Why I attend all the talks: Prof Racaniello

This week I was lucky to meet Prof. Vicent Racaniello, who is a professor at Columbia University’s College of Physicians and Surgeons. He has been working on virology for about 40 years, which really impresses me (the time, not the topic). His talk was by far the better attended talk of this seminar series and the way Prof Racaniello structured it was very simple, with a very catchy title “One brain, three viruses, and one podcast”: he gave a historical perspective of his life work and dedicated quite some time to talk about science communication and why it is important. This, non-research aspect of his work is incredible time-consuming and according to himself not always brings the expected return. However, Prof Racaniello is happy if he manages to get 10 followers per travel, which I find very humbling coming from a person that has been doing science communication for 14 years, hosts five podcasts, has one blog and is present in all social media.

A few of us stayed for lunch with the speaker: listen to Prof Racaniello talk was very comforting as most of his ideas resonated with mine, in great opposition to those of the established scientists I have around me most of the days.

I did not know Prof Racaniello, his scientific work, or his science communication work before last Wednesday, but now I know which podcast I will be listening to on those long sessions in the FACS machine.


I have very recently been to a conference in Australia. As a PhD student, attending conferences is a big perk: I get to go out of my department bubble and see what is done and more importantly how it is done in other labs. In my specific case, it’s even a bigger advantage as I am the only person studying animal models of multiple sclerosis, and the only person studying so many innate immune receptors in my building (and, as far as it feels, in this city). I got some interesting warnings regarding the technical difficulties of my research and I have learned a few things that in hindsight are not that new but I had never given much thought about.

I have attended this conference series before. In fact, I have been there for the last 3 editions: in Germany, Israel and Australia. Organisation wise it does not differ much from event to event and the poster sessions are also always quite a mess. Small rooms, no space between posters, too many posters per session, you name it. This time I had to pitch my poster just before my allocation session. Where did this fail? There were three groups pitching at the same time, in the same hallway, during apero. Regardless to say that 1) no one could hear each other, 2) no one make an effort to go watch the pitches. I felt quite useless there. No feedback on the pitch either.

The positive aspect of this conference was location. Most people find it very far to travel to Australia from Europe, therefore most Americans who would collaborate with European labs also did not attend. This brought the challenge of finding topics and speakers to fill in the program. Most new topics were presented by Canadian and Australian researchers. There are a few interesting things going on, specially when it comes to the immune component of psychiatric and other neurological diseases. Will stay tuned to this in the following years.


Does Aspirin protect against Alzheimer’s?

For this week I had decided to read and summarise a paper published in J Neuroscience, about the protective role of aspirin in Alzherimer’s Disease (AD). I saw a link highlighting this paper on Facebook and could not immediately get access to the full text (paywalls). I did not even read the full title, but I already had 1 question: “did they check by correlation studies if there’s less people chronically taking aspirin, among those with AD? When I finally saw the full title of the paper, I realised that the study was more precise: “Aspirin induces Lysosomal biogenesis and attenuates Amyloid plaque pathology in a mouse model of Alzheimer’s disease via PPARα”.

Here I got a bit doubtful if I should still read the paper for this blog. But I persisted. My main concern being: why this particular pathway? The answer, which gets obvious mid-paper is: the authors seem to have worked on this one molecule before. But the rational to have chosen Aspirin as treatment seems to have a logical argumentation.

Stepping back: Alzheimer’s disease is caused by accumulation of Amyloid-beta (Abeta). This is a protein that can aggregate with others of the same type in such unbreakable structures that the aggregates are known as plaques. In general, protein aggregates would normally be broken down by the cell machinery: e.g. proteasome and lysosomes, but in AD it seems that this machinery is not working, or is not efficient enough. The authors point out that aspirin has been shown to increase the production of lysosomes.

For this study, the authors follow up on this increase of production of lysosomes by aspirin.

They started by adding Abeta to cells and then aspirin and check that indeed, cells treated with aspirin have less Abeta because 1) they take in less Abeta from the solution and 2) they get rid of the previously taken in Abeta, faster. To check the cause of this clearance, the authors checked for lysosome numbers and saw that it was increased in cells treated with aspirin. Moreover, they hypothesised this was due to new production of lysosomes and therefore looked into known transcription factors (TF) that promote lysosome biosynthesis. Having identified TFEB, they looked for enhancers of this TF and this was when they identified the PPRE site, and went on to study PPARs (Peroxisome proliferator-activated receptor), the pathway they have been familiar with. The next step in this paper is to distinguish between PPAR alpha, beta and gamma, by coupling Abeta-incubation with aspirin treatment in the presence and absence of inhibitors of these 3 molecules: meaning that the authors were able to see that when PPAR alpha was blocked, then aspirin treatment did not lead to increase lysosome production and Abeta was not broken down efficiently.

Pathway-studies done in cells is very useful to pin-point all the players in a certain process but it is not always a good model of the real situation. Therefore, the authors have used a mouse model of AD to test aspirin treatment in vivo: mice with 5 mutations in the Abeta production pathway, that are known to cause familiar AD in humans. These mice develop AD “spontaneously” after a certain age. Thus, once the mice had developed AD, the authors have treated these mice with aspirin, as well as a new set of mice that have the 5 mutations associated with AD and are deficient for PPARalpha. The conclusion is that in the group of mice that are deficient for PPARalpha, the accumulation of Abeta plaques is higher, because the same aspirin treatment is not effective anymore.

So, my question remains: is there a correlation in humans between those taking aspirin and decrease risk of Alzheimer’s disease? This study does not address that.

What this study points at is to a link between PPAR alpha mediated increased lysosome production after treatment with aspirin.


Notes & References

Transcription Factor (TF) – protein capable to bind to a sequence of DNA and therefore regulate gene expression.

Enhancer region – sequence of DNA to which TF bind.

How I chose it

I am (still) interested in the role of the immune system as the poisonous environment in which ageing and neurodegenerative diseases (e.g. Alzheimer’s disease) develop. Thus, to me it was very important to do a PhD in the field of Neuroimmunology. There are a few places in the world where this field thrives, and I had excluded outside Europe (the daily live seems to be quite different from here) and South Europe (don’t like the weather). Scandinavia is too dark in winter, and I am a person that tends to see the world through darker glasses anyway. Central Europe comes as solution.

But I refused to work one more day for free (did that for 10 months) or for a stipend: I wanted to pay taxes! Paying taxes makes me believe I have a proper job, a job that will secure my retirement, my illnesses and even my maternity leave. This is only possible in a few countries: Switzerland is one of them.

There’s a hub for neuroimmunology in Zurich, so I applied here.

When I came for the interview I did not ask

  • How many PhD/master students are you supervising?
  • What do you expect from me in the next 4 years?
  • What do you expect from this project for the next 4 years?
  • How big do you expect the lab to become?
  • What techniques can I learn that will distinguish me from the competition when applying for a new job?
  • What can I bring to this lab, from all the things I have learned?
  • Who is my “go-to” person in case I have questions with data analysis?
  • How reliable is this model? How appropriate is this model to study this condition?

I just read the project description. I talked to the Professor, the 2 junior PIs, and the PhD student that I would replace. I was very happy with what I heard and I had a very positive feeling that our way to go about life matched. I chose the lab because it was quite small (but it tripled in 2 years) and because the project was pretty solid (actually, the model was not working), and just needed some molecular characterisation that I could do (still haven’t done it) and eventually I could starting exploring other angles and putting together bits and pieces.

For some of those questions I got to see/experience the answers in real life and the others I have eventually asked. Other important questions are not to be asked, but to be answered by the PhD candidate:

  • What do you want from your PhD?
  • Do you want to supervise a master student?
  • What do you want to learn that will make you a suitable candidate in your next job? This could be a super-fancy technique, a coding language, or organizational skills.
  • What do you have in your portfolio that you are confident with, and can use as a “reliable, always working method”?

In my case, I wanted to learn immunology, specially about the immune system. This is difficult on a daily basis because the project tends to be way more narrow than the general knowledge in immunology I would like to acquire. I don’t mind supervising master students, but I do not have a structured project they can pursue on their own. I would like to acquire good knowledge of FACS and CyTOF and my go to method is qPCR. It always works.

Adventures around the water bath

The expression of proteins in cellular models or bacteria is quite common in order to study their function and structure or for large scale production.

To make a bacteria express a certain protein, the cells must take up the DNA sequence that encodes for that protein. Because proteins can be modified after production by some cell-specific mechanisms, the final protein produced by a bacteria is not necessarily exactly the same as we would find in the original environment.

Not all bacteria can take up DNA, the bacteria who can do this are called competent bacteria, and to the process of inserting foreign DNA into a bacteria, we call transformation.

About a year ago, I had to do transformation of some competent bacteria. I did not want to express the protein, I needed to be able to distinguish a sequence of DNA that had been mutated from its normal version. We were hoping that in every four bacteria: one would have taken up the mutated version, one would have the control version and the other two would have  a mix of these two sequences. Because bacteria grow very fast, overnight I would have more than 100 colonies, from which I could select 10-20 and hoping “Mendel was on my side” send this for sequencing and voilà: I would know the exact mutation I had introduced in that DNA.

I have discovered the hard-way that even competent bacteria are not so competent if the scientist is not bright. After two months of failing to see bacterial colonies, asking for help from everyone I could find in the lab, making them read my protocol, comparing it to their protocol to no avail, the issue started bugging the professor. On a hallway conversation he says “Are you sure the water bath is at 42 C?” To which I reply “which water bath? I am using the thermo-block.”. It so seems that there is a preference for water baths over heating-blocks among the older generation of scientists. Is this well supported? The short answer is: of course not!

But there are differences between water-baths and heating blocks

Water baths need a very big amount of water to function, thus once they reach a certain temperature they will be very stable in maintaining it constant throughout the entire surface. This means that every single sample will be exposed to the same temperature as the one next to it and that every single particle of a given sample will be at that temperature.

Heating blocks on the other hand are constantly giving away heat, the temperature along its surface varies a bit (I never measured but I wouldn’t expect it to be much more than a few tenths of degree), also the temperature from the bottom of the tube to the upper part should vary a bit.

Is this sufficient to make a difference in getting colonies of bacteria growing?

In our lab, we will never know: people that used to use water bath still use them, people who used heating blocks still use them. And I, I have converted! Working experiments are better than non-working ones!

In any case, how does putting bacteria at 42 C help them taking up DNA?

Basically, we “heat-shock” them: the bacteria are on ice for 30 minutes, all cold and stiff. Then they go directly into 42 C, which makes them more comfortable. Suddenly, after 50 seconds, they go back to ice. As bacteria membranes are made of lipids, and lipids are very fluid, the changes in temperature allow for pores to be open in the cell membrane, through which DNA can move in without resistant.