A RESPONSE FROM CHAD MIRKIN’S GROUP [follow up #1/n]

Some readers might wonder why I am going on about this, so let me tell you: this is a considerably more important story than Stripy Nanoparticles Revisited. If, as I am arguing, some of this science is shaky, then it is not only the way we evaluate scientists and spend public money which are put into question, but the foundation of ongoing clinical trials.

Back to basics: in the section of Mirkin’s group PhD dissertation (previous post) that respond to our critique of their work on Spherical Nucleic Acid / SmartFlare / StickyFlare, they wrote the following:

Additionally, since the commercialization and sale of the nanoflare platform under the trade name Smartflare (Millipore), dozens of researchers around the world have participated in successful sequence-specific gene detection.[80]

Reference [80] correspond to six (half a dozen) articles, 80a to 80f (see below for details and links). Out of these six, only two are actual research papers, and, for both, the SmartFlares are a very minor addition to the work. Out of these two, only one is completely independent of Mirkin/EMD Millipore (the other one comes from Northwestern).

80a) is not primary research; it is an advertorial produced by EMD Millipore.

80b) is not primary research: it is a 300 words congress abstract (no figure). A follow up paper by the same group is discussed here.

80c) is a review and it is a collaboration between Northwestern (Mirkin’s University) and EMD Millipore. CoI statement from the paper: “D. Weldon is the R&D Manager at EMD Millipore responsible for the production of SmartFlares. Patents related to therapeutically targeting Nodal in tumor cells have been awarded to E.A. Seftor, R.E.B. Seftor, and M.J.C. Hendrix.

80d) is a research paper. It does not show in any way that SmartFlares work. It assumes it does. The SmartFlare is a minor part of the article.

80e) is not primary research: it is an advertorial in a magazine funded by company advertising (including EMD Millipore in that very issue). The author is a journalist working for the magazine, not a practicing scientist.

80f) is a research paper. It does not show in any way that SmartFlares work. It assumes it does. SmartFlares are a very minor part of the article. The authors are from Northwestern, i.e. Mirkin’s University.

 

A response from Chad Mirkin’s group

Well nearly. Possibly as close as we might get.

If you have followed the Spherical Nucleic Acid / SmartFlare / StickyFlare story on this blog, you will know that we have raised doubts about the endosomal escape of these nanoparticles which are supposed to reach the cytosol of cells where they could detect mRNAs. We have even published an article on this topic. The Mirkin group has developed the technology and it has been commercialized four years ago for the live cell detection of mRNAs by EMD Millipore.

One PNAS paper on the topic was Briley et al (see here for letter to PNAS and what happened to that letter). In his PhD dissertation (Briley, W. E. (2016), William Edward Briley respond to our criticism. I reproduce the relevant section below. One might note that there is an incredibly simple way to determine the localisation of these particles inside the cells: electron microscopy, a technique which has been used for this exact purpose for over 50 years. We have used it. The results were unambiguous.

2.3 Commentary on the Endosomal Escape of SNAs
Though the endosomal escape of SNA nanostructures such as the Nanoflare and stickyflare is evident based upon their ability to provide sequence-specific information regarding RNA levels and locations within cells, one researcher has concluded that SNAs cannot escape from endosomes.[75] That researcher [That’s me!] is ignoring the many papers now that use such architectures for sequence-specific cell-sorting experiments. Indeed, if these architectures, which are taken up by scavenger-receptor mediated endosomal pathways,[68a] do not escape the endosome, then it is difficult to understand the reports by the many groups who have documented the sequencespecific function of SNAs (all of which require endosomal escape), in antisense gene regulation,[12, 44-45, 53] siRNA gene regulation,[20, 68b, 76] nanoflare gene detection,[47, 67a, 67c, 77] and sticky-flare gene detection.[78] Perhaps the best demonstration of this sequence specificity is in the function of the multiplexed Nanoflare.[67a] This nanoconjugate, discussed in chapter 1, contains two targeting sequences specific to two different genes (actin and survivin). When cells treated with multiplexed nanoflares were subjected to siRNA that specifically knocked down the expression of survivin, a corresponding decrease of fluorescence was observed in the nanoflare’s survivin-associated fluorescence (cy5), but not the actin-associated fluorescence (cy3) compared to control.[67a] Likewise, when cells were subjected to actin-targeting siRNA, a decrease in actin-associated fluorescence was observed, with no decrease of survivin-associated fluorescence.
These results indicate that the detection by nanoflares is unique to the targeted mRNA transcript. To rule out any possible effects of the fluorophores, the Cy5 and Cy3 dyes were switched to the other gene (actin-cy5, survivin-cy3), and again the corresponding sequence-specific responses were observed. Importantly, since the development of the multiplexed nanoflare, other research groups have independently developed nanoflare-like structures capable of sequence-specific detection of 3,[57] and even 4[79] genes simultaneously. Additionally, since the commercialization and sale of the nanoflare platform under the trade name Smartflare (Millipore), dozens of researchers around the world have participated in successful sequence-specific gene detection.[80]
Further evidence of SNA endosome escape can be seen visually through analysis of the sticky-flare construct. If sticky-flares were incapable of escaping endosomes, one would expect consistent colocalization with endosomes. However, this is not the case. The patterns of β-actin targeted sticky-flares, when used in HeLa cells, instead exhibit localization around mitochondria. If such structures were limited to endosomes, it is inconceivable that they would co-localize with specific organelles. This is consistent with mitochondrial localization of multiple RNAs which
has also been identified by other groups, using multiple techniques, in HeLa cells.[74] Sticky-flare release from the endosome was further confirmed by designing sticky-flares targeting a second sequence, a U1 short nuclear RNA (snRNA). U1 snRNA is known to traffic from the cytoplasm to the nucleus. Indeed, cells treated with U1-targeting sticky-flares exhibited specific fluorescence within the nucleus. The pattern observed indicates nuclear localization through endosomal escape and sequence-specific tagging of an RNA that is actively transported into the nucleus. Again, this would not be possible if such structures were confined exclusively to endosomes. Speculation that SNAs do not escape endosomes has been fueled by the observation that the fluorescence pattern of β-actin in MEFs is punctate, which has been interpreted as an indication that the sticky-flares are simply trapped in endosomes. However, β-actin is well known to exhibit punctate fluorescence in many cases, an observation made by others in multiple cell lines, including MEFs.[81] Punctate fluorescence is very common in RNA-labeling studies and well known by researchers familiar with FISH.82 This is due to the fact that RNA is often packaged into large RNA-containing granules, which facilitates transport and translational control of the included transcripts.[82b, 83] Such packaging into granules has been extensively studied using β-actin mRNA. Thus, the fact that sticky-flares targeting β-actin were packaged into granules as previously observed, while U1-targeting sticky-flares were specific to the nucleus in the same cell line, demonstrates the functionality of the construct.
Taken together, the success of the many groups who use flare architectures for the detection and knockdown of RNA in cells, and the work of dozens of labs studying related nanoparticle constructs, provide unambiguous evidence of the ability for such architectures to escape the endosome and participate in reactions exclusive to the cytosol. The mechanism of endosomal escape for nanoparticle-based vehicles is currently unknown and is an interesting and important question that is actively being investigated by many in the field.[84]

ref2ref3

Briley, W. E. (2016). Investigation and manipulation of the local microenvironment of spherical nucleic acid nanoconjugates (Order No. 10117274). Available from ProQuest Dissertations & Theses Global. (1795522748). Retrieved from https://search.proquest.com/docview/1795522748?accountid=12117

Quantum dots for Immunofluorescence

Guest post by Dave Mason

In modern cell biology and light microscopy, immunofluorescence is a workhorse experiment. The same way antibodies can recognise foreign pathogens in an animal, so the specificity of antibodies can be used to label specific targets within the cell. When antibodies are bound to a fluorophore of your choice, and in combination with light microscopy, this makes for a versatile platform for research and diagnostics.

Most small-dye based fluorophores that are used in combination with antibodies suffer from a limitation; hit them with enough light and you irreversibly damage the fluorochrome, rendering the dye ‘invisible’ or photobleached. This property is the basis of several biophysical techniques such as Fluorescence Recovery After Photobleaching (FRAP) but for routine imaging it is largely an unwanted property.

Over 20 years ago, a new class of fluorescent conjugate was introduced in the form of Quantum Dots (QDots); semiconductor nanocrystals that promised increased brightness, a broad excitation and narrow emission band (good when using multi-channel imaging) and most importantly: no photobleaching. They were hailed as a game changer: “When the methods are worked out, they’ll be used instantly” (ref). With the expectation that they would “…soon be a standard biological tool” (ref).

So what happened? Check the published literature or walk into any imaging lab today and you’ll find antibodies conjugated to all manner of small dyes from FITC and rhodamine to Cyanine and Alexa dyes. Rarely will you find QDot-conjugated antibodies used despite them being commercially available. Why would people shun a technology that seemingly provides so many advantages?

Based on some strange observations, when trying to use QDot-conjugated antibodies, Jen Francis, investigated this phenomenon more closely, systematically labelling different cellular targets with Quantum dots and traditional small molecule dyes.

Francis_et_alFig3_GM

Figure 3 from doi:10.3762/bjnano.8.125 shows Tubulin simultaneously labelled with small fluorescent dye (A) and QDots (B). Overlay shows Qdot in green and A488 in Magenta. See paper for more details. See UPDATE below.

The work published in the Beilstein Journal of Nanotechnology (doi: 10.3762/bjnano.8.125) demonstrates a surprising finding. Some targets in the cell such as tubulin (the ‘gold standard’ for QDot labelling) label just as well with the QDot as with the dye (see above). Others however, including nuclear and some focal adhesion targets would only label with the organic dye.

2190-4286-8-125-graphical-abstract.png

The important question of course is: why the difference in labelling when using Quantum Dots or dyes? This is discussed in more detail in the paper but one explanation the evidence supports is that it is the size of the QDots that hinder their ability to access targets in the nucleus or large protein complexes. This explanation further highlights how little we really know about the 3D structure of protein complexes in the cell and the effect of fixation and permeabilisation upon them. Why for example, can tubulin be labelled with QDots but F-actin cannot, despite them both being abundant filamentous cytosolic structures? At this point we can’t say.

So why is this study important? Publication bias (the preferential publication of ‘positive’ results) has largely hidden the complications of using QDots for immunofluorescence. We and others have spent time and money, trying to optimise and troubleshoot experiments that upon closer study, have no chance of working. We therefore hope that by undertaking and publishing this study, other researchers can be better informed and understand when (or whether) it might be appropriate to use Quantum Dots before embarking on a project.

This paper was published in the Beilstein Journal of Nanotechnology, an Open Access, peer-reviewed journal funded entirely by the Beilstein-Institut.

UPDATE [2017-06-13]: in response to a comment below, I’ve updated the overlay figure to use green/magenta instead of green/red. The original figure can be seen in the paper or here.

Lettre d’Angleterre: Souveraineté

English version below.

Le Front National veut soi-disant redonner à la France sa souveraineté en la sortant de l’Union Européenne. Aux oreilles d’un résidant en Angleterre, ce discours est familier : c’est celui, répété ad nauseam par Leave.eu et les supporters du Brexit : reprenons le contrôle de nos lois et de nos frontières (‘Take back control’). C’est aussi une illusion. Pour deux raisons.

La première raison est que le parlement du Royaume-Uni était souverain. Ce n’est pas moi qui le dis mais le livre blanc produit par le gouvernement de Theresa May lui-même : « La souveraineté du Parlement est un principe fondamental du Royaume-Uni. Bien que le Parlement soit resté souverain durant toute la période où nous avons été membre de l’Union Européenne, on n’en pas toujours eu le sentiment. »

La deuxième raison, peut-être contre-intuitive, est que, au-delà de la souveraineté du processus législatif, la capacité du Royaume-Uni à prendre en main son destin de manière indépendante est mise à mal de manière spectaculaire par le Brexit. Son gouvernement, isolé, sera encore moins capable de résister aux lobbys, comme l’explique admirablement… Rupert Murdoch répondant à la question pourquoi êtes-vous tellement opposé à l’Union Européenne : « c’est facile, quand je vais au 10 Downing Street, ils font ce que je leur demande, quand je vais à Bruxelles, ils m’ignorent complètement. »  On voit mal un Royaume-Uni isolé faire face à Microsoft, Google ou Apple. De plus, quittant le marché commun, le Royaume-Uni doit négocier des accords bilatéraux avec d’autres pays, et, ces pays, vont imposer leurs conditions, d’autant plus qu’il est clair que le Royaume-Uni est en position de faiblesse. Theresa May a choisi d’aller en Inde pour l’un de ses premiers voyages après le referendum. L’Inde insiste que tout accord économique devra s’accompagner d’un assouplissement des règles d’immigration et de visa. Theresa May devra donc choisir entre le contrôle de l’immigration (‘Take back control’) et un accord commercial avec l’Inde. Chaque pays (y compris ceux de l’Union Européenne) aura ses propres demandes auxquelles le Royaume-Uni devra bien souvent se soumettre.

Le Brexit est un abandon de souveraineté.

*********

The National Front is pretending that it will give France its sovereignty back from Brussels. To my ears, this is familiar discourse: it is the one peddled ad nauseam by Leave.eu and other Brexit supporters: Take back control (of laws, borders). It is an illusion. For two reasons.

The first reason is that the UK Parliament was sovereign. Not my word, but the white paper produced by the government of Theresa May herself: “The sovereignty of Parliament is a fundamental principle of the UK constitution. Whilst Parliament has remained sovereign throughout our membership of the EU, it has not always felt like that.

The second reason, maybe counter-intuitive, is that, beyond the sovereignty of the legislative process, the UK capacity to shape its own destiny is badly damaged by Brexit. Its government, isolated, will be even less able to resist pressures from lobbies, as explained transparently by…  Rupert Murdoch, who, replying to the question of why he was so opposed to the European Union, replied. ”That’s easy, when I go into Downing Street they do what I say; when I go to Brussels they take no notice.’ It is hard to imagine the UK, isolated, courageously facing Microsoft, Google ou Apple. Furthermore, leaving the common market, the UK must negotiate new bilateral agreement with third countries, and, these countries will impose their conditions, especially since it is clear the UK is in a weak position. Theresa May chose to go to India for one of her first travel after the referendum. India insists that any agreement will need to include a softening of immigration and visa rules. Theresa May will therefore need to choose between control of immigration (‘Take back control’) and a commercial agreement with India. Each country (including those of the EU will have their own demands to which the UK will often need to submit.

Brexit is a loss of sovereignty.

Lettre d’Angleterre

J’ai cru que la doctrine et l’histoire d’un peuple si extraordinaire méritaient la curiosité d’un homme raisonnable.

Voltaire, 1734, Lettre d’Angleterre.

Je ne suis certes pas Voltaire, mais, peut-être puis-je prétendre être un homme raisonnable?

J’habite Liverpool depuis 15 ans. Les événements politiques en Angleterre durant les 12 derniers mois sont extraordinaire, et, particulièrement important à considérer à trois jours du premier tour de l’élection présidentielle française.

Ce qui se passe ici, c’est une dislocation de la société, travaillée par les mensonges et la haine. Avec la légitimation toujours plus grande d’un discours qui fait appel à « la volonté du peuple » pour « écraser les saboteurs » . Un discours violent qui rejette toute discussion rationnelle et informée, au profit d’un tribalisme viscéral, émotionnel et souvent xénophobe.

C’est pourquoi je tremble lorsque j’entends ce même appel au peuple, « le gros animal, un monstre qui ne connaît ni la vertu ni la raison » (Philippe Val/Socrate) chez deux candidats qui peuvent se qualifier pour le second tour (Au-Nom-Du-Peuple-LePen et La-Force-du-Peuple-Mélenchon): je suis affolé.

Ces deux candidats qui ont des amitiés particulières pour divers dictateurs.

Ces deux candidats qui partagent la volonté explicite ou implicite de détruire l’Europe et ses institutions. L’Europe qui a contribué énormément à la prospérité retrouvée de cette belle ville de Liverpool et de beaucoup d’autres régions défavorisées sur le continent. L’Europe qui a garanti la paix pendant les derniers 70 ans (un ex-dirigeant du parti conservateur a proféré des menaces de guerre contre l’Espagne à propos de Gibraltar. En Avril 2017). L’Europe qui, seule, nous donne une voix suffisamment forte pour défendre nos principes démocratiques, parler d’égal à égal avec les autres grandes puissances, qu’elles soient des états ou des multinationales, et, affronter le défi majeur du changement climatique.

L’enjeu ne pourrait être plus grand.

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.

vessels_neubias_banner

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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