In silico versus in vitro #2

There is no adequate excuse for not keeping up with regular blog entries. Arguably, working in the lab from 9 a.m. to 8.30 p.m. – in a mad rush to collect some data before the lab meeting next week – in addition to preparing for seminars and the imminent badminton match against Oxford help to explain the recent scarcity of cell/science-related news. Especially when a statistically significant number of experiments in the lab end up looking like this Western blot on the right (image copied from here):

notpub-three kinds of westernsHowever, on the more positive side of things I have learnt a couple more cell/molecular biology techniques since I started working in the lab several weeks ago. Beforehand I only knew about the theory of these methods and how to interpret results, but not how to actually carry out the experiments:

  • Immunohistochemistry (IHC): this technique allows specific staining of (mouse) tissue sections. One can, for example, compare specific markers of proliferation or cell death in healthy skin versus tumours/melanoma lesions.
  • Quantitative reverse transcription polymerase chain reaction (qRT-PCR): this allows analysis of gene expression (specifically transcription) in tumour samples or cultured cells under different conditions. For example, I have been comparing the expression of certain transcription factors in cells that are sensitive to a melanoma drug versus cells that have acquired resistance to that drug.
  • Cell cycle analysis by fluorescence-activated cell sorting (FACS): lastly, this method is used to quantify the proportion of cells within a population that are actively dividing, non-dividing or dead. To do this, the amount of DNA in each cell is stained using a dye such as propidium iodide, or newly synthesised DNA can be labelled using bromodeoxyuridine.

Now on to the simple task of analysing the data…

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In silico versus in vitro

For the past five days at least I have felt bad for not updating my blog with some fact about CRISPR or the latest controversy concerning “cancer stem cells”. The reason for this involuntary hiatus is, of course, lab work. All the lab work. All the time. How should I put this; there are distinct benefits to working in silico: leaving the lab/office whenever is convenient, being able to continue work at home, and actually getting home on time for dinner.

Here I am, genuinely happy to be doing work in cell culture (isn’t the dark blue of that lab coat excellent?):

image3

And then running samples in pre-cast 20-well gels ready to do a Western blot after having treated melanoma cells with a plethora of small molecule inhibitors:

image1

But then disaster had to strike. It was all going too well. Due to the bubbles rising from the electrodes in the above picture I didn’t have a clear view of the second gel running behind it. What a disappointment:

image2The sad smiley face accurately represents my emotions concerning this gel. Well, at least I know what I’m doing tomorrow.

So I’ll end with a mini CRISPR update: Tsai et al. (2014) developed a new method last year to reduce non-specific cleavage of DNA during genome-editing. They require expression of RNA-guided FokI nucleases, which also cleave DNA, but are only active when dimeric. Each single FokI molecule is guided to its target by a guide RNA, but only when two guide RNAs each bring a FokI to the desired locus do the enzymes become active. This drastically reduces off-target effects because both the sequence and spacing has to be correct. The original CRISPR/Cas9 system has considerable off-target effects, as shown by Lin et al. (2014), for example.

Reference:

Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research 42: 7473-7485

Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotech 32: 569-576

Departmental Research Day & M. Sci. Symposium

The beginning of Lent term was far from gentle. For three days I have been sitting in lecture theatres and seminar rooms. Firstly, for a full day we listened to several professors/group leaders of the biochemistry department describing their research. And secondly, we had two days of the so-called Part III symposium, that is a twenty-minute research update from each of the 31 biochemistry M. Sci. students in the department. (Since then I have started working in a lab again and I have to admit I had forgotten how strenuous it can be.)

First things first. The “departmental research day” was hosted at Robinson College, because the lecture theatre within the department is actually too small to seat all the members of staff and students. The introduction was given by Chris Smith and his most interesting point was probably that the department received an Athena SWAN bronze award last year, which “recognises and celebrates good practice in recruiting, retaining and promoting women in Science, Technology, Engineering, Mathematics and Medicine (STEMM) within Higher Education”. So three cheers for the department!

AthenaSWANBronezAward

The actual research talks by the various professors ranged from mildly piquing to downright riveting. There were several talks on cancer (the head of the department, Gerard Evan, is a cancer biologist so this is hardly surprising): at one end for example, Helen Mott explained how basic biology, crystallography and peptide chemistry are being exploited to research a new class of drugs based on alpha-helical peptides, which are meant to block activity of some small GTPases (sometimes known as cellular switches because they can turn signalling pathways on and off). At the more clinical end, Kevin Brindle demonstrated how techniques such as dynamic nuclear polarisation magnetic resonance imaging (MRI) are progressing to better image biology/cancer in (live) patients. However, the department is also strong in the field of structural biology, since the crystallographer Tom Blundell used to be the head of the department. Furthermore, there is an increasing number of lab groups working on single-celled eukaryotes such as trypanosomes and Toxoplasma.

Additionally, there were at least two overt political references to keep us on our toes. The first one was this:

Screen Shot 2015-01-14 at 20.14.58And I have to say that I wholeheartedly agree. Perhaps unsurprisingly, the professor who used this image in her slides is originally from the Czech Republic and probably quite vehemently opposes the idea of having an in/out referendum in the UK. [Eukaryotes, by the way, are organisms whose cells contain a nucleus and would include plants, animals and fungi, but also single-celled eukaryotes such as trypanosomes and Toxoplasma.]

The second political reference was a quote by Donald Rumsfeld: “As we know, there are known knowns; there are things we know we know. We also know there are known unknowns; that is to say we know there are some things we do not know. But there are also unknown unknowns – the ones we don’t know we don’t know.” He said this in response to questions about Iraq’s involvement in the supply of weapons to terrorist groups. In the context of science this was a comment on the inherent difficulties of modelling biological processes: Steve Oliver uses yeast as a model organism to study metabolic pathways, and in contrast to the qualitative modelling I have been doing, his models are quantitative (i.e. use differential equations and enzyme kinetic data). Interestingly however, both types of model can suffer from similar problems; for example they can be plain wrong, or incomplete, or based on faulty assumptions. And sometimes when we know they are wrong that doesn’t mean we know how to improve them. Not knowing that they are wrong/incomplete (i.e. the unknown unknown) is arguably the most comfortable position to be in.

The following two days were filled with project reports of all the biochemistry M. Sci. students. It is worth noting that several of these talks were possibly more interesting and of better quality than some of those given by the professors. There was an extremely wide variety of topics including: cancer research, developmental biology, disease biology (including the rare lysosomal storage disease called Krabbe disease), in vitro enzyme evolution, structural biology (including taking trips to the x-ray source near Oxford, the Diamond Light Source), stem cell biology and research into the origins of life. This latter research project is investigating how the first RNA molecules may have come together to form larger, catalytic molecules of RNA (“RNA world hypothesis”), and to do this the reactions are carried out at -9ºC in the eutectic phase of water-ice, a condition thought to mimic prebiotic chemistry.

Lastly, what would a blog post be without the mention of CRISPR. At least two of the M. Sci. projects involve the use of this genome-editing technology. In one case it will be used to knock-out a microRNA that may be involved in the regulation of bicoid mRNA during Drosophila (fruit fly) development. And in the other case it is being used to target a transcription factor that is implicated in the regulation of stem cell fate. Interestingly, the strategy here involves using two guide RNAs simultaneously, both targeted to within the gene of interest, with the aim of creating a large deletion rather than just a small insertion/deletion.

Needless to say, the progress of all our projects is far slower than we (and probably our supervisors) would have hoped.

Michaelmas Term Round-Up

This past Michaelmas term – the first term at several British universities including Cambridge and Oxford, which runs between late September/early October to Christmas – was an exceedingly exciting time.

The master’s thesis project I am working on progressed from being an unmanageably complex undertaking to a concise and interesting project. At this stage, the melanoma model I have constructed includes not only the most basic cellular phenotypes (e.g. proliferation and cell death) as readouts, but also an indication of motility/metastatic potential. This addition seemed important to us, because in the case of melanoma the metastases cause mortality. The model accurately recapitulates a range of data from the available literature and can now be used to make simple predictions about the phenotypic readouts when the starting conditions are altered. Next term (Lent term here in Cambridge, but Hilary term in Oxford) I will actually get to test some of these new hypotheses in the lab. For example, I will be carrying out immunohistochemistry staining of tumour samples from mice with genetically different melanoma lesions and checking whether the differential activity of various signalling pathways predicted by the model actually occurs in vivo. Additionally, we will perform analyses on a cell line (from Girotti et al. (2013)) that has become resistant to vemurafenib (the drug described here) and try to confirm the mechanism(s) of resistance as predicted by the model.

Apart from the project the biochemistry course also consists of taught elements, including lectures and weekly workshops. The lecture module I took was entitled “cell fate”; we learnt about stem cells, somatic cell reprogramming, neurodegenerative disorders and cellular ageing and cell death. I’m not sure what this says about me, but one of the things I look forward to most during the upcoming holidays is being able to sit down with a cup of tea and (re)consolidate the content of these lectures, and also tackle some essay questions, such as, “Is it accurate to describe embryonic stem cells as stem cells? In what ways do they differ from adult stem cells?”

Interestingly, I think the combination of starting to use Twitter, blogging and learning more and more about the latest experiments and scientific discoveries in a particular area has made me much more aware of the current scientific literature. A particularly helpful resource is the Nature News & Views section, which provides short commentary articles from where one can follow up by reading the actual papers. For example, just a couple of days ago this led me to read about a new type of pluripotent stem cell (Tonge et al. (2014)) termed the F-class cell (F stands for “fuzzy” because of their morphological appearance). The authors conducted various experiments, including in vitro differentiation tests and teratoma formation assays, to show that the F cells truly are pluripotent. On the one hand, unlike embryonic stem cells (ESCs), they cannot form part of a chimaeric embryo when injected into the blastocyst (early stage of embryo developments). On the other hand, however, due to their morphology and decreased adherence to each other and the Petri dish they may be more suited to being grown in stirred suspension culture. And this may be useful for the testing of new drugs or in clinical applications which require reprogramming of large quantities of patients’ somatic cells. (This might be an interesting development worth mentioning in the essay I will write once I stop procrastinating by blogging.) Tonge et al. summarise their findings in this diagram (copied from the paper):

Screen Shot 2014-12-14 at 14.01.26

Lastly, this term also marked the beginning of the quest for a PhD position starting next year. Since the last update about this I have been offered a place to study type 2 diabetes at the MRC Clinical Sciences Centre! Another meeting with the supervisor in January will hopefully shed more light on the precise project to be undertaken and also help in the decision-making process.

All in all, despite (or maybe precisely because?) the differences between this term and the previous years here I feel like I’ve had a successful and worthwhile last Michaelmas.

References:

Girotti MR, Pedersen M, Sanchez-Laorden B, Viros A, Turajlic S, Niculescu-Duvaz D, Zambon A, Sinclair J, Hayes A, Gore M, Lorigan P, Springer C, Larkin J, Jorgensen C, Marais R (2013) Inhibiting EGF Receptor or SRC Family Kinase Signaling Overcomes BRAF Inhibitor Resistance in Melanoma. Cancer Discovery 3: 158-167

Tonge PD, Corso AJ, Monetti C, Hussein SMI, Puri MC, Michael IP, Li M, Lee D-S, Mar JC, Cloonan N, Wood DL, Gauthier ME, Korn O, Clancy JL, Preiss T, Grimmond SM, Shin J-Y, Seo J-S, Wells CA, Rogers IM, Nagy A (2014) Divergent reprogramming routes lead to alternative stem-cell states. Nature 516: 192-197

Project Update #3 and PhD?

Now that I’ve read what feels like most of the current literature on melanoma, I’ve been able to construct a model in Microsoft’s BioModelAnalyzer. The model currently recapitulates the main outputs observed experimentally upon changing various parameters, such as the mutational status of various genes implicated in melanoma. If I showed you a picture/screenshot of what the model looks like then some of you might recoil in fear at the apparent complexity of the network, whereas others will very quickly start to point out all the simplifications and omissions I am making. Since it is a model and is not trying to depict exactly what it “looks like” within a skin cancer cell, some of the abstractions are valid. However, the model will certainly benefit from inclusion of more signalling pathways, such as those involved in cell motility and metastasis, in the future.

Apart from making a little bit of progress with the model itself, it is becoming increasingly likely that next term I will get to do actual experiments on melanoma cells to test whether the in silico model can make accurate predictions.

Lastly, it is worth mentioning that a lot of students on the biochemistry course, myself included, are currently slightly stressed out about the prospect of applying to various PhD programmes across the country and further afield. The deadline for the first programme I have sent an application to is on Monday, and their interviews are at the beginning of December – exciting times ahead!