Behrens lab retreat 2016

Imagine spending a weekend in these idyllic surroundings in the Peak District with nothing to do but talk about and discuss science.

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The Peak Mermaid Inn – taken at sunrise on November 13th 2016

Well, that’s exactly what we, the Behrens lab, did last weekend. We invited a keynote speaker, Roland Rad, and Dieter Saur’s group from the Technical University of Munich to join us. Each of us gave a talk about the most interesting or exciting aspects of our projects and in between we drank copious amounts of coffee. In the evenings we cooked enough food to feed a small regiment, drank beer, played pool, darts or table football, all punctuated by heated debates about science. Although this wasn’t a relaxing weekend by normal standards, it was motivating and inspiring and a good reminder of why I enjoy being a scientist: a combination of rational and logical thinking, curiosity and the drive to learn new things for their own sake, all shared with people who, by and large, know more than I do and think differently.

Of the talks I just want to highlight one in particular, because my project also uses one of the techniques mentioned. Dieter Saur is a medical doctor and has his own lab group, which studies mainly gastrointestinal diseases, including pancreatic cancer. In a recently published paper (Schönhuber et al, 2014) they describe an experimental system in mice called the “dual recombinase system“. This is a genetic system that allows the study of complex diseases such as cancer. Until recently it was only possible to simultaneously switch on a gene that drives tumour progression and switch off a gene that prevents tumour formation in a cell type or organ of interest (e.g. in the pancreas). Using the dual recombinase system it is possible to make genetic alterations sequentially. For example, in the beginning of a mouse’s development one can activate a potent tumour driver called Ras and delete an important tumour suppressor called p53. And then, once a tumour has formed, one can additionally delete genes that may be important to maintain the established tumour. Alternatively, the dual system also makes it possible to make genetic changes to the normal cells surrounding the (pancreatic) tumour. If all goes well then I will be able to use these tools to conduct experiments like this in the next year or so.

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Oh and admittedly we did have an activity scheduled that was slightly less scientific: we got all geared up and went on a GoApe outing. Secured by a harness and after some rigorous safety instructions we got to fly down zip lines, balance over gaping abysses and jump over the void below.


Lastly, the following week saw Queen Mary University London and Barts host the 11th UK cancer stem cell symposium. There were several interesting talks, including by group leaders at the Crick Institute, but the most unusual talk was given by a philosopher called Lucie Laplane. She did her PhD in philosophy and combined this with a research master’s in stem cell biology. Putting the two fields together she came up with a classification of (cancer) stem cells using definitions and guidelines borrowed from philosophy, applied to biology. [In general, researchers agree that stem cells are cells that can self-renew (i.e. generate new copies of themselves) and can produce differentiated/specialised daughter cells.] The most important point was how to pin down what kind of characteristic “stemness” is or what makes a stem cell a stem cell:

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Framework for defining (cancer) stem cells – copied from Lucie Laplane’s talk at the symposium

For instance, in some cases a stem cell might always be a stem cell no matter what the environment is like (i.e. categorical); other stem cells may be dispositional in nature, meaning that they always have the potential to act as a stem cell but only do so in a permissive environment. Alternatively, being a stem cell might not be property of a single cell at all but rather an attribute of an entire organ (i.e. systemic). Laplane argued that the way we define (cancer) stem cells has a huge impact on how we try to treat diseases such as cancer. For example, if cancer stem cells are “systemic” then even the best therapies targeted against these cells will fail because the system/the tumour will make new cancer stem cells from other tumour cells. Hans Clevers, one of the Gods in the stem cell field, wrote a glowing review of the book here.

References:

Laplane, Lucie. Cancer Stem Cells: Philosophy and Therapies. Harvard University Press, 2016.

Schonhuber N, Seidler B, Schuck K, Veltkamp C, Schachtler C, Zukowska M, Eser S, Feyerabend TB, Paul MC, Eser P, Klein S, Lowy AM, Banerjee R, Yang F, Lee C-L, Moding EJ, Kirsch DG, Scheideler A, Alessi DR, Varela I, Bradley A, Kind A, Schnieke AE, Rodewald H-R, Rad R, Schmid RM, Schneider G, Saur D (2014) A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer. Nat Med 20: 1340-1347

Cancer Research UK – PhD Student Meeting

Cancer Research UK (CRUK) is the world’s largest independent cancer charity (according to Wikipedia)  and funds thousands of scientists across the UK. In the latest annual report they state that more than £340 million were spent on research. Take a look at the fancy infographic here to see a break-down of how that money was spent:

annual report

The reason for writing this post on CRUK is that today the charity held its first-ever first year PhD students’ meeting in London, at the Quaker Friends House near Euston station. The attendees came from all over the UK: from as far afield as Aberdeen and Manchester to Oxford and Cambridge and finally us lazy Londoners who could afford to get up later than on a normal lab day.

The main aims of the meeting were to get to know some of the people working at CRUK’s head office in London and how, in future, we might apply for their funding. One of the first things I learnt today was that CRUK has made four cancer types – brain, lung, pancreatic (!) and oesophageal – “strategic priorities”, because the survival rates for these are still low and lagging behind those of, say, breast and prostate cancer. We also heard, from the senior research funding manager, Richard Oakley, how CRUK spends its money and what we can and are meant to do to help. Among other things this involves wearing branded t-shirts and participating in fundraising events. So tomorrow morning I will wear this to run in the park in preparation for the 10 km Race for Life happening at the end of June – please feel free to fund me and/or the maybe pink team and/or join the run! [We can start a separate conversation on the topic of the martial language used by CRUK, and other charities, to help raise the money. N.B. The back of the t-shirt reads, “Ask me about my life-saving research.”]

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Since doing a PhD is all about the learning experience, most of the morning was filled with one of three workshops on either a) assertiveness, b) time management, or c) having an effective working relationship with your supervisor. I chose the first option, and although some people (especially in my lab) will argue that it would be better if I were a bit more quiet on occasion, I thought it would be interesting to see what it could offer. The basic message was, of course, quite clear: effectively communicate your needs whilst appreciating other people’s needs. Easier said than done for sure. The only thing that helps is practising being in potentially awkward situations and putting oneself outside one’s comfort zone, which is where learning can happen. Possibly the most helpful information was to realise what isn’t assertive behaviour (e.g. being too passive, or too (indirectly) aggressive) and making sure to recognise those behaviours in oneself and learning to avoid them in future. We’ll see how that goes.

Over lunch we got to browse a few posters. I particularly enjoyed the ones on intestinal stem cells and a potential preventative treatment for breast cancer using the diabetes drug Metformin. And lastly, we were politely, with the help of beer and/or wine, coerced into networking…

Of mimiviruses and making progress

It’s been so long since I’ve written anything about CRISPR that I feel completely rusty. Luckily, I spotted some interesting new research on an “immune system” found in giant viruses called mimiviruses. Levasseur et al. (2016) propose that mimiviruses – large viruses that can be seen with a light microscope and have a genome that is bigger than that of some bacteria – can defend themselves against so-called virophages. In analogy to the viruses that infect animals, bacteria can be infected by viruses called bacteriophages, and mimiviruses can be infected by virophages. Although generally viruses are regarded as non-living these giant viruses do possess some genes that code for proteins that help build the virus. Normally, viruses hijack the infected host cell’s machinery to replicate and are completely dependent on the host cell. Only last year a group reported (Ekeberg et al., 2015) using high power X-rays to study a single virus and being able to look inside the particle. There is a nice 3D reconstruction video in the Nature News & Views article.

One of the virophages that infects some mimivirus lineages, but not others, is called Zamilon. Levasseur et al. guessed that mimiviruses  might incorporate stretches of DNA from Zamilon and use them to recognise infecting virophages. This would be in complete analogy to many bacteria and archaea that protect themselves from their attackers using CRISPR – clustered regularly interspaced short palindromic repeats. The DNA “spacers” correspond to the sequences found in the invading DNA  of bacteriophages, for example. To test their idea, Levasseur et al. sequenced the genomes of 45 mimivirus strains and did indeed find a repeating region that corresponded to Zamilon DNA. They called it MIMIVIRE, for mimivirus virophage resistance element (the figure is copied directly from the paper):

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Schematic of the mimivirus MIMIVIRE locus – copied directly from Levasseur et al., 2016

Nearby in the mimivirus genome the researchers also found (Cas-like) genes that might be involved in this defence mechanism: when those genes were genetically silenced the viruses that were normally resistant became sensitive to Zamilon infection. Furthermore, these Cas-like genes code for proteins that can bind and modify DNA and are therefore perfect candidates for destroying invading DNA. More experiments will need to be carried out to show exactly how the MIMIVIRE system works, but it is likely to be different from the CRISPR/Cas system. However, it is interesting to see how immune systems are pervasive in all domains of life (and almost life) and although I am no evolutionary biologist I think this may be an example of convergent evolution. [Thoughts on this would be appreciated!]


To end today’s post on a personal positive note: this year’s birthday present was being able to image the cells I am studying by electron microscopy. Electron microscopes are, in principle, similar to light microscopes, which most of us have used at school, except that they utilise electron beams instead of light rays as the source of illumination. Since electron beams have a much shorter wavelength (i.e. are higher in energy) than visible light they can visualise objects with much higher resolution, making it possible to get sharp images at greater magnification. Some of the images we – the extremely knowledgeable and helpful scientists at the electron microscopy facility in our institute and I – took were magnified 60,000 times!

Although I can’t share those images at the moment, here is a transmission electron micrograph of a normal pancreatic ductal cell (copied directly from here):

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Pancreatic ductal cell

In the centre of the cell is the large nucleus, clearly defined by its double membrane. The darker patches within the nucleus are parts of the DNA that are more tightly compacted than others. In the cytoplasm (the area that isn’t the nucleus) there are some mitochondria, where a lot of the cell’s metabolism takes place, and so-called endocytic vesicles, membrane-bound compartments that are involved in recycling and housekeeping. At the top left are microvilli, short protrusions of the cell into pancreatic duct. Although I’m not sure I can spot it here, the images we took also revealed the rough endoplasmic reticulum where ribosomes sit and produce proteins. Can you believe I saw ribosomes?!

Lastly, and this goes out mainly to all the other PhD students, I’ve recently been finding it helpful and refreshing to relish the feeling that, as a student, there is always so much more to learn. To really bask in the glory of one’s own ignorance and then pester and listen to more knowledgeable and more experienced people to learn something new. For example, you can plan a decent experiment that has the proper controls and would give you some useful information. Then you tell your supervisor about it and s/he suggests doing that little extra something that suddenly turns the experiment into real science. On the one hand, I find these occurrences humbling. But, on the other hand, they are also exciting because I’m finding it easier and easier to recognise what makes a really good experiment and I can see, still beyond my reach but tantalisingly close, the potential of making that last mental leap myself. Needless to say that doesn’t at all mean that an experiment will technically work…


References:

Ekeberg T, Svenda M, Abergel C, Maia FRNC, Seltzer V, Claverie J-M, Hantke M, Jönsson O, Nettelblad C, van der Schot G, Liang M, DePonte DP, Barty A, Seibert MM, Iwan B, Andersson I, Loh ND, Martin AV, Chapman H, Bostedt C et al. (2015) Three-Dimensional Reconstruction of the Giant Mimivirus Particle with an X-Ray Free-Electron Laser. Physical Review Letters 114: 098102

Levasseur A, Bekliz M, Chabrière E, Pontarotti P, La Scola B, Raoult D (2016) MIMIVIRE is a defence system in mimivirus that confers resistance to virophage. Nature advance online publication

First PhD Checkpoint

In December, we – the (mostly) young and innocent first-year PhD students at the Francis Crick Institute – gave our first formal talks. Each student had to present the outline of their project to all the other students in a ten minute slot. This was probably intended mostly for our own benefit to ensure that we had at least a rough idea of what we will be working on for the foreseeable future. Here I’d just like to mention a few of the talks that I found particularly interesting, but it’s worth saying that I thought the overall level of presentations was very high and the questions we ended up asking each other were well thought through. Overall a very enjoyable experience.

  1. To begin with there were a couple of talks from students in the same lab studying the interactions of cancerous cells with the immune system. In particular, they are trying to find out how dendritic cells – cells that normally alert effector cells of the immune system that something is wrong (e.g. an infection is happening) – can sense the presence of dead/dying tumour cells and relay this information to so-called T cells. The two students are looking at both the molecular mechanism by which this happens, but also whether precursors of dendritic cells in the bone marrow have similar abilities.

  2. A few students in the programme are working on mathematical/computational projects and will never have to wear a lab coat. For example, one lab is interested in understanding how non-cancerous cells near a tumour interact with the cancer cells and influence their ability to move. To do this one can mathematically model the movement patterns of the “cancer-associated fibroblasts” and how they interact with extracellular proteins to form tracks for the cancer cells to move along. In the simplest terms (and that’s the only level at which I understand this), the model relies on the Morse potential, which is normally used to understand how atoms interact but can be scaled up to model interacting cells. Here is a video of a fibroblast interacting with a breast cancer cell; the accompanying text is maybe overly simplistic, but you get the gist:

    Another student is studying how sheets of cells move together, both during embryonic development and tumour formation. This relies (roughly) on modelling cells as polygonal shapes that stick together via their vertices. Yet another “dry” project is investigating how cancers evolve over their lifetime: this is done by collecting DNA sequencing data from cancer cells at various stages of their development and inferring which changes happened when.

  3. Since it is generally the metastases that are the deadly part of cancers it is important to  understand how cells move. There is, of course, a lot of information about this already but here the aim is to find out more about how different cancer cells (e.g. breast cancer and skin cancer) share certain features in their movement patterns.

  4. Not all labs in the institute study cancer. Some labs focus on basic research using yeast as a model organism. Both yeast cells and our cells contain a lot of DNA that is not translated into protein; for a long time all this DNA was termed “junk” and nobody bothered with it too much. It is becoming increasingly clear that this so-called non-coding DNA can still play various roles in the cell and some of these may be deleterious. Therefore one student is studying how cells prevent the activation (transcription) of some of these stretches of DNA.

  5. Another major branch of the institute deals with infectious diseases and the immune system more broadly. Two talks that I enjoyed on this front were given by students again working in the same lab. They are studying “neutrophil extracellular traps” (NETs), which I had never heard of before and sound quite cool. Neutrophils are a cell type of the immune system and are the first to react to infectious agents. By releasing very broad-acting antimicrobials they try to quell an infection in its infancy, but by doing so they also cause the four main symptoms of inflammation: pain, heat, redness and swelling. NETs are made of DNA and proteins from the neutrophils and are sticky. One of the students is looking into how NETs can exacerbate atherosclerosis, while the other is finding out how NETs physically trap invaders, such as the fungus Candida albicans.

  6. Lastly, and because it would pain me not to mention CRISPR, one student is trying to find a way to control the sex ratio of offspring in laboratory animals, specifically mice. While at first glance this might seem dangerous or cruel, it is actually part of an effort to reduce, replace and refine the use of animals in research. For example, if you are studying prostate cancer or ovarian cancer half of the experimental animals born are completely useless and end up being “wasted”. At the moment, some agriculture relies on physically sorting sperm cells into those carrying X or Y chromosomes and using mainly those with X chromosomes for in vitro fertilisation (because far fewer male animals are needed). Although this is a very accurate method it is expensive and time-consuming. Since CRISPR is precise and can be genetically encoded it would virtually work by itself once established.

This is by no means an exhaustive list of the topics covered by our projects, but hopefully it’s an interesting glimpse into what we are currently spending (almost all?) of our brains and energy trying to figure out.

These talks will be complemented with a so-called thesis committee meeting later this month: here each student will present a very similar talk to three professors or group leaders, who will be advising the project from an outside perspective. Hopefully being locked in with three clever and knowledgeable people will conjure up constructive criticism as well as (even more) new ideas!

A PhD Student for a Month

After the week of induction I have now spent three weeks in the lab. I have cells to look after, so that is most definitely a good thing. Mainly I am trying to figure out what kind of experiments to do that will actually give some sort of result and that is of course a lot harder than most papers will have you think. Until now I have just been finding my bearings in the lab (e.g. where do you keep the protease inhibitors? What? You freeze down your protein ladders/markers in the -80 °C freezer? Why can’t I find the antibodies in the boxes they’re meant to be in according to the Excel sheet? Etc.)

Luckily, people in the lab are all friendly and willing to help out. Despite that I still managed to crush a bottle in the centrifuge, by spinning it at 10,000 times the gravitational force. Turns out that it doesn’t need to be spun that quickly and quite evidently can’t take those forces. And this was already the second try. The first time I actually managed to rupture the bottle, losing some of my sample…

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Then of course there are the always unpredictable Western blots, as already alluded to earlier, in which one can investigate whether particular cells express certain proteins of interest. Ideally, this sort of experiment should yield a straightforward answer (more or less), as on the left of the following picture. But who even knows what happened on the right.

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Slotted between these attempts at lab work I’ve attended training to use the fancy flow cytometers, which can analyse single cells and even sort them according to different properties, and confocal microscopes. And so, let the next week of lab commence!