open science

How to Elucidate the Structure of Peptide Monolayers on Gold Nanoparticles?

I have recently submitted my PhD thesis and we have now pre-printed on bioRxiv the work constituting its major chapter. Together with the pre-print, the data have been made publicly available in an online repository of the University of Liverpool. Well isn’t it perfect timing that this week is open access week? 😉

This work has been conducted nearly entirely during the 2 years of my PhD spent at the A*STAR Institute of Materials Research and Engineering (IMRE) and at the A*STAR Institute of High Performance Computing (IHPC) in Singapore.

In this study, peptide-capped gold nanoparticles are considered, which offer the possibility of combining the optical properties of the gold core and the biochemical properties of the peptides.

In the past, short peptides have been specifically designed to form self-assembled monolayers on gold nanoparticles. Thus, such approach was described as constituting a potential route towards the preparation of protein-like nanosystems. In other words, peptide-capped gold nanoparticles can be depicted as building-blocks which could potentially be assembled to form artificial protein-like objects using a “bottom-up” approach.

However, the structural characterization of the peptide monolayer at the gold nanoparticles’ surface, essential to envision the design of building-blocks with well-defined secondary structure motifs and properties, is poorly investigated and remains challenging to assess experimentally.

In the pre-printed manuscript, we present a molecular dynamics computational model for peptide-capped gold nanoparticles, which was developed using systems characterized by mean of IR spectroscopy as a benchmark. In particular, we investigated the effect of the peptide capping density and the gold nanoparticle size on the structure of self-assembled monolayers constituted of either CALNN or CFGAILSS peptide.

The computational results were found not only to well-reproduce the experimental ones, but also to provide insights at the molecular level into the monolayer’s structure and organization, e.g., the peptides’ arrangement within secondary structure domains on the gold nanoparticle, which could not have been assessed with experimental techniques.

Overall, we believe that the proposed computational model will not only be used to predict the structure of peptide monolayers on gold nanoparticles, thus helping in the design of bio-nanomaterials with well-defined structural properties, but will also be combined to experimental findings, in order to obtain a compelling understanding of the monolayer’s structure and organization.

In this sense, we would like to stress that, in order to improve data reproducibility, enable further analysis and the use of the proposed computational model for peptide-capped gold nanoparticles, we are making the data and the custom-written software to assemble and analyse the systems publicly available.

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Snapshots of the final structure of the simulated 5 (left) and 10 (right) nm CFGAILSS-capped gold nanoparticle, illustrating different content and organization of secondary structure motifs.

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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/

Gold nanorods to shine light on the fate of implanted stem cells

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Joan Comenge

This is a guest post by Joan Comenge

Our work regarding the use of gold nanorods as contrast agents for photoacoustic tracking of stem cells has been just published (or here*). You can find all the technical details of the work there, so I will try to explain here the work for the readers who are not very familiar with our field.

It is important to have the appropriate tools to evaluate safety and efficacy of regenerative medicine therapies in preclinical models before they can be translated to the clinics. This is why there is an interest in developing new imaging technologies that enable real time cell tracking with improved sensitivity and/or resolution. This work is our contribution to this field.

To distinguish therapeutic cells from the patient’s own cells (or here from the mouse’s own cell),  the therapeutic cells have to be labelled before they are implanted. It is well known, that biological tissue is more transparent to some regions of the light spectrum than others. This fact is very easy to try at home (or at your favourite club): if you put your hand under a green light, no light will go through it, whilst doing the same under a red light the result will be very different. That means that red light is less absorbed by our body. Near infrared light is even less absorbed and this is why this region of the spectrum is ideal for in vivo imaging. Therefore, we made our cells to absorb strongly in the near infrared so we can easily distinguish them.

Gold nanoparticles of different sizes and shapes (synthesis and picture by Joan Comenge).

Gold nanoparticles of different sizes and shapes (synthesis and picture by Joan Comenge).

To do this, we labelled cells with gold nanoparticles. Interestingly, the way gold nanoparticles interact with light depends on how their electrons oscillate. That means that size and shape of the nanoparticles determine their optical properties, and this is one of the reasons why we love to make different shapes of nanoparticles. In particular, gold nanorods strongly absorb in the near infrared and they are ideal contrast agents for in vivo imaging.

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Figure reproduced from: The production of sound by radiant energy; Science 28 May 1881; DOI: 10.1126/science.os-2.49.242

We have now cells that interact with light in a different way than the tissue. The problem is that light is scattered by tissue, so resolution is rapidly lost as soon as you try to image depths beyond 1 mm. Obviously, this is not the best for in vivo imaging. Luckily for us, Alexander Graham Bell realised 130 years ago that matter emits sounds when is irradiated by a pulsed light. This is known as the photoacoustic effect and it has been exploited recently for bioimaging. Photoacoustic imaging combines the advantages of optical imaging (sensitivity, real-time acquisition, molecular imaging) and the good resolution of ultrasound imaging because ultrasounds (or phonons), contrarily to photons, are not scattered by biological tissue.
GNR-35.2Si3 in cells_16

Silica-coated gold nanorods inside cells

To optimise the performance of our gold nanorods, we coated them with silica. Silica is glass and therefore it protects the gold core without interfering with its optical properties. This protection is required to maintain gold nanorods isolated inside cells since nanorods are entrapped in intracellular vesicles, where they are very packed. The absence of a protective coating ultimately would result in a broader and less intense absorbance band, which would be translated to a less intense photoacoustic signal and consequently a lower sensitivity in cell detection. This of special importance in our system, a photoacoustic imaging system developed by iThera Medical which uses a  multiwavelength excitation to later deconvolute the spectral information of the image to find your components of interest. Thus, narrow absorption bands helps to improve the detection sensitivity even further. With this we demonstrated that we were able to monitor a few thousand nanorods labelled-cells with a very good 3D spatial resolution for 15 days. This allowed for example to see how a cell cluster changed with time, see how it grows or which regions of the cell cluster shows the highest cell density. In addition, this work opens the door to new opportunities such as  multilabelling using gold nanorods of different sizes and consequently different optical properties to observe simultaneously different type of cells. We also believe that not only stem cell therapies, but also other fields that are interested in monitoring cells such as cancer biology or immunology can benefit from the advances described in our work.

You can find the original publication here (or here*).
All the datasets are available via Figshare.

This work was supported by the UK Regenerative Medicine Platform Safety and efficacy, focusing on imaging technologies. Joan Comenge was funded by the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme. The in vivo imaging was done in the Centre for Preclinical Imaging, the Electron Microscopy in the Biomedical EM unit and the Optical Microscopy in the Centre for Cell Imaging.

* the alternative link is to 50 free e-prints; the link will be removed once the paper is fully open access (in a couple of days).

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Cluster of gold nanorod-labelled cells imaged by photoacoustic imaging three days after implantation in mice.

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.

 

 

 

 

 

The Spherical Nucleic Acids mRNA Detection Paradox

We are publishing today “The Spherical Nucleic Acids mRNA Detection Paradox“, the outcome of an open science project which started with an Hns student last year. In the last 12 months, we have reported in quasi real-time our experiments, protocols and analyses in an open science notebook and shared the data on our repository. The data are also stored at FigShare (e.g. Electron Microscopy results).

In addition to being exciting scientifically (he says!), this has been an experiment in how do science in the open using the tools of the 21st century to share information and solicit feedback. It is therefore fitting to publish it on a platform that challenges conventional modes of peer review.

We have chosen ScienceOpen where publication happens immediately (a couple of hours from submission to publication), followed by open peer review. In the coming weeks and months, I hope that many scientists will provide their expert evaluation of our article. In particular, Chad Mirkin will be invited to provide a review.

This article is important to all scientists who are using nanoparticles for imaging and sensing inside living cells. It should also be particularly relevant to past, current and prospective customers of the SmartFlares. Here is the abstract:

From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy.  In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently-labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.

The paper will be typeset in the next few days and open peer review will be open from that point. Comments are already possible. Thank you to Dave Mason, Gemma Carolan, Joan Comenge and Marie Held for their contributions to this work.