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The stem cell trachea scandal

Yesterday night, BBC Newsnight broadcasted an investigation by journalist Deborah Cohen featuring interviews with my colleague Prof Patricia Murray as well as extremely moving testimony by the mum of Shauna Davidson. Shauna’s mum had been misled on the nature of the intervention on her daughter and even on the cause of her death.

Patricia is a stem cell expert and we’ve had a long term (and ongoing) collaboration on using imaging to track stem cells for evaluation of their safety and efficacy. When, a few years ago, she started to get interested in the Macchiarini scandal and realised that similar experiments on patients had been done in the UK (and continued to be done), she contacted me knowing my interest in ethical issues and scientific misconduct. I am proud to have supported her sterling work in uncovering this scandal – I am also pleased that legal threats have failed to silence us. The extent of conflicts of interest and unresolved matters involving major institutions in the UK is massive. So much so that we have had to go all the way to Parliament (see our two contributions to the Science and Technology committee on Research Integrity inquiry) to get (so far unsatisfactory) responses. We hope that the BBC reporting will help bring much needed clarity, but the obfuscating responses of GOSH and MRC to Deborah’s questions are deeply worrying.

See also the excellent and detailed work of Leonid Schneider on this same scandal.

 

The war on (scientific) terror…

Thank you Philip for this post and your support.

Symptoms Of The Universe

I’ve been otherwise occupied of late so the blog has had to take a back seat. I’m therefore coming to this particular story rather late in the day. Nonetheless, it’s on an exceptionally important theme that is at the core of how scientific publishing, scientific critique, and, therefore, science itself should evolve. That type of question doesn’t have a sell-by date so I hope my tardiness can be excused.

The story involves a colleague and friend who has courageously put his head above the parapet (on a number of occasions over the years) to highlight just where peer review goes wrong. And time and again he’s gotten viciously castigated by (some) senior scientists for doing nothing more than critiquing published data in as open and transparent a fashion as possible. In other words, he’s been pilloried (by pillars of the scientific community) for daring to suggest that we do science…

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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.