PhD – 21 months in 

Do you remember my optimistic blog post about finding my bearings in the lab after a month of the PhD? I also included pictures of a failed western blot and slightly crushed centrifuge tubes.

Well, twenty months later and I’m still making mistakes. Often they’re new and different mistakes, which could almost be exciting. But today I made the same mistake and lost a lot of plasmid-growing bacteria (bacteria I am using as work horses to produce specific DNA for me) in a centrifuge (which I subsequently cleaned!)…

Photographic evidence attached.

March for Science

London, Saturday April 22nd 2017

The weather is changeable as I leave the flat in the late morning. Sunny spells – dazzling my eyes clad in contact lenses – are abruptly overtaken by the English drizzle that leaves me damp and puzzled because the sun has already regained its prominence. I’m on the Westbound Piccadilly line wearing a Cancer Research UK t-shirt that reads, “I’m a researcher fighting cancer”, and I can’t tell whether I’m getting more looks than is usual on the Tube. I alight at South Kensington to meet a friend of mine, the bubbleologist Li Shen. (And yes, that is now a technical term. Li, who has a degree in mathematics, is a PhD student studying the physics of bubbles, which has far-reaching implications: from the amount of bubbles generated by different types of beer to the undesired foaming of lubricants used in oil extraction.) But we’re not just here to catch up, although it is conveniently close to his lab/office at Imperial College. No, we’re here to join the March for Science. [All of the following images were taken either by Li or by me.]

science march banner.jpg

According to the BBC, “thousands of people” joined the march, the first of its kind taking place on the annual Earth Day and organised around the world. I think the event probably got part of its boost from the Women’s Marches that took place on January 21st, the day after Donald Trump’s inauguration. Certainly, the protesters on both occasions had much in common.

destroy the patriarchy, not the planet

One of the most notable differences between the two events, however, was that this second protest was certainly smaller and also much quieter. I suppose it’s true that scientists – and yes, the marchers were mainly scientists and their relatives, partners and close friends – are a little bit shy and socially awkward. Amongst the stewards, one was trying to get the following chant off the ground, with little success, “Scientists are good at generating questions, not so good at slogans”…

french embassy

Here’s a blurry Li in the foreground, with a sharp French embassy in the background. Walking by I couldn’t help but send what’s known as a “Stoßgebet” in German to the high heavens; roughly translates as a quick (secular) prayer. For now we can breathe a brief sigh of relief after the first round of the presidential elections. Hopefully Europe, science and European Research Council funding will be able to continue to prosper.

knowledge trumps ignorance

Speaking of Trump, the March for Science event emanated from Washington DC, where it started as a protest against fake news, alternative facts and a world in which experts are regarded as worthy of derision. Honestly, as with the Women’s March, I don’t know and can’t tell how much impact marches like these actually have in politics, but as a start there was significant media coverage. Even Buzzfeed compiled its list of top banners and slogans (some scientists do have a sense of humour). My personal favourite was this one, of course.

big brains

I do know that within three months I went to two marches, the first two of my life. Ideally, I won’t have to go to any more and will be able to spend my Saturdays in the lab, where a diligent PhD student should be (and where I know some of my colleagues were). Lastly, let’s give reason, described by Wikipedia as being “the capacity for consciously making sense of things, applying logic, establishing and verifying facts, and changing or justifying practices, institutions, and beliefs based on new or existing information”, a big thumbs up.

reason

CRISPR Digest #11

By this point my interest in CRISPR research is apparently well known to the people around me: a friend shared a research article with me on Facebook by Kaminski et al. (2016) published earlier this month in the journal Scientific Reports. In this paper the researchers used the CRISPR/Cas9 genome-editing technology to eliminate the human immunodeficiency virus (HIV) genome from infected cells.

HIV is the causative agent of acquired immune deficiency syndrome (AIDS): the virus infects some of the so-called T cells in our immune system and integrates its genetic material into the DNA of the host cell. The infection causes a lot of T cells to die and eventually – over a period of years or often even decades – this makes the immune system less and less efficient. Therefore the causes of death of AIDS patients are usually infections, which would normally be fought off by a healthy immune system, or rare types of tumours. Currently there are efficient retroviral therapies available to treat HIV infection. However, these therapies do not remove the virus DNA from the patient, rather they keep the virus at bay (it is said to be “latent”). Therefore the treatment is usually lifelong and expensive.

Kaminski et al. mainly used a human cell line to test whether they could design guide RNAs that would specifically guide the Cas9 protein to the DNA sequences at either end of the HIV genome. The cell line is called 2D10 and has been well characterised and has a single HIV genome inserted at a known location, making it a good model to test their experimental tools. Since CRISPR is such a ubiquitous tool in the lab already a lot of the paper actually focusses on making sure that cutting out the HIV genome – which they manage successfully – does not have any unintended consequences. In particular, the researchers checked that Cas9 does not introduce mutations elsewhere in the host genome.

Having established these controls Kaminski et al. then go on to show that 2D10 cells with Cas9 and the guide RNAs (gRNAs) are less susceptible to a new HIV infection. To further test their system the researchers used human T cells from healthy individuals to show that these cells can also be made more resistant to infection when given the Cas9/gRNAs. Lastly, the paper shows that the technique can also target HIV DNA in human T cells from infected patients. However, here the Cas9 was not able to entirely excise the HIV genomes. Partly this can be attributed to the fact that human cells are much more heterogeneous than the 2D10 cell line: the virus will have integrated at different sites in the host genome in different T cells and there may also be several integration sites per cell.

This is an impressive study and a good step towards being able to treat patients with HIV using genome-editing technology, but there are still some shortcomings. To me one of the main problems seems to be the way in which the Cas9 and guide RNAs are delivered into the infected T cells: often this is done by putting the DNA that codes for the Cas9 protein and the gRNAs into a lentivirus, which belongs to the same group of viruses as HIV itself. The lentivirus would therefore itself integrate into the genome of the host cell and this might cause problems in itself, for example, by disrupting important genes. Furthermore, and the authors allude to this, not all HIV genomes are exactly the same and so for each patient one might have to design individual gRNAs.


Since we are on the topic of HIV/AIDS I would like to mention something another friend has brought to my attention. Some countries, such as the USA, Canada and France, have programmes to make so-called pre-exposure prophylaxis (PrEP) available to people who are HIV-negative but at high risk of contracting the infection. The National Health Service in England has recently released a statement explaining that it will no longer pursue this avenue although the once-daily pill has been shown to decrease the relative risk of becoming infected by over 90% in men who have sex with men (see, for example, Grant et al., 2010). The National AIDS Trust has therefore started a campaign for people to write to their local MPs so that this issue can be raised in parliament. And I did just that right now and realised that my MP is none other than Jeremy Corbyn.


On a slightly more upbeat note, here is a wallpaper design by Nature for their special CRISPR issue (downloaded directly from their website):

crispr wallpaper

And regarding the CRISPR patent dispute, there was a good News & Views article on the topic a few weeks ago and the take-home message is that it will probably take several years for it to be decided.

Lastly, happy Easter to all those who celebrate it in one way or another. Instead of (chocolate) eggs I will share with you a slightly abstract art image that I inadvertently took on the microscope a couple of months ago. With a little bit of imagination the organoids could be mistaken for Easter eggs.

organoids


References:

Grant  RM, Lama  JR, Anderson  PL, McMahan  V, Liu  AY, Vargas  L, Goicochea  P, Casapía  M, Guanira-Carranza  JV, Ramirez-Cardich  ME, Montoya-Herrera  O, Fernández  T, Veloso  VG, Buchbinder  SP, Chariyalertsak  S, Schechter  M, Bekker  L-G, Mayer  KH, Kallás  EG, Amico  KR, Mulligan  K, Bushman  LR, Hance  RJ, Ganoza  C, Defechereux  P, Postle  B, Wang  F, McConnell  JJ, Zheng  J-H, Lee  J, Rooney  JF, Jaffe  HS, Martinez  AI, Burns  DN, Glidden  DV (2010) Preexposure Chemoprophylaxis for HIV Prevention in Men Who Have Sex with Men. New England Journal of Medicine 363: 2587-2599

Kaminski R, Chen Y, Fischer T, Tedaldi E, Napoli A, Zhang Y, Karn J, Hu W, Khalili K (2016) Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing. Scientific Reports 6: 22555

(Almost) One year later

With large parts of continental Europe currently enduring a severe heatwave, maybe some people will remember, with fond memory, the ice bucket challenge. A year ago the challenge went viral on social media and my inaugural post featured a friend of mine doing it too. People were pouring buckets/pots/canisters of ice-cold water over themselves to raise awareness and donations for a rare disease called amyotrophic lateral sclerosis (ALS), a type of motor neuron disease (MND). One person went to even greater lengths and had glacial water released over him from a helicopter. Celebrities joined the challenge too and even Homer Simpson poured a cup of cold water over himself.

At the time, my main concern was that not enough of the donated money – a whopping $115 million in the USA and £7 million in the UK – would go into (basic) research. However, according to ALS Association approximately two thirds of the money is being channelled into various research projects. For instance, a $5 million chunk went to a collaboration between several labs based in California that investigate how nerve cells/neurons can be isolated from patients and grown as stem cells in the lab. Once this is achieved, these cells can be used to study the properties of affected neurons in more depth and eventually to test new drugs before giving them to patients. Another approach will be using $3.5 million to sequence the genomes of patients suffering from ALS and thereby find out what genetic signatures are associated with the disease. Overall therefore, my initial scepticism seems to have been unfounded since a large chunk of the money is indeed being invested in research. [To put these figures in perspective, however, let me just add that, on average, it takes ten years to produce a new drug with an associated cost of at least $1 billion.]

Pie chart showing where the $115 million went. - Image obtained from the video of this CNN article.

Pie chart showing where the $115 million went. – Image obtained from the video of this CNN article.

Apart from research, other funds are going into patient care, such as providing improved intravenous nutrition and better mobility aids, such as walking sticks, rollators and wheel chairs. Additionally, the ALS Association is investing in education and spreading the awareness for MND even further and lastly, it is trying to motivate people to re-do the ice bucket challenge this August. And if that all goes to plan I’ll write again in a year, hopefully reporting on advances in the field and maybe with some news on the early stages of development of a new drug.

Further reading:

A newspaper article in the Guardian.

A CNN article.