by one all of my plates became infected and I literally lost every single culture I had. Now, I know that this is probably a pretty extreme case, but in any event, it demonstrated what can happen when antibiotics are battling a bacterial infection. Since the infection was not obvious, I continued to expose my cells to contamination unknowingly and therefore contaminated everything.

Secondly, using an antibiotic can mask mycoplasma infections. Usually, mycoplasma infections are accompanied by other infections—or rather, when the sterility of your cultures is breached, mycoplasma can also be introduced, and they are typically introduced with other infections such as bacteria. If the antibiotic successfully fights off the bacterial infection, your cells will still have the mycoplasma infection, which is typically only detected by using specific mycoplasma detection tests (by taking spent media and testing it). Last year, our iPS facility tested positive for mycoplasma. This was a total disaster. We had to throw away all cell cultures, close down the core for fumigation, and literally throw away all disposable materials in the room including reagents and media. It left us out of commission for a whole month. Not only was this an unbelievably expensive endeavor, it also made us lose valuable time, resources, and in some cases permanently lose cell lines. At the time when this happened, we were all using antibiotics in our media; since then, we have made it a room rule to not use them. Since we have been antibiotic free, we have also been mycoplasma free. Not using antibiotics also helps reinforce good practices in sterile technique, forcing you to be ultra careful with your cells and keep your surroundings very clean. This helps eliminate some variables in culturing, since you have more control over your environment and therefore over culture conditions.

Last notes
I think one of the most important things to remember with iPS cell culture is to be patient. Especially if you are just thawing cells for the first time! Even if it looks like there are no colonies, I would be willing to bet that if you keep feeding and wait, you will see at least one. Sometimes this can be a slow and frustrating process, but just keep at it and you’ll eventually get some great cultures. I was the first person in my lab to do human iPS cells work, so I truly understand how difficult it can be getting things up and running. There are a lot of helpful resources online, and as the stem cell community grows, the resources grow also. The HSCI iPS core has their protocols available online (http://www.hsci.harvard.edu/ipscore/node/8), which I have found to work well. WiCell has many helpful resources and protocols available online as well. I use their protocol for teratoma assays, and it pretty much works without fail (https://www.wicell.org/home/stem-cells/support/stem-cell-protocols/-home-stem-cells-support-stem-cell-protocols-stem-cell-protocols-cmsx-.cmsx).

So, to wrap things up, if you are new to hES/iPS culture, I hope this has lifted the curtain a bit on culture techniques, hopefully helping to eliminate at least a couple variables while you get started. If you have tips of your own now (or later!), please do share! Pooling our secrets, we can help each other out and make some real scientific progress.

Christine Miller is Research Assistant at Harvard University, Joslin Diabetes Center, Amy Wagers Lab.

=================

Standard Operating Procedures (SOP) for the culture of human iPS

In general, caring for hiPS cells is identical to caring for hESCs, which you are assumed to have experience with prior to requesting these cell lines. While there are different ways of culturing these cell lines, the general concepts are the same. Provided are the general instructions we have used to culture hiPS cells (primarily adapted from the protocols from WiCell):

Media

Standard hES Media (500 ml)

  • 400 ml DMEM/F12
  • 100 ml KOSR
  • 5 ml L-Glutamine
  • 5 ml P/S (optional)
  • 5 ml MEM-NEAA
  • 3.5 ul 2-Mercaptoethanol
  • 5 ug bFGF (although WiCell suggests 50 ug, we found that 5 ug is sufficient)

MEF Media (500 ml)

  • 450 ml DMEM
  • 50 ml FBS
  • 5 ml P/S
  • 5 ml L-Glutamine

2X Freezing Media (10 ml)

8 ml defined FBS 2 ml DMSO

Plating MEFs

The iPS cells are typically maintained on 0.1% gelatin coated plates with MEFs. One can alternatively plate these cells on Matrigel and use mTeSR1 media for a feeder-free condition.

Commercial irradiated MEFs may be obtained from Global Stem Cell (CF-1; 6001G) containing about 4-5 million MEFs per vial. It is recommended to plate 1 million MEFs per 10 cm plate (~170,000 per well of a 6-well plate). You should optimize your MEF density as you see appropriate, judged by the level of differentiation of your iPS cells. MEFs should be given 8 hours minimum to settle after plating, although overnight is best (ideally used within 2 days).

Thawing Shipped hiPS Vial

Each vial shipped should be thawed in 1 well of a 6 well plate. The passage number and the name of the cell line can be found on the vial. All other information on the label can be ignored.

These cells should be kept in as large of clumps as possible to increase survival efficiency so one must minimize the amount of pipetting when thawing these vials.

  1. Set up 2x 15 ml conical tubes. In tube 1, add 1 ml of pre-warmed hES media. In tube 2, add 9 ml of pre-warmed hES media.
  2. Partially thaw the frozen vial of iPS cells at 37ºC, until there is a small piece of ice remaining. Spray the vial with 70% ethanol to sterilize.
  3. Taking 1 ml of media at a time from tube 2, slowly add the pre-warmed media dropwise to the vial and transfer the liquid content with cells into tube 1. Repeat until all 9 ml used.
  4. Spin at 1000 RPM for 2 min.
  5. Meanwhile, wash with PBS one well of a 6 well plate that was plated with MEFs atop gelatin one day prior. Add 2 ml hES media. Although not required, it is highly recommended that you add 10 μM ROCK inhibitor Y-27632 (both to 9 mL thawing media in tube 2 and to the final 3 ml of plating media) to improve survival efficiency. Do not add this ROCK inhibitor to any subsequent feeds. (The ROCK Inhibitor Y-27632 Enhances the Survival Rate of Human Embryonic Stem Cells Following Cryopreservation; Li et al.)
  6. Aspirate the media from the spun down tube 1, and gently resuspend the pellet with 1 ml of hES media. Pipet slowly 1 or 2X maximum, trying to avoid disrupting the chunks of cells, and transfer to one well of a 6-well plate.
  7. Change the medium after 36 to 48 hours.
  8. Feed cells daily with 2 ml medium. Colonies should emerge anywhere from 5 to 10 days.
  9. The first split should be mechanical (ratio depending on cell density observed).

NOTE: It is highly recommended that you perform a mycoplasma test upon successful thawing of these cells. It is also recommended that you karyotype the line about every 10 passages.

Passaging hiPS cells

Note: These instructions are for passaging cells grown on MEFs. For cells grown on Matrigel, one should use Dispase in place of Collagenase IV. Trypsinization is not recommended.

1. Before splitting, remove differentiated colonies under a microscope in sterile conditions (i.e. via slow-vacuum aspiration or pipet scraping). Be careful not to leave plate out too long and make sure cells do not dry out if using vacuum method.
2. Wash cells with either warm hES medium or PBS
3. Add 1 ml of Collagenase IV per well of a 6 well plate and incubate at 37ºC for 5-10 minutes (expect to see visible curling or thickening of colonies around the edges).
4. Aspirate off the enzyme and add 1 ml of hES medium. Using a cell lifter (i.e. Corning #3008), scrape the entire well to lift the colonies.
5. Pipet the solution into a conical tube; wash the well with an additional 1 ml hES medium and combine into tube.
6. Centrifuge 1000 RPM (200xg) for 2 min.
7. Aspirate off the media, and resuspend pellet in 1 ml media per well of a 6 well plate that you wish to plate (ratio depends on cell density just prior to splitting). Triturate to get medium-small fragments (~50-200 cells per fragment). Avoid over-triturating since that will lead to cell death, especially when colonies are broken down to single cell suspensions.
8. Plate 1 ml each into a well of a 6 well plate of MEFs that was pre-washed with PBS and containing 1 ml of hES media.
9. We recommend splitting 1:3 if the cells are close to confluency.

Freezing Cells

Note: For cells grown on Matrigel, the cells should be frozen in same manner, except using 500 ul of mFreSR per 6-well.

As with thawing, it is very important to minimize the amount of pipetting to ensure cell survival later on.

  1. Prepare the cells as described in steps 1-6 of “Passaging hiPS cells.”
  2. Aspirate the media and carefully add 250 ul of hES media for every vial you intend to freeze (should freeze either 1 vial per well of a 6 well plate, or 5 vials per 10 cm dish).
  3. Add 250 ul of 2X freezing media for each vial you intend to freeze, and carefully resuspend the pellet in the combined media (keeping cells in as large of chunks as possible; generally pipetting 2x should be enough).
  4. Quickly transfer 500 ul per cryo-vial, and place inside isopropanol-containing freezing container (ie Mr. Frosty; VWR 55710-200). Store 24-48 hrs at -80C and then transfer to liquid nitrogen. (Once DMSO in contact with cells, work quickly and ideally get the cells at -80 within 3 min of contact).

For further information, please consult the following protocols:

  1. WiCell protocol
  2. Lerou et al. Nature Protocols 2008; 3:923-33
  3. Boston Protocols:  http://www.bu.edu/dbin/stemcells/protocols.php
  4. mostoslavskylab: http://www.mostoslavskylab.com/papers/Park_Mostoslavsky_CPSTB_2018.pdf
  5. Doug Melton protocol

Vendor list:

  • DMEM/F12: Invitrogen cat# 11330-057
  • KOSR: Invitrogen cat# 10828-028
  • L-Glutamine: Invitrogen cat# 25030-156
  • Penicillin/streptomycin: Invitrogen cat# 15140-155
  • MEM-NEAA: Invitrogen cat# 11140-050
  • 2-Mercaptoethanol: Sigma cat# M-7522
  • bFGF: Millipore cat# GF-003
  • DMEM: Invitrogen cat# 11965-118
  • FBS: Invitrogen cat# 16000-044
  • Defined FBS: Hyclone cat# SH30070.01
  • DMSO: Sigma cat# D-2650
  • Irradiated CF1 MEFs: GlobalStem cat# 6001G
  • Collagenase IV: Invitrogen cat# 17104-019
  • Dispase: Invitrogen cat# 17105-041
  • Rock inhibitor Y27632: Calbiochem cat# 688000
  • 0.1% gelatin: Millipore cat# ES-006-B

If you have any question, please contact us at: Laurence_Daheron@harvard.edu

Types of grant: for 千老 and postdoc

Research Grants (R Series)
R01 (Traditional Research Grant) – The Research Project (R01) grant is an award made to support a discrete, specified, circumscribed project to be preformed by the named investigator(2) in an area representing the investigator’s specific interest and competencies, based on the mission of the NIH. R01s can be investigator-initiated or can be in response to a program announcement or request for application. All of the NIH institutes and centers support R01 awards.These grants are for investigators who have some proven ability to manage external funds, a strong publication record, pilot data, and support for theproposed project. It is recommended that applications are made while eligible as a new investigator and early stage investigator.US citizens and non-US are eligible to apply.

R03 (Small Research Grant) – The R03 grant mechanism will support small research projects that can be carried out in a short period of time (two years) with limited resources ($50,000 per year in direct costs.) Examples of the types of projects that NIH Institutes or Centers support with the R03
include the following:
• Pilot or feasibility studies
• Secondary analysis of existing data
• Small, self-contained research projects
• Development of research methodology
• Development of new research technology
These grants are recommended as a first grant, if pilot data needs to be generated, for proof of concept or methods development, and to provide bridge funding to an R01.
US citizens and non-US are eligible to apply.

R21 (Exploratory/Developmental Research Grant) – The R21 grant mechanism is intended to encourage exploratory/developmental research by providing support for the early and conceptual stages of project development. These awards are for up to two years, have a combined budget for direct costs for the two-year project period that does not exceed $275,000, and require no preliminary data. The R21 can not be renewed. These grants are recommendedfor novel scientific ideas or new model systems, tools, or technologies.
US citizens and non-US are eligible to apply.

Career Development Awards (K Series)
The NIH has developed a chart that compares K awards across the various institutes and centers. It is online at http://grants.nih.gov/training/K-Awards_Across_ICs.xls.

K01 Mentored Research Scientist Award – The purpose of the omnibus K01 program is to provide support and “protected time” (3-5 years) for an intensive, supervised career development experience in the biomedical, behavioral, or clinical sciences leading to research independence. Awards are not renewable, not are they transferable from one principal investigatorto another.
Only US citizens and permanent residents are eligible to apply.

K02 Independent Scientist Award – This omnibus NIH K02 program provides
support for newly independent scientists who can demonstrate the need for a period of intensive research focus as a means of enhancing their research careers. The K02 is intended to foster the development of outstanding scientists and to enable them to expand their potential to make significant contributions to their field of research.
Only US citizens and permanent residents are eligible to apply.

K22 Career Transition Award – The Career Transition Awards provide support to an individual postdoctoral fellow in transition to a faculty position. This award is not offered by all of the NIH institutes and centers, but for those that do offer it it provides an opportunity for postdocs to apply for independent research and career development support. Some of these awards support and additional period of postdoc training followed by a period of support as an independent researcher. Others just include an independent segment.
Only US citizens and permanent residents are eligible to apply.

K25 Mentored Quantitative Research Development Award – The purpose of the omnibus K25 is to attract to NIH-relevant research those investigators whosequantitative science and engineering research has thus far not been focusedprimarily on questions of health and disease.
Only US citizens and permanent residents are eligible to apply.

K99/R00 NIH Pathway to Independence (PI) Award – The primary, long-term goalof the Pathway to Independence (PI) Award program is to increase and maintain a strong cohort of new and talented NIH-supported independent investigators. The PI award program is for postdocs with no more than five years of postdoctoral training who need one or two more years of mentored training. It is designed to facilitate a timely transition from a mentored postdoctoral research position to a stable independent research position with independent NIH or other independent research support at an earlier stage than is currently the norm.
US citizens and non-US are eligible to apply.

Research Training and Fellowship Awards (F Series)
F32 Individual Postdoctoral Fellowship – The purpose of this individual postdoctoral research training fellowship is to provide support to promisingFellowship Applicants with the potential to become productive, independent investigators in scientific health-related research fields relevant to the missions of participating NIH Institutes and Centers.
Only US citizens and permanent residents are eligible to apply.

NIH提供的各种模板:https://www.niaid.nih.gov/grants-contracts/sample-applications#r21

博后和千老可以申请的grant:

https://research.columbia.edu/funding-opportunities-postdocs

https://www.med.upenn.edu/postdoc/postdoc-funding.html

自然科学基金评审人谈标书的撰写

一份好的国家自然科学基金申请书,首先是“项目名称、关键词与400字摘要”三部分。因为,这三项对于获得评审人印象分至关重要。项目名称要带点新意,我曾经为每一个基金项目名称苦思冥想多日,而且有时在本子写好后依然对题目名称做过更改,当然一般情况下项目名称是越改越好,但也有改糊涂的时候,最后又决定采用原先的项目名称。在我的印象里,提炼出一个很有创意的基础研究项目题目取决于很多因素,既与你的科研能力有关,也与你的文字功底及对大量前人文献调研与理解相关。项目名称与一篇重要论文的题目有点相似,过于俗套题目会像一杯白开水无味,好的题目就是与众不同。

在我的印象里,基金申请书中的400字摘要有时可能决定了一个项目的命运。因为,现在很多中-青年评审人平常工作很繁忙,尤其对那些从事热门领域研究的牛人们,他们每年会收到几十份基金本子(我自己有记录的10多年来最多年份只有14份),要他们认真审读每份申请书全文(尽管这是应该的事情)不太现实。所以,往往首先仔细审阅400字摘要的内容。这也是“先入为主”重要一步,只有400字内容引起了评审人的注意,能让他的眼睛一亮,他们才会认真审读后面的内容。相反,当400字摘要内容写得平淡无奇,就像一杯白开水那样没有一点滋味,阅读正文内容就主要属于形式上的事情了。

关于一个项目的创新性是一个让人非常纠结的事情,因为如何准确评价一个项目的创新性在很多情况下只是“凭着感觉走”。其实我们大可不必为选题的“创新”性过于纠结,我认为,只要你的选题但凡有点新意就可能打动评审人。我们不要奢望提出一些“很刺激”的很强原始创新科学选题。我们更不要想象提出一些“前无古人后无来者”的“空前绝后”选题,对于我们大多数“凡人”,还是老老实实对同行研究成果进行全面认真调研。例如首先海量(泛读)阅读同行文献,并在此基础上选择精读代表性文献,这样确定的项目选题就比较靠谱了。涉及多学科交叉选题项目要阐述清楚科学(技术)问题需要下点功夫,一定要让评审人看到这种学科交叉绝不是简单的学科原理之间捆绑,而是相互渗透,找出交叉学科之间内在本质联系,并在此基础上提出选题,除了花时间琢磨内容外,要让评审人看出“门道”,对文字表述给予很高要求。

当我对一份基金本子内容比较熟悉时,我会注意申请人对国际同行中代表性人物发表成果的引用与评论。有时我还会针对性地查询几个主要文献数据库,以便核实相关内容。如果申请人对选题相关的一些重要论文没有引用,我会对申请人本子中“立项依据”的国内外研究成果调研和评论质量与遴选的“科学问题”水平大打折扣。因为,及时追踪与恰当评述专业学术同行成果是一个从事基础科学研究人员的“基本功”。其实,一份国家自然科学基金项目申请书的主题内容与一篇重要学术论文的“前言”内容有点相似。两者都需要全面认真阐述“为什么这个选题值得研究,这个选题的创新性如何?前人做的怎么样?”等共性要点。

随着学科发展与新兴学科出现,专业小同行会越来越少,因此,凭知识面与感觉评审项目的现象在所难免,否则就会有一些“有创意”项目找不到合适的评审人。“凭知识面与感觉”评审对基金本子的书写要求实际上更高,那种传统老套文字陈述的基金本子就会被认为没写清楚科学问题而遭遇不好命运。作为一份好的基金本子,从形式上尽量做到:400字内容要让人看得“赏心悦目”“颇有新意”“不落套俗”,具体内容与结构我在相关博文中做过介绍。在第一部分“立项依据”中尽量做到:“选题较新颖;科学问题定位准确;追踪分析国内外同行研究状况到位;评述同行研究状况准确恰当,既不可随意抬高同行成果水平(拍马屁之嫌),更不能贬低同行成果(骄傲自大之嫌)。

下面是一些细节问题:

国家基金重点在探索与创新,求新,但许多申请书试图通过“目前研究很少”,“无人问津”来说明创新性。殊不知,研究很少有几种可能:
1、研究遇到了无法克服的困难,如量子计算机;
2、意义不大;
3、已经解决;
4、根本行不通,如水变油。

创新点主要是指独特的研究特色(如独特的视角、解决问题的方式等),或有何理论创新或技术创新。创新点一般不大,如将创新点写成“应用实验与理论计算分析相结合,探索…规律”,或“通过…问题的研究,建立…的关系”,很难让评审人认可。
摘要通常包含五方面内容,通常是:
1、…在…方面非常重要;
2、将用…研究方法;
3、重点研究…内容;
4、实现…目标;
5、会有…科学意义” 。

但一些申请书的摘要几乎全是研究方法和内容,很难让大同行看到研究的必要性与创新性,而评审人全是小同行的概率较小。
立项依据通常包括三个方面:
1、从重要性引出科学问题;
2、由研究动态说明别人的研究瓶颈,存在的问题及你的创新思路;
3、如果实现项目预期目标后的重要意义(价值)。

但一些申请书用过多篇幅介绍内行均知的背景,降低了评审人的评审效率,在研究动态介绍上却过于简单,没有讲清楚别人的研究现状与遇到的共性问题,又要浪费评审人很多时间来判断你的创新性是否可靠。

研究内容写的过多过大是常犯的错误。对国家以及多数评审人来讲,不指望用30-40万就能解决好多问题,只要把一个小的问题解决清楚就可以了。一些申请书的题目像一本专着的名称,研究内容面面俱到,这时就要警惕了。除了求新,国家基金撰写的另一个核心是求有限目标。如果“新”有了,但因研究内容过多没批下来,就亏大了。

一些申请书简历写得过于简单,只写了何时取得学位与工作/职称变化。实际上简历的内容可包括:取得学位简历、研究工作(方向与成果)简介、人才培养与学术兼职、重要学术论文与成果。特别是对于青年基金,主要看申请人的科研潜力与项目的创新思路。而科研潜力则主要通过简历与工作基础来体现。如果主持人成果较弱,只有3、4篇文章时,不妨写写这些文章的意义及别人给出的评价。评审人也害怕人们说“现在的评审只看发表文章多少,就是1906年的爱因斯坦也未必能拿到中国的面上基金”这一说法。所以,你可以用别人的评价、引用、甚至审稿意见来证明你两三篇文章的重要性,侧面说明你的研究潜力。

八点建议

1、科学问题的把握是关键。一份申请,最重要的是把握准科学问题,也就是要研究的问题是什么,为什么要研究这个问题,研究这个问题有什么价值。
2、有限目标。新入道的人写申请,往往会列很多研究内容。
3、文题相符。一些申请谈科学问题谈的是一个问题,研究内容规划解决的是另外一个问题,文题不符。
4、摘要千锤百炼。400字摘要很关键,许多人评申请可能只细看摘要。摘要要提供尽可能多的信息,既要讲清楚科学问题、研究意义,还要讲清楚研究内容。
5、注意细节。任何事情都可能出错。在一些人看来的小问题,在另外一些人,从不同的视角看就是严重的问题。
6、让“外行”看明白。申请书写完后最好请一个非同行的专家看一下,如果他能看懂,能理解你的idea,你的申请就有希望中标,否则,你最好想法改进你的表述(如果你坚信你的科学问题把握准确的话)。
7、不要有意忽略。一些申请的”idea”是从国外文献借鉴来的,但有意不列关键性文献,冒充新东西。这一招在数年前还行,现在稍认真点的人只要Google一下就很易发现,发生这种情况基本上都会招致封杀。
8、写申请就是在做研究。写基金申请的过程就是一个深刻的研究过程,年轻人只有多写才能提高,无论中与不中都会有收获。如果没有中标,多从自身找原因,最好不要从关系、名气等方面找自我安慰的理由。自然科学基金的评审还是比较客观公正的。没有获得资助最好不要妄怪评委,最好反思自己是否真的了解研究领域、把握了科学问题,是否表达清楚你的认识与思路。

指导思想篇

1、 追求卓越,在知识上要绝对专业,坚决反对侥幸心理。
2、 相信NSFC申请是公平的,大家靠实力竞争,必须花大力气写标书;如果你认为NSFC只有关系,你就不用继续往下看了。
3、 NSFC是一个系统工程,需要花很多时间和精力,而不仅仅是几页标书,是智慧沉淀的结晶。
4、 不要把NSFC看的高不可及,你要相信自己的创意,哪怕你只是一名一年级硕士。
5、 机会主义是有的,但我们没有什么其它的资本,只能消灭标书里一切可能的失败因素,加上完美的选题和课题设计,彻底征服评委,不给评委任何黑掉你的机会。
6、 基金申请不同于实际研究课题设计,必须把个人兴趣与NSFC兴趣结合一致,投其所好。

选题立项篇

1、 基金成败关键还是选题,提前半年,刚入行的提前一年进行课题搜索。
2、 老板指定的题未必是好题,最好自己选题,如何立项应该是研究生学习最重要的一课,毕业后你会发现,没有人会指点你什么课题有价值了,在中国学术的沙漠里,只剩下你自己了。
3、 好课题是对学科深刻理解的条件下产生的,大量翻阅文献吧,汲取知识的同时千万别忘了思考,你发现别人存在漏洞的时候,好课题就离你不远了。
4、 选题最好以问题为导向,不要以技术为导向,找到问题了,课题就找到了。而拿着新技术去找能解决的问题,效果多数不好,但还是大有人在,比如RNAi。
5、 解放思想,发散思维,多方法多学科交叉,一般都会比较受人青睐,容易申请到基金,但不能为了交叉而强行交叉。
6、 创新性新技术、新理论的课题要有一定的理论与技术基础,最好有工作基础,没有你也要东拼西凑,这是在中国,NSFC似乎讨厌空中楼阁。
7、 临床课题研究最好别选临床应用方向,而选应用基础研究。
8、 选择自己熟悉,有工作基础的领域,别跨越太远。
9、 重要科学问题的切入点准确,切忌过宽、过大,只要体现一定的新意和研究价值就行了,能得诺贝尔奖的课题NSFC是不给钱的。
10、 没有人做过的课题不能做为立项的依据,但NSFC资助的项目必须是国际上没人做过的,而不是国内空白。当然,如果国际上有同类结果,你不说,地球上的中国人也许也不知道,但一旦被识破,你死定了。
11、 如果是捕捉科研前沿性的课题,最好设计周密,尤其是目的和结果的一致性、可获得性和可预期性,通过课题实施所获得的结果必须能充分支持与研究目标相一致的结论。
12、 热点课题不一定是好课题,热点上的人也很热。但在还没热起来的热点,一定是一个好课题,标书评审滞后半年呢,比如最开始的一批SARS课题。有时也不妨设计一些非热点但是对与科研有价值的课题,发挥出奇不意的效果。
13、 临床课题可以是当前没有好办法治疗的疾病,急需解决的临床问题,而在国际上检索的文献只有几篇的那种。
14、不主张以最新的重量级文献做指导,你会发现,很多人跟你的想法惊人的一致。有人特别反对跟风。
15、 一定要到NSFC检索一下类似课题的历年资助情况,太多、太少都不好。最好是最近二年逐渐增加的资助领域。

立题依据篇

1、 题目要有新意,吸引人,既要概括主题,容易懂,又要有些少见的新词或缩写,调胃口。
2、 5000字左右,最多两页,不包括文献,行距字体大小适中。
3、 国内外研究现状及分析一定要准确,甚至是中庸,绝不能偏激,不然不同意你的专家会带着逆反心理看你的标书。
4、 课题研究的具体问题和研究意义,则必须说的清楚。当然如果有实力,可以解决关键的科学性问题,那再好不过。然而课题意义不是最重要的,但常常被撰写得份量过重,课题总体构想、大体实施方案及可能的预期结果才是人们最关心的。
5、 要把复杂的事说简单。既要论述充分,写作又要简练,最多两页半(不算文献)。剔除所有不必要的知识细节、理论和概念,要舍得割肉才行。越简单,出错越少,专家不懂的越少。写出来的理论,要让人家能欣赏。写出来的理论,要让人家看不懂,这份申请书很危险。
6、 立论依据要非常突出:理论性课题一定要有新观点,应用性一定要实用,与现有理论或方法具有明显的先进性,总之要让人感觉到有意义。
7、 一定要有可预见的成果,至少画一张大饼,但看上去要象真的才行。
8、 任何重要的论点都要有文献标注,有文献就等于没有疑问。参考文献要新,最好是当年的,增加自己立论依据的权威性。最好包括已有工作基础,将已有相关结果以及发表的杂志列上,可以增加可信度。
9、 一定多让本实验室的人修改,特别是中过基金的前辈,要改15遍以上才行。
10、 标书的评委参差不齐,评审意见也差异悬殊。好的标书最容易受到高水平评委的赏识,只要你的题好,这些评委是好征服的。难就难在如何让水平差的评委通过你的标书。我认为除了运气好,少碰到一些这样的评委之外,最关键的一点就是让他们看懂你的标书;第二就是标书不能太长,他是看不下去的;第三就是实验设计在不失科学性、先进性的条件下,尽可能简单,千万别让他觉得你比他高很多,那样你死定了。所以一份好的标书是在高水平教授和低水平教授之间的平衡,写的非常玄妙的标书通常中不了。
11、 评审专家通常是本专业的,也可能不是,尤其是交叉学科投递的项目,评审专家未必对你的研究领域特别熟悉。所以尽可能少引入非常专业的概念,如果不可避免,也要解释清楚。
12、 文字写作要有适当的弹性,不能把话说死,留有余地。

研究方案篇

1、 研究目标要明确要精,提法要准确、恰当;内容要详细但文字不宜过多,且一定不能写得太具体。关键的问题要突出,一定要准确。
2、 可行性分析是你说服评委的第二次机会,可按成熟的理论基础(理论上可行)、研究目标在现有技术条件下的可实现性(技术上可行)、本单位现有技术设备实验材料的完备(设备材料可行)、课题组成员完成课题能力(知识技能上可行)等几方面分层论述。
3、 创新点要切合实际,又要有所发挥,指出国际国内研究的先进性和创新性,点明理论和现实意义。
4、 研究内容要集中,与研究目标紧密一致,只作支撑课题最关键最必要的内容。不可为多作实验显示劳动量或增加预算而使研究内容过泛。
5、 实验方案和技术路线合理、可靠、可行,没漏洞是最重要的。思路好,材料独特,方法独特新颖,会增加获得资助的机会。技术当然是越新越好,但未必需要采用最时髦的研究手段,不能为了技术而研究。
6、 研究内容及方案切忌复杂,步骤最好有一流程图。研究方法、技术路线、实验方案不能太具体化,容易出漏洞。但你必须让评委认为你十分了解实验技术的整个过程,可以尽可能多的应用技术术语和技术缩写,写出主要实验材料和实验过程。
7、 技术方法一定是本实验是已经建立的,至少是有相关实验基础。所有关键技术要有文献出处,最好是自己实验室发表的,有文献就等于没有疑问。如果本单位力量弱,可挂靠较强的研究机构,从而使评审相信你能完成课题。关键实验材料必须已经具备,或可以获得。

预期研究结果

1、 预期结果要考虑对基础和实用双重的价值。
2、 以发表论文和申请专利结题比较容易。最好突出SCI收录杂志的影响因子,给基金委的专家们觉得,您的实力确实不一般。因为最终结题情况,是基金委专家们最关心的事情,他们当然愿意把基金支持能发表高水平文章的人。

工作基础篇

1、 工作基础是你说服评委的第三次机会。课题科学先进、技术路线新颖合理可行、工作基础雄厚这三方面表述要紧密联系、前后呼应。
2、 一定要有基础。把实验室发表的所有文章搜集起来,找出与你设计课题相关的,只要沾边,都列上。
3、 预实验结果很重要,而且是有硬data的结果,一定附上。但一定要慎重掌握,不要写的太多,评委会认为你的工作做的差不多了,没必要再申请基金了。只预期你的课题肯定有好的结果就行了。
4、 有针对性地把研究队伍的相关工作经历、论文、成果等展示出来。

人员组成篇

1、 主要成员6-10名,结构合理。
高级研究人员(1-2人)
中级研究人员(2-3人)
技术人员及研究生(3-5人)
2、 参加人员技术力量的配备要合适,必须保证一定的劳动力。
3、 1名高职足够,多了浪费资源,现在NSFC限项很死的,我们的高职资源快耗竭了。
4、 中级人员是骨干,但在职的不要太多,1-2名。
5、 技术员2名左右,很重要呦,这是专业技术保障。
6、 研究生不能少于2名,这是主要劳力,地球人都知道。但也有人认为而不应将研究生列为主要人员,这样NSFC会认为人员稳定,富有干劲。
7、 成员介绍要紧扣课题的研究内容和技术路线,既注重梯队、比例、技术力量等科研综合实力的展示,又注意与本课题相关。

个人简历篇

1、 申请者和项目组主要成员的学历和研究工作简历,近期已发表与本项目有关的主要论着目录和获得学术奖励情况及在本项目中承担的任务,所有复印件一定要附上,眼见才为实。
2、 申请者和项目组主要成员正在承担的科研项目情况,包括自然科学基金的项目,要注明项目的名称和编号、经费来源、起止年月、负责的内容等。完成的可以都列上,没结题的一定不要写了。
3、 中级技术职称的推荐信或在职研究生申请项目的导师推荐信一定不要忘了。
4、 个人简历一定有针对性的倾向于课题方向,并与课题中各人的分工相一致。所从事的研究项目可适当给出,但不能过多,保证课题组有充足时间完成基金课题。

摘要写作

1、 摘要字数少,但最忌讳写得平淡无奇。
2、 一定要语气坚定,旗帜鲜明。
3、 摘要字数有限,资源宝贵,惜字如金,因此要特别注意重点突出,讲明现状、课题意义、课题构想和预期结果。
4、 防止“头重脚轻”,削减一般性细节描述,多用概括性语句,讲明现状、课题意义、课题构想和预期结果部分要相互平衡。

学科选择篇

1、 申报的方向和学部很重要,往往结果天壤之别。尽量避重就轻,在竞争不很激烈的领域申请,除非您有充分的把握。
2、 投到你老板能量比较集中的学科,是第一选择。
3、 仔细研读基金申报指南,洞悉各专业领域倾斜性项目和优先资助方向。
4、 仔细分析NSFC历年与你课题相关资助项目在各学科的分布,发现隔年资助或近几年资助递增的学科,你基本找到钱在哪了。
5、 学科交叉鼓励,但尽可能投到你熟悉的学科。

善后工作

1、 版面调整,清晰,层次分明,使版面简洁、易于阅读。
2、 坚决消灭错别字。
3、 合理行使基金委赋予的权利—-回避制度自我保护。
4、 仔细审查自己的申请人资格是否达到NSFC要求。
5、 仔细审查自己的项目组成人员(包括自己)有没有超项。

Cas12a和Cas13a在诊断领域的应用

CRISPR-Cas系统背景回放

面对噬菌体的威胁,细菌进化出了一套专门针对噬菌体或外源性遗传物质的CRISPR-Cas免疫系统。CRISPR全称为“簇状,规律间隔的,短回文重复序列”(Clustered Regularly Interspaced Short Palindromic Repeats),是由众多短而保守的重复序列区(repeat)和间隔区(spacer)组成。如图1所示: Repeat是细菌固有序列,能够同时结合Cas蛋白和spacer的序列,而spacer则是细菌(或是其祖先)感染过的病毒序列。一旦噬菌体感染发生,绝大多数的细菌死亡,极少部分的细菌由于其基因变异得以生存。这些细菌中的一部分,将噬菌体的DNA序列切割后,插入repeat区域中,形成spacer,从而获得类似高等生物“免疫记忆”的能力。

随着CRISPR-Cas机理的逐步揭示和新的Cas酶(Cas12/Cas13/Cas14)的发现,科学家们发现这个系统非常强大,可以精准高效地实现基因编辑,比如对某个基因的敲除、插入和替换等。而CRISPR系统的序列特异性识别能力已经被应用在越来越多的领域,如医药,食品,农业和工业生物技术等,这些应用很大程度上都是以Cas9为基础进行开发的,而新发现的Cas12/Cas13/Cas14不同于Cas9,使得CRISPR系统在病原体的快速诊断和肿瘤基因检测领域的应用成为可能。

Cas12a-单链DNA的“新魔剪”

今年4月,有CRISPR女神之称的Jennifer Doudna 教授在《Science》撰文指出Cas12酶家族在gRNA的引导下与目标序列结合以后,便会切换为激活状态,疯狂的切割体系内其它的单链DNA。Cas12a这一特点可被用于分子诊断领域,实现对肿瘤基因或特定病原体的检测。在体系内加入含有报告基团的单链底物后,如果Cas12a识别到靶序列(目标病原体或肿瘤基因)的存在,就会切割单链底物从而释放荧光报告基团。

Cas12a可以实现HPV的准确分型

但是如果样本中的目标基因含量非常少,Cas12a与gRNA复合物匹配到需检测靶序列的概率很低。此时就需要先扩增靶序列,提高需要检测底物的丰度。PCR(聚合酶链式反应)是常用于这一目的扩增手段,但是需要专门的PCR仪进行温控反应。而另外一种信号扩增技术——重组聚合酶扩增(RPA),可以在恒温状态下实现信号的扩增,而不需要复杂的升温降温过程。

Doudna教授创新性的将Cas12a靶向切割单链DNA的特性与RPA技术联合起来,开发了一种名为DETECTR的技术(DNA Endonuclease-Targeted Crispr Trans Reporter)。研究结果表明,肛拭子取样后等温扩增10分钟,使用Cas12a系统对扩增产物进行检测,可以在1小时内检测到人乳头瘤病毒(HPV)并准确区分两种相似的亚型,HPV16和HPV18。DETECTR技术的开发为实现病原体或肿瘤基因的即时检验(POCT) 又提供了一个强有力的支撑平台。

那CRISPR-Cas12a与CRISPR-Cas9有什么区别呢? Cas9是最早发现的Cas酶之一,也是目前为止研究最深入和应用最广泛的Cas酶,在基因编辑、疾病治疗等方面的应用前景巨大。然而CRISPR-Cas9缺乏切割单链核酸的酶活结构域,无法用于体外检测。而Cas12/13/14则普遍存在第二个酶活结构域,当蛋白正确结合到靶向序列时,能够激活这一结构域,切割探针小分子,实现从待检序列信息到荧光信号的转化。基于它们的这一特点,在医学检测领域需要靶向检测已知序列的情况下,能够实现普通实时定量PCR所无法达到的灵敏度,摆脱对实时定量PCR仪的依赖。

CRISPR13a:“异于常酶”的RNA切割机

2016年6月,张锋实验室与Eugene koonin实验室等合作在《Science》杂志上发表文章,首次描述了一种RNA靶向的Cas酶—C2c2(后称 Cas13a)。文章指出,在大肠杆菌中导入 CRISPR-Cas13a系统,由于Cas13a含有两个称为HEPN的保守RNA核酸内切酶结构域,该系统可以成功地切断噬菌体的核酸序列,帮助大肠杆菌抵御噬菌体的入侵。他们还发现,体外实验中,Cas13a与单链靶标RNA底物结合后,还可以 “附带切割”反应体系中其它的单链RNA分子。

9月份, Jennifer Doudna 教授在《Nature》发文,进一步阐述了Cas13a的分子切割机制。他们指出与Cas9不同,Cas13a具有将crRNA前体切割为成熟crRNA和切割靶向RNA的双重酶活性。当Cas13a正确与靶向RNA序列结合以后,其非特异性切割的特性便会被激活,进而切割体系中荧光报告基因,实现待检序列信息从荧光信号的转化。

“夏洛克”实现登革热、寨卡单分子检测

2017年4月,张锋团队在《Science》再次发表Cas13a的研究成果,根据Cas13a与靶标RNA结合后的“附带切割效应”,将其与反转录,重组聚合酶扩增(RPA)以及体外转录三项技术结合,开发了名为“SHERLOCK”(Specific High-sensitivity Enzymatic Reporter unLocking的缩写)的检测技术,实现靶标序列扩增后检测,进而显著提高该技术的灵敏度。“SHERLOCK”取自大众广为熟知的英剧《神探夏洛克》,寓意在该技术的协助下,医学检测能够像大侦探夏洛克的探案能力一样精准。

与Cas12a检测策略类似,两位科学家不约而同地使用了RPA进行靶标序列扩增,可以有效解决样本中的靶标序列少的问题。而不同点在于Cas12a检测的是DNA靶标,Cas13a检测的是RNA靶标,因而Cas13a必须在反应体系中引入体外转录系统。无论是Cas12a还是Cas13a,是否加入逆转录系统,取决于待检靶标是DNA还是RNA,如果是后者,则需要首先将靶标序列信息逆转录成为DNA,才能进行后续扩增与检测。

“夏洛克”+“哈德逊”实验室走向临场检测

今年4月,张锋参与,布罗德研究所Paridis Sabeti主导的关于CRISPR-Cas13a的研究成果在《Science》杂志以封面形式刊登。他们将“SHERLOCK”与一种新的技术“HUDSON”(Heating Unextracted Diagnostic Samples to Obliterate Nucleases)联合,实现了登革和寨卡病毒的即时检测,满足了脱离实验室的临场快速检测需求。在《神探夏洛克》里,房东哈德逊(Hudson)太太总是对夏洛克关爱有加。同样,对“SHERLOCK”检测技术而言, HUDSON技术就像哈德逊太太一样协助“夏洛克”,通过对临床样本的两步快速热处理和化学处理,实现灭活核酸酶和病毒的同时释放病毒核酸。

两种技术的联合使灵敏度增至1个拷贝/ml,不需要复杂的核酸提取技术,2小时内便可完成临床多种样本中的登革病毒检测。夏洛克与哈德逊技术的强强联合可实现如此令人叹服的快速,准确的便携式诊断。技术的革新浪潮也激起了我们更深一步的反思。在基因技术和万物互联的洪流之中,以技术为工具,以伦理为度量,以实现整个人类健康为使命,或是我们最期望到达的终点。CRISPR编辑婴儿基因为时尚早,体外诊断正当其时。

参考文献

1.Abudayyeh, O. O., Gootenberg, J. S., Konermann, S., Joung, J., Slaymaker, I. M., Cox, D. B., … & Severinov, K. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, 353(6299), aaf5573.

2.East-Seletsky, A., O’Connell, M. R., Knight, S. C., Burstein, D., Cate, J. H., Tjian, R., & Doudna, J. A. (2016). Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature, 538(7624), 270.

3.Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., … & Myhrvold, C. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, eaam9321.

4.Zuo, X., Fan, C., & Chen, H. Y. (2017). Biosensing: CRISPR-powered diagnostics. Nature Biomedical Engineering, 1(6), 0091.

5.Myhrvold, C., Freije, C. A., Gootenberg, J. S., Abudayyeh, O. O., Metsky, H. C., Durbin, A. F., … & Garcia, K. F. (2018). Field-deployable viral diagnostics using CRISPR-Cas13. Science, 360(6387), 444-448.

6.Sashital, D. G. (2018). Pathogen detection in the CRISPR–Cas era. Genome medicine, 10(1), 32.