The bust of Nelson Mandela and the Professor

nelson-mandela-sculpture-june-20133

Bust of Mandela commissioned by Ken Livingstone in the 1980’s

Foreword/disclaimer: as for all posts here (except when noted otherwise), these are my words and I take full responsibility/credit for them. This post is a bit different from most as it is not about science nor the fabric of science. 

The past week has seen antisemitism making the headlines and the former Mayor of London Ken Livingstone suspended from the Labour party. Here is an anecdote which illustrates why it is so difficult for the left to understand and fight this pervasive prejudice.

The Professor in my anecdote is David Colquhoun, a hero of mine as an advocate for open science, defender of the NHS  and indefatigable fighter against quackery and p-hacking. As many, he tweeted about the antisemitism row (he tweets quite a lot; the few tweets I quote here do not claim to be representative). Here is an early one that caught my attention (and gave this post its title).

What strikes me is the sheer irrationality of the comment. Somehow Ken’s support for Mandela in the 1980’s is seen as relevant to evaluate whether his comments last week were antisemitic. The Professor does not think Ken could be racist. It is simply unimaginable: he is one of the good guys who supported Mandela. Unfortunately, it is impossible to confront that one can not imagine.

In another thread, the Professor asksGenuine Q. Is it “anti-semitic” to deplore treatment of Mordechai Vanunu? Or deplore not signing nuclear non-proliferation?“. The answer is obviously no… and no one had suggested this (and possibly no one has ever suggested this ever). A classical example of the Livingstone formulation, a displacement from a concern about antisemitism to one about “silencing criticism of Israel”. Since the Professor seemed to have a genuine desire to find ways to express his views while avoiding antisemitism, I suggested this piece and noted that:

I suppose my tweet was slightly clumsy: the Professor thought I had accused him of antisemitism. Now, if I am accused of prejudice, any form of prejudice, my reaction will be to seek to understand the charge so that I can learn what I could have done better, and, if necessary, apologize or dispute the charge. Well, that is not how the Professor reacted. Instead, he quoted my tweet to his 12.5K followers:

Antisemitism is not a prejudice, it is an insult. I clarified the misunderstanding (and the Professor accepted the clarification). In the following few days, the Professor would go on to quote various (thoroughly unrepresentative) Jewish voices that would reassure him that no, there really isn’t a problem with antisemitism in the left, and, the Tories are much worse anyway, etc. And then this retweet; a good old conspiracy: the “antisemitism” is within brackets and the story has been “concocted”.

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Some resources:

 

Time-resolved Microscopy and Correlation Spectroscopy

This is a guest post from Jennifer Francis, PhD Student in the group. When group members go to conferences or courses, they have to write a travel blog post.

I recently attended the 8th European Short Course on “Time-resolved Microscopy and Correlation Spectroscopy” at PicoQuant headquarters in Aldershof-Berlin; specifically to learn about the principles and application of FRET, FLIM, and FCS to the Life Sciences. A day prior to this microscopy course, I also attended the 14th SymPhoTime Training Day. As well as discussing problems and applications of the SymPhoTime64 software with other users, I also had the opportunity to speak with a programmer of the software, who demonstrated many new features and assisted with analysis of my own FCS measurements at a computer station. My mornings generally started with passing an incredible sculpture of two heads, before arriving at the Max Born Institute for lectures delivered by scientists in the field of time-resolved microscopy, including Professor Jörg Enderlein.

two heads

Landmark of Berlin-Aldershof: Kopfbewegung – heads, shifting.

 

After a Flammkuchen, I participated in afternoon practical microscopy sessions, where I got the chance to experience the commercially available super-resolution microscope: MicroTime 200 STED, which was awesome. Not only did we see this cutting-edge instrument in action, resolving structures below the diffraction limit, but we also had a peek inside the operating laser boxes! Whilst at the STED station, members of GATTAquant informed us about nanorulers, which are new standards for super-resolution microscopy. Another highlight of this workshop was the demonstration of the Zeiss LSM 880 with Airyscan, which boasts 32 detectors. There were a total of 42 participants on this course, both from academia and industry, including microscope representatives from Olympus, Zeiss, Leica, and Nikon. The consensus take home tip from all participating microscope companies was to always match the refractive index of the objective with that of the sample and to adjust the correction collar to take into account coverslip thickness.

 

SmartFlare Maths

SmartFlare are nanoparticle sensors which are sold by Merck and are supposed to detect mRNA inside live cells. The technology has been developed by Chad Mirkin. In his papers, the nanoparticles are called Nano-Flares or Spherical Nucleic Acids. I am saying “supposed to” because the central question of how those sensors could possibly reach the target that they are supposed to detect has not been addressed by Mirkin nor by Merck.

After evaluating the SmartFlare, we published recently our conclusions at ScienceOpen. We ran this research as an open science project, sharing our experimental results, analyses and conclusions in quasi real time using an open science notebook. All of the imaging data can also be consulted via our online Open Microscopy Environment repository.

Gal Haimovich, who reviewed our paper, first on his blog and then at ScienceOpen, suggested we should do some SmartFlare Maths (point 4 of his list of comments). This had been at the back of my mind for some time. There are various ways to look at this problem, but all those I have tried lead to the same conclusion that the protocols, results and conclusion published do not add up. Here is what I believe the simplest way to think of the SmartFlare Maths problem. As usual, comments and corrections would be very much appreciated.

Estimation of the number of SmartFlares per cell

SF-figure adapted from Giljohann

Adapted from Giljohan et al, Figure 1b

Estimate 1. SmartFlares are added to cells at a final concentration of 0.1 nM (following Merck’s protocol). For 400,000 cells and 20 μL (following Merck’s protocol), this would result in 150,000 SmartFlares per cell, assuming that all nanoparticles are uptaken.

 

Estimate 2. Giljohann et al  (Mirkin’s group) published a quantitative study of uptake of SmartFlares in various cell lines in 2007. From their Figure 1b, we can see that in the lower concentration range tested, there is a linear correlation between SmartFlare concentration in the medium and number of particles per cell. For cells exposed to a medium concentration of 0.1 nM, this would result in an uptake of 75 000 SmartFlares per cell. In the following discussion, we will use this lower estimate. With ~50 oligo probes per SmartFlare, this would give 3,750,000 oligo probes per cell.

Oligo probes per cell versus mRNA per cell

The copy number of any specific mRNA per cell depends on sequence, cell types, signalling events etc, but typically it ranges from a few copies to a few thousands of copies. Our estimate above indicates an excess of oligo probes of at least three orders of magnitude over the most abundant mRNA.

If just 0.1% of these probes would bind their target, it would block 3,750 mRNA resulting in silencing. However, Merck and Mirkin both report that there is no silencing effect in the conditions of these experiments. It follows that more than 99.9% of the SmartFlares do not bind their target mRNA.

Fluorescence background

Seferos

Reproduced from Seferos et al, Figure 1.

Seferos et al (2007, Mirkin’s group) show that in the absence of release of the probe, fluorescence value of ~30% of the total value after release is observed (in ideal test-tube conditions, i.e. in the absence of nucleases). This is presumably due to a non-complete quenching of the fluorescence. For the SmartFlares to work, we would therefore have to detect a variation of less than 0.1% over a background of ~30%.

 

Lab Times: “Flare up over SmartFlares”

Stephen Buckingham interviewed me for Lab Times

On the face of it, Millipore’s SmartFlares are meant to be a tool cell biologists dream of – a way of measuring levels of specific RNA in real time in living cells. But does it really work? Raphaël Lévy and Gal Haimovich are in doubt.

Raphaël Lévy, Senior Lecturer in Biochemistry at the University of Liverpool, UK, was so unconvinced about SmartFlares that he decided to put the technique directly to the test (The Spherical Nucleic Acids mRNA Detection Paradox, Mason et al. ScienceOpen Research). As a result, Lévy has found himself at the centre of a row; not only over whether the technique actually does the job but as to whether it can actually work, at all – even in principle. Lab Times asked Lévy why he is in doubt that SmartFlares really work.

Lab Times:  What’s all the fuss about SmartFlares?

Read it all here (page 50-51).

I can’t resist also quoting this bit of pf the final paragraph…

In interview, Lévy is reasonable and measured in tone. But he is no stranger to controversy and can deliver fierce polemic with style.

If you have not yet, you should also check Leonid Schneider’s earlier and more complete investigation.

Nanoparticles & cell membranes: history of a (science) fiction?

One of the reason scientists, journalists and the general public are excited about nanoparticles is their supposed ability to cross biological barriers, including, the cell membrane. This could do wonders for drug delivery by bringing active molecules to the interior of the cell where they could interact with key components of the cell machinery to restore function or kill cancer cells. On the opposite side of the coin, if nanoparticles can do this, then there are enormous implications in terms of their potential toxicity and it is very urgent to investigate. But is it true? What is the evidence? How did this idea come into the scientific literature in the first place? I have been intrigued by this question for some time. It is the publication of an article about stripy nanoparticles magically crossing the cell membrane that led me to engage in what became the stripy nanoparticles controversy. It is this same vexing question that led me to question Merck/Mirkin claims about smartflare/nanoflare/stickyflare.

In the introduction of our article “The spherical nucleic acids mRNA detection paradox“, we describe the long history of the use of gold nanoparticles (“gold colloids”) in cell biology and conclude that

…, more than five decades of work has clearly established that nanoparticles enter cells by endocytotic mechanisms that result in their entrapment inside intracellular vesicles unless those nanoparticles are biological in nature and have acquired through evolution, advanced molecular tools which enable them to escape.

In the paragraph that followed, we were trying to make the point, in part using citation data of one of these 1950s pioneering articles, that this solid knowledge has been ignored in some of the thousands of recent articles on interactions of nanoparticles with membranes and cells that have appeared in the past 15 years. In his review of the first version of our article, Steve Royle criticises that latter paragraph (in his word, a “very minor” point):

I’m not a big fan of using number of Web of Science search results as an argument (Introduction). The number of papers on Gold Nanoparticles may be increasing since 2007, but then so are the number of papers on anything. It needs to be normalised to be meaningful. It’s also a shame that only 5 papers have cited Harford et al., but it’s an old paper, maybe people are citing reviews that cover this paper instead?

This is a fair point. While normalisation as well as more detailed and systematic searches might shed some light, it is rather difficult to quantify an absence of citation. Instead, I have tried to discover where the idea that nanoparticles can diffuse through membranes comes from. Here are my prime suspects (but I would be more than happy to update this post to better reflect the history of science and ideas so please leave comment, tweet, email), Andre Nel and colleagues, in Science, 3rd of February 2006, “Toxic Potential of Materials at the Nanolevel” :

“ Moreover, some nanoparticles readily travel throughout the body, deposit in target organs, penetrate cell membranes, lodge in mitochondria, and may trigger injurious responses.”

This claim is not supported by a reference, but later in the article Nel et al refer to an earlier paper entitled “Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells” by Marianne Geiser and colleagues. These two papers, Nel et al, and, Geiser et al, have been cited respectively 5000 times and 850 times according to PubMed.

As early as 2007, Shayla Banerji and Mark Hayes had already challenged this idea of transport of nanoparticles across membranes in an elegant experimental and theoretical study which was a direct response to the two papers cited above “Examination of Nonendocytotic Bulk Transport of Nanoparticles Across Phospholipid Membranes“:

In accordance with these health concerns, Nel et al. have described some phenomena that can only potentiate fear of the negative health risks associated with nanotechnology.

[…]

Non-endocytotic transmembrane transport of large macromolecules is a significant exception to what is presently known about cell membrane permeability. Most early studies show that lipid bilayers are essentially impenetrable by molecules larger than water under physiological conditions: transport of most molecules across cell membranes is specifically cell-mediated by endocytosis.34 Endocytosis, unlike proposed passive, non-endocytotic transport, is an active cell-mediated process by which a substance gains entry into a cell. Specifically, a cell’s plasma membrane continuously invaginates to form vesicles around materials that originated outside the membrane: as the invagination continuously folds inward, the cell membrane constituents simultaneously reorganize in such a way that the material being transported into the cell is completely enclosed in a lipid bilayer, forming an endosome.35,36

[…]

The results suggest that a diffusive process of transport is not likely.

Figure 8 is particularly telling (!).

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The article by Shayla Banerji and Mark Hayes has been cited 44 times.

 

Opening up peer review: the peculiar case of PNAS contributed papers

Proceedings of the National Academy of Sciences (PNAS) has two paths for submission of research articles, one standard and one less so, the famous contributed track where the submitting author has to be a member of the National Academy of Sciences of the United States of America. Peter Aldhous reviewed in 2014 this inside track and those who use it more often. He describes the contributed track as follows: “This unusual process allows authors to choose who will review their paper and how to respond to those reviewers’ comments“.

There are two elements of transparency and accountability to counterbalance this conflict of interest of having an author acting as the editor of their own article: it is recorded on the paper that it is a contributed paper, and, the names of the referees (chosen by the author) are also published. It is interesting (maybe) to note that this ancient house of PNAS has a system there which is pretty similar to what has been recently proposed as a disruptive innovation in scientific publishing by Jan Velterop and implemented by ScienceOpen, i.e. peer review by endorsement (PRO). There are a couple of differences. The first one is that PRO at ScienceOpen is open to everyone, not just National Academy members. The second one is that not just the names of the reviewers, but also the content of the reviews that is shared in PRO.

As readers of this blog will know, David Mason and I have recently challenged a particular contributed PNAS paper by the Mirkin group on StickyFlares. The discussion can be found at PubPeer (the authors did not engage). We requested some data which (after some efforts) we eventually obtained. We wrote a letter to the Editor. Which was eventually rejected by Inder Verma editor of PNAS. The letter is available at BiorXiv.

Given that neither PubPeer nor the PNAS letter to editor enabled to get any answers from the authors to our substantial criticism, we were curious to know if any of the referees had maybe raised similar issues and, if yes, how the authors had replied. Dave therefore wrote to the referees to ask whether they would share their reports. The response was negative; they could not share their reports because “referee comments from peer review have to be kept confidential as it is an essential part of maintaining the integrity of the peer review process“. I was rather surprised by this response and was moved to write the following:

01/10/2015

Dear Shana, Chris

I am Raphael; Dave is a member of my group. Apologies for pitching in and for a rather long response!

Thank you both for your replies. [paragraph edited out about the issue of whether the PNAS guidelines on choice of reviewers were followed; see the PubPeer discussion for more]

I fully share your commitment to defend the integrity of the review process, but I would urge to you to reconsider your decision to keep your comments confidential, precisely because it does not serve that very commendable aim.

It is worth considering for a moment what is the role of confidentiality and anonymity in the peer review process, and also who is in charge of guaranteeing that confidentiality and anonymity. In a traditional peer review process, the reports are confidential and anonymous: the justification is the protection of the reviewers from potential reprisals if they were to write a very critical review. The editors are in charge of protecting the confidentiality and anonymity: this is part of the contract between the editor and the reviewers. If the editor was to publish the reviews or/and, worse, reveal their identity, he/she would breach that contract and this would significantly affect the trust between future reviewers and the journal. I have myself pondered on publishing on my blog the reviews of a (rejected) submission of one of my papers, and was eventually convinced (though I am still entirely sure this was the right decision) not to do so by the detailed comments of an editor who did point out that the reviewers expected their reviews to remain confidential and that I would therefore breach their trust by doing so [1]. It is however a completely different matter for reviewers who can decide to forego their right to anonymity both immediately at the stage of the review process (“signing reviews”, usually, precisely with the motivation of increasing transparency and integrity of the review process), or later, for various reasons (nearly 200 000 examples at Publons, a site that enables and encourages reviewers to share their reports [2-3]). A recent prominent example of a reviewer sharing her reviews (on PubPeer) is Vicki Vance, who had reviewed several of the papers of Olivier Voinnet and noted serious problems (they were nevertheless published) [4]. I have never heard anyone suggesting that a reviewer who would decide to share their reviews of a paper after publication, i.e. their own scholarly evaluation of published work, would be damaging the integrity of the peer review process. I also really fail to see by what mechanism it could do so.

Obviously, the PNAS “contributed submission” path is another can of worms. Many would argue that it is in itself damaging to the integrity of the peer review process with this very unusual situation where an author chose its reviewers. In this specific case, it is hard to see any justification at all for the confidentiality of the reviews: it does not serve to protect the reviewers from potential reprisals from the author since the author has chosen its referees in the first place. The only thing it does is prevent the public (and in particular other scientists) to benefit from the insights that would be provided by sharing the reviews. I would argue that here even more than in any other case, sharing the reviews would be the best way to protect the integrity of the peer review process and therefore I hope you will reconsider,

Best wishes

Raphael

[1] https://raphazlab.wordpress.com/2013/01/03/nature-materials-peer-view-of-stripy-revisited-july-to-september-2009-confidential-or-not/ (see first comment in particular)
[2] https://publons.com/
[3] The scientists who get credit for peer review, Nature, 2014
[4] http://www.lab-times.org/editorial/e_600.lasso

Unfortunately, I did not get a reply.

Hot (biochemistry-related) topics

I am in charge of a module entitled “Advanced Skills for Biochemistry“. Our third year Biochemistry (Honours) students take this course. One of their tasks is to prepare and present a poster on a hot topic or technique. I have therefore asked them, the world (via Twitter) and my colleagues at the Institute of Integrative Biology to come up with suggestions of topics for these posters, as well as references that students could use as a starting point. The first suggestions can be found below and I will keep updating as I get new ones. I need a few more topics so get in touch via Twitter (@raphavisses, #LivUniL301), email or in the comments below.

  1. Genome editing with CRISPR/Cas9, suggested by Jerry Turnbull, Dada Pisconti and Pat Eyers:  perhaps this is a useful guide paper for its potential in a disease: ‘Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.’
  2. Cellular Thermal Stability Assay (CETSA) for drug target identification, suggested by Pat Eyers: good starting points are: The cellular thermal shift assay for evaluating drug target interactions in cells and Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay.
  3. Quantification of proteins in organisms, suggested by Pat Eyers; e.g. recent publication from here on ‘Direct and absolute quantificaion of over 1800 Yeast proteins via Selected Reaction Monitoring‘.
  4. Amyloid diseases (in this case Alzheimer’s disease) are possibly transmissible, suggested by Hannah Davies: the paper and some commentary articles and media coverage. [also some comments at PubPeer ; added by RL]
  5. More amyloids, but with an NMR flavour (and oxidative stress is always in vogue), suggested by Marie Phelan or Behaviour meets molecular structure, also suggested by Marie with some Liverpool Darcinrelated papers.
  6. Lattice light sheet microscopy, suggested by David Stephens, from Bristol, via Twitter and by Violaine Sée; Betzig’s article.
  7. Ion mobility–mass spectrometry (IM-MS), suggested by Jerry Turnbull + relevant papers selected by Claire Eyers: Claire wrote a review on IM-MS and a research paper and she also points to this one from Carol Robinsons (Oxford)
  8. Selective Plane Illumination Microscopy (SPIM), suggested by Dave Mason; two good places to start with SPIM and some nice variants adding more planes.
  9. Open science, suggested by Dave Mason; application to big data in c elegans:
    cross over with SPIM (lots on the website: http://openspim.org/Publications )
    some nice discussion of pros and cons (for genomics).
  10. Signalling controlled by frequency modulation, suggested by Violaine Sée, e.g. this article.
  11. Organoids cultures, suggested by Dada Pisconti, e.g. this review Modeling mouse and human development using organoid cultures
  12. Tissue clearing techniques for optical microscopy, suggested by Marco Marcello, exemple paper here.
  13. The potential and challenges of using recombinant spider silk in biomedical applications, suggested by Roger Barraclough, e.gTo spin or not to spin: spider silk fibers and more, and, Controlled assembly: a prerequisite for the use of recombinant spider silk in regenerative medicine?
  14. CryoEM – suggested by Steve Royle via Twitter; advances in electron detectors and software has led to explosion of new fascinating structures. Pat Eyers agrees and provides these examples of CryoEM of the anaphase promoting complex.
  15. XFELs open a new era in structural chemical biology, suggested by Svetlana Antonyuk, with these two additional references.
  16. Dynamics of outer membranes in bacteria (completely discounts ‘lipid raft’ hypotheses) suggested by Marie Phelan.
  17. Predicting contacting residues, within and between proteins, purely from sequence information (large alignments), suggested  by Daniel Rigden . This allows fold prediction, prediction of modes of interaction and many other applications. Review + amazing papers on predicting complexes and structures for uncharacterised Pfam entries.