FSA及其使用

Putting money into a flexible spending account is a smart way to reduce your taxable income. Now more companies are giving workers additional time to spend their 2018 funds—but the deadline is coming up. 

An FSA must be sponsored by an employer, so you’ll have one only if you have job-based health insurance. In the past, employers commonly had a use-it-or-lose-it policy requiring you to spend the money by Dec. 31.

But in a growing trend, more companies are giving workers a grace period that extends spending deadlines till March 15. Now 37 percent of companies give FSA users that mid-March deadline to spend 2018 FSA money on eligible expenses, up from 32 percent in 2017, according to the Society for Human Resource Management’s annual employee benefits report (PDF).

Whether your deadline for FSA spending is Dec. 31 or March 15, you have until Mar. 31 to submit the claims for that spending.

So if your company is giving you more time and you haven’t spent all your FSA funds from last year, make sure to use the money soon. With an FSA, you put pretax money away to spend on qualifying medical expenses not covered by your health insurance.

That means you never pay taxes on the money from the FSA you spend for eligible healthcare costs. In 2018, you could fund up to $2,650 in an FSA. In 2019 you’ll be able to put away an additional $50, for a total of $2,700 pretax dollars. If you’re in the 24 percent tax bracket, you’ll avoid $648 per year in taxes if you put away the maximum amount. You can estimate your savings with this calculator offered by WageWorks. 

If you have job-based insurance, you’re likely to have an FSA option. Half of all firms with 25 or more workers that offer health insurance offer it, and among companies with 200 or more employees, three-quarters provide FSAs, according to the Kaiser Family Foundation’s 2018 Employer Health Benefits Survey. 

Here’s what you should know to get the most out of your FSA.

What You Can Spend FSA Money On

People often rush at the end of the calendar year to make doctor’s appointments (copays are an FSA-eligible expense). Buying eyeglasses and going to the dentist in December are also common ways to use up the funds. But the list of what you can pay for (PDF) with FSA money is long, and if you have extra time to spend the funds but not enough time to get a doctor’s appointment, you can use the money up by simply making a trip to the drugstore. Items such as arch supports, breast pumps, and other medical devices are eligible. So are lip balm and sunscreen. 

And if you have a prescription or a doctor’s note, there are additional items and treatments that are covered, such as acne medicine and sleep aids.

An easy way to see what qualifies is to go to FSAstore.com. Everything sold on the site is eligible for FSA dollars, and items that need a doctor’s approval are clearly marked.

FSA vs. HSA

Your employer may also offer a health savings account (HSA), another way to put away pretax money for future healthcare costs. 

An HSA is an option only for people with a high-deductible health plan (HDHP), which have an individual deductible of at least $1,350 per year, or $2,700 for a family. A deductible is the amount you must pay before insurance begins to cover some of your healthcare bills. (Some preventive services are covered at 100 percent before the deductible.) You can’t have both an HSA and an FSA with employer insurance.

But an HSA is more flexible than an FSA. Unlike an FSA, there is no deadline to spend the money in the account, and if you leave your employer, the account goes with you. You can put more in an HSA, too: up to $3,500 annually for individuals or $7,000 for families in 2019. You can contribute an additional $1,000 per year if you are 55 or older.

Employers don’t have to set up HSA accounts for their employees in high-deductible plans, but about half of those that do set them up make a contribution to the account. You can also choose a different HSA if you don’t like your employer’s option. And if you buy your own insurance and have a high-deductible plan, you can always open one on your own. 

RNA提取过程中的基因组DNA污染控制

Genomic DNA is often co-extracted with RNA and can serve as a template in downstream processes such as PCR. However, if your TaqMan® MGB probe spans an exon-exon junction, genomic DNA can be excluded as a template in a real-time PCR reaction.
In contrast, if both primers are designed within one exon, then genomic DNA could serve as a template for PCR amplification. In these cases, the user has to decide if the genomic DNA is sufficiently negligible.

RT reactions without reverse transcriptase (No RT controls) can be used to evaluate levels of genomic DNA in a RNA preparation. A No RT control is a reaction that has been prepared for reverse transcription (including RNA, dNTPs, buffer and so on) but no reverse transcriptase is added. One can estimate the amount of amplification in their samples that is attributable to genomic DNA templates by running No RT controls. For example, if a No RT control sample has a CT value 10 cycles higher than an RT test sample, then the No RT control sample started out with approximately 1000-fold less target sequence (assuming 100% efficiency, 1 CT ≈ 2-fold difference in initial template amount). Since the target template in this No RT control would exclusively be genomic DNA, one may conclude that 0.1% (1:1,000) of the amplification in the RT sample is attributable to the genomic DNA template. You will then have to determine if the PCR amplification attributable to the genomic DNA is sufficiently negligible compared to the amplification of the cDNA sequence.

via：

https://www.ncbi.nlm.nih.gov/pubmed/26545322

点击以访问 1125331_ABI_-_Guide_Relative_Quantification_using_realtime_PCR.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3281124/

做好学术性演讲的几个要点

In my first year of graduate school, I was terrified of giving presentations. I would put too much information on my slides, talk too fast and constantly forget or trip over certain words. Unsuprisingly, the reception was lukewarm at best. 

As I progressed, my adviser and committee members were kind enough to point out which areas of my seminars needed the most improvement. I also paid closer attention to other people’s seminars to identify the aspects that I liked and wanted to emulate. Now, my presentations are met with enthusiasm and praise. 

There are two key aspects to focus on improving: you and your slides. Your slides should function as a roadmap, helping you and your audience to follow the main ideas. Here’s how to build a great roadmap. 

Focus on one idea at a time. The biggest mistake that most speakers make is putting too much data on a slide. No one wants to hear, ‘This slide is busy, but let me walk you through it’. Use animations to build ideas: introduce a single point on a slide, then gradually bring in the rest of the elements one at a time or on separate slides. 

Do not write paragraphs. A presentation is not the right forum for the written word. Don’t introduce a paragraph in a slide and read it out loud: people can read faster in their heads than you can on stage. Also, if you put up a paragraph, but talk about something else entirely, everyone will be too busy reading to pay attention to what you’re saying. Use bullet points with a minimal amount of text. 

Reel the audience in. Seminars usually last anywhere from half an hour to a full hour. To keep the audience’s attention for such a long period, make sure you provide an answer to the question, ‘Why should we care about this’? Addressing this right at the beginning should help you to capture your audience’s attention for the rest of the talk. 

Follow the ‘question, experiment, result’ format. Research is messy, but your presentation doesn’t have to be. For example, when I first began my thesis project, the proteins that I was studying had no obvious role. Because there were several possibilities about what their function could be, I had to address each one of those hypotheses individually. My experimental data showed that most of my hypotheses were wrong. Eventually, I worked out the role of those proteins. When the time came to present what I’d done, I didn’t subject my audience to all the data because a large portion of it has no bearing on my story.

To achieve clarity, always emphasize to the audience the question you were trying to answer early in your talk: what was your hypothesis? Then talk through the experiment that helped you to answer that hypothesis. After that, show your audience the results. Following this formula ensures that, by the time you get to the result, your audience will be invested in the outcome, have an idea of what to expect and be delighted when they see that your results matched their thinking.

Paint a big picture. It is nice to have a summary slide at the end of the talk that captures all of your key ideas. However, many in the audience will forget most of those ideas after a few days. If you want them to remember only one thing, what would it be? Put your research in the larger context. How has your work furthered the field? People are more likely to remember the big picture than the minute details of your work. 

Now that your roadmap is ready, what other steps can you take to make your seminar a success? Remember, a presenter is not so different from a performer. As a presenter, you’re constantly trying to win over your audience and convince them that your work is important and interesting. Here are a few guidelines that will help you to become a more effective communicator: 

Pace yourself: breathe, let the audience soak it in. Rushing through a seminar is an easy mistake to make. People tend to speak faster when they’re nervous and, although this is natural, it damages a presentation. An audience needs time to understand the results and their implications. Racing through the slides will only disorient them, especially if the results are nuanced and complicated. So, remember to breathe. After every important result, stop. Give it a few seconds, and then continue. This will help your audience to follow the talk, and it will calm your nerves. 

Convey passion. How the audience reacts to a particular piece of information depends on how you present it. If you are lukewarm about particular results, the audience will think that those results are not worthy of their attention. By using phrases such as, ‘These results were surprising’, ‘We found a really cool phenomenon’ and ‘The implications of these findings are exciting’ — and by matching those expressions with your tone of voice — you are giving the audience cues on how to respond. Remember that these phrases sound dull if they aren’t said with enthusiasm. 

Point the laser correctly. There is one aspect in which people behave exactly like cats. If you point the laser haphazardly all across the slide, human eyes will follow the laser. Use the laser judiciously. Point to one thing at a time, and hold your arms steady so that the audience can focus on that one thing. 

Modulate your voice. It can be easy to speak in a monotone when we’re nervous and don’t want to sound squeaky. Learn to use intonation. For example, when you ask a question, use a high note at the end. When you make a declarative statement, finish on a low note. These things usually happen naturally, but they can get lost when you’re nervous. 

Practise, practise, practise. The key to a good performance is practice. Practise your pitch, your pacing, your body language, how you walk around, how you use your hands and where you point the laser. Although practising can be time consuming, it will boost your self-confidence. My rule of thumb is: be so familiar with your talk that you won’t miss a beat even if the computer fails and none of your slides can be seen.

From： https://www.nature.com/articles/d41586-019-01574-z?utm_source=twitter&utm_medium=social&utm_content=organic&utm_campaign=NGMT_2_SJH_Nature

选购割草机经验谈

工欲善其事，必先利其器。如何选择割草机呢？我自己用过电动的、手推的，今年刚买汽油的。下面介绍一下使用后的心得。

电动割草机（Electric mower）

利：不用考虑加油，不用维护，不担心启动，噪音较小。

弊：

1、我的电动割草机有蓄电池，但要提前充电。常常是我想要割草的时候没有电，充好了电又没时间割草了。

2、不到两年，我的电动割草机电池就坏了。蓄电池的寿命一般都不长，换一个并不便宜。

3、电动的也可以扯线，因怕割到线，要多花时间。割草的时候，往往不能很规律的一排一排的走，有些地方割得不好，要再推回去割。

4、电动的容易烧电机。虽然都很小心，还是免不了烧了。

手推割草机（Reel mower）

利：价格较低、除了磨刀片其本不用维护、运营成本低、没有噪音。

弊：

1、小的草坪，手推式割草机非常好用。如果院子大，或有上坡，就特别累人，一会儿就一身汗。若说锻炼身体，林间漫步，打打球等比枯燥的割草快乐多了。

2、手推式割草机割出的草地不是很美观，有些地方草倒伏或太长就割不好。

汽油割草机（Gas mower）

汽油割草机也分为两种，一种是手推式（Push-type）需要人力往前推动前进，适用于面积不大，没有坡度的草地。一种是自走式（Self-propelled）自动往前进，人只需掌握方向即可。适合面积大、有坡度的草地，如果是力气不足的老人或妇女需要割草，Self-propelled的割草机就很省力。有些Self-propelled割草机只有一种移动速度，通常为每小时4公里左右，有些则配备多种速度可供选择，通常为每小时0到8公里。

利：

1、修整同样的一块草坪，底座较小的割草机需要多割几个来回才能完成割草工作。电动式割草机切割宽度为18-19英寸。汽油Push-type的切割宽度在19-21英寸之间，而汽油Self-propelled的切割宽度在21-22英寸之间。因此汽油割草机会为你节省更多的时间，享受夏日时光。

2、汽油的马力大，割起草来干净利落，更加美观。

3、后轮驱动比前轮驱动的割草机，贵些，但使用体验大大地不同。后轮驱动的，非常省力。上坡的时候重量都压在后面了，前驱的在有坡度和潮湿的时候可能会打滑，推上去费力。

4、我的草地上种著枫树，秋天挂在树上的枫叶固然美丽，但才下枝头，又上心头，得花不少功夫耙起这些枯叶，烦恼了。自行式割草机也有不同的档次，有的功能齐全，可以将枯叶吸入、打碎、装袋，这就是树下人家的理想选择了。HONDA217HYC，就有此功能。HONDA是目前割草机市场上比较好的品牌，不光是引擎好，后轮驱动，且轻便、给力、耐用、噪音小。当然，一分钱一分货，价格也贵些。

弊：

1、汽油式割草机需要加油、换机油。

2、比电动和手推的割草机噪音大。

温馨提示：

1、西人销售员往往认为华人更注重价格，所以会推荐你便宜的割草机，却忽略了你的真正需求。所以在购买之前，一定要搞清楚，哪个功能会使自己的生活轻松很多，是必须的。然后再考虑质量和价格。质量其实非常关键，它让你在一段较长的时间内，投入较少的金钱和时间。比如我以前买了一台微波炉，花了200多加元，用了10年，平均下来每年20加元。而第二次，花了70加元的微波炉，用了1年多一点就坏了，平均每年费用60加元，还多花了采购的时间。

2、如果需要对割下的杂草、树叶进行装袋处理，最好选购Self-propelled割草机，有些割草机能够装载超过40磅重的树叶，会让你有物超所值的感觉。因为它大大提高了你的生活品质。

3、轮子稍大的割草机使用起来更为灵活。

4、后轮驱动割草机更省力。

5、与气门侧置发动机的割草机相比，气门顶置发动机的割草机效率更高、扭力更强、占用空间更小。

6、有些型号的汽油割草机，只需转动钥匙即可启动发动机，价格要高些。

7、新机器都有磨合期，一开始不好用，只是你还没了解它，所以不要遇到难题就退缩。等你掌握了机器用法，一切都那么简单。

8、割草时不建议将割草机刀片调到很低，草坪太低不容易保湿，很容易好草枯死杂草丛生，切记！另外刀片太低，阻力大容易憋死割草机。

9、清晨露水大、雨后、草湿等情况不宜割草，湿草容易粘在割草机上，阻力很大，不易收集碎草，容易把割草机憋死。

10、打边机打边时，只用塑料绳尖端抽打草或土地就可以，切勿用中部和根部，否则阻力大导致断线。再好的线，如果不懂使用技巧，都会很快断掉。

11、长时间割草，建议带口罩，杂草或许有有害物质，尘土也对呼吸不好。有次割草没戴口罩，肺和气管难受了半个月多。

12、有杂草立即除，每年要给草坪施肥和除杂草，建议用这款weed&feed，如果有螃蟹草，那建议用拔草神器立即拔除，否则扩散很快。7、汽油割草机本身带一罐机油，不需要买了，但汽油桶还是要买的，绝对不能用牛奶桶代替。买桶的时候，一定要看说明，确定那个桶是可以装汽油的。避免安全隐患。

汽油桶有铁罐的，需要再另买一个漏斗。也有塑料的，盖子上自带一个长管子漏斗。两者价格算下来差不多。但加油的时候，铁罐多多少少会漏一些。自带管子漏斗的塑料桶就没有漏油的问题，又和漏斗是一个整体，节省了找东西的时间。

13、没有什么比捍卫自身的健康更重要的了。汽油机割草时，戴上噪音耳套或者耳塞还是需要的，因为长时间的噪声对听力损害很大。

14、割草的时候，偶尔会飞崩出石头，对于小孩子是危险的，应让孩子远离，自己也要带眼镜保护眼睛，防止意外的发生。

下图是2019年美国消费者杂志（Consumer Reports）对当前市场主流的汽油式割草机的评价。

怎样充分利用学术会议的资源？

So you’ve done the research, gathered up your data into an exciting story, and are ready to present your findings at a conference. But what you get out of a conference depends on what you put into it before, during, and after the meeting. Let’s break it down into the following: the elevator pitch, talks, the poster session, networking, and social media.

The elevator pitch

Before the conference

Practice your “elevator pitch,” the 30 – 60 second blurb you will use to introduce yourself and what you do. You might want to include your university and your advisor’s name for some context and maybe common ground. The people you meet may all be scientists but not all will be familiar with the specifics of your research. Try to avoid jargon in this initial introduction.

During the conference

When you meet someone new at a conference, one of the first things you might want to know is what their project is. Or maybe you want to introduce yourself to a scientist whose work you are familiar with. You can tweak your elevator pitch slightly if you know a bit more about the other person. If you have a poster or talk at the conference, you can end by asking them to come to your poster or talk.

After the conference

After using your elevator pitch several times at the conference, you might notice some trends. Were there parts of your elevator pitch where the other person seemed confused, bored, or particularly excited about? If you tried tweaking your pitch throughout the conference, what worked? Use this information to prepare for the next meeting.

Navigating talks

Before the conference

Is the conference you are attending on the larger side? At large conferences, chances are that several talks will be going on at once in different rooms. Before arriving at the conference, plan out your agenda. Some conferences even have apps you can use to build a schedule. If there is a list of poster titles or abstracts available before the conference, comb through that list and note which posters you’d like to go to.

During the conference

Take notes during talks. If you have questions during the talk, jot them down for the Q&A immediately after. For one conference I went to, I made it a personal goal to ask one question each day (this may be more or less difficult depending on the size of the conference). This made sure I didn’t zone out and kept me engaged throughout the day.

After the conference

If there’s a cool new method that you learned about at the talk, bring it to your next lab meeting. Don’t be afraid to follow up with the speaker from the conference if you have additional questions. If you or your lab identifies a potential collaborator from the meeting, reach out to them, pointing out that you heard their talk at the meeting and why you think a collaboration would be beneficial.

The poster session

Before the conference

If you’re presenting a poster at a conference, be sure to practice beforehand. Generally, two types of people will visit your poster: those that want you to walk them through the entire poster, or those that want to look through themselves and then ask questions. Practice your presentation for both instances. If your poster material is nearing publication, consider uploading your work to a preprint server (and include this information on the poster) so your visitors can have a DOI to go to.

During the conference

Arrive at the poster session early to get your poster up on the board and meet your poster session neighbors. During the session, if someone comes up to my poster, I usually greet them and say something like “Let me know if you have questions or if you want me to walk you through the poster.” Have a notebook handy so you can jot down notes or questions from your poster viewers and trade contact information if your conversation is particularly fruitful.

After the conference

Bring back what you’ve learned from the poster session to your lab. Perhaps some of your poster visitors had a great idea or interesting questions worth discussing with your lab. If you made a connection during the poster session, send them a follow up email thanking them for their conversation and connect with them on social media.

Social media

Before the conference

Networking can be made easier through social media. Use social media to find out who’s going to the conference and who you’d like to talk to there. Check out the conference website for any relevant hashtags and handles to help you find other scientists attending. Reach out if you’d like to meet someone and set aside a time to meet. Some conferences may also have a dedicated app with a social media component, but if it doesn’t seem like this is being used, it’s best to turn to an established social media platform.

During the conference

If the conference has a social media policy, this should be on their website or the program booklet. Make sure you’re familiar with the policies before posting something on social media. If someone is presenting unpublished data they may ask the audience to not share their research on social media. Use the hashtag if you are live-tweeting any sessions that the presenter doesn’t mind sharing on social media. If you could not attend some of the sessions, you can see if others have live-tweeted as well. In addition to using social media for sharing new scientific information from the conference, many conferences also have “tweep ups” where you can meet those you’ve interacted with on science Twitter in real life. If there is not a tweep up, create one yourself. Pick a time (ex: lunch, happy hour) and use the conference hashtag to set one up. It’s also helpful to write your Twitter handle on your nametag for the tweep up (and for the entire conference, in general).

After the conference

Review your favorite conference moments on social media and if there was something you missed during the conference, you can try getting up to speed on social media. If you haven’t already, connect with those you’ve met at the conference.

Networking

All of the things we’ve mentioned above could be considered networking, but here are some more general tips.

Before the conference

Networking is key even if you are not looking for a job – those connections can help you later on. Update your LinkedIn and your resume and prepare business cards. You might not consider being a student or postdoc a “business,” but having business cards is a good way to share your information quickly.

During the conference

Arriving at the conference can be overwhelming when you are presented with a sea of strangers. Try things like sitting at a table for lunch with people you don’t know or attending social events. I like to set goals for myself like meet ten new people each day. Exchange business cards. When you’re done networking for the day (or whenever you have a small break), write down some notes on the card so you remember who they are and what you’ve talked about.

After the conference

Follow up with those that you’ve met. Connect with people on LinkedIn or Twitter, for instance. If you’ve had a particularly meaningful conversation, and you have their email address, you can send them a note.

After reading this, you probably notice a trend: networking happens everywhere! Even when you aren’t “trying to network.” For some more networking tips not covered here, head over to the “How to Make Friends and Meet People at a Scientific Conference” blog post.

There’s so many great tips for getting the most from your conference experience so we unfortunately couldn’t cover them all. What are your top tips for getting the most out of your scientific conference?

本文来源：https://blog.addgene.org/tips-to-make-the-most-of-a-scientific-conference

怎样的求职信才打动HR？

I’ve read a lot of cover letters throughout my career. When I was a fellowship program manager, I reviewed them in consideration for more than 60 open positions each year. So I saw it all–the good, the bad, and the standout examples that I can still remember.

As a result, I’ve become the go-to friend when people need feedback on their job applications. Based on my own experience putting people in the “yes” (and “no”) pile, I’m able to give these cover letters a quick scan and immediately identify what’ll turn a hiring manager off.

While I can’t give you insight into every person’s head who’ll be reading your materials, I can share with you the feedback that I give my own loved ones.

1. THE BASICS

First things first, I skim the document for anything that could be disqualifying. That includes typos, a “Dear Sir or Madam” or “To Whom It May Concern” salutation, or a vibe so non-specific that it reeks of find-replace. I know it seems harsh, but when a hiring manager sees any one of these things, she reads it as, “I didn’t take my time with this, and I don’t really care about working here.” So she’s likely to pass.

You don’t need to thank the hiring manager “so incredibly much” for reading your application–that’s his job.

Another thing I look for in this initial read-through is tone. Even if you’re applying to your dream company, you don’t want to come off like you think someone entertaining your candidacy is the same as him offering you water at the end of a lengthy hike. You don’t need to thank the hiring manager so incredibly much for reading your application–that’s his job. If you align considering your application with the biggest favor ever, you’ll make the other person think it’s because you’re desperate.

So, skip effusive thanks and demonstrate genuine interest by writing a cover letter that connects the dots between your experience and the requirements of the position. Telling the reader what you’ve accomplished and how it directly translates to meeting the company’s needs is always a better use of space than gushing.

2. THE OPENING SENTENCE

If your first line reads: “I am writing to apply for [job] at [company],” I will delete it and suggest a swap every time. (Yes, every single time.) When a hiring manager sees that, she won’t think, “How thoughtful of the applicant to remind me what I’m reading!” Her reaction will be much closer to, “boring,” “meh,” or even “next!”

Compare it to one of these statements:

I’ve wanted to work in education ever since my third-grade teacher, Mrs. Dorchester, helped me discover a love of reading.

My approach to management is simple: I strive to be the kind of leader I’d want to work for.

In my three years at [prior company], I increased our average quarterly sales by [percentage].

See how these examples make you want to keep reading? That’s half the battle right there. Additionally, it makes you memorable, which’ll help when you’re competing against a sea of applicants.

To try it out for yourself, pick a jumping-off point. It could be something about you or an aspect of the job description that you’re really drawn to. Then, open a blank document and just free-write (translation: write whatever comes to mind) for 10 minutes. Some of the sentences you come up with will sound embarrassing or lame: That’s fine–no one has to see those! Look for the sentence that’s most engaging and see how it reads as the opening line for your cover letter.

3. THE EXAMPLES

Most often, people send me just their cover letter and resume, so I don’t have the benefit of reviewing the position description. And yet, whenever a letter follows the format of “I am skilled at [skill], [skill], [skill], as evidenced by my time at [place].” Or “You’re looking for [skill], and I am a talented [skill], ” I could pretty much re-create it. Surprise: that’s actually not a good thing.

If you write a laundry list, it’ll blend into every other submission formatted the same way.

Again, the goal isn’t just to show you’re qualified: It’s to make the case that you’re more qualified than all the other applicants. You want to make clear what distinguishes you, so the hiring manager can see why you’re worth following up with to learn more. And–again–you want to be memorable.

If you write a laundry list, it’ll blend into every other submission formatted the same way. So, just like you went with a unique opener, do the same with your examples. Sure, you might still include lists of skills, but break those up with anecdotes or splashes of personality.

Here’s a real, two-line excerpt from a cover letter I’ve written before:

If I’m in a conference room and the video isn’t working, I’m not the sort to simply call IT and wait. I’ll also (gracefully) crawl under the table, and check that everything is properly plugged in.

A couple lines like this will not only lighten up your letter, but also highlight your soft skills. I got the point across that I’m a take-charge problem solver, without saying, “I’m a take-charge problem solver.” Plus the “(gracefully)” shows that I don’t take myself too seriously–even in a job application. If your submission follows the same list-type format all the way through, see if you can’t pepper in an example or anecdote that’ll add some personality.

You want your cover letter to stand out for all the right reasons. So, before you click submit, take a few minutes to make sure you’re putting your best (and most memorable) foot forward.

via： https://www.fastcompany.com/3064221/i-review-hundreds-of-cover-letters-these-are-the-ones-i-instantly-rej

美国护理专业的申请

优势：

美国移民局特为外国护士设立特殊优惠的移民签证种类“i-140（也叫EB－3;专长移民是EB-1，投资移民是EB-5）”，从属于急需专业人士签证，不是劳工签证。护理学这个专业前几年的移民倾向太严重了，就算学校收了你，签证官也不肯放人 可是大概从06年开始吧，护士移民的优势就没有了，所以有兴趣读这个专业的同学还是应该能拿到F1签证的~

美国注册护士RN(RN，Registered Nurse 的简称)的收入多数为4－80000美元，工作五年可申请加入美国籍，并可申请家长移民美国。

区别：

中国的医院运行机制大家都比较熟悉了，护士由护士长管，护士地位不高。美国医院是因注册护士管理和运作的，是护士（管理层）负责人医生“干活”，指引医生进行手术治疗，医生与医院仅是签署合同关联，如此既调低了医院的管理成本，又能保证医生集中精力在私人诊所从事专项的、高水平的医疗服务。我国香港、澳门、台湾的医院大都也是因注册护士管理。

就业：

工作十分辛苦，不同等级的工作内容职责差异:

护士和医生一样，由于直接对病人负责，所以其营业执照是受州政府和联邦政府的控制的(一般是由州政府控制)。

护士分为好几个层次，最底层的是护士助理(Nurse Assistant)，在医院或者医疗单位直接听从注册护士 (Registered Nurse) 或者更高层次的护士或者经理的指派，一般做最低级最脏最累的活，例如喂饭、帮病人洗澡等，而且薪水也最低 (薪水有地方差异，一般情况下每小时10美元)。这类护士助理并不需要从政府那里取得执照，也不需要特定的技能，一般有一定的背景就可以做相关的工作。

比 Nurse Assistant 稍微高一个“层次”的护士称为 PCT (Patient Care Technician)，字面翻译为“病人护理技工”，不知对应于国内医院什么样的头衔 。PCT 也无须州政府颁发营业执照，但是通常需要一定的培训，取得相应的证书。PCT 薪水也比护士助理要高些，通常在 12-20 美元/小时。

第二种就是注册护士 (RN，Registered Nurse 的简称)，这类护士是美国目前最紧缺的，一方面是因为人的寿命在延长，人口老龄化很快，医院等需要大批的护士，另一方面这类护士必须取得营业执照方可上岗，而取得营业执照的先决条件就是先通过州政府的相关考试，而考试的前提就是至少有护理专业的专科文凭。事实上许多拥有护理博士文凭的专业人士的头衔也是注册护士。美国是个在许多方面很死板很保守的国家。比方说，某个PCT (病人护理技工) 很聪明能干，能够胜任注册护士的工作，但是他/她是不能做注册护士相关的工作的，否则被视为违法行为，如果被政府发现了，会得到严厉惩罚的。目前美国政府承认中国的文凭，但是这并非意味著你有护理专业的专科或者本科文凭就有报考的资格 (说句题外话，一般的医学专业不是护理专业，哪怕你有硕士文凭，也是不能报考的)，因为政府对非美国的护理专业所学习的课程内容会审查的，一般与其要求总会有所差别。

第三类护士称为开业护士 (Nurse Practitioner)，地位大体和医生差不多，但是还是属于护士的类别。报考开业护士的先决条件就是需要护理专业的硕士或者以上的文凭。单独开业的护士必须要有 Nurse Practitioner 的营业执照。这类护士通常做很专业的技术工作或者管理工作，刚开始比较难以取得，所以这里略过不谈。

以上三种中；

注册护士 (Registered Nurse)，这是目前最紧缺的护士，原因之一就是这类护士必须要拥有政府部门开发的执照。新招的注册护士薪水每小时约 25 美元，俱备多年经验的护士一般可以挣到 30 多美元。由于工作的特殊性，很多护士每周工作三天。每天 12 小时，如果加班或者在法定的节假日上班，工资更高一些。所以有些不怕吃苦工作玩命的护士每年可以挣到 10 万美金，当然，这些都是血汗钱。一般每个注册护士得管理好几个病人，通常是4个，重症病房一般是两个，直接向医生汇报，很大程度上相当于医生助手；

每个注册护士会配置一个或者几个护士助理。由于属于长时间的体力劳动，所以工作相当辛苦 (这也是美国为什么护士短缺的另一个理由)。有意取得这类护士执照的人，如果确实拥有护士类文凭，有相应的工作经验，最好在国内直接报考取得执照 (外加英语语言考试，例如托福)。

那么到底又有哪些途径能够在美国作为护士身份就业呢？

可以在国内就报考，也可以直接申请美国大学的护理专业。

中国学生怎么考国外注册护士？

关于国外注册护士考试措施及待遇:

凡在中国国内高中毕业后并且得到三年正式护理教育的毕业生，都可以在全世界多个考点考中美国cgfns或登记护士执照（nclex-rn，简称rn），非英语国家的护士再提供托福83分、口语26分（雅思a类6.5分、口语7分）的成绩，有美国雇主的聘用，就可以拿到签证去美国就业，并可全家移民。一般注册护士首年的年收入约为50,000美元，以后能达到6－80,000美元，开业护士薪酬100,000美元以上，还有医疗保险等其他福利。

报考有两个步骤：第一是取得考试资格 (有护理专业大专或者以上的文凭，并且修过所报考州的卫生局规定的课程。每个州的规定略有差别，但是大同小异);必须注意的是，有些专业知识更新比较快的课程，或者很重要的课程，例如生理与解剖，有效期一般在5到7年，亦即如果你这样的课程是在7年之前修习的，那么它是无效的，你必须重修。俱备一定的工作经验有助于得到雇佣，有时，工作经验还能抵消一到两门课程。从州政府取得报考资格后，第二步就是通过州政府的考试，也就是取得上班执照。这个考试据说是很不容易的，在国内护理专业学到的知识肯定是不够的，通常报考者得自学一些教材(至于学习什么教材，我就一窍不通了)。如果能通过这个考试，一般情况下你就能得到雇佣。目前有一些直接从大陆过来的护士 (以女性为主)，他们都是通过这一途径走来的。

美国大学的护理专业招生情况？

     有些不俱备报考条件又希望以后当护士的，往往只能先读护理专业。尽管护士紧缺，但是美国的大专院校有限，招生有限，教师资源有限 (因为教师也得取得执照)，每个班的规模是有限制的 (例如 30 人，不能因为生源很多就扩大班级，这是违法的，同时也是学校主动降低教学质量的表征)，所以录取比率很低。

    优秀大学和名牌大学的护理专业通常只招收本科或者以上学位的学生，报考这类大学的护理专业往往还容易被录取一些，因为这类学校的学费较贵 (特别是私立大学，不受政府的资助，更贵)，而且要求也高，所以淘汰率也高，学习也比较辛苦。所以竞争最激烈的往往是那些社区学院 Community College) 的专科，因为学费低廉，每个学分只要几十美金 (当然这些学费远不足以维持学校的运转，这些公立学院的主要资金通常来自当地政府，特别是县政府)。如果你只有志当个注册护士的话，社区学院专科和名牌大学的本科是一样的，而且社区学院学习相对来说也稍微轻松一些。这些社区学院大体上相当于我们的地方专科学校，没有任何学术目的，大部分教师都是兼职，规模也小 (但是条件一般都不差，例如电脑设备，很多还强过好大学)。在加州、东北部等经济比较发达的州的大城市，因为就业机会较多，报考护士的相对来说就少很多，竞争也就小很多，在那些经济不太发达的州，例如南部和西部山区各州，竞争就很残酷，有的学生 (包括美国本土的) 的先修课都修完了好几年了，眼见就要失效 (例如生理解剖就有个有效期的问题)，还是没有录取;如果还想下年继续申请的话，那么这门快要失效的课程就得重新选修，否则连资格也没有。

一个专科文凭一般需要修60到72学分 (每个学校不一样)，通常学生应该在两年时间内完成。由于录取者一般得俱备不少先行课程的基础，那些先行课很多是可以计入学分的，所以，实际上这两年大部分学生只需修习40-60学分，这样平均下来，每个学期就只需修习 12 学分的课程；大部分人起早贪黑，整天念书做作业，还时常担心考试通不过。倒不是这些课程有多难，但是课程内容太多太杂，作业很多，有些课程有几本教材，教材动不动就是六、七百页或者一千多页。那些学生身材强壮也好，瘦弱也罢，每天都得背着那二、三十斤的书包在校园穿梭 (美国的教科书大抵是精装，一般很重，一本书往往有好几斤)。除此之外还有医院实习，从第一学期就有，每周得花两个全天或者三个半天，而这些是不计学分的 (但是学生必须通过实习，否则就会被除名。

这样的专业的毕业生因为最终都得参加州政府的考核 (拿取执照)，如果考核通过率不佳，州政府就怀疑学校的教学质量，进而影响经费的申请，也影响学校的声誉。所以哪怕是社区学院这样不入流的学校，象护士这样的热门专业，其教学质量也是抓得很严的。一般说来，如果有门课程考核达不到75分，学生就会被劝退或者直接除名。所以每个学期下来 (特别是第一学期)，总有一些学生遭到淘汰。。一般说来，从第一个学期开始能坚持到最后一个学期毕业的，一般只有 30-40% 的比例。其余的 60-70% 的学生，有的得“留级”重修某门课程，有的则干脆被踢出去了 (比方说如果你的某门功课太糟糕，或者有两门功课需要补考)。

举个具体的实例，一个学生韩娜 (Hannah，一个韩国人，在读护士专业)说，她们年级有个中国男同胞，似乎是白求恩医学院 (似乎位于吉林吧?我不太清楚) 毕业的，而且有临床经验，也在这里的社区学院读护士专业，应该说基础相当不错吧?这位仁兄第一个学期某门课程第一次考试据说就砸锅了，可能是看书学习不刻苦，悠忽了一把，结果就得了五十几分。这下麻烦了，虽然那门课程有四次考试 (外加作业)，理论上他还可以得到 75 分，是不是?可是学校不这么认为，而是将他叫到了办公室，声明第二次考试必须拿到 A (亦即 90 分以上)，否则除名。这是真的，因为有对黑人双胞胎考试就得了四十几分 (据说这对双胞胎学习十分刻苦，每天凌晨六点就到学校，只是据说似乎智力有些问题)，连警告也没有，立即被除名了。当然学校很善良，发出警告后估计这位仁兄会焦虑，所以学校出钱给他找了个心理医生 (强制性的) 咨询了一次。这位仁兄当然急了，以后自然夹着尾巴做人，可是有次忙中出错，去医院实习时竟然迟到了，而那个带队的老师是个俄罗斯籍贯的。俄罗斯人向来以死板著称，这还了得?立即将这位仁兄带到办公室又来个警告：以后实习报告迟交或者实习迟到的话都算违规，这次算口头警告，第二次违规由学校开俱书面警告，第三次除名，哪怕你的学习再好也除名。还好，这位仁兄第二次考试40个选择题对了36个，殊不知他们班上28人就三人上了90分。只是可惜得很，这位仁兄最后还是被学校刷了下来，毕不了业。

提供的护理专业学位有：

本科：BSN(Bachelor of Science in Nursing)

硕士：MSN(Masterof Science in Nursing）

开业护士NP(Nurse Practitioner)或者Ph.D.

Basic的BSN是四年，前两年是基础课，GPA够好的话可以申请进入后两年的专业program，毕业前后参加登记护士执照（NCLEX-RN考试，通过后获得RN资格;

加速的BSN是你已经有一个本科学位了，想再拿一个护理本科学位的课程。一般是三年。

护理这个专业很讲究临床经验，所以很多学校招MSN或以上的学历都要求有临床的工作经验.

靶向大脑的基因治疗策略

Genome editing has rapidly transformed biomedical research and has demonstrated therapeutic promise via successes in tissue culture, ex vivo, embryonic editing, and animal models of human disease. For successful translation, a genome-editing therapeutic must be safe, effective, and ideally straightforward to manufacture. DNA encoding the RNA and protein components of a CRISPR-derived genome editing enzyme such as Cas9 can be delivered by adeno-associated virus (AAV) with high efficacy, but safety may be a concern and the manufacturing burden is substantial [1]. One emerging alternative is the delivery of genome-editing enzymes in the form of a pre-assembled ribonucleoprotein (RNA and protein, or RNP) complex. This approach is appealing because it ensures a tight therapeutic window: the RNP will be degraded in less than 24 h. By contrast, viral expression can result in prolonged expression of the genome-editing enzyme that persists for days. This has been associated with an increased prevalence of unintended off-target edits compared with RNP-based editing [2]. The nuclear localization signal (NLS) has routinely been used to ensure transport of RNP from the cytosol to the nucleus, but transporting a large genome-editing enzyme from the cell exterior to the cytosol presents a distinct challenge. Several strategies have been successful in promoting the cellular import of Cas9 RNP, such as modification of the Cas9 protein to include incidentally membrane-disrupting NLS sequences [3], or appending negatively charged domains to the Cas9 protein to promote its interaction with polymers that promote cellular entry [4]. The Murthy lab has developed an approach that uses a nucleating gold nanoparticle conjugated to single-stranded DNA to recruit Cas9 RNP, all of which is coated in a cationic polymer that facilitates delivery across the cell membrane, dubbed CRISPR-Gold [5].

The brain is an appealing site for initial forays into therapeutic genome editing because it is anatomically insular, allowing straightforward surgical access and providing an immunoprivileged status that ameliorates the risks associated with introduction of viral vectors [1] and/or genome-editing enzymes [6]. In a recent study by Lee and colleagues [7], the Lee and Murthy labs collaborated in using CRISPR-Gold to deliver either Cas9 or the analogous Cas12a (Cpf1) to the mouse brain. CRISPR-Gold carrying either Cas9 or Cas12a was stereotactically injected into the mouse hippocampus or striatum, performing efficient genome editing as detected by fluorescent reporters. A mouse model of FXS, based on an Fmr1 knockout (KO), was used for experiments probing the ability of genome editing to treat autism. FXS is a common, inherited single-gene form of autism spectrum disorder (ASD), and drug treatments are largely inadequate. Importantly, the mGluR5 gene has emerged as a promising candidate for genetic therapy, since it can contribute to FXS as well as other ASDs. To test whether reduction of mGluR5 could diminish autism-associated phenotypes in the FXS model mice, CRISPR-Gold bearing a Cas9 RNP targeting mGluR5 was injected into the striatum. In treated striatal cells, 15% of mGluR5 loci were disrupted, leading to a ∼40% reduction in mGluR5 mRNA or protein abundance, via qPCR or immunostaining, respectively. Behavioral studies of edited FXS model mice showed a marked reduction in two established hallmarks of mice with autistic phenotypes: marble-burying and spontaneous jumping. This promising result was bolstered by the observation that CRISPR-Gold treatment had no discernible impact on mouse locomotion. Other tests for toxicity showed that CRISPR-Gold treatment was not associated with cell death in vivo or changes in the properties of cultured neurons.

It is illustrative to evaluate these CRISPR-Gold results in comparison with AAV-mediated delivery, which was quickly adopted by pioneering genome editors for use in the brain (Figure 1). Delivery of AAV encoding Cas9 and its single guide (sg)RNA (Cas9/AAV) has been particularly successful in generating models of neurodegenerative diseases and other diseases of the brain and nervous system. A 2016 report from the Zhang laboratory reported Cas9/AAV-mediated editing of the MCP2 gene in the brains of mice, resulting in disruptive edits in a majority (68%) of the cells in the injected tissue. The observed robust viral distribution throughout the tissue and editing in postmitotic neurons allowed generation of a mouse model of Rett’s syndrome bearing the corresponding behavioral phenotype [8]. AAV has also been applied in therapeutic models; for example, the Davidson laboratory edited the disease-causing allele in a transgenic mouse model of Huntington’s disease, observing reductions in the levels of mutant huntingtin protein of up to 80% following an injection of Cas9/AAV into the brain [9]. A similar approach was recently reported for in vivo editing of the mutant alleles of the APP gene that underlies Alzheimer’s disease, another condition with dominant inheritance [10]. Cas9/AAV vectors were injected into the hippocampus of transgenic adult mice expressing multiple copies of the human mutant APP allele, and selectively generated indels (1.3%) in the mutant allele allowing a decrease in pathogenic amyloid-β protein levels in the brains of the mice [10]. Neither example of Cas9/AAV editing disease-causing mutant alleles demonstrated an associated therapeutic phenotype in mice, as was convincingly demonstrated with the CRISPR-Gold phenotype in the recent report by Lee and colleagues. However, the model systems differ, and it is reasonable to anticipate that Cas9/AAV-mediated editing might perform comparable editing in an FXS model system.

One apparent advantage of AAV-mediated delivery is that the viral particles spread throughout the brain in mice and primates. By contrast, RNP as delivered in isolation [3] or by CRISPR-Gold [7] tends to edit only cells within an area of several cubic millimeters. This suggests a potential hurdle for translation. Another potential concern related to the use of CRISPR-Gold in humans is its introduction of heavy metal, which is known to be toxic. However, this issue is tempered by the knowledge that the gold constitutes a miniscule fraction of the nanoparticle assembly by weight, and that genome editing is ideally a one-time treatment that avoids the accumulation of gold that would be associated with a treatment that is repeatedly dosed. With additional development, RNP delivery may prove itself as a leading strategy for therapeutic genome editing of the brain.

【References】
1. Colella, P. et al. (2018) Emerging issues in AAV-mediated in vivo gene therapy. Mol. Ther. Methods Clin. Dev. 8, 87–104
2. Kim, S. et al. (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012– 1019
3. Staahl, B.T. et al. (2017) Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat. Biotechnol. 35, 431–434
4. Wang, M. et al. (2016) Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 113, 2868–2873
5. Lee, K. et al. (2017) Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat. Biomed. Eng. 1, 889–901
6. Leong, Chew Wei (2017) Immunity to CRISPR Cas9 and Cas12a therapeutics. Wiley Interdiscip. Rev. Syst. Biol. Med. 10, e1408
7. Lee, B. et al. (2018) Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndromefrom exaggerated repetitive behaviours. Nat. Biomed. Eng. 2, 497–507
8. Swiech, L. et al. (2015) In vivo interrogation of gene func- tion in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33, 102–106
9. Monteys, A.M. et al. (2017) CRISPR/Cas9 editing of the mutant huntingtin allele in vitro and in vivo. Mol. Ther. J. Am. Soc. Gene Ther. 25, 12–23
10. György, B. et al. (2018) CRISPR/Cas9 mediated disruption of the Swedish APP allele as a therapeutic approach for early-onset Alzheimer’s disease. Mol. Ther. Nucleic Acids 11, 429–440

Via: https://www.sciencedirect.com/science/article/pii/S1471491418301473?via%3Dihub

靶向RNA的小分子药物研究进展

昨日，专注于开发靶向RNA的小分子药物的生物医药公司Arrakis Therapeutics宣布完成数额为7500万美元的B轮。同时，该公司创始人、总裁兼首席执行官Michael Gilman博士在访谈中披露，该公司的药物发现平台，针对编码著名”不可成药“的致癌蛋白myc的mRNA，已经取得出色的筛选结果。这一新闻将靶向RNA的小分子药物再一次推到了聚光灯下。

那么我们为什么需要开发靶向RNA的小分子药物？发现和开发靶向RNA的小分子药物需要注意些什么问题？最近在这一领域又有什么新的进展？今天药明康德的微信团队将结合公开资料，与读者共同探索这些问题的答案。

为什么要开发靶向RNA的小分子药物？

小分子疗法绝大部分的靶标都是蛋白质，这一策略在过去数十年来也带来了大量好药和新药，据估计，接近99%的口服药物靶向的是致病蛋白。然而，这需要小分子药物能够与致病蛋白中的特定位点或“口袋”相结合，而对于大部分（接近85%）的蛋白来说，它们没有适合小分子药物结合的位点。这意味着它们用传统手段“无法成药”。

而且，蛋白只占了基因组信息的极少部分。人类的基因组中，只有1.5%的序列编码了蛋白质，和疾病相关的蛋白更是只占其中的10-15%。毫无疑问，如果小分子药物的靶点能超越蛋白质，将给新药研发带来新的变革。

▲靶向RNA，将给我们带来更多的成药选择（图片来源：参考资料[8]）

RNA就是这样一种潜在的靶点。在正常细胞中，RNA有着重要的生理功能——mRNA携带了基因的遗传信息，指导蛋白质的合成；非编码RNA则调控基因的表达。靶向RNA有着多种好处：由于处于蛋白质的上游，靶向RNA有望直接对蛋白质的翻译效率进行上调或下调，解决蛋白“不可成药”的难题；RNA在人类基因组中极为丰富，产生非编码RNA的序列更是占到了基因组的70%，丰度比编码蛋白质的序列高出一个数量级；而近期一些概念验证性试验的成功，也让我们看到了希望。

开发靶向RNA的小分子药物的原则

根据《Nature Reviews Drug Discovery》 上的一篇综述，目前靶向RNA的小分子药物根据靶向的RNA结构，可以被分为三大类，它们分别靶向RNA中：

1. 多个密集螺旋结构（multiple closely packed helices）
2. 不规则的二级结构（irregular and usually bulge-containing secondary structures）
3. 或是三联体重复序列（triplet repeats）

第一类药物中已经出现了多种候选化合物，包括四种分子——linezolid、ribocil、branaplam、以及SMA-C5。这些分子“靶向了复杂的RNA结构模块，且每一个均有很高的QED（quantitative estimate of drug-likeness）分值”，表明其具有潜在的成药潜力。值得一提的是，这些分子都是通过表型筛选找到的结果，之后才被确认具有RNA结合的属性。因此，尽管它们开了一个RNA靶向新药研发的好头，但我们尚无法从中推导出明确的RNA靶向方针。

▲第一类药物中已彰显出潜力的四款分子（图片来源：参考资料[8]）

在另两类药物中，研究人员们最初的筛选过程，就是为了找到能靶向RNA的小分子药物。这些筛选方法包括高通量筛选、使用专注这一方向的化合物库、受结构启发的设计、基于片段的筛选方法、以及计算机模型。这带来了大量不同的潜在RNA靶向分子，让我们对靶向RNA的原则与方针有了更深的认识。这也是像Arrakis等新兴生物医药公司开发的方向。

如何靶向RNA？

由于RNA与蛋白质结构上的显著不同，因此，RNA是否具有“可成药性”是一个需要解决的问题。与蛋白不同，RNA主要由四类核苷酸组成，带有大量电荷，也比蛋白质更为亲水。然而，RNA在折叠后的具有复杂的三维结构。这些复杂结构有望带来足够的成药构象，让小分子药物结合与识别。

▲小分子药物也可识别RNA结构（图片来源：参考资料[8]）

过去“意外发现”的小分子RNA靶向药物支持了这一观点。上文中提到的linezolid和ribocil从传统药物化学的角度看，都是非常杰出的分子——它们符合经典的“里宾斯基五规则”，有较小的极性总表面积（tPSA），也有较好的细胞膜穿透性。此外，它们没有明显的毒性。最关键的是，它们能结合RNA靶点结构上的“口袋”。这与许多靶向蛋白质的小分子药物如出一辙，也支持了靶向RNA的小分子药物的研发。

如同蛋白质靶向药物一样，靶向RNA的药物也需要对RNA结构的了解。该综述的作者们指出，良好的RNA靶点，结构上应有足够的“信息量”。目前，具有成药潜力的几个分子都靶向复杂的RNA结构。而倘若靶向简单的RNA结构，可能会影响到靶向分子的亲和力与特异性。

▲好的RNA靶点应有足够的“信息量”（图片来源：参考资料[8]）

筛选靶向RNA的小分子药物的八大指导方针

在了指导未来的新药筛选，研究人员们整理了几大方针，供后人参考：

• 专注于具有足够复杂度，结构独特的RNA模块。这些模块有望带来高质量的“口袋”，方便小分子药物结合。这些模块在大型RNA分子中较常见。
• 谨慎决定靶向RNA的小分子基本结构。在定义这些结构前，我们还需要更多的研究。过去几十年来，靶向蛋白质的经验或许有用。
• 在核糖体RNA上取得的成功，未必能旁推到其他领域。这些RNA在细胞中高度富集，靶向它们的分子可能有特殊的性质。
• 对于那些高度碱性、具有插入特性、或是高度疏水的小分子化合物，对实验结果的解读要尤其谨慎。这些化合物可能与RNA有很高的亲和力，但可能会有严重的脱靶效应。
• 留意那些能够靶向RNA-蛋白质相互结合的分子，譬如branaplam和SMA-C5。它们有望带来出色的特异性与亲和力。
• 找到那些具有“高信息量”的结构。相比蛋白质，RNA的一大优势在于我们有许多定量的化学方法去帮助我们完成这些工作。
• 在现有工具的基础上，开发全新的工具，去更好地了解RNA的复杂结构。
• 找到那些具有明确治疗机制的靶点，并针对它们寻找潜在的新药。

这一领域的最新进展

我们高兴地看到，这一领域的新兴生物医药公司的研发方向与这些建议不谋而合。前文提到的Arrakis公司在过去两年多的时间里，构建专属于靶向RNA的小分子筛选和优化平台。它包括名为Tryst的RNA靶标筛选系统，能够预测RNA序列上适于小分子药物结合的位点和结构。以及帮助分析小分子药物与RNA之间的结合机制，并且选出最佳候选化合物进一步开发的Pearl-seq系统。

根据Michael Gilman博士的描述，这一药物开发技术平台目前已经可以开始大规模地针对多种不同RNA进行药物筛选。该公司聚焦的第一个RNA就是编码myc蛋白的mRNA。Myc是最初发现的致癌基因之一，在接近40年前就被发现了。它编码的转录因子myc蛋白与多种人类癌症相关。Arrakis公司已经通过多种筛选手段寻找与myc RNA结合的小分子化合物，用Michael Gilman博士的话来说，发现了靶向这一RNA的不少奇妙的化合物。Arrakis公司靶向myc RNA的策略是找到能够与RNA结合并且阻止RNA翻译生成蛋白的小分子药物。RNA在与核糖体结合翻译蛋白时，需要解开折叠的三级结构变为线状结构，而如果使用小分子药物将RNA的三级结构固定住，让它们无法被解开，就可以防止蛋白的产生。

而由Scripps研究所（The Scripps Research Institute） 教授Matthew D. Disney博士创建的Expansion Therapeutics公司，在开发靶向三联体重复序列的小分子化合物方面也获得了出色的进展。Expansion Therapeutics公司的药物开发工具能够将上千个不同的RNA结构与共价固定在芯片上的小分子化合物库进行相互筛选。这一筛选结果能够发现与特定RNA折叠结构结合的高质量的小分子化合物，它们可以被传统的药物化学手段进一步优化。

▲Expansion公司的筛选平台（图片来源：Expansion公司官网）

Matthew D. Disney博士领导的研究团队还开发出了能够切断富含CUG三联体重复序列的RNA的小分子化合物。CUG重复序列是导致1型强直性肌营养不良（DM1）的基因变异。DM1是一种无法治愈的神经肌肉疾病。在近日发表在《PNAS》的一项研究中，这种靶向CUG重复序列的小分子药物cugamycin，能够有选择性地靶向导致疾病的RNA结构，并且在临床前动物试验中改善小鼠的DM1症状。

在探索靶向RNA的小分子药物领域还有其它新兴生物技术公司。例如，去年，Skyhawk Therapeutics在麻省正式成立，力图开发靶向RNA的小分子药物，修复RNA剪接时出现的外显子跳跃错误，治疗神经疾病和癌症。该公司去年与新基（Celgene）公司达成为期5年的研发合作协议。

目前，这些疗法仍然处于临床前开发阶段，它们仍然需要在人体中临床试验的验证。我们期待这一天尽快到来，让我们进一步疗法靶向RNA的小分子药物在治疗人类疾病方面的潜力。

参考资料：

[1] Expansion Therapeutics. Retrieved April 19, 2019, from https://www.expansionrx.com/expansion-repeat-disorders/

[2] Arrakis Therapeutics. Retrieved April 19, 2019, from http://arrakistx.com/

[3] Drugging the undruggable and other challenges on the road to targeting RNA with pills. Retrieved April 19, 2019, from https://www.statnews.com/2019/04/19/michael-gilman-rna-arrakis/

[4] Ribometrix. Retrieved April 19, 2019, from http://www.ribometrix.com/index.html

[5] RNA研发热！这12家公司正在推进靶向RNA的小分子药物. Retrieved April 19, 2019, from https://mp.weixin.qq.com/s/df8pOurBsAeXs57CBHuJOQ

[6] Nature深度综述：小分子靶向RNA，这些原则你都知道吗？ Retrieved April 19, 2019, from https://mp.weixin.qq.com/s/kpIoktgNTmlXNCfz_mCosQ

[7] RNA-targeting Compound Shows Ability to Limit Muscle Damage in Early Myotonic Dystrophy Type 1 Study. Retrieved April 19, 2019, from https://musculardystrophynews.com/2019/04/08/rna-cutting-compound-shows-ability-to-limit-muscle-damage-in-myotonic-dystrophy-type-1-in-early-study/

[8] Warner et al., (2018). Principles for targeting RNA with drug-like small molecules. Principles for targeting RNA with drug-like small molecules. Nature Reviews Drug Discovery. https://doi.org/10.1038/nrd.2018.93.

从“死胡同”到绽放光彩——Cre-Lox神经遗传技术的20年

本文转自：https://zhuanlan.zhihu.com/p/26257864

撰文 | 钱卓 (美国奥古斯塔大学佐治亚医学院大脑与行为研究所所长、云南西双版纳生物医学研究院院长)

翻译 | 王德恒（云南西双版纳生物医学研究院大脑破译中心研究员）

一百多年前，西班牙神经解剖学家、1906年诺贝尔奖得主Ramón y Cajal在显微镜下观察到大脑不同神经元的微观结构，惊叹于各种脑神经细胞如同夜空闪烁的星星一样美丽。近代神经科学的新篇章由此开启[1]。

20世纪70、80年代，随着单细胞标记和膜片钳技术的出现，神经元结构与功能的研究得到了飞速的发展[3,4,5,7]。分子生物学技术也进一步把神经科学领域推到了更深层次的基因和蛋白质水平[8,9,10,11,12,13,14,15,16]，大大推动了突触可塑性机理的研究[17,18]以及利用遗传工程技术增强大脑学习记忆和认知功能的研究[19,20]。

20世纪80年代后期和90年代初，在Mario Capecchi, Oliver Smithies和Martin Evan三位先驱基因打靶和胚胎干细胞技术（该工作使他们在2007年共享诺贝尔生理学或医学奖）工作的基础上[21]，几个著名的实验室开始尝试创建突变小鼠用于基因在发育、肿瘤和免疫方面的功能研究[23,24,25]。随后，Alcino Silva, Seth Grant和Thomas O’Dell利用基因的敲除技术来研究CaMKII或Fyn蛋白激酶在突触和记忆功能 [26,27,28]。这些工作为基因和大脑功能的研究开辟了新的思路。然而，大家很快发现，许多全身基因打靶（基因在单个受精卵时就被敲除）构建的小鼠模型，由于遗传补偿缺少表型，或由于被敲除的基因在许多器官发育中的重要作用，发生了产后夭折或发育不良的情况。比如，在CaMKII基因敲除的小鼠身上，人们观察到了自发性的癫痫症状，而在Fyn基因敲除的小鼠中，大脑海马中的齿状回明显存在神经发育缺陷。这两种情况立即引起了关于如何解释模式小鼠脑功能缺陷的激烈争论：到底是先天发育缺陷，还是该基因在成年大脑思维过程中的调节作用，引起了小鼠的学习记忆功能下降？鉴于NMDA受体在可塑性中的关键作用，麻省理工学院的李育庆等人[29] 着手去证明其在学习行为上的开关作用，但是他们发现NMDAR1基因敲除的新生幼鼠由于大脑发育的缺陷（如吸吮反射的缺失），出生15小时后就全部死亡了。这些夭折的小鼠丘脑的固有触须桶状结构（stereotypic whisker barrels）没有形成，说明了NMDA受体在脑结构发育中的关键作用[29]，对发育神经科学家来说，是一个令人兴奋的发现。相反，这样的结果对于认知和行为神经科学家来说，却是令人失望的，他们无法利用全身基因敲除技术来研究成年大脑的思维活动与神经机理。大家渴望能有一种新的遗传工程技术，不仅能避开发育过程，而且还能把任何一个基因只在特定的脑区的某一类细胞中进行条件性删除。

年轻而喜欢冒险的岁月

我对研发新一代遗传工程技术的兴趣起源于1990-1993年在哥伦比亚大学的那段经历。1990年，我从明尼苏达大学博士毕业，来到Eric Kandel（2000年诺贝尔生理学或医学奖得主）实验室作博士后。密歇根大学Bernie Agranoff教授曾在1964提出长时记忆需要新的蛋白质合成和基因表达。因为我看了一本描述Kandel在海兔研究中“争斗内幕”的科普书（Explorers of the Black Box: The Search for the cellular Basis of Memory），我在Kandel实验室主动避免用海兔做研究，而是选择大鼠为研究对象，寻找并克隆能被大脑活动所调节的基因。在我之前，Kandel两个极为优秀的博士后（在其他实验室读博期间就已发过一两篇Cell文章）由于技术原因，都徒劳而归。1990到1993年，我利用蛋白抑制所引进的即早基因“超表达”的现象，首次成功地差异克隆了一批被大脑活动所调节的新基因，包括大脑特异的即早基因BAD1 (Brain Activity-Dependent)、组织型纤溶酶原激活剂（tPA）和一种促细胞分裂的原活化蛋白激酶磷酸酶[30,31]。后来, BAD1基因也被约翰霍普金斯大学的Paul Worley实验室分离并另命名为Arc (Activity-regulated cDNA)[32]。对我来说，分离出大脑新的基因固然令人兴奋，如何检测这些基因在脑认知记忆中的功能更是一个巨大的挑战。大家已经知道基于反义寡核苷酸的“knock-down”方法既不精确也不可靠，而全身基因敲除看似是个不错的选择，但也有如上所说的局限性。

后来，我偶然发现杜邦公司的Brian Sauer博士的一篇论文报道了Cre重组酶在哺乳动物培养细胞株中对环形质粒转染时成功切除标记基因的现象[33]。该文在结语提出了一个很重要的问题：“不知Cre是否也能引起哺乳动物细胞染色体上内在基因的lox位点重组？” 这引发了我尝试用它来开发脑区和细胞类型特异性的基因敲除和/或转基因过表达新技术的念头。我知道，所有的教科书和文献描述DNA复制和重组与细胞分裂密切相关（如减数分裂或有丝分裂; 图1）。这个基本的学说深深地印在每个人的脑中，从Sauer的论文开篇句不难看出，他也对此深信不疑：“近年来，有丝分裂期的哺乳动物的细胞DNA重组的控制过程已成研究的热点……有丝分裂重组在免疫系统的发育和功能中起着核心作用。” 当时大家都知道大脑的神经元在出生后就不再进行有丝分裂（除了嗅球和海马体中的齿状回）。因此，任何人打算在成年大脑上进行DNA重组的工作，也意味着其科研生涯必将走入死胡同。大自然已经明确表明，所有的脑肿瘤都不是发生在神经元，而是在还能分裂的胶质细胞。因此，要想利用DNA重组的方式在大脑中进行区域和神经细胞特异性基因敲除显然是不可行的。

►图1

但我很想弄明白大脑基因的功能，只是当时只有少数的实验室拥有用来制作传统基因敲除小鼠的基因打靶设施和胚胎干细胞。因此我放弃了申请大学Faculty（教职）的机会（那时大家一般只做一个博后），而联系了犹他州的Mario Capecchi和麻省理工学院的Susumu Tonegawa两位大师，表达了想做第二个博后的愿望。高兴的是他们都同意了。于是，我去向Kandel寻求指导和建议，他建议我去Tonegawa的实验室，这让我有些诧异（Tonegawa因发现免疫抗体多样性的遗传机理在1987年诺贝尔生理学或医学奖，然后转行闯入了Kandel所在的学习记忆领域这一地盘，因此他俩关系很僵)。我也取得了Kandel实验室另一博士后Mark Mayford的同意使用他克隆的CaMKII启动子，该启动子将在小鼠出生2到3周后激活并只在前脑的主要细胞如锥体细胞上表达[34]。对于Kandel的指点和Mayford的慷慨我万分感动。直到数年后Mayford喝了酒后才偷偷地告诉我，原来Kandel当时看中了我的想法会注定失败，正好来个一石二鸟，为消耗掉一个现在的和一个将来可能的竞争对手，“绅士般”地顺水推了这一舟。

1993年秋天，我来到麻省理工学院。在我向Tonegawa做了只有15秒的简短介绍后（大名人们都太忙了），免疫专家的他并没有对我想研发的Cre-loxP神经遗传技术有什么看法，而是放任自由。我惊讶地发现这个有40个博后和学生的大实验室弥漫着丛林动物生存法则的氛围。不过总的来说，麻省理工学院还是一个非常令人兴奋的地方。

开发Cre-loxP神经遗传技术，面临着三大风险：

（1）当时的教科书中认为，DNA重组只能发生在分裂细胞中，而我的直觉是，疱疹病毒（引起唇疱疹）感染外周神经节并以某种方式进行自身复制（迄今为止，该机制仍然是未知的），而神经节的神经元“可能”没有分裂；

（2）实验的周期长而且程序复杂，两年内没有反馈，何况这是第二个博士后，哪怕在原理上能成立，如果在技术上出了差错，我仍将求职困难。我必须构建一系列质粒并创建出至少三种不同的小鼠品系，然后着手开始多年的交叉繁殖工作（图2A，B）。在那个年代，能够得到一只基因突变的小鼠，已属非常难得。事实上，因为复杂的程序、冗长的项目周期和昂贵的成本，再加上得到的可能是没有任何表型或非己想要的结果，学术界的一个残酷现实就是只有少数几个成功的人能得到一份大学的教授工作，另一大批没有成果的、不幸的博士后们只能从学术界消失。

（3）我给自己挖的另一个坑是：我选择NMDAR1而不是BAD1/Arc作为条件性敲除的基因。我们知道，如果在插入LoxP的位点在某种方式上干扰了NMDA受体的表达，那么数年后得到的将是生下就夭折的小鼠[29]。相反，如果在LoxP插入位点时，无意中把Arc基因弄坏，至少我还能发表一篇全身基因敲除的文章。但是我说服了自己，相信自己的想法不仅有希望，而且还能得到好结果：不成功，便成仁，到时，我也会因不相信教科书的教义，而得到一枚“荣誉傻瓜勋章”。

►图2

真正开始实验时，亟需把纷繁而又复杂的事项进行合理的安排。刚到新的实验室，一切还没理顺，幸好身边的几个同事都很友好，都愿意帮忙。David Gerber和Toshikuni Sasaoka分别教我受精卵和囊胚的微注射；陈东风在免疫抗体和染色方面给予指导；李育庆(现在佛罗里达大学任教)分享他对NMDAR1质粒构建的见解；徐明（现在芝加哥大学任教）为我提供胚胎干细胞并教我如何进行完美的胚胎干细胞培养；同时我也非常感谢加州理工学院的David Anderson提供的LacZ报告基因和杜邦的Brian Saucer提供的Cre-loxP质粒。

1994年初，我完成了所有的质粒构建并开始转基因小鼠的繁育和floxed NR1胚胎干细胞打靶。更幸运的是，我还有一个勤奋的本科生Cindy Tom和技术员Jason Derwon协助进行基因分型和脑组织切片的工作。

7月，德国的Klaus Rajewsky在Science上报道了他们在T细胞的RNA聚合酶中取得了50%的敲降[35]，一团黑云飘到了我的头顶。因为这意味着，即使在分裂的T细胞中Cre重组的效率也不高：或是50%的T细胞可以100%敲除RNA聚合酶，或100%的细胞只删除一个基因的拷贝，或更糟的是，两种情况相互混合。尽管如此，我仍“奢望”（在进一步的文献搜索后）他们使用的瞬时激活启动子可能是T细胞发育过程中的Cre重组效率低下的罪魁祸首。在随后的岁月里，我把自己变成了鸵鸟，不顾外界的情况，一头扎进了我的Cre-loxP神经技术研发的沙堆里。

当年的深秋，我终于从实验中第一次得到了反馈。

在一个阳光明媚但寒冷的早晨，我仍然记得在显微镜下，我看见 LacZ大脑染色切片时的那种无法言喻的惊喜，在第一条的 Cre 转基因品系小鼠大脑中，我就发现海马CA1区锥体细胞中发生了特异的基因重组 (图 2)。因为海马 CA1区 是许多可塑性和记忆研究者眼中的“宇宙中心”，这样的好运使我坚信上帝不仅存在，而且一直就在我的身旁。随后，另外的几个 Cre品系也确认了类似 CA1区域特异性的重组；然后其他的Cre品系小鼠还显示具有前脑的特异性。当Tonegawa从日本出差回来，我向他汇报了我的发现。可能由于他在倒时差的缘故，他也没有想起我这几年在做的是什么课题。当他醒悟过来后，非常兴奋，立即打电话给加州大学洛杉矶分校的Alcino Silva（把Tonegawa引入神经领域的第一个博后，已成为该校的助理教授），与对方分享了这一惊喜。

与此同时，我在思考为什么本来应该在前脑表达而不是CA1区特异性的CaMKII 启动子会有如此的特异性作用。陈东风和我通过对Cre的染色找到了原因——我们发现 Cre在CA1区锥体细胞上的表达更高，因此基因顺利而有效地重组了。在之后的6到8个月的时间里，我通过交杂Cre转基因T29-1品系获得了floxed NR1纯合子小鼠，确认了CA1区锥体细胞特异性 NMDA 受体敲除。拿到3-5周的小鼠，Pato Huerta和我就各自推进脑片记录和组织学实验。我们亦留出一组小鼠与Tom McHugh, Kenny Blum和Matthew Wilson进行合作研究，在NR1突变小鼠上记录海马位置细胞的放电活动模式。现在，我们知道T29-1的Cre-lox重组在出生两个月的小鼠上保留了CA1区特异性，不过随着Cre表达时间的积累，如同预期的CaMKII启动子的动态，大概在小鼠出生第8周后开始，特异性重组将逐步扩散之整个前脑。

1996年的秋天，我们准备给Cell杂志投3篇连续的文章。我们决定将报告Cre-lox神经遗传技术的可行性作为第一篇，CA1区特异性NMDA受体敲除研究作为第二篇，位置细胞的特性作为第三篇。因为在工作中使用了Kandel实验室的CaMKII启动子，最初和Eric Kandel商议的是，Cre-lox的工作万一成功，我们将把他和Mark Mayford放在第一篇投稿中, 作为共同作者。这个起初不被看好的项目, 如今得到了出乎意料好的实验结果，也因此带来了诸多烦恼，令人头痛。Kandel认为神经科学界对CA1特异性的NMDA受体敲除在学习和记忆研究具有更为重要而广泛的意义，他要求将他的署名从第一篇的方法论文章中移到第二篇，但是Tonegawa也认为第二篇文章可能更重要，坚决反对。一个多月来来回回的争吵让我夹在中间左右为难，头痛不堪，但也让我亲身体验了什么叫做脑袋被挤夹在石缝里。最终Kandel的名字还是被Tonegawa硬放到了第一篇文章里，而最终这三篇连续文章在没有通讯作者的情况下，与1996年的12月底发表在了Cell杂志上[36，37，38]（第一篇引用次数为921，第二篇为1526，第三篇514）。

神经科学的Cre-driver资源

Cre-lox神经遗传技术可行性的成功验证在神经科学领域引发了一个新的热点。我也于1997年夏天在普林斯顿大学分子生物学系建立了自己的实验室（施一公几个月后也来到来该系，我们成为系里仅有的两位华人教授）。美国国立卫生研究院意识到Cre-lox神经遗传技术对神经科学的独特而重要的作用，启动了神经科学研究蓝图Cre-driver (Cre-表达体系)项目，为认知行为学创建一批小鼠品系，以便更好地鉴定特定的细胞类型和神经回路的功能。我也在立项、评审过程中提供了一些指南和建议（我回避了申请，而专注创构转基因“聪明鼠”， [39]）。美国国立卫生研究院最终遴选了三个中心，启动了神经系统表达Cre重组酶的转基因小鼠的创建及繁殖工作。三个研究中心分别由贝勒医学院的Ronald Davis博士（现在佛罗里达州Scripps研究所），冷泉港实验室的Z. Joshua Huang博士（与Brandeis大学的Sacha Nelson一起）和Scripps研究所的Ulrich Mueller博士领导。这几个项目创建了数百条Cre-driver的小鼠品系[40]。现在，这些品系的小鼠获得的途径有突变小鼠区域资源中心（MMRRC）或美国最大的动物供应商Jackson实验室的Cre库（见表1）。

美国国立卫生研究院的神经科学研究蓝图还资助了洛克菲勒大学的GENSAT项目。该项目由Nathaniel Heintz领导，创建BAC-Cre重组酶驱动的小鼠品系[41]。现今，已经创建了总计288个Cre品系。除了官方的努力，许多个人实验室和科研院校也创建了多种Cre-drivers[42,43]。迄今为止，Jackson实验室已经收集了600多个Cre品系的小鼠模型，提供给大家研究。

欧盟也跟随美国国立卫生研究院的倡议推出以CREATE命名的Cre-driver项目（为等位基因突变小鼠条件表达的资源协调）（表1）。CREATE联盟代表的大多数欧洲、国际小鼠数据库持有人以及研究团体的核心，建立Cre-driver品系的一体化战略，并应用在小鼠上建模来研究复杂的人类疾病。各国的研究人员可以通过联系EMMA (European Mouse Mutant Archive, Italy) 或者 MSR(International Mouse Strain Resource; Table 1)来获得Cre的不同品系。另外，英国、加拿大和日本也启功和资助了他们自己的Cre-driver项目（表1）。

►表1

脑研究的一个重要通用平台

在过去的十年中，Cre重组基因技术应用最令人激动的一个方向是采用光刺激操纵神经元的光遗传学[44,45]。研究人员通过在丰富的Cre品系小鼠上或Cre病毒表达光感应离子通道，刺激或抑制某类神经细胞的放电，使其成为耀眼的明星（这一系统被Karl Deisseroth简称为光遗传学）。亲眼看到光感应离子通道能充分地利用Cre重组基因技术的原理及这些Cre品系动物[46]，为神经科学界更多的同行服务，对我来说是个莫大的乐趣。

在神经科学方面，并非仅有表达光感应离子通道受益于Cre-LoxP神经遗传技术这一平台。Cre-LoxP神经遗传技术在化学遗传学中已显现出强大作用，使得科学家能够激活或抑制特定的神经元。例如，在化学–转基因修饰过的蛋白上进行过表达，如蛋白激酶[47,48]或在特定的神经元或脑区设计药物激活专门受体[49]。研究人员还可以通过Cre品系使用floxed白喉毒素片段A（DTA）来删除（毒死）特定的细胞，然后观察所产生的表型。

此外，Cre-lox遗传技术也应用于逆行病毒对大脑各种神经元的示踪，如斯坦福大学骆利群教授等小组示踪锥体细胞或多巴胺细胞[50,51]，或被用在透明大脑（Clarity）特异神经元环路的示踪方法中。哈佛大学的Jeff Lichtman和Joshua Sanes还巧妙地利用Cre-loxP系统的独特性质在单个神经元中，随机表达不同比例的红、绿、蓝色的绿色荧光蛋白（GFP）的衍生物，这种技术被称为“脑彩虹”[52]。这种技术使他们能够以一种独特的颜色标记每一个神经元，对神经科学连接组学领域来说，无疑是巨大的贡献。

Cre-lox遗传技术平台也被用于电压敏感的蛋白质来监测神经元的活动变化。基因编码的钙传感器，如GCaMPs，使得许多实验室通过对钙的瞬态来推断神经元活动的变化[53]。研究人员也在开发其他基因编码的电压敏感的荧光蛋白，以便可以离体或在体的方式来研究神经元的放电反应[54，2，6]。

去年，Stuart Ibsen和Sreekanth Chalasani报道了一个有趣的方法，采用低压超声刺激产生的微气泡可以增强机械的形变，从而激活力传导通道（TRP-4）促发神经元放电[22]。作者发现，通过在特定的神经元中过表达TRP-4，可以增强神经元对超声刺激的敏感性。另外，科研人员还报道了另一种非侵入性的方法：磁遗传学（magnetogenetics），可以通过磁刺激在体激活神经元。比如，神经元的激活是可由表达并刺激阳离子通道（TRPV4）与顺磁性铁蛋白的混合体来实现[55,56]。或外源磁受体铁硫簇组装蛋白1（Isca1)[57,58]。可以想象，随着各种新蛋白功能的开发，脑科学家们可利用Cre-lox技术平台，利用它们操纵特定的神经元，进一步了解大脑的工作原理。

回首过去的20年，研发Cre-lox神经遗传技术曾被同行断定为是一条死胡同，但凭着直觉和初生牛犊不怕虎的冲劲，我用头撞穿了“南山”，幸运地闯出了一条生路。如今在神经科学领域，几乎每周都会有科研人员利用Cre技术的学术成果报道。当学生们问起时，我会感慨地鼓励大家：年轻时当个愣头青，说不定就是上帝给你的一大宝器！

本文来自：Tsien JZ (2016) Cre-Lox Neurogenetics: 20 Years of Versatile Applications in Brain Research and Counting… Front. Genet. 7:19. doi: 10.3389/fgene.2016.00019

https://www.frontiersin.org/articles/10.3389/fgene.2016.00019/full

Proximity-CLIP — close encounters of the RNA kind

“Proximity-CLIP can be used to simultaneously map the compartment-specific landscape of RBPs, the transcriptome and RBP-occupied RNA loci”

The spatial compartmentalization of RNA is pivotal to gene expression and its regulation. However, the study of RNA localization has been hampered by a lack of high-throughput methods for mapping the transcriptomes of different cellular compartments. A new method called Proximity-CLIP combines proximity-labelling of proteins with ultraviolet (UV) crosslinking and next-generation sequencing to map the subcellular locations of RNAs.

The team built on the previously established APEX-RIP, a technique for the high-throughput mapping of RNA localization in intact cells that combines compartment-specific protein biotinylation with protein–RNA formaldehyde crosslinking. The engineered peroxidase APEX2 functions as a labelling tag that is genetically targeted to subcellular compartments through fusion with compartment-specific localization signals. Addition of biotin-phenol, a small-molecule substrate for APEX2, results in the biotinylation of endogenous proteins in close proximity to the enzyme. Proteins can subsequently be identified by mass spectrometry, whereas crosslinked RNAs can be identified by RNA sequencing (RNA-seq).

In Proximity-CLIP, RNAs are first labelled with 4-thiouridine (4SU) in living cells that express specifically localized APEX2. RNA and proteins are crosslinked in vivo by UV light, rather than by chemical crosslinking, during the quenching step that follows the biotinylation of APEX2-proximate proteins. Localized, biotinylated and crosslinked ribonucleoprotein complexes are then isolated using streptavidin affinity chromatography. While the proteome is determined using mass spectrometry, the RNA can either be analysed by standard RNA-seq to identify local transcripts or undergo RNase treatment to reveal RNase-resistant ‘footprints’, which reveal the occupancy of these sequences by RNA-binding proteins (RBPs). Sequencing of cDNA libraries generated from footprints enables the identification and quantification of RBP-occupied cis-acting regulatory elements on transcripts.

The UV crosslinking of RNA labelled with 4SU leads to a T-to-C mutation in the corresponding cDNA libraries. This mutation can be exploited to bioinformatically remove contaminating RNAs that bind nonspecifically to the affinity chromatography matrix, thereby increasing the specificity of Proximity-CLIP. UV crosslinking also avoids some pitfalls of formaldehyde crosslinking, such as the crosslinking of long-range or indirect interactions.

Applying their technique to HEK293 cells inducibly expressing APEX2 fused either to a nuclear export signal or to histone H2B, Benhalevy et al. determined both the proteome and transcriptome of the cytoplasm and nucleus, respectively. Functional enrichment analysis showed that proteins biotinylated by H2B-APEX2 were involved in transcription or belonged to nuclear categories, such as ‘nucleus’ and ‘nucleoplasm’. Moreover, the protein profiles resembled those previously reported in a study that used mass spectrometry to determine nuclear and cytoplasmic proteomes.

Transcriptome profiles of the cytoplasmic and nuclear compartments obtained using Proximity-CLIP matched RNA profiles obtained after biochemical fractionation. Furthermore, sequencing of RBP-protected fragments from both nucleus and cytoplasm revealed a footprint profile that reflected each compartment; that is, the vast majority of RBP footprints on mRNA in the cytoplasm were found on mature mRNA, whereas 43% of mRNA footprints in the nucleus resided in introns.

Taken together, Proximity-CLIP can be used to simultaneously map the compartment-specific landscape of RBPs, the transcriptome and RBP-occupied RNA loci. Its high throughput compared with imaging-based techniques makes it particularly appealing for identifying the subcellular localization of transcripts and potentially interacting RBPs.

【REF】

Benhalevy, D. et al. Proximity-CLIP provides a snapshot of protein-occupied RNA elements in subcellular compartments. Nat. Methods 15, 1074–1082 (2018)

Koch L. Proximity-CLIP – close encounters of the RNA kind. Nat Rev Genet. 2019 Feb;20(2):68-69

A representative statistical analysis

Boxplots were created using Prism7 (GraphPad). The box extends between the values for the 25th and 75th percentiles of the data. The whiskers extend 1.5 times the difference between the values of the 75th and 25th percentiles of the data from the top and bottom of the box. Values lying outside the whiskers were defined as outliers, and the mean was computed excluding these outliers. The median is indicated with a black horizontal line inside the box, while the mean is indicated with a red horizontal line. Violin plots were created using the statistical software package R.

Data in all groups were tested for normality using the D’Agostino–Pearson omnibus normality test. Parametric statistics were used only if the data in all groups in the comparison were normally distributed.

The statistical significance of differences between multiple groups was determined using one-way ANOVA followed by Tukey’s multiple comparison correction for normally distributed data, and using one-way Kruskal–Wallis ANOVA followed by Dunn’s multiple comparison correction for data with non-normal distribution. To compare baseline and post-treatment measurements at multiple time points with non-normal data, Friedman one-way repeated measures non-parametric ANOVA followed by Dunn’s multiple comparison correction was used.

Statistical comparisons between two groups were performed using Student’s t-test or paired t-test for normally distributed data, or with the Mann–Whitney test or Wilcoxon matched-pairs test for data with non-normal distribution. P <0.05 was considered statistically significant. All statistical analysis was performed using Prism7 (GraphPad).

We use a standardized set of significance indicators across all figures in this manuscript. For comparisons between groups: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For matched comparisons before and after treatment: +P < 0.05, ++P < 0.01.

From:  https://www.nature.com/articles/s41593-018-0329-4

Python代码编写规范

一 代码编排
1 缩进。4个空格的缩进（编辑器都可以完成此功能），不使用Tap，更不能混合使用Tap和空格。
2 每行最大长度79，换行可以使用反斜杠，最好使用圆括号。换行点要在操作符的后边敲回车。
3 类和top-level函数定义之间空两行；类中的方法定义之间空一行；函数内逻辑无关段落之间空一行；其他地方尽量不要再空行。

二 文档编排
1 模块内容的顺序：模块说明和docstring—import—globals&constants—其他定义。其中import部分，又按标准、三方和自己编写顺序依次排放，之间空一行。
2 不要在一句import中多个库，比如import os, sys不推荐。
3 如果采用from XX import XX引用库，可以省略‘module.’，都是可能出现命名冲突，这时就要采用import XX。

三 空格的使用
总体原则，避免不必要的空格。
1 各种右括号前不要加空格。
2 逗号、冒号、分号前不要加空格。
3 函数的左括号前不要加空格。如Func(1)。
4 序列的左括号前不要加空格。如list[2]。
5 操作符左右各加一个空格，不要为了对齐增加空格。
6 函数默认参数使用的赋值符左右省略空格。
7 不要将多句语句写在同一行，尽管使用‘；’允许。
8 if/for/while语句中，即使执行语句只有一句，也必须另起一行。

四 注释
总体原则，错误的注释不如没有注释。所以当一段代码发生变化时，第一件事就是要修改注释！
注释必须使用英文，最好是完整的句子，首字母大写，句后要有结束符，结束符后跟两个空格，开始下一句。如果是短语，可以省略结束符。
1 块注释，在一段代码前增加的注释。在‘#’后加一空格。段落之间以只有‘#’的行间隔。比如：
# Description : Module config.
#
# Input : None
#
# Output : None
2 行注释，在一句代码后加注释。比如：x = x + 1 # Increment x
但是这种方式尽量少使用。
3 避免无谓的注释。

五 文档描述
1 为所有的共有模块、函数、类、方法写docstrings；非共有的没有必要，但是可以写注释（在def的下一行）。
2 如果docstring要换行，参考如下例子,详见PEP 257
“””Return a foobang

Optional plotz says to frobnicate the bizbaz first.

“””

六 命名规范
总体原则，新编代码必须按下面命名风格进行，现有库的编码尽量保持风格。
1 尽量单独使用小写字母‘l’，大写字母‘O’等容易混淆的字母。
2 模块命名尽量短小，使用全部小写的方式，可以使用下划线。
3 包命名尽量短小，使用全部小写的方式，不可以使用下划线。
4 类的命名使用CapWords的方式，模块内部使用的类采用_CapWords的方式。
5 异常命名使用CapWords+Error后缀的方式。
6 全局变量尽量只在模块内有效，类似C语言中的static。实现方法有两种，一是__all__机制;二是前缀一个下划线。
7 函数命名使用全部小写的方式，可以使用下划线。
8 常量命名使用全部大写的方式，可以使用下划线。
9 类的属性（方法和变量）命名使用全部小写的方式，可以使用下划线。
9 类的属性有3种作用域public、non-public和subclass API，可以理解成C++中的public、private、protected，non-public属性前，前缀一条下划线。
11 类的属性若与关键字名字冲突，后缀一下划线，尽量不要使用缩略等其他方式。
12 为避免与子类属性命名冲突，在类的一些属性前，前缀两条下划线。比如：类Foo中声明__a,访问时，只能通过Foo._Foo__a，避免歧义。如果子类也叫Foo，那就无能为力了。
13 类的方法第一个参数必须是self，而静态方法第一个参数必须是cls。

七 编码建议
1 编码中考虑到其他python实现的效率等问题，比如运算符‘+’在CPython（Python）中效率很高，都是Jython中却非常低，所以应该采用.join()的方式。
2 尽可能使用‘is’‘is not’取代‘==’，比如if x is not None 要优于if x。
3 使用基于类的异常，每个模块或包都有自己的异常类，此异常类继承自Exception。
4 异常中不要使用裸露的except，except后跟具体的exceptions。
5 异常中try的代码尽可能少。比如：
try:
value = collection[key]
except KeyError:
return key_not_found(key)
else:
return handle_value(value)
要优于
try:
# Too broad!
return handle_value(collection[key])
except KeyError:
# Will also catch KeyError raised by handle_value()
return key_not_found(key)
6 使用startswith() and endswith()代替切片进行序列前缀或后缀的检查。比如：
Yes: if foo.startswith(‘bar’):优于
No: if foo[:3] == ‘bar’:
7 使用isinstance()比较对象的类型。比如
Yes: if isinstance(obj, int): 优于
No: if type(obj) is type(1):
8 判断序列空或不空，有如下规则
Yes: if not seq:
if seq:
优于
No: if len(seq)
if not len(seq)
9 字符串不要以空格收尾。
10 二进制数据判断使用 if boolvalue的方式。

via： https://www.cnblogs.com/niansi/p/7751374.html

Buffering transition

Aberrant aggregation of normally soluble proteins into insoluble amyloid is involved in the onset and the progression of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). In the neurons of ALS patients, aggregation of prion-like RNA-binding proteins (RBPs) usually occurs in the cytoplasm rather than the nucleus. To investigate what prevents prion-like RBPs from forming solid-like aggregates in the nucleus, Maharana et al. used in vitro phase-separation assays and fluorescence correlation spectroscopy to show that RNA blocks the liquid–solid phase transition of prion-like RBPs, which is a prerequisite of aggregation. In living cells, assemblies of prion-like RBPs are observed by reducing the concentration of nuclear RNAs, by increasing intranuclear protein expression, or by impairing the RNA-binding ability of proteins. Photobleaching experiments showed that RNA kept condensates formed by prion-like RBPs in a dynamic state and prevented the formation of solid pathological assemblies. Overall, the findings suggest that nuclear RNA buffers the phase separation behavior of prion-like RBPs, provide insight into the chemical characteristics of this type of protein, and deepen the understanding of the pathological cause of prion-like RBP-related neurodegenerative diseases.

https://www.ncbi.nlm.nih.gov/pubmed/29650702

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和引用数据。

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。

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。

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。

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」。

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。

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。

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。

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。

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

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。

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。

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。

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

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。

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。

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）。

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）。

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。

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）。

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.

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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).

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For further information, please consult the following protocols:

1. WiCell protocol
   • http://www.wicell.org/index.php?option=com_content&task=section&id=23&Itemid=273
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
   • http://www.mcb.harvard.edu/melton/hues/

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.

Tips for choosing academic mentors

Science: Having good mentors is critical in any career. In academia, most researchers train under just one or two graduate and/or postdoctoral mentors; however, little is known about how this mentorship affects the mentee’s career. Liénard et al. analyzed an open-access database of 18,856 researchers to determine if graduate or postdoctoral mentors have a greater impact on trainee careers. Results show that although postdoctoral mentors were more influential in trainees’ success, the breadth of training between graduate and postdoctoral mentors was also predictive. Trainees working under mentors with disparate expertise, who were then able to integrate both sets of expertise into their own work, had higher levels of academic success. Advice to future scientists: Consider mentors who will teach you diverse, yet complementary, skill sets.

Nature: Researchers who incorporate ideas and techniques from multiple mentors while still forging their own paths are the most likely to succeed in academia, according to a study of 18,865 biomedical researchers published in Nature Communications1.

The authors also suggest that mentoring received during postdoctoral training had a bigger impact than mentoring received during graduate school.

The study analysed data from the Academic Family Tree, an online database of academic relationships that launched in January 2005. The authors identified ‘triplets’ — trios comprised of a scientist, their graduate mentor and their postdoctoral mentor — dating back to 1970.

Professional success was gauged in part by the number of trainees a researcher mentored per decade, and an analysis of terms used in abstracts made it possible to track similarity of scientific approaches.

The results give empirical evidence to support some popular career strategies, says study co-author Stephen David, a neuroscientist at the Oregon Health & Science University in Portland. For example, the most successful scientists transferred concepts they learnt in graduate school to their postdoctoral work, suggesting that prospective postdocs should try to join labs that lack their particular skill set.

“You want to be able to offer something new,” David says. That requires stepping beyond the shadow of a graduate mentor without becoming a facsimile of a postdoctoral mentor. “You have to stake out some unique territory, which is always a challenge for postdocs,” he says.

The study found that joining the lab of a prolific mentor — one who has trained many researchers over the years — also increases a scientist’s chance of success. This held true for both graduate and postdoctoral mentors, but a closer look at the data revealed that the qualities of a postdoctoral mentor were especially predictive of success. “You can get a graduate education just about anywhere,” David says. “Postdoc labs are where you establish professional relationships and develop collaborations.”

Researchers should be especially discerning when accepting postdoctoral positions, David says. “You can take a data-driven approach to choosing your mentor.”

[REF]

https://www.nature.com/articles/s41467-018-07034-y

 

Novel Vaccine Technologies: Essential Components of an Adequate Response to Emerging Viral Diseases

The availability of vaccines in response to newly emerging infections is impeded by the length of time it takes to design, manufacture, and evaluate vaccines for clinical use. Historically, the process of vaccine development through to licensure requires decades; however, clinicians and public health officials are often faced with outbreaks of viral diseases, sometimes of a pandemic nature that would require vaccines for adequate control. New viral diseases emerge from zoonotic and vectorborne sources, such as Middle East Respiratory Syndrome coronavirus and Chikungunya, and while these diseases are often detected in resource-rich countries, they usually begin in low- and mid-income countries.¹ Therefore, part of the timeline for a vaccine involves surveillance and detection of new pathogens in remote areas and transfer of specimens to laboratories capable of vaccine development.

Development of vaccines for viral infections has historically been an empirical and iterative process based on the use of attenuated or inactivated whole virus. This requires unique methods of cultivation for each virus, development of animal models for vaccine testing, and a prolonged process of fine-tuning product formulation and immunogenicity, and for live-attenuated vaccines, pathogenicity. Thus, preclinical vaccine development can take years, followed by several more years of early-phase clinical testing and defining of dose and schedule. Moreover, efficacy testing and registration with regulatory agencies often takes another 5 to 10 years. In total, 15 to 20 years would be a typical timeframe from virus discovery to vaccine availability if the process proceeds smoothly and there are no major biological or logistical challenges.

Fortunately, during the last decade, there have been substantial technological advances for conceiving, developing, manufacturing, and delivering vaccines. Rapid genetic sequencing allows both early identification of new pathogens and the identity of the genes encoding structural proteins that can form the basis for vaccine immunogen development. Also, rapid isolation of human monoclonal antibodies has proven to be extremely helpful in defining epitopes that are the targets of protective immunity.

Additional tools of modern vaccinology include (1) delineation of atomic-level structures of viral proteins that facilitates structure-enabled immunogen design and protein engineering; (2) cell sorting and sequencing technologies that allow single-cell analysis of immune responses; and (3) genetic knock-in technologies that allow construction of animal models with human antibody genes for vaccine testing. These tools have already provided the potential not only for solving long-standing problems in vaccinology, such as the development of a new candidate vaccine for respiratory syncytial virus, but they have facilitated rapid development of new candidate vaccines for emerging pathogens such as the Zika virus and pandemic strains of influenza virus. Synthetic vaccinology and platform manufacturing are important innovations that can speed the initial vaccine immunogen design and vaccine development process, and shorten the time needed for manufacturing and initial regulatory approval to begin phase 1 testing.

Synthetic vaccinology is the process of using viral gene sequence information to accelerate vaccine development.² For example, if a new influenza virus emerges anywhere in the world and is identified through genomic sequencing, the digitally transferred information can be used to synthesize nucleic acids encoding the viral surface proteins (hemagglutinin and neuraminidase). The process of gene synthesis is now extremely rapid and relatively inexpensive. Thus, within a few weeks, DNA plasmids encoding viral proteins can be available for preclinical testing. These genetic vectors (DNA and mRNA) can be used directly for immunization whereby intramuscular immunization leads to muscle cells producing the viral proteins. Alternatively, the genetic vectors can be used to express recombinant protein antigens, in vitro, that can be used for immunization.

Similarly, if an outbreak of a new flavivirus becomes an epidemic or even a pandemic threat, as with Zika in 2015, the gene sequences that encode the viral surface proteins premembrane and envelope can be rapidly identified and form the basis for vaccine immunogen design strategies, based on prior knowledge of flavivirus structure and mechanisms of neutralization.³ Once a structurally authentic immunogen is available, the protein or genetic vectors encoding the protein can be used to immunize animals. In addition, the vaccine proteins can be used as probes to identify monoclonal antibodies secreted by B cells of convalescent humans. Such antibodies are valuable not only for refining vaccine immunogen designs, but also for development of diagnostic assays and potentially for use in passive transfer as therapeutic agents. Thus, development of reagents, diagnostics, candidate vaccines, and immune assessment assays can be done without having the actual virus in hand. This has particular value for viruses with extreme pathogenicity because it avoids the need for high-level containment in laboratory and manufacturing facilities.

Platform manufacturing technologies allow more rapid production and clinical implementation once the vaccine immunogen design is established. The term platform is used in many ways; however, in vaccine production, it implies that the method for generating and presenting a vaccine immunogen can be applied across multiple pathogens. In essence, the cell substrates, production approach, purification processes, and analytical assays used as release criteria for products made under current Good Manufacturing Procedures are the same even though the immunogen may change. DNA or mRNA nucleic acid vaccines are good examples of how platform manufacturing can shorten timelines from pathogen identification to phase 1 clinical trials.⁴ DNA vaccine delivery and immunogenicity have evolved and improved over the last 2 decades, making it a viable platform for vaccination.

For DNA plasmid vaccines, the manufacturing process is well established, and their toxicity profile is well understood. The National Institute of Allergy and Infectious Diseases Vaccine Research Center has developed candidate DNA vaccines for several viral disease threats during outbreaks, including SARS coronavirus in 2003, H5N1 avian influenza in 2005, H1N1 pandemic influenza in 2009, and most recently for Zika virus in 2016. Once these pathogens were identified, the time from viral sequence selection to initiation of the phase 1 clinical trial was shortened from 20 months to slightly longer than 3 months .

Other examples of vaccine platform technologies include viral vector–based approaches where genes encoding viral proteins are incorporated into viral vectors (eg, adenovirus, poxvirus, vesicular stomatitis virus, or paramyxovirus vectors) for gene-based immunogen expression and delivery, or chimeric replication-competent viruses in which the vaccine antigens of one virus are expressed in a common replication-competent virus allowing uniform manufacturing processes (eg, yellow fever or other flavivirus antigens expressed in dengue virus, or human parainfluenza or pneumovirus antigens expressed in bovine parainfluenza or Sendai virus vectors).

Traditional approaches, such as live-attenuated virus vaccines (eg, Sabin polio) or whole-inactivated virus vaccines (eg, Salk polio) would not qualify as platform approaches because the requirements for growth in cell culture and purification are usually different among virus families. Protein-based approaches are also likely to have different requirements for purification and formulation, and they may not be amenable to platform approaches unless the display of proteins on nanoparticles or other carrier systems brings more uniformity to downstream manufacturing approaches. Having a standard manufacturing approach reduces the time needed for current Good Manufacturing Procedures process development and simplifies regulatory approval because the safety database that has accumulated for a given platform can be applied to multiple vaccine products.

In summary, emerging viral diseases with pandemic potential are a perpetual challenge to global health. The time-honored approach to vaccinology, which depends predominantly on isolating and growing the pathogen, has not adequately met this challenge. To effectively prepare for and respond to these continually emerging threats, it will be critical to exploit modern-day technological advances, preemptively establish detailed information on each family of viral pathogens, and invest in more infrastructure for surveillance in developing countries to expedite pathogen identification and jump-start the process of vaccine development using these new technologies.² Failure to do so will result in the untenable situation of not optimally using vaccinology in the response to newly emerging infectious disease threats.

via: JAMA. 2018;319(14):1431-1432. doi:10.1001/jama.2018.0345

[Refs]

[1] Jones KE, Patel NG, Levy MA, et al. Global trends in emerging infectious diseases. Nature. 2008;451(7181):990-993.
[2] Graham BS, Sullivan NJ. Emerging viral diseases from a vaccinology perspective: preparing for the next pandemic. Nat Immunol. 2018;19(1):20-28
[3] Dowd KA, Ko SY, Morabito KM, et al. Rapid development of a DNA vaccine for Zika virus. Science. 2016;354(6309):237-240.
[4] Ulmer JB, Geall AJ. Recent innovations in mRNA vaccines. Curr Opin Immunol.2016;41:18-22.

Cell发布“相分离”研究指南

原文链接：

https://www.sciencedirect.com/science/article/pii/S0092867418316490

细胞是生物体结构和功能的基本单位，细胞内的各种组分如何在正确的时间以及空间上聚集以执行其相应的功能，是细胞在一系列基本的生命活动中需要解决的问题。为此，细胞进化出了一系列的细胞器，包括有膜包裹的（比如线粒体，细胞核，溶酶体等）和无膜包裹的细胞器（核仁等）。有膜包裹的细胞器将特定蛋白、核酸等物质包裹起来，以在特定的空间内执行其功能，如果这些蛋白或者核酸脱离特定的位置，将导致严重的后果（比如，细胞色素C释放到细胞质，将导致细胞凋亡，核酸释放到细胞质，将导致innate immune signaling pathway 的激活）。另一类无膜包裹的细胞器是如何形成，以及其物理化学本质，是困扰了大家多年的问题。

Hyman和Brangwynne 2009年在Science发表了题为：Germline P granules are liquid droplets that localize by controlled dissolution/condensation 的文章【1】，提出了细胞内通过“相分离”，可以提供一种特定的方式让细胞内的特定分子聚集起来，从而在“混乱的”细胞内部形成一定“秩序”，为困扰了大家多年的问题，提供了全新的思路。近几年的研究表明，液-液相分离（LLPS：liquid-liquid phase separation）可能是细胞形成无膜细胞器的物理化学基础,比如细胞内的p granule, nucleolar, stress granule等（图1）。

图1：细胞的的相分离结构。（引自参考文献【2】）

LLPS也被报道在一些疾病（如：癌症，以及神经退行性疾病等）的发生发展过程中起着非常重要的作用。相分离领域已经成为生命科学领域研究的热点，相关的文章近年来呈现井喷似的增长（见文末的延伸阅读：Bioart 系列解读）。（另外，Brangwynne在iBiology上面有三期的讲座，对这个领域的历史，以及其实验室相关的此方面的开创性工作做了非常具体的介绍，题目为：Liquid Phase Separation in Living Cells。链接：https://www.ibiology.org/biophysics/liquid-phase-separation-in-living-cells/  ）

Cliff Brangwynne (Princeton & HHMI)在iBiology上的介绍相分离研究

然而，像许多新兴的研究领域一样，该领域的研究对于实验的设计，以及实验方法还没有统一的标准和相关的指南，对于该领域的研究造成了一定的困扰，亟需建立一个从理论体系到具体实验设计的统一的标准。近日Simon Alberti, Amy Gladfelter, 和 Tanja Mittag联合在Cell 杂志发表了题为：Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates的文章，较为系统地阐述了LLPS相关的理论基础，提出了LLPS的体内体外的实验设计方法的具体指南。

相分离具体的作用可以参考另一篇由Hyman以及Michael Rosen执笔的发表在Nature reviews molecular cell biocology上的题为 Biomolecular condensates: organizers of cellular biochemistry的文章，这篇文章更加系统的介绍了相分离的原理，以及其目前所报道的生物学功能【2】。另外，Simon Alberti等人在2018年，发表了另一篇题为A User’s Guide for Phase Separation Assays with Purified Proteins，非常细致地阐述了体外相分离实验的纯化蛋白的考虑以及一些相关的tips【3】。建议读者在具体的实验中可以参考此文章。这篇Cell文章更倾向于整体实验设计指南，为相关领域的研究提出一些标准化的实验步骤，也提出了一些这个领域还需要进一步阐述的机制，以及相分离的研究的根本目的，即要研究其生物学的具体功能。

相分离的基本概念，相分离的形式，以及如何预测一个蛋白是否会形成相分离现象

LLPS的发生，高度依赖溶液中生物大分子（比如：蛋白，DNA以及RNA等）的浓度、物理化学性质，以及溶液所处的环境，比如：温度、pH、盐离子浓度、盐离子类型以及溶液中存在其它的生物大分子。作者使用如下的Phase Diagram来描述这些与相分离相关的条件与该溶液是否发生相分离的关系（图2）。

图2：Schematic Phase Diagram

简单来讲：当溶液中所处的大分子浓度低于一个特定的值c时，这一体系无论在什么样的温度，pH等条件下都不能发生相分离。当高于这一浓度后，在合适的pH以及温度等条件下，就能形成相分离现象，形成相分离后，该生物大分子便有两种存在形式，一种是在溶液中的低浓度状态，一种是形成的“液滴”中较高浓度的形式存在。随着相关条件的变化，两种形式可以相互转化。也就是说，相分离是一种高度动态的过程。

另外，LLPS不仅能形成液滴状的结构，还能继续转变为胶状物的形式。凝胶状态的相分离经常不可逆转，这也为阿尔兹海默症等体内形成的amyloid-like fibers的形成，提供了全新的思路，为相关药物的设计提供了全新的理念。已有研究表明一些蛋白的突变会加快这种LLPS向凝胶状态转化的过程。

液-液相分离的发生是蛋白质和核酸在某种特定的情况下的一个普遍特性，然而多数相分离根本不可能发生在一个正常的细胞中。正如仅有一小部分蛋白质能够在生理状态下发生淀粉样改变那样，仅有一小部分蛋白质的序列具有在活细胞内形成相分离的能力。截至目前为止，我们对控制相分离的基因学及生物学特性仍所知甚少。因此，在断定相分离的发生时，我们应该尤其谨慎。

近几年涌现了许多对在生理状态下能够发生相分离的分子的普遍特征的研究。其中之一即是支架及客户蛋白的理论。支架分子被认为是相分离的驱动分子，而在相分离形成以后参与到液滴当中的则被称为是客户蛋白。支架蛋白与客户蛋白的相分离需要一个互作网络的形成，该网络常由蛋白质-蛋白质互作及蛋白质-RNA互作构成。

两种蛋白质类型参与促进此类互作网络的形成：一类以多个折叠的结构域为特点，如Nck蛋白中的SH3结构域，该结构域能够与短的线性模块如SLiMs相结合；另一类蛋白则以内部无序区（IDR）为特点。两类蛋白有多处相似，其中最重要的一点是：蛋白间都通过多个结构域或模块相互作用。因此，通过从基因上初步推断蛋白的化合价可以判断该蛋白形成相分离的能力及饱和浓度。对RNA而言，特定的RNA可驱动相分离的发生，而一些含有IDR的蛋白包含多个RNA结合结构域、其目的RNA也包含多个蛋白结合序列。因此，蛋白和RNA能够通过多种方式形成多价互作，这些互作决定着特定蛋白及核酸形成相分离的能力。

多价的蛋白质互作网络如何发生相分离这一问题，可以从高分辨率的结构数据中得到结果。然而，IDR是如何驱动相分离的则相对不那么容易理解。

IDR是相分离蛋白中一种常见的结构域，常常不含有芳香族及脂肪族氨基酸，且不能够形成一个相对能量较低的、单一的折叠结构。相反，这些蛋白的构象能量往往与其一级序列所含有的能量相同。一级序列往往决定了这些蛋白的相变能力、相变的驱动因素、相变的临界浓度及黏弹性。能够影响相分离的序列特征包括IDR的长度、数量、模式、及IDR与IDR间序列的特征。典型的决定因素包括疏水性氨基酸的组成、模式等。尽管IDR中疏水性氨基酸的含量相对较少，他们代表了相分离中的粘附成分、并根据温度变化调控相分离的浓度。带电氨基酸也能够影响相分离的形成，成对的、带相反电荷的蛋白能够以复合凝聚的形式形成沉淀。

IDR的另一个共同特征是由低复杂度序列区（LCR）构成，如单一氨基酸的重复序列。一个典型的LCR是朊病毒样LCR，富含极性氨基酸如丝氨酸、酪氨酸、谷氨酰胺、天冬酰胺，不含有带电氨基酸。另一个典型的LCR是RNA结合蛋白中常见的RGG结构域，富含精氨酸，且能够调控LCR/RNA间的相互作用。LCR间相互作用的基础是电荷-电荷、共轭-共轭、阳离子-共轭互作。

因此，目前常应用预测算法推测蛋白质中的IDR以分析其发生相分离的能力（图3）。

 

图3：蛋白序列分析以及相分离预测工具

以FUS为例，IUPred算法鉴定前250个氨基酸、aa365-420、aa450-C端为潜在IDR，PLAAC算法鉴定到FUS N端含有QGSY-及G-重复序列，D2P2算法则区别了FUS的可折叠结构域与无序结构域。Anthony A. Hyman教授的团队则对这一预测算法的结果进行了实验验证。

体外重构相分离

相分离可以在体外通过纯化的蛋白以及核酸等在特定的条件下发生，体外重构的相分离实验对于该领域具有重要的作用（2012年，美国西南医学中心Michael Rosen和Steven McKnight独立发现在试管中这些分子通过微弱的作用力形成液滴，即首次证实了相分离能够通过简单的生化实验在体外重复【4, 5】。清华大学李丕龙教授便是Michael Rosen 这篇文章的一作）。

体外的相分离现象可以非常简单的使用普通的光学显微镜观察，发生相分离的特点是溶液会从澄清变得浑浊，镜检时会看到在溶液中会存在一些如水中的油滴状态的液滴。作者建议使用PEG或者lipids包被载玻片，以更好的观察和记录相分离现象。另外，可以将蛋白或者RNA使用荧光标记，或者将不同的组分通过不同的荧光分开标记，以方便使用荧光显微镜或者激光共聚焦显微镜拍摄出更好的图片，同时可以做FRAP等实验，以及持续拍摄获得LLPS动态变化过程。但是在具体应用此方法的时候要注意，一些RNA与RNA结合蛋白之间可能会被拍摄中的激光照射所交联，在拍摄的时候需要注意此类问题。

除此以外，可以使用检测溶液浑浊度的方法检测相分离，也可以使用离心沉淀的方法检测相分离现象。实验相关的具体的细节，推荐大家阅读香港科技大学张明杰教授题为Cell Phase transition in postsynaptic densities underlies formation of synaptic complexes and synaptic plasticity 的Cell文章中图4中的实验【6】。（另：Bioart专门邀请了张明杰教授过去的博士，现为复旦大学生物医学研究院PI 温文玉教授对张明杰教授的工作进行了系统解读，见延伸阅读）

另外，体外相分离的体外实验存在一定的局限性。体外相分离实验最大的优势是在于其各个组分以及各个组分的浓度，以及各种外在条件（比如：温度，pH等条件）可以被严格控制，更加方便我们去研究相分离现象。因此，需要注意的是，相关的蛋白以及RNA、DNA等各个组分的纯度是至关重要的。在此，作者提出了用于体外相分离实验的蛋白以及核酸样品的表达纯化，保存以及样品处理的标准。

蛋白纯化的考虑

用于体外相分离实验的蛋白可以在E.coli，酵母或昆虫细胞中表达纯化，或者通过体外转录/翻译系统得到。在能够发生相分离的蛋白中普遍存在无序区域（IDR：Intrici Disordered Region）在纯化的过程中非常容易被降解，因此在纯化此类蛋白时需要特别加以注意。含有IDR的蛋白质通常需要在纯化体系中加入蛋白酶抑制剂，需要快速纯化以防止蛋白的降解和蛋白聚集。另外，纯化此类蛋白可以让目的蛋白大量表达进细菌包涵体里面，这种方式可以防止被宿主体内的蛋白酶降解。但是，需要注意的是，在进行相分离实验之前，必须将蛋白复性到生理条件的缓冲液中。另外，表达蛋白的时候带上一些助溶的标签，比如MBP等，可以帮助得到较好的蛋白，但是在进行相分离实验的之前，需要将这些标签切掉，以免这些标签带来一些影响。

另外需要注意的是，蛋白质的翻译后修饰（PTM）对于相分离是非常重要的，在细菌表达纯化出来的蛋白一般翻译后修饰非常少，在真核细胞纯化的蛋白一般具有较好的翻译后修饰，建议真核表达的蛋白在做实验之前，最好先使用质谱等方式鉴定其翻译后修饰，以保证实验的可重复性以及利于更深入地了解该蛋白发生相分离的具体的分子机制。

在纯化的过程中，一般要避免目的蛋白发生相分离现象，但是如果发生相分离后，可以被一定的条件重新溶解，也可以通过利用这种反复发生相分离，溶解的方法来纯化该蛋白。纯化出来的蛋白需要用SDS-PAGE以及质谱等方式鉴定是否确实是目的蛋白以及鉴定其纯度。我们建议将蛋白保存在不发生相分离的Buffer中，保存Buffer通常在中性的pH, 使用高浓度或者非常低浓度的盐离子来防止发生相分离现象。同时需要加入还原剂。常用的Buffer体系如下（pH 缓冲体系：50mM  HEPES pH7.5, 盐离子：300 mM NaCl或者500 mM KCl, 或者不加盐离子，还原剂：1mM TCEP 或者5mM DTT ）。我们建议将蛋白分装后使用液氮速冻以后保存在低温的条件下，不要冻融蛋白，冻融会导致蛋白的聚集以及部分变性，影响其性质。在做相分离实验的时候，需要注意，对于每一个蛋白而言，都需要优化相应的PH，盐离子浓度以及温度等条件，尽量使其接近生理条件。详细的一些tips, 建议读者阅读另外一篇文章（Alberti et al., 2018）A User’s Guide for Phase Separation Assays with Purified Proteins

RNA来源

许多相分离需要使用RNA。 RNA可以使用体外转录或者直接化学合成。体外转录是较长的RNA非常好的来源，短链RNA可以直接从一些公司购买。 可以将RNA使用荧光标记，从而更方便的检测RNA与蛋白相分离的现象。

Macromolecular Crowders

另外，相分离对物理化学条件的变化非常敏感。温度，蛋白，核酸或盐浓度的微小差异也会导致不同的结果。因此，体外相分离实验应精确控制缓冲液的体系和蛋白浓度。

在相分离的实验中，经常使用到一些Macromolecular crowders，比如：PEG，Dextran或Ficoll。很多情况下，添加到实验中的crowder的量可能超过存在于细胞内的环境。目前，crowder是如何促进相分离发生的具体的机制仍不明确，因此，应当慎重使用crowder。如果使用crowder，我们建议使用多种crowder做实验，以排除由于Crower带来的假阳性的结果（私底下听到过有老师提到，可能80%左右的蛋白在有Crower存在的时候都可以发生相分离）。

小分子对于相分离的影响

在体外相分离实验体系中，可以测定LLPS是否改变酶的活性，从而研究相分离的生物学意义。在一些体外实验中，通常需要将高浓度的小分子化学物质（例如激酶抑制剂，甲基转移酶抑制剂等）添加到相分离体系中。此时应该考虑到，这些高剂量的小分子化学物质，除了本身对酶活的影响外，还可能对相分离产生直接作用，从而影响了酶活。

在体外，IDR通常足以介导相分离的发生。在高浓度下相分离的IDR的例子：Ddx4，LAF-1，FUS，hnRNPA1和Whi3的IDR。这些结果提示了这些IDR能够自主地通过homotypic interactions驱动相分离的发生。越来越多的证据显示，与同一多肽或其他蛋白其他区域的heterotypic interactions也可以驱动相分离的发生。另外，简单的单组分或双组分系统建立的理论如何扩展到细胞中的更复杂的混合物中，是亟待解决的问题。

相分离的物理特性

相分离液滴的物理状态变化极大。从液滴样直至多孔固体或胶体，其特性取决于相分离中的分子构成、时间、液滴的稳定程度、淬灭深度等。RNA的参与也可以影响相分离液滴的物理状态，但由于RNA既提供了多价结合位点、又贡献了静电，尚不清楚RNA究竟使其更流体化还是更固态化。由于不可逆的固态常被认为是一种病理状态，因此调控相分离的物理状态的因素仍有待进一步探究。体外重现相分离时，有多种方法可对液滴的物理性质进行具体描述。最直接的方法即测量表征表面张力的反毛细管速度，辅以被动微流变学，可推测液滴的表面张力。液滴表面与盖玻片间的接触角亦可表征液滴表面张力及表面的化学性质。

被动微观流变学通过在液滴内部置以珠子、测量该珠子的均方位移的方法表征液滴的物理性质。该方法受到多种因素的影响如液滴的组成、珠子的材料及大小、珠子表面的钝化程度、显微镜设备的漂移等。当相分离液滴极其粘稠乃至类似于胶体时，则可使用原子力显微镜或光镊来测量液滴内部的硬度。此外，还可使用荧光标记的右旋糖苷来检测相分离液滴中聚合物网格的孔径大小，以描述液滴的物理状态。

荧光漂白恢复实验（FRAP）常用于测量液滴的流动性，且其恢复时间因蛋白/RNA的不同而表现出很大的差异，尤其当对比蛋白质与RNA形成的相分离时，由于RNA的结构相对较刚性，其相分离液滴的FRAP时间往往更长。尽管FRAP是一个非常常见的实验，其应用的限制性却常常被忽略了。如FRAP的恢复时间不仅仅取决于液滴的稀释度，还取决于被光漂白的液滴大小、内部流动性、光漂白区域的大小等。如果条件允许，半荧光漂白恢复实验可以提供更多的关于液滴内部流动性的信息。此外，FRAP还可以用于评估液滴内部的同质性，但不能作为确定某一结构的形成机制是相分离的依据。

为更准确的估计相分离液滴内部某一分子的扩散能力，荧光相关光谱可作为检测手段之一。此外，偏振荧光显微镜可检测相分离液滴内部纤维性或固态样结构的各向异性成分。

检测细胞内的相分离现象

目前相分离领域的一个难点就是如何去鉴别细胞内的一些特殊的结构是否真的是相分离形成。在体外特定条件下，一些蛋白和RNA在足够的浓度或者合适的Buffer的情况下会发生相分离现象，通常通过在细胞内过表达这些蛋白，观察到形成较大的，球状的结构，以此来推测细胞内低浓度的该蛋白仍然会形成相分离，只是在普通的光学显微镜下无法检测到而已。然而，相分离需要足够浓度才能发生，因此，在使用过表达蛋白检测相分离的时候一定要考虑到外源过表达带来的影响。同时应该致力于寻找除过度表达之外的其他方式去证明细胞内确实发生了相分离现象。

目前被大家所接受的认为是相分离结构的的标准：形成球状结构，能够融合，同时使用FRAP技术，证明其能够发生荧光漂白恢复（图4）。但是FRAP实验并不是证明LLPS发生的金标准，仍然存在很多问题。

 

图4：检测相分离的方法

体内相分离液滴的物理特性

体外重现相分离现象时，对液滴物理特性的研究手段不胜枚举：如可利用延时显微镜测量液滴的反毛细管速度、通过FRAP测量液滴的流动性。然而如何探究体内相分离液滴的物理性质仍是一个难题。目前已有的手段如基因编码的纳米粒（GEMS）、荧光共振能量转移技术（FRET）、定量相显微镜（QPM）、折射率层析成像技术及更先进的布里渊显微镜等都可对体内相分离液滴的物理特性进行初步描绘。此外，体内相分离液滴的生物学功能也是研究的重点之一，如何改变液滴的性质以观察其功能，是未来的研究重点。

应用高分子化学来指导相分离研究的可行性及局限性

液液相分离研究的目标之一，是建立能够解释和预测大分子相分离现象的理论体系，并通过一级序列预测相分离的饱和浓度、刺激因素、液滴状态。高分子化学中的弗洛里赫金斯理论描述的是由焓介导的均聚合物从贫溶剂中析出的化学基础，其扩展理论考虑到了这一过程中的静电力作用。无规相近似方法则仅仅考虑了带电氨基酸的序列特征对杂聚体的形成的影响。此外，通过近似模拟也可以对相分离现象的机制进行进一步发掘：对单分子蛋白的模拟已能够较准确的说明其序列和功能间的关系，然而对成百上千个分子构成的相分离现象的建模及分析仍是目前的难点之一。对多组分相分离系统的粗粒化模拟初步解释了多层无膜细胞器如核仁形成的物理机制：蛋白-蛋白间、蛋白-RNA间的相互作用由序列决定；而不同细胞组分之间的相互作用呈互斥或亲和的状态，则可最终导致非随机的多层结构的形成。

算法模拟及理论体系可作为相分离现象实验数据的有力补充。反之，体内相分离现象的实验描述可作为算法模拟的数据库、并从中产生能够描述多分子复杂相分离现象的新的理论。

相分离到底意味着什么？研究相分离生物学功能

相分离研究的重点还是在于对其生物学功能的阐述。作者对目前报道的生物学功能进行了归纳总结（图5）：

图5：相分离功能总结

1. LLPS可以感知环境的变化，并对环境的变化做出快速响应。这种响应比通过细胞内的转录以及翻译过程更加快速。目前的一些研究已经证明，LLPS可以感知温度以及pH, 另外，还可以用于感知细胞内外源的DNA（cGAS相分离）

2. LLPS可以用来调节相关蛋白在细胞内的浓度。LLPS可以将高浓度的蛋白以液滴的形式储存起来，在细胞需要的时候将该蛋白释放到细胞环境中。

3. LLPS可以形成局部的高浓度蛋白，从而激活一些生化反应，激活相关信号转导途径以及促进细胞骨架的形成。

4. LLPS可以将一些蛋白与其底物隔离，从而抑制细胞内的一些生化反应过程。

5. LLPS可以介导一些蛋白定位到已经存在的一些无膜包裹的细胞器中。

6. LLPS的特殊结构可能对于细胞的形态起着重要作用。

7. LLPS可以介导形成一些孔状结构，比如核孔。

近年来，相分离领域已经成为生命科学领域研究的大热点，更多的相关的文章还在持续不断的发表，该领域对于揭示一些细胞的基础生物学问题以及对一些疾病的发生发展都将提供全新的思路。就在解读这篇文章的过程中，注意到Michael Rosen组在生物学预印本bioRxiv上online了一篇题为Organization and Regulation of Chromatin by Liquid-Liquid Phase Separation文章，说明了相分离在染色体的结构上起着重要的作用。

另外，去年12月份在深圳举行的的华人生物学家双年会上(The 12th biennial meeting of Chinese Biological Investigators Society )，诺奖得主Yoshinori Ohsumi 介绍了其实验室最新的研究进展， ATG13与ATG17结合能够发生phase separation的现象，揭示了autophagy中几个关键的conjugation system形成的物理学基础，作者发现突变了关键位点的 ATG13 与 ATG17 不再能形成相分离结构。从体外纯化的蛋白以及在细胞内都验证了这一理论。另外，去年清华大学俞立教授与李丕龙教授今年在 Cell research 上发表 了 Polyubiquitin chain-induced p62 phase separation drives autophagic cargo segregation的文章，报道了autophagy的adaptor protein P62能够发生相分离现象， 而且此现象能够介导 autophagy 对于底物的选择性。这些研究都将进一步拓展我们对于autophagy等领域的认识。

1.    C. P. Brangwynne et al., Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729-1732 (2009).

2.    S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen, Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18, 285-298 (2017).

3.    S. Alberti et al., A User’s Guide for Phase Separation Assays with Purified Proteins. J Mol Biol 430, 4806-4820 (2018).

4.    M. Kato et al., Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753-767 (2012).

5.    P. Li et al., Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336-340 (2012).

6.    M. Zeng et al., Phase Transition in Postsynaptic Densities Underlies Formation of Synaptic Complexes and Synaptic Plasticity. Cell 166, 1163-1175.e1112 (2016).

本文来自bioart，编译丨QY、赤贞

What 50 principal investigators taught me about my failure to land tenure

Getting a tenure-track academic research post is like winning a raffle — it happens for some, but the chance of success for any one individual is low, because the number of ticket holders far exceeds the number of prizes. It is fair to tell you here and now that I did not make it to the tenure track.

But I was curious about why I didn’t, and I learnt from interviewing more than 50 principal investigators (PIs) that my mindset might well have presented obstacles. I’m not claiming to know the formula for how to get tenured (if I had that, I would have used it for myself). Instead, I would like to offer the advice I’ve gathered.

At the beginning of my career, I did not think that I’d encounter any obstacles securing tenure. I had a great mentor for my PhD programme in structural biochemistry, which I completed in 1995, and another excellent one for my first postdoc position. They gave me achievable goals, taught me the joy of discovery, lifted me up when I lost hope. Most of all, they looked out for me — they cared about my career, introduced me to other leaders in the field and helped me to get ahead. But I became complacent: I started to believe that if I did a good job by my supervisor and solved the research questions placed in front of me, my career would take care of itself.

I was wrong, of course.

I went from postdoc to postdoc, four in all, working on research questions that interested me and that were relevant to human disease. I worked hard — I had a great time doing experiments and producing data, and I felt relevant and useful to society. But time goes by quickly. I became “too expensive” to stay in academia as a postdoc. A PI can hire a person half my age for less money if I don’t come with my own fellowship, and I had been a postdoc for so long (23 years, all told) that I was no longer eligible for many fellowships.

I did try to get on the tenure track. At the age of 41, after working in several postdoctoral positions over the course of 12 years, I started applying for tenure-track and other permanent positions. At this point, I had 19 peer-reviewed publications, including 5 as first author; 2 book chapters; and 10 published meeting abstracts. Perhaps it was not a hugely impressive record, but I was hoping it would be enough.

In the next 3 years, I sent out more than 100 applications to academic institutions, to the private sector and to government agencies (predominantly in the United States, where I was living at the time).

I applied for 57 academic tenure-track positions at first-tier research universities, second-tier teaching colleges and two-year ‘community’ colleges (which do not confer four-year degrees). Four institutions offered me a phone or in-person interview. I think I could have secured a position in Hungary, but I was not ready to move to another continent, and the university wanted me to start immediately.

Between 2007 and 2009, I also applied for 22 positions in the biotechnology–pharmaceutical sector and for 25 positions at government organizations, including various institutes and agencies connected with the US National Institutes of Health, the US Department of Defense, US National Laboratories. I received no offers, not even an interview.

Then I moved back to Europe to start a new family, and I took another postdoc. Finally, I lost interest in becoming a PI. I had a brief but pleasant engagement at a biotechnology company (brief, because it was in the wrong country for me), and then became an innovation manager at a university. This position turned out to be a poor fit for me, so I left after 2.5 years. These days, I am spending my time collecting more PI interviews, taking data-science courses and writing job applications. I hope to publish the results of my interviews as a book on mentoring.

Hard work

Over the years, I have attended many seminars and workshops on career development. At such events, the takeaway is usually that you need to work hard and network to get ahead. I was doing both. After a while, I gave up on my dream of becoming a PI, but I didn’t understand why others succeeded at something I hadn’t. It seemed to me that I was as smart as they were and worked as hard as they did. I could not ascribe my failure to get on the tenure track to just bad luck. I was a researcher, after all, and I wanted to understand my failings.

Around this time, I learnt a few things about cognitive psychology — for one, that emulating my successful colleagues’ ways of thinking might be a helpful step towards achieving the overall goals that they had managed to reach. So, I decided to interview tenured or tenure-track PIs who were leading their own laboratories to find out more about their thought processes.

Many of the 50 PIs I interviewed mentioned being lucky — being in the right place at the right time, knowing the right people. Of course, “be lucky” is not a very useful piece of advice. But some of their advice might help you to recognize and take advantage of your luck. I chose the tips that stuck in my mind because I knew I had not followed them. I hope that they might be useful for you, regardless of the sector you work in. Use them like a catalogue of chess moves: none of these tips will guarantee that you win the game, but they might help you to avoid some pitfalls along the way.

Accept your data. According to one PI, success depends more on the people doing a project than on the topic of study; some people make their project work no matter what the topic is. Others block their own way to success because they require too much evidence. I, for one, have always been excessively critical of my own data. Although I am a meticulous experimenter, whenever my results seemed to confirm my hypothesis, I was afraid to accept them because I knew I was biased (it’s funny, I know). I was spending too much time looking for alternative explanations instead of accepting that I was on the right track and moving on to the next step. We have all heard about the reproducibility crisis — and I am not advocating the reckless publication of unverified data, or recommending being so careful that you end up publishing nothing — but there is a difference between being reckless and shooting yourself in the foot.

Own your project. Another PI told me that the best researchers take ownership of their projects. Or, as he put it, the best researchers do not ask for permission to do something, they just tell their PI what they have done. Another PI echoed this thought when she told me that postdocs should work on important questions, instead of sticking to the questions put in front of them. I have been guilty of violating this advice, too — I stumbled once on a huge effect of a group of enzymes on the favourite protein of our lab. I even managed to persuade myself that the effect was not an artefact. My supervisor acknowledged that the size of the effect was remarkable, but pursuing a new direction did not fit in with the lab’s plans — and I let it go. I worked on continuing projects and dropped this exciting new avenue. Had I insisted, I think he would have come around — few PIs will say no to a promising publication.

View yourself in your desired role. When I asked another interviewee how long he had been a PI, he said, “I have always been a PI — in somebody else’s lab.” Others have voiced similar sentiments; they always treated their supervisors as future colleagues, not as their superiors, establishing a relationship of two equal scientists working towards common goals.

Ward off despair. When your results seem to contradict your PI’s hypothesis, you might fall into despair (I did), but this is not good for productivity. It might help to know what one PI told me — “students should know that PIs are wrong 90% of the time”. You must generate your own motivation in science and work out how to pick yourself up, because nobody else will do it for you. Working on questions that are important to you and taking ownership of your project will help you to keep your motivation level high.

Maximize your time. Several PIs emphasized the importance of keeping your eye on timelines. “In science,” as one of them put it, “you have to be productive in a short time.” I know I have messed this up, too. When I started my last project, the two years of my brand-new fellowship seemed to stretch out ahead of me like the ocean. But they went by much more quickly than I thought they would. You should always keep your eye on the calendar and make sure that you will have publishable results when your fellowship is ending. Funding is tight worldwide for both research grants and postdoctoral fellowships, and it is far from certain that a lab will keep postdocs on board to give them time to complete and publish their projects. (I still have the data from that last project and am hoping to publish them eventually.)

Outline your goals. In line with this, other PIs told me that they always had a plan for the next step in their career. When they started their PhDs, they were already thinking about where they would do their postdocs. When they started their postdocs, they already knew where they would apply for assistant professorships. Of course, their plans changed over the years — but they always had a plan to work towards.

Trust your intuition. Most of the PIs I interviewed told me that they made quick decisions with their ‘gut’. I had been doing quite the opposite. I always felt that being a scientist meant making well-informed decisions and, if you couldn’t come to a decision, collecting more information. But most PIs acknowledged that they made decisions — from everyday minor choices to more major decisions involving the direction of a study — knowing that they would find out only later if they had made the right choices. Instead of working on one or two well-thought-out projects, some PIs start ten half-baked ones. Even the most carefully planned projects can fail, and the chances are that you will learn more from an experiment that you conducted in one day than you would from three days of only thinking about it.

Finish. There is lot of truth to the maxim “finished is better than perfect”. If you do not publish something that you have worked on, you have wasted your time and your supervisor’s time and money (guilty there, too). It is as if you had never done the work, as far as everybody else is concerned. If you can’t prove your hypothesis because there is not enough time to do all the required experiments, it is better to prove and publish only part of it, rather than trying to go for the complete story and ending up with an unfinished, unsubmitted manuscript (I have a couple of those).These few pieces of advice stuck with me — I hope they will be useful to you.

https://www.nature.com/articles/d41586-019-00560-9?utm_source=twitter&utm_medium=social&utm_campaign=crs-&utm_content=150219v2

To win at gene therapy, companies pick viruses with production credentials

Nature Biotechnology volume 37, pages 5–6 (2019)

Is a spontaneous chemical change on the proteins that coat an adeno-associated virus (AAV) a problem for gene therapy developers? According to a recent controversial paper from the lab of gene therapy pioneer Jim Wilson, professor of medicine and pediatrics at the University of Pennsylvania, the hitherto overlooked phenomenon of protein deamidation can affect the capsid of AAV, the vector most widely adopted in gene therapy, reducing the efficiency with which vectors enter their target cells. Because the reaction is unpredictable it may impair lot-to-lot consistency in manufacturing. “My first response to the paper was here we go again,” says Michael Linden, newly appointed CSO of Hampton, UK-based Touchlight Genetics. “Let’s see if this one explodes.”

The findings (Mol. Ther. https://www.sciencedirect.com/science/article/pii/S1525001618304544) have not been universally accepted. But at the same time, Linden adds, the paper serves to highlight the lack of standardization in gene therapy vector analytics and manufacturing. Every lab has its own way of measuring vector titer and purity. “These things have probably a much bigger effect on the potency of the vector than deamidation,” he says.

In protein science, deamidation is a well-known post-translational modification. As it can affect the potency and stability of monoclonal antibodies, it is included as a design consideration in monoclonal antibody development . But for gene therapy vectors, its potential impact has been largely ignored. “At a minimum, the study is important because it is something that has to be explored more fully,” says Mark Kay, professor of pediatrics and genetics at Stanford University. Isolating the precise contribution that deamidation may—or may not—make to vector yield and potency during production is a difficult undertaking, given the complex matrix of parameters that influences the final product. “There is a lot that is unknown about manufacturing and how that affects potency,” says Kay. “You can use the same manufacturing method and have two lots of vector that give you similar levels of titer but different biological activity.”

One significant parameter is formulation—the precise composition and concentration of the excipients used to package the vector. “This causes huge variability between different manufacturing groups,” Kay says. Formulation specialists, such as Martinsried, Germany-based Leukocare, have developed algorithms to optimize the selection of excipients in order to maximize stability and avoid issues such as aggregation and oxidation. “We are able to substantially stabilize viruses regarding their functionality,” says CEO Michael Scholl. Purification methods influence vector potency as well. The final product may also contain DNA from the bacterial plasmids used to transfect the producer cell line, as well as residual components of those cells, both of which can also influence activity.

All of these issues are beginning to take on added importance as the gene therapy sector moves into its early stages of maturation. The first therapies are now on the market, and approvals for several others are in the offing. “Manufacturing expertise is becoming 80% of the question; [clinical] efficacy is 20%,” according to a recent report from a roundtable session hosted by the investment bank Jefferies Financial Group. Gene therapies that require systemic delivery, for conditions such as hemophilia, Duchenne muscular dystrophy and spinal muscular atrophy, need far higher production efficiency than does a product for localized use—to treat the eye, for example. Luxturna (voretigene neparvovec-rzyl), Spark Therapeutics’ FDA-approved AAV2-based gene therapy for retinal dystrophy (Nat. Biotechnol. 36, 6, 2018), is administered at a dose of 1.5 × 1011 vector genomes per eye, several orders of magnitude lower than some of the high-dose AAV-based therapies that are now in or approaching phase 3 trials. Even that metric can itself be unreliable: methods for measuring the amount of vector DNA present, such as quantitative polymerase chain reaction (PCR) or digital droplet PCR, provide an indirect measure of the actual level of viral activity. “We dose based on genome copy [number], which is about the only assay we can reliably perform,” says Wilson. The cell-based assays in use are suboptimal, Linden notes. “They’re not reflective of the bioactivity of the virus. What you see in tissue culture cannot be extrapolated into animal models,” he says.

Notwithstanding the challenges, gene therapy manufacturing is obviously not stuck. Industrial scale-up and optimization are proceeding on all levels, from cell transfection to produce the virus vector to formulating the finished product. One shift underway is a migration from adherent cell culture to suspension cell culture, which allows more rapid expansion. Engineered insect cell lines employing baculovirus-based expression systems are growing in popularity for that reason. “There’s a preference for insect-cell-based manufacturing at the moment because once you can get it tweaked and working, you get higher yields,” Kay says. In contrast, traditional mammalian cell lines, such as HEK293, grow more poorly in suspension culture. Valoctocogene roxaparvovec, Biomarin’s AAV5-based hemophilia A gene therapy, which is in a phase 3 trial, is one high-profile example of an insect-cell-grown vector. The preference is not universally held, however. “I’m personally of the opinion that mammalian systems are better suited to make mammalian viruses,” says Linden. Alain Lamproye, CEO of the gene and cell therapy contract manufacturing organization Yposkesi, agrees. The insect-cell-based approach “is restricted to certain serotypes,” he says. It does offer cost benefits, he says, because the process dispenses with the need for bacterial plasmids to introduce the vector components to the producer cell line, but consistency can be a problem.

Yposkesi, located in Evry, France, claims a ten-fold improvement in vector production using the conventional HEK293 cell process. This yield resulted from replacing polyethylenimine with an as yet undisclosed transfection agent, which improves transfection efficiency by up to fivefold, in a highly efficient HEK293 cell line. “We have identified a subpopulation which are high producers,” he says. The company, a spin-out from the not-for-profit gene therapy research organization Généthon, can achieve up to 70% vector purity without the need for cumbersome and expensive ultracentrifuges. “Ultracentrifugation is not an easily scalable manufacturing technique,” he says.

For all their challenges, viral vectors remain the most effective way of delivering large quantities of DNA to target cells. The repertoire of available capsids, particularly AAV vectors, has expanded in the past two decades—an effort spearheaded by Wilson following the death of Jesse Gelsinger in a trial of an adenovirus-based therapy for ornithine transcarbamylase deficiency, a trial Wilson led. The 11 available AAV serotypes can be further extended by pseudotyping—mixing and matching different viral genomes and capsid proteins—so the resulting constructs allow selective, if not specific, targeting of particular organs or tissue types. But what remains largely unchanged are the DNA payloads the vectors carry—for the most part they involve a transgene under the control of a strong viral promoter, as well as the sequences encoding capsid assembly functions. “The capsid [only] gets you so far,” says David Venables, CEO of Edinburgh-based Synpromics. An AAV9-based vector, for example, will efficiently deliver its payload to the central nervous system, but, he adds, it will not do so exclusively. “You’ve still got exposure elsewhere.”

Synpromics is developing promoters that allow greater control of transgene expression in terms of both location and strength. It’s not the first company to develop cell-selective promoters, nor is it the first to develop inducible switches that can dial up or down the level of gene expression required. But it is attempting to overcome the shortcomings of existing systems, which, says Venables, are “leaky” in terms of their expression profiles and which require the coexpression of additional factors, such as transcriptional activators or repressors, to work. They are neither able to shut down expression completely nor able to crank it up sufficiently when required. “The amplitude of the dial-up tends to be quite low.” The company has not yet unveiled the workings of its inducible expression switches, but it has secured commercial agreements with six of the ten leading gene therapy firms, he says. One of them is UniQure, of Amsterdam. It recently reported preclinical data in a nonhuman primate model indicating that a liver-directed gene therapy, under the control of a Synpromics-developed liver-selective promoter, resulted in an eightfold increase in gene expression compared with current approaches.

Touchlight has developed a method to obtain viral vectors using in vitro DNA amplification, which eliminates the need for plasmid transfection and avoids the packaging of unwanted bacterial DNA into viral capsids. “Part of your drug product is contaminated by antibiotic resistance genes,” says Linden. “In the traditional system, you can’t get around it.” Touchlight avoids the problem by using a dual-enzyme system, comprising a phage DNA polymerase and protelomerase. Via rolling-circle DNA replication, a circular template containing the sequence of interest is replicated into a concatemer, a long continuous sequence, which is then processed into individual ‘doggybone’ DNA (dbDNA) molecules. So-called because of their shape, these double-stranded, covalently closed DNA molecules can be introduced into producer cells lines for capsid assembly and packaging using the same triple transfection method currently used for bacterial plasmids. “You do exactly the same, except you don’t package the crap,” says Linden. What’s more, the generation of dbDNA vectors is rapid. “You can make gram amounts of DNA within two weeks at scale in GMP [good manufacturing practice],” he says.

The technology can be used to produce any DNA-based medicines, including gene therapies, DNA vaccines and what Linden terms “DNA-launched” products, such as antibodies. Touchlight recently entered a collaboration with the Janssen Biotech arm of Johnson & Johnson, which is evaluating the technology for undisclosed genetic therapies in infectious disease and oncology. Linden, who, as Pfizer’s former vice president of gene therapy, helped to develop the pharma firm’s gene therapy strategy, says a dbDNA-based AAV gene therapy could reach in the clinic within one to two years.

Linden expects gene therapy to progress in a hybrid fashion in which new ideas and innovations are bolted onto the existing technologies. Because they are potentially curative, AAV and other viral vectors are here to stay, he says, “and what we have to do is address the limitations.” Mark Levi, senior consultant with Parexel, regards this as being feasible, given the incentives involved, “I think you’ll see manufacturing rise to the occasion and deliver,” he says. “Where there’s money to be made it’ll get done.”

如何做一次优秀的学术演讲？

Courtney是爱尔兰利莫瑞克大学的三年级博士生，研究金属与二维半导体材料的相互作用。2017年7月，微观科学显微镜大会（MMC）在英国曼彻斯特举行，她在思考如何在会上展示自己的研究成果时，突然“觉醒”了。

许多青年科学家都感受过这种因为设想即将登台演讲而引起的发自内心的恐惧。不管是受邀参加国际会议、学术团体会议或者公众参与活动都会这样，甚至是面试过程中最重要的陈述环节也不可避免。

尽管演讲的听众和目标可能有所不同，但完成演讲所需的技巧和技术是相似的。那么，好的演讲和坏的演讲有什么区别？如何提高在台上演讲的水平？能给听众留下深刻印象真的那么重要吗？

最后一个问题的答案是肯定的，加州斯坦福大学的神经生物学家Susan McConnell说。关于“如何进行演讲”，她已经演讲了十多年。“做科研的关键是能够与他人交流自己的研究，”McConnell说，“无论是对我们的亲密同事，或是对我们领域有一定兴趣的其他科学家，还是对非科学家人士，清晰的沟通都是必不可少的。”

以下是她的十大秘诀，她计划在即将出版的电子书中更详细地进行说明。

1 表现真实的自己：真实最能打动人。
2 准备、练习和完善：去掉无谓的辅助词，比如“嗯”和“你知道”。
3 描述你想传达的事物：用生动的文字帮助听众想象出对应的画面。
4 说话抑扬顿挫：改变你的声调、音量和音高来保持观众的注意力。
5 向优秀的演说家学习：观察真正优秀的演说家是怎么演讲的。
6 寻求反馈：练习时，找一个听众有助于你找出错误。
7 外在形象：如果你看起来很好，你会感觉很好，这将有助于你进行一场优秀的演讲。
8 停顿：给听众时间思考，增加他们的参与感。
9 身体语言：使用肢体语言，利用空间来传达信息。
10 自信：用你的面部表情、肢体语言和站姿来掌控舞台。

并非所有的研究人员都认识到，在实验室之外花些时间与同事们交流一下自己的工作自有其价值。“有些人有这样的想法：你花时间演讲，只是在做宣传推广，而把这些时间用来做科研可能会更有价值。”Dave Rubenson说。他是总部位于洛杉矶的nobadslides.com的联合创始人之一，该公司提供有关如何有效进行幻灯片演示的课程。

“事实上，进行一场引人入胜并让听众理解的演讲，可以同时提高你和听众对你的工作的理解，这对科研本身具有重要意义。”Rubenson说，除此之外，在会议上发表演讲是一个很好的吸引合作者的方式，这些合作者可以帮助你在职业生涯中取得突破和进步，但只有当他们听明白你想表达的内容才可能提出合作。

一个良好的开端是站在观众的角度思考。需要尽早让他们知道为什么他们应该关心你所说的内容，你演讲的核心是什么。Rubenson说一开始简明扼要地总结你的演讲内容，之后在此基础上做一进步的延伸扩展，胜过花时间去琢磨你的500张幻灯片有哪些可以省略掉。

演讲者经常演讲失败，因为他们试图传递太多复杂的信息。通常而言，语言和内容的设计必须考虑到非相关专业的科学家。Rubenson说：“你必须考虑到你关注的那些听众中知识面最窄的人。”

另一个常见的错误是将幻灯片当作“数据垃圾站”。你还记得眯着眼睛看那些满满当当塞了八张小图表的幻灯片，并好奇为什么演讲者只提到其中一张的时候吗？在设计自己的幻灯片时，请牢记这一点。有些动画软件可以让你在演讲的过程中提到什么添加什么，这很有用。

最重要的是：保持听众的注意力。

不同的方法适用于不同的人。下面是Eileen Courtney在讲台上保持冷静的秘诀。

1 在与实际演讲环境相似的地方练习。
2 根据提纲记住关键句子，而不是逐字逐句地背诵。
3 确保你在规定的时间内可以完成演讲，这样你就少了一个需要担心的事情。
4 穿一些看起来专业又舒适的衣服，而不是新衣服。
5 避免在演讲当天暴饮暴食，同时限制咖啡摄入量。

你可以通过在每一段的末尾添加总结幻灯片来帮助你避免思维混乱。“你可以把演讲看作是一系列数据潜水。”McConnell说，“你需要定期浮上来呼吸新鲜空气，并说‘这是我们刚刚了解到的，这是结论，这是它与下一部分的联系’。”

McConnell在一个大受欢迎的42分钟在线视频中描述了这一点以及更多有助于研究人员提高科学演讲能力的方法。2013年美国神经学家Matt Carter的《设计科学演讲》（Designing Science Presentations）一书也提出了很多建议。这些都很有帮助，而且大多数人发现，需要进行公共演讲和报告时，多多练习，即使不能达到完美，也肯定会有所完善。

这一观念正是国际演讲会（Toastmasters International）的核心理念所在。国际演讲会是一个非营利性组织，通过遍布143个国家或地区的16000多个分支机构，帮助个人提高公共演讲能力。在每周或每两周一次的会议上，成员们练习演讲并互相提供反馈。

前年夏天，Eileen Courtney意识到需要增进自己的演讲技巧，于是参加了当地的一个国际演讲会分支机构。她的决定似乎得到了回报。5月，她在伦敦物理研究所举办的“3分钟奇迹”竞赛中获得亚军和最受观众欢迎奖。这是一个科学传播挑战赛，参赛者需要在180秒内，仅通过一张幻灯片向非专业人士展示他们的研究。

“我最近要进行其他的演讲，我已经淡定了很多，因为我既参加了国际演讲会，又完成了作为博士学习部分的教学环节。”Courtney说，“当你积累了较多的公共演讲经验后，你就不会那么害怕了。”

starting the PhD – learning new vocabulary — patter

Scholarly work often involves learning new words. You k […]

通过 starting the PhD – learning new vocabulary — patter

siRNA/ASO转染效率低？试试Two-step Trasnfection

Via: https://mbio.asm.org/content/9/3/e00716-18.long#sec-12

Human membrane trafficking gene siRNA screen.The Human siGENOME siRNA Library, which targets 140 membrane trafficking genes (4 siRNAs per gene; Dharmacon, Inc.), and 16 other selected genes were included in the primary screen. In brief, NHDFs were seeded in a 96-well plate a day before siRNA transfection. Next day, cells reached 90 to 95% confluence and were transfected with siRNA twice (4 h apart between first and second transfections) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. Transfected NHDFs were incubated for 48 h and then infected with GFP-expressing TB40/E virus at an MOI of 3. GFP intensity was monitored every 24 h with a Synergy HT microplate reader (BioTek). The entire screen was performed in duplicate and repeated twice. At 7 days postinfection, 5 µl supernatant was transferred to fresh untransfected cells and GFP levels were monitored as described above.

Via: https://www.nature.com/articles/s41593-018-0293-z

The neuroblastoma cells (ATCC) were cultured in DMEM/F12 (Gibco) supplemented with 10% fetal-bovine serum (Omega) and 1% penicillin-streptomycin (Gibco) at 37 °C with 5% CO2. For knockdown experiments, cells were transfected with SMARTpool ON-TARGETplus siRNA targeting TDP-43 (L-012394) or control siRNA pool (D001810–10) (GE Dharmacon) at a final concentration of 50 nM, for 96 h in two doses (0, 24 h), after complexing with Lipofectamin RNAiMAX (Invitrogen) in Opti-MEM (Gibco) for 20 min.

细胞裂解中应该注意的问题

You’ve cultured your cells and completed your treatments, now it’s time to harvest them and proceed to the downstream effects. Cell lysis is the crucial stage that determines if your experiment has a chance of producing the data that you have been waiting for. Part of the starting biological material is inevitably lost on each step of your experiment, so it’s critical to extract it all at the beginning. Often, the amount and quality of resulting biological molecules depend on the chosen lysis method (1).

1. Frozen vs. Wet Cells for Lysis
If you work with cytoplasmic enzymes or DNA, it probably doesn’t matter if you are going to do your cell lysis on “just-off-the centrifuge” cells or frozen cells. However, keep in mind that any method that involves wet, non-frozen cells does not completely stop biochemical reactions (2). If you need to get a snapshot of “normal” cellular activity, the sooner you freeze your cells and stop stressing the cells and thereby altering normal transcription and translation, the better.

After you decide on the starting state of your material, there are several things to consider when planning your lysis:

2. Are You Dealing with a Cell Wall?
You are lucky if you work with mammalian or insect cell lines, as they are only surrounded by a thin plasma membrane that doesn’t require mechanical effort to rupture. Gram-negative E.coli is a slightly bigger problem because of multiple rigid peptidoglycan layers . However, if you work with algae, fungi (that include yeast), archaea or plant cells, you need to get rid of at least parts of the cell wall, so cell lysis will require more effort than just adding a detergent and heating cells up.

There’s a difference in cell wall thickness and composition within groups as well. Gram-positive bacteria have many more layers of the main cell wall component peptidoglycan than E.coli and it will require extra effort to lyse them. “Algae” is a diverse group with cell walls of various composition, so it’s worth checking recommended methods, if not for your species than at least for the algal family.

3. Consider Culture Volume
Significantly upscaling you prep without changing the lysis method often creates problems. For example, you were always doing your yeast preps for the western blot on a tabletop bead beater that takes Eppendorf-size screw-top tubes. Now you need to do a large pull down experiment where you need to lyse tens of grams of cells. It would be a mistake to just split your culture into smaller volumes. The smaller the volume, the more material you lose. But even leaving aside the time you will spend aliquoting and pooling you samples, it’s worth exploring lysis options for larger culture volumes: sonicator (suitable for medium-size preps), French press (not the coffee one) or cryogenic tissue grinder.

4. Choosing the Method
You choose your lysis method depending on your downstream application. It can be either mechanical or enzymatic.

• Mechanical lysis methods include boiling cells with a detergent, vortexing with glass or ceramic beads, grinding cells in liquid nitrogen or sonication. Grinding with beads is suitable for isolation of soluble cytoplasmic proteins or the cell walls but not for preparation of intact mitochondria or plasma membranes. In these cases, enzymatic lysis or protoplast lysis are more appropriate.
• Enzymatic methods are based on using specific enzymes (no surprise there) to strip off cell wall and osmotic shock to release the cell content. This method is less harsh than the mechanical grinding, so suitable for isolating intact cell structures such as mitochondria.
• Detergent methods are quick but you need to make sure that you know how to get rid of the detergent afterwards. The leftover detergent can interfere with your downstream biochemical assays.

Considering your lysis options before starting the experiment will save you time and reagents, and help you get the answers you were looking for the first time around!

Literature:
Sohrabi M. et al. (2016) The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology. J Microbiol Methods. 122:64-72. doi: 1016/j.mimet.2016.01.013.

Lee S. J. et al. (2017) Cost-effective and rapid lysis of Saccharomyces cerevisiae cells for quantitative western blot analysis of proteins, including phosphorylated eIF2?. Yeast. 34(9):371-382. doi: 1002/yea.3239.

Tetbow:一种神经元多色标记的方法

Stochastic multicolor labeling is a powerful solution for discriminating between neurons for light microscopy-based neuronal reconstruction. To achieve stochastic multicolor labeling, Brainbow used the Cre-loxP system to express one of the three fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene cassette are introduced, stochasticity will result in a combinatorial expression of these three genes with different copy numbers, producing dozens of color hues (Livet et al., 2007; Cai et al., 2013). However, the brightness of Brainbow was inherently low. This is because the stochastic and combinatorial expression of fluorescent proteins is only possible at low copy number ranges, resulting in low fluorescent protein level.

The principle of Tetbow

In modern neuroscience, plasmid or viral vector-mediated gene delivery is more common than conventional transgenic strategies. In such vector-based gene delivery systems, the stochastic expression of XFPs is possible by simply introducing a mixture of XFP genes. Stochastic multicolor labeling is possible when each of the XFP genes is introduced at ~2 copies per cell per color, following a Poisson distribution. To overcome the limited expression levels of XFPs in the original method, we used the Tetracycline trans-activator system to boost the expression levels, and we named the system Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018) (Figure 1). Once introduced, fluorescent proteins were best imaged after tissue clearing with SeeDB2 (Ke et al., 2016, 2018).

How to use Tetbow for neuronal labeling

To use Tetbow, you can introduce the four Tetbow plasmids (Addgene #104102 – #104105) using in utero electroporation or with AAVs (prepared with Addgene #104110 – #104112) (Figure 2). We’ve also created constructs with containing chemical tags such as the SNAP-tag, Halo-tag, and CLIP-tag (Kohl et al., 2014) (Addgene #104106 – #104108) (Figure 3) that can be introduced using in utero electroporation. Then at your preferred time points, sacrifice the animals and fix the samples. When you use AAVs, it typically takes a few weeks to achieve optimal expression of XFPs. Any longer and the cells may start to show morphological abnormalities due to the excessive amount of XFPs expressed. Then, clear the samples and visualize.

For more details on this procedure, find the protocol here.

Tips and troubleshooting

1. Plasmid concentrations and AAV titer – The XFP genes have to be introduced at ~2 copies per cell per color to achieve the highest color variations. Adjust the plasmid concentrations or AAV titer for the best results.
2. The timing of sacrifice when using Tetbow AAVs – You will have to wait a few weeks to achieve an adequate expression level of the Tetbow AAVs. However, you should not wait too long, as too much XFP is toxic to the cells.
3. Inadequate expression levels of XFPs – Reduce the amount of tTA. Paradoxically, too much tTA leads to a reduced expression level of XFPs, most likely by suppressing transcription. It is critical to express a minimal amount of tTA to achieve highest expression levels.
4. Clearing large tissues – Fluorescent proteins are very stable in SeeDB2. However, SeeDB2 is not powerful enough for large brain samples. For large brain samples, we recommend pretreatment with ScaleCUBIC1 (Susaki et al., 2014), before clearing with SeeDB2.

To see Tetbow in action, watch the video below depicting dendrite wiring of mitral cells.

---

via addgene: https://blog.addgene.org/tetbow-bright-multicolor-labeling-for-neuronal-tracing

 

RNA-Seq的序列比对：哪种算法最合适？

RNA-Seq has replaced microarrays for many applications in the area of biomarker discovery. The prices have been fallen substantially in recent years. The sequence data allows to extract more information than gene expression only. And there is no requirement that a reference genome must exist. However, the analysis of the resulting data is much more challenging and requires more ressources than other approaches.

RNA-Seq read alignment

One of the most ressource-intensitve steps during a NGS data analysis is the alignment of the sequence reads to the reference genome. Therefore, a common question is about choosing the best NGS alignment tool. As we show in the referenced article, finding the best tool is not possible without in-depth examination of your use case.

Finding an optimal alignment of NGS sequence reads is already a challenging task, and for RNA sequencing data is has to be carried out millions of times. Compared to the alignment of DNA sequences, tools aligning sequences from RNA transcripts have to cope with intronic sequences that lead to large gaps in the alignment.

Our method of comparing RNA-Seq mappers

In order to compare different short read aligners, we use a published, real-life RNA-Seq dataset. All optimal alignments (also multiple mapping loci) of 100,000 read pairs of each sample were calculated with the full sensitivity mapping tool RazerS 3. In the benchmark shown below, we measured the performance in finding all optimal hits of different NGS mappers with default parameters. True positives are reads with up to 10 multiple mapping loci, allowing up to 10 errors (mismatches and indels). Note that we explicitely want to find all multiple mapping loci in this benchmark and not only unique mapping loci or just one random hit of several. We have used the publicly available SRR534289 dataset. Please find more information in the benchmark details here.

Sensitivity and accuracy

The following comparison addresses the question: how accurate do the tools report alignments when compared to the known truth. On-target hits means how many of the reported alignments do actually map to one of the true locations for this sequence. False positives counts the number of reported alignments that do not map to any of the true positions.

sensitivityOn-target hitsmRNA-Seq99.1%100.0%99.1%97.8%100.0%99.7%98.8%Bowtie2(v.2.1.0)segemehl(v0.1.7)BWA(v.0.7.4)BWA-MEM(v.0.7.4)STAR(v.2.3.0e)GEM(v.1.376)BBMap(v.34.41)979899100

sensitivity: 99.95%

false positives

False positive hitsmRNA-Seq3261461212021580333Bowtie2(v.2.1.0)segemehl(v0.1.7)BWA(v.0.7.4)BWA-MEM(v.0.7.4)STAR(v.2.3.0e)GEM(v.1.376)BBMap(v.34.41)05001000150020002500

Runtime and memory requirements

Next, we tracked the computational ressources that are beeing used by running the different tools. Note that several tools need significant more memory than a typical desktop computer has.

timeUser time [s] *mRNA-Seq97.45175.163.2430.087.0315.6458.01Bowtie2segemehlBWABWA-MESTARGEMBBMap050100150200250300350400450500

BBMap● user time: 458.01 s

GBMemory consumption [GB]mRNA-Seq3.7670.053.855.7528.124.7389.24Bowtie2segemehlBWABWA-MESTARGEMBBMap **0102030405060708090100

segemehl● memory consumption: 70.05 GB

* The time shown includes the (for some tools dominating) index loading step, which will be less influential (or even negligible) when mapping real-life datasets (>10 Mio reads).
** By default BBMap takes as much memory as the system provides. The minimum requirement for the used genome is 24GB.

Other common decision factors for choosing an RNA-Seq aligner

Further criteria that people commonly use for selecting an aligner are

• Additional information provided, for example on splicing
• Addtional functionality, e.g. soft clipping
• Interplay with other upstream or downstream tools
• Maturity level and development activity
• Number of citations of the respective publication

一抗标记的5种方法

1. NHS (Succinimidyl) Ester Method

This method is useful for the conjugation of antibodies with widely available fluorescent dyes such as rhodamine derivatives. It is typically performed in a phosphate buffer with subsequent on-column separation from the unlabeled dye. The main disadvantage is that the esters are unstable because they are moisture-sensitive. The labeled antibody should be used immediately after the end of the reaction.

2. Isothiocyanate Method

You may used this method to make fluorescein isothiocyanate (FITC), which is very popular in the preparation of fluorescent proteins and antibodies. If you’ve worked with fluorescent microscopy, you’ve dealt with FITC. Isothiocyanate analogues of different standard dyes are also available.

Isothiocyanate is more stable than NHS but it is harder to make and your labeling reaction will likely be less efficient with this method. As with NHS, the excess dye should be removed after the reaction by chromatography.

3. Carbodiimide Method

Carbodiimide-derived compounds convert carboxyl groups on proteins into reactive intermediates that can react with lysines. The high reactivity of carbodiimides means that they can be used to label antibodies with relatively inert materials such as magnetic or gold particles. The most commonly used carbodiimide is EDC. NHS is sometimes added to the reaction to aid the formation of relatively stable intermediates.

The method is simple, but similarly to NHS, EDS is hygroscopic, so you will need to use your antibody immediately after labeling.

4. Two-Tag Method

Here, you label your antibody with another protein that serves as a catalyst, for example HRP or alkaline phosphatase. As both of the molecules contain numerous amino-groups, direct cross-linking (as with the NHS method) would lead to polymers of the same molecule. Therefore, you would perform the linking in two separate steps. You cross-link the antibody to molecule X and the catalyst to molecule Y. You need to choose X and Y carefully because they should interact to form the conjugate. For example, you could choose maleimide as X and a thiol as Y.

While the resulting conjugate will more stable that what you obtain with the NHS method, this method will require three times as much work; separate labeling and purification steps for the antibody, catalyst, and the conjugating reaction. The good news is that you can buy pre-labeled catalytic molecule and then label your antibody accordingly.

5. Periodate Method

This method is useful for generation specific HRP-antibody conjugates. Periodate activates HRP by creating aldehyde molecules that interact with lysine residues. The HRP itself has only a few lysine residues, so enzyme polymerization is not a significant concern. The bonds between HRP and the antibody are reversible unless stabilized by adding sodium cyanohydride.

That was my run down of the top home-made antibody labeling methods. There are a lot of commercial kits on the market with similar underlying chemistry that may make your life much easier. If your lab has the money.

Do you have another antibody labeling method that you’d like to share with us? Get in touch by writing in the comments section.

Literature:

Meyer JP, Adumeau P, Lewis JS, Zeglis BM. Click Chemistry and Radiochemistry: The First 10 Years. Bioconjug Chem. (2016) 27(12):2791-2807. doi: 10.1021/acs.bioconjchem.6b00561. Epub 2016 Nov 22.

如何审稿及撰写同行评议

我们建议每篇论文读三遍，每次只关注一个点，并且分主要缺陷和次要缺陷来撰写每个点的评语。主要缺陷会需要大量时间进行解释或纠正。

读第一遍可以对全文有个大致了解，并弄清楚论文目的。一边读一边做记录。确保论文内容属于期刊范围内。虽然很少发生错投期刊的情况，但强迫自己回答这个问题，能让你更好地理解整个研究和研究目的：也就是研究想要达到的结果。

在你最有专业发言权的部分多做一些记录。编辑并不要求你在论文涉及的所有方面都很专业，但编辑也不希望你是个完全的新手。提前开诚布公地向论文作者和编辑说明你的审稿意见将侧重于哪几个方面的科学内容。

第一遍读完后，试着详细写下你对论文研究内容的理解——“映射”这篇论文。这能告诉作者，从你，也就是一个读者的角度，是如何看待他们的研究目的、结果和创新性。如果作者不同意你的分析，至少让他们从你的评语中明白这不是你的问题。你的分析将给出一条明确信息，即作者需要在传达研究目的方面再下功夫。

第一遍通读时，你可能会发现某个致命缺陷。先忘了次要和主要缺陷吧：致命缺陷足以让你停下审稿的脚步。如果实验方法具有致命缺陷，论文缺失了某一整段内容，或论文根本就无法阅读，那么继续审稿的意义也不大了。在这种情况下，你可以在评语中说明所有缺陷，然后提交。视不同的期刊而定，有的期刊会让你选择“拒稿，但可重新提交”。要不然，直接拒稿就行。没什么不好意思的！

如果你没有发现致命缺陷，那就开始读第二遍。这一次，你仍然需要花费一点时间，安安静静地投入其中。

读第二遍能让你关注到研究的细节：方法、分析和结论。记得注意区分主要和次要缺陷，并按时间顺序阅读。这时要问自己以下问题：

• “摘要”和“引言”是否清晰地描述了研究的必要性和相关性？

•  研究主要问题（可能不止一个）的“方法”是否合适？

• “结果”是否表述清晰，逻辑严密，是否有数据支撑？数据是否清楚、描述完整？

•“结论” 是否合理回答了作者在“引言”中提出的问题？

要确保在“引言”中提出的问题在“结论”中得到了恰当回答，这一点非常重要。此外，注意寻找文中有“水中捞月”嫌疑的论点，论文应该远离捞“月”的尝试，除非这是篇天文学期刊投稿。

到了这个阶段，一种不错的做法是把这篇论文放几天，让自己从这些细枝末节中抽离一会儿。

第三遍，也就是最后一遍阅读时，你应该关注写作和陈述方式。论文中的科学内容或许很棒，但长篇累牍和结构混乱容易遮蔽主要信息。如果你想对写作方式进行评价，请确保自己言之有物。不要只评注“写得很差”，而是要给作者提出具体建议，让他知道如何写得更连贯、缜密。比如，这篇文章读起来很累是不是因为段落衔接不通顺？作者是不是在文中用了太多让人混淆的缩略语？

你不需要对论文进行排版润色，这通常是请你审稿的期刊的工作。但是，能从整体上提高文章语言质量的建议也是受欢迎的，它们也是同行评议的一个重要部分。

现在，你已经罗列了每一条评语和建议，足以形成一篇完整的审稿意见。完整的审稿意见可以分为以下几个部分：

1. 引言：先“反映”论文，说明你的专业领域，以及论文是否可以发表，或是否存在致命缺陷；

2. 主要缺陷；

3. 次要缺陷；

4. 其它，少量建议和最终意见。

最后，仔细通读一遍你的意见，最好大声朗读：如果你在朗读自己的文章时结结巴巴，那你的读者肯定也会遇到相同的问题。这种做法还能让你注意到你的批评在作者听来是什么感受。确保你的批评具有建设性，而不是攻击性；有帮助，而不是有害处。有时你的确需要提交一篇尖刻的评论，但绝不是粗鲁的评论。记住同行评议的黄金法则：“用你希望别人审你稿的方式为他人审稿。” 

原文以How to write a thorough peer review为标题

发布在2018年10月8日的《自然》职业专栏上

RNA-Seq的序列分析及质控

RNA-seq is based on next-generation sequencing (NGS) and allows for discovery, quantitation and profiling of RNA. The technique is quickly taking over a slightly older method of RNA microarrays to get a more complete picture of gene expression in a cell.

Data generated by RNA-seq can illustrate variations in gene expression, identify single nucleotide polymorphisms (SNPs), profile transcription and identify new genes. RNA-seq is better suited for following rapid changes in cellular transcriptomes, finding post-transcriptional modifications, gene fusion, and other changes in transcripts. Modern NGS methods have made these discoveries faster to come across.

Key Metrics in RNA-Seq

A number of key data points have been found to be valuable for interpreting RNA-seq results. These include:

• Total, mapped and transcript-associated reads: Reads (cDNA fragments, often produced in tens of millions) are mapped to the genome or transcriptome. More reads will indicate a deeper analysis and discovery of lower-expression genes. Percentage of mapped reads will indicate the accuracy of sequencing and rule out contaminating DNA. And transcript-associated reads will reveal the existence of regulatory and expression regions.
• Aligned reads: Matching the reads to a reference sequence, or known genome, will show similarities and differences.
• Strand specificity: Some library preparation approaches allow for the retention of strand-specific information so that aligned cDNA-derived reads correspond to the original mRNA.
• Normalization: Methods used to remove technical biases from sequencing and improve comparability of test sequences to references. These include spike-in controls such as the Invitrogen ERCC controls, and a number of mathematical adjustments described below.

Tools for RNA-Seq Data Analysis

Methods for evaluating how RNA-based mechanisms impact gene regulation and disease and phenotypic variation include comparisons to sequences collected by the ENCODE Consortium, an international collaboration of genetic scientists funded by the US Human Genome Research Institute, and/or comparisons to reference transcriptomes, the number and variety of which are growing rapidly and largely available online. Other analysis software, such as the Partek Genomics Suite, analyzes microarray, qPCR, and pre-processed NGS data from a desktop computer. The Galaxy Project community hub posts a course adapted from Weill Cornell Medical Center on how to use these analytical tools.

Spike-In Controls

A mistaken assumption in sequencing is that all RNA yields are equal. Cells from different experimental conditions, however, do not yield identical amounts of DNA and RNA, reducing comparability of sequences.

Spike-in controls must be added proportional to the number of cells for data normalization, allowing accurate interpretations of true increases (or decreases) in signals. The Invitrogen external RNA control consortium (ERCC) spike-in control mix provides a blend of synthetic transcripts that mimic the lengths of natural eukaryotic mRNAs.

The more abundant a unique read is, the more likely fragments from it are going to be sequenced. But counts need to be normalized, so they can compare with other reads, samples and experiments. A number of mathematical adjustments make this possible:

• RPKM: Reads Per Kilobase Million, this adjusts comparisons of shorter and longer isoforms (since longer isoforms will have more reads). In this case, this is done by dividing the number of reads by the kilobase number, and then compared to the total number of fragments (usually in the millions).
• FPKM: Fragments Per Kilobase Million, this is similar to RPKM, but accounts for the fact that two reads can map to one fragment and avoids counting that fragment twice.
• TPM: Transcripts Per Million, this helps analyze RNA-seq data from two different tissues. RPKs will be the identical in each sample for the same isoform, TPM will compare to total number of transcripts to identify differences between tissues.

Analyzing Stop Sites

Identifying transcription stop sites and polyadenylation poly(A) can often require a special type of sequencing. PolyA tails are important because they are part of the process leading to transcription stops and the creation of mature mRNA. They are often added to the 3’ terminal of RNA to stabilize the RNA in eukaryotic cells, making translation more efficient. The Invitrogen^TM Collibri^TM Stranded RNA Library Preparation kit for Illumina^TM systems can help to sequence the poly(A) tail and identify these sites and alternative adenylation more easily without additional sequencing steps.

RNA-Seq Provides New Avenues for Research

RNA-seq is quickly helping gain understanding of the complexities of gene expression —complexities that may help develop new ways to diagnose and treat cancer and a host of other diseases and determine genetic solutions in applications ranging from agriculture to health to industry. But much of this complexity invites the risk of observational bias, including assumptions of rates of RNA expression yields, and comparisons of reads. Fortunately, many tools are available that can help normalize RNA-seq data and help make meaningful conclusions from different experimental conditions.

 

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最近火狐浏览器特别慢，打开gmail，看twitter和facebook，简直让人难以忍受。因此决定彻底删除，看看哪个扩展造成的。

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RNA-Protein互作的一些基本概念和技术

Techniques for Analysing RNA–Protein Binding

Capture Hybridization Analysis of RNA Targets (CHART)
CHART has been used to identify RNA-bound DNA or protein partners in Drosophila cells. This technique is a hybridization-based strategy that specifically enriches endogenous RNAs along with their targets with complementary oligonucleotides from reversibly crosslinked chromatin extracts.

Chromatin Isolation by RNA Purification (ChIRP)
Similar to the CHART assay, the ChIRP method is based on affinity capture of target lncRNA-associated DNA or proteins by biotinylated and tiling antisense oligos . The probe design requires prescreening and validation for maximum hybridization efficiency.

RNA Antisense Purification (RAP)
Similar to the CHART and ChIRP methods, RAP uses biotinylated antisense oligos and was first used to determine the localization of Xist . RAP uses 120-nucleotide antisense RNA probes to form extremely strong hybrids with the target RNA.

Crosslinking Immune Precipitation (CLIP) and Its Derivatives (HTS-CLIP, PAR-CLIP, iCLIP, etc.)
CLIP and its derivatives have been used to map the RNA sequences bound to an RNA-binding protein in vivo with high resolution and specificity. Essentially, after UV crosslinking between RNA and its binding protein, the RNA–protein complex is immunoprecipitated using an antibody. The bound RNAs are ligated to an RNA linker, purified, and analysed by sequencing.

Techniques for Mapping RNA Bound to Chromatin

Mapping RNA–Genome Interactions (MARGI)
This technique has been used for mapping global RNA–chromatin interactions in human embryonic stem cells and human embryonic kidney cells. After extraction of formaldehyde/DSG crosslinked chromatin, RNA–DNA is bridged with a biotinylated double-strand oligo by proximity ligation and is purified and converted to a sequencing library for paired-end sequencing.

Global RNA Interactions with DNA by Deep Sequencing (GRID-seq)
Similar to the MARGI method, the GRID-seq method applies proximity ligation RNA to DNA with a biotinylated linker in situ on fixed chromatin, which reduces nonspecific interactions, in human, mouse, and Drosophila cells. Biotin-purified products are cleaned with native polyacrylamide gel electrophoresis and are analysed using single-end 100-bp sequencing.

ChAR-seq
Similar to the MARGI and GRID-seq methods, ChAR-seq uses proximity ligation with a biotinylated oligo in situ on fixed chromatin in Drosophila cells. ChAR-seq prepares relatively longer RNA and DNA fragments (20–100 bp) than the methods above and uses 152-bp single-end reads to sequence across the entire junction of the bridge.

Chromatin-Enriched RNAs (cheRNAs)
Stringent nuclear fractionation coupled to RNA sequencing. Purified nuclei from HEK293 cells are extracted with a 0.5-M urea/0.5%-NP-40 buffer to yield a soluble nuclear extract and insoluble chromatin pellet, and both pools are sequenced. Tightly chromatin-associated lncRNAs identified from insoluble fraction are termed cheRNA.

DNA:RNA Immunoprecipitation (DRIP-seq)
DRIP-seq has been used for mapping DNA:RNA hybrid across the genome in human pluripotent Ntera2 cells using the monoclonal S9.6 antibody recognising DNA:RNA hybrids in a sequence-independent manner.

via: https://www.sciencedirect.com/science/article/pii/S0962892418302174

Citizen AT 9010-52E的时间调整

这个表按理应该是自己调整时间，但是最近发现不太正确，时间多出了2个多小时。每次都要计算一下，及其不方便。

操作视频：https://www.youtube.com/watch?v=KvTKzZm03C0

中文说明：https://www.citizenwatch-global.com/support/pdf/h820/sc.pdf

手动调整时间方法：

1、把齿轮拨到1档，摁住B键,直到橘黄色的秒针指向30秒为止

2、把齿轮拨到2档，橘黄色秒针会归零。

3、拨动齿轮调整：摁住B键2秒，松开，依次可以调整：

3.1 小时和分钟，调整好后，摁住B键2秒，继续调整：

3.2 日期， 调整好后，摁住B键2秒，继续调整：

3.3 星期几，调整好后，摁住B键2秒，继续调整：

3.4 年份和月份。

4、把齿轮归位，调整完毕。

怎样在科技论文中写好讨论部分？

A strong Discussion section provides a great deal of analytical depth. Your goal should be to critically analyze and interpret the findings of your study. You should place your findings in the context of published literature and describe how your study moves the field forward.

It is often easy to organize the key elements of a Discussion section into distinct paragraphs (or groups of paragraphs).

• • Paragraph 1: This paragraph provides a “big picture” perspective for readers to remind them of the importance of your study.
    • Summarize the major gap in understanding that your work is attempting to fill. What was the overarching hypothesis? In the first few sentences of the Discussion, state the main problem that you were trying to address. Although this should relate to the information that you provided in the Introduction, this paragraph should not repeat statements that have already been made.
    • Why is filling this gap important? How will answering this question move the field forward? After identifying the problem, state the main reason that this study was needed. Describe how answering this specific research question will make a significant contribution to your field.
    • In the following example, we state the problem (bold) as well as the significance (underline), the ultimate “big picture” reason for performing the study.

    Example: EGFR-overexpressing cancers are highly aggressive and have a higher tendency to metastasize. Currently, available drugs specifically target the EGFR and elicit high response rates. However, the majority of patients eventually develop progressive disease. The mechanisms through which cancers escape EGFR-targeted therapies remain unclear. Identification of specific molecules that mediate resistance to EGFR-directed treatments will facilitate the development of novel therapies and may improve responses to currently available therapies.

 

• • Paragraph 2: This paragraph provides a critical analysis of your major finding(s).
    • What was your overall approach for studying the gap? In one or two sentences, state the main models or strategies that you used to study this specific research question. This should recapitulate whether the work included animals, cell culture, human subjects, or other novel techniques. (Some investigators prefer to place this section at the end of the first paragraph. This decision may vary depending on the specific study.)
    • What was the most important result of your study? The focus of this paragraph is to highlight the most important contribution that your study has made. Explicitly state this result. Additional findings (major and minor) can be described in subsequent paragraphs. Do not repeat detailed results that can be found in the Results section. In general, specific figure numbers do not need to be re-stated in the Discussion unless you feel that doing so would substantially enhance your argument or discussion point. A schematic of your proposed mechanism or model can often be helpful for clearly and concisely summarizing your major result(s).
    • How does your result(s) fit with existing literature? This is an important part of the paragraph and may require multiple paragraphs depending on the number of key studies that exist on your topic. This paragraph should be well-rounded, meaning that contrary reports must also be discussed. In the case of a contrary report, you should state your interpretation of how and why the results of the two studies differed. For example, did the approaches differ or were there major differences in sample sizes that may have affected results? (Depending on how much information is available in the literature, a critical analysis of your major finding may require multiple paragraphs.)
    • In the following example, we state the approach (bold) and the main result (underline).

    Example: In this study, we measured secreted factors in the media of sensitive and resistant cell lines to identify differentially expressed cytokines that may mediate resistance. Through a combination of ELISAs and mass spectrometry-based assays, we identified cytokine A as being significantly up-regulated in resistant cells. Cytokine A is a major activator of the ABCD signaling cascade (literature citations). The ABCD cascade is a known target of EGFR signaling and is usually blocked in response to EGFR inhibition (literature citations). A previous study demonstrated that exogenous stimulation of ABCD signaling reduces the response to EGFR-targeted drugs (literature citations). This report is consistent with our finding that a major stimulus of ABCD signaling is overexpressed in resistant cells. Based on these data, we propose that hyperactive ABCD signaling is a major mechanism of resistance to EGFR-targeted therapies (Figure XX, schematic of proposed mechanism of resistance). This section will be greatly expanded in a real Discussion section to place your finding in the context of multiple published studies.

 

• • Paragraph 3: Discuss additional findings and how these fit with existing literature.
    

    Most studies yield multiple results. After you discuss your main result in the paragraph above, discuss additional major or minor findings. Unexpected and intriguing findings may be especially important to convey to readers. In addition, if a finding is contrary to what has been suggested in the literature, acknowledge this, and offer explanations based on your study. Even if a result was not statistically significant, it can be helpful to discuss a potential trend that may be important to assess in a future study. If these additional findings relate to your main finding, discuss the associations.

 

• • Paragraph 4: Discuss the limitations of the study.
    

    Discuss potential limitations in study design. For example, how representative was your model? Did sample size affect your conclusions? Consider how these limitations affect the interpretation or quality of data. Do they affect the ability to generalize your findings?

 

• Paragraph 5: Discuss future directions.
  • What major follow up studies are indicated based on your results? Most studies yield new discoveries that prompt additional studies. Consider what new directions are supported by your findings. For example, do your experiments suggest that a specific molecule should be tested as a new drug target or that tissue-based studies or clinical investigations should be performed to translate your animal studies to patients? Making recommendations for follow-up studies is an important part of a Discussion.
• Paragraph 6: Discuss your overall conclusion and the major impact of your study.
  • What is the main take-home message of your study?
  • What is the main contribution that your study makes to your field?
  • Relate this section to the first paragraph of the Discussion. In other words, how does your study fill “the gap” or address the problem that you presented in the Introduction and re-stated earlier in paragraph 1 of the Discussion?

In summary, a strong Discussion includes a concise summary of the problem you are investigating and a critical discussion of major and minor findings in the context of published literature. The limitations should also be acknowledged, and future directions should be discussed. A strong ending is important; discuss the significance, overall conclusion, and major impact of your study.

via： http://www.biosciencewriters.com/How-to-Write-a-Strong-Discussion-in-Scientific-Manuscripts.aspx

研究RNA-protein互作的新方法： OOPS

Researchers from the University of Cambridge have developed a new method to capture protein-RNA complexes which is compatible with downstream proteomics or RNA-Seq.

The new method is based on the acid guanidinium thiocyanate-phenol-chloroform (AGPC) which is commonly used to extract RNA. In this method, RNA is partitioned into an aqueous phase, protein to the organic phase, DNA the interface and organic phase, and lipids the interface. Applying 254 nm UV crosslinking generates protein-RNA adducts which are “pulled” in both directions and thus end up at the interface. Extraction of this interface and repeated rounds of AGPC phase separation yields an interface enriched in protein-RNA adducts without free protein or RNA. Subsequent treatment of the adducts with proteinase K or RNase digests one half of the adduct and a final round of AGPC returns the remaining RNA or protein to the aqueous or organic phases, respectively. This simple method yields much more material than current techniques to enrich RNA-protein complexes and can be used to quantitatively study either protein-bound RNA or RNA-bound protein.

In their recent Nature Biotechnology publication, Queiroz, Smith & Villanueva et al demonstrate that all long RNAs are bound by protein and sequence the RNA to show that OOPS recovers all cross-linked RNA. After proteinase K digestion, a few amino acids remain on the RNA at the site of crosslinking which in turn inhibits reverse transcription across the crosslink site. Whilst this means slightly more RNA is required to generate RNA-Seq libraries, an upshot is that the RNA-Seq read coverage profiles inform the site of crosslinking.  By comparing the profiles for crosslinked and non-crosslinked samples, protein occupancy sites can thus be detected. Since OOPS recovers all RNA, protein occupancy can be assessed across the entire transcriptome, including non-coding RNAs. The Nature Biotechnology publication is a proof of principle that OOPS can be used to quantify protein binding systematically and modifications to the RNA-Seq library preparation would be expected to yield improved resolution. This approach could help identify global changes in RNA binding between healthy and disease states, or changes in RNA binding across biological conditions.

Having demonstrated that OOPS recovers all crosslinked RNA, the researchers interrogated the bound protein to generate the first full RBPomes of human cell lines, identifying 926 putative novel RBPs. Importantly, OOPS is 100 times more efficient than current methods, thus replicate samples can be easily obtained for multiple conditions. This allowed them to assess differences in RNA binding between nocodazole arrested and released cells. Finally, since OOPS is not dependent on any RNA feature, they were able to obtain the first draft RBPome catalog for a bacterium, the model organism E. coli.

OOPS has the potential to be very useful to further our understanding of the role of RNA binding in biological processes, and to elucidate the etiology of neurological diseases known to involve perturbations in RNA binding, such as amyotrophic lateral sclerosis.

利用Image J分析条带浓度

其实这类资源，网络上遍拾即是。但是很多有一定的误导性。跟几个labmate探讨了一下这个过程中可能碰到的问题，在此总结一下总体流程。

工具：
Photoshop CC 20.0
Fiji (Fiji is a distribution of ImageJ which includes many useful plugins contributed by the community.)

参考网页：https://di.uq.edu.au/community-and-alumni/sparq-ed/sparq-ed-services/using-imagej-quantify-blots

点击以访问 ImageJ.pdf

https://alfresco.uclouvain.be/alfresco/service/guest/streamDownload/workspace/SpacesStore/62eef827-f095-4bfd-b607-e0688df2317c/ImageJ%20-%20western%20blot%20quantification.pdf?a=true&guest=true

步骤：

1. 用PS打开图片，点击View-Show-Grid
2. Edit-Free Transform, 这个操作可以任意角度旋转图片，根据第一步的方格，调整所有带处于水平位置，方便定量。
3. 保存调整好的图片为jpg格式。

4. 打开image j，导入保存好的jpg图片。
5. 点击软件第二栏中的第一个方框，然后在图中选择第一条泳道。技巧：把框选长一点，这样拖动时比较容易操作。因为如果拖动方框过程中如果改变了方框形状，只能从头开始。

6. Analyze-Gel-Select First Lane

7. 拖动方框到第二泳道，在键盘上敲击“2”。重复此操作，每次操作都输入“2”。这样软件会自动按序对每个泳道进行标记。

8. Analyze-Gel-Plot Lanes
9. 这时每个泳道都会出现一个对应的峰形。按住shift，截取single peak。技巧：线条必须跨越峰的两侧，否则后面定量时，会高亮方框内所有面积。

10. 点击Analyze-Measure，再点击软件第二栏中的”A” 左边的小棒，然后回到刚才截取的single peak里点击一下，就会在右侧出现对应的浓度。

11. 导出到excel表格，计算。计算方法： http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/

美国安居：买房之基础和必需知识

一、准备买房
买房是一个人一生中最大的投资之一，所以要做充分准备(planning)，三思而后行。尽可能多地了解买房知识是绝对必要的。如果你正准备换更大一些的房子以下这些问题将帮助你决定你是否已经准备好换更大或在更理想的位置的家。如果大多数问题的答案是肯定，就表明你已经准备好了。

1、你现在的房子是否为你创造了庞大的资本？

请看看你每年贷款公司给你的付款和利息清单或打电话给你的贷款人便能知道。通常，在买房子的最初几年你的抵押贷款每月支付的主要是利益，但如果你已拥有你的住宅5年或以上，那么你的房屋很可能已经为你储存了一定的资本。

2、你的收入或财政状况好转？

如果你现在能赚更多的钱，使你可以支付更高的按揭付款和支付搬迁费用。

3、你是否想搬到更理想的地区？

你的第一个家可能不在您理想的地区内。例如，你可能已经意识到，你希望家离你的工作地点更近或者为了你孩子的将来想搬到一个更好的学区。

4、你现在的房子可以改建或加建吗？

有时你可以加建一个新的房间或在原来的土地上重新建造一栋新的房子。但如果你的土地不够大，你所在的地方政府不允许，或者你根本不感兴趣重建，那么搬到一个更大的房子就可能是你最好的选择。

5、目前房地产市场情况允许你更换更大的房子吗？

如果现在的房市很好，你的房子可很快出售并且可以卖个好价钱，但你要买的房屋
也相对更加昂贵。如果现在的市场不景气，寻找买家可能需要更长的时间，但是你在寻找新的房屋时也有更多的选择和更好的价格。

6、利率是否有吸引力？

低利率不仅可以帮你更容易地找到买主，还能帮助你买到一个更大的房屋。

二、了解买房全过程 (Home Buying Steps)

（一）在这个过程中，你要做许多事情：

1、看房子（Show House）；

2、签合同（Sales Contract）；

3、检查房子（Home Inspection）；

4、申请贷款（Loan Application）；

5、购买保险（Home Insurance）；

6、过户（Settlement）；

7、搬家（Moving）。

（二）在这个过程中，你还要接触到许多人：

1、房地产经纪人（Real Estate Agent）；

2、房屋检查师（Home Inspector）；

3、贷款经纪人（Mortgage Loan Originator or Broker）或银行家（Baker）；

4、保险经纪人（Insurance Agent）；

5、过户律师（Settlement Lawyer）；
6、搬家公司员工（Mover）。

你可以到图书馆去借几本关于买房和贷款的书看看。当地中文报纸房地产专栏也是不错的买房全过程知识来源。

三、确定房价范围 (Price Range)
你要知道你能买得起多少钱的房子。一个简便的计算方法是最高贷款额为家庭年收入的四到五倍（假定贷款利率是6%到8%）。

如果你家的年收入是$100,000，你能买得起$400,000 到$500,000 的房子。
如果你有$80,000 现金做头款(Down Payment)，你就能买$480,000 到$580,000 的房子。
如果你没有房价5%的现金做头款，最好不要买房子。一是因为贷款难借，得不到好利率。二是防止万一钱不凑手，不能按月还贷款，房屋被银行没收。

如果有房价20%的现金做头款，就可以省下1.5%的贷款保险(Mortgage Insurance)费。

如果你的父母能给你一些钱做头款，要注意税法。税法是每年每人可给任何人$10,000 的现金做礼品不用付税。如果你的父母各给你$10,000，你就有$20,000。如果你的父母再各给你的配偶$10,000，你们就有$40,000。如果你配偶的父母再如法炮制，你们就可以有$80,000 不用报税的钱做头款。这些钱最好提前四个月存到你们的银行账户，贷款时就可以看作是你们自己的钱，不用再填贷款礼品表格(Gift Letter)。

四、决定买房地点 (Location)
上班族当然是希望住房离上班地点越近越好。问题是离大都市越近的地方房价越贵。其实真上了高速公路，不堵车时10英里已也只要10分钟。如果公司有弹性上班时间，可争取躲开高峰期上班。还可以在高峰期车流相反方向的地方买房。如果一家两个人上班地点相去甚远，房子就要买在一个折中的地点，或是买在离女主人上班较近的地方。因为大多数人家女主人主内，家里或孩子有急事可迅速赶回家。也可以买在离会做饭的一方上班较近的地方。会做饭的下班先到家把饭做好，等开长途的进门坐倒就吃。看到这里恐怕两个人都要抢着开长途了。

同样的地方也会有不同的社区(Community)，有的社区房价贵，有的社区房价便宜。

完全是独居屋的社区，房价比有很多公寓的社区贵。

在中国是人以群分，在美国是人以钱分。你用多少钱买房子，你邻居的收入也就在同样的范围内。

有孩子的家庭还会考虑孩子上学的问题。美国的公共教育有严格的学区划分，从小学到高中，学生们只能在居住地所属学区(School District)的学校上学。由于学生来源和师资质量等因素的影响，造成学校标准考试成绩及其他评估结果的差异，从而出现好学区，不好学区一说。每个学区都有自己的网站，打进一个地址就可以查到所属学校。还可以查到每个学校的一览表，包括从考试成绩到学生比例的统计数字。网址可向经纪人要。从房地产角度来看，好学区的房价比其它学区要贵。许多人希望在好学区买房，给子女提供好的学习环境。但是由于高昂的房价，一些人根本买不起好学区的房子。一些人勉强买得起，也要把住房条件降一个等级。在其他学区能住独立屋，在好
学区只能住连栋屋。如果你只有一两个小孩，可以考虑私立学校。如果孩子太多，每个孩子私立学校的学费加起来，就会超过好学区和其它学区房子的差价，这样还是在好学区买房较省钱。另外许多郡从小学到高中都有重点班，所有住在郡内的学生都可以考。孩子如果考上了重点班，就不必在好学区买房子。还有一个方法是把在好学区买独
立屋的钱分成两笔，用一笔钱在好学区买或租一个连栋屋或公寓给孩子上学用，用另一笔钱在其它地方买一栋独立屋全家享受。一个好的社区对您的生活方式有很大的影响。但是怎样才能找到可以安家的理想社区呢？请按照下列步骤来找找看：

1、是否接近你最喜爱的景点？

列一个你常去的活动场所的清单，电影院、健身俱乐部、教堂和你经常光顾的商店等。看看你每天或经常出入的这些地方离你考虑购买的房子到底有多远。

2、检查一下当地的学区。

如果你有孩子这一点尤其重要，而且它也可以影响你房屋的转售价值。你所在城市的教育部门或许可以提供关于此学区的考试成绩，班级人数，学生上大学的百分比，和资优班计划等资料。如果你有学龄儿童，你应该去参观你正在考虑的社区的学校。家长可以上www.schoolmatters.com 网站查询。

3、了解此社区是否安全。

可以要求警察部门提供社区犯罪统计数据。不仅要考虑犯罪的数量，而且类型也要考虑：例如是盗窃还是武装抢劫、犯罪的趋势是增加还是减少。此外，犯罪中心是只在部分街区，如零售店面附近还是在很多的方都有发生？

4、确定社区附近的经济是否稳定。

请向当地市经济开发办公室查询，看看此社区附近居民的收入和房屋的价值是否稳定或上升。独立屋和公寓的百分比是多少？公寓不一定减少价值，但却意味着更多的流动人口。此地区的商业楼宇或房屋空置几个月才租或卖掉？

5、看看你买房之后能否赚钱。

咨询当地的房地产经纪人或致电当地房地产经纪人协会，获取附近有关房价的信息。虽然过去的表现不能保证将来的结果，但这些信息可以给您买房时作为参考会。从当地房地产经纪人或政府规划机构也可以了解到当地的发展计划或其他变化，例如要建一所新学校或公路等等，这些很可能会影响你房屋的价值。

6、你要注意观察，你的个人意见更重要。

一旦你的注意力集中到两个或三个街区，去那里走走。看看是否有整洁的家园和良好的维护？安静的街道？你的感觉怎么样？选择温暖的一天，这样你可以与在外面工作或玩耍的人交谈。

五、选择房型(House Type)
同样的价钱，在有的地方可以买独立屋（Single Family House），在有的地方只能买连栋屋(Tome House)或者公寓(Condo)。为了将来房子好卖，我建议两卧室买公寓，三卧室买连栋屋，四卧室买独立屋。从投资的角度看，独立屋最保值，其次是连栋屋，最后是公寓。新房子的升值速度大于旧房子。从居住的角度看，怕听邻居噪音的最好买独立屋。不愿割草和收拾院子的最好买连栋屋。买不起连栋屋的只好买公寓。严格地说公寓不是房型，只是一种组织形式。买独立屋和连栋屋时，你是连房子带地一起买(Fee Simple)。买公寓时，你只买你住的那一个单元，房顶和地都不是你的。因为公寓的公摊面积大，修缮管理费用高，所以公寓费(Condo Fee)比独居屋的社区管理费(Home
Owner Association Fee)贵得多，一般是每月$200 到$400，有的高达$900。有的公寓费包括水电费，如果自己住也还划算。但如果是出租，万一空着，还要照付公寓费，就划不来了。市场上有连栋屋型的公寓，注意不要弄错了。

六、选经纪人
买房一定要找经纪人。

首先经纪人比你有经验。如果你是初次买房，一定有数不清的问题，经纪人会耐心地为你解答这些问题。

第二经纪人比你信息灵通。她可以根据你的要求为你设置自动搜索，每天将符合你要求的新上市或降价的房子自动寄到你的电子信箱。

第三经纪人比你了解行情。她知道什么房子在什么地方应该卖多少钱,因而可以据理力争地为你砍价。

第四经纪人可以为你把关。她会告诉你买新房时哪些东西是必不可少的，哪些东西是可以后来再加的。她会告诉你买旧房子时要注意哪些东西。

最后，经纪人为买主的服务是免费的。惯例是卖主付买卖双方经纪人的佣金。加上许多经纪人会给买主一些过户费，何乐而不为？

有些朋友怕给别人添麻烦，不好意思老让人家带着自己去看房子。往往一去就是大半天时间，何况现在汽油又这么贵。但如果你是真的要买房子，经纪人在帮你买了房子之后，可以从卖主那里拿到佣金，所以你大可不必客气。但为了确保你的经纪人能从卖主那儿拿到佣金，最好与你的经纪人签一份独家买方经纪人合约（Exclusive Buyer Agency Agreement），这样你就可以心安理得地让你的经纪人为你服务了。如果你怕受独家买方经纪人合约的太大限制，你可以注明此合约仅限于该经纪人带你看的房子，甚至仅限于某一栋房子。合约有时间限制，如果不放心，可以先签一个月的合约。经过一个月的试用，对经纪人的服务满意，可再签三个月甚至六个月的合约。

随着网络的日益普及推广，人们掌握各种房屋资讯的途径不断拓宽，特别是在波士顿地区，不论是政府部门、军队或地方企业，进来的都是高素质、高科技人材，对各种信息的收集更是及时准确。因此有些人认为，不用经纪人自己查找房屋，自己与卖主谈判，这样卖主可以把需要花在买方经纪人身上的佣金，返还在房价中，买主可以得到更便宜的房价，在实际交易中真是这样吗﹖

1、买方经纪人怎样保护你的利益呢﹖
如果你找了一个有经验的经纪人代表你，同时你也和你的经纪人签了合同，当你找到了一栋你中意的房子，你的经纪人会帮你准备合同，而这些合同大多是以保护消费者为前提的，合同中会有很多的条款会给你一段时间，允许你对这栋房子进行检验，对社区进行调查，对週围的环境、学区、发展潜力进行整理分析，而在这段时间内你能以合法的理由选择退出，或是继续这个合同，无须承担任何风险，而当你在房检或其他检查中发现了问题又不想退掉这个合同时，你的经纪人会代表你与卖方讨价还价，为你争取最大的利益。

2、卖主自售房屋(For Sale by Owner)
如果是卖主自己卖房，你很有可能与卖主商量降低价钱，自己不找经纪人，把经纪人的部份佣金(因为没有经纪人，卖主也需要承担风险，增加交易过程中的工作量)返还在房价中，但你同时也失去了保护。合同一旦签成，你无法退出，无法讨价还价。

3、经纪人代卖的房子
近来由于房地产市场低迷，大多数屋主都会僱用经纪人帮助卖房。在房子上市之前卖主一定要和经纪人先签订一份合同(Listing Agreement)。在这份标准的合同中卖主授权给经纪人同时也同意在房子卖掉时付多少佣金给买卖双方的经纪人。如果这时你贸然进入，要求卖主分些佣金给你，可能性就不大了。省下来的佣金都进了卖方经纪人的口袋。美国法律规定卖方经纪人衹能代表卖方的利益，不能一个经纪人同时做买卖双方的代理(在维州同一个经纪人可以同时代表买卖双方，但一定要在代理之前向买卖双方声明是他们双方的代理人)。当你没有买方经纪人时，卖方的经纪人可以帮助你做很多事情以完成这笔交易。他可以帮你写合同，可以帮你找人做房检、白蚁检查等一切事情。但当你的权益和卖主的利益有冲突时，他必须要维护卖主的利益。

4、新房买卖
还有一个误区是很多朋友认为如果在建筑商手裡买新房就不需要经纪人了，因为建筑商都有自己专门的销售人员，这些销售人员大多数训练有素，态度和蔼，在签合同前对你有求必应，给你的感觉好像他就是你的代理人，而且有些销售人员还告诉你如果不用买方经纪人，他们会在价格上和成交费上给你些好处，或是给你免费完成地下室等，请不要被这些现象迷惑，羊毛出在羊身上，一旦你签了合同，他们会按照他们的应许去做不错，但他们给你的衹是你应得的一部分，而非全部。而你的经纪人了解市场知道行情，会帮你争取到更好的价格，会在合同中加入保护你的条款，在房子建造期内如果有任何意外的事情发生，可以让你安全退出合同。

5、怎样才能找到满意的经纪人呢？你可以试用以下的途径。

（1）当地中文报纸或网站房地产专栏
一些经纪人会在当地中文报纸房地产专栏发表文章。见字如面，文如其人。如果你读了数篇文章后觉得作者房地产知识丰富，人品正直，不访打电话或去电子邮件做进一步的了解。如果文章内容能引起你的共鸣，你与这位作者能谈得来的可能性就大。

（2）房地产公司和经纪人广告
一些公司和经纪人会在报纸或杂志上刊登广告。从广告上可以了解公司和经纪人的基本情况。比如擅长的服务地区，历年的销售业绩，经纪人的宗旨等等。

（3）亲戚朋友介绍
向买过房子的亲戚朋友打听，他们用过的经纪人水平可高，人品可好。

（4）初步选定经纪人后，可对经纪人做以下的测试:
A、经纪人说话和写作是否有说服力
在买房过程中要谈判房价，房屋维修等事宜。一个讲话没有说服力的人很难胜任。光会说话还不够，还要会写。就算你的经纪人能把死的说成活的，一下就把卖方的经纪人说服了，卖方经纪人不一定能说服卖主。这时候你的经纪人就要发挥她的写作能力。把你报价的原因1，2，3，4 写出来用电子邮件寄给卖方经纪人。卖方经纪人只要印出来拿给卖主看就行，或者直接把电子邮件转给卖主即可。

B、经纪人英文口头和书面表达能力是否合格
除非卖主和卖方经纪人都是中国人，你的经纪人就要用英文谈判。所以经纪人的英语口头和书面表达能力必须要好。口语你可以听出好坏，书面表达能力从英文电子邮件可见一斑，也可让经纪人给你一份她的英文代表作。

C、经纪人房地产知识是否丰富
经纪人要对买房过程了如指掌。在带你看房时，能指出房子的优缺点，不同的房子应该怎样做比较。比如说如果地下室少个厕所，要花7000 到10000 去修一个。如二楼少一个厕所，花再多钱可能也修不了。报价时，会使用市场分析为你定价，会用附加条款帮你在必要时合法地取消合同，拿回定金。房屋检查后，会为你与卖主谈判，或是让他们修理，或是让他们给钱。还会为你介绍贷款和过户公司，提供搬家公司信息。

D、经纪人是否能运用网络和现代通讯技术
人的脑系胞的数量是有限的，能储存的信息也是有限的。经纪人不一定能立即回答你所有的问题。但好的经纪人可以运用网络这个巨大的资料库迅速为你找到问题的答案。用传真机传递信息已逐渐过时，用电子邮件传递电子扫描文件省钱省时。你的经纪人如果能熟练运用计算机新技术，对你的买房过程肯定会有很大帮助。

E、经纪人是否敬业
聪明能干的经纪人还要肯敬业能吃苦。你可以问他最多带一位客户看了多少房子，我的纪录是113。你可以问他最早什么时间能看房子，我的纪录是早上6：30 出门去接客户，8：30 看房子。你可以问他最迟什么时候可以给他打电话，我的手机是7天24小时从不关机，伏首甘为孺子牛。

F、经纪人是否住过新房
如买新房，要问经纪人是否给自己买过新房，住过新房。新房的标价都是基本价，有很多其它的选择(Option)都要另外加钱。选择之多，另初次买新房的人眼花缭乱，无从下手。一定要有一个自己买过新房，住过新房的经纪人为你把关。我给自己买过三次新房，住过三次新房。每一次的选择都比前一次有所改进。

七、贷款预审
据美国房地产经纪人协会2009 年统计，90%的人是贷款买房。如果你是10%用现金买房的幸运儿，就不需要贷款，更不需要什么贷款预审。但不管是现金买房还是需要贷款，经纪人都会让你填写一份买主经济状况表(Financial Information Sheet)。你要如实填写这份表格，特别是姓名，住址，职业，工作单位，年收入(Gross Annual Income)和欠债部分（Liabilities）。欠债包括汽车贷款，信用卡欠额，以及其它债务。如果你不愿意透露你所有的财产(Assets)，倒是没有关系，但你填写的存款额不能低于你的头款(Down Payment)数。你的经纪人要根据你的经济状况向你建议房价范围。很多卖主也要求买主在报价时附上买主经济状况表。如果是现金买房，买主经济状况表
就是卖主唯一的依据，所以在在报价时一定要附上这份经济状况表。

如果需要贷款，经纪人会根据你的经济状况建议你向有关贷款公司要一封贷款预审批准书(Loan Pre-approval Letter)。因为贷款公司要查你的信用分，还会要你的社会安全号。你如果已经与其它贷款公司或者你自己的银行联系过，你就可以用你自己的贷款公司或银行的贷款预审批准书。不管你用谁的，你在正式贷款时还是可以货比三家。贷款预审批准书的目的是决定你的房价上限。贷款预审批准书也是报价合同的附件之一。它告诉卖主，你已经与贷款公司或银行联系过了，信用分和收入都查过了，银行初步同意借钱给你。贷款预审批准书贷款额越高越好，不用担心卖主会因为你有钱而提高房价。卖主担心的是在接受报价合同后买主申请不到贷款，不能过户，浪费卖主的时间。在房屋买卖中，时间就是金钱。一栋$500,000 的房子，如果利息是3%，一个月的利息就是$1,250，卖主更愿意接受经济状况好的买主的报价。

1、申请贷款预审批准书还需要以下的信息：
（1）买主姓名(Buyer Name)
（2）社会安全号(Social Security Number)
（3）房价(Sales Price)：实际房价或最高房价
（4）贷款额(Loan Amount)
（5）贷款种类(Program)：一般是30 年固定利率
（6）定金(Deposit)
（7）过户日期(Settlement Date)：写实际日期或待定
（8）买主地址(Buyer Address)
（9）买主电话(Buyer Phone)
（10）买主年收入(Income)

2、最高贷款额取决于家庭总收入和其它债务
大部分银行允许买主将月收入的28%用于与住房有关的花费，比如还贷款本金(Principal)，利息(Interest)，房地产税(Real Estate Tax)和屋主保险(Home Owner Insurance)。银行还会考虑家庭其它债务，比如汽车月款(Car Loan)，学费贷款(College Tuition Loan)，信用卡欠款(Credit Card Debt)等。这些债务加上住房花费
不能超过月收入的36%。如果你的其它债务占月收入的12%，你只能用24%做房款。所以在买房时，要把其它债务限制在8%获更低。如果你没有其它债务，你可以用36%做房款。在目前房价超过绝大部分人所能承受的情况下，联邦住房和城市发展部(HUD)允许家庭其它债务加上住房贷款高达月收入的45%。

贷款种类有二百多种，但大多是从两个基本类型衍生出来的：浮动利率(Adjustable Rate Mortgage, ARM)或固定利率(Fixed Rate Mortgage)。

浮动利率贷款的开始利率比固定利率贷款低，以后的利率会随着一个指数(Index)而浮动。开始浮动的年限和浮动的幅度事先都有规定。如果贷款时利率高于5%，并且呈下降趋势，可用浮动利率。常见固定利率贷款有15 年或30 年的。15 年的利率比30 年的低。

如果贷款时利率低于3%，可选固定利率。从贷款来源可以把贷款分成三种，一般性贷款(Conventional)，低收入政府补助贷款(FHA)，和美国退伍军人贷款(VA)。从贷款的数额又可以把贷款分成三类，小额贷款，普通贷款，和大额贷款(Jumbo Loan)。十万以下的属小额贷款。贷款额超过$417,000 的属大额贷款。从2008 年3 月起，华盛顿地区贷款额超过$562,000 的才算大额贷款。大额贷款的利率比普通贷款高。如果你不清楚，可以问贷款经纪人。如果问了以后还是不清楚，干脆就选30 年固定利率。

3、定金(Deposit)。是买主在报价的同时，交给买方经纪人公司的一笔钱。如果买主在没有正当理由的前提下单方撕毁买房合同，定金就拿不回来了。定金越多，说明买主诚意越大，卖主越放心。大多数公司规定定金不得低于$5,000。卖主为了保护自己的利益，还会要求更高的定金。

4、过户日期(Settlement Date)。可订在合同生效后的30 天到45 天。计算机自动化大大缩短了贷款审批的时间，使30 天或更短时间过户成为现实。一些银行没收的房子明确规定30 天之内过户。

5、贷款预审批准书。内容包括贷款种类(Program)，头款额（Down Payment），贷款额(Loan Amount)，利率（Interest Rate）。它还会有以下的字样：

（1）该预审批准书的依据是已核实的买主信用史(Credit History)；

（2）经济状况(Financial Information)和职业史(Employment History)；

（3）贷款的最终批准还有待于认可的房地产合同(Ratified Contract)和房屋估价(Appraisal)。
预审批准书虽然不注明作废日期，却是有时间性的。这是因为人的信用分，经济状况和职业都会有变化。如果预审批准书是六个月以前写的，就要去重新写一张。

 八、选房看房
经纪人会根据你的要求为你设置房屋自动搜索。只要有符合你要求的房子上市，信息就会自动送到你的电子邮箱。搜索的标准可以是房价范围，房型（独立屋，连栋屋或公寓），城市名或邮政编码，建筑年代，等等。虽然学区也是搜索标准之一，但很多经纪人为了保护自己，不填学区。所以你要把学校所在地的房子都找出来，然后根据地址到学区网站去确认是不是在你要的学校。从电子邮件的链接中可以看到地址，房价，房型，以及一到三十张照片。如果看到满意的，可自己开车去看周围的环境，包括离高速
公路和主要公路的距离。离高速公路四英里之内比较好，再远就要多走小路，交通灯多，费时费油。离高速公路也不能太近，太近会听到车流的声音。有人不喜欢附近有高压线。虽然高压线对人体健康的影响尚有争异，但如果有别的选择，就尽量少找麻烦。还有房子的后院是否开阔，背向树林或空地当然比背对别人的后院要好。如果是独立
屋，旁边最好是独立屋。如果是连栋屋，旁边最好不是公寓。对外部环境不满意的，可以排除。比如我带客户看房，就碰到过以下的情况，没有进门：

（1）通向房子的路很难开，又陡又窄，开了一身汗。一下车大家的第一句话是，如果下雪怎么办？

（2）刚到房门口就听到暄哗的车流声。有些房子虽然离高速公路不是很近，但由于山谷的回音效果，车流声听得特别清楚。

（3）房子旁边就是坟场或殡仪馆。美国人对这些不再乎，有些中国客户比较忌讳。

初选过关，再约经纪人带你去看内部。在同一地区的房子一般20 分钟可以看一个，一次可看五到六栋房子。下面是不同房型要注意的事项。

1、公寓：除非你准备一辈子单身，最好不要买一间卧室的公寓，自己很快就不够住了，将来也难卖。许多人不喜欢一楼或最高层。两间卧室的公寓最好有两个浴室，一个在主卧室内，另一个最好紧挨着第二个卧室，可供家人和客人使用。最好有一到两个停车位。

2、连栋屋：如果你需要三间卧室，就买连栋屋。最好有一到两个车库。楼上最好有两个浴室，一个在主卧室内，另一个可供第二和第三个卧室使用。一楼最好有个卫生间。

3、独立屋：如果你需要四间或更多的卧室，就买独立屋。一楼会有客厅，起居室，餐厅，和厨房。楼上两层在三千英尺以上的，还会有一个书房。楼上最好有三个浴室，第一个在主卧室内，浴缸最好有喷水孔，还有单独的淋浴。第二个在第二卧室内，第三个可供第三和第四卧室使用，如能连接这两个卧室更好。中国人喜欢厨房的抽
油烟机通向室外。地下室能完全平地走出最好，能向上阶梯走出也行。地下室如果已经完成，最好有一个全浴。如果有一个卧室，最好紧接着浴室，晚上使用方便。地下室如果没有装修，最好有预留排水管，将来好建浴室。如果要放乒乓球桌，至少需要24×13（长x 宽）英尺的场地。
看房时，可用一张看房比较表（看房比较表列出了 26 各个可供参考的项目，需要的读者可以在文章下面的网站下载）来记录并比较不同房子的优缺点。比如每个房间的大小，布局是否合理，状况如何等等。
地址(Address)
要价(Listing Price)
政府估税价(Tax Assessment)
几个卧室 (# Bedrooms)
几个浴室 (# Bathrooms)
几个车库 (# Garage)
居住面积 (Living Area)
占地面积 (Lot Size)
建筑年代 (Built Year)
客厅 (Living Room)
起居室 (Family Room)
餐厅 (Dining Room)
厨房 (Kitchen)
书房 (Library)
主卧室 (Master Bedroom)
其它卧室 (Other Bedrooms)
衣橱 (Closet)
储藏室 (Storage)
地下室 (Basement)
阳台 (Deck)
前庭 (Drive Way, Side Walk)
后院 (Backyard)
状况 (Condition)
学区质量 (School Quality)
上班开车时间 (Commute Time)
购物方便 (Shopping)
总评 (Overall Opinion)

九、签署合同
选定满意的房子，经纪人会为你全面查询有关信息，确定要价。有关信息包括: 房子上市历史，市场分析 (Comparative Market Analysis, 简称 CMA)，市场统计数据（Market Statistics），和政府估税价(Tax Assessment)。

1、房子上市历史：房子倒过几次手，每次售价是多少，在什么时间。房子在市场上有多少天了，有没有降过价。卖主为什么要卖房子。如果卖主买价较低，这次卖只是赚钱多少的问题，可能好谈价一些。如果房子刚上市不久，或者刚降过价，或者不急于出售，卖主可能不大愿意你砍价。

2、市场分析：因为同样的房子在不同的地方(Location)房价相差很大，所以市场分析实际上是看类似的房子在同一社区同一时间（6个月之内）卖多少钱。如果能找到同样大小，同样多的卧室和浴室，同样的地下室的房子，那最好。否则要调整价格或者向稍远的地方寻找类似的房子。

3、市场统计数据：房地产电脑网络每月都会公布售房统计数据，其中一项是房屋所在邮编售房价与要价比例平均数。经纪人会参照这个比例平均数报价。比如 2008 年2 月邮编20850 的售价与要价比例平均数是93.58%，报价时则可砍价6%。如果给卖主留还价的余地，还可以砍得更低。

4、政府估税价：这是当地政府为了决定房地产税额而给房屋和土地估的价，应该是一个比较客观的数据。在卖方市场时，房屋买卖价格可能高于估税价。在买方市场时，房屋买卖价格能低于估税价。在同一时间同一社区，如果其它的房子要价都低于估税价，你要买的房子的要价却高于估税价，那就是要价偏高，你可以以此为由而砍价。

5、报价合同还要注明定金(Deposit): 这是买主在报价的同时，交给买方经纪人公司的一笔钱。如果买主没有正当理由单方撕毁买房合同，定金就拿不回来了。定金越多，说明买主诚意越大，卖主越放心。大多数公司规定定金不得低于$5,000。卖主为了保护自己的利益，还会要求更高的定金。一般定金额是报价的2%到3%。

6、除了确定报价、定金外，经纪人还会帮你选择一些附加条款，以保证你在必要时能合法取消合同，拿回定金。下面是常用的附加条款：
（1）房屋检查(Home Inspection)；
（2）氡气检查(Radon Inspection)；
（3）白蚁检查(Termite Inspection)。
在报价合同中，可以附上这三项检查的附加条款。条款的有效期一般是7 到15天。在有效期内，买主有权以任何一项检查不合格为由取消合同或要求卖主出钱维修或处理。

7、房屋估价(Appraisal)和贷款批准(Financial)：在报价合同中，还可以附上房屋估价和贷款批准两项附加条款。条款的有效期一般是15 天。但由于次级贷款的影响，银行比较挑剔，出现房屋估价低于合同售价的情况，影响到贷款批准。为安全起见，可把贷款批准附加条款有效期增加到20 天。如果房屋估价低于合同售价，买主可
要求卖主把合同售价降到房屋估价,或取消合同。如果买主申请不到贷款，买主可在附加条款有效期内要求取消合同。

8、空调(Air Conditioning System)或游泳池(Swimming Pool)检查：如果在冬季买房，空调或游泳池的功能不能检查。在报价合同中，可以选择在过户后5 月31 日之前检查空调或游泳池。如果不工作，可要求卖主出钱修理。

9、在报价合同中，还可以加上各种各样的附加条款，用于不同的目的。比如逐步升级条款(Escalation Clause) 在卖方市场用得比较多，在买方市场很少用。但我在买方市场为客户抢购银行没收的房子时用过。具体方法是买主选择逐步升级的数额和最高报价。如果其他报价高于我的报价，我的报价就自动按逐步升级的数额增长。如果其他报价也有逐步升级条款，那最高报价额高者取胜。

10、报价时还要确定过户日期，一般在30 天到45 天左右。最好定在星期二到星期四。如果在星期一，过户公司星期日不上班，不能提前一天拿过户清单，看要开多少钱的银行支票。如果在星期五，又怕万一当天不能过户完毕，要等到下个星期。时间最好定在下午一点。

上午可以做个最终房检(final walk through)，到银行去开个支票。报价合同加上附加条款和必需的文件，有50 页左右，买主要签很多字。如果卖主拒绝报价，报价合同就是一堆废纸，也浪费大家的时间。一个省时省事并有利于环境保护的方法是先写一张报价意向书，在意向书的基础上谈判。等价钱谈妥后再出正式报价合同。报价意向
书可包括以下的内容：
房屋系统编号（MRIS# ）：
地址（Property Address）：
要价（Listing Price）：
日期(Date)：
买主姓名（Buyers’ Names）：
报价（Offer Price）：
过户日期（Closing Date）：
贷款额（Loan Amount）：
订金（Deposit on Sale）：$10,000
房屋检查条款（Home Inspection）：合同批准后7 天
氡气检查条款（Radon Inspection）：合同批准后7 天
白蚁检查条款（Termite Inspection）：合同批准后7 天
房屋估价条款（Appraisal Contingency）：合同批准后15 天
贷款批准条款（Financial Contingency）：合同批准后20 天
房屋买卖人税（Transfer Taxes）：买卖方各 50%
买主年收入（Buyers’ Income）：
买方经纪人（Buyer Agent）：
为了防止卖主拿着你的报价合同与其他的买主讨价还价，你可以在报价合同上注明，此报价在1 天内有效。卖主可能会拒绝你的报价，或者还你一个价。你可以拒绝还价，或提高你的报价。如果双方能达成协议，合同就签字生效了(Ratified)。

十、房屋检查
房屋检查(Home Inspection)是报价合同不可缺少的附加条款(Contingency)之一。

1、当买卖双方在购房合约上签字生效(ContractRatified)后，买主要做的第一件事就是请有执照的房屋检查师（Home Inspector）来检查房子。检查费由买主付，价钱根据房价而定，从$200 到$600 甚至更高，房价越高检查费越贵。买方经纪人会推荐两到三个检查师，供买主挑选。一般房屋检查大概要两到三个小时。

2、买主在房检时跟着检查师走，可以学到很多东西。检查师会一边检查一边向买主解释注意事项或问题所在。检查内容包括房屋基本结构(Structure)，地基(Foundation)，屋顶(Roof)，地板(Floor)，墙壁(Wall)，门窗(Doors and Windows)，地下室(Basement)，供电系统(Electrical Systems)，供排水管(Plumbing)，冷暖设备(Heating and Air Conditioning Systems)，烟雾报警器(Smoke Detector)等。

3、检查师要填写一份详细的检查报告，包括房子的状况，哪些设备可能出问题，剩余寿命是多少，应该怎样维护，等等。除了详细的检查结果，还会有一份总结，列举卖主要修理的部分。如果买主要求，检查师还会帮着估价。好的检查师还会附上彩色照片，使问题所在一目了然。

4、买方经纪人会根据检查报告在房屋检查附加条款有效期内（一般是七天）为买主写一份房屋检查附件(Home Inspection Addendum)交给卖方经纪人。一般银行没收的房子写明按现状出售(AS IS)，房检问题概不负责。买主在房屋检查发现问题后，只能要求退房或自己修理。正常售房时如果房子的主结构有大问题，如地基断裂，房屋有倒塌危险等，买主可以要求取消合同，其它的问题可以要求卖主修理。

5、卖主在接到房屋检查附件时，要在三天之内做出答复，是同意修理还是出相应的修理费。有些东西是卖主必须修的，象房屋基本结构，地基，屋顶，供电系统，供排水管，冷暖设备，烟雾报警器等有问题或不工作。供电系统和供排水管的问题还要有执照的电工或水管工来修理。其它的东西卖主可以不修，象墙上有洞，地板或地毯破损等等。

6、但卖主不修买主可以不买，所以要双方协商解决。买主在接到卖主的回应后，也要在三天之内做出答复。双方达成协议后，房屋检查的附加条款就取消了。卖主答应修理的东西，要有修理工的发票做依据。

PS:

1、氡气是一种含放射性元素的气体，是致癌物质。户外空气中的氡气含量是每升0.4 微微居里(pCi/L) 。美国环境保护署（Environmental Protection Agency, EPA）规定室内氡气含量不能超过4.0 微微居里。氡气由土壤中存在的铀分裂产生，会通过地基的裂缝或地下室的排水泵进入室内。没有地下室的房子不需要做氡气检查(Radon Inspection)。氡气检查也可以是报价合同的附加条款之一，可以请房屋检查师一起做。检查费也是由买主出，价格在$100 元到$500 元。如果检查结果表明氡气指标超过标准，买方经纪人会为买主写一份氡气检查附件(radon Inspection Addendum)，要求取消合同或要求卖主采取降氡气措施(Radon Mitigation System)，花费在$800 到$2000。卖主接到氡气检查附件后要在三天之内做出答复，是同意降氡气还是出相应的处理费。买主在接到卖主的回应后，也要在三天之内做出答复。双方达成协议后，氡气检查的附加条款就取消了。

2、美国住宅的主要建材是木料，白蚁是损坏木质结构的害虫。白蚁检查(Termite Inspection) 可以是报价合同的附加条款之一。如买房要贷款，银行一定要求白蚁检查。独立屋，连栋屋，公寓楼三层以下都要检查。费用在$80 元-$150 元，由买主、还是由卖主出，各州不一样。检查公司会把检查报告和账单寄到过户公司。检查报告的日
期必须在过户的30 天之内。如检查发现白蚁损害，检查公司会在检查报告单中注明处理的费用。买方经纪人会为买主写一份白蚁检查附件(Termite Inspection Addendum)，要求卖主请专业人员处理。处理帐单也会寄到过户公司，由卖主承担。卖主接到白蚁检查附件后要在三天之内做出答复。双方达成协议后，白蚁检查的附加条款就取消了。

3、如果在冬季买房，空调(Air Conditioning System)或游泳池(Swimming Pool)的功能没法检查。可以在报价时加上过户后进行空调或游泳池检查的附加条款。这两项检查费也由买主出。检查时间不能超过过户后的5 月31 日之前。检查师必须有相关执照。买主在检查前不能试开空调。如果检查出问题，可要求卖主出钱修理。

4、1978 年前建的房子，可能使用含铅的油漆。铅进入人的血液，会影响神经系统，肾脏和造血机能。含铅油漆对小小孩影响较大，因为他们可能会把带有铅尘的东西放进嘴里吃进肚里。美国1978 年宣布禁止生产和使用含铅油漆，但有些地区到1979 年才执行（包括马里兰州）。买1979 年以前建的房子，买主可要求对室内墙壁油漆进行
含铅检查（Lead-based paint inspection），作为报价合同的附加条款之一。条款有效期一般为十天，检查费大约50 元-450 元，由买主出。现场化学检查比较便宜，但只能测出有没有铅，不能测含量。送30 到70 个样本到实验室检查比较麻烦，还会损坏墙壁。X 光检查准确又无破坏性，但收费较贵。如果检查出油漆含铅，买主可以取消合同或要求卖主处理。卖主必须请专业人员处理，并出示处理证明书。

一个房屋检查师对我说过，1979 年到现在墙一定重新油漆好多次了，应该不会有铅尘掉下来。如果窗户也换过了，就更好了。如买新房，建筑商会给一年保修，所以在过户前可不做房检。建议在十个月时做个房检，如查出问题可让保修公司修理。如果盖房子时在地基回填土中掺了化学药粉，防止白蚁进入室内，则新房在五年内不会出现白蚁，因为这种药粉的有效期是五年。新房在地基四周也可能会装排气管道，然后在水泥地面打孔或利用地下室小水井，与排气管连接将氡气排出室外。

5、房地产交易时间性很强(Time Is Of The Essence),房屋检查，氡气检查和含铅油漆等附加条款有效期到期日晚9：00 前买主如果不向卖主送检查结果附件，这些附加条款就自动取消了。如果买主在有效期内向卖主送通知，卖主要在三天之内给答复。买主在收到卖主答复后，也要在三天内做出回应。任何一方在接到对方通知后三天之内不做答复，就等于接受对方最后条款。双方对每项检查结果处理方法达成协议后，经纪人会准备一份最后的附件，让买卖双方签字。

十一、申请贷款
如果不是现金交易，贷款就是最关键的一步，借不到钱就买不成房。买主在合同生效七天之内要去申请贷款。你可以找银行，也可以找贷款公司。如果你知道哪家银行利率低，你可以直接到银行申请。否则你只有找贷款公司，因为贷款公司可以在众多银行中寻找利率最低的一家，或寻找适合你情况的贷款。在美国买房流程第七、预审贷款时，你已经与一家贷款公司接触过。如果你信任那家公司，可以向它申请。如果你还想货比三家，也可以再资询两家公司。要注意的是，查询当天得到的利率不是你最后锁定的利率，因为利率每天都在变化。

1、与贷款有关的开销包括以下费用：
手续费(Loan Origination Fee): 一般是贷款额的1%
点数(Discount Point)：一个点是贷款额的1%，一个点可降低利率0.25%
房屋估价费(Appraisal Fee): $250-$500，房价约高，收费越贵。
信用报告费(Credit Report): $20-60$
处理费(Processing Fee)：$200-$500
纳税服务费（Tax Service Fee）: $50-$100
洪水证明费(Flood Certificate): $10-$20
审批费 (Underwriting Fee): $200-$500
文件准备费(Document Preparation) $200-$500
以上的费用在每个公司可能稍有不同，你可以找一家收费少的。每家公司都会让你挑选无点数贷款。加点数的好处是最终还款额会降低，缺点是过户时要多交钱。因为有点数无点数贷款在七年时还款额相等，如果你能保持贷款七年以上，可以加点数。否则不要加点，特别是在过户费很紧的情况下。

2 、选定贷款公司后， 首先要填一份贷款申请表(Uniform Residential Loan Application)。贷款申请表有五页纸，包括以下内容：
贷款种类和期限(Type of Mortgage and Terms of Loan)
房地产信息和贷款的目的(Property Information and Purpose of Loan)
贷款人情况(Borrower Information)
工作情况(Employment Information)
月收入和支出(Monthly Income and Housing Expense Information)
财产与欠债(Assets and Liabilities)

3、房地产交易细节(Details of Transaction)
贷款申请表一定要如实填写，并将所有文件准备好，比如30 天之内的工资单，两年的W-2, 最近三个月的银行清单，等等。如果没有W-2, 要提供两年的税单。贷款员收到你 的申请表后，要在三天内给你一份过户费预估表(Good Faith Estimate, GFE)，事先告诉你借这笔贷款你最终要花多少钱。许多初次贷款的人在看到30 年总共还款
额是贷款额的两到三倍时，都大吃一惊。有人不理解为什么过户费预估表上会有两个利率，一个低，一个高。低的那个是利率(Interest Rate)，高的那个是年百分率(Annual Percentage Rate, APR)。年百分率是利率加上所有与贷款有关的开销后算出来的，更能体现贷款的真实利率。所以在比较两家银行贷款利率时，应该比谁的年百分率低。
买主递交贷款申请后，贷款公司或银行会请估价师给房屋估价(Appraisal) 。在报价合同中， 可以附上房屋估价(Appraisal Contingency)和贷款批准(Financial Contingency)两项附加条款。条款的有效期一般是15 天。但由于次级贷款的影响，银行比较挑剔，出现房屋估价低于合同售价的情况，影响到贷款的批准。为安全起见，可把贷款批准附加条款有效期增加到20 天。
如果房屋估价低于合同售价，买主要在房屋估价附加条款有效期内向卖主递交买主房屋估价通知书(Buyer Appraisal Notice)并附上房屋估价报告，要求卖主把合同售价降到房屋估价。如果买主没有在有效期内向卖主递交通知，房屋估价附加条款仍然延续，除非卖主书面通知买主，房屋估价附加条款有效期到了，应该取消了。如果买主在接到通知后三天之内还不向卖主递交买主房屋估价通知书，房屋估价附加条款在第三天晚上9 点就正式取消了。
买主要在贷款批准附加条款有效期内向卖主递交取消贷款批准附加条款的通知书(Financial Notices)。如果买主没有在有效期内向卖主递交通知，贷款批准附加条款继续延续。这时卖主有权宣布取消合同，如买主在三天之内不做答复，合同在第三天晚上9:00 就取消了。如果买主不愿失去房子，必须补交取消贷款批准附加条款的通知书。如果买主在贷款批准附加条款有效期内收到拒绝贷款申请的书面通知，只要把通知寄一份给卖主，合同就取消了。
贷款批准后买主还要锁定利率(Rate Lock-In)。利率锁定期有30 天，45 天，60 天或更长。时间越长，利率越高。一般在过户前30天内锁定。
贷款额上限是由贷款人的经济状况决定的，能不能贷到款或利率好坏是由贷款人的信用分(Credit Score)决定的。信用分低于620 或者没有信用分很难贷到一般性贷款 (Conventional Loan)。低收入政府补助贷款(FHA)和美国退伍军人贷款(VA) 最低信用分是600。信用分越高，利率越好，但740 以上利率都是一样。另外头款越多，利率越好。

4、怎样才能提高信用分呢? 让我们来看看信用分的影响因素和所占比重。
过去付款情况(Past Payment Performance) 35%
信用卡使用(Credit Utilization) 30%
信用史(Credit History) 15%
信用种类(Types of Credit in Use) 10%
信用查询(Inquiries) 10%
因为过去付款情况所占比重最大，所以你只要按时交水电费，信用卡及各种账单，信用分就不会差。如果夫妻有一方没有信用分，贷款时会按低于620 来算。所以最好把一些账单转到无信用分的一方，让他/她建立信用分。信用分高的人给信用分低的人要一张信用卡副卡，也会提高信用分低的人的信用分。

十二、购买保险
除非你用现金买房，否则贷款公司一定要你在过户之前购买屋主保险(Home Owner Insurance or Hazard Insurance)。这种保险主要保赔火灾对房屋造成的损失，所以有人叫它火险(Fire Insurance)。
其实屋主保险还保赔许多其它原因对房屋或个人财产造成的损失，以及客人甚至路人发生的意外。居民屋主保险有以下几类: 下面的HO 是英文屋主(Home Owner)的缩写：
1、HO-1 基本险(Basic)：保赔由于火灾、雷击、烟熏、风灾、冰雹、抢劫、偷窃、爆炸、玻璃破碎、飞机或汽车、暴乱或骚动、以及故意破坏对房屋造成的损坏。基本险还保赔人体受伤，损坏他人财产，律师费，医药费，室内个人财产，房屋损坏后的住房花费。

2、HO-2 扩充险(Broad)：在基本险的基础上加保由于电击、冰雪重压、水管冻坏、天空落体、热水器或空调系统断裂或烧坏、室内漏水造成的损坏。

3、HO-3 全保险(All Risk)：在基本险和扩充险的基础上加保除战争、水灾、地震和地陷之外几乎所有的内容。还保赔客人在室内或室外发生的意外，路人在室外发生的意外。家里的狗咬伤人后还要赔偿医药费，甚至律师费。

4、HO-4 房客险(Renter’s)：保赔公寓楼房客室内的个人财产。

5、HO-5 综合险(Comprehensive)：比全保险保赔的范畴更广，保费更贵。

6、HO-6 公寓险(Condominium Coverage)：保赔公寓屋主的单元和室内个人财产。客人或路人在室内或户外的意外事故、火灾、盗窃、或漏水带来的损失。屋主一定要仔细阅读公寓章程来决定必须构买的保险费额。

7、HO-7 活动房险(Mobile Home)：适用于活动房屋主。

8、HO-8 老房险(Older Home)：老房子的市场价格要低于重建价格。老房险允许屋主以低于重建价格的市场价来保险。

9、另外还有一种房东火险(Dwelling Fire)，适用于非商业用房的房东。除火灾外，还保赔冰雪，风暴和霉菌带来的损失。

10、除了提高自付费，还有一些其它的方法来减低房屋保险费:
（1）减低保险额：如果你买了一栋$500,000 的房子，土地值$200,000，房子本身只值$300,000。那你只要买$300,000 的房屋保险，因为$200,000 的地不会受到火灾或暴风的损坏。不过如果你的首付不到$200,000，贷款银行可能会要求你的保险额不能低于贷款额。
（2）房屋与汽车同保：如果房屋与汽车在同一家保险公司，可能会得到15%-20%的折扣。
（3）不要换保险公司：有些保险公司会给3 到5 年的老客户5%的折扣，6 年以上的老客户10%的折扣。
（4）增加房屋安全性：安装烟雾报警器(Smoke Detector)或防盗报警器(Alarm)可能会得到5%的折扣。

十三、交接过户
请过户律师的主要目的是保证买主得到合法产权，所以买主有权选择过户律师。如果你不知道找谁，经纪人会向你推荐。律师在过户前会检查产权史(Title Search)，查明卖主确实拥有产权，可以把房子卖给你。最长要查40 到60 年的历史，各个州有自己的规定。如果查出问题，要把问题解决了才能过户。产权检查还能发现与房产有关的债务(Lien) ，比如拖欠税金(Real Estate Tax Lien) ， 贷款(Mortgage Lien)，装修费(Mechanic’s Lien)等等。这些债务会跟着房子走，所以在过户前一定要卖主把它们清除掉。如果没有问题，过户后律师会到当地土地管理局(Department of Land Record)把房契登记在买主名下。为了防止意外，贷款银行还会要求买主为贷款银行买产权保险(Title Insurance Lender’s Coverage)。屋主自己的产权保险(Owner’s Coverage)，买主有权决定买不买。

过户前一个星期要落实贷款。如果利率还没锁定(Lock)，要马上锁定，以免过户时措手不及。还要根据过户公司给的过户费清单(HUD-1)草稿，把过户费准备好，并通知银行什么时候要多少钱。

过户前几天买方经纪人会向卖方经纪人要房屋检查时查出来的问题的修理账单(Repair Invoice)。一般修理应有90 天的保修期，修理账单也可以做为保修凭据。买主在过户前还要把房子再检查一遍(Final Walk Through)。除了看该修的东西都修好了，所有设备都正常运行，卖主该留下的东西都在，还要记录水电气表的数字。如果发
现没修好的东西，过户时还要与卖主交涉。
在过户的前一天，过户公司会给一张精确的过户费清单，买主可根据清单上的过户费，到银行开现金支票(Casher Check)。现金支票可以开给买主自己，过户时在反面写上付给过户公司既可。过户时还要带一张私人支票，如果实际过户费超过现金支票数额，再开一张私人支票补齐。
过户费包括五个部分：与贷款有关的费用、贷款银行预收款、中间账户预扣款、与产权有关的费用、以及过户税和产权登记税。各种费用加起来是房价的2%到4%，这还不包括首付部分。

1、与贷款有关的费用（Items Payable in Connection with Loan）:[见美国买房流程十一、申请贷款]
贷款银行预收款（Items Required by Lender to be Paid in Advance）
预付一个月的贷款利息；

贷款保险的总数大约1.5%，可按月付，首付达20%的可不用买贷款保险；
一年房屋保险，约每$1,000 收费 $1.0 到$2.0；
中间账户预扣款（Reserves Deposited with Lender）；
预扣两个月的房屋保险费；
预扣两个月贷款保险；
预扣两到七个月的房地产税，取决于过户月份。

2、与产权有关的费用（Title Charge）：
过户律师费（Settlement Fee）$200-$500；
产权检查费（Title Search）$100-$200；
银行产权保险(Title Insurance Lender’s Coverage) 每$1,000 房价的$2.4 左右；
屋主产权保险(Owner’s Coverage) 每$1,000 房价$1.8 左右；
公证等其它费用；
政府登记税和过户税（Government Recording and Transfer Charges）；
登记费（Recording Fee）$80市/郡过户税/房产证印花税（City/County Tax/Stamps）为房价的1%
州过户税（State Tax/Stamps）为房价的0.5%，在美国第一次买房，买方的 0.25%减免
州登记税（State Recordation Tax），麻州为每$1,000 房价$6.90。如果是自住房，前五万不付登记税。华盛顿特区40 万以下的房子为房价的1.1%，40 万以上的1.45%。维州为每$1,000 房价$3.34麻州的惯例是买卖方分担政府登记税和过户税，但新房建筑商不付这些税，全由买主付，所以买新房比买旧房要多付1.1%的税钱。华盛顿特区的惯例是卖主付过户税（1.1%），买主付登记税。华盛顿特区第一次买房虽然不减免过户税，但在买房当年报收入税时，自住屋可减免5000 收入税。如果调整后的家庭收入高于11 万（或一个人收入高于七万），只能减免2500，高于13 万（或一个人高于九万）则没有减免。
过户律师还会建议买主购买地界勘察图（Survey）。有人问为什么每次卖房都要重花钱做地界勘察图，就用卖主的那张不是可以省钱吗。律师的回答是，重做可保证地界图的精确性和真实性，否则可能会有人钻空子给假图。简单的地界勘察图（House Location Drawing）是画有房子位置和地界的平面图，费用在$150 到$300。复杂的还有三维房屋与地界定位（Boundary Survey），费用在$450 到$2,000。做一张最简单的就可以了。过户费清单还会有POC 的字样，意思是在过户前付过了（Paid Outside of Closing），比如房屋检查费等等。白蚁检查费一般在过户时付。房屋保险费可以先付，也可以在过户时付。
过户时要带有效证件，比如驾照或带照片的身份证。如果没有，绿卡或护照等也行。还要带银行开的现金支票和私人支票。一般过户要一个小时，主要的任务就是签字交钱，要签很多文件。过户律师先把过户费清单（Settlement Statement）过一遍，如无异议，就把所有的文件拿出来，让卖主和买主签字。签完字后，买主把支票交给过
户律师，过户律师把所有签了字的文件拿去复印，交给签字人一份。最后卖主把钥匙交给买主，房子就是买主的了。

十四、乔迁搬家
过户的最后一步是卖主将钥匙交给买主，所以过户后买主就可以搬家了。如果买的是新房子，直接就可以搬进去。如果买的是旧房子，卖主的责任仅限于把房子扫干净。所以你可能要重新油漆墙壁或清洗地毯。在搬家前4 到5 天联系水电气公司，问清要做的事情。有的公司要你自己读仪表，有的公司不需要。搬家当天再去电话确认将水电
气账户转到买主名下。
电话，电视，网络服务可能要提前10 到20 天预约。如果你的生活依赖网络, 不能上网就不能过日子，一定要提前约好网络公司来接通线路的时间。
如果请人搬家，要提前两周予约搬家公司。一般收费是一小时$80到$100，三到四个人，最少收费三个小时，再加$50 左右的卡车费。
如果自己搬，也要提前一周到租车公司定好哪天要用车。卡车有不同的尺寸，根据家具的多少来决定车的大小。一到两个房间的家具，可租十英尺的卡车；三到四个房间，可租16 英尺的；五到八个房间，可用24 英尺的。小一点的车好开，自动挡的比手动的好开。有升降机便于上下东西。没有升降机的话要选车身离地面距离较近，并有上
下货跳板的。
搬家前列一张家具清单，按房间排列，注明什么家具放哪间房间。包装箱也要注明搬到哪间房间。这样搬起来就比较省时间。搬家前要到邮局或在网上（https://moversguide.usps.com/?referral=USPS）填写换地址的表格（The Official Change of Address Form）。可能要七到十天才能收到从旧地址转来的信件。
搬家后要做的第一件事大概是装窗帘。沃尔马（Walmart）有价廉物美的窗帘和杆子。窗帘的价格在$10 到$30。杆子的尺寸有24-48，48-84，84-120 英寸的，价格在$16 到$32。每根杆子都带有安装用的钩子和锣丝钉，很好安装。挂杆子的钩子要装在离上窗框四到六英寸的地方（为的是躲开窗子的木质框架，在石灰墙上好打锣丝钉洞）。
先把钩子放在墙上，用铅笔标上锣丝钉的位置。然后用电钻在墙上钻个洞，你可以感觉到电钻钻透了石灰墙。接着把塑料空心钉用锤子打入钻好的洞中，再用锣丝钉把钩子固定在塑料空心钉上，主要的任务就完成了。这时你只要把窗帘穿到杠子上，挂在钩子上，再把钩子上的锣丝钉拧紧，拉窗帘时，杆子就不会动了。
搬家后要注意的一件事是查收前住宅的账单并及时交付，否则造成过期迟付，会影响信用分数。不管你以前是不是自动付款，最后一份账单不能自动付，会寄到你给的地址。你一定要注意查收，及时交付。

新住宅的账单也要妥当处理。首先上网找各家公司的参加自动付款的说明，然后按说明办理。
搬家后要通知所有亲戚朋友和有关公司更换地址，比如工作单位、信用卡、银行等等。如果你有给亲属申请绿卡，别忘了给移民局或签证处写信告诉他们你的新地址，否则他们无法通知你绿卡进展状况。
过户时会预付一个月的贷款利息，所以过户后的第一个月不用还贷款。因为每月还贷款的小本子一时半会儿寄不来，第一个月的付款单就在过户文件中，千万不要忘了付，否则会因迟付而罚款。如果能自动付款，就办理自动付款。但每个月要保证自动付款的账户有足够的钱。
过户费清单上有些东西是可以减税的，象预付的利息，为减低利率而交的点（Points），和贷款保险（Mortgage Insurance）。所以要把过户文件收好备查。年终还会收到贷款银行给你的贷款利息清单（Form1098, Mortgage Interest Statement）。这些东西报税时要用。

美国买房经验谈：详细流程，房贷注意事项和攻略

无论身在美国还是中国，身为中国人，买房子这个槛总是要绕不过去的。对于买房好还是租房好这样问题，归根到底其实是什么时候买房子的问题。

租房的好处是省心，只需按月交好房租水电，诸如灯泡坏了，房屋漏水、空调坏了等等什么的完全不需自己操心，报给管理处他们会负责派人搞定。不好的地方则是租房一般要求签一年的合同，中途走人违约金很吓人；房租年年涨，而且涨多涨少也是公寓公司说了算，觉得价格离谱了就只能走人；此外，搬走的时候，move-out check多少会被割一刀，就看是大刀还是小刀了。

买房呢，美国的房子都是木头造的，维修问题少不了费钱费心的，买了房后，水电气垃圾等utilities的开销必然增大，还增加了房屋保险(Homeowner’s Insurance)，草坪剪草，保安系统(Security System)，小区物业费(HOA)等等各项开支；再就是房产税(Property Tax)也跟着来了，根据房子所在的城市和地区，大概为房子估价的1％~3％。以加州旧金山湾区为例，房产税为1%，比例不算高，但一栋房子动辄上百万，算下来房产税就很高了。此外，有的地区还有市政建设费(Mello Roos)等。

当然，买房的好处也是多多，比如在某些高房价的地方，一套两居室的公寓租金和一整栋3房2.5卫的Single Family House贷款按揭的费用几乎不相上下，买房不仅房子本身会不断增值，还款中利息的部分及所交的房产税在每年报税的时候还可以用来抵税的，此外，房子维修投入的发票都可以先收着，以后若要卖房，这部分花费是可以用来抵增值税的。与完全打水漂的租金相比，买房确实更加实惠。

买房流程中美大不相同

在美国，从报价被接受签下购房合同开始，其后大概还需一个月的时间才能完成整个流程，这其中包括房屋估价，房屋检查，白蚁检查，贷款核准，房屋过户等，涉及的参与方有贷款机构/银行(Mortgage Company/Bank)，贷款中介（Loan Agent），房屋估价机构(Appraisal)，买方中介(Selling Agent/Buyer’s Agent)，卖方中介(Listing Agent/ Seller’s Agent)和 第三方交易托管机构(Escrow)，房屋检查服务机构(Home Inspection／Termite Inspection)，任何环节出问题都有可能导致购房交易失败，在此过程中支付的某些不可返还的费用也就白白损失掉了。

贷款买房准备工作

现金购房者请跳过这段。

①贷款预批准(Pre-approval/Pre-qualification)

贷款预批准的作用是让想要贷款买房的购房者了解自己能否贷款及能贷到多少钱。

购房者首先要确认自己的身份，美国公民和绿卡居民自然不成问题，工作签证(E1, E2, H1B, H2A, H2B, H3, L1及G1-G4等)持有者一般也OK，但其余的外国人则会有较多的限制，例如要支付较高比例的首付(Down payment)和利息(Interest Rate)，必须购买贷款保险(Mortgage Insurance)和产权保险(Title Insurance)，并开设Escrow Account等，有的人甚至无法贷款买房。

想要知道自己是否可以贷款买房，最简单的办法就是联系贷款中介(Loan Agent)了解一下情况。Loan Agent最好多联系几个，也不要轻易相信他们的打包票说一定能过，很多Loan Agent 为了把你的单子拿到手，会先承诺一堆东西，低利率，快速Close什么的，但其实贷成贷不成不是他们能控制的，银行会有一群叫Underwriter的人来审核你的各种资料，而Underwriter才是最终能过给你发放通行证的人。Loan Agent只是凭自己的经验作出判断，最后若是过不了的话，他最多是没有佣金可拿，而你在购房过程中付出的时间付出的钱，例如房屋检查费，房屋估价费，甚至定金（Deposit）等很可能就这样打水漂了。

记住，不同贷款公司/银行的要求(Guideline)各不相同，这家被拒了另外那家可能就行了，多问问总是好的。

也是这个阶段，可以向有意向的贷款机构/银行申请贷款预批准，由贷款机构/银行给你出具一张证明信(Pre-approved /Pre-qualified Letter)，上面申明你已获得XX机构/XX银行的XX万的贷款预批准。

拿到预批准信并不代表一定会拿到贷款，因为正式的贷款审核会比预批准要严格很多很多。预批准信的作用是：1) 让你了解自己可以购买什么价位的房子；2) 附在购房offer上，向卖家证明你有购买下这套房子的资金实力。

预批准是需要Hard Pull的，对信用分数会有负面影响，因此预批准信找一家开就好。开具的时候，可以先不用太在意利率(Interest Rate)，推荐找Citi Bank，Chase，BOA，Wellsfargo等大银行。大银行审核比较严格，也因此在下购房Offer的时候更有分量。等到正式要申请贷款的时候，可以再货比三家，选择利率最好的那家。总之，千万千万不要觉得Pre-approved Letter在哪里开的就必须也要在哪里贷款。

②打理好信用分数

在美国，建立好的信用历史非常重要，小到申请宽带电视，租住公寓，大到买车买房，都需要查询信用记录。

信用分数有一套很复杂的计算逻辑，简单说来就是你先借钱，然后按期还款，这样周而复始，信用机构会根据你的还款情况给你记分，按期还款的加分，拖欠迟交则扣分，借钱给你的人越多加分，欠的钱越多则扣分等等。但是如果你从不借款，那么信用分数也就无从打起了。

决定买房了，就千万不要再做拖欠房租等事情，信用卡也请尽量全额还款，并至少半年内不做例如申请新信用卡，贷款买车，开通宽带、AT&T账户等会带来Hard Pull的事情。信用分数(Credit Scores)的高低将直接影响贷款利率的高低，一般说来，分数在740+的人能拿到最好的利率。

对于非居民和在美信用历史不足两年的人来说，通过信用审核比较棘手。好在还有部分银行认可国际信用报告，在申请贷款之前，请与你的Loan Agent确认你的银行是否在此范围内。所谓国际信用调查，是指银行将指定一家第三方机构来负责你的国际信用调查，这个第三方机构会要求你提供在中国的信用卡、保险、水电气公司和租房房东的联系电话，然后他们的员工会和你约好时间以三方通话的方式一起给这些机构/个人打电话来核实你在过去两年的还款情况，包括是否有欠款或迟交的情况。因为国际信用调查要求至少提供三条时间跨度有2年交易线（Trade Lines）。我在国内的时候一直住在家里，水电费都是父母缴交，信用卡也是在直到出国前才开通了一张，所以我提供给他们的是公积金中心、社保中心、支付宝以及我爸（作为房东）的联系方式。

③申请贷款前三个月银行账户不要有大额现金存款或来自他人转账

贷款买房的资金，除了准备好首付(Down Payment)之外，还需要应对购买过程中的其他开销，其中较大笔的是购房成交费用(Closing Cost/Closing Fee)，小笔支出则包括房屋估价费，房屋检查费等这种。在贷款流程启动之初，贷款机构会预估一个Closing Cost，Underwriter只有在确认了你当前的存款足以应对所有这些开销之后才会给予批准。

美国银行特别害怕洗钱，一般说来，他们会追溯你最近三个月的银行账单(Bank Statement)，如果在此期间有大笔的现金存款，或是来自他人的转账，你需要就这些钱的来源给出合理解释，例如，如果是来自父母的资助，将需要父母提供这笔款项的赠予信(Gift Letter)。所以，为了避免横生枝节，请最好提早三个月将购房资金存入自己的银行账户。

来自工资的存款(Direct Deposit)，或是从自己的其他银行账户转入的存款则没有关系。

购房详细流程/步骤/指南

①你需要先看中一套房子

建议在可能的情况下，尽量选择交通便利，位于好的Neighorhood和学区的房子。因为这样的房子，转手相对容易，并且在市场好的时期，增值空间比较大，在市场不好的时候，也比较保值，不至于贬值的太惨。

寻找房子传统的办法是到当地的房屋中介机构一坐，找个地产中介(Realtor Agent)聊一聊，告诉TA你想要的地点、价格，面积，户型，学区等要求，然后中介就会把手上符合条件的房源列个清单给你，选出你感兴趣的他就会带你过去看房。这个中介就成为了你的房产中介，也就是传说中的买方中介(Buyer’s Agent/Sales Agent)。

在美国，除非买新房可直接与建筑商(Builder)交易，二手房基本是一定需要中介的：1) 非Open House的房子是必须由买房中介陪同去看的；2) 看好房后，出价Offer和后续的购房合同等都必须由双方中介来协助进行，整个交易过程，买卖双方甚至于是完全不碰面的。

房产中介的佣金，也就是中介费，一般是成交价的2.5%~3%，而在一宗房产交易(Real Estate Transaction)中，会同时存有两个中介角色：卖方中介(Listing Agent/ Seller’s Agent)和买方中介(Buyer’s Agent/Sales Agent)。他们分别代表房主和购房者进行沟通谈判及推动交易进行，所以在中介方面总的支出大概为房屋成交价的5%~6%。这笔钱都由房主，也就是卖方，在房子成交后支付。

现在互联网这么发达，像Redfin，Zillow这些网站都提供了非常强大的线上找房功能，输入地点(Zipcode)等要求，网站就会把符合条件的房源列出，有很详细的照片，房子描述，建造年份，当前估价，卖方中介的联系方式，所在区域的房产市场热度，学校等等等等信息。如果你还没有自己的中介，你可以使用网站推荐的中介，也可以直接用卖方中介充当双重中介(Dual Agent)。双重中介为买卖双方提供服务，佣金当然也就拿双份的。

自行在房产网站上看房，除了时间上比较自由，还有一个好处就是可以要求你的中介将他的部分佣金返现给你(Rebate)。这很好理解，中介不用东奔西跑的带你去看房，他的服务成本下降了，自然佣金也就可提供折扣了，而你也相当于变相的得到了购房折扣。像Redfin网站，就直接标明了若使用它的中介完成这笔交易，你可以获得多少的返现。返现的比例从1%到2%的都有，一般说来，华人中介给的返现会高一些，老美中介有的甚至完全不给Rebate，就看你和中介如何谈了。

你和中介之间关于Rebate的约定，请谨记口说无凭，一定要求白纸黑字的写下来并签字。

②报价Offer

看中了房子后，就该给房主下Offer了。

首先，房主会给出一个他的期望售价，你和你的中介商量后，会以正式的文件的形式，提出你们的报价(Offer)及其他一些要求，例如房内的冰箱洗衣机烘干机要留下，由卖方支付Escrow费用等等。如果房主同意了你的Offer，那么你们双方就会签署购房合同，正式进入购房流程；如果房主不满意你的报价，他可以不做回复，或通过Listing Agent在你的Offer的基础上回复一份Counter Offer，提出他的要求，双方可以这样你来我往直到达成一致。如果没有达成一致，这个购房当然也就终止了。

所有这些Offer往来及后续购房合同文件(Residential Purchase Agreement, 简称RPA)都是有标准模版的。流程每进行一步，双方中介会在相应的文件模版上填好条款内容，然后交由卖家和买家签字以确认生效。你需要在签字画押前仔细核对其中的条款是否描述到位，防止中介有所遗漏甚至犯错误。在美帝，无论何时，一定要认真对待签字画押这个事情，因为即使是你的中介搞错了，签下字后，你就是对这个签字负责的人，承担损失的也将是你自己。

至于回价多少合适，无法一概而论。在房地产市场火热的地区，例如加州旧金山湾区，因为竞争对手太多，很多Offer都是直接在卖家的报价上加价出的，而在市场不温不火的地区，回价一般是对报价进行砍价。

前面提到的贷款预批准证明也就是在此刻派上用场。一旦卖方接受了你的Offer，房子就进入一种叫做Pending的状态，这意味着在这为期一个月左右的交易过程中，买卖双方都将受到购房合同的约束，卖方不能再接受其他人的Offer。所以如果最终没有交易成功，卖方相当于白白浪费了一个月的时间，尤其在卖方需要这笔钱急用的情况下，交易延误的代价可能是极大的。因此，在Offer中附上贷款公司/银行出具的Pre-approval letter已经基本是约定俗成的要求了。

当买卖双方就Offer的价格及条件达成一致，签署了确认文件后，购房合同就正式生效了，接下来你就要合同约定的时间内交付定金，提交贷款申请，和请专业人士来对房子状况进行检查。

③签署RPA – Residential Purchase Agreement购房合同

对多数人来说，买房是件大事，因此在签署合同之前，请仔细考虑自己的情况，用好其中的保障条款，为风险预留足够空间。

购房合同生效意味着双方就不能随意的退出此次交易了。好在有一项叫做Contingency的条款，它们是买家不损失定金(Deposit)合法退出购房交易的利器。要知道，定金一般为购房价格的1%~3%，一般会要求在签署购房合同后三天内将它交给Escrow公司。

Contingency一般有三种：Inspection Contingency，Appraisal Contingency和Loan Contingency/Mortgage Contingency。翻译过来就是，房屋检查附带条款，房屋估价附带条款和房屋贷款附带条款，它们是买家不损失定金合法退出购房交易的三大利器:

An Inspection Contingency (also called a “due diligence contingency”) gives the buyer the right to have the home inspected within a specified time period, such as 5-7 days. It protects the buyer, who can cancel the contract or negotiate repairs based on the findings of a professional home inspector.

房屋检查附带条款－－买家有权在合同约定的时间内对房子进行检查，并可根据检查结果决定是否要取消房屋交易。

An Appraisal Contingency is an exit for a buyer. If a property is under contract for $600,000 and the appraisal comes in at $590,000, then the buyer has an option (in addition to other contingencies) to exit the deal.

房屋估价附带条款－－如果专业的房产估价机构对房子的估价结果低于合同价格，买家可以选择是否需要取消房屋交易。

A Loan Contingency/Mortgage Contingency is a provision in the home purchase contract saying that if the prospective buyer cannot get a mortgage within a fixed period of time with the specified terms, the buyer can call off the whole deal and get back his deposit.

房屋贷款附带条款－－如果在合同约定的时间内房屋贷款还没有获得批准，买家可以选择是否取消房屋交易。

这些Contingency是买房者的安全屏障，每个Contingency在购房合同中都有明确的时间期限，以Home Inspection Contingency为例，假设时间期限设置为17天，这就是说从购房合同生效的那天起，17天内，买方要完成房屋检查并明确是移除这项附带条款(Remove Home Inspection Contingency)呢还是退出购房交易。但是，即使超出了17天，Contingency是不会自动移除掉的，必须要买方签署移除文件方可(Contingency Removal Form)，这个17天的限制在于：若时间到了，买方不肯签署移除文件，卖方是有权力终止购房交易的。

以加州为例，标准购房合同中，各Contingency的默认时间期限如下：

– Home Inspection Contingency – – 17天 – – Appraisal Contingency – – 17天 – – Loan Contingency – – 21天 –

Contingency的约定时间到了，房主并不能强迫你签署Contingency移除文件，他只能终止购房交易，不过，常见的手段是，房主以终止交易来威胁你签署移除文件。

从买家的角度出发，Contingency保留的时间越长越好，至少尽量不要对默认的做删减，Loan Contingency建议至少增加到30天，能够保留至银行放款最安全。

④Escrow公司

整个购房交易将由一种叫做Escrow的第三方机构进行监管。买方所交的定金也好，后续贷款通过后银行的放款也好，都将被存放在Escrow公司为这宗交易开设的账户里，在房子过户登记之前，并不会进到卖家手里。同时，卖方这边也需要把房子的产权等相关资料交到Escrow公司手上。Esrow公司会在收到银行的放款后的一个工作日内，到房子所在的县政府(County)进行房屋产权转让的登记，然后才分钱给各方。

当然，Escrow公司的服务也是要收费的，通常是由买卖双方各出一半。也并不是所有的州都要求在购房交易中使用Escrow，但例如加州，Escrow公司则是必须要使用的。

⑤提交贷款申请

在美国，贷款买房的审核流程比中国要严格的多。

首先，银行会直接指定一家房屋估价机构来对房子进行估价(Appraisal)，估价结果若高于购房合同上商定的价格自然没有问题，一旦估价结果低于合同价格，那么银行只会批给估价金额的贷款，价格溢出的部分买家要自己补齐。如果买家不能够或者不愿意自己补足购房款，而卖家也不肯降低售价，那么买家可以使用Appraisal Contingency拿回定金退出交易。

其次，银行将通过调取你的信用报告(此时会再Hard Pull一次)，并且查询你过去三个月的银行账单(Bank Statement)，以及过去两年的报税记录和房租缴纳情况等信息，以评估你的贷款偿付风险。这个过程中，你需要按照银行的要求提供各种资料，最近三个月的银行账单和工资单，最近两年的W-2表格，房租支票记录，非公民/绿卡居民还需要提供护照，Visa，I-94等身份证明材料。

贷款审核工作是由银行的Underwriter负责。若信用报告上显示你最近三个月有过Hard Pull，Underwriter就会要求你就Hard Pull的原因给出合理解释。若银行账单上显示你有大额进账，Underwriter会要求你解释这些钱的来源。若你的租房记录显示你有过迟交历史(Late Payment)，没有合理的解释，Underwriter很有可能因此拒掉你的申请。

你的Loan Agent是你与Underwriter之间的中介，他只能协助你提供资料，以及与Underwriter沟通以推动审核进程，对申请能否通过并无决定权。为了避免贷款进度延误，请务必精确的按照Underwriter的要求提供资料，不要心存侥幸，Underwriter一定只会在你的所有资料都符合了要求后才会给予放行。

记住，虽然Loan Agent没有能力决定你的贷款能否获得批准，但是一个好的Loan Agent可以根据自己的经验，判断你获得贷款的难易程度，提早告诉你需要准备哪些材料，准备的材料是否合格等，以帮助你降低贷款被拒的风险，减少与Underwriter来回沟通的时间从推动整个流程在约定时间内完成。

贷款核准是整个购房交易流程中最为耗时耗力的环节，通常至少需要30天的时间。不过，据说也有两周内完成的案例存在。

整个申请过程中，根据Underwriter的审批进度，会依次出现三种状态： 1) Conditional Approval/Loan Commitment：有条件的批准，你会拿到一封叫Commitment Letter的文件，上面会列出Underwriter还需要你补足的材料，在这些材料被审核通过后，你才可获得贷款批准； 2) Final Approval/Clear to Close ：贷款申请获得批准，你会拿到一份叫Loan Doc的文件，上面会列明贷款的总额，期限，利率以及Closing Cost的各项细节。请仔细核对这些数字都没有问题后再行签字； 3) Funding：银行放款给到Escrow，此时贷款才是最终到手，而在此之前的任何阶段，都无法100%保证你能拿到贷款。

Loan Contingency/Mortgage Contingency的作用就是在贷款不幸被拒的情况下，赋予你退出购房交易，取回全部定金的权力。有些Buyer Agent或Loan Agent会在Conditional Approval阶段就建议你移除Loan Contingency，但于你自己最最安全的做法就是Funding后再进行签字移除。这也是为什么我会强烈建议在RPA购房合同中尽量争取把Loan Contingency的时间保留到Closing Date。

这里推荐一个Citibank做Mortgage的华人小姑娘吧。当初在我在网上做买房调研的时候，看到她在天涯上写的有关在美国买房的文章，非常详细，就加了她的微信。此外，从我个人的经历来看，BOA，Wellsfargo，Chase和CitiMortgage四家报价之中，Citi给出的利率是最低的，当然，如果想拿到好利率，多找几家报价是一定要做的，银行之间也会互相match利率的。小姑娘会中文，有兴趣的朋友可以加她微信聊聊看：sunsunmianbao。也可以使用邮件联系她，她的Citibank的工作邮箱是：sunny.duan@citi.com。

⑥房屋检查

在等待贷款申请批准的同时，买方可以开始房屋检查。

你肯定不会希望买完房子，入住后发现里面一大堆东西需要维修，尤其是像屋顶，空调，热水器这类非常耗钱的维修，再加上美国的房子都是木头做的，感染白蚁如果不及时处理，后果十分严重，这些都使得常规房屋检查(Home Inspection)和白蚁检查(Termite Inspection)成为了二手房交易过程中买家必做的事情，其他还有Mold Inspection，Radon Inspection等，是否做就视情况而言了。

大部的Home Inspector都是应付了事，真正好的Home Inspector并不多见。而口碑好的Inspector的工作往往非常多，预约有可能要等上两个星期，甚至是一个月。此处强烈建议各位已然决心买房的小主们，在下offer之前，可以先预约好房屋检查的时间，大不了之后再改时间或者取消预约，也比临到头约不到只得寻找其他不靠谱的Inspector要好。

房屋检查完成后，Inspector会出具一份详细的图文并用的检查报告，拿到这份报告后，你可以和房主协商维修的事宜，譬如要求他在交房前找人解决掉其中比较大的问题，或者让房主直接返给你一些费用(Credit)，之后你自己找人维修。

Inspector查的越仔细，越有利于你与房主的讨价还价，这就是为什么要尽量找认真负责经验丰富的Home Inspector。

Home Inspection Contingency就是在这个环节起作用的，如果房屋检查的结果太过糟糕，或者无法就维修补偿与房主达成一致，你有权退出购房交易，取回全部定金。

房屋检查的费用范围大概是$300~$500之间，和购买产权保险(Title Insurance)类似，同属于那种不做不能安心，但做了后又经常感觉没啥大用的购房支出。

⑦房屋过户(Close Escrow)

银行在你的贷款申请Final Approval后，会将贷款文件邮寄给Escrow公司，叫做HUD-1的表格，之后Escrow就会分别通知买方去签署贷款文件，卖方去签署产权转让文件等。

银行收到Escrow公司寄回的签署过的贷款文件后，就会进行放款，Escrow收到银行放款后，一般当天就会去County登记房子的产权转让，一旦产权转让登记在案了，就表示房屋过户完成了，之后Escrow会通知你去取新家的钥匙，然后你就可以享受你的新房子了。

大概会在房屋过户后一个月左右，你会收到一份由County Clerk-Recorder寄给你的平信：Grant Deed，俗称转让契约，类似房产证。如果没收到也没有关系，随时都可以去County再要一份。

后续
在房屋买卖结束后的一段时间内，你陆续会收到各种和房子相关的文件、信件。这其中也包括各种骗局的信件，例如让你付好几十或者上百刀以获得房子产权转让纪录的拷贝，但其实这个拷贝完全可以自己去County的办公室免费打印，因此在浏览各种信件，尤其是需要回复个人信息或支票的信件的的时候不妨多留个心眼。

Via: http://oaklandchina.com/%E7%BE%8E%E5%9B%BD%E4%B9%B0%E6%88%BF%E7%BB%8F%E9%AA%8C%E8%B0%88%EF%BC%9A%E8%AF%A6%E7%BB%86%E6%B5%81%E7%A8%8B%EF%BC%8C%E6%88%BF%E8%B4%B7%E6%B3%A8%E6%84%8F%E4%BA%8B%E9%A1%B9%E5%92%8C%E6%94%BB%E7%95%A5.html

用于ELISA 实验的细胞裂解方法

You may also use your own lysis buffer, such as RIPA or other formulations optimized for immunoprecipitation for ELISA assay..

Please note the following guidelines on lysis buffer composition:

1. Avoid using >0.1% SDS or other strongly denaturing detergents. In general, non-ionic detergents such as Triton X-100 or NP-40 are best, although zwitterionic detergents such as CHAPS, or mild ionic detergents such as sodium deoxycholate will work.
2. Use no more than 2% v/v total detergent
3. Avoid the use of sodium azide
4. Avoid using >10 mM reducing agents, such as dithiothreitol or mercaptoethanols

We strongly recommend adding a protease inhibitor cocktail to the lysis buffer prior to homogenization. Most general biochemical supply companies including Roche, Sigma-Aldrich, Pierce, and Calbiochem stock a wide variety of these products. Since susceptibility to proteolytic cleavage and the type of proteases present in the lysate vary, we do not recommend a specific product. Instead, your choice of which combination of protease inhibitors to use should be based upon a literature search for your protein(s) of interest and/or tissue or cell type. Phosphatase inhibitors may be used but are not necessary unless the antibodies used in the kit specifically recognize phosphorylated forms of the protein.

Choices of the method for lysis and homogenization include glass-bead “smash,” douncing, freeze-thaw, sonication and crushing frozen tissue with a mortar and pestle, or even a combination of these. There is no best method for all sample types; your choice of method should be made following a brief search of the literature to see how samples similar to yours have been prepared in previous investigations.

After homogenization, centrifuge the lysates to remove cell/tissue debris (5 min @ 10,000 x g or 10 min @ 5,000 x g) and save the supernatant. Unless testing fresh, lysates should be frozen as soon as possible and stored at -20°C (or -80°C, if possible). Centrifuge them again before incubating with any immunoassay. Next, determine the protein concentration of your lysates using a total protein assay not inhibited by detergents (such as the Bicinchoninic acid (BCA) assay) and normalize the volume of each sample used to deliver the same amount of total protein for each assay.

Note: The Bradford assay is not recommended as it can be inhibited by the presence of detergents.

Since different cells and tissues may contain different amounts of protein, as starting point, we suggest using 500 µL of lysis buffer per 1×10⁶ cells or 10 mg tissue. You may have to adjust this based upon your results. Your target total protein concentration of the homogenate should be at least 1,000 µg/mL, but 2,000 µg/mL or more would be better.

Regardless of the sample type you have, it is strongly recommended that you sub-aliquot all samples after preparation to minimize protein degradation from multiple freeze-thaw cycles. This also ensures availability of sample for further experiments.

Single-cell RNA-seq—now with protein

Two new methods simultaneously measure epitope and transcriptome levels in single cells.

The molecular understanding of the cell has been greatly advanced by single-cell RNA-seq, a technique that generates a library of all transcripts (the ‘transcriptome’) in a single cell. The technique has revealed surprising heterogeneity in cell populations previously considered homogeneous, identified new and rare cell types, and extended our understanding of cellular development. The transcriptome is, however, only a proxy of the ‘proteome’, the collection of proteins in a cell that defines how the cell looks, acts, and reacts. Although the transcriptome provides valuable information, it does not necessarily reflect protein abundance in the cell. And while flow cytometry is an established strategy for profiling populations at single-cell resolution according to surface-protein levels, it cannot access the rich phenotypic information available in the full cellular transcriptome.

Now, independent efforts led by Marlon Stoeckius at the New York Genome Center (NYGC) and Vanessa Peterson at Merck have yielded approaches to measure levels of both gene and protein expression in single cells on a large scale.

Both CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), developed by the NYGC group, and REAP-seq (RNA expression and protein sequencing assay), designed by the Merck group, use a similar approach. Proteins are detected by using antibodies conjugated to a tripartite DNA sequence that contains a primer for amplification and sequencing (PCR handle), a unique oligonucleotide that acts as an antibody barcode, and a poly(dA) sequence. The poly(dA) sequence allows for simultaneous extension of antibody-specific DNA sequences and cDNAs in the same poly(dT)-primed reaction. This generates a protein readout that is captured and sequenced along with the cell’s transcriptome. The two approaches differ in how the DNA barcode is conjugated to the antibody. While antibodies used in CITE-seq are conjugated to streptavidin that is noncovalently bound to biotinylated DNA barcodes, REAP-seq relies on covalent bonds between the antibody and aminated DNA barcode.

In a proof-of-principle study, Stoeckius and colleagues monitored ten surface proteins and the transcriptomes of 8,000 single cells from cord blood mononuclear cells. The CITE-seq analysis revealed cell profiles similar to those established by flow cytometry. In addition, the multimodal data from CITE-seq enhanced the phenotypic characterization of a specific type of immune cell, the natural killer cell, compared with single-cell RNA-seq alone.

REAP-seq was used to characterize the effect of a CD27 agonist on human naïve CD8+ T cells by employing 80 barcoded antibodies and monitoring the expression of more than 20,000 genes in a single workflow. The transcriptome data analysis identified several differentially expressed genes in treated versus untreated cells. But REAP-seq’s ability to quantify cell surface proteins also led researchers to determine that ICOS, an immune checkpoint protein, is increased on the surface of treated cells, regardless of the fact that this protein’s mRNA does not differ in abundance between treated and untreated cells. REAP-seq also identified a small and previously unknown cell population within the enriched naïve CD8+ lymphocytes.

While both CITE-seq and REAP-seq add to established methods for transcriptome analysis without affecting the quality of the data, the main limitation of both approaches is the quality of the antibodies used and the epitope location, which is currently restricted to the cell surface. Both research groups anticipate that the use of these tools will soon be extended to measure intracellular proteins.

References

Stoeckius, M. et al. Simultaneous epitope and transcriptome measurement in single cells. Nat. Methods 14, 865–868 (2017).

Peterson, V.M. et al. Multiplexed quantification of proteins and transcripts in single cells. Nat. Biotechnol.

5’RACE & 3’RACE原理

雖然 RACE 為 RT-PCR 的變奏版，在原理跟步驟上和 RT-PCR 有很多相似，但 RACE 卻能解決很多特別的問題。例如，分析兩端序列未知的 RNA、界定啟動子 (promoter) 下游、調查部分胺基酸序列已知的基因等…

快速擴增 cDNA 末端 (Rapid Amplification of cDNA Ends, RACE)，不要被這長長的名稱嚇到了！其實簡單來說，就是一種可以用來幫助你尋找 RNA 末端未知序列的技術。一般來說，我們會依照分析的端點而將RACE 分成 3’ RACE 和 5’ RACE 兩種。下面以幾個簡單的例子分別說明：

3’ RACE 及 5’ RACE 的流程：

3’ RACE

圖二. 3’ RACE 分析真核生物 mRNA

以分析某個真核生物特定基因的 mRNA 3’ 端為例，首先會藉由 poly-T 引子將具有 poly-A tail 的 mRNA 做為模板，反轉錄成第一股 cDNA (first-strand cDNA)。poly-T 引子在設計上 3’ 端有時會多帶一個 V (V = A or C or G)，藉此增加黏合在 poly-A 與基因交界處的機率，以免 poly-T 引子黏合在離基因太遠的 poly-A 區段上。

隨後利用目標基因的 Gene-Specific Primer (GSP)，並以first-strand cDNA 為模板，合成第二股 cDNA (second-strand cDNA)。取得含 3’ 端未知序列的 cDNA 後，我們可以利用 poly-T 引子與 GSP 擴增產物，定序先前未知的 mRNA 3’ 端。說到這，不知大家會不會想到一個問題：如果我們的目標 RNA 沒有 poly-A tail該怎麼辦呢？像是例如非編碼 RNA (non-coding RNA, ncRNA) 或原核生物的mRNA……等。

這時就可以利用 poly-A polymerase 將目標 RNA 3’ 端加上 poly-A，用來仿照真核生物的 mRNA 3’ 端，就可以解決這樣的問題囉！另外，一般的反轉錄酵素在面對太長的 RNA 目標時，往往會後繼無力而無法合成整段 first-strand cDNA。所以如果設計的 GSP 位置距離 RNA 3’端太遠，以 poly-T 引子合成的 first-strand cDNA 可能就無法包含能被 GSP 辨識的區域。

想要解決這樣的問題，其中一種解決的策略是：先使用隨機引子 (random primers) 合成多種 first-strand cDNA，這些 first-strand cDNA 當中可能有些是包含部分未知序列且能被 GSP 辨識的。搭配 random primer 配合 GSP 擴增產物並定序取得部分未知序列的資訊後，就能設計更靠近 RNA 3’ 端的 GSP 來分析剩下未知的區域。

——

5’ RACE

 

圖三. 5’ RACE 分析原核生物 mRNA 範例圖

這部分，我們以分析某個原核生物特定基因的 mRNA 5’ 端為例。

首先用 GSP 合成 first-strand cDNA 後，通常會以末端脫氧核苷酸轉移酶 （Terminal deoxynucleotidyl transferase, TdT） 在 first-strand cDNA 3’ 端加上一段能夠被引子辨識的標靶序列 (adapter)。

所謂的TdT 是一種不需要模板就能合成 DNA 的聚合酶，因此可以直接在 first-strand cDNA 3’ 端加上一段序列，這段序列可以依照我們給予的 dNTP 原料來決定，例如如果給予 poly-C。之後就能以 poly-G 引子來合成 second-strand cDNA。

poly-G 引子在設計上 3’ 端有時會帶一個 H (H = A or T or C)，藉此增加黏合在 poly-C 與基因交界處的機率。取得含完整 5’ 端的雙股 cDNA 產物後，以 poly-G 引子與 GSP 擴增目標產物，就可以將未知的 5’ 端序列定序出來了！

如果 GSP 的位置距離 RNA 5’ 端太遠而無法合成含完整 5’ 端的 first-strand cDNA，也能繼續以互補 adapter 的引子先合成 second-strand cDNA，再用 GSP 配合能互補 adapter 的引子擴增含部分未知序列的產物，定序取得更多資訊後就能設計更接近 5’ 端的 GSP。

——

RACE 在實作上也有相當多的花招，當我們覺得 RACE 的產物不夠專一時，可以藉由巢式聚合酶鏈鎖反應(Nested PCR) 的概念，將原先使用的 GSP 替換成另一種新的 GSP 並進行第二次擴增 (詳見“房屋仲介”有巢氏”，PCR也有巢式!”)。或是當我們覺得用 TdT 製造 adaper 的步驟太繁瑣時，亦可選用能在產物 3’ 端額外添加 adapter 的反轉錄酵素產品。

在應用面上，RACE 也佔有重要的地位。除了能夠藉由 5’ RACE 來分析啟動子外 （promoter），也能以某個蛋白的特徵序列設計退化性引子 (degenerate primer) ¹作為 GSP，再藉由 RACE 將某個系列的蛋白從 RNA pool 釣出來。

另外，實驗設計上，RACE 產物的取向較 RT-PCR 集中，因此能夠建立較小的 cDNA 文庫 (cDNA library)，利於我們進一步的篩選。而當目標 RNA 過大而無法以 RT-PCR 分析全長時，我們也能以 RACE 逐步分段地對整條目標 RNA 進行分析。見圖四：

圖四. 以 RACE 對 Long-RNA 逐步進行定序。

雖然目前具有高通量的次世代定序分析，奪去了 RACE 的部分風采，但秉持著能夠穩健、輕巧分析的優勢，RACE 至今仍被許多實驗室應用。經過小編今天的介紹之後，大家對於 RACE 有沒有比較熟悉了呢？

名詞解釋：

退化性引子 (degenerate primer)：用已知的胺基酸序列逆推可能的核酸序列，再以此資訊設計出對應的引子群。例如：TENGIGA 胺基酸序列可對應到的 DNA 序列為 ACN GAR AAY GGN AUH GGN GCN：

T (ACU, ACC, ACA, ACG)

E (GAA, GAG)

N (AAU, AAC)

G (GGU, GGC, GGA, GGG)

I (AUU, AUC, AUA)

G (GGU, GGC, GGA, GGG)

A (GCU, GCC, GCA, GCG)

參考資料：

1. 維基百科：https://en.wikipedia.org/wiki/Rapid_amplification_of_cDNA_ends
2. Michael J. McPherson, Simon Geir Møller (2006). PCR (Second Edition) pp 47-50. In US: 270 Madison Avenue New York, N Y 10016, In UK: 4 Park Square, Milton Pa. Taylor & Francis Group.
3. http://download.bioon.com.cn/view/upload/201201/18181845_7261.pdf

防止细胞抱团的方法

General tips:
Firstly, if they are adherent or even if they grow in suspension but in
clumps, make sure that they are in as good a single cell suspension that you
can make before fixation
A biodegradable anticoagulant named ACD-A at 0.6% in PBS prevents cell
clumping of mononuclear blood cells and some stromal cells.
Also it can be a good idea to filter the cells through a 60-70um mesh to get
rid of the larger lumps (although clearly this will lose cells).
Keep cells suspended in 1 to 5 % BSA/media helps prevent clumping.
Try adding some EDTA (0.02%)
Try adding DNase at 50U/mL in all preparation media.
If you are using an alcohol fixation, try to resuspend the cells in the last
remaining volume after centrifugation and then add the alcohol drop wise
with mixing. Another problem we have run into is over trypsinization. It
sounds as if you are getting free DNA into your prep which will string
everything together. We have experienced this also. Final tip may be to
assure yourself that the cell number is kept below 1X10e6/ml. The cells
tend to clump less for some reason.
Where visible clumps are seen in the sample tubes, our users use nylon mesh
or Falcon 2270 or 2235 tubes with the mesh in the caps to filter the
samples. If the cell type they are using tends to clump, Accutase sold by
Phoenix Flow Systems o San Diego, CA. USA prevents clumping. They also sell
Accumax, an enzyme based product used for creating single cell suspensions
from tissue samples.

Ethanol fixation step:
Inadequate ethanol fixation is a primary cause of clumpage – it’s a good
idea to vortex while fixing (careful though if looking for apoptotic cells
as they could explode at this point!). Also make sure that the pellet is as
free as possible of residual PBS before adding the ethanol.
Use EDTA or Dnase:
Secondly you could try using either EDTA or a low level concentration of
DNase in your PI solution as it is DNA from blown-up cells that can cause
clumps as well.
Use Anti-clumping agents:
I have read about a firm that’s called TCS CellWorks in the UK, that sells
Accumax. It is used to disaggregate clumpy cells and viability seems to be
very good after treatment. You can read about it on
http://WWW.innovativecelltech.com <http://WWW.innovativecelltech.com&gt; for
additional information. You can also contact the office manager at TCS
CellWorks Ltd :Fay Crook : Fay at tcsgroup.co.uk <mailto:Fay at tcsgroup.co.uk>
PAA sell a product called Accutase, that’s for prevent cell clumping. I
never used it, so I can’t tell you if this product works or not

Use Nuclei:
A variation on this assay utilizes hypotonic lysis to isolate individual nuclei instead of analyzing whole cells. It is described in: J Immunol Meth, 139: 271-279, 1991. This should eliminate cell/cell binding.

Protocols:
Protocol 1: APOPTOSIS AND CELL CYCLE ANALYSIS:
Courtesy of Nigel Miller

1. 1 million cells in 200ul PBS
2. Add with stirring 2ml ice cold 70% ethanol 30% PBS.
3. Leave 30 mins. On ice.
4. Centrifuge 2000 rpm 5 mins.
5. Resuspend pellet in 800ul PBS. If cells are clumped pass through a 25 gauge needle.
6. Add 100 ul Rnase (1mg/ml-boiled 10′ to destroy DNAse).
7. Add 80 ul Propidium Iodide (0.5mg/ml).
8. Incubate at 37 deg C for 30 mins.
9. Analyse using the FACSCalibr..
10. Use MODFIT to calculate DNA.

Protocol 2:
Courtesy of Dennis Young
Fix cells by adding the 10^6 cells (or less) in 1 ml to 10 ml ice cold 70% EtOH while vortexing.
Use detergents (0.1% Triton-X 100)
Use filters (20 – 75 microns should work)
Use 25 gauge needle to resuspend cells right before sampling (Lastly) Lyse cells and measure nuclei instead.

Protocol/Advice 3:
Courtesy of Kent Claypool
If preparing a single cell suspension is producing aggregates remove the cytoplasm through a pepsin digest (references below) and look at bare nuclei. Also the PI for a sub-G1 peak does not always work. The example at the below web site had cell death that didn’t produce a sub-G1 peak until a positive control with Fas ligand was used. Then produced a great profile.
Cells have to have Fas receptor to trigger this apoptotic pathway. Might want to also look at gel electrophoresis for DNA laddering, Bcl-2 by western and/or flow, as well as the ‘TUNEL’ assay which can be done with nuclei

also. http://sciencepark.mdanderson.org/flow/files/DNA_PI.html

Further reading: https://www.sigmaaldrich.com/technical-documents/articles/biology/cell-culture-troubleshooting-cell-clumping.html

如何报考ASCP

以下的内容都是针对IMT（international Medical Technologist）的，其他类别的请参照本文。另外，说明以下，国家医学考试网上有对于ASCPi的介绍文章http://www.nmec.org.cn/ksfw/11051701.htm，其主要内容就是ASCP官网上的内容并附翻译。但是对于这一段内容的翻译值得商榷，

需要指出，MLT和MT分别是medical laboratory technician 和medical technologist的缩写。在台湾蔡宗仁检验师的介绍文章中对此的翻译是“国际医检生”和“国际医检师”。我认为，对于大陆目前的情况还是翻译为国际医学检验技士和国际医学检验技师来的恰当。检验医师应该是Laboratory Physician，和技师technologist还是有差别的。这个情况其实也是检验医学的乱象之一，不光中国有，美国加拿大也有。像美国现在把本土的MT改为MLS-Medical Laboratory Scientist，而加州的称谓是CLS-Clinical Laboratory Scientist，纽约州的交CLT-Clinical Laboratory Technologist。而加拿大对于检验技师叫做MLT-Medical Laboratory Technologist，而技士叫做MLA/T-Medical Laboratory Assistant/Technician。真是各有各的叫法。甚至连检验医学（Laboratory Medicine）还是医学检验（Medical Laboratory Science/Technology）都无法统一，这点还需我们全体检验工作者继续努力。

言归正传，对于iMT的报名，ASCP有5个Route可以报考，原文请见ASCP官网http://www.ascp.org/Board-of-Certification/International/Certification#tabs-1及国家医学考试网http://www.nmec.org.cn/ksfw/11051701.htm

Route1，在经过认证的教育机构的医学技术，生物学，或化学领域获得学士学位，完成医学技术训练课程（即实习），该课程必须包括血库，化学，血液学，及微生物学

Route2，在经过认证的教育机构的医学技术获得学士学位，在认证的实验室所有方面完成3年的工作经验，经验需包括血库，化学，血液学，及微生物学

Route3，在经过认证的教育机构获得学士学位，及完成至少2年的医学检验课程，实习课程必须包括血库，化学，血液学，及微生物学

Route4，在经过认证的教育机构的任何生物学或化学领域获得学士学位，在认证的实验室所有方面完成5的工作经验，经验需包括血库，化学，血液学，及微生物学

Route5，在经过认证的教育机构的获得学士学位，和最少2年的大专文凭或在生物学或化学方面取得相等的文凭，以及在认证的实验室所有方面完成5的工作经验，经验需包括血库，化学，血液学，及微生物学.

翻译的不好，因为这个本身比较拗口和复杂。简单而言，就是如果你有医学技术的学士学位的，完成了实习就可以考。有其他专业学士学位的，学过2年检验也可以考。非检验专业学位的，如果有5年的检验工作经验，也可以考。对于Medical Technology，ASCP有说明*Degrees/Diplomas in Medical Technology include Medical Technology, Medical Laboratory Science, Clinical Laboratory Science, and Biomedical Laboratory Science.就是说，包含医学技术，医学检验学，临床检验学和生物医学检验学。如果没有学士学位，是大专及以下的专业人士，就只能报考MLT了。

我再以我自己的经历说明一下报考的流程：

1. 到所在学校取得中英文成绩单，密封盖章；
2. 在中国学位与研究生信息网http://www.cdgdc.edu.cn/申请认证，这个就是俗称的清华认证，认证费英文学历260加英文学位260加英文成绩单360共880元；
3. 连同毕业证和学位证寄到清华认证中心，认证的结果要让他们寄到美国的WES认证中心，这个过程要2周左右；
4. 在WES网站上申请Course-to-Course认证,认证费200美金左右，时间1周，结果要寄到芝加哥的ASCP International；
5. 在ASCP上申请考试，申请费200美金，现在好像有优惠了。类别iMT，route2学历加经验；
6. 让主任签几份文件，并寄到ASCP。注意应该要寄航空信，但是我最后一份工作经历是扫描后发Email给他们，也承认。

a.Reference Letter

推荐信自己写，主要讲自己的学习经历和工作情况，必须要有医院台头。没有这种有台头信纸的话可以在推荐信的上部打上医院名称和地址，并改为红色，再打出来就可以了

b.Letter of Authentication这是确认主任授权

c.Work Experience工作经历证明

以上步骤都完成后，就等待ASCP的考试确认信Admission Letter吧。

关于考试：

1.整个考试时间是120分钟，题目是100题，每道题的时间应该是1分钟。但我其实考了1小时多点就考好了。

2.全部是单选题，有些题的题干很长，会给你一个病例，叫你分析。

3.如果你做对了一题，下一题会变难。就是说，如果你觉得题目越做越简单，那就形势不妙了。

4.各部分的比例不会像BOC上写的一样。我的感觉是体液方面蛮多的，光尿液的题目就出了好几道。

5.对于真菌和寄生虫的英语单词，我真的无法完全记住，所幸考的不多，随便瞎猜罢了。

6.可以mark题目，到最后再review。但我没改答案，因为经验告诉我第一感觉往往是对的。

7.考完点击over就结束了，结果是当场就出来的。这比较好。

8.我当时pass了以后非常兴奋。但是后面还有一个survey，有许多问题让你回答。像你用什么材料复习，单位里的情况之类的。

总结：

1.这个考试难不难？

考过了当然感觉并不难，但是没考过的肯定觉得很难了。从准备到考试，我前后加起来大概花了将近1年的时间，了解情况如何报名，上论坛了解需要哪些复习资料，托朋友从美国带书回来，到自己报名，这期间还有移民的事情。实际的看书时间加起来的话不超过5个月。我考中级只看了2个月。

2.这个考试报名怎么报？

这个比较复杂，我会在下一篇里专门讲讲怎么报名。我是，检验5年本科，医学学士，10年工作经验。

3.费用怎么样？

考试费200刀，但据说现在有优惠。WES认证费200刀。清华认证1000+。

4.那一本书最好？

我买了3本书：BOC Study Guide 5th Edition；Clinical Laboratory Science Review；Quick Review Cards。感觉最有用的是CLSR，才30多刀，题目多，讲解细，适用面广；BOC骗钱，80多刀真心贵，讲解太少，只用来熟悉题型，它上面的题目是不会出现在正式的考试中的；Cards有些总结的很好，但有些没讲到，毕竟是用来背重点的，覆盖面不广。我自己还下载了很多电子书，但没怎么看，又伤眼睛又没重点。所以我是先快速看一遍BOC，然后结合CARDS和本科的书本，有些网上百度谷歌的东西，重点看CLSR。把重要的知识点做成笔记，到最后考试前把笔记记熟。

5.对英语的要求怎样？

要求很高，一分钟一题的阅读，还有专业词汇，英语不好很吃力。我是先考过雅思，把一般的英语先过了，至少题干阅读没大问题了。然后碰到一个专业词汇就查字典，看到多了就熟悉了。最好不要背专业词典，一是效果差，而是没必要。因为这些词你多看几遍就记住了，在阅读中记的词才能真正记得住。细菌的英语还可以，真菌和寄生虫，哎，我是放弃了。

6.有了这个证书能在美国工作吗？

很不幸，ASCP网站上说了，有证书的人士也必须走Visa Screen才能得到签证，到美国工作。直接以ASCPi证书找美国工作是行不通的。但是随着国内对国际认证越来越重视，如果你有这个证书相信对你的事业发展也有相当的好处。

本文来自：https://labmedguru.wordpress.com/2012/12/24/%E5%8E%9F%E5%88%9B%EF%BC%9A%E5%A6%82%E4%BD%95%E6%8A%A5%E8%80%83ascp/

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实习一共是52周，一整年。周一到周四都是实习。周五去学校上课。实习一年只有五天大的节假日，感恩节，圣诞节，元旦这些，但是赶上是周五了，连这一天假期都没有了。如果有事不能来，理论上是要找另外的时间补上的。不过后来我们老师说可能有几天的缓冲。就是一共少几天没关系。但是我比较谨慎，一天也没有请过假，连有一次感冒也带病去。我的搭档请过几次病假和事假，我知道他没有周末来补上。好像也没事。我只是担心LFS最后批准license的人，万一因为这个时间不够被拒岂不是太悲剧了。所以安全点吧。实习就是在所有的部门轮转一遍，有的一个星期，有的两个星期。最多的是细菌六个星期，血液和血库各有5个星期。应该包括免疫，血液，微生物，化学，血库所
有方面。不过不同的医院或者公司有一些特别的部门，所以在不同的实习单位也会有些不同。实习过的东西学的时候会比较容易理解，容易记住。没有接触过的可能就不容易背出来。我实习的单位四个同学，关系还比较融洽。其他医院的，有很多人有矛盾，经常听说什么闹矛盾之类的。因为搭档都是安排的，不是自己挑的，但是不会跟人相处也说明你有点问题，所以还是尽量不要闹矛盾。听老师说以前有一个人因为和医院里别的人喜欢同一个女孩，吃醋跟别人打架，后来就给开除了。还有去年一个学生实习了一半，出了很严重的车祸，在医院里呆了很久，后来康复了，今年又接着完成剩下的一半实习，已经工作了。

周五的上课，也是免疫，血库，血液，微生物，化学所有的科目都重复一遍，等于是学过的课再复习一遍，以前学过的就容易些。之前没学过的就得费劲一点。之外还有research和management两门课。我以为有research的经验还免修了这一门。不过周五的上课和实习的部门不是同步的，所以两个都得同时学。实习每周或者两周，三周会有些小测验。有的医院比较严格，都是闭卷考试，所以一直很紧张。我实习的单位还比较好，大部分都是开卷考试。所以我这个单位据说是最轻松的。周五上课的也是一个月就有一个科目结束的考试，每次考试前都很紧张。这样每个科目等于上课和实习都学一遍。到了最后几个月，还得自己有计划地复习了，不然最后一个月复习还不够。

我们是六月底实习结束。五月初已经开始申请ASCP考试了。我们是要给ASCP寄评估的成绩单，证明有本科学历。其他的是program的coordinator会给他们寄别的材料。大概五月二十号email通知批准了，可以预约考试时间了。考试是考试中心，是很多考试都在那
里，位置有限，所以还要尽早预约。我约了七月下旬的。还要申请州里的，就是LFS。以
为以前申请过Trainee license，所以他们已经有大部分材料了。所以这次我没有寄任何
材料。只有老师寄这一年的材料就可以了。我六月十号申请的。到七月20号收到合格，可以考试的email，还有一个连接，要做一个quiz。

六月底实习结束，就天天在家复习。考试当天提前到，考试中心还提供计算器，一张纸，一支笔。电脑考试，100道题。两个半小时。结束后一提交，就会告诉你通过了。加州
还有一个十道题的小quiz，是在家自己电脑上就能做，很简单的，是关于加州的Law的。
然后就等着了。

还要提一下找工作的情况。加州这里，这方面比较缺人。实习还没结束，很多人都已经有两个以上Offer了。不少人都已经定了工作。我这个实习单位，所以得学生都有offer。我知道有的地方，比如UCLA，8个学生听说是要了3-4人，剩下几个要自己再找工作。Cedars sinai也是所有实习学生都要了，还有我的搭档也要了。Children’ hospital也比较缺人，也给了我们好多学生offer。工资方面，加州比较高，在$35-42/hour。大医院比较高，但是同时会压力大，工作比较紧张。小的地方会工资低一点，不过可能工作轻松一点。还有就是好的地方大部分得先做夜班，白班有空位才能换过来，想直接白班比较难。工资低的地方就有可能直接做白班。所以自己考虑想要怎么样的生活。我感觉对很多要照顾小孩的妈妈，这样的比较轻松的工作，工资又还可以的，工作内容跟以前生物博后还比较类似，应该算是比较理想了吧。

面向药企的简历撰写终极指南

Tips for a Winning Resume In Sales, R&D, and Management

Today, there are currently an estimated 287,000 people employed by the medical device and pharmaceutical industries, according to figures by the United States Bureau of Labor and Statistics. Bill Lawhorn, a staff economist for the United States Department of Labor at the bureau, says these industries are expected to shrink slightly over the next decade, meaning an increase in competition for these jobs.

Whether you are breaking into pharmaceutical sales, desire a position as a research and development engineer, or you want to move up to management in medical devices, here are some tricks of the trade from experienced resume writers and experts who specialize in these industries. With these tips, you can freshen up your resume like a pro and beat out your competition!

Tips for the Sales Resume

1. Include Your Target Title

Always include the job title or position you want on your resume and bold it. Human resources persons typically scan resumes and this is one of the first things they look for. Include the specific title or position you are targeting such as Sales Representative, Field Representative, or Customer Service Representative rather than a general title. This is particularly important if you are new to the pharmaceutical or medical device industries.

2. Tailor Your Sales Resume

Just as each customer has different needs, so do potential employers. You will need to customize your resume for each and every position that you apply for. That isn’t to say you can’t have a general resume on file as a starting place. You will just need to read the job description of each sales role for which you apply and mold your resume to the specific needs of that position at that company to give yourself the best shot at getting an interview.

3. Nix the Objective

Remove “Objective” as a section title in your resume. The person who reads your resume will know that your objective is to get a job offer from them. This is an old resume style that you should avoid. Instead, start with your “Career Summary.”

4. Lead with Numbers

Melissa Orpen-Tuz, a certified professional resume writer for Great Resumes Fast, says those wanting to enter the medical device or pharmaceutical industries need a different kind of resume. For these industries, specifically in sales, she says you should include solid numbers that demonstrate your performance.

“This is truly different from any other career field,” says Orpen-Tuz. “On a sales resume you want to lead with numbers and quantifiable results to show what you have done.”

Include sales numbers, details of how you have grown the client pool for your current employer, or explain the more extreme lengths you have taken to make a sale such as taking medical training to better know your target industry. This is a place you can really set yourself apart from other candidates.

5. Feature Relevant Classes & Training

The medical device and pharmaceutical industries are very closed, meaning hires from outside the industry are uncommon, according to Debra Boggs, a professional resume writer for Great Resumes Fast. With that in mind, she advises any coursework or medical experience you can include in your resume will be beneficial.

“On your resume, do whatever you can to show your knowledge of the industry or in sales and marketing,” says Boggs. “If you have been a top performer in another industry, you are more likely to have success in the transition. Because they are so selective, they aren’t going to take someone from outside the industry unless they are a high performer. These are the hardest industries to get into.”

Highlight relevant courses you have taken to demonstrate your preparedness and ability for success in sales. This is especially true if you have attended seminars or industry-specific courses that apply to the position you want. Mention any classes that have specifically prepared you for pharmaceutical or medical device sales and how that education translates to an added value for your potential employer.

6. Boast About Your Relationships

In addition to quantifiable results and numbers, Orpen-Tuz says anyone seeking work in the medical device and pharmaceutical industries should also tell stories about how they have gone above and beyond to make a sale and develop long-term relationships with customers.

“It’s also really important to be able to build relationships with highly educated medical clients,” she says of these industries. “Be personable in your resume and show you want to build partnerships with doctors and others in the medical field.”

7. Tell Your Individual Career Story

Every resume is a narrative of a person’s career past and this is where you should flaunt what you have done to add value in your previous roles. Building this picture of you as a strong contributor makes you a better candidate for a sales role. This is where you’re going to highlight certain aspects of these [medical device and pharmaceutical] resumes you may not showcase in other industries, according to Orpen-Tuz.

“Show how you took the opportunity to contribute to a company on a national level, to build consistent sales strategies, and be able to shine as an individual contributor but also as a member of a team,” says Orpen-Tuz, who also emphasizes the importance of training. “Look for ways you contributed to team projects, training, experiences you have had to work across regions. I have done several sales resumes where they have done great in an individual contributor role which then led them to get invited to other high-visibility initiatives within an organization. Also, think about what is motivating you to enter this field so you can articulate that on your resume.”

8. Tout Your Applicable Skills

Sales is a very broad category. Any position where you have had to convince others or even just explain the features or qualities of a product counts as sales experience. Whether you had a retail job in the summer after graduation or have sold other products in the past, these are transferable sales skills.

In addition, where pharmaceutical and medical device sales are concerned, you also need to demonstrate analytical skills so you are able to easily share data and statistics with potential clients and existing customers. Being able to show that you are competent in identifying trends or sorting data is incredibly useful in pharma and medical device sales.

Science skills are also valuable when discussing the science behind the drugs or devices you are selling. Any experience you have had in a lab, working on science projects, or work involving any type of technical element can also give you a leg up.

9. Using Saleable Keywords

For sales resumes, you have to identify the correct key terms to use. I highly recommend focusing on industry-specific hard skills over generalized soft skills as they are more likely to be picked up by an applicant tracking system (ATS).

For example, if you are leaving accounting and are transitioning to pharmaceutical sales, you may wish to use keywords such as sales trends, market share, customer target plan, or deliverables. Research keywords for hard skills you will be employing in pharmaceutical or medical device sales. You can find abundant lists of keywords simply by googling key terms for a sales resume.

Under your position title, include industry-specific key terms that relate directly to your target position and specific skill set. Bold these key terms in italics. For pharmaceutical or medical device sales, you might want to include terms such as:

• Ownership Mentality
• Results-Driven
• Strong Presentation Skills
• Active Listener
• Customer-Focused
• Initiative
• Business Acumen
• Organizational Skills

10. It’s About the Bottom Line

Most employers, especially in medical device and pharmaceutical sales, want to know how hiring you would positively impact their bottom line. While you may not have facts, figures, and metrics that apply directly to the position for which you are applying, you can incorporate such numbers from your current position to demonstrate your value. The general assumption is that if you have been successful in the past, you can be successful in the future. To figure out numbers and facts to include in your resume, ask yourself the following questions:

• How much revenue did I generate this year?
• How did this year’s sales compare to last year’s or last quarter’s?
• Did I cut costs? By how much?
• Did I increase productivity? By how much?
• Did I impact client satisfaction?
• Did I increase efficiency?
• Did I save time? How much?

Consider ways you impacted the bottom line of your company and include that information in the bullet points under each employer on your resume.

11. How Do You Add Value?

Ask yourself the questions below to uncover the benefits you bring to the sales table. Mold the responses into your personal brand:

• What benefit or contribution do you add?
• What key sales accomplishments or successes have you delivered time and time again in your present or past roles?
• What would you say is distinct about yourself and how you do what you do?
• What are your greatest strengths?

12. Details Matter

Hiring managers love details. While your resume offers limited space, you still want to ensure there are details in it that recruiters and human resources personnel want to see. For example, you will want to include your ranking, quotas, call points, and the positions you have previously held. You will also want to include the types of products you have sold such as the names of particular drugs or medical devices. You will also want to mention clients you have worked with in the medical industry especially those with big names such as Johns Hopkins or the Mayo Clinic.

13. Share Your Website & Blog URLs

Be sure to share the URLs for your professional website or personal blog in your resume under your contact information. Websites and blogs are a wonderful means of sharing your work with potential employers. This is especially true if the work relates to the medical device or pharmaceutical industries such as tips for producing better sales numbers or crafting killer emails to garner attention for a product.

14. Keep It Short and Sweet

Orpen-Tuz says you can use as much space as needed to say what you need to say but resumes shouldn’t go beyond two pages long except with executives who may have resumes of up to three pages in length.

“Sales resumes give you the opportunity to be very succinct and less wordy,” she says. “The goal of the resume doesn’t have to describe the totality of what you have done. It’s to get you a face-to-face interview for a specific goal.”

With this in mind, your sales resume should be at least one full page in length.

15. Format and Style Count

When it comes to sales, you want a neatly formatted, clean, and easy-to-read resume. Your resume should be visually appealing. Stick with a basic font such as Arial or Times New Roman. This also ensures your resume works well with an applicant tracking system (ATS).

16. Tap Your Network

While many candidates start with applying through pharmaceutical or medical device company websites, this can be a slow process. A better strategy may be to reach out to people within the company through your existing network or through social media such as LinkedIn. By using your network to obtain the names of contacts where you can send your resume, it gives you an extra push to get your foot in the door that other candidates may not have.

Tips for the Management Resume

17. Lead with Leadership

Leadership skills are important in any management role across all fields but this is especially true in the medical device and pharmaceutical industries where competition for positions is fierce. This is particularly the case for someone transitioning from another industry. Focus your resume on skills that are highly valued in your target industry.

The article The Science of the Job Search, Part I: 13 Data-Backed Ways To Win by TalentWorks states that the use of the right leadership words can boost your resume by more than 51 percent over your competition. The article recommends using words such as:

• Communicated
• Coordinated
• Leadership
• Managed
• Organization

While hard skills take precedence, some soft skills can be added. Think in terms of your leadership ability, especially when seeking a management position, as well as team-building, problem-solving, and other invaluable soft skills. For each of the job experiences you include on your resume, tout your skills and professional accomplishments including numbers. Some soft skills, such as leadership, cross over industry and title. Use them to your advantage in your professional summary.

18. Plot by Numbers

As mentioned above, numbers are worth their weight in gold and are imperative to any management resume. Remember, your resume markets your brand, grabs attention, and demonstrates your abilities and results. It’s imperative you include measurable numbers and data if you want to be considered as a serious candidate for a management position.

For example, if you boosted sales 70 percent, added more than 200 clients, or improved productivity of your department by 1/3 in your last role, that information should be included in your resume. Numbers like these are attention-getters for recruiters who want to see the value you offer even if it’s in a different role or industry.

In the aforementioned piece by TalentWorks, resumes that demonstrate results with numbers were boosted more than 40 percent over competitor resumes that lacked such stats. To get the most bang for your buck, the blog recommends using at least one number every three sentences to demonstrate your positive impact and leadership ability.

19. Match Your Skills

Job candidates should always match their skill set in their resume to each specific job description. Employers know what they are looking for and use those key terms in the job description when they post it. Make sure that when you are adjusting your management resume to fit each position for which you are applying that you employ the words used in the job description. You can even use them more than once.

20. A Specific Career Summary

When applying for a management role, realize your career summary is a space of opportunity. This is where you can really set yourself up to shine which is important given you are looking for a management position in one of the most competitive career fields out there. While many people think of this area on their resume as a career overview, you should use it to set yourself apart from other candidates.

Include specific positions you have held and your most significant accolades. This part of your resume is a career snapshot where you should include prominent client names, major accomplishments, and bottom-line numbers, especially if you are seeking a position in sales management.

“Pharmaceuticals and the medical device industries are very numbers-driven, so any time you can show numbers such as new product launches, market share growth, or quota attainment, those are all really helpful, particularly for sales and marketing,” says Boggs.

You can also feature names of prominent Fortune 500 companies you have worked with. This grabs attention and demonstrates your ability to work with industry leaders, even if it’s in a different industry, as well as your potential to bring in big clients with your former connections.

21. Use Industry-Specific Buzzwords

Most employers now use ATS to sort resumes so using the right keywords is imperative to having your management resume seen by an actual person. Research and select three to five industry-specific key terms to use in your career summary that apply directly to the role of management. According to The Science of the Job Search, Part I: 13 Data-Backed Ways To Win, using industry jargon and buzzwords every three to six sentences can bump your resume over other applicants by more than 29 percent. Look at job postings for industry-specific management terms you can use.

Monster offers a list of key terms to use for management positions in its blog 15 Keywords You Need on Your Executive Resume. Ensure that the key terms you use apply to your individual skill set and the industry to which you are applying. Some of the key terms it recommends for those seeking an executive-level position include:

• Strategic Planning
• Performance Optimization
• Budgeting and Finance
• Crisis Management
• Multi-Site Operations
• Profitability Improvement
• Decision-Making
• Joint Ventures & Alliances
• Consensus Building & Teaming
• Best Practices & Benchmarking

22. Ask Others What They Think

Asking others what they think of you can help you identify and build a personal brand that is authentic. Ask friends and colleagues the questions below and use their responses to help you build a personal brand.

• How would they describe you?
• What do others see as the value you add?
• Read your LinkedIn recommendations and past performance evaluations and look for themes. When you put similar words and phrases together, what picture do you get?
• What do others say are your greatest strengths?
• How do others describe you?
• What do your boss, team, direct reports come to you for on a regular basis?

If you are introverted like I am, questioning others about yourself can create some anxiety. If you are someone who struggles with asking such questions, I highly recommend the Reach Personal Branding Survey. This tool allows you to gather the email addresses of those you wish to request feedback from and the program sends it out. All you have to do is watch the responses roll in. It’s anonymous so people can respond without worrying about your reaction and the survey does all of the work of searching for common themes in your personal brand.

23. Reveal Yourself

Think about the new management role you are targeting. What qualities does the perfect candidate demonstrate? What are their professional attributes? A CFO may be extremely analytical or focused on the bottom line. The best sales manager may challenge the status quo of how to sell a particular product. Think about your own experiences in the work force. In what situations did you display professional attributes and what were the results of your leadership?

By taking this time, you’ll see yourself from the view of a potential employer. These are the very things recruiters and human resource managers will consider when reviewing your management resume. Center your resume on your management career goals rather than simply adding a boring list of tasks others may perform.

24. Cash in on Past Experience

If you are applying for a management position, you obviously have some previous experience that you can capitalize on in your resume. Many employers will be delighted to have someone with demonstrated success in their previous work experience, especially if you can tell your story in a way that applies to the position you want. This is particularly true if you are changing industries. Consider how your past experience applies to the management role you want, focusing on sharing applicable highlights and numbers to set you apart. Any previous experience in biotech, pharmaceuticals, or medical devices will give you a leg up.

25. Show, Don’t Tell

Recruiters, human resources personnel, and hiring managers don’t want to hear about your soft skills. They want to see them in action through your accomplishments in your resume. Rather than writing in your resume that you are a “people person,” explain how your ability to work well with others won you a large contract or gained you additional clients for your current or previous employer.

26. ATS for Everyone

Managers, executives, and C-level applicants often assume that they don’t need to prepare their resume for an ATS. Everyone should have a resume to use with ATS as many companies use them for regulatory compliance with fair hiring practices. This means that despite the level of the position to which you are applying, an ATS scan might still be part of the process.

27. Addressing Abbreviations

When it comes to abbreviations in your resume, just spell them out on first reference. For example, you would spell out “Regional Sales Manager” on first use but could then abbreviate the term to RSM thereafter. This is important as some ATS won’t recognize such abbreviations.

28. Do Your Homework

Before you start working on updating and fine-tuning your resume to a specific position, make sure you have done your research on the company to which you are applying. If you are looking at a medical device or biotechnology company, you need to know about the products that company sells as well as the industry. By knowing what the company produces, sells, and who the target clientele is, you can adjust your resume accordingly and you’ll do much better in the interview.

29. Be Thorough and Succinct

Orpen-Tuz recommends being thorough in your management resume but not using more than two pages unless you are applying for an executive role. If you are applying to an executive management position, your resume may be up to three pages in length.

Do not include your entire work history. Only use the past 10-15 years of your work experience unless something further back is highly relevant to the position you want. A resume is not meant to be a complete summary of your life’s work. Orpen-Tuz says the goal is to get a face-to-face interview where you can go more in depth about who you are as a candidate.

Tips for the Research and Development Resume

30. All the Right Stuff

There are certain sections that should go into any solid resume and this is also the case for research and development. For the field of research and development, which is more scientific in nature, there are sections to include in your resume that may not appear in other industries or for other positions. Your resume should include the following sections and relevant information under each:

• Personal/Contact Information
• Professional/Career Summary
• Education
• Technical Skills/Key Skills
• Publications & Patents
• Leadership Experience
• Awards & Accolades
• Affiliations
• Licenses

31. The Career Summary

Your career summary will go just under your name and contact information on your resume. This is a concise paragraph that focuses on and emphasizes your key skills and accomplishments that are relevant to the position for which you are applying.

32. Key Skills to Kill It

Speaking of bullets, the article The Science of the Job Search, Part I: 13 Data-Backed Ways To Win by TalentWorks highly recommends adding a key skills section since you can’t drop enough industry buzzwords. The piece says adding this section of 15-20 skills using bullets can increase your hireability by more than 58 percent above your competition that fails to include this section in their resume.

Technical skills are incredibly important in research and development. Some key skills you may use in your research and development resume include:

• Ability to handle large datasets
• Perform high-level data analysis
• Analytical thinker
• Critical problem-solver
• Time management
• Independent worker
• Teamwork
• Technophile
• Multi-disciplined
• Understanding of legal and regulatory issues

33. Keywords for R&D

The most successful candidates for research and development, science-related positions, have a comprehensive resume. As someone in research and development, you must demonstrate superior written communication skills and part of that means identifying the right key terms for the title you are pursuing. Some key terms you may consider using, if they apply to you, include:

• Advancements
• Manufacturing
• Scientific Research
• Scalable Design
• Deliverables
• Project-Focused
• Consumer Goods
• Development
• Patented Devices
• Solutions

34. The 30-Second Rule

Professional resume writers often apply what is known as the 30-second rule. The rule states that you have—at most—30 seconds for your resume to grab the attention of the person reading it. That’s why the career summary needs to be compelling and the key skills bullets succinct yet applicable. These are the two sections hiring managers will skim to see if you have what they are looking for. If your resume passes the 30-second test, they will read the remainder of your resume.

35. Check out Online R&D Resumes

There are a ton of sample resumes available online to look at, especially for entry-level scientists in research and development. Review some of these sample resumes and use the portions most relevant to the position you want. Be sure to include the sections mentioned above in “All the Right Stuff,” and leave off “References Available Upon Request” as this is a waste of space. It is assumed by recruiters that you will be able to provide references upon request.

36. Connect Former Work and New Goals

You’ll want to identify aspects of your previous work that relate directly to research and development that you can share in your resume. Topics that translate well across virtually any industry include:

• Communicating with stakeholders to define business requirements
• Responding to business needs with new solutions
• Enhancing operational performance
• Cutting costs
• Improving customer experience and satisfaction

All of these items also relate well to research and development when it comes to meeting the needs of the business, developing new products, and keeping costs low. Think about how you have accomplished these goals in your current or previous roles so you can connect them in your research and development resume.

37. Bullets to Hit the Mark

Make a list of your top achievements in your current and past careers. Include successes such as leading a challenging team of individuals, expanding research, or increasing sales through the development of new products. Recall and enjoy the highlights of your career which have brought you accolades and recognition from others.

Include these in your research and development resume, ensuring you also add the impact your work had outside your company such as honorable mentions, distinctions, or awards given by professional industry organizations. Use bullet points to effectively sum up career highlights. Orpen-Tuz warns that bullets should be no longer than two lines of text and never three.

38. Share Your Achievements

In your R&D resume, share stories of success from some of the bulleted achievements above. Use the Challenge-Action-Result format to describe challenges you faced, the action you took, and most importantly, the results of your efforts.

These stories become the foundation of your scientific resume. These achievements should replace the classic list of tasks performed in prior positions as they provide hiring managers proof of your performance that can translate to your new position and distinguish you from other candidates. If a particular achievement is not applicable to the new role or industry you’re pursuing, leave it out.

39. Highlight Relevant Education

I always recommend listing your education at the top of the resume if a degree or certification is required for the position you want and it has been recently obtained or will soon be completed. This is especially true in research and development where many companies look for R&D scientists who have a degree in genetics, biology, biochemistry, chemistry, or a closely-related field.

Recruiters also search for candidates who have a strong background in next-generation sequencing (NGS) methods, products, and applications in addition to excellent documentation and communication skills so they can effectively present data. Be sure to highlight your educational background so recruiters know you have the necessary training and knowledge for the role you want.

40. Branding with Quantifiable Metrics

Just as numbers are important for grabbing attention in sales and management resumes, they are also a distinguishing factor in research and development. Metrics, facts, and figures are essential to your resume.

When building your personal brand, facts and figures are paramount but can be tricky to identify in scientific terms. Think data loads you have analyzed, an increase in sales due to your product development, and the number of patents to your credit. If you are having trouble identifying metrics or numbers to use in your resume or branding, or even if you feel you have none, read Using Metrics in a Resume When You Have None for help.

41. Talk to People in R&D

Client Services Manager for Great Resumes Fast Chelsea Kerwin recommends talking to people who work in the medical device or pharmaceutical industries in research and development. By doing so, you will learn more about the role you want, the industry you are pursuing, and can identify potential companies to which you may want to apply. She says this is also particularly helpful when building your personal brand for your resume and online profiles. Such conversations may also help you identify keywords and mold a resume to get you where you want to go.

42. A Focused Format

While a great-looking resume won’t get you a job, an unappealing resume can knock you out of the running for an interview. Your resume should look nice and include bullets to make for easy reading where possible. Avoid fancy fonts, borders, or anything that might impede readability. Make sure your resume is sent in an easily read format such as Word. Those sent in a pdf format often cause import issues.

43. A Great Subject Line

When you do send your R&D resume, make the subject line of the email stand out. Find an interesting way to phrase it rather than just “My Resume.” For example, you might send a subject line that reads, “Top 5% Researcher Seeking Position in Chicago.” Using statistics and numbers is just one great way of catching a recruiter’s eye.

Tips for Every Pharmaceutical and Medical Device Resume

44. Your Name Matters Most

The name is the very first thing a recruiter scans on a resume, according to eye-tracking surveys. For better readability and to grab attention make sure your name is:

• In larger print than the rest of your resume
• In bold font
• At the top of your resume and cover letter either left-justified or centered

Some ATS have difficulties “reading” post-nominal titles or abbreviations attached to names such as Ph.D, RSM, and CFO. Leave these qualifications off of your name. To avoid having your resume dismissed by the ATS, simply include them in your career summary or education section.

45. Keys to a Professional Email Address

You may need to create an entirely new email address if your current professional email includes a former title or business name for a different industry. The creation of a professional email address should include your initials and a combination of numbers, or your first name and last name with the addition of numbers if necessary to distinguish your address from the other “John Smiths” out there. Keep it simple.

46. Use Your Address

Recruiters will sometimes check proximity of an applicant when running a search so include your address. If you leave your address off, your resume may not register in an employer database when they search for candidates by location. Zip codes are especially important when it comes to applicant tracking systems (ATS). If you have concerns about privacy, just use your city, state, and zip code on your resume so it can still be found by recruiters using an ATS.

47. Use Your LinkedIn Profile

When it comes to personal branding, Kerwin advises marketing yourself fluidly between your resume and LinkedIn profile. Whether a recruiter or hiring manager is looking at your resume or your online profile, it’s important to deliver a consistent message of who you are professionally and where you want to be.

“Make sure your resume and LinkedIn profile are positioning you for the industry and jobs you want to target now,” says Kerwin.

The fact is recruiters are more likely to connect with candidates who have common connections and LinkedIn automatically shows visitors who you are both linked to. This is a terrific way to get your foot in the door with a medical device or pharmaceutical company, especially if you are venturing into one of these industries for the first time. You can get even more resume tips for changing career fields in the blog 101 Resume Writing Tips for Career Change Resumes.

48. Your Branding Statement

Your branding statement goes at the top of your resume just beneath your contact details where the old objective statement used to be. This statement should be hyper-focused on the position you want, set you apart from other candidates, address the value you offer potential employers, and speak to problems you can solve for them. For example, I always tell people my heart’s desire is to use my 12+ years of HR experience to help job seekers create interview-winning resumes for those who don’t have the time, experience, or expertise.

If you are making a switch into the medical device or pharmaceutical industries, Boggs recommends adding a little narrative in this spot on your resume. The narrative should explain why you are making the transition and what you bring to the position.

For those already in the industry, Boggs says to mention your area of specialty (neurology, biologics, etc.) and any specific information you can give about drugs or technologies you have worked with. She says this is helpful because it makes you attractive to other companies as you already have that background.

49. Ask, “Why Do I..?”

When it comes to personal branding, one of the questions to ask yourself is “Why do I…?” Why do you want to be in a particular industry or position? Why do you do the work you do? If you are having trouble answering this question, or some of the others, I highly recommend Start with Why. This link can help you figure out the why behind what you do. Many people know what they do and how they do it, but not the why. Knowing the why is imperative to formulating a personal brand that will help you achieve your career goals.

50. Job Post Pointers & Making Career Connections

Kerwin recommends looking at sample job posts to learn which qualifications are required for the industry and position you want. She says by looking at job posts for the industry and career you are targeting, you can determine which required skills and experience you have to offer. Incorporate these into your resume and LinkedIn profile in a way that is relevant to the position you are seeking.

Whether you are in a career transition or are just starting out, Orpen-Tuz suggests those seeking a career in the medical device or pharma industries read position descriptions carefully to see where they can draw connections from their prior work experience.

“Researching specific companies and their approach to medical devices and pharma [is a good place to start],” according to Orpen-Tuz. “You may not have the necessary specific requirements but you can see the job connections between what you have done and how that can connect.”

51. Keywords to Capture Attention

Boggs also recommends paying special attention to the keywords in the job description for which you are applying. Keywords that are in the requirements for the job need to be in your resume. Make sure you are tailoring your resume to the job for which you are applying.

To be effective, keywords should be used throughout your resume. In the various sections of your resume from the career summary to your individual work experience, sprinkle in key terms and use the most important ones more than once. As you adjust your resume to each job post, you can easily add in extra keywords to the Key Competencies/Areas of Expertise section which is essentially just a list of keywords anyway.

If you are struggling to develop a branding statement, simply select three keywords related to your personal brand. A thesaurus is a great resource for this as is the Google Keyword Planner. After you have selected your three words, center them across the top of your resume beneath your contact information. I also advise first researching industry-specific key terms to use. Job postings are a great place to start this research.

52. Optimize Your Resume for ATS

The number one thing Boggs recommends when it comes to resumes for the medical device and pharmaceutical industries is to ensure your resume is ATS-optimized. She says nearly every job seeker in these industries will encounter an ATS so your resume needs to be up to snuff.

53. Avoid Double-Duty Resumes

Orpen-Tuz says if you are looking at pharmaceutical or medical devices as a new field, don’t try to use an old resume that does double duty. Be very focused on breaking into the medical devices and pharma idustries with an updated resume specific to the field you wish to enter.

54. Shun Personal Pronouns

When it comes to your resume, words such as “I,” “my,” or “me” should be removed. Recruiters are always pressed for time so they find options to systematically sort resumes and this is one way they do it. A tiny error such as using first-person pronouns can land your resume in the garbage. As a matter of fact, the use of personal pronouns can drop your chances of getting a call back for an interview by more than 54 percent, according to the online article The Science of the Job Search, Part I: 13 Data-Backed Ways To Win by TalentWorks.

55. Make Your Action Verbs Work

Start sentences with action verbs to pump up your resume. Action words demonstrate actively what you have done and how you added value to your past employers and/or industry. The List of 100 Action Verbs is a convenient and helpful reference for incorporating action into your resume. This is especially important when competing in industries where there are more candidates vying for fewer positions such as in the medical device and pharma fields. Utilizing the right verbs demonstrates your personal brand, energy, and value.

56. Avoid Unnecessary Adjectives

“I recommend that you are really, really cautious about using adjectives,” says Orpen-Tuz. “Rather than using adjectives like ‘I am a go-getter, dynamic, etc.,’ use those words that tell a story about a time that exemplified those characteristics.”

Write something more specific using one adjective that describes you but not every other candidate. For example, you might write, “Lead a departmental team of 10 who increased sales 20 percent under my supervision.” A thesaurus is a good resource for finding adjectives beyond the ordinary.

57. Pass on Overused Phrases

In every industry, certain phrases have become tired because recruiters have seen them time and again. These include “team player” and “excellent communicator.” Avoid these overused phrases at all costs. Using the term “team player” reduces your hireability by 51 percent, according to the online piece The Science of the Job Search, Part I: 13 Data-Backed Ways To Win.

Other words I recommend avoiding on a resume that fail to add value include “detail-oriented,” “responsible for,” “successful,” and “results.” Eliminate overused words and phrases in your resume such as “accomplished professional,” “demonstrated excellence,” or “proven ability.” Find other phrases to avoid at 10 Overused Resume Phrases Damaging Your Job Search. This short blog also provides options for replacing such tired old terms.

58. Talk to People in the Industry

Boggs suggests reaching out to professionals who have the job you would like to arrange informational interviews where you can ask for advice. This will also help with introductions to others in the industry you are pursuing before you even start working on your resume.

59. Experience Outside of Work

Kerwin says the experience you share on your resume doesn’t have to be all work-related. Consider including accomplishments outside the workplace such as volunteer work and opportunities you’ve taken outside of your company. When it comes to your job search and resume, all relevant experience counts. Whether it is volunteer, academic, or older experience, include it if it is relatable to the job you want.

60. Gauge Your Graphics

While graphics can add to the appearance of your resume, Orpen-Tuz warns against using too many graphics. She says it is important that graphics support what is in the text.

“I always stress with people that we want something that is clean and easy to read,” she says. “Sometimes flashy is ok but we don’t want formatting flairs to eclipse the content. I personally tend to use those (graphics) with higher-level resumes.”

61. Contemplate Company Culture

Research and consider company culture when crafting your resume. You can research each company on their website and on sites such as Glassdoor as well as on social media including Facebook and LinkedIn.

Once you learn about a company’s culture, adjust your resume accordingly. For example, if the company you wish to work for expects employees to give back to the community, you’ll want to include your volunteer efforts both inside and outside the workplace on your resume. You want a resume that fits with the company for a better shot at getting called for an interview.

62. Downplay Irrelevant Education

If the degree or education you have isn’t required or directly related to the position, put it at the end of your resume. Another reason for placing this information at the end of your resume is if you want to share with the employer that you have some education, but didn’t complete your education. This is not something you want to draw attention to. If you are not planning on finishing your degree or certification, check out the article What Should You Put on Your Resume When You Didn’t Finish your Degree?

63. Formatting Formulas

You may choose to use a chronological or hybrid format for your resume. A chronological resume simply lays out your career history in order starting with your most recent or current position and moving backwards over the last 10 to 15 years.

While the chronological resume is the most common, a hybrid can downplay job-hopping in your resume. This is ideal for those who have switched positions every year for the past several years or for those who have significant employment gaps. Keep the focus on relevant achievements and push the chronology to the end of the resume. While a hybrid format is similar to a standard resume, the section for relevant experience comes after the career summary. You’ll need to be strategic about employment dates and keep your current position brief. Read more about these strategies in 4 Tips to Downplay the Appearance of Job Hopping on Your Resume.

For more resume writing tips on using fonts, design, and formatting, and more, read 131 Resume Writing Tips – The Most Comprehensive List of Resume Writing Tips on the Internet.

64. Review Your Resume

As always, I highly recommend running a spell check and grammar check of your resume. When this is done, give your resume a final read. You want to catch any potentially embarrassing grammar and spelling errors that could take you off the interview list.

找工作，没有合适的推荐人怎么办？

来美国这么多年，一直赖在一个单位，而且和当前老板相看两生厌。想跳槽，又没有合适的推荐人。近期看到这篇文章，有点参考价值：

The conventional sequence for getting a new job in science (or anywhere else) goes like this: 1) Apply for job 2) Get an interview 3) Ace the interview 4) Pray that your references hold up.

So if you had a bad relationship with your last boss, you’re in trouble. Because no matter how well you do at interview their bad reference will sink you. Well, that’s what the conventional wisdom says anyway. But the conventional wisdom is wrong.

If you get to step 2) and are shortlisted for an interview, you already have a good chance of getting the job. And when making decisions after the interview, your potential employer will put more weight on their personal experience of you than on a reference from an unknown person with a potential conflict of interests.

That said, they will still normally ask for a reference. But if you think you can get a bad one from an old boss, there are a number of ways to make sure it doesn’t hurt your prospects. The main thing is to just be honest why you don’t want to ask for a reference from your previous supervisor or PI.

Here are three cases from my experience that illustrate the possibilities.
Case 1 – the PhD Student With the Unhappy Supervisor

I know a PhD student (not his real name), who had a huge row with his supervisor at the end of his postgraduate studies. It was something to do with the author order on a paper, but the details are not important. What is important is that he was able to get a postdoctoral position without a good reference from the unhappy former supervisor.

After the interview he was asked about the references and told the truth, without going into details, that the supervisor was not happy with him. He also mentioned that he could supply references from his second supervisor and his Masters Project advisor.

The interviewing PI was happy to accept his explanation and alternative references. And PhD Student got the job.
Case 2 – the Young Postdoc Who Found Himself in the Wrong Job

An acquaintance of mine, Newly Minted PhD (also not his real name), had papers in good journals and a stellar references from his PhD supervisors. He got a postdoc in one of the best UK universities, the topic looked good on paper, and everything was rosy.

But when we started in the post, he discovered that his new PI was unsupportive and that no-one was willing to help him start working on a new topic. He needed to get out. But – urgh – that would surely incur a bad reference from his new boss, wouldn’t it? Well, no!

He asked his former PhD supervisor what to do and was advised to leave ASAP. He did as his former supervisor advised and instead of asking for a reference from his short-lived boss, Newly Minted PhD used his previous references (from his PhD supervisor and previous tutors) to get another job in a different institution.
Case 3 – the Mature Postdoc Who Just Wanted to Stay in the Same City

A friend, we’ll call him The Mature Post-Doc (you’ve guessed it; not his real name), was desperate to stay in the same city. A half-paid mortgage on his house, children in a good school and other unnecessary luxuries were keeping him there. So in desperation he accepted a job with a bad history; two previous postdocs had left the same job.

Unsurprisingly, he got the job. And just as unsurprisingly, he didn’t get along with the PI and left with the sounds of “you’ll never work again!” ringing in his ears.

Well he did get another job. And he did it without even trying to get a reference from the Angry Supervisor. He just asked for one from his previous long-term employer, and that was good enough for his new boss.
Case 4 – the Post-Doc With a History Who Still Got the Job

If you think those guys above were battling against the odds, then the example of the Post-Doc with a History will knock your socks off! He not only had bad references from his two consecutive previous jobs, but he was actually fired from the second job in a middle of a grant. Ouch.

But did he let his history drag him down? No! He rose from the ashes to grab a new postdoc position, relying only on the references from the time of his PhD.
The Moral of These Stories

The moral of these stories is that you don’t need your previous employer’s reference to get a job. Even as a PhD student you have several people to ask for a reference if you suspect your last employer will be a problem. It could be your tutor, summer project/master/second PhD supervisor, even the internal seminar organizer who has seen you presenting your work and answering questions. Or if you are a bit longer in the tooth, just go back to your last satisfactory employer for a reference.

The important factor is – as it is even if you have the best reference ever – how you come across as a person in the interview.

Cases 2 and 3 give us another important rule of thumb: Do not stay in an obviously dead-end position for more than a year. Everybody understands that not all employer/employee relationships are made in heaven, but if you worked more than a year this makes people (including you) wonder why you didn’t quit if it was really that bad.

Case 4 gives us all hope. If he was able to find a job with two strikes and current horrible job market, I am sure you can do it too!

一个Ph.D的转行经验

1. 前言

拿到了人生中第一份工作offer！从无到有，从编程小白到数据科学。希望自己的经历能够给其他正在探索职业发展的同学们一点点启发。

2. 探索

哥大的生物科学博士项目是一个综合的科研项目，项目内各个实验室的方向相差甚远，有做传统分子生物学，细胞生物学的，也有做计算生物，神经科学，和生物物理的。我所在的实验室是一个神经科学和物理的交叉领域，既有前沿神经生物的实验训练，也有少量的物理建模和计算。虽然我一直对神经科学的科研非常感兴趣，也很想继续在科学前沿进行探索，但是神经科学这个领域的竞争空前惨烈，大牛众多，跟很多博士后聊了之后，发现自己并不适合目前这样的科研环境，对一年到头写科研经费和管理学生也没有太多兴趣。作为一个自然科学的博士生，面对学术界越来越激烈的竞争环境，越来越意识到自己一来科研能力并不出众，二来这些年的科研进展也不算顺利，三来也是读书二十多年读得太腻，于是毅然决然地决定毕业之后不继续在学术界做科研了。

身边大多数博士转行无非是这几条路。1）药厂，2）教书，3）金融，4）编程，5）科学交流，6）咨询，7） 其他

2.1 药厂

大多数生物专业的学生去了药厂，前几年还是要做实验的，虽然可能工作时间和收入比做博士后好一些，但是我是不想再做周期很长的实验了。而更多的药厂职位，倾向于经验丰富的博士后，而非刚毕业的博士，这就意味着想要进药厂工作，还是绕不过博士后的几年。了解了就业行情之后，就放弃了药厂这条路。

2.2 教书

身边确实是有博士毕业之后去高中或者大学做讲师的，不过一方面我个人并不是特别喜欢教书育人，另一方面要想成为老师，也要做很多准备，比如积累教书经验，这对于在实验室长时间工作的博士生可能并没有那么容易。我曾经在博士二年级的时候去做了一个学期纽约公立小学的志愿讲师，虽然小朋友们都很可爱，但是我觉得还是更喜欢一些更前沿更具有探索性质的工作。于是也就放弃了教书这条路。

2.3 金融

略微了解之后发现并没有直觉上的好感，那些金融词汇听上去就像是另一种语言。试着看了看CFA（特许金融分析师）的书，也并没有觉得特别喜欢，大概是因为从小就没有被培养出财务管理的技能。

其他与金融投资相关的行业，比如行业研究员（equity research），可以用上一些自己的专业知识进行股票分析，但是听说工作时间特别长，而且压力也不小。更重要的是，这些行业分析和投资理财顾问更倾向于经验丰富的人，比如已经干了几年的管理咨询。毫无经验的菜鸟一开始就做这个，机会也不是很多。

所以很快就放弃了金融这条路。

2.4 编程

我第一次接触编程是本科时期的C语言，当时觉得简直就是在听天书，什么二进制、指针、二叉树，完全不理解它们存在的意义，于是觉得自己大概并没有跟计算机打交道的天分。这令人不开心的第一次接触，让我一直觉得编程语言都非常难学，并且很难用得上。在PhD阶段也几度打起精神学习编程，但是都很不幸地半途而废。先是学完了一遍Google的Python教程(https://developers.google.com/edu/python/)，感觉很容易上手，却还是觉得什么都不会，不觉得这些基础的内容在自己的工作中会有什么用处，也不知道学会这个能干什么。自己科研中最多也就是用MATLAB处理一些图像，并没有专业系统的编程训练。编程什么的就这么搁置了下来。几年前隔壁化学实验室一个博士师兄自学编程，然后去了华尔街做金融量化分析师（quant），他当时还鼓励我可以考虑一下做金融工程，说是只要会编程数学好，金融专业知识不懂也可以做。我看了一下他的C++教科书，真是一句话都看不懂，马上就放弃了。而传统意义上的编程工作，基本就是软件工程师了。身边有一些博士转软件工程师然后去了Google的例子，不过大多都是在博士毕业之后又读了一个计算机的硕士，或者博士没读完就中途转了计算机的硕士，总而言之，都是受过专业训练的。而完全靠自学转软件工程师的真的是少之又少。于是我还一度想着要不要申一下哥大的计算机硕士专业，但是看到高额的学费，想到还要再考一次入学考试GRE，就打了退堂鼓。

2.5 科学交流

如果对写作特别有兴趣，也可以考虑进行科学交流(scientific communication)。身边已经有不少PhD成功转行到科学交流行业，从事科学写作。他们的工作内容跟科研紧密联系，虽然不用自己做实验，但是还是需要经常阅读科学文献，然后写综述，做PPT。需要的不仅仅是写作能力和技巧，还有信息搜集和概括的能力，以及把复杂的科学概念说清楚的沟通能力。我对于写作本身虽然有很大的业余爱好，但是如果是当成工作，大概也谈不上喜欢。在跟已经转行科学交流的学长学姐交流之后，作罢。

2.6 咨询

从第一次听说咨询（大概是博士二年级），到在Coursera上学习经济学、战略分析，到选修哥大商学院的课，参加一些社交活动，到2016年加入哥大咨询俱乐部，开始申请咨询的工作。咨询是我前期投入非常大的方向。身边有很多中国同学都拿到了麦肯锡或者BCG在中国办公室的工作，而想要留在美国从事咨询工作却鲜有成功的案例。咨询业的职位需求本来就不多，在国内在美国的竞争都非常激烈，毕竟咨询已经成为很多博士转行的默认选项，特别是top N的那些学校，也就是咨询公司常说的target school。非target的学校申请难度要更大。美国公司能够提供工作签证的就更少，想要在美国工作不仅要英语特别好，主要看气质。而所谓的咨询气质是什么，准备过咨询的同学应该都能理解，无法言传。

我在面试的过程中，发现自己的性格跟咨询业的整体风格和工作模式并不是很合得来，特别是在穿正装分析商业案例的时候，心中总是有很强的表演错觉，感觉那个人并不是真正的我。至于咨询业强调的领导力和解决问题的能力，确实很难客观地去衡量，以至于被拒都不知道为何被拒。有些人觉得咨询不用怎么准备，target school的同学练两个月case就可以面试了。然而，借用我一个朋友的话，“咨询确实不怎么需要学习hard skill，但是要重新学习做人。”技术可以学习，但气质和性格却是经年累月的。所以，咨询公司在面试时，case做得及格之后，性格和眼缘非常重要。关于hard skill和soft skill，我后来才发现，不只是咨询，很多行业包括数据科学都是如此。hard skill决定入门资格，而soft skill决定入门之后能走多远。

在申请咨询的暑假项目失败之后，不甘心的我去哥大的职业教育中心CCE做了MBTI性格测试，当然我之前也在网上测过自己的性格，CCE测出来的结果跟我在网上测出来的一样，更适合从事运筹工程之类目的明确，行动清晰的工作，所以我大概并不适合做咨询吧。于是，由于种种原因，摩拳擦掌近两年的我，在经历了无数难以入睡的焦虑夜晚之后，在2016年6月即将申请的时候，放弃了咨询这条路。

而这个时候，已经博士5年级的我马上就要迎来博士的第6年了。我还是不知道自己想要做什么。

2.7 其他

之所以说是其他，因为PhD转行的方向非常多，身边也有成功的例子。有去读法学院JD的，有去做专利的，有去医学院读MD的，有去医学院读医师PA的，有去做学校行政工作的，当然还有创业的，等等。我或多或少都了解了一些可能的方向，也都或多或少地谈不上喜欢，也不觉得自己能坚持下来。比如法学院，一想到要考LSAT入学考试，要回到啃书的日子，就觉得压力太大。

3. 发现

本科同一个实验室的师姐，神经科学博士毕业之后去了Facebook做数据科学家，跟她聊了之后发现这个工作非常有趣，用到一些编程，也用到一些数据分析，还需要解决问题和思考。在跟其他师兄师姐的聊天中，我逐渐了解了数据科学(data science)这个行业，也逐渐发现自己似乎找到了自己喜欢的方向。在网上（知乎，一亩三分地BBS，Quora）读了很多介绍数据科学的文章和如何准备之后，我踏上了数据科学之路。

4. 准备

说干就干。2016年的6月底，我开始了转行之路。

我个人比较推荐通过系统的课程学习数据科学，而不是速成或者刷题，不是看几页别人总结的cheat sheet，也不是跟着大牛们做几个kaggle项目刷个名次。系统学习的时间虽然很长，也可能学着后面忘了前面，但从知识掌握的角度，自己一步一步慢慢学，一边学一边琢磨，然后自己融会贯通自己进行总结，掌握基本的理论和概念，知其然也知其所以然。这样之后再去刷题，参考别人的cheat sheet，看kaggle上别人的项目流程和思路，不仅事半功倍，也能够更好地理解数据分析。说到底，数据科学是门硬功夫，需要持续不断地学习新知识，反思旧概念。如果不能付出足够的时间和汗水，而只看到所谓数据科学的光鲜外表，可能很难转行成功吧。

因为需要投入很多 (平时晚上和周末基本都要学习)，所以决定需谨慎。

4.1 编程

编程的乐趣在于搭建项目，这样一来有成就感，二来有可视化的结果。

4.1.1 Python入门(1-2月）

对于零基础或者有一些基础但没有系统训练的人来说，Udacity的Python课程非常有帮助。

1. Intro to Computer Science
   • https://www.udacity.com/course/intro-to-computer-science–cs101
   • 非常浅显易懂的入门课程，有大量的上手编程训练，最后的项目也非常有趣。
2. Programming Foundations with Python
   • https://www.udacity.com/course/programming-foundations-with-python–ud036
   • 涉及到class的构建和使用，调用函数

4.1.2 数据结构和算法（2-3月）

软件工程师的面试算法和数据结构考的比较多，而数据分析的职位大多数并不在意算法，但是有一些公司还是会问简单的算法，比如二叉树和搜索算法。

1. Design of Computer Programs
   • https://www.udacity.com/course/design-of-computer-programs–cs212
   • 难度中等，对于已经学了上面两门课有Python基础的人来说，是非常不错的算法入门课。特别是从来没有接触过算法的人，课上的例子都非常贴近生活，妙趣横生
2. Essential Data Structures in C/C++
   • http://www.cs.columbia.edu/~jae/3136/
   • 这是哥大的一门课，老师Jae是个非常非常有趣的人。对于从来没有接触过C++也没有系统学过数据结构和算法的人非常有用。
3. Introduction to Algorithms
   • https://ocw.mit.edu/courses/electrical-engineering-and-computer-science/6-006-introduction-to-algorithms-fall-2011/
   • 这是MIT公开课的算法课。网上有很多算法课的资源，只有好好学完一门算法课，就可以了。
4. LeetCode
   • https://leetcode.com/
   • 学完基本的算法之后，可以用这个来练一下手。

4.2 数据分析

在网上查了一圈之后，发现还是Udacity的课程比较全面，而且是项目导向，也就是说每上完一门课，就可以做一个项目。我最终选择的是Data Analyst Nanodegree (https://www.udacity.com/course/data-analyst-nanodegree–nd002)。

这个Nanodegree的课程包括以下几个领域。所有的课程都可以在Udacity上免费学习。而Nanodegree的好处是有人进行远程指导和项目反馈。

• Statistics
• Intro to Data Analysis
• Data Wrangling with MongoDB
• SQL For Data Analysis
• Data Analysis with R
• Intro to Machine Learning
• Data Visualization and D3.js
• A/B Testing

全部课程学习完成，并且把作业的大项目做完，基本要4-6个月。我个人非常推荐。

当然，nanodegree价格也不算便宜（跟学校的学分比起来也不算太贵）。是否值得投资，全看个人喜好。我跟Udacity也没有任何利益关系，只是分享下自己的学习经验。

4.2.1 概率和统计（~1月）

偏理论。Udacity的这几门课非常基础，适合入门。当然很多人本科已经修过概率和统计，所以看视频只是巩固一下知识，顺便了解一下一些专业的单词英文怎么说。

• Intro to Statistics
  • https://www.udacity.com/course/intro-to-statistics–st101
• Intro to Descriptive Statistics
  • https://www.udacity.com/course/intro-to-descriptive-statistics–ud827
• Intro to Inferential Statistics
  • https://www.udacity.com/course/intro-to-inferential-statistics–ud201
• Time Series Forecasting
  • https://www.udacity.com/course/time-series-forecasting–ud980

这些课可以应付大多数的面试，而少数公司比如Google（参看glassdoor和一亩三分地的面试经验），对统计知识的要求更为深入。

YouTube的这个英国助教的A full course in econometrics，一共有271个视频，我觉得讲得深入浅出，特别对于没有上过研究生统计课的人，帮助非常大。里面涉及regression assumption和time series的部分，在我面试的时候就用到了。

https://www.youtube.com/user/SpartacanUsuals/playlists

4.2.2 A/B testing （~2周）

根据公司不同，一些职位可能会要求懂什么是A/B test，怎么设计实验。这对于实验出身的博士并不是很难理解，但是涉及到一些网上实验的细节和统计的计算，还是需要多加练习。Udacity这门课是由Google的员工讲授，获益匪浅。https://www.udacity.com/course/ab-testing–ud257

4.2.3 Numpy & Pandas（2-4周）

如果使用Python进行数据分析，那么Numpy和Pandas这两个函数包是必不可少的。

Udacity的Intro to Data Analysis讲了如何使用这两个包，非常好学，好用。https://www.udacity.com/course/intro-to-data-analysis–ud170

4.2.4 R（2-4周）

一般来说，只要熟练掌握一门语言Python，就可以申请大多数数据科学的职位。之所以把R单独列出来，是因为R有很多统计相关的函数和包，在调用的时候比较方便。

Nanodegree里Data Analysis with R是非常不错的R入门教程。https://www.udacity.com/course/data-analysis-with-r–ud651

这门课只是介绍了基本的作图和探索性数据分析EDA，而不涉及其他的R的功能。所以如果想要深入学习R的话，还是要自己多加练习。

4.2.5 SQL（2-4周）

同样的，Nanodegree里Data Wrangling with MongoDB是非常不错的SQL入门教程。https://www.udacity.com/course/data-wrangling-with-mongodb–ud032

除此之外，SQL zoo也是很好的练习资源。http://sqlzoo.net/wiki/SQL_Tutorial

4.2.6 大数据（1-2 月）

有些职位可能会要求有大数据处理的经验。

• Intro to Hadoop and MapReduce
  • https://www.udacity.com/course/intro-to-hadoop-and-mapreduce–ud617
  • 很基础的Hadoop介绍和应用
• Udemy有一些Hadoop的课也可以看一看。
• 其他类似于Spark这些技能如果找的职位需要就学，不需要的话可以入职之后需要再学。

4.2.7 机器学习（1-2月）

Udacity的机器学习课程偏操作，对于理论和计算的要求不高。而Coursera的课更偏理论的推导和数学。两门课同时学习，效果更佳。

• Intro to Machine Learning
  • https://www.udacity.com/course/intro-to-machine-learning–ud120
• Machine Learning
  • https://www.coursera.org/learn/machine-learning

4.2.8 人工智能（2-6月）

大多数数据科学的职位并不要求会人工智能，但是如果需要或者感兴趣的话，可以自学。

• Intro to Artificial Intelligence
  • https://www.udacity.com/course/intro-to-artificial-intelligence–cs271
  • 强烈推荐的AI入门课
• Deep Learning
  • Udacity有自己的AI nanodegree，课程设计和内容都很充实，但是价格略贵，个人感觉性价比一般。
  • Coursera的deep learning听说评价不错，我还没有上过https://www.coursera.org/specializations/deep-learning

4.3 商业思维

根据职位不同，有些数据科学的面试会有类似于咨询的案例分析，而有些则只是强调编程和数据分析技能的匹配。

强烈推荐咨询的案例面试。比如Victor Cheng的免费视频https://www.caseinterview.com/ 对于理解如何站在商业角度看问题，还是很有启示的。由于大多数案例分析没有所谓的标准答案，表达能力和思考问题的角度就非常重要了。

举两个例子：

1. 问：如何预测谷歌引擎2020年在比利时的搜索量？因为搜索量直接决定公司需要购买的服务器数量，所以预测很重要。

思路：这是很明显的时间序列预测问题。建几个time series prediction model 不难，但在涉及到任何实际数据分析之前，最好先进行案例分析。从大局着眼，分析问题，提出框架(MECE思想, mutually exclusive collectively exhaustive)，最后再建模。毕竟建模简单，关键是思考的过程。以下是一些可能的思路。

1) customer 我们的用户是谁，市场多大，近些年趋势如何，客户用什么设备搜索(电脑，手机，ipad)。每月每周每天趋势如何，是否具有季节性或周末高峰？

2) competitor，其他搜索引擎。

3) company, product, cost。如果搜索有周期性，多余的服务器资源如何利用？租赁或公司内其他的服务？

4) 建模。estimate range，上面的因素如何影响到各个参数的设定。是overestimate还是underestimate更糟？为什么？

总结：案例分析是个讨论交流的过程，一定要避免个人独白。建模是最后一步，在此之前，还是要多多分析。

2. 问：如何提高某保单的销售额(revenue)？

思路：revenue在咨询中是个很经典的问题。准备过咨询的人可能可以很快地列出框架，没有类似经验的则可能想到哪儿说到哪儿，一会说要降价，一会又说要促销，一会又说要增加销售渠道。但从分析问题的框架来看，MECE思想，可以大体分为内因外因。

1) 外：该类保单市场多大？趋势如何？主要竞争者是谁？客户是谁？客户年龄收入等状况(customer segmentation)？价格敏感度如何？

2)内：该保单是啥？车还是人？在公司占比多少？是主打产品还是副线？该产品特色如何？是价格还是品质？与其他保单是否冲突？

3) 要提高销售额，无非是提高价格或者提高销量。如果该产品价格敏感度低，而且属于独特的刚性需求产品，可以提价。如果该产品是generic，也可以通过降价提高销量，但要考虑是否有其他竞争者的价格战。也可以针对不同客户进行不同定价。

4) 行业相关。这里保单是保险业，如果对保险业或者任何面试的行业不熟悉，可以直接问面试官，保单的销售额是如何计算的？是保费投保人越多越好，还是说保费减去保险公司理赔的数量，算差价。这样就需要进行风险评估定价。因为我并没有深入了解过保险业，所以也不清楚这个行业相关的知识。但是如果你拿到了保险公司的面试，提前了解该行业的基本情况，特别是盈利模式，对面试很有帮助。

总结：如果实在不懂，一定要问面试官。在进行任何建模和数据分析之前，一定要确认自己理解对了问题。不然就南辕北辙了。

此外，大多数博士并没有什么商业的实践经历，如果有可能，可以参加一下data hackathon或者case competition，了解一下思考问题的一些方法。

5. 做项目

除了Udacity的项目，也可以自己在Kaggle（https://www.kaggle.com/）上做项目。总之是要有内容可以写，面试的时候可以聊。

订阅一些数据科学的邮件，比如https://www.datascienceweekly.org/，里面经常有其他人做的项目，可以借鉴一下。

如果有时间，可以参加数据科学领域的线下聚会https://www.meetup.com/,我就在一次线下聚会的时候遇到了神经科学转行的同志。

项目是简历的重中之重。

然而，我个人不建议在基础没有打好的情况下去刷项目，因为那样很容易陷入调参的大坑。毕竟有时候机器学习是玄学，参数模型选对了，准确率刷得很高。然而，从知识的角度，只刷项目并不能很深入地理解。在学习课程之后，再有针对地刷几个项目，了解工作流程和思路，比不停调参有意义得多。

6. 简历

网上和学校的职业教育中心已经有很多教如何写简历的攻略了(https://www.careereducation.columbia.edu/topics/resumes-cvs)，Udacity的Nanodegree也有教人写简历的课程和反馈。这里就强调一下可能会被忽略的几点。

1. 格式
   • 一页PDF。
   • 避免用花哨的格式，比如竖列的格式，看起来虽然新奇，但是确实没什么大用。
   • 避免用图标和新奇的字体，避免在电脑自动筛选简历的时候无法识别格式。
2.  Summary
   • 大多数应届毕业生并没有太多的东西可写，也很难通过自己的经历体现自己的优势。在一位职场达人学姐的指导下，我在简历最前面加了Summary的部分，针对所申工作的要求，高度概括总结自己的优势和经历。这样在HR筛简历的时候，可以一目了然地看到优点。
3. Projects
   • 量化。数据量有多大? 用了多少个特征？用的是什么具体的模型？结果提高了多少？改变了什么？
   • 独立项目还是合作项目？个人做的内容是什么？
   • 细节越多，越独特，也越能够吸引人。如果写得很宽泛，就显得缺乏个人的思考。

7. 软实力

7.1 沟通能力

推荐一本书吧，卡耐基的how to win friends and influence people。

https://www.amazon.com/How-Win-Friends-Influence-People/dp/0671027034

不少人说这是一本鸡汤软文，充斥着传销一般的励志故事……我之前也是这么想的，所以一直不想读。去年一个我认为人际能力很好的朋友推荐我去读，他表示自己刚来美国的时候也是没啥朋友，后来就按照书里的一些建议改变自己的行动，刚开始就是跟人尬聊，碰上路人或者街边的流浪汉都尬聊，给予对方最大的注意力和兴趣，后来逐渐就自然起来。我读了之后，真的是获益良多。最大的收获是，大多数人做决定，并不是100%理智的，相当多的时候，瞬间的决定取决于当时的情势，自己的喜好，和运气。人性并不是我们想象中那么直截了当，不是跟数学公式一样精准科学。

 

7.2 英语

交流起来应该没啥问题，口音的话推荐一下American Accent Training 里面讲了很多中国人发音的问题，美式英语的特点。如果对自己的口音很在意，可以用这本书短时间强化一下。

https://www.amazon.com/American-Accent-Training-Audio-CDs/dp/1438071655

8. 申请

8.1 投简历

大概10个月-2年之后（取决于个人的进展），就可以投简历找工作了。

2018年的暑假实习基本2017年的8月就有公司开始招人了，一直持续到2018年年初，取决于公司的招人情况。而全职工作很多公司都是全年招人，应届毕业生的招收主要集中在校内的秋招或者春招，把握好时机。

如果有可能一定要投实习，积累业界经验，这样在找全职工作的时候也更加有所准备。如果实在没办法实习，那就尽量做一些项目。在投简历的时候尽量内推，增加自己获得面试的几率。不管是通过LinkedIn直接找校友，还是在论坛上找人帮着递简历，在不惹人烦的情况下多多联系。

当然，很多公司可能还是会自己网上海投。如果可能，最好能够联系公司里的员工出来喝茶进行informational interview，了解一下公司情况。海投的话，建议广撒网，反正投简历不收费。

8.2 培训项目

除了投工作的简历，还可以申请一些免费的培训项目，这些项目基本都是针对转行的PhD，进行3-4个月的高强度培训，做项目，然后跟其他公司直接面试。

• Insight Data Science
  • http://insightdatascience.com/
  • 我所接触的Insight fellow对项目的评价都很高，也基本都通过Insight找到工作了。当时我同时拿了实习公司和Insight的offer，因为考虑到实习可能有return offer以及是真实的工作环境，最终并没有参加Insight的培训。
  • 我个人的面试经历是，Insight确实是要求申请人本身有一定的编程和数据处理经验，最好已经会用Python，R，或者MATLAB。除了PhD的项目，申请人最好有自己的小项目，比如很多人做过的twitter自然语言分析，纽约citi bike之类的。如果只是写自己PhD阶段处理过的数据和项目，其实并不是特别充实，也不太容易过简历这一关。
  • 面试的时候，需要申请人自己讲述一个数据处理的项目。这个项目不一定要非常复杂，或者非常高深。我之前有听别人说一定要有很炫酷的机器学习或者自然语言之类，但根据我个人的经验来看，Insight更看重申请者个人对数据本身的热情和对问题的思考，特别是表达能力。如何把一个项目讲得绘声绘色，让听众也能够很感兴趣，非常engaging，如何把一些复杂的编程语言说清楚，如何解释自己编程的逻辑（比如为什么选择决策树，而不是其他机器学习的模型）。如果只是进行一个简单粗暴的grid search，用TensorFlow建个神经网络跑几百个epoch，却不解释清楚“为什么”选择一个metrics，“为什么”要多次抽样，甚至于“为什么”对这个问题感兴趣，那么可能即使你的机器学习项目最后的准确率非常高，但也很难过得了面试。
  • 我当时把面试的项目整理成三篇博客（http://www.juyang.co/shared-ride-efficiency-data-wrangling/），项目基本是数据探索和解释，并没有预测分析和机器学习。在面试的时候不仅给面试官展示了Python代码，也讲述了一个完整的故事，得到了一个比较有用的结论，申请的同学们可以作为一个参考。
• Data Incubator
  • https://www.thedataincubator.com/
  • 在Insight之前，我也申请了这个项目，但在code challenge环节被拒。后来想想被拒的原因非常明显，我的项目做得不好。就如我上面所说，我本以为一定要做炫酷的自然语言分析啊机器学习啊时间序列分析啊神经网络啊，这些看上去很“高端”的项目，却忽略了自己本身的技术和专长，以及时间和资源。俗话说，没有金刚钻，别揽瓷器活。我当时想做的项目是用自然语言分析纽约地区Yelp上的餐厅review，从而给用户推荐个人化的餐厅。这个想法可能是好的，但是项目工程太大，而且数据并不能简单获得，Yelp的API限制下载流量，所以我花了大半天才下好数据，然后发现这个API并不提供完整的review，而只是一句话简介。而我本身对自然语言（NLP）的了解只是停留在知道怎么用的阶段，并没有深入的理论学习，在分析数据的时候并不能很快地抓住重点。最后，因为时间有限加上数据不完整，这个项目我做得非常仓促。不过在Data Incubator失利之后，我吸取了教训，好好准备了Insight的项目。

投完简历就是面试了！这个时候的你，对数据科学行业有了基本的了解，自己的技能树也打好了基础。面试就是另一个阶段的事情了，以后有机会再写。

9. 结语之前

据我有限的道听途说，今年data scientist招人基本都是跳槽的experienced hire，新人大多要求博士学位。之前跟一些统计硕士聊过，他们抱怨说自己专业对口，花了几年时间专门学统计，难道还比不过那些个从物理生化机械转行的博士么？而现实是，除了某些打着data scientist名号把data scientist和data analyst放在一块招的，大多数data scientist真的倾向于博士，哪怕专业不那么对口。至于原因，问过一些面试的人，大概是说data scientist整体因为算是个不太成熟的领域，很多东西都要自己去探索，去research，自己去想idea，然后设计实验，分析结果，解释不清不白的数据，还要做ppt报告，几年博士的训练在广义的科研能力上可能更加适合，特别是在失败和不顺时候的韧性。当然就不提大多数博士年龄比硕士大，所以在人情世故上可能更通达一些。至于知识啥的，他们觉得你博士都读下来了，智商应该够，啥都可以以后再学。所以我其实觉得quit phd可能是比较冒险的行为，如果可以的话，最好是一边读phd，一边学习转行技能。说到底，学历还是很重要的。

在市场竞争激烈的现在，申请的时候别管工作名称，data scientist和data analyst，business intelligence，如果编程算法能力不错的话也可以申一下engineer职位。总之能申的都先申了，入门再说。与其抱着不切实际的理想，不如脚踏实地地从头做起。

10. 结语

感谢这一路上帮助我的家人、导师、朋友、同学、同事，以及在我焦虑崩溃的找工作过程中坚持不懈打Dota2并一直陪伴鼓励我的PT。

本文来自：http://www.juyang.co/phd%E8%BD%AC%E8%A1%8C%E4%B9%8B%E8%B7%AF/