The #IceBucketChallenge Two Years On

August two years ago saw our facebook feeds flooded with footage of friends and acquaintances dousing themselves in ice-cold water to raise awareness and money for amyotrophic lateral sclerosis (ALS; or motor neuron disease (MND)) charities. It was the topic of the inaugural blog post and a follow-up one year later. My inherently slightly cynical and skeptical nature questioned whether all this social media craze (and £87.7 million raised) would actually make a difference. Well, facebook came to the rescue and linked me to articles from the BBC and The Guardian alerting me to a paper published recently in Nature Genetics (Kenna et al., 2016).

The researchers contributing to this study work in eleven countries across the world. (Who ever thought science benefited from international collaboration? Am I still frustrated by Brexit? No, not at all…) Large proportions of the funding were provided by the National Institute of Health in the USA as well as  ALS and MND Associations in the USA and UK. Kenna et al. sequenced those parts of the genome that are actively expressed – a technique known as whole exome sequencing – in over 1000 familial/inherited ALS patients and over 7000 controls. Since sequencing technologies are becoming better and cheaper all the time, this is the less impressive part of the study. Next, all this data was processed using so-called gene burden analyses. This is where I stop understanding what is done with the data, but in essence it was possible to use previously known genetic risk factors of ALS to infer overlooked genes that are also associated with the disease. In the figure below the genes indicated in blue are genes that were already known to confer ALS risk (e.g. SOD1 and FUS), whereas those in black are the new genes, and everything above the red dotted line was considered statistically significant.


Graph depicting genes associated with ALS risk – copied directly from Kenna et al., 2016

As you can see, the researchers identified mutations in the NEK1 gene through these sequencing and data analysis experiments. However, only about 10% of people with ALS have the familial/inherited form of the disease. Therefore Kenna et al. then went on to check whether these NEK1 mutations could also be found in samples from patients with sporadic ALS and indeed they could. Overall approximately 3% of ALS patients have abnormal NEK1 genes.

After all this data analysis the paper ends with a description of what the NEK1 protein normally does and what it might not be doing in ALS patients’ cells: NEK1 helps to repair damaged DNA and contributes to the formation of an organelle called the cilium. Now future experiments will have to focus on exactly why and how mutations in NEK1 contribute to ALS. And since only 3% of ALS patients have NEK1 mutations there are still many other genes to discover.

The Project MinE aims to do just that – with headquarters in the Netherlands, this collaborative DNA sequencing project is analysing samples from even more ALS patients and controls. Their website says that a donation of €75 enables sequencing and analysis of a single chromosome! Anyone fancy another cold shower?

Lastly, I’ve found an interesting article combining two of my favourite things (science and Impressionist art) – so look forward to that in the next post.


Kenna KP, van Doormaal PTC, Dekker AM, Ticozzi N, Kenna BJ, Diekstra FP, van Rheenen W, van Eijk KR, Jones AR, Keagle P, Shatunov A, Sproviero W, Smith BN, van Es MA, Topp SD, Kenna A, Miller JW, Fallini C, Tiloca C, McLaughlin RL et al. (2016) NEK1 variants confer susceptibility to amyotrophic lateral sclerosis. Nature Genetics advance online publication.


A Second Word on Evolution

Life as a PhD student is busy and doesn’t leave much time for other activities, including this blog. So last time, about a month ago, I left you with the question of how the genetic code may have evolved over time.

For decades some scientists have hypothesised that the genetic code evolved by a so-called direct templating mechanism (also known as the stereochemical hypothesis). That is, the strings of ribonucleotides that make up an RNA molecule could physically interact with amino acids, the building blocks of proteins. This interaction would promote the reaction of adjacent amino acids to start forming a longer polypeptide chain. For a review on the different hypotheses see Koonin & Novozhilov (2009).

One of the proponents of the stereochemical hypothesis is Bojan Zagrovic and his research group at the Max F. Perutz Laboratory in Vienna. They have published several papers on this topic and almost a year and a half ago I went to a symposium where Bojan Zagrovic gave a talk on exactly this topic. I wrote about the various presentations I heard there and then several months later a friend I had met during the Cold Spring Harbor Laboratory (CSHL) undergraduate research programme sent me a message saying he had been inspired, by the blog post, to do some research of his own.

In particular, John wanted to investigate whether there was a pattern behind the observed interactions between the amino acids in proteins and the ribonucleotides in RNA. To do this he and Rachel (another student from CSHL) used computational biology approaches to study a large published dataset of protein-RNA complexes. They found that there is a correlation between these physical interactions and the way the genetic code is laid out.

Once these findings had been made they wrote up a draft manuscript, including some figures, which were produced by Grace, a colleague of John’s at Carleton College in the USA. John asked whether I would mind reading the manuscript to give feedback and of course I was happy to do that. We started e-mailing back and forth and decided to extend the computational experiments, and I edited and expanded the text.

The most interesting result was that we could use the knowledge derived solely from the interaction data (blue and red bars) to predict, significantly more accurately than expected by chance (yellow bars), the amino acid sequence of a protein from its mRNA precursor:

Screen Shot 2015-12-07 at 23.00.28

Combining amino acid-nucleobase affinities with mRNA nucleobase content to predict amino acid sequences without universal genetic code. Copied directly from our paper.

In particular, the proteins that form the ribosome – the molecular machine that translates mRNA into protein in modern-day cells – were more accurately predicted than a random protein from our dataset, possibly suggesting that direct interactions between RNA and amino acids led to the formation of the first primitive ribosomes. However, as you can see, the prediction accuracies do not exceed 15% so all results from this paper need to be taken with a pinch of salt; I think the best we can do is say that our results strengthen the stereochemical hypothesis but by no means prove it. [In any case, the scientific method is only good at disproving theories.] Since the journal, Scientific Reports, is an open access journal anyone can read the paper here.

Overall, I am just proud that we managed to publish our work after a long and iterative process, including one revision. All of this was done long-distance via Skype and e-mail. We were all working or studying full-time at the same time and moreover, we did this without the help of a professor/group leader. In fact, none of us even has a PhD (yet).

Lastly, I have noticed a mini-surge in views of my blog posts pertaining to PhD interviews. Clearly the invitations for the next year have been sent out and I hope whoever is reading this is finding it helpful and: good luck!


Cannon JGD, Sherman RM, Wang VMY, Newman GA (2015) Cross-species conservation of complementary amino acid-ribonucleobase interactions and their potential for ribosome-free encoding. Scientific Reports 5: 18054

Hlevnjak M, Zagrovic B (2015) Malleable nature of mRNA-protein compositional complementarity and its functional significance. Nucleic Acids Research 43: 3012-3021

Koonin EV, Novozhilov AS (2009) Origin and evolution of the genetic code: the universal enigma. IUBMB Life 61: 99-111

Polyansky AA, Zagrovic B (2013) Evidence of direct complementary interactions between messenger RNAs and their cognate proteins. Nucleic Acids Research 41: 8434-8443

de Ruiter A, Zagrovic B (2015) Absolute binding-free energies between standard RNA/DNA nucleobases and amino-acid sidechain analogs in different environments. Nucleic Acids Res 43: 708-718

The Joys of Revision #2

Exams are drawing closer and that is both a relief – at this point I really just want to be done with this taking exams business – and a consternation – they do seem to spring up on one every year rather unpleasantly and seemingly out of the blue. To deal with my mix of boredom and quiet anxiety it has been useful to seek out various different places to do revision. A friend of mine even has a blog dedicated to describing several Cambridge libraries.

To begin with there is of course my “home” library at Pembroke College. Large desks and sitting between volumes of classics (Greek, Latin, English, you name it) make for a good working environment. However, the bell in the clock tower, which strikes every half hour, is a rather unpleasant reminder of how quickly time is ticking. [Photo copied from the Pembroke College website.]


Just across the street is the Colman Library, the library of the Department of Biochemistry. Again this is quite an old building with large wooden desks or smaller, personal booths to sit at. It is rarely busy, often giving it a feeling of calm, but the people who do study there are often my (stressed?) colleagues, which could become a bit unpleasant the closer we get to exams.

If one is feeling particularly brave as a science student one can make one’s way to the Sidgwick site where several art/humanities and social science departments are situated. The main recollection I have of the law library is its coldness (temperature-wise). In the economics library people were generally better dressed, although I hear that this is also deteriorating as  exams approach. The Judge business school has an “information service” instead of a formal library, in which people can chat and bring in drinks, which makes for a more relaxed atmosphere, but when I heard some students (?) talking about their newest great idea in which they’ll move from the production to the service sector to make “massive profits” I started feeling out of place.

The winner of the library competition is undoubtedly the Faculty of Education: it’s bright and full of light wood, the staff are helpful and kind (there was free cake on Friday afternoon) and one can sit looking out onto a green garden. [Photo copied from their website.]


CRISPR Let Loose

Most people reading this blog today will probably consider this slightly old-fashioned, but I received a newspaper clipping from my mother in the post yesterday (yes, I mean a physical piece of paper that arrived in an envelope with a postage stamp on it). The title of the article (online here), which was published in the Austrian quality daily newspaper “Die Presse”, roughly translated to, “Genetic engineering is spiralling out of control”.

The article was referring to an experiment conducted by Gantz & Bier (2015) in which they invented a “mutagenic chain reaction” (MCR) based on the CRISPR/Cas9 technology in the fruit fly Drosophila melanogaster. Their experimental rationale is illustrated in this figure, which I copied directly from the paper:

MCR scheme

The idea was to create homozygous insertional deletion mutations of a target gene, in their case the yellow gene in Drosophila. When yellow is mutated heterozygously in females (i.e. in only of the two genomic copies) the flies look normal/wild-type because mutations in this gene are recessive and the gene is located on the X chromosome; only once both copies are mutated does the phenotype manifest: albino flies. Since males only have a single X chromosome they are always albinos if they inherit the mutated yellow gene. Gantz and Bier built a DNA construct that expresses Cas9, the DNA endonuclease, and the guide RNA targeting yellow in both somatic and germ-line cells. However, the special feature of their construct was the flanking homology arms (HA; depicted in red): these facilitate the insertion of the construct at the site of cleavage by Cas9 via homology-directed repair. This means that the genome now stably expresses one copy of the Cas9/gRNA combination. Next, the second copy/allele will be targeted and again the construct is inserted in the DNA, disrupting the yellow gene. If this approach didn’t work you would expect first generation female flies, progeny from a cross between wild-type flies and flies containing the DNA depicted as a circle in panel A above, to all be phenotypically normal. However, they actually found female albino flies in these crosses. When these white flies were crossed to wild-type males they found that almost 100% of those offspring were white, although one would expect only the males to be. So their MCR approach clearly worked, and in a way it violates the age-old rules of Mendelian inheritance.

In their conclusion they stated that MCR may, “provid[e] a potent gene drive system for delivery of transgenes in disease vector or pest populations, and potentially serv[e] as a disease-specific delivery system for gene therapy strategies”. Gene drive “involves stimulating biased inheritance of particular genes to alter entire populations of organisms” (Pennisi (2014)). This approach may rapidly disseminate mutations that inactivate virulent traits in various disease vectors, such as the Anopheles mosquito, which acts as a vector for the malaria parasite Plasmodium falciparum.

Gantz and Bier also wrote that they are, “keenly aware of the substantial risks associated with this highly invasive method. Failure to take stringent precautions could lead to the unintentional release of MCR organisms into the environment”. In a comment on the paper their security precautions are mentioned: the flies are kept in three layers of tubes and boxes, locked behind five doors operated by fingerprint recognition to ensure they do not escape into the wild. However, just as Baltimore et al. (2015) called for caution and open discussions concerning CRISPR applications with regards to (human) genome-editing in general, Esvelt et al. (2014) demanded extreme caution in the use of CRISPR-based gene drive systems. They especially emphasised the need to both molecularly and ecologically contain these systems: at the molecular level, for example, the targeted gene must not be present in wild species; at the ecological level experiments should only be attempted in inhospitable environments where organisms will not easily find mating partners. Their hesitation to fully embrace this new approach is understandable. For example, what if the guide RNA becomes mutated and the nuclease starts targeting other genes as well? Furthermore, such gene drives employed by the government of a country, for instance, to combat a disease vector will almost inevitably cross political borders. If introduced into endangered species by accident they could eradicate them entirely. It seems to me that it will be quite difficult to test such approaches (in the manner of a clinical trial, say) without actually conducting the experiment in the wild. And somehow inexplicably these articles entirely slipped under my radar, whereas the recent human genome-editing experiment caused much more uproar although it is arguably a lot less prone to “spiral out of control”.


Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J, Sternberg SH, Weissman JS, Yamamoto KR (2015) Biotechnology. A prudent path forward for genomic engineering and germline gene modification. Science 348: 36-38

Esvelt KM, Smidler AL, Catteruccia F, Church GM (2014) Concerning RNA-guided gene drives for the alteration of wild populations DOI:10.7554/eLife.03401

Gantz VM, Bier E (2015) The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science 348: 442-444

Pennisi, E (2014) U.S. researchers call for greater oversight of powerful genetic technology. Science Insider: Accessed May 3rd 2015.

The Joys of Revision #1

Please immediately note the hint of sarcasm in this title. What academic year would be complete without the harrowing prospect of end-of-year exams, which, this year, happen to also be finals? … Well, exactly. Any old academic year would be fine without. But alas, that is not how the system works. So on top of being judged on the basis of our lab project/dissertation/viva voce examination we also need to take two written exams, which are worth 50% of our mark.

To ease the transition from dissertation writing and editing into revision I thought I would start with something fun/potentially useless that can be classed as elaborate procrastination:

Landmark timeline

As mentioned previously, some of our teaching was provided in the format of seminars discussing landmark papers – papers that have shaped the way biologists think about their areas of research and that have had lasting, even if sometimes subconscious, effects. Several of the professors emphasised the importance of understanding these papers within their historical contexts, which is, I would contest, not something we scientists normally think about. Therefore I decided to look for a tool that would allow easy creation of a timeline like the one shown above and the only free app I could find in the store is simply called Timeline 3D. Right, well, and I just realised that on top of procrastinating by colour-coding different landmark papers I’ve now also spent a good fifteen minutes writing this blog post. If anyone else has well-disguised procrastination tools, please do share…

P.S.: Another way to pretend like you’re doing something useful – attend the departmental seminar given by a Nobel prize laureate, Jules Hoffmann. I was pleased to hear that he was appreciative of all of his collaborators and acknowledged his students and post-docs; something he seems to share with another Nobel prize winner, Christiane Nüsslein-Volhard, who gave a seminar a few weeks ago. Based on these examples I was about to draw the conclusion that a couple of the marks of great scientists are a) their willingness to acknowledge co-workers and b) to some extent their modesty (granted, this one doesn’t apply to other Nobel laureates I’ve encountered [e.g. James Watson]). However, as I was reading about Jules Hoffmann I very quickly came across this blog written by Bruno Lemaitre, a former research associate in Hoffmann’s lab, claiming that most, if not all, the award-winning work was conducted by him without encouragement/help from Hoffmann. Now what to believe?!