Author: Raphaël Lévy

Happy holidays, the preprint is out there!

This is a guest post by Marie Held. Credits are given to Andrew Plested for the title/timing of this post (see here).

It had been a long time coming but the preprint of our manuscript Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy is now available on BioRxiv. Together with the preprint, the associated large body of imaging data has been made publicly available in the online Image Data Resource (IDR) repository. Through the power of OMERO you can have a play with the data or even download it and then re-analyse it.

The imaging has been conducted on the Zeiss Z.1 Lightsheet microscope at the Centre for Cell Imaging at the University of Liverpool, whose ever helpful staff enabled us generating lots and lots of image data and providing an efficient infrastructure for data storage as well as support for image and subsequent data analysis. Of course this is only half the story because the imaging would not have been possible without generating the samples first: A big thank you also goes to the students and academic staff in the Institute of Translational Medicine at the University of Liverpool.

In this study we have generated organoids from dissociated and re-aggregated mouse embryonic kidney tissue and imaged them with a light sheet fluorescence microscope. The microscope optically sections the samples, therefore preserving the three-dimensional context of the sample throughout imaging. We have found organotypic kidney structures in the organoids and evidence for the maturation of cells to the point of forming glomeruli, the basic filtration unit of the kidney. A functional assay showed that the developed tubules display secretory function.

Most importantly though, we have also performed live imaging of organoids made from genetically tagged fluorescing cells. The light sheet microscopy setup combines an illumination that is perpendicular to the detection. Therefore, full frame images can be recorded rapidly and only the section of the sample that is recorded is illuminated, thus vastly reducing photobleaching and phototoxic effects that limit long-term live fluorescence imaging in wide field and in particular confocal scanning fluorescence microscopy. We have then tracked the fluorescing cells with the help of an automated algorithm and subsequently analysed the generated tracking data. We have {started to} analyse the tracking data and can now quantitatively compare between experiments.

Yet, there is so much more that can be done with the images and data and we would love to see which ideas and approaches others might have, so please do not be shy to dig in and have a play. Be sure to let us know though.

Featured image caption: Organoid of mouse embryonic kidney cells formed following dissociation and re-aggregation of embryonic kidney rudiments. Yellow: Pax2+ cells indicating the metanephric mesenchyme, a prerequisite for nephron development, Red: Peanut-agglutinin staining basement membranes of the ureteric tree and developed nephrons.

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Mind-boggling plagiarism of this blog

In January 2015, someone went to the effort of creating a fake raphazlab blog as the stripy nanoparticles controversy was descending from a scientific debate into the gutters of online discussions.

Fast forward three years. The Spherical Nucleic Acids controversy is slowly heating up. Chad Mirkin continues to win prizes after prizes, but he is unseemly asked to comment on the failing SmartFlare technology (the commercial name of the Spherical Nucleic Acids) by Dalmeet Singh writing for Chemistry World.

Dalmeet writes:

But Chad Mirkin, a chemist at Northwestern University in the US, who developed the precursor to SmartFlares, nanoflares, pointed Chemistry World to more than 30 papers, which, he says, have successfully used the technology.

Chemistry World contacted a number of groups that have used SmartFlares. Hirendranath Banerjee, a molecular biologist at Elizabeth City State University in North Carolina, describes SmartFlares as a ‘very novel and effective technique’, noting that it has been helpful in evaluating gene expression experiments in his lab.2

Now comes the mind-boggling part.

The introduction of Hirendranath’s paper (reference 2 above) is largely plagiarized… from this very blog. From the very first SmartFlare post on this blog, entitled How smart are the SmartFlares?

Below, is an excerpt from my post with, in red, the sentences that reference 2 copied.

RNA molecules play crucial roles in cells such as coding, decoding, regulation, and expression of genes, yet they are much more difficult to study. SmartFlares are nanoparticle-based probes for the detection and imaging of RNA in live cells. Could they become the GFP of the RNA world?

 Many certainly believe this to be the case. SmartFlare ranked second in TheScientist top ten 2013 innovations, with one of the judges, Kevin Lustig, commenting “These new RNA detection probes can be used to visualize RNA expression in live cells at the single-cell level.”  The following year, SmartFlare won an R&D100 award. The technology comes from Chad Mirkin’s lab at Northwestern University. Chad Mirkin is the winner of numerous prestigious prizes and a science adviser to the President of the United States. The scientific articles introducing the SmartFlare concept (under the name of Nano-Flare) were published in the Journal of the American Chemical Society in 2007, ACS Nano in 2009, etc. In 2013, the SmartFlare technology was licensed to EMD Millipore. Here is one of their promotional video:

For a molecular sensor to work, it needs a detection mechanism. The principle of the SmartFlare is explained from 0:45. A capture oligonucleotide (i.e. DNA) is bound to the gold nanoparticles. A reporter strand is bound to the capture strand. The reporter strand carries a fluorophore but that fluorophore does not emit light because it is too close to the gold (the fluorescence is “quenched”). In the presence of the target RNA, the reporter strand is replaced by the target RNA and therefore released, quenching stops, and fluorescence is detected.

This is plagiarism, with, in addition, a clear intent to deceive: whilst the article entire point appears to be the celebration of the SmartFlare technology, e.g concluding sentence (Thus Smart Flare novel gold nanoparticles could revolutionize the field of differential gene expression studies and drug discovery), the 2015 blog post was already doubting the validity of the technology.
I wrote to the Editor of the Journal who said that they would evaluate the claims and take some form of action. I contacted Hirendra Banerjee who declined to provide a statement; instead he issued a legal threat against the publication of this post.
Meanwhile, on Twitter
Capture
Capture
Capture
Conclusion of this exchange?
Capture

Drug Discovery 2017

This is a guest post by Marie Held reporting from the ELRIG conference held last week.

On 3rd-4th October I attended ELRIG’s flagship event, Drug Discovery 2017, in Liverpool. With around 250 participants, it was the largest of the ELRIG conferences yet. The spacious arrangement of the vendors and posters in the exhibition hall was a refreshing change. There was ample space to mingle, chat and discuss equipment on show.

On day one, I attended the Advances in Imaging stream (one of three parallel streams). The keynote lecture by Tony Ng covered a broad range of the spatial scale, stressing the importance of whole body imaging in cancer in combination with investigating the tumour microenvironment down to super resolution imaging of individual molecules. He outlined their attempts in predicting tumour metastasis enabled via immune system hijacking by the cancer cells. An important conclusion was that with the wealth of imaging methods and tracers being developed, we need standardisation and validation across facilities to bring them closer to the clinic, ultimately improving the lives of patients. The imaging methods discussed in the following six talks ranged from man to molecule, focussing on ever smaller features as the day went on. A transpiring theme was the generation of large amounts of data from different techniques and the associated challenge of deriving meaningful information. Machine learning and artificial intelligence were mentioned time and again as being part of that quest. The last scientific presentation, by Charlotte Dodson, focussed on twinkling enzymes, studying the conformational changes of kinases in disease and after treatment via single molecule spectroscopy. Throughout the imaging stream, twelve men contributed to the presentations, vendor snapshots and poster tasters and three women contributed to the stream. The other streams were a bit more gender balanced but only the workshop on Tuesday achieved a 50/50 split.

On the second day, I attended the Lab of the Future workshop presented by SiLA and ELRIG. The general consensus was that the lab of the future (whether you call it Lab 4.0, Industry 4.0 or something else) is an interconnected space in which smart machines are communicating with each other, running fully automated cycles of fabrication, screening and/or testing. Machinery that can be monitored if not controlled remotely via mobile device apps was mentioned multiple times. Smart products are uniquely identifiable, may be located at any time and “know” their own history, current status and alternative routes to achieving their target state. It left some of the audience wondering where innovation is going to come from. A lot of innovation is not based on a “Eureka” moment but rather lucky accidents or not quite sticking to the protocol and making mistakes. These instances are near on excluded in an automated lab. Another doubt that was raised was: Where is the space, if not need, for the scientist is in this fully automated lab? “He” has more time to think about the science and efficiency gains rather than processing the work. Unfortunately, the scientist was exclusively referred to as a “he” throughout the whole workshop, which irritated myself and another female member of the audience to the extent that it seemed appropriate to clarify that the scientist can be a female scientist. Unconscious discrimination is one of the reasons why there are still so few leading women in science. There was a conspicuous lack of women, both in the audience and in particular in the selection of session leaders, which were all male. It would be nice to see some female panel members in the future. Also, this year only one out of 12 session chairs throughout the whole conference were female.

Near on every panel member in the lab of the future workshop voiced that the interconnectivity should be down scalable to medium and small labs. As a member of the academic research community and a small lab, I felt somewhat left out though. We do not generally use automated machinery, never mind machinery connected to the internet of things. Often enough there is a piece of equipment, that has to be taken off the net entirely because the software is so outdated (and not supplier maintained anymore) that it has to run on an obsolete operating system posing a risk to the University network. That means we are in fact taking a step away from the lab of the future. The audience saw the responsibility with the industrial sector to come up with a solution and I am looking forward to seeing a change in the future. Also, electronic notebooks (find the same presentation here with audio comment) are already a standard in the industrial sector but the academic sector is severely lagging behind. Not all universities have specific guidelines on how to keep a paper lab book, never mind having a system of electronic lab books in place. The responsibility here lies in the academic sector to catch up but it might have to be a bottom up approach to induce a change.

The high point of the second day and probably the conference as a whole was the plenary keynote by Dr Nessa Carey asking whether we can fix big pharma. Her keynote was eloquent, inspiring and also entertaining. We can all do our bit to help fix big pharma. It is not the evil it is often made out to be. Millions of lives have been saved by pharmacological advances and still are being saved, however it does suffer from the worst PR there is.

Overall, I enjoyed the ELRIG Drug Discovery 2017 and am looking forward to the next instalments in London in 2018 and back in Liverpool in 2019.

 

Three little (nano) controversies and their morals

This post is a translation of an article originally published in French in Médecine/Sciences. The Editorial of the same issue (also in French) by Pierre Corvol is entitled Scientific integrity: the need for a systemic approach (open access).  You can download a pdf of my article, or, read at the publisher’s website (subscription). For citation, please refer to the original article as follows:

Trois petites (nano) controverses et leurs morales; Raphaël Lévy; Med Sci (Paris), 33 8-9 (2017) 797-800; Publié en ligne : 18 septembre 2017; DOI: https://doi.org/10.1051/medsci/20173308027

« Selon que vous serez puissant ou misérable, les jugements de cour vous rendront blanc ou noir » [1] [Depending on your social height, The law will see your crime as black—or else as white.] Thus concludes the Fable, by Jean de La Fontaine, The Animals sick of the plague : the donkey, guilty of the theft of a few blades of grass, is condemned to death, whilst the Lion and other powerful animals guilty of much more serious crimes are treated to praise and flattery. It is tempting and comforting to think that scientific judgments are of an altogether different nature. Seen in this light, science would reside outside of power struggles and the few mishaps (mistakes, frauds, conflicts of interest) would be rapidly corrected since the reality of the material world would quickly come back to us through experimental results if we were to try to ignore it for too long. The truth is however very different. A large fraction of published scientific results cannot be reproduced. It is not a few mishaps but structural problems which affect the foundations of the scientific enterprise [2, 3]. Peer evaluations seems to encourage the publication of extraordinary stories in high impact factor journals rather than careful and rigorous experimental studies. Contradictory or “negative” data are rarely published: scientific journals are not really interested, and us, scientists, are not particularly motivated by publicly stating our doubts on the work of colleagues who could be in charge of evaluating our next article or grant application. It is particularly urgent to repair our knowledge production system because science is at the center of numerous challenges critical for the future of human beings and the planet. The (real) problems of reproducibility have already been harnessed by lobbies to attack the credibility of scientists [4]. After the election for president of the largest scientific and military power of the world of a man who denies climate change, is very positive about the use of the atomic bomb, and, more broadly wages an open war against science and truth [5], we have a paramount need for science to be open, robust, capable of defending its independence, integrity and universal values. This seems a distant prospect.

The near absence of critical discussion in the scientific literature in many areas of science could make us forget that controversies are an essential aspect of the quest for knowledge, allowing to identify weak points of experiments and theories, thus enabling to consolidate or invalidate them [6]. They are consubstantial to the scientific practice [7]. The analysis of controversies is also a tool to “symmetrically map” the actors to better understand the roles of individuals and social processes [8]. In this piece, I describe three recent controversies in my area of research: gold nanoparticles applied to biology and medicine. This is no “symmetric map”: I am not a neutral observer but a scientist active, to various degrees, in each of those. I am trying nevertheless to draw some lessons and suggestions to improve the ways we work as scientists.

Stripy Nanoparticles

In 2004, Francesco Stellacci’s group at the Massachusetts Institute of Technology (MIT) published in the prestigious journal Nature Materials an article describing gold nanoparticles covered by a mixture of two molecules that self-assemble to form stripes that are observed by scanning probe microscopy [9]. This article and the numerous other ones that will follow in the same journal and in others just as prestigious such as the Journal of the American Chemical Society [10], Science [11] and Proceedings of the National Academy of Sciences (PNAS) [12], suggest that, thanks to their stripes, these nanoparticles have unique properties in terms of wetting, self-organization, interaction with proteins, penetration in cells, with lots of potential applications for biomolecular sensing, or even drug delivery. These articles certainly contributes to the progress of their authors’ careers, but the stripes are an experimental artefact well known by users of scanning probe microscopy. How to explain then that more than 20 “stripy” articles were published between 2004 and 2012? It is obvious that specialists (and even enlightened amateurs) had identified the problem as early as 2004. Yet, the articles and reviews of that period show no sign of it. One now knows that Predrag Djuranovic has been the first to engage into a scientific investigation aiming at testing, and eventually, contesting, the evidence for the the existence of the stripes. In 2005, this rigorous and brave scientist was a student in Francesco Stellacci’s lab. His experimental results and numerical simulation showing how the stripes originate from a poorly adjusted feedback control system were unambiguous but MIT ensured that these results would remain secret [13]. In 2007, I submitted a technical comment responding to the Science article. This first attempt, limited in its scope to the Science article itself, was unsuccessful: Science did ask Francesco Stellacci to respond but then decided not to publish the exchange of views [14]. In 2008, a new article from the MIT group, again in Nature Materials, report that, thanks to their stripes, these nanoparticles can cross the cell membrane and directly access the cytosol [15]. This is accompanied by a commentary entitled “Particles slip cell security” [16]. After discussions with several of my students, we decide to propose a more exhaustive answer. A few months later, the article “Stripy Nanoparticles Revisited” is ready. It includes a new analysis of the stripy images concluding that the stripes are a scanning artefact as well as a critical discussion of the physicochemical and biological properties which, together with experimental results, contradict the claim of direct access to the interior of cells. The article is first submitted to Nature Materials (rejected), then NanoLetters (rejected), and, finally, Small… where it is published after an editorial process that lasted three years [17-19]. The publication of our article, in November 2012, does not end the controversy. Instead, it expands in the scientific literature (a little) and it also takes new forms (in particular on my blog and others [20-23]). Problems with the reuse of images in different publications emerge and eventually lead to two corrections ([12] and [15]). After a number of requests, Philip Moriarty and Julian Stirling (School of Physics and Astronomy, university of Nottingham, UK) are given access to the original data of the 2004 article. They demonstrate, among other things, that the stripes are present in the entire image, i.e. even between the gold nanoparticles [24], a conclusion still rejected by Francesco Stellacci [25].

Homeopathic nanoparticles

The laboratory of Molly Stevens at Imperial College is one of the most prestigious in the field of biomaterials. In 2012, two articles from the group relate the particularly interesting properties of nanoparticles for diagnostic applications. The first one, published in Nature Materials, reports a phenomenon which is entirely extraordinary in which the signal detected increases when the concentration of molecules to detect decreases (“inverse sensitivity”) [26]. Even more incredible, this phenomenon extends to the point where there is less than a molecule of enzyme, on average, in the volume under study. The second article published in Nature Nanotechnology goes further : no need for instruments, the detection of concentration of analytes in the same range is achieved thanks to a colour change visible with the naked eye [27]. Detailed critiques of these articles are available on the website PubPeer [28, 29] as well as in a preprint authored by Boris Barbour; the objections are both simple and profound but the authors have chosen not to respond. One can note that the Avogadro number includes lots of zeros (630 000 000 000 000 000 000 000) and that the detection of a macroscopic change of property due to the presence of a single molecule is therefore an achievement that requires extremely solid proofs. One of the posts on PubPeer indicates that someone contacted the Editor of Nature Nanotechnology in January 2013, but, four years later, no doubts are expressed on the journal website, in the traditional scientific literature nor in the newspapers that had covered this story (e.g. Le Monde and the Daily Mail) when it was initially published [30, 31].

Spherical Nucleic Acids

The laboratory of Chad Mirkin at Northwestern University (USA) is one of the most prestigious in the field of nanosciences applied to biology and medicine. One major theme of their research are the Spherical Nucleic Acids (SNAs), a term introduced by Mirkin to describe gold nanoparticles functionalised with DNA (or RNA) strands. These SNAs are supposed to have properties very different from linear DNA [32]. In particular, they can access the cytosol of live cells, where they could detect and regulate, the presence and quantity of mRNAs. One could ask why this solution did not appear during evolution : to access the cell machinery, viruses and bacteria would have only needed to package themselves within their genetic materials. The first articles (in Science [33], the Journal of the Americal Chemical Society [34], NanoLetters [35], ACS Nano [36]) proposing this surprising theory do no mention the mechanism of the SNAs into cells whatsover. The following one, e.g. [37], propose that the particles enter by endocytosis, but do not explain the mechanism by which the SNAs would escape endosomes. After several dozens of articles on this topic, the proportion of particles reaching the cytosol is still to be measured and reported (in spite of the fact that gold nanoparticles have been used since the 1950s to study intracellular trafficking; such a study would not be difficult). One article from the Mirkin group suggests that SNAs are degraded in the endosomes and that a “small unquantifiable portion escapes […]” [38]. Nevertheless, the particles are now commercially available under the name SmartFlares (Merck Millipore) to detect RNA inside cells. We have studied the entry of nanoparticles in cells and their ability to detect RNA. Given our difficult experience with the publication of Stripy Nanoparticle Revisited, we decided to adopt a different strategy. The project has been open and we have shared our results in quasi real time on our blog. In contradiction with the descriptions made by Mirkin and by Merck Millipore, we have observed that the SmartFlares were degraded in endosomes and were not able to detect mRNA.  Mirroring the tale of Predrag Djuranovic and the stripy nanoparticles, we were not the first to have doubts about the technology: Luke Armstrong, who had been in charge of developing the SmartFlares at Merck Millipore in California (before leaving the company) had reached the same conclusion [39]. To ensure speedy publication and transparency, we published our article on the (not so prestigious) ScienceOpen platform where peer review occurs after publication [40]. We invited comments by Mirkin to no avail. Another article by the same group in PNAS describe a new version of the SmartFlares [41]. Our analysis of the raw data (obtained after multiple insistent requests) show that the signal comes from endosomes. Our letter submitted to PNAS has been rejected by the editorial board because it “[did] not contribute significantly to the discussion of this paper” [42].

 Morals

Access to raw data is essential and guaranteed by clear rules adopted by Universities, scientific journals and funding agencies. It is therefore generally possible to access data with some efforts. It is obviously preferable to publish data at the same time as the articles. This is already the norm for some categories of results and it should become generalised. Researchers should also adopt the Manifesto for reproducible research [43]. The tools are in place to improve the practice of science.

Evaluations of science and scientists must imperatively be based on a critical analysis of their work and the robustness or their results, not on the prestige of the institutions or journals. This requires a change of mind and a clear commitment from researchers who are in positions of power, i.e. everyone who features on promotion or recruitment committees. To say that an article is good because it has been published in a prestigious journal is a moral and logical error which needs to be challenged.

Institutions and scientific journals are not motivated by the quest for scientific truth. The decisisons taken by MIT (keeping Predrag Djuranovic’s findings secret), by Nature Materials (not publishing the exchange with Francesco Stellacci [14]), and by PNAS (not publishing [42]) have directly impacted progress of knowledge. These institutions have commendable principles but, in practice, they aim first at defending their reputation and finances [44]. The latter objective only partially aligns with scientific progress which requires rapid and open discussion of results and conclusions. The Worldwide Web, invented for the sharing of science, enables this discussion. Researchers therefore should embrace the following tools: 1) Pubpeer to comment on articles; 2) Preprints to publish rapidly, minimise the influence of editors, and, dissociate publication, i.e. sharing of information, from evaluation, i.e. peer review; 3) Social networks, e.g. Twitter and blogs, which constitute an ongoing scientific conference to discuss experiments, results, methods, analyses, and new publications.

Acknowledgements: I thank Marianne Noel (IFRIS) for her critical reading of this piece, and, Marianne Lévy for comments on the grammar and style [French version] very necessary after 14 years in an English-speaking country…

Conflicts of interest: The author declares that he has no conflict of interest related to this article.

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Guest post: SmartFlares fail to reflect their target transcripts levels

Czarnek&BeretaThis is a guest post by Maria Czarnek and Joanna Bereta, who have just published the following article in Scientific Reports entitled SmartFlares fail to reflect their target transcripts levels

We got the idea of using SmartFlare probes when working on generating knockout cells. In the era of CRISPR-Cas9 genome editing, the possibility of sorting out knockout cells based on their low target transcript content (mRNAs that contain premature stop codons are removed in a process called nonsense-mediated decay) instead of time-consuming testing of dozens or thousands of clones would be a great step forward. SmartFlare probes seemed to be just the ticket: no transfection, lysis or fixation needed; moreover, the probes were supposed to eventually leave the cells. We were full of hope as the first probes arrived. (more…)

Hot topics in Biochemistry

Every year our 3rd year Biochemistry students have to produce posters on hot topics in Biochemistry. And every year I ask colleagues to contribute some ideas for this list of topics. You can find last year’s list here; as it is still pretty current, we will just extend it below. And you can contribute to this year’s list via Twitter or in the comments below.

#22

 

At the 254th ACS conference in Washington DC

This is a guest post by Sumaira Ashraf.

Last month, I got the opportunity to attend and present my work at the 254th ACS conference held in Washington DC. The session was organized by Raphaël Lévy, Niveen Khashab, and Zhihong Nie. I presented preliminary data regarding my work on multimodal imaging probes for stem cell tracking. I enjoyed the conference but was disappointed by some of the talks. Many speakers  exaggerate their results to impress their audience and hype the worth of their work. I did wonder if it is productive to point out mistakes as no one seems interested in correcting them, but, instead, they might become your lifelong rivals. Here is an example. I know from practical experience that liposomes loaded with small molecule dyes cannot be used as long-term imaging modality because within 24 hrs of loading ~99% signal intensity is lost due to leakage from the liposomal cavity. But speakers presented quantitative imaging data based on these approaches. I wanted to point this problem out but I did not want to get involved in never ending discussion where no one will be ready to admit their errors. Eventually, I did not say anything.

Of course, my own work with polylelectrolyte capsules has some limitations as well: while they are good candidates for protecting imaging probes from intracellular degradation and intracellular species from potentially toxic probes, the limit of detection depends on the amount of contrast agent delivered which still remain low. In my current experiments, I need to overcome this limitation by modifying or choosing an alternate approach.

The Kavli lectures were really very informative… but I could not attend the full talk of the 2nd speaker because the air conditioning was set at a temperature so low that I could not tolerate it: I was forced to leave this interesting lecture. The industry exhibition downstairs the convention centre was massive and fun. I particularly liked the Bruker stall which had good explanation of NMR, mass spectrometers, etc., (covering theoretical and experimental data evaluation). There were other advantages to the visit of the exhibition: chocolates, bags, T- shirts, ice cream (prepared in liquid nitrogen within 3 minutes), lunch, free L’Oréal products, and lucky draw (especially when you win; I won a Swag Bag of merchandise from the ACS Store worth USD$75.00). I was lucky to see the solar eclipse through the cover glasses provided by the conference.

I missed the opportunity to visit the reflecting pool near Lincoln memorial and some interesting museums due to time limitation. I am looking forward to some forthcoming opportunity to see them.

I came back with some thoughts to work with new nanocomposites (recent work presented in one of the talk by Amit Joshi; based on doping materials rather than combining them inside big capsules or attaching via linkers) for multimodal imaging which might have potential to overcome the flaws associated with above mentioned materials. But I need to try… without trying one cannot be sure of anything.