Into The Great Wide Open

We have a new paper out! You can read it here.

I thought I would write a post on how this paper came to be and also about our first proper experience with preprinting.

Title of the paper: Non-specificity of Pitstop 2 in clathrin-mediated endocytosis.

In a nutshell: we show that Pitstop 2, a supposedly selective clathrin inhibitor acts in a non-specific way to inhibit endocytosis.

Authors: Anna Willox, who was a postdoc in the lab from 2008-2012, did the flow cytometry measurements and Yasmina Sahraoui who was a summer student in my lab, did the binding experiments. And me.

Background: The description of “pitstops” – small molecules that inhibit clathrin-mediated endocytosis – back in 2011 in Cell was heralded as a major step-forward in cell biology. And it really would be a breakthrough if we had ways to selectively switch off clathrin-mediated endocytosis. Lots of nasty things gain entry into cells by hijacking this pathway, including viruses such as HIV and so if we could stop viral entry this could prevent cellular infection. Plus, these reagents would be really handy in the lab for cell biologists.

The rationale for designing the pitstop inhibitors was that they should block the interaction between clathrin and adaptor proteins. Adaptors are the proteins that recognise the membrane and cargo to be internalised – clathrin itself cannot do this. So if we can stop clathrin from binding adaptors there should be no internalisation – job done! Now, in 2000 or so, we thought that clathrin binds to adaptors via a single site on its N-terminal domain. This information was used in the drug screen that identified pitstops. The problem is that, since 2000, we have found that there are four sites on the N-terminal domain of clathrin that can each mediate endocytosis. So blocking one of these sites with a drug, would do nothing. Despite this, pitstop compounds, which were shown to have a selectivity for one site on the N-terminal domain of clathrin, blocked endocytosis. People in the field scratched their hands at how this is possible.

A damning paper was published in 2012 from Julie Donaldson’s lab showing that pitstops inhibit clathrin-independent endocytosis as well as clathrin-mediated endocytosis. Apparently, the compounds affect the plasma membrane and so all internalisation is inhibited. Many people thought this was the last that we would hear about these compounds. After all, these drugs need to be highly selective to be any use in the lab let alone in the clinic.

Our work: we had our own negative results using these compounds, sitting on our server, unpublished. Back in February 2011, while the Pitstop paper was under revision, the authors of that study sent some of these compounds to us in the hope that we could use these compounds to study clathrin on the mitotic spindle. The drugs did not affect clathrin binding to the spindle (although they probably should have done) and this prompted us to check whether the compounds were working – they had been shipped all the way from Australia so maybe something had gone wrong. We tested for inhibition of clathrin-mediated endocytosis and they worked really well.

At the time we were testing the function of each of the four interaction sites on clathrin in endocytosis, so we added Pitstop 2 to our experiments to test for specificity. We found  that Pitstop 2 inhibits clathrin-mediated endocytosis even when the site where Pitstops are supposed to bind, has been mutated! The picture shows that the compound (pink) binds where sequences from adaptors can bind. Mutation of this site doesn’t affect endocytosis, because clathrin can use any three of the other four sites. Yet Pitstop blocks endocytosis mediated by this mutant, so it must act elsewhere, non-specifically.

So the compounds were not as specific as claimed, but what could we do with this information? There didn’t seem enough to publish and I didn’t want people in the lab working on this as it would take time and energy away from other projects. Especially when debunking other people’s work is such a thankless task (why this is the case, is for another post). The Dutta & Donaldson paper then came out, which was far more extensive than our results and so we moved on.

What changed?

A few things prompted me to write this work up. Not least, Yasmina had since shown that our mutations were sufficient to prevent AP-2 binding to clathrin. This result filled a hole in our work. These things were:

  1. People continuing to use pitstops in published work, without acknowledging that they may act non-specifically. The turning point was this paper, which was critical of the Dutta & Donaldson work.
  2. People outside of the field using these compounds without realising their drawbacks.
  3. AbCam selling this compound and the thought of other scientists buying it and using it on the basis of the original paper made me feel very guilty that we had not published our findings.
  4. It kept getting easier and easier to publish “negative results”. Journals such as Biology Open from Company of Biologists or PLoS ONE and preprint servers (see below) make this very easy.

Finally, it was a twitter conversation with Jim Woodgett convinced me that, when I had the time, I would write it up.

To which, he replied:

I added an acknowledgement to him in our paper! So that, together with the launch of bioRxiv, convinced me to get the paper online.

The Preprinting Experience

This paper was our first proper preprint. We had put an accepted version of our eLife paper on bioRxiv before it came out in print at eLife, but that doesn’t really count. For full disclosure, I am an affiliate of bioRxiv.

The preprint went up on 13th February and we submitted it straight to Biology Open the next day. I had to check with the Journal that it was OK to submit a deposited paper. At the time they didn’t have a preprint policy (although I knew that David Stephens had submitted his preprinted paper there and he told me their policy was about to change). Biology Open now accept preprinted papers – you can check which journals do and which ones don’t here.

My idea was that I just wanted to get the information into the public domain as fast as possible. The upshot was, I wasn’t so bothered about getting feedback on the manuscript. For those that don’t know: the idea is that you deposit your paper, get feedback, improve your paper then submit it for publication. In the end I did get some feedback via email (not on the bioRxiv comments section), and I was able to incorporate those changes into the revised version. I think next time, I’ll deposit the paper and wait one week while soliciting comments and then submit to a journal.

It was viewed quite a few times in the time while the paper was being considered by Biology Open. I spoke to a PI who told me that they had found the paper and stopped using pitstop as a result. I think this means getting the work out there was worth it after all.

Now it is out “properly” in Biology Open and anyone can read it.

Verdict: I was really impressed by Biology Open. The reviewing and editorial work were handled very fast. I guess it helps that the paper was very short, but it was very uncomplicated. I wanted to publish with Biology Open rather than PLoS ONE as the Company of Biologists support cell biology in the UK. Disclaimer: I am on the committee of the British Society of Cell Biology which receives funding from CoB.

Depositing the preprint at bioRxiv was easy and for this type of paper, it is a no-brainer. I’m still not sure to what extent we will preprint our work in the future. This is unchartered territory that is evolving all the time, we’ll see. I can say that the experience for this paper was 100% positive.

References

Dutta, D., Williamson, C. D., Cole, N. B. and Donaldson, J. G. (2012) Pitstop 2 is a potent inhibitor of clathrin-independent endocytosis. PLoS One 7, e45799.

Lemmon, S. K. and Traub, L. M. (2012) Getting in Touch with the Clathrin Terminal Domain. Traffic, 13, 511-9.

Stahlschmidt, W., Robertson, M. J., Robinson, P. J., McCluskey, A. and Haucke, V. (2014) Clathrin terminal domain-ligand interactions regulate sorting of mannose 6-phosphate receptors mediated by AP-1 and GGA adaptors. J Biol Chem. 289, 4906-18.

von Kleist, L., Stahlschmidt, W., Bulut, H., Gromova, K., Puchkov, D., Robertson, M. J., MacGregor, K. A., Tomilin, N., Pechstein, A., Chau, N. et al. (2011) Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell 146, 471-84.

Willox, A.K., Sahraoui, Y.M.E. & Royle, S.J. (2014) Non-specificity of Pitstop 2 in clathrin-mediated endocytosis Biol Open, doi: 10.1242/​bio.20147955.

Willox, A.K., Sahraoui, Y.M.E. & Royle, S.J. (2014) Non-specificity of Pitstop 2 in clathrin-mediated endocytosis bioRxiv, doi: 10.1101/002675.

The post title is taken from ‘Into The Great Wide Open’ by Tom Petty and The Heartbreakers from the LP of the same name.

Give, Give, Give Me More, More, More

A recent opinion piece published in eLife bemoaned the way that citations are used to judge academics because we are not even certain of the veracity of this information. The main complaint was that Google Scholar – a service that aggregates citations to articles using a computer program – may be less-than-reliable.

There are three main sources of citation statistics: Scopus, Web of Knowledge/Science and Google Scholar; although other sources are out there. These are commonly used and I checked out how comparable these databases are for articles from our lab.

The ratio of citations is approximately 1:1:1.2 for Scopus:WoK:GS. So Google Scholar is a bit like a footballer, it gives 120%.

I first did this comparison in 2012 and again in 2013. The ratio has remained constant, although these are the same articles, and it is a very limited dataset. In the eLife opinion piece, Eve Marder noted an extra ~30% citations for GS (although I calculated it as ~40%, 894/636=1.41). Talking to colleagues, they have also noticed this. It’s clear that there is some inflation with GS, although the degree of inflation may vary by field. So where do these extra citations come from?

  1. Future citations: GS is faster than Scopus and WoK. Articles appear there a few days after they are published, whereas it takes several weeks or months for the same articles to appear in Scopus and WoK.
  2. Other papers: some journals are not in Scopus and WoK. Again, these might be new journals that aren’t yet included at the others, but GS doesn’t discriminate and includes all papers it finds. One of our own papers (an invited review at a nascent OA journal) is not covered by Scopus and WoK*. GS picks up preprints whereas the others do not.
  3. Other stuff: GS picks up patents and PhD theses. While these are not traditional papers, published in traditional journals, they are clearly useful and should be aggregated.
  4. Garbage: GS does pick up some stuff that is not a real publication. One example is a product insert for an antibody, which has a reference section. Another is duplicate publications. It is quite good at spotting these and folding them into a single publication, but some slip through.

OK, Number 4 is worrying, but the other citations that GS detects versus Scopus and WoK are surely a good thing. I agree with the sentiment expressed in the eLife paper that we should be careful about what these numbers mean, but I don’t think we should just disregard citation statistics as suggested.

GS is free, while the others are subscription-based services. It did look for a while like Google was going to ditch Scholar, but a recent interview with the GS team (sorry, I can’t find the link) suggests that they are going to keep it active and possibly develop it further. Checking out your citations is not just an ego-trip, it’s a good way to find out about articles that are related to your own work. GS has a nice feature that send you an email whenever it detects a citation for your profile. The downside of GS is that its terms of service do not permit scraping and reuse, whereas downloading of subsets of the other databases is allowed.

In summary, I am a fan of Google Scholar. My page is here.

 

* = I looked into this a bit more and the paper is actually in WoK, it has no Title and it has 7 citations (versus 12 in GS). Although it doesn’t come up in a search for Fiona or for me.

hood

 

However, I know from GS that this paper was also cited in a paper by the Cancer Genome Atlas Network in Nature. WoK listed this paper as having 0 references and 0 citations(!). Does any of this matter? Well, yes. WoK is a Thomson Reuters product and is used as the basis for their dreaded Impact Factor – which (like it or not) is still widely used for decision making. Also many Universities use WoK information in their hiring and promotions processes.

The post title comes from ‘Give, Give, Give Me More, More, More’ by The Wonder Stuff from the LP ‘Eight Legged Groove Machine’. Finding a post title was difficult this time. I passed on: Pigs (Three Different Ones) and Juxtapozed with U. My iTunes library is lacking songs about citations…

Some Things Last A Long Time

How long does it take to publish a paper?

The answer is – in our experience, at least – about 9 months.

That’s right, it takes about the same amount of time to have a baby as it does to publish a scientific paper. Discussing how we can make the publication process quicker is for another day. Right now, let’s get into the numbers.

The graphic shows the time taken from submission-to-publication for papers on which I am an author. I’m missing data for two papers (one from 1999 and one from 2002) and the Biol Open paper is published online but not yet “in print”, but mostly the information is complete. If you want to calculate this for your own papers; my advice would be to keep a spreadsheet of submission and decision dates as you go along… and archive your emails.

In the last analysis, a few people pointed out ways that the graphic could be improved, and I’ve now implemented these changes.

The graphic shows that the journey to publication is in four eras:

  1. Pre-time (before 0 on the x-axis): this is the time from first submission to the first journal. A dark time which involves rejection.
  2. Submission at the final journal (starting at time 0). Again, the orange periods are when the manuscript is with the journal and the green, when it is with us. Needless to say this green time is mainly spent doing experimental work (compare green periods for reviews and for papers)
  3. Acceptance! This is where the orange bar stops. The manuscript is then readied for publication (blank area).
  4. Published online. A purple period that ends with final publication in print.

Note that: i) the delays are more-or-less negated by preprinting provided deposition is before the first submission (grey line, for Biol Open paper), ii) these delay diagrams do not take into account the original drafting/rewriting cycle before the fist submission – nor the time taken to do the work!

So… how long does it take to publish a paper?

In the top right graph: the time from first submission to being published online is 250 days on average (median). This is shown by the blue bar. If we throw in the average time it takes to go from online to print (15 days) this gives 265 days. The average time for human gestation is 266 days. So it takes about the same amount of time to have a baby as it does to publish a paper! By contrast, reviews take only 121 days, equivalent to four lunar cycles (118 days).

My 2005 paper at Nature holds the record for the most protracted publication 399 days from submission to publication. The fastest publication is the most recent, our Biol Open paper was online 49 days after submission (it was also online 1 day before submission as a preprint).

In the bottom right graph: I added together the total time each paper was either with the journal, or with us, and plotted the average. The time from acceptance-to-publication online is shown stacked onto the “time with journal” column. You can see from this graphic that the lion’s share of the delay comes from revisions that we must do in order for a paper to be published. Multiple revisions and submissions also push these numbers up compared to the totals for reviews.

How representative are these numbers?

This is a small dataset at many different journals and so it is difficult to conclude much. With this analysis, I was hoping to identify ‘slow journals’ that we should avoid and also to think about our publication strategy (as much as a crap shoot can have a strategy). The whole process is stochastic and I don’t see any reason to change the way that we navigate the system. Having said this, I can’t see us doing any more methods/book chapters, as they are just so slow.

Just over half of our papers have some “pre-time”, i.e. they got rejected from at least one other journal before finding a home. A colleague of mine likes to say:

“if your paper is accepted at the first journal you send it to, you sent it to the wrong place”

One thing for sure is that publication takes a long time. And I don’t think our experience is uncommon. The pace of scientific publishing has been described as glacial by Leslie Vosshall and I don’t disagree with this. I think the 9 months figure is probably representative for most areas of biology. I know that other scientists in my field, who have more tenacity for rejections and for slugging it out at high impact journals, have much longer times from 1st submission to acceptance. In my opinion, wasting even more time chasing publication is crazy, counter-productive and demotivating for the people in the lab.

The irony in all this is that, even though we are working at the absolute bleeding edge of science with all of this technology at our disposal, our methods for reporting science are badly out of date. And with that I’ll push the “publish” button and this will be online…

The title of this post comes from ‘Some Things Last A Long Time’ by Daniel Johnston from his LP ‘1990’.

My Blank Pages

Books about the MRC Laboratory of Molecular Biology are plentiful. If you haven’t read any, the best place to start are the books written by some of the Nobelists themselves: “I Wish I’d Made You Angry Earlier” by Perutz, “My Life in Science” by Brenner. Also, “Sequences, Sequence, Sequences” by Sanger, “What Mad Pursuit” by Crick and even Watson’s “The Double Helix” cover ‘how it was done’ and ‘what the place is like’. After that are the biographies of the Nobelists and their associates. Then comes the next layer, the comprehensive but rather dry “Designs for Life: Molecular Biology after World War II” by de Chadarevian and hell, even “The Eighth Day of Creation” by Judson is substantially about the LMB, since so many major discoveries in Molecular Biology happened there.

If your appetite is not sated after wading through all of those, then there are the books for the insiders.

John Finch wrote a book “A Nobel Fellow on Every Floor” which was enjoyable, if rather selective on who and what was included. The latest book from the LMB Press is a collection of essays entitled “Memories and Consequences: Visiting Scientists at the MRC Laboratory of Molecular Biology, Cambridge”. It was edited by Hugh Huxley and was made available last summer (around the time of his death).
You can get it here

 

memories

The premise of Memories and Consequences is that there were a large number of postdoctoral fellows, mainly from the USA, who spent time at the LMB (in the 60s, mainly) and then went away and had hugely successful scientific careers. At one point in the book, Tom Steitz writes that, of his friends during this period, 40% are now NAS members! The essays cover the time of these visitors in England and how it shaped their subsequent careers.

This is definitely a book to dip in and out of. The experiences are actually pretty repetitive: yes, we drive on the other side of the road; Cambridge is a very stuffy place and Max Perutz liked to be called Max. This repetition is amplified if the chapters are read one-after-the-other. Overall however, the essays are nice reminiscences of a booming time in Molecular Biology and many capture the magic of working at the LMB during this period. Brenner and Crick come to life and even Sir Lawrence Bragg looms large in many chapters filling the authors with awe.

When I first downloaded the book, I read the chapters by those whose work I am most familiar. I didn’t even know that Dick McIntosh had spent not one but two sabbaticals at the LMB. Tom Pollard, Harvey Lodish etc. followed. I then read the other chapters when I had more time.

The best chapters were those by Harry Noller and by Peter Moore who gave the right amount (for my taste) of personal insight to their stay at the LMB. I would recommend that the reader skips the chapter by William Dove and Alexandra Shevlovsky, who tried to be a bit clever and didn’t quite pull it off. Sid Altman’s chapter has previously been published and I actually witnessed him read this out (more-or-less) verbatim at the DNA50+1 celebrations – which was far more enjoyable than it sounds.

In short, I enjoyed the book and it’s worth reading some of the chapters if you have a leaning towards the history of science, but there are plenty of other books (listed above) where you should start if you want to find out what life is like inside the Nobel Prize Factory.

I’ll leave you with three quotes that I enjoyed immensely:

“I remember seeing copies of the journal Cell, where we all yearned to publish (though, I noticed, not the really great scientists, like John Sulston or Sydney Brenner). I would shudder and turn away; Cell was for other scientists, not for me.”
Cynthia Kenyon

“Like many others who worked at the LMB in that era, I still think of its modus operandi as exemplifying the blueprint that all scientific research establishments should aspire to emulate. Pack the very best scientists you can find into a building, so densely that they cannot avoid talking to each other, and encourage them to interact in every other way you can. A canteen or dining room might be a good idea. (The facility itself need not be luxurious, and indeed, it is probably better if it is not.) Give those scientists ample staff support, and all the money they need to get on with the job. Stir well, and then be patient because good science takes time. My subsequent career has taught me that this recipe is much harder to execute than it is to describe. I still wonder how the MRC managed to do it so well for so long.”
Peter Moore

“I learned that protein chemistry didn’t need me, that King’s College High Table was for tougher folk than I, and that Sydney talked but Francis conversed.”
Frank Stahl

A comprehensive guide to LMB books is available here

Don’t worry, book reviews will be a very infrequent feature as I hardly have any time to read books these days!

The post title is from My Blank Pages – Velvet Crush from their LP Teenage Symphonies to God. Presumably a play on the Dylan/Byrds song My Back Pages.

A Day In The Life

#paperoftheday #potd

A common complaint from other PIs is that they “don’t read enough any more”. I feel like this too and a solution was proposed by a friend of a friend*: try to read one paper per day.

This seemed like a good idea and I started to do this in 2013. The rules, obviously, can be set by you. Here’s my version:

  1. Read one paper each working day.
  2. If I am away, or reviewing a paper for a journal or colleague, then I get a pass.
  3. Read it sufficiently to be able to explain it to somebody else, i.e. don’t just scan the abstract and look at the figures. Really read it and understand it. Scan and skim as many other papers as you normally would!
  4. Only papers reporting primary research count towards #paperoftheday.
  5. If it was really good or worth telling people about – tweet about it.
  6. Make a simple database in Excel or Papers – this helps you keep track, make notes about the paper (to see if you meet #3) and allows you to find the paper easily in the future (this last point turned out to be very useful).

I started this in 2013 (for one full year) and am trying to continue in 2014. I feel that this is succeeding in making me read more than I would have otherwise done.

My stats for 2013 were:

  • 85% success rate. Filling that last 15% will be tough.
  • Stats errors in 48% of papers! Most common error was incorrect use of Student’s  t-test.
  • 68% of papers were from 2013 and 22% were from 2009-2012.

The big surprise was which journals I read most:

  1. J Cell Biol 13
  2. PLOS One 12
  3. Nat Cell Biol 10
  4. PNAS 10
  5. Curr Biol 9
  6. Mol Biol Cell 8
  7. Nature 8
  8. Dev Cell 7
  9. eLife 7
  10. Nature Methods 7
  11. Cell 6
  12. Neuron 6
  13. Traffic 6
  14. J Cell Sci 4
  15. Science 4

I thought that Cell would be much higher and PNAS would be much lower. Since where we publish is dictated by who is likely to see and read the paper, this list was thought-provoking.

*I think this was a colleague of @david_s_bristol who suggested it, sometime in 2012.

The post title is of course from A Day in The Life – The Beatles from the LP Sgt Pepper’s Lonely Hearts Club Band. For the first line…

So Long

How long does it take to publish a paper?

I posted the picture below on Twitter to show how long it takes for us to publish a paper.

papers

The answer is 235 days. This is the median time from submission at the first journal to publication online or in print. The data are from our last ten papers.

The infographic proved popular with 40 retweets and 22 favourites. It was pointed out to me that the a few things would improve this visualisation:

1. Showing the names of the journals

2. Showing when the 1st submission was relative to the 1st submission at the journal that finally accepted the paper

3. What about reviews and other types of publication.

I am working on updating the graph to show all of these things… watch this space.

My point was really to show (perhaps to non-scientists) how long the process of publishing a paper can be. There is other information that can be gleaned from this, e.g. what proportion of time is at the journal’s side and how much is at our end?

The people who are eager to see which journals perform badly (slowly) will be disappointed: this is a very small subset of papers from one lab. I’d be interested in scraping the information on journal tardiness on a larger scale and synthesising this so that it can inform journal choice. Recently though major publishers have taken steps to make this information less accessible so don’t hold your breath.

The title of this post is from So Long by Cian Ciarán from the LP ‘Outside In’