Mechanotransduction in Collective Cell Migration and its Synthetic Mimic

Written by: Raman van Wee

Speaker:  Joachim P. Spatz

Department: Biophysical Chemistry

Subject: Mechanotransduction         

Location: Applied Sciences  

Date: 08-09-2017   

 

Text:

Spatz kicked off by showing us several videos of collective movement, both at the population level and at the cellular level. The latter included wound healing and formation of lateral line in zebrafish. Next a video of the motion of an epithelial monolayer of upper skin came by, it was very chaotic and movingly, which actually surprised me, I was expecting a rather static situation. Quantitively speaking groups of 10 cells up to 200 micrometer showed to behave as a collective, tuning the direction of the force to the group. In contrast there are leader cells, which go into a space on their own, followed up by the rest of the group. By reversing videos, behavior of leader cells could be investigated before it became apparent that the cell would become a leader cells. These cells seem to be predetermined, as they are at least 1000 micrometers apart of each other. The system regulates itself as shown by putting several leader cell too close to each other leading to the system eliminating those that are unwanted and thus leading to a stable, well distanced situation of leader cells. Remarkably if a group of followers, following a leader cell, threatens to exceed 10 followers, a new leader is brought forward from the group. I would say this requires extensive communication and coordination to execute well.

The second part of the seminar was about making a synthetic cell. This begins by making droplets having bilayer of membrane. Using sequential pico-injection microfluids, proteins and lipids could be inserted into the future cells. This sequential addition is very important, blending it all together at once doesn’t do it. Although once in the synthetic cell, the different components can not be separated with the eye, they do actually collaborate into larger structures. Interestingly, including myosine leads to the cell rotating around its axis. The need/strength for this rotating became clear when rotation was blocked by attaching a bead. In that case tension would build up until the cell would lose contact with the glass.

Afterwards we had the opportunity to speak to Spatz directly and ask him a few questions. We came to realization that he thinks in one year from now his synthetic cells will have some sort of mitochondria like energy factories. For the duplication part of life he thinks other research previously done can be very beneficial, drastically lowering the time required to integrate it in his cell. Besides, what actually surprised me was that his seminar was primarly based on 2 papers, whereas I expected more.

 

Conclusion:

Collective migration is mechanically regulated by the length up to which cells collectively integrate forces together. The cells follow this rule to select and follow new leaders. In synthetic cells, components have to be inserted one by one although they eventually entangle. Forces in a cell can lead to the cell rotating around its axis.

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Information of the structure of the universe

written by: Raman van Wee

Speaker:      Robbert Dijkgraaf

Department: Physics dept. Princeton

Subject:       The relation between the extremely large & the tiny world

Location:     Applied Sciences

Date: 07-09-2017   

Professor Dijkgraaf told us about the various scales scientists work at. We concluded that life (& Nanobiology) is centered at the middle of this sizescale.
According to Dijkgraaf this makes studying life extra challenging. In the outer sizes a different set of rules and equations hold, but in the middle, where life happens to be, the collide, making it extra hard. I found this both surprising and intriguing. Surprising because it posed quite a contradiction with my
instinctual beliefs, intuitionally I felt the center of scale is the relatively easy part and going out into space or into the world of quarks would be more challenging. It intrigued me as well, because as a Nanobiologist I am working right around this center!
The concepts in the introduction were well imaginable and thus comprehensible, as the seminar continued, it became less and less logical for me. A lot of concepts were new for me and were touched on only briefly, hence I lost parts of the seminar. Just like Dijkgraaf said one could easily get lost in H space while I didn’t even know what is comprised. Despite me not following all the content, it still widened my horizon, as it became apparent how much there is around I still don’t know anything about. A lot of those concepts and theories are also more or less related to my field of study and are therefore worth taking a closer look at some time. Although slightly off topic, when Cees Dekker introduced Dijkgraaf, he told us about some of the many committees, boards or councils Dijkgraaf has working in. I found this interesting as well, besides his own research he is also looking at science from different levels and angles, which I believe is anything but boring.

Conclusion:
All in all life is at the center of the sizescales. Beyond the cellular and organelle level there is yet so much to be discovered. In the future a beautiful goal
would be to implement concepts in the universe scale into the atomic scale and vice versa. Personally I am most interested in some of the quantum related theories and concepts.

What you can learn from computer modelling

Speaker: Christine Jacobs-Wagner
Department: Casimir Research School
Subject: How to achieve cellular replication without fail: lessons from bacterial cells
Location: Delft
Date: October 13, 2017

Although Christine Jacobs-Wagner studied biochemistry in college, she would describe herself more as a cell biologist. According to her, one is not defined by education, but rather by the questions they pose themselves. That’s why her lab is very multidisciplinary, which made her seminar really interesting. I liked the way she didn’t just come to Delft to lecture us about what’s she’s done, but rather to get feedback from the scientists here, who might have a stronger modelling background than hers. This is how science should work, and it’s a great feeling to be a part of it.

She spoke about the importance of distributing plasmids evenly during cell division. If one of the daughter cells doesn’t inherit an essential plasmid for some reason, it will not survive. That’s why it is crucial to have a method in place to guarantee the survival of both daughter cells. The ParA and ParB proteins make up such a system. This was the focus of her research.

Untitled
Plasmids should be distributed like these green dots

The ParA proteins bind to the nucleoid of a bacterium, and the ParB associates with plasmid DNA. When they interact, ParB induces a reaction which breaks the ParA-DNA bond. This is all well understood, but how would this have the desired effect of distributing all plasmids evenly? Well, it might be easier to observe what happens when there’s only one plasmid in the cell. It starts to oscillate along the long axis, but why?

 

 

Then she proceeded with an in silico model, a computer simulation. Unfortunately, the first model didn’t show the desired behaviour. The plasmid made a random walk, so the Par proteins didn’t do anything. At least, not until she adapted the model to make the DNA non-static. She could model ParA molecules as tiny springs, which oscillate around an equilibrium position. This was a very small change, but it had a great impact. It was all she needed to accurately model the oscillatory behaviour.

It was really cool to see such a small change make such a big difference. For further research and exploration of the ParA-B system, an analytical model would be useful, but that will also be a big challenge. I’m excited for the future!

Joachim Spatz: How wounds heal

Speaker: Joachim Spatz
Department:
Casimir Research School
Location: Delft
Date: September 8, 2017Transmission electron microscopy image of a biomimetic nuclear pore complex. Stefan Kowalczyk

Subject: Mechanotransduction in Collective Cell Migration and its Synthetic Mimic

Wound healing is a very important part of the medical sciences, so it is not very surprising that scientists have been studying it for a long time. Some 20 years ago, when looking at the way cells in the dermis – a specific layer of the human skin – closes a wound, they noticed a peculiar pattern. Although there is a lot of movement and fluctuations in this layer, some ‘leader cells’ will inevitably arise, evenly spaced and venturing into the void of the wound. These would be followed by many other cells and thus does healing begin.

 

In his talk, Joachim Spatz explained how he discovered the mechanism behind electing these leader cells by means of traction force microscopy. Essentially, he prepared a glass plate, covered with an elastic hydrogel containing a matrix of fluorescent beads to which cells adhere. Following their movement, one can measure the forces these cells exert on each other. The cells themselves were grown in a rectangular stencil on top of this gel. When he removed this stencil, he essentially created a wound, a void for the skin to grow into.

They made two important discoveries. Firstly, the cells are constantly moving around and fluctuating with respect to density and tension forces. They saw areas with high and low forces. This led them to measure the ‘force correlation length’. It’s a distance of about a 160 μm over which the beads are pulled in the same general direction and with about the same force. Secondly, just before a leader cell becomes visually apparent, there is an area of high forces just behind it. This means it is not a single cell’s autonomous decision to become a leader, but rather a consequence of the general population dynamics.

Along a long stretch of skin boundary, multiple leaders will emerge, and the spacing between them is always the same. This is explained by the fixed force correlation length. Next to the area of high tension behind the leader cell, there has got to be an area with low tension in which a leader cannot form. This means that the number of leader cells to form, important for the efficiency of the healing process, does not need to be actively regulated but is a consequence from the apparent chaos of the cells’ movements.

The second part of his seminar treated the creation of a synthetic cell. A subject that is being actively researched in Delft as well. It reminds me of Feynman’s famous quote: “What I cannot create, I do not understand”. The idea being, of course, that we’ll never completely understand how a cell works, until we can create it ourselves. Spatz’s approach was based on forming tiny water droplets in an oil solution. Since the two don’t mix such a water droplet can be the foundation for a synthetic cell. He then injected a mixture of hydrophilic and hydrophobic proteins into the droplet, which formed a membrane, further separating the cell from its environment.

Of course a normal cell has a membrane made of two layers of phospholipids, so he had to find a way to get these and other molecules into the cell. He ended up using an electrode to make the protein membrane porous allowing him to inject whatever he fancied. To make the lipid membrane, he injected a number of smaller vesicles, small membranes of lipid bilayers, and positive ions to destabilize them and fuse them together until they were big enough to form a membrane for the whole cell. FRAP experiments confirmed that these were similar to those of biological cells! Basically, they made the lipids fluorescent so they could see them under a microscope. Then they bleached an area by shining a very bright light on it. A property of biological membranes is that they are ‘liquid’, meaning that the individual phospholipid molecules are constantly moving around. This means that the extinguished phospholipids should diffuse into the greater, non-bleached, area and that other ones (still fluorescent) should be able to take their place. So the bleached area should disappear after some time. This was new to me and I find it very interesting.

But what is the link with the first part of his talk? Well, Spatz was also able to inject myosin and actin into the cell. These are essential to the movement of a cell and can be found in high concentrations in muscle cells, for example. When the cells floated up in the oil, they hit the glass plate and adhered to it. It meant that the cell suddenly had a fixed point of contact and that allowed the myosin to exert forces on the plate, which made the cell rotate. This can be seen through a microscope. If you also fix a little bead to the outside of this membrane, it can stop the rotation when it hits the glass. That is, until a sufficient force is generated to make the cell ‘jump’ over the bead, thereby actually moving the cell instead of rotating in place.

In conclusion, these artificial cells aren’t nearly as complex as their biological counterparts, but the technology is advancing and so does their complexity and capabilities. Joachim Spatz already succeeded in extracting them from the oil into an aqueous medium and he is working on an artificial mitochondrion. Lots of interesting research opportunities. I’m excited to hear what more will be discovered, or to work on it myself.

How to achieve cellular replication without fail: Lessons from bacterial cells

Speaker:     Christine Jacobs-Wagner

Department: Molecular, Cellular and Developmental Biology, Yale University

Subject:       How to achieve cellular replication without fail: Lessons from bacterial cells

Location:    TU Delft (BN)

Date:           13-10-2017

Author: Antoine Rolland

This talk was given by Christine Jacobs-Wagner. She started her talk by saying that most of the work in her lab is based on different phases in the self-replication of the cell. The research is mostly done on bacterial cells, since these are for example simpler than eukaryotic cells. In this talk, she introduced a mechanism that made sure that some low copy number plasmids in bacterial cells are evenly distributed among the two daughter cells after replication.

This mechanism actually consists of a pretty simple biochemical cycle, where two molecules are the most important: parA and parB. Both of these factors are encoded by the plasmids themselves. ParB can bind to the plasmid and cover it almost completely and parA can, after it has bound an ATP,  bind to the chromosomal DNA, which is spread almost all around the bacterial cell. ParB has a high affinity for parA that is bound to the DNA, and in this way the plasmid, which is covered with parB, can be connected to a given loci of the chromosomal DNA where parA is bound. After a small time step, the parB unbinds and the parA loses its ATP, after which the cycle can be repeated. Experiments have shown that this cycle creates a near-perfect distribution of the plasmids  along the long axis of bacterial cells. In case all the plasmids are merged, so only one large plasmid is present, this plasmid oscillates along the long axis over time, following the gradient of the parA concentration. However, in a first model in which only this cycle was introduced, this behavior wasn’t observed. This was caused by the fact that it was assumed that the parA molecules, once they were bound to the chromosomal DNA, would be static. This is not the case, since loci oscillate from its equilibrium position in the cell, because of a certain spring force.

When this part was introduced to the model, exactly the same behavior was observed as in the in vitro and in vivo experiments. Also, changing some parameters, like the amount of parA or the cell shape or size, still produced a nice distribution of the plasmids. What I would be interested in, is which parameters would disturb this distribution, and how this is done.

model parA parB

One of the models of a bacterium cell which was used, with in green the parB covered plasmid and in red parA

What I conclude from this talk is that a simple system, like the biochemical cycle of parA and parB, can still create a very nice distribution of the plasmids before replication. Before this talk, I would think that some really complex processes with a lot of components would be needed to create such a distribution. Also, I think it is nice to see how the model first didn’t work at all, but after one small change, namely having the parA molecules experience a spring force, it made the model work perfectly.

Mechanotransduction in Collective Cell Migration and its Synthetic Mimic

Speaker:     Joachim P. Spatz

Department: Cellular Biophysics at Max Planck Institute for Medical Research,                                  Biophysical Chemistry at University of Heidelberg   

Subject:       Mechanotransduction in Collective Cell Migration and its Synthetic                                Mimic

Location:    TU Delft (BN)

Date:           8-9-2017

Author: Antoine Rolland

The lecture was divided into two parts. The first part was about the dynamic movement of skin cells, which is influenced by different forces acting on them. The second part was about how this behavior could potentially be mimicked in synthetic cells.

In the first part, Joachim Spatz began by saying that group movement is a very interesting topic in biology. From birds flying together to cells moving in groups, it is very exciting to study the way they move. Joachim Spatz has been looking at the way skin cells move when there is no boundary on one side of the cells. This is comparable to the way skin cells move in the process of healing a wound. What is well known is that there are so-called leader cells that begin to move into the open space, and that a group of cells go and follow that leader cell. What was found out is that these leader cells are already determined before they go and lead the other cells. This was discovered by measuring the force that was on the cells. What was observed was that there were strong forces behind the leader cells, when that cell cannot be distinguished yet. What was also very interesting, was that a leader cell always ‘recruited’ nearly the same amount of cells. This can be explained in a mechanical way, because this is the amount of cells that a leader cell can reach with the force that it exerts.  What I found the most interesting and surprising about this part is how mostly physical properties determine the way the cells move, rather than the biological properties of the cells. Joachim Spatz explained that there are sensors in the cells that can sense the force on them, and that in this way the leader cells can be determined. Moreover, the density had a big impact on the way the cells moved into the open space. The process went a lot faster when the density was lower. A higher density required to first eject some cells into the upper layer, before the process could start properly.

In the second part, Joachim Spatz explained how this movement of the cells could potentially be mimicked in synthetic cells. For this, a stable cell environment is needed. This can be achieved by creating nanodroplets with a polymer outside layer. To make an equivalent of the cell membrane, a lipid bilayer was added that bound to this polymer. It was proven that by adding components to this nanodroplet one after another, the result was better than when adding pre-assembled components. These nanodroplets also showed forms of adhesion, making it possible to apply and let them exert forces to potentially make the system from the first part in a synthetic manner. After the talk, we discussed some things with Joachim Spatz. What was interesting was the following question that arose: what exactly is life? To Joachim Spatz, for his cells to really become artificial life, they should be able to replicate, to move by their own and to create energy for themselves from the environment.

Leader cell with its follower cells

What really stood out to me from this talk is that physics and forces inside cells are way more important than I thought. Also, analyzing group movements of different kind of cells, could be very interesting for future research. Maybe we would discover that in group movement forces are often a very important factor. For me, this group movement is very interesting, and I wouldn’t mind learning more about this. Also, I think we are very close to creating synthetic life. Of course, ethical questions will rise if eventually real synthetic life could be created. I think this will be a big challenge for the future.

Information, from the structure of the universe to life

Speaker: Robbert Dijkgraaf
Department: Kavli institute
Subject: Information, from the structure of the universe to life
Location: TU Delft
Date: 07-09-2017
Author: Renée van der Winden

For the Kavli Day on September 7th Robbert Dijkgraaf gave the opening lecture. In this seminar he did not talk about a particular research subject, but rather tried to give us an insight in the assumptions and dimensions we work with in science. In order to do this he discussed several ways in which we can distinguish different types of science and gave examples of many scientists and the different views they have on this matter.

The first thing Dijkgraaf discussed was the scale on which we operate. The smallest things in life we can explore are on the 10-35 m scale, while the biggest structures are about 1025 m. The things we are studying, life and cells, are right in the middle at around 10-5 m. When considering beauty and elegance in science, you can find it at the ends of this spectrum. Those are the places where the messy, everyday life structures crystallize into simple and clear-cut laws and phenomena. The fact that you see this at each end is a reflection of two ways to approach science: reductionism and emergence. Reductionism starts at bigger phenomena and tries to uncover the laws that underlie it. For example, going from a tree to photosynthesis. Emergence starts out at laws of nature and tries to see what they bring about. For instance, going from collisions of water molecules to fluid dynamics. Whichever approach you choose, the end product seems to be more elegant from that point of view, though not from the other, which is interesting. This realization is of the same order as one of the quotes Dijkgraaf used in his presentation, this particular one coming from John Wheeler: “Every law of physics is approximate, not precise”. Dijkgraaf used this quote to illustrate that no matter how elegant a theory might seem, it is still something we came up with to explain our data.Seminar 9

Figure 1: Exerpts from the movie ‘Powers of Ten’

Another thing Dijkgraaf illustrated is that what we consider to be truths, might not in fact be true completely and always. He pointed out the many theories Einstein has come up with to explain how relativity emerges from quantum theory, all while ignoring the possibility that things might be the other way around. Moreover, while discussing black holes, Dijkgraaf mentioned that something strange happens when you move within a certain zone around it, called the horizon. After the horizon, time flips and stops being endless, rather it moves to the center of the black hole, where time stops. What we consider as a given (the flow of time), turns out to be false under certain conditions.

Dijkgraaf ended his talk by saying that the scale of the powers of ten that he started his talk with, might not be the endpoint. He stated that it might be possible there is an even smaller layer below it, a so-called basement layer, which may be concerned with quantum information.

I had hoped to get a chance to see Robbert Dijkgraaf speak for a while, so I was very excited when that opportunity came around. His talk was easy to follow and interesting. I think Dijkgraaf is good at explaining even difficult topics concisely and clearly, so I would very much like to hear him speak about his own ongoing research as well at some point.

Information from the structure of the universe to life 

Aïsha Mientjes 

aishamientjes@gmail.com 

4460960 

Seminar 6:  

Speaker:  Robbert Dijkgraaf               

Department: Institute for advanced study (Princeton) 

Subject: Information from the structure of the universe to life 

Location: TU Delft            

Date: 07-09-2017         

On the 7th of September 2017 I attended a seminar given by Robbert Dijkgraaf in de new AS building. Robbert Dijkgraaf started by giving us some information on scales and ‘the power of ten’. He explained that 10 to the power of –35 is the smallest (Planck) scale and the largest scale is 10 to the power of 25 (the Hubble scale). He also elaborated that life occurs right in the middle of this ‘scale line’. Which he finds quite remarkable. He also said that we have discovered approximately three quarters of this ‘line’. 

powers of ten

He continued by explaining a little bit about reductionism and emergence, and that the concepts that we work with are not fundamental but emergent. He also explained the phenomenon of rescaling in physics, where we take a building block of something as a new base. Zooming in and zooming out is a very interesting and intuitive way to look at physics. Life simplifies at both ends. 

Then we arrived at the more technical part of the lecture, which concerned quantum theory and black holes. We were given an explanation about the Einstein-Rosen bridge, which connects spacetimes. We were also told a bit about the horizon of a black hole, where the direction of time is reversed. The amount of information a black hole contains is determined by the size of its horizon. Robbert Dijkgraaf also explained to us that black holes collide and form larger black holes.  

The lecture ended with a small not on quantum information, which is even beyond the smallest scale. It is quickly becoming a universal ‘currency’ in science. 

I found it very nice to attend a lecture of such a famous figure in the world of science. The building was very full which was extra confirmation for me that attending something like this is truly a special experience. I found the talk very interesting and generally fairly easy to follow. There were some more complicated bits, but most of the talk I could understand well. The topic was very appealing to me as well, even though I found it a little abstract at some places. All in all I really enjoyed attending this seminar.

Linear and nonlinear cascade structures as auditory filter models and machine hearing front ends

Speaker: Richard F. Lyon
Department: Neuroscience
Subject: Linear and nonlinear cascade structures as auditory filter models and machine hearing front ends
Location: Erasmus MC
Date: 04-09-2017
Author: Renée van der Winden

In this seminar Richard Lyon came to talk to us about his work on machine hearing. When proposed with the problem of speech recognition, Lyon realized he did not even know how humans performed that task. So he went and studied how the human auditory system works in order to try and replicate that in machines.

The human auditory system contains a structure called the cochlea, which has a spiral-like form. The sound travels along this spiral as a waveform and the structure vibrates in specific places depending on the frequency of the sound. Lyon found the cochlea can be modelled as a cascade of filters through which the sound passes. If these filters are linear, that means the system is not signal dependent. If they are non-linear, the system is signal dependent and that is thus the more accurate representation of the real-life situation. Now on to the type of filter. It turns out that the best model for the human auditory system is a pole-zero filter cascade. You can still distinguish between different scenarios here; you can have either stable or unstable zero crossings. When there are stable zero crossings, the zero crossings of signals with different frequencies align, while for unstable zero crossings they do not. The model that was eventually chosen by Lyon is termed CARFAC and is somewhere in between the pole-zero models with stable and unstable zero crossings. It turned out to be an efficient cochlear model and also an efficient machine front end. Moreover, CARFAC can adapt the gain of the filters to model desensitizing.

Seminar 8

Figure 1: Model of waveforms travelling along the cochlea (Lyons, 2017)

There is a big drawback to CARFAC though, since it is very expensive. Lyon discussed two solutions to this problem. The first one is to use a linear model instead of a non-linear one. This would then be named CARL (Cascade of Asymmetric Resonators, Linear). The second solution is an idea that Lyon came up with. He proposed to think of sound not as a waveform, but as particles. He called these particles ‘sound atoms’. If you combine several of the sound atoms you can also get sound molecules. You can then model these sparse events instead of the waveforms and that is also cheaper. Lyon termed this model CARLA (Cascade of Asymmetric Resonators, Linear with Atoms).

I thought Lyon’s approach  to the speech recognition problem is a great one. It is a very logical approach when you think about it, but apparently people around him had not thought of it. I could not quite understand the details of the model and its workings, but I was interested in how to model a biological system to fit machines. I would like to know a bit more about how it was determined that a filter cascade was a good starting point for a model in this case.

Targeting Epigenetic Changes in Immune Cells: Implications in Disease

Speaker: Esteban Ballestar
Department: Bellvitge Medical Research Institute (IDIBELL), Barcelona Spain
Location: Erasmus MC
Date: July 13, 2017
Author: Teun Huijben

Esteban Ballestar got his Bachelor and Master degree at the University of Valencia in Spain, followed by a PhD. Afterwards he did a Postdoc abroad and returned to start his own research group at the same university. The main interest of his group is the DNA methylation, and especially in the context of diseases involving the immune system.

The first part of Estebans talk was about mapping DNA methylation in immunological diseases to understand which proteins are involved in the disease. The group of diseases they studied were Common Variable Immunodeficiencies Diseases (CVID) in which the body has not enough primary antibodies. These diseases are mostly caused by severe deficiencies in the number of switched memory B-cells. With switched B-cells we mean activated B-cells that start producing the antibodies in high quantities after recognizing the antigen. By mapping the DNA methylations of these B-cells, they hope to find genes that are differently methylated and are mostly likely causing the disease.

Methylation of DNA means that a methyl group is added to the 5-prime end of a cytosine (5mC) nucleobase. This can only be done if the cytosine is next to a guanine (see Figure 1). DNA methylation is maintained by de-novo DNA methyltransferases (mostly DNMT1, DNMT3A and DNMT3B). DNA methylations can be actively removed by demethylation, in which the 5mC is oxidized to a 5hmC or 5caC. Adding or removing methyl group to the DNA has an effect on the gene expression of that particular gene.

zakhari01

Figure 1: DNA methylation. The cytosine of the CG-pair gets methylated by a de-novo methyltransferase (DNMT). [S. Zakhari 2015]

To identify disease causing genes, Estebans group did DNA methylation profiling of the B-cells from a CVID patient. To eliminate as much side effects as possible, thy only investigated twins of which one sibling had CVID and the other was healthy. After collecting the B-cells, they performed DNA methylation profiling and looked at genes with different methylation profiles between the two brothers. They found 230 genes that are more methylated in the CVID patient and 81 genes that are less methylated. Gene ontology analysis showed that most of the genes were related to immune responses, indicating that changing the gene expression of these genes can cause an immune related disease.

All the genes that showed different methylation profiles between healthy and CVID patient, were taken into further research. The B-cells are sorted on the fact whether they were naive (not yet switched to active) or switched. They found that in healthy persons most genes got demethylated after the transformation from naive to switched. On the other hands, the same genes in CVID patients showed no decrease in methylation, a second indication that these genes are involved in the disease.

However, the question remains whether the different methylation profile itself causes the disease, or is it a downstream effect caused by other factors. To investigate this, more research needs to be done on this subject. Also, more methylation profiles of twins are needed to draw real conclusions about the disease causing genes, since one set of results of the statistically valid enough. Overall, the talk of Esteban was interesting and he is a very good speaker. Despite using many difficult immunology term, he explained very clear the research his lab is doing.