stripy revisited

A welcome Nature Editorial

I reproduce below a comment I have left on this Nature editorial entitled “Go forth and replicate!“.

Nature Publishing Group encouragement of replications and discussions of their own published studies is a very welcome move. Seven years ago, I wrote a letter (accompanying a submission) to the Editor of Nature Materials. The last paragraph of that letter read: “The possibility of refuting existing data and theories is an important condition of progress of scientific knowledge. The high-impact publication of wrong results can have a real impact on research activities and funding priorities. There is no doubt that the series of papers revisited in this Report contribute to shape the current scientific landscape in this area of science and that their refutation will have a large impact.” [1]

The submission was “Stripy Nanoparticles Revisited” and it took three more years to publish it… in another journal; meanwhile Nature Materials continued to publish findings based on the original flawed paper [2]. The ensuing, finally public (after three years in the secret of peer review), discussions on blogs, news commentary and follow up articles were certainly very informative on the absolute necessity of changing the ways we do science to ensure a more rapid discussion of research results [3].

One of the lessons I draw from this adventure is that the traditional publishing system is, at best ill suited (e.g. Small: three years delay), or at worst (e.g. Nature Materials) completely reluctant at considering replications or challenges to their published findings. Therefore, I am now using PrePrints (e.g. to publish a letter PNAS won’t share with their readers [4]), PubPeer and journals such as ScienceOpen where publication happens immediately and peer review follows [5].

So whilst I warmly welcome this editorial, it will need a little more to convince me that it is not a complete waste of time to use the traditional channels to open discussions of published results.

[1] The rest of letter can be found at https://raphazlab.wordpress.com/2012/12/17/letter-to-naturematerials/
[2] The article was eventually published in Small (DOI:10.1002/smll.201001465

2 comments on PubPeer

); timeline: https://raphazlab.wordpress.com/2012/12/20/stripy-timeline/
[3] https://raphazlab.wordpress.com/stripy-outside/
[4] https://raphazlab.wordpress.com/2015/11/16/pnas-your-letter-does-not-contribute-significantly-to-the-discussion-of-this-paper/
[5] https://raphazlab.wordpress.com/2015/11/17/the-spherical-nucleic-acids-mrna-detection-paradox/

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 (!).

Capture

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

 

Featured Image -- 2038

Do nanoparticles deliver? Merck’s SmartFlares and other controversies

Leonid Schneider’s article starts with a summary of the stripy controversy and then moves on to the SmartFlare. Of particular interest is the quote from Luke Armstrong, formerly at EMD Millipore, which demonstrates that the company ought to be well aware that the probes detect nucleases rather than mRNAs. This begs the question of why they are still selling and advertising this product. Unfortunately, they did not provide a statement to Leonid. [Picture above is from Leonid’s post]

For Better Science

A large body of scientific nanotechnology literature is dedicated to the biomedical aspect of nanoparticle delivery into cells and tissues. The functionalization of the nanoparticle surface is designed to insure their specificity at targeting only a certain type of cells, such as cancers cells. Other technological approaches aim at the cargo design, in order to ensure the targeted release of various biologically active agents: small pharmacological substances, peptides or entire enzymes, or nucleotides such as regulatory small RNAs or even genes. There is however a main limitation to this approach: though cells do readily take up nanoparticles through specific membrane-bound receptor interaction (endocytosis) or randomly (pinocytosis), these nanoparticles hardly ever truly reach the inside of the cell, namely its nucleocytoplasmic space. Solid nanoparticles are namely continuously surrounded by the very same membrane barrier they first interacted with when entering the cell. These outer-cell membrane compartments mature into endosomal and then…

View original post 2,353 more words

Towards the end of the stripy controversy?

Last week saw the publication in PloS One of Quy Khac Ong and Francesco Stellacci’s response to Stirling et al “Critical Assessment of the Evidence for Striped Nanoparticles” published a year earlier (November 2014, I am one of the co-authors).

The controversy had started with our publication of Stripy Nanoparticles Revisited after a three year editorial process (2009-2012) and was followed by a large number of events at this blog, on PubPeer and a few other places.

Here is a short statement in response to Ong and Stellacci. Since theirs  was a response to Stirling et al, Julian Stirling was invited to referee their submission (report).

We are pleased that Ong and Stellacci have responded to our paper, Critical assessment of the evidence for striped nanoparticles, PLoS ONE 9 e108482 (2014). Each of their rebuttals of our critique has, however, already been addressed quite some time ago either in our original paper, in the extensive PubPeer threads associated with that paper (and its preprint arXiv version), and/or in a variety of blog posts. Indeed, arguably the strongest evidence against the claim that highly ordered stripes form in the ligand shell of suitably-functionalised nanoparticles comes from Stellacci and co-authors’ own recent work, published shortly after we submitted our PLOS ONE critique. This short and simple document compares the images acquired from ostensibly striped nanoparticles with control particles where, for the latter (and as claimed throughout the work of Stellacci et al.), stripes should not be present. We leave it to the reader to draw their own conclusions. At this point, we believe that little is to be gained from continuing our debate with Stellacci et al. We remain firmly of the opinion that the experimental data to date show no evidence for formation of the “highly ordered” striped morphology that has been claimed throughout the work of Stellacci and co-workers, and, for the reasons we have detailed at considerable length previously, do not find the counter-claims in Ong and Stellacci in any way compelling. We have therefore clearly reached an impasse. It is thus now up to the nanoscience community to come to its own judgement regarding the viability of the striped nanoparticle hypothesis. As such, we would very much welcome STM studies from independent groups not associated with any of the research teams involved in the controversy to date. For completeness, we append below the referee reports which JS submitted on Ong and Stellacci’s manuscript.

Julian Stirling, Raphaël Lévy, and Philip Moriarty November 16 2015

 

 

The death of Nanonymous

Before you worry: nobody died.
But it is really with great regret that I relay here Nanonymous’ decision “to retire the moniker” [see below]. Nanonymous was a clever handle and his/her comments have constituted important and enjoyable contributions to the stripy nanoparticles controversy. We first encountered Nanonymous in October 2013 at ChemBark commenting on the Response to ACS Nano Editorial on Reporting Misconduct where he/she defended post-publication peer review as a legitimate form of scientific discussion and urged authors to engage, notingIf this keeps up, “nano”science will get the reputation it deserves. What a shame.
Nanonymous became interested in the controversy; he/she was at the beginning undecided and looking at the scientific issues with an open mind, asking, still in the ChemBark thread

Raphael,
Do you have a response to (what I understand to be) the latest Stellacci article?
http://pubs.acs.org/doi/pdf/10.1021/la403546c

I mean, as someone who knows very little about this field I have a hard time understanding how one could demonstrate “stripiness” as a a scanning artifact…unless a bunch (more) people were wrong:
http://pubs.acs.org/doi/pdf/10.1021/la403546c

Since then Nanonymous made numerous contributions here and at PubPeer, with great scientific insights, many questions to me/Julian/Philip, forcing us to clarify our thinking further, as well as outstanding dissection of “unreg”[…] arguments and argumentative strategies.
Below is the last comment of Nanonymous, left this morning on the previous post.
nanonymous |

Raphael,
The latest post on pubpeer:

https://pubpeer.com/publications/58199FBEA31FB5755C750144088886#fb23615

falsely attributes authorship to my handle. It is certainly the same troll who has been spamming recently. My response isn’t showing up on pubpeer yet, I post it below. My, this is getting quite silly. I’m afraid the “encyclopedia dramatica” aspects will detract from the serious nature of the debate. I will probably sign up as registered peer to avoid this.
——————————————————————————————

The above post (Unreg February 1st, 2015 6:04pm UTC ) was not written by the real nanonymous (me). The poster (who is quite clearly associated with all the gish galloping) uses a deliberately sacchrin tone, perhaps for the purposes provoking a “spot the impostor” cliche:

http://tvtropes.org/pmwiki/pmwiki.php/Main/SpotTheImposter

The persistence of anonymous handles is a measure of how untrustworthy a forum can be, the fact that the above Unreg has approriated a handle that is not his/her own (this was already done once on the fakerapha site, there is no doubt that it is the unreg above is the same identity that is responsible for that site).

This is an attack against the utility of PubPeer since we must presume that the vast majority of comments that do not mention Russian brides, Nigerean banks or viagara are written in good faith and that no one would appropriate an established handle in good faith. This clearly demonstrates that we need to figure out ways around these attacks, we all knew this was inevitable. While the PubPeer admins certainly don’t have time, one could envision some sort of digital signature type scheme in PubPeer 2.0. (there are probably a few good reasons we don’t want mere registered usernames).

Suppose I don’t have much choice but to retire the moniker, any further posts labelled “nanonymous” are not written by me, the identity that assumed the (relatively) novel name during the course of learning about the stripy saga. I may consider joining as a regular Peer with my academic address, but will need to consider carefully for a bit.

I always thought that no one could possibly vest so much time and effort into trying to obfuscate facts about (what are to that vast majority of people) obscure subjects like nanoparticles but fakerapha/unreg/bionanochair/ProfSTM/gustav/weichen/etc. has proven me wrong. Learning that one is wrong anout something is perhaps the part that is most fun about internet discussions.

Identity theft: a new low in the stripy nanoparticles controversy

If you are familiar with the controversy and wonder why a PloS One comment thread disappeared today, skip directly to recent events.


Jackson et al, Nature Materials 2004

Jackson et al, Nature Materials, published 18 April 2004

In 2004, Alicia Jackson (now deputy director of the Biological Technologies office at DARPA), Jacob Myerson and Francesco Stellacci (now Constellium Professor at EPFL) mis-interpreted a common scanning probe microscopy artefact for a real structure. They submitted their results to Nature Materials, which published the work. This error was to become the starting point of a controversy which, more than 10 years later, is still unraveling.

Predrag Djuranovic

Predrag Djuranovic was a PhD student in Stellacci’s group at MIT

In 2005, concerns were raised by Predrag Djuranovic, then a student in Francesco Stellacci’s group. According to Lauren Wolf writing for Chemical & Engineering News last year:

he discussed his concerns with his department head at MIT […]. A few months later, an official investigation of Stellacci launched at MIT and Djuranovic left Stellacci’s research group. MIT closed its review three years later, in 2008, finding Stellacci not guilty of academic misconduct. The university did, however, suggest that Stellacci should do additional work to substantiate his 2004 findings.”

MIT's Building 10 and Great Dome; adapted from this  pic

MIT’s Building 10 and Great Dome; adapted from this pic

MIT was not convinced by the published data but instead of sharing those concerns with the scientific community, they kept them secret and Stellacci was asked to “substantiate” what he had published. This –trying to substantiate a structure which had been postulated to explain an experimental artefact– is what he has now been doing for more than 10 years… with over 30 articles published.

stripy nanoparticles revisited

Stripy Nanoparticles Revisited, published 23 Nov 2012

The controversy entered the public domain in 2012 with the publication of our article entitled Stripy Nanoparticles Revisited in Small: I was not aware of Predrag’s work but had come to the same conclusion. Francesco Stellacci published his response alongside our paper. He, and co-authors, also published several other articles around the same period. At the end of last year, Julian Stirling, and co-authors, published in PloS One an extensive Critical Assessment of the Evidence for Striped Nanoparticles which demonstrates that there is no evidence for the existence of the stripy nanoparticles. A response from Francesco Stellacci is still expected at PloS One.

Scientists interested in the details of the scientific arguments can refer to the articles linked to above (and references therein) as well as the blog posts and PubPeer discussions.

I wish this was all about science and differences of interpretation. I have touched previously on the re-use of figures in different articles. This eventually led to two corrections, one at Nature Materials and one at PNAS.  An EPFL investigation was triggered, opened, and, eventually, following the report of an international independent panel, closed. A third article, pretty much entirely based on data re-use, has not been corrected.

I decided to start blogging about the controversy after the publication of our article in 2012. I used the blog to present our paper, discuss the response (without having to wait three years), publish an invited post from Predrad Djuranovic, other experts (Philip Moriarty, Mathias Brust, and Quanmin Guo), and many other things . Other blogs and the post-publication peer review platform PubPeer also became important fora.

Unfortunately, just as the traditional literature has its flaws and can be gamed by scientists who do not respect the rules, so do these new modes of scientific communication. I have argued earlier that the stripy controversy was a window into the scientific process revealing the serious failures of self correction mechanisms. It is also a window into the frightening flaws of online post-publication peer review – I say this as a strong and determined supporter of post-publication peer review.

Those who believe in stripes have never engaged openly in the online debate (they have even vigorously complained that it was taking place, likening it to cyberbullying). Instead, we’ve had, in chronological order:

  • a troll posting over a period of two days a large number of comments on this blog… he eventually was identified as an Editor of the journal.
  • A fakerapha blog (that still exists) and a fakerapha twitter (the account has been removed) who impersonated me on the Chembark blog.
  • A text book case of Gish Gallop by “unreg” (a debating technique named after, and often used by, creationists) at PubPeer, It is to their immense credit that Julian Stirling, Philip Moriarty and several anonymous peers have engaged, repeatedly and honestly with “unreg”… until it became absolutely beyond doubt that this was no ordinary – if a little heated – discussion. Maybe best to let Nanonymous, an anonymous but insightful and regular commentator of the controversy describe that thread:

    Wow, the same thing again and again, in spite of being addressed again and again.

    and later

    […] it is interesting to reflect on the motivations for comments like these. They seem to come from an identity that either: 1)Does not understand basic scientific reasoning.2)Is vested in obfuscating some fairly straightforward arguments against clear and obvious errors in scientific work.Perhaps both. It could be just some random troll, but clear evidence that there are people acting in very bad faith out there.   Nanonymous

  • Possibly that thread also includes sockpuppetry with “unreg” and “ProfSTM/BioNanoChair” being the same person.

These things happened on blogs or at PubPeer, i.e. platforms which accept anonymous writing. It is hard to think of how they could be completely avoided while retaining anonymous comments.  The discussions of the costs/benefits of anonymous post-publication peer review is ongoing (I agree with Peer 1 in that thread).

In the last few weeks, the stripy nanoparticles discussion has moved to PloS One which enables comments directly on the article. Plos One has a clear policy requiring contributors to “unambiguously identify themselves with their first and last names, their geographic location, […] Any registered user who is found to have provided false name or location information will have their account suspended and any postings deleted.” In spite of this:

  • A “Gustav Dhror” posted over 10 comments (Gish Gallop again) about “Problems with Fig 4”. There is simply no doubt that Gustav is not a real person but instead a sockpuppet. There is no person with that name who has ever published any article on anything, let alone STM, nanoparticles or surface science. Even first year PhD students get their names listed on some website in academic institutions but  “Gustav Dhror” draws zero hits on Google, or more precisely two: the PloS One thread and a baby name website. After I posted a comment (and contacted PloS One) raising the issue of false identity, that thread stopped… but a few days later…
  • A “Dr Wei Chen” started another thread entitled “Multiple flaws with this paper“. The user profile linked to a real and existing Wei Chen, Professor at Suzhou Institute of Nano-Tech and Nano-Bionics in China. Given the context and previous events, I eventually took the liberty of calling Wei Chen to check whether he was “Wei Chen”, commenter at PloS One. And he was not. He had no idea. He had never posted anything at PloS One. Someone had stolen his identity to post these comments. Someone who wanted to discredit our paper (with flawed pseudo-scientific arguments), was not prepared to do it under their own name, but was prepare to steal someone else’s identity to do it.

PloS One has been contacted and has now removed the “Dr Wei Chen” thread. They have been in contact with “Gustav Dhror” and should post a statement regarding that thread tomorrow. In the paragraphs above, I have used links to WebArchives where you can see how the pages looked like this morning.

Nanostripe controversy in new twist

Simon Hadlington, writing for Chemistry World, reports on our new paper:

A simmering controversy over whether certain nanoparticle structures are merely instrument artefacts has boiled over into a bitter dispute, with a senior scientist alleging that he has become the victim of a personal vendetta, something that is strongly denied by researchers on the other side of the argument.

A significant section of the report deals with such accusations. These are not new. More interestingly, given the very small number of scientists who have expressed a public view on the controversy, it was notable to read the following from Paolo Samori, from the University of Strasbourg:

In my laboratory we have imaged Professor Stellacci’s particles and found that these particles indeed have stripes on them. The images show clear features that are invariant with imaging parameters (scan rate, scan angle, feedback loop, etc) and hence they can ascribe to true tip sample interactions […] (he then goes to discuss power spectral analysis, this is thoroughly address in Stirling et al, e.g. see fig 10)

I would like to invite Prof Samori to share these images “that indeed have stripes on them”, e.g. using FigShare. In the meantime, we can only relie on the published evidence. To my knowledge, the only image of stripy (?) nanoparticles published coming from Samori’s lab is from Biscarini et al and is reproduced below next to stripy nanoparticles from the original 2004 Jackson et al paper.

left from Fig 1a of Jackson et al (2004), right from Biscarini et al (2013)

left from Fig 1a of Jackson et al (2004), right from Biscarini et al (2013), fig 1d

Reactions to this 2013 Biscarini et al were somewhat incredulous:

Mathias Brust: “And yet there are stripes

Quanmin Guo: “Where are the stripes

Philip Moriarty: “The Emperor’s new stripes