SmartFlare controversy: independent confirmation of endosomal localization

Check this previous post for a quick summary of the SmartFlare controversy, or read all SmartFlare-related posts if you are really passionate.

At the centre of the SmartFlare controversy is the rather simple question, from an experimental point of view, of how many Spherical Nucleic Acids (to use Chad Mirkin’s terminology), if any, escape the endosomal pathway.

In contradiction with Chad Mirkin’s many peer reviewed articles and EMD Millipore marketing material, we concluded (Mason et al, 2016) that the Spherical Nucleic Acids do not escape endosomes and do not detect cytosolic mRNAs.

A few days ago, Sven Budik et al, an Austrian group published their evaluation of the SmartFlare in the context of equine embryo development. They write: “In all positive cells,
regardless of whether they occurred in equine conceptus, trophoblastic vesicle or fibroblast cell culture, the fluorescence signal showed a spotted pattern that is in accordance with the observations of Mason et al. (2016).

They also used electron microscopy to look at the intracellular localization of the particles. Here is the relevant part of their discussion and conclusion (emphasis mine):

The present study indicates that the intracellular process of nanogold particle uptake is endocytic and endosomal with a lysosomal sorting after longer incubation periods. This finding is in agreement with results from HeLa cells in vitro (Gilleron et al. 2013). Similarly, nanoparticles injected intravenously were taken up by endocytosis and later
clustered in lysosomes primarily in macrophages (Sadauskas et al. 2007). The incorporation time of lipid nanoparticlecontaining short interfering RNA gold particles in HeLa cells was similar (Gilleron et al. 2013) to that demonstrated in equine trophoblast vesicles in the present study. Accumulation of SmartFlare probes in residual bodies may be a consequence of increased stability of the immobilised oligonucleotides adjacent to the nanogold particles due to enhanced nuclease resistance (Rosi et al. 2006). In accordance with the results of Mason et al. (2016), we observed no or very few nanogold particles free in the cytoplasm, confirming a primarily endosomal and lysosomal localisation.

This observation raises the question how a specific SmartFlare probe is able to detect its target mRNA located in the cytoplasm. One possible explanation for the generation of lysosome-located specific fluorescence signals by SmartFlare probes could be the existence of specific RNA sequences imported for subsequent degradation into lysosomes (Fujiwara et al. 2013). Further studies using qRT-PCR investigating the isolated lysosomal fraction before and after incubation with specific SmartFlare probes are necessary to confirm this hypothesis. An 18S RNA nano-flare probe had a dose-dependent cytotoxic effect on porcine fetal fibroblasts (Fu et al. 2016). In contrast, no cytotoxic effects or changes in morphology after incorporation of antisense oligonucleotide nanogold particles in a mouse endothelial cell line were observed by Rosi et al. (2006). In addition, in the present study, there was no evidence that incubation with the SmartFlare probes had a toxic effect on the equine cells tested, even at higher concentrations. This is in accordance with the results of Pan et al. (2009) demonstrating that 15-nm gold particles have only low cytotoxic effects compared with the detrimental effects of small 1.4-nm gold particles.

In conclusion, SmartFlare probes pass into early equine conceptuses at stages used for embryo transfer, as well as trophoblast vesicles and cells cultured in vitro. In these early ZP equine conceptuses, the time frame (.5 and ,24 h) for SmartFlare uptake would be suitable for practical applications in commercial embryo transfer programs. Therefore, these probes are suggested to be applicable to pre-implantation genetic diagnosis before transfer of these conceptuses to the recipient.

In summary, the authors’ results are entirely consistent with our observations. They conclude, quite reasonably, that if SmartFlares detect mRNAs whilst being in endosomes, they cannot directly detect cytosolic mRNAs. This is in direct contradiction with Mirkin et al and EMD Millipore. Then, they propose that if the SmartFlares work, they maybe detect mRNAs which are in endosomes. This an interesting hypothesis that will require further study and is very different from anything published by Mirkin and EMD Millipore (the relevant reference is here). Since Budik et al do not provide any evidence that the SmartFlares actually detect mRNAs in the first place, maybe a simpler explanation is that the SmartFlares signal is unspecific and result from the probe degradation by nucleases in endosomes.

Thoughts on #LiveTweeting

Dave Mason on why you should be live-tweeting at conferences

Blog and Log

As a part of the Centre for Cell Imaging and a member of the Microscopy and BioImage Analysis community, I occasionally get away to conferences like the recent NEUBIAS training school and symposium in Portugal.

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Since having joined Twitter last year (@dn_mason), this is the second conference that I’ve been to, and as a result, was the second time I tried (with reasonable success) to Live Tweet at the conference.

Live What Now?

Going right back to basics, Twitter is a platform for broadcasting small messages (of ~140 characters). Some describe it as micro-blogging. To many, the brevity of each tweet is both it’s greatest strength and also one of the most frustrating features.

Live tweeting, is basically the act of providing a running commentary of a seminar, event or even a whole conference. All of the tweets associated with such an event can be tied together using…

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Publication bias. Grant bias.

All academics writing grants will tell you this: if you want to be successful when applying to a thematic research grant call, you must tick all of the boxes.

Now, imagine that you are a physicist, expert in quantum mechanics. A major funding opportunity arises, exactly matching your interest and track record. That is great news. Obviously you will apply. One difficulty however is that, amongst other things, the call specifies that your project should lead to the “development of highly sensitive approaches enabling the simultaneous determination of the exact position and momentum of a particle“.

At that point, you have three options. The first one is to write a super sexy proposal that somehow ignores the Heisenberg principle. The second option is to write a proposal that addresses the other priorities, but fudges around that particular specification, maybe even alluding to the Heisenberg principle. The third option is to renounce.

The first option is dishonest. The second option is more honest, but, in effect, is not so different from the third: your project is unlikely to get funded if you do not stick to the requirements of the call, as noted above. The third option demonstrates integrity but won’t help you with your career, nor, more importantly with doing any research at all.

And so, you have it. Thematic grant calls that ask for impossible achievements, nourished by publication bias and hype, further contribute to distortion of science.

OK, I’ll confess: I have had a major grant rejected. It was a beautiful EU project (whether BREXIT is partly to blame I do not know). It was not about quantum mechanics but about cell tracking. The call asked for simultaneous “detection of single cells and cell morphologies” and “non-invasive whole body monitoring (magnetic, optical) in large animals” which is just about as impossible as breaking the Heisenberg principle, albeit for less fundamental reasons. We went for option 2. We had a super strong team.

How to Characterize Gold Nanoparticles’ Surface?

Guest post by Elena Colangelo

Our Topical Review on the characterization of gold nanoparticles (GNPs) has just been published in the Bionconjugate Chemistry Special Issue “Interfacing Inorganic Nanoparticles with Biology”.

The literature is abounding in works on GNPs for applications in biology, catalysis and sensing, among others. GNPs’ appeal resides in their optical properties, together with the well-developed methods of synthesis available and the possibility of functionalizing their surface with small molecules of interest, which can readily self-assemble on the GNPs’ surface forming a monolayer.

However, allegedly the structure and organization of self-assembled monolayers (SAMs) at the GNPs’ surface are in fact aspects too often neglected [though surely not on this blog; RL]. Such elucidation is challenging experimentally, but it is crucial not only to ensure reproducibility, but also to design nanosystems with defined (bio)physicochemical and structural properties, which could then be envisioned to assemble in more complex systems from a “bottom-up” approach.

Our Topical Review gives an overview of the current knowledge and methods available to characterize the GNPs’ surface with different molecular details.

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Cartoon illustrating the different levels of GNPs’ surface characterization discussed in the Topical Review.

First, the experimental methods commonly used to provide the basic characterization of functionalized GNPs, such as identification and quantification of the ligands within the monolayer, are detailed with the aid of some examples.

Second, the experimental methods providing information on the monolayer thickness and compactness are reviewed.

Third, considering that the SAM’s thickness and compactness do not only depend on the amount of ligands within the monolayer, but also on their conformation, the experimental methods that can provide such insights are recapitulated. However, we also stressed on the limitations intrinsic to these methods and on the challenges associated to the determination of the structure of SAMs on GNPs.

Fourth, we summarized some of the approaches used to give insights into the organization of different ligands within a SAM. Indeed, mixed SAMs on GNPs are useful since they can impart to the nanoparticles different functionalities and offer a way to tune their stability.

Fifth, highlighting again the limited insights into the SAM’s structure and organization that can be gathered with experimental techniques, we detailed some examples where a combination of experimental and computational approaches was able to provide a compelling description of the system and to assess molecular details that could not have been revealed experimentally.

Overall, this Topical Review gives emphasis on the importance of GNPs’ surface characterization and on fact that even though a number of experimental techniques are available, they are intrinsically limited and they cannot provide a fully detailed picture. Hence, it is advantageous to combine experimental and theoretical approaches to design nanoparticles with desired (bio)physicochemical properties [such as, e.g., our paper under review, currently available as a preprint; RL].

Time to reclaim the values of science

This post is dedicated to Paul Picard, my grand dad, who was the oldest reader of my blog. He was 17 (and Jewish) in 1939 so he did not get the chance to go to University. He passed away on the first of October 2016. More on his life here (in French) and some of his paintings (and several that he inspired to his grandchildren and great-grandchildren). The header of my blog is from a painting he did for me

A few recent events of vastly different importance eventually triggered this post.

A  (non-scientist) friend asked my expert opinion about a campaign by a French environmental NGO seeking to  raise money to challenge the use of nanoparticles such as E171 in foods. E171 receives episodic alarmist coverage, some of which were debunked by Andrew Maynard in 2014. The present campaign key dramatic science quote “avec le dioxyde de titane, on se retrouve dans la même situation qu’avec l’amiante il y a 40 ans {with titanium dioxide, we are in the same situation than we were with asbestos 40 years ago}” is from Professor Jürg Tschopp. It comes from an old media interview (2011, RTS) that followed a publication in PNAS. We cannot ask Professor Tschopp what he thinks of the use of this 5 years old quote: unfortunately he died shortly after the PNAS publication. The interpretation of this article has been questioned since: it seems likely that the observed toxicity was due to endotoxin contamination rather than the nanomaterials themselves. There is on the topic of nanoparticles a high level of misinformation and fear that finds its origins (in part) in how the scientific enterprise is run today. Incentives are to publish dramatic results in high impact factor journals which lead many scientists to vastly exaggerate both the risks and the potential of their nanomaterials of choice. The result is that we build myths instead of solid reproducible foundations, we spread disproportionate fears and hopes instead of sharing questions and knowledge. When it comes to E171 additives in foods, the consequences of basing decisions on flawed evidence are limited. After all, even if the campaign is successful, it will only result in M&M’s not being quite as shiny.

I have been worried for some time that the crisis of the scientific enterprise illustrated by this anecdote may affect the confidence of the public in science. In a way, it should; the problems are real, lead to a waste of public money, and, they slow down progress. In another way, technological (including healthcare) progress based on scientific findings has been phenomenal and there are so many critical issues where expertise and evidence are needed to face pressing humanities’ problems that such a loss of confidence would have grave detrimental effects. Last week, in the Spectator, Donna Laframboise published an article entitled “How many scientific papers just aren’t true? Enough that basing government policy on ‘peer-reviewed studies’ isn’t all it’s cracked up to be“. The article starts by a rather typical and justified critique of peer review, citing (peer-reviewed) evidence, and then, moves swiftly to climate change seeking to undermine the enormous solid body of work on man-made climate change. It just happens that Donna Laframboise is working for “a think-tank that has become the UK’s most prominent source of climate-change denial“.

One of the Brexit leaders famously declared that “people in this country have had enough of experts”. A conservative MP declared on Twitter that he”Personally, never thought of academics as ‘experts’. No experience of the real world. Yesterday, Donald Trump, a climate change denier was elected president of the USA: “The stakes for the United States, and the world, are enormous” (Michael Greshko writing for the National Geographic). These are attacks not just on experts, but on knowledge itself, and, the attacks extends to other values dear to science and encapsulated in the “Principle of the Universality of Science“:

Implementation of the Principle of the Universality of Science is fundamental to scientific progress. This Principle embodies freedom of movement, association, expression and communication for scientists, as well as equitable access to data, information and research materials. These freedoms are highly valued by the scientific community and generally well accepted by governments and policy makers. Hence, scientists are normally able to travel to international meetings, associate with colleagues and freely express their opinions regardless of factors such as ethnic origin, religion, citizenship, language, political stance, gender, sex or age. However, this is not always the case and so it is important to have mechanisms in place at the local, national and international levels to monitor compliance with this principle and intervene when breaches occur. The International Council for Science (ICSU) and its global network of Members provide one such mechanism to which individual scientists can turn for assistance. The Principle of the Universality of Science focuses on scientific rights and freedoms but implicit in these are a number of responsibilities. Individual scientists have a responsibility to conduct their work with honesty, integrity, openness and respect, and a collective responsibility to maximize the benefit and minimize the misuse of science for society as a whole. Balancing freedoms and responsibilities is not always a straightforward process. For example, openness and sharing of data and materials may be in conflict with a scientist’s desire to maintain a competitive edge or an employer’s requirements for protecting intellectual property. In some situations, for example during wars, or in specific areas of research, such as development of global surveillance technologies, the appropriate balance between freedoms and responsibilities can be extremely difficult to define and maintain. The benefits of science for human well-being and development are widely accepted. The increased average human lifespan in most parts of the world over the past century can be attributed, more or less directly, to scientific progress. At the same time, it has to be acknowledged that technologies arising from science can inadvertently have adverse effects on people and the environment. Moreover, the deliberate misuse of science can potentially have catastrophic effects. There is an increasing recognition by the scientific community that it needs to more fully engage societal stakeholders in explaining, developing and implementing research agendas. A central aspect of ensuring the freedoms of scientists and the longer term future of science is not only conducting science responsibly but being able to publicly demonstrate that science is being conducted responsibly. Individual scientists, their associated institutions, employers, funders and representative bodies, such as ICSU, have a shared role in both protecting the freedoms and propagating the responsibilities of scientists. This is a role that needs to be explicitly acknowledged and embraced. It is likely to be an increasingly demanding role in the future.

It is urgent that we, scientists, reclaim these values of humanity, integrity and openness and make them central (and visibly so) in our Universities. To ensure this transformation occurs, we must act individually and as groups so that scientists are evaluated on their application of these principles. The absurd publication system where we (the taxpayer) pay millions of £$€ to commercial publishers to share hide results that we (scientists) have acquired, evaluated and edited must end. There are some very encouraging and inspiring open science moves coming from the EU which aim explicitely at making “research more open, global, collaborative, creative and closer to society“. We must embrace and amplify these moves in our Universities. And, as many, e.g. @sazzels19 and @Stephen_curry have said, now more than ever, we need to do public engagement work, not with an advertising aim, but with a truly humanist agenda of encouraging curiosity, critical thinking, debates around technological progress and the wonders of the world.

 

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.