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.

 

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.

Looking for a beautiful PhD?

Scientific research can be dispiriting at times, when experiments won’t work and even the coffee machine decides not to cooperate. And I can’t promise such times won’t happen in this project ever.

But, in addition to great science and collaboration with an active and interesting industrial partner, this PhD is about beautiful images (full of informative scientific content that we will exploit of course!). In preliminary work which form the basis of this collaborative project, Aurelia Bioscience Matt Vassey prepared samples which were imaged by Marie Held with our light sheet microscope in the Centre for Cell Imaging:

MED-72-T-P-stack_MIP

Human-induced Pluripotent Stem Cells growing on an electrospun scaffold. Sample prepared by Matt Vassey (Aurelia Bioscience Ltd, Nottingham), imaging by Marie Held (Institute of Integrative Biology, Liverpool)

You can find the formal advert here:

Our understanding of the biology of the cell has been informed by decades of studies of cells growing in two-dimensional cell layers. Real biological systems however are 3-dimensional and it has become clear that there are significant differences in terms of gene expression and response to treatments. This project explores new opportunities for growing and imaging cells in 3 dimensions and their impact on the biology of the cell. It offers an array of opportunities to the student for scientific development and close collaboration between an academic and industrial partnership. The extensive multidisciplinary nature of the project would involve interaction with biologists, materials scientists, biophysicists, pharmacologists and industrial biotechnologists and would suit an ambitious applicant with a strong interest in technology development and biotechnology.

At this stage, you should, either apply by sending me an email explaining why you are interested with your CV attached (yes, I know it is obvious, but still), or, help me find a lucky enthusiastic student for this project! [unfortunately, nationality/residency funding conditions apply]. Deadline for application is 12/02, however, apply as soon as possible!

Other things we do in the group here.

We are anonymous (or not). We need you to join. We are (mostly) making scientific discussion in the open possible and easy.

The last thing you probably want to read is one more article about anonymity in (post-publication) peer review. The topic has been covered recently by Bastian, Blatt, Lawrence, Oransky, Moriarty & PubPeerNeuroskeptic, Schneider to name just a few. I am sorry. I’ll keep it short.

I have decided to sign the peer review reports I write as a referee. Yet, I insist that attacks against anonymity in post-publication peer review are unfair, misguided and counterproductive. These two positions might seem contradictory. Bear with me to the end of the post and, hopefully, you might agree they are not.

Social media (and in particular PubPeer) have played a role in pretty much all recent scientific controversies, in part because the traditional channels are at best inefficient and at worst useless. Journal editors and some authors do not know how to react when criticism of articles appear on these platforms. One possible reaction is to shoot the messenger. If it is anonymous, call them anonymous cowards and question the motives. If it is not, call it cyber bullying.

Philip Moriarty, a strong supporter of PubPeer, has nevertheless titled his blog contribution to this debate “We are anonymous. We are legion. We are (mostly) harmful.” With friends like this, who needs enemies… I, and others, have responded in the comments section.

We can argue all of 2016, but the key practical question are the following. Your colleague/student has read a paper and has some interesting comments that she would like to share with the world;

Say you have chosen the first option and your colleague/student has now posted her critique on PubPeer (anonymously). You happen to also know the authors.

These are very simple questions. How we answer them has implications. Peer review is central to our practice, yet publicly engaging in scientific discussion on someone else’s work is often seen as not nice. This is the cultural barrier that we need to break. Many authors make the choice of not responding to carefully crafted criticism of their work (whether the criticism is anonymous or not). A colleague recently contacted me querying my opinion on a paper. We exchanged a few emails. We agreed there were several problems and possible errors of interpretation. I suggested to share our critique on PubPeer thereby giving the original authors the opportunity of a reply (and the rest of the community the opportunity to contribute to the discussion). He replied (SIC, smilies included): “Sorry, not for me :) I do not like sharing :)“. This is the culture we have to change. We did not even get to discuss the possibility of anonymity.

Leonid Schneider argues that most of PubPeer is not really post publication peer review. Instead, it is calling out fraud. While anonymity is OK when you call out fraud, it would problematic in cases of scientific arguments. Even if we accepted the latter (which I do not), the distinction is artificial. There is a continuum of practices from outright fabrication to cherry picking of data and extremely optimistic interpretation of results (Twitter convo on this point here), e.g. it is common that extensive statistical analysis of data is necessary to demonstrate fraud. Any analysis of published data is post-publication peer review, whether it results in new hypotheses, questions and clarifications, or suspicions of fraud.

I disagree that anonymity is a problem and I can see plenty of valid, honest, reasonable (and even anodyne) reasons why you might choose to comment anonymously. If you insist it is a problem, fine, but I hope you will agree that it is secondary to getting valuable critiques of published work in the open. And, most importantly, do not side with Blatt who is calling for authors not to respond to criticism of their work.

It is sometimes argued that anonymity in peer review is fine because there is someone, the editor, who knows the identity of the reviewer. The power asymmetry, and therefore the potential for abuse, is however much larger in formal peer review than in post-publication peer review. At PubPeer, an anonymous comment stands purely on its merits; a scientifically strong response from the authors will bring credit to the authors. In formal peer review, the anonymous referee comes with the prestigious vetting of the editor. Furthermore, editors who are often not experts and always stretched with time, rely largely on these reports for their decisions; those have an impact on career progression, eventually grant funding, etc. The accountability of reviewers is little to none. I have started to sign my reviews during the course of 2015 with some hesitations, but my commitment is now firm. I have had some positive feedback and in one case (where I had rejected the paper), an email from an author asking me for further advice on their revised ms before submitting elsewhere. For more reasons to sign your reviews, check this.

So, to conclude, here is my advice for 2016: contribute to PubPeer (anonymously, or not, I care little), sign your peer reviews… and publish them when possible.

 

 

 

 

 

#socialmedia4academics

I ran today a one hour training session for researchers at the University of Liverpool about online presence. About 20 researchers from very different backgrounds (from language to physics, chemistry  ecology, etc) mostly at the post-doctoral level attended. We started with a round table where I asked each participants to tell which social media they use and what they expected from the workshop.

Many were Facebook users, mostly for personal networking, while a few had started to use it for professional networking too. Research Gate and LinkedIn were prominent as well (often with low level of usage). Google+ had one mention. One or two had limited experience of Twitter. One question that came several times was the personal versus professional limit. How much should we keep private? I don’t think there is any easy answer to this question, except that it is useful to understand how each tool you use work and therefore how to control what you are actually sharing or not. In that context, Facebook is a bit of a pain while Twitter is simple: everything is public so don’t share what you want to keep private.

Does it mean though that everything on your Twitter feed has to be serious professional stuff devoid of any personal aspect? I asked this question to Twitter during the event itself

Vladimir Teif responded immediately

I don’t actually agree with Vladimir (you can check my reply to him on Twitter), but thanks to him for this nice demonstration of the power of real-time conversation and crowdsourcing of  information.

When preparing this session, 12 hours before the event, I had asked on Twitter suggestions on of posts an points on social media for academics. I got a number of responses:

 

 

 

 

 

 

 

 

 

I ended up talking too much, mostly advertising the benefits of Twitter. Whether I have convinced them or not will be seen in the number of them that join and tweet me in 2015. Or participate in the comments section below. So far so good:

//platform.twitter.com/widgets.js

Please read this leaflet carefully before taking to Twitter

Please read this leaflet carefully before taking to Twitter

1. Name of the medicinal product

TWITTIVIR 5% w/w cream

2. Qualitative and quantitative composition

TWITTIVIR 5% medical grade w/w cream (cis:trans isomer 95:5)

3. Pharmaceutical form

Cream for topical application (usually to the finger tips).

4. Clinical particulars

 4.1 Therapeutic indications

TWITTIVIR 5% w/w cream is indicated for the treatment of Anemic Network Infection, Grant Blood Clot, Publication Circulatory Virus and Altmetric Intestinal Flu

4.2 Posology and method of administration

TWITTIVIR 5% w/w cream is suitable for adults, children of 13 years  of age and above, and the elderly. TWITTIVIR 5% w/w cream is for external use only and should not be applied to broken skin, mucous membranes or near the eyes.

4.3 Contraindications

TWITTIVIR 5% w/w cream is contra-indicated in subjects with known hypersensitivity to the product and its components. (group 1)

TWITTIVIR 5% w/w cream is contra-indicated in highly obsessive subjects. (group 2)

TWITTIVIR 5% w/w cream is strongly contra-indicated in subjects that cannot resist a Twitter spat with Louise Mensh. (group 3)

4.9 Overdose

There are rare cases of overdosage of TWITTIVIR 5% w/w cream, usually in patients from group 3 above. The effects can be serious, leading to grumpiness and even, in extreme cases (in parents), child neglect. In such cases, the treatment should be immediately stopped.