Molecular and functional heterogeneity in the human haematopoietic stem cell compartment

Speaker: Elisa Laurenti
Department: Cambridge Stem Cell Institute, University of Cambridge, Cambridge
Location: Erasmuc MC Rotterdam
Date: Juni 12, 2017
Author: Teun Huijben

Elisa Laurenti has been interested in stem cells during her entire academic carrier. After doing a PhD and PostDoc in this field, she now has her own lab at Cambridge, where she studies the heterogeneity in the human haematopoietic stem cell (HSC) compartment. In this one hour she introduced us to the field and explained the research she has performed in the last years.

The main point of Elisa’s talk was that where we all think of stem cells as just stem cells, there is actually a large heterogeneity between them. By quantifying the differences between the different HCSs, she hopes to define distinct subsets with different functions, characteristics and detectable markers within the broad HSC-pool.

By single-cell analysis, Elisa found two distinct subsets of haematopoietic stem cells: long-term HSCs (LT-HSC) and short-term HSCs (ST-HSCs). They are characterized by just two surface markers: LT-HSCs express high levels of CDf49 and low levels of CD90, where ST-HSCs have opposite expression levels. The functional difference between them is that LT-HSCs divide very rarely, and ST-HSCs divide more often.

Transcriptional analysis of LT-HSCs and ST-HSCs didn’t give any results, they both showed the same expression landscapes. One explanation for this could be that both cells are very quiescent and therefore not transcriptionally active. The solution Elisa and her colleagues found was to activate the cells and then analyse their transcriptomes. Once activated, the cells start in quiescence, which is a ’sleeping’ state, and are then activated. The ST-HSCs are activated earlier than LT-HSCs, which is another functional difference between them.

To activate the in vitro cultured HSCs, they are transplanted into living mice or into in vitro cultured tissues. Both activated cells are analyzed by single RNA-seq and microarrays. By doing this, they found at least 34 genes that are differently expressed between two subsets. CDK6 appeared to have to most distinct difference in expression between the two groups and was the best gene to indicate whether a cell is ST-HSC or LT-HSC. Surprisingly, treatment with CDK6 determined the state of the cells: over-activation of CDK6 resulted in a faster activation and a CDK6 inhibitor resulted in slower activation.

However, next to this direct effect by changing the expression level of CDK6, also long-term effects were measured. When CDK6 was over-expressed, LT-HSCs gained a positive competitive advantage over SC-HSCs over the long term. In other words, they outnumbered the ST-HSCs. This can be explained by the fact that CDK6 stimulates activation of the cells. ST-HSCs already activate quite fast, so stimulating activation results in activation of all ST-HSC. They all start differentiating and no ST-HSC will be left. LT-HSCs on the other hand, activate more slowly and will remain abundant in the HSC-pool, and will eventually dominate over the ST-HSCs in number.

In the remainder of the time, Elisa told about her current research in further defining subsets of haematopoietic stem cells by finding new markers that characterize distinct groups. Her talk emphasized once more the difficulties we face when looking at stem cells, or molecular biology in general; tissues are very heterogenous and we do not yet know a lot about all their differences. However, her talk was very clear and she is obvious an important person in this field.

Light harvesting and photo-protection in photosynthesis

Speaker: Lijin Tian
Department:
 Biophysics of photosynthesis
Subject:
Light harvesting and photo-protection in photosynthesis
Location: TU Delft
Date: 24-05-2017

Author: Kristian Blom

At the 24th of June I visited a talk given by dr. Lijin Tian, a postdoc candidate for the Nynke Dekker lab who is currently working in the lab of Prof. dr. Roberta Croce in the department of biophysics of photosynthesis at the VU. The main research goal of Prof. dr. Croce is to get an understanding of the light reactions of photosynthesis at the molecular level, with particular emphasis on light absorption, excitation energy transfer and photo-protection.

Dr. Lijin Tian started the talk by introducing photosynthesis and the main actors involved in this process. Photosynthesis can be divided in two separate processes: light-dependent and light-independent. In the light-dependent process the solar energy is harvested by chlorophylls and thereafter converted into chemical energy. In the light-independent reactions, called the Calvin cycle, carbohydrate molecules are assembled from carbon dioxide using the chemical energy harvested during the light-dependent process. For the light-dependent process there are two multiprotein complexes, called Photosystem I and II, who catalyze the process of light harvesting and conversion of light energy into chemical energy. Both these complexes can on their turn be divided into two parts: an antenna system that harvest the light energy and is responsible for the energy transfer to the reaction center, and a core complex in which charge separation and electron transfer takes place.

For the harvesting of light energy there is an critical value regarding the amount of energy influx into the reaction centers. Above this value irreversible damage to the photosynthetic system can be caused. To circumvent this problem, photo-protective mechanisms in the antenna system can reduce the energy influx by quenching excess excitation energy as heat in a process known as nonphotochemical quenching (NPQ). In their latest research paper, Prof. dr. Roberta Croce and her lab show that LHCII, the main light harvesting complex of algae, can only switch to a quenched conformation as a response to a pH change when LHCSR1 (light-harvesting complex stress related 1) is present in low concentration.

Figure 1: Fluorescence traces of LCHII-only and LCHII+LHCSR1 cells. (A) LHCII-only, (B) LHCII+LHCSR1 cells with (red)/without (black) nigericin. The signal was collected at 680 nm. Nigericin (100 μM) addition and pH changes are indicated by arrows.
Image from: E Dinc, L Tian, LM Roy, R Roth, U Goodenough, R Croce (2016) “LHCSR1 induces a fast and reversible pH-dependent fluorescence quenching in LHCII in Chlamydomonas reinhardtii cells”

Regarding the talk itself, I didn’t found it that interesting. For me it was hard to follow the story, since the speaker his English wasn’t that good. Also, and this is not the first time that I notice this, the slides were overcrowded with images and data. It was quite surprising for me that there isn’t any research lab in BN that focusses on photosynthetic systems, since it is such a fundamental field in cell biology. At the end of the talk one of the PI’s of BN asked a lot of questions regarding the status of the research field in photosynthetic systems. From my point of view it almost looked like he/she was planning to start a new lab with a specialization in the biophysics of photosynthesis. Perhaps that within a few years from now we have a new lab in the BN department.

 

EMT-controlled phenotype switching drives malignant progression

Speaker:              Geert Berx

Department:      JNI Oncology

Location:            Erasmus MC Rotterdam

Date:                    24-5-2017

Author:                Katja Slangewal

Dr. Geert Berx was one of the people how helped discovering E-cadherin as aberrantly regulated in cancers. E-cadherin is for instance lost or partially lost in invasive lobular breast cancers. However, this type of breast cancer only represents 15% of all the breast cancer types. Dr. Berx went on in studying the transcriptional regulation of the protein. He found that mutations in specific E-boxes right before the transcriptional start site can lead to the loss of E-cadherin expression in epithelial cells. This brought him to a protein called ZEB2, an important transcription factor.

ZEB2 is, together with ZEB1, part of a small but evolutionary conserved family. Both ZEB1 and ZEB2 have multiple interaction partners. One of which is E-cadherin. ZEB2 represses E-cadherin expression. This has been shown by inducing a knock-out of ZEB2. This resulted in an upregulation of E-cadherin, leading to mis-expression. On the other hand, a conditional up regulation of ZEB2 leads to the loss of E-cadherin expression. The same is true for another transcription factor called Snail. Dr. Berx and his group have focused on ZEB1, ZEB2 and Snail as transcription factors regulating E-cadherin expression. The loss of E-cadherin has a cancerous effect. The hallmarks of invasive cancer cells are occurring. This means that cells are undergoing EMT (epithelial mesenchymal transition, figure 1). This transforms the cells in invasive cells.

seminar 12

Figure 1: Epithelial to Mesenchymal transition. The main processes and related proteins are indicated in the picture. http://www.ijournalhs.org/article.asp?issn=2349-5006;year=2015;volume=8;issue=2;spage=77;epage=84;aulast=Angadi

EMT is a process controlled by four major interconnected regulatory networks:

  • Post-translational control
  • Transcriptional control
  • Differential splicing
  • Non-coding regulation

So far it was mainly thought that cells are either in a stable epithelial state, an unstable transition state or a stable mesenchymal state. However, dr. Berx has shown eight metastable transition states.

EMT is not only associated with invasive cancers. Dr. Berx also shows a connection to stemness of cells. This can be explained by taking a look at the reprogramming of iPSCs (induced pluripotent stem cells). When firoblasts are being reprogrammed towards iPSCs, they undergo an epithelial intermediate. This state is reach by a decrease of for instance Snail. Snail has also been shown to regulate stem cell maintenance in the intestines. ZEB2 on the other hand has been shown to be important for stem cell maintenance in embryonic hematopoiesis. When ZEB2 is knocked- out, stem cells accumulate and leukocytes, erythrocytes ad platelets are reduced. This means that ZEB2 is not necessary for stem cell maintenance, but it is necessary for faith determination.

In the recent months, dr. Berx has mainly focused on ZEB2 in melanoma cancer types. Melanomas are a product of EMT, so this makes it interesting for ZEB2 expression. A ZEB2 knock-out mouse has no pigment. This indicates that ZEB2 functions in melanocyte differentiation. A very interesting observation was made during this experiment. When ZEB2 levels decrease, ZEB1 levels automatically increase. An immunostaining revealed that ZEB1 mainly localizes in the stem cell compartments, whereas ZEB2 is mainly found in the differentiated states of melanocytes. Also, an overexpression of ZEB1 leads to a dedifferentiated gene signature in primary melanocytes. A double knock-out of both ZEB1 and ZEB2 lead to growth arrest, indicating that at least one of them is necessary for a cell to function.

In short, an increase of ZEB1 leads to a highly invasive signature and almost no proliferation. An increase of ZEB2 leads to a low invasive signature and high proliferation. This observation led to the following model:

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Figure 2: The oscillating model proposed by dr. Berx and his group.

In the primary tumor ZEB2 is high. When invasion starts and EMT is happening ZEB1 takes over. Then in the metastasis ZEB2 expression increases again. So ZEB2 drives melanoma proliferation and differentiation. Enhancing the ZEB2 expression does not lead to an increased melanoma frequency. However, it does lead to an increased metastasis formation. For this to happen an oscillation between ZEB2 and ZEB1 expression is necessary. This is regulated on protein level by TNF. TNF probably marks one of the ZEB proteins for degradation. However, how this regulation exactly takes place is not known.

After reading this you will probably have noticed this: increase of ZEB2 leads to increase of metastasis. So, shouldn’t there just be a drug that inhibits ZEB2 expression? Unfortunately for drug design, biology is not that simple. ZEB2 does not only have an important role in E-cadherin expression. It is also required for natural killer cell maturation. This means that an inhibition of ZEB2 will cause an immature immune system, leading to many more problems. Luckily, there are many more options to investigate. Recently, it has been shown in a different cell line, that an increased expression of ZEB2 marks the cells sensitive to a specific drug. This observation is useful to prevent giving unnecessary medicine to patients. Dr. Berx and his group will focus on this feature in melanomas in the near future.

My final seminar, was an interesting one. Dr. Berx had a clear story. Just like my very first seminar, there were a lot of technical terms embedded in the talk. I am very happy to see the difference in how far I could understand this talk compared to two years ago. It makes the talks a lot more interesting if you can understand them properly.

EMT controlled phenotype switching drives malignant progression

Speaker: Geert Berx
Department: Molecular and Genetic Oncology Lab, Ghent University, Belgium
Location: Erasmuc MC Rotterdam
Date: May 24, 2017
Author: Teun Huijben

Geert Berx is introduced by one of our teachers Riccardo Fodde as one of the pioneers in the field of the epithelial-mesenchymal transition (EMT). After this introduction he starts to give us an introduction on EMT.

As the name implies, EMT is the transition of an epithelial cell to a mesenchymal cell. The epithelial cells are polar and very tightly connected to there surrounding cells, thereby forming a clear boundary between the underlying tissue and the outside world. To become a mesenchymal cell, the mesenchyme is the connective tissue lying underneath the epithelium, the cell has to loose its polarity and connections to other cells. This is done by losing the cell-cell interaction protein E-Cadherin (epithelial Cadherin). E-Cadherin is down-regulated as a result of binding of transcription factors to specific E-boxes near the promotor.

The most important transcription factor doing this, and discovered by Geert Berx himself, is ZEB2. If ZEB2 is high in expression the E-cadherin is down-regulated, resulting in less cell-cell interactions enabling EMT. Control experiments in different mouse models and human cell lines showed that knocking-out ZEB2 resulted in more E-cadherin and no EMT, proving this theory.

The reason why EMT is widely studied is because of its importance in cancer. When malignant epithelial cells undergo EMT, they can travel through the mesenchyme to the blood and travel then to new places to form metastases. For a long time, people thought of EMT in a very binary way; a cell is either epithelial or mesenchymal. However, Geert and his colleagues proved that there are also multiple transitional states between epithelial and mesenchymal cells, and they showed at least 8 different metastable intermediates. The distinct states differ in levels of amongst other things E-cadherin, EpCAM and ZEB2. In both normal tissue as tumors, a wide variety of these states is found, indicating that the EMT system is way more difficult than thought.

Further research into the importance of ZEB2 in EMT and tumor formation resulted in many new insights. ZEB2 appeared also important in the maintenance of stem cells, spontaneous tumor formation and the p53 pathway. However, in the study of ZEB2 importance in human melanoma cell lines they found something interesting. When ZEB2 was knocked-out the tissue didn’t differentiate anymore, and high levels of its counterpart ZEB1 were measured. Indicating that ZEB2 is important in differentiation and proliferation. Further studies showed that ZEB1 is important in stem cell maintenance and tumor invasion. This resulted in a clear model where either ZEB1 of ZEB2 is present, supported by experimental data.

However, when ZEB2 was over-expressed, they found more metastases, which contradicted the current model. Further investigation resulted in the finding that TNF (tumor necrose factor) down-regulates the ZEB2 protein, resulting in higher ZEB1 levels and thereby creating more metastases. All of this knowledge together resulted in an oscillating model of ZEB1 and ZEB2 levels during tumor progression (see Figure 1).

Schermafbeelding 2017-05-24 om 13.43.33Figure 1. The levels of ZEB1 and ZEB2 oscillate during the progression of cancer. In the primary tumor ZEB2 is highly expressed, resulting in a high proliferation. During the transient state, ZEB2 is down-regulated paving the way for ZEB1 to be active and facilitate invasion. In the metastases again ZEB2 is present to stimulate proliferation and tumor outgrowth.  

After all, I found the talk by Geert Berx very interesting. Although it made very clear how many players are important in the progression of cancer and how difficult it is to do research on it.

The Pathways Traveled: Structural Studies of Virus Assembly

Speaker: Dr. Elizabeth Wright
Department:
Department of Pediatrics, Emory University
Subject: The Pathways Traveled: Structural Studies of Virus Assembly
Location: A1.100 TU Delft
Date: 19-05-2017

Author: Kristian Blom

On the 19th of May I visited a BN colloquium given by Elizabeth Wright, principal investigator at Emory University. The Wright lab is interested in the use of cryo-electron microscopy (cryo-EM) and molecular biology approaches to explore the three dimensional structures of viruses and cells. The goal is to use this information to aid in the development of novel antimicrobials, therapeutics, and vaccines.

Dr. Wright started by mentioning the benefits and methods in cryo-EM. One of the benefits is that samples stay in their ‘native’ state because all the molecules within the sample are frozen and thus do not move over time. The other benefit, which I think is even better than the first, is that with conventional cryo-EM specimen preparation artifacts are eliminated. While I’m writing I now realize that the cause of this benefit wasn’t mentioned during the talk, but I think it has to do with the cooling of the sample.

Within the realm of cryo-EM, there are different methods one can use to analyze your sample. The most extensively used methods are single particle analysis, electron crystallography, helical reconstruction and tomography. The latter method is imaging by sections. From these sections it is possible to make a 3D image by stacking the individual 2D images. During the talk dr. Wright showed us one example of a 3D image constructed by cryo-EM tomography.

After a short review of the different methods we moved to the recent advances in cryo-EM. These advances can be separated in three different areas: Sample preparation, data collection and data processing. Especially the data collection part has made some big improvement in 2008, when Direct-Electron introduced the large-format Direct Detection Device (DDD®). In traditional transmission electron microscopy (cry-TEM) cameras use a so-called scintillator. This is a material that produces a flash of light by the passage of a particle through it. For cryo-TEM this particle is an electron that causes a photon to be emitted by the scintillator to the CCD sensor. In contrast, the DDD directly detects image-forming electrons in the microscope without the use of a scintillator. This direct electron sensing results in better resolution, signal-to-noise ratio and sensitivity. The data processing improvements do mainly come from faster computing, better algorithms and auto-segmentation.

Figure 1: The difference between traditional transmission electron data collection and DDD data collection. Image from: http://www.directelectron.com/products/de-series

The second part of the talk was devoted to the current studies of the Wright lab. Besides the research, there is also a big interest to the technological developments of cryo-EM. One of the recent innovations is the correlation of fluorescence microscopy with electron microscopy. This allows one to improve the resolution and identify certain parts of the sample by staining them with a specific fluorophore.

To be honest, I didn’t found the talk that interesting. The slides that dr. Wright used during her talk were overcrowded with text and sometimes lacking important information. Therefore it was quite hard for me to keep my focus. Every time I was halfway through reading one slide, dr. Wright already went to the next slide.  Also I couldn’t found any structure in her presentation, and that is even more annoying for me because I always need some structure if I want to understand the complete picture of the talk. What I do like about here presentation is that she knew a lot about her field of research. All the questions she got from the audience were answered in a very nice way.

The ‘Nano’ in the huge Universe

Speaker:              Prof. Sir Vincent Icke

Department:      Theoretical astrophysics University of Leiden

Location:            TU Delft

Date:                    17-5-2017

Author:                Katja Slangewal

The universe is immensely huge. So, it might be a bit surprising that describing Nano-scale processes are necessary to describe our universe. Until a few years ago the field of Cosmo-chemistry, which focusses on the Nano-scale processes happening in the universe, seemed unnecessary. However, nothing is less true.

The universe exists for 99% of hydrogen and helium. All the other elements we know are filling the final percent. Most of the material is concentrated at stars and solar systems. The rest of the universe is quite empty compared to these dense regions. However, small dust grains, once formed after the dying of a star, are ‘floating’ around in space. The surface of these small dust grains contains attractive and repulsive parts. Which can for instance bind a glycolaldehyde molecule. This event might seem very unlikely to happen considering the density in which the dust grains and glycolaldehyde molecules are found in space. However, one should not forget we are talking about immense timescales. The reaction taking place at the surface of dust grains happen at approximately 50 Kelvin. Also, there is no source of energy anywhere near. So, when a particle binds and the energy excess is released in form of radiation, the changes are small that the particle will leave the dust grain anytime soon. This means that several molecules can spontaneously form at dust grains. This forms the basis for Cosmo-chemistry.

Nearby a star, the conditions are very different from the ‘empty’ universe. A star forms a reserve of energy and a source for water. When stars are just starting to get formed a huge ring of dust will surround the star. The dust is attracted by gravity. Because of the excess of water, the dust grains will be covered in ice. As soon as the star grows and starts irradiating, the ice will melt of for dust grains close enough to the stars. A so called ‘ice line’ is formed. This line determines the difference between the nearby rocky planets that will form and the more far away gas giants. This has happened in our solar system (compare Mercury, Venus, Earth and Mars to Jupiter, Saturn, Neptune and Uranus), but this process is also true for exoplanets. As mentioned before, as soon as molecules bind to the surface of dust grains there will be an excess of energy. This energy is turned into radiation, which can be measured. This has allowed us to image exoplanets and see the difference between the icy ad rocky ones.

The question is, can there be life on the exoplanets. To go deeper into this question prof. Icke told us more about the Miller experiment (figure 1). In this experiment, a primeval soup was made, which has shown to produce complex molecules (like amino acids) within a couple of weeks. The primeval soup contained substances which were present on early earth. The same conditions can be found in black smokers on the bottom of our oceans. Prof. Icke stresses that according to physics life is unbelievably simple. It only contains six elements, some relatively easy molecules and the more complex structures found in a cell are just made of chains from the easy molecules. All the elements needed are synthesized by stars, so no complex processes on a planet are needed. And even from the simple molecules we don’t need much. Take for instance the amino acids. We use only 20, while there are over 500 found in nature. So exo-life is very probable, planets are everywhere and the recipe for life is quite simple.

seminar 11

Figure 1: Millers experiment. http://www.smithlifescience.com/millersexp.htm

After this very interesting talk on the importance of Nano-processes happening on dust grains, the formation of solar systems and the probability of finding exo-life, prof. Icke continued his talk about Nano-pills, which he calls a spin-off of astrophysics. He thinks DNA specific medication in the future will read of the genetic code and determine which medicine to release in patients. He thinks this will prevent misuse of medicine, since you cannot buy pills and use them for someone else anymore.

To my opinion, the last part of the talk really brought down the rest of the talk. I really liked the main part. It contained a new view on where Nano-processes can be interesting. Also, I think the question of how life originated is very interesting and it gave a fresh look to consider life as something easy and simple. However, I thought it was a real pity how prof. Icke ended his talk. The part about DNA specific medicine was brought as if we had no idea about biology. The talk was given at the Nanobiology symposium, so I think he should have known what kind of study Nanobiology is. He showed us pictures of DNA with an attitude as if we were seeing it for the first time. Also, I am wondering if prof. Icke has any idea about DNA imprinting, translational regulation and other mechanisms which won’t enable a quick DNA readout to decide on medication. This was really a disappointment after such a good start of the talk.

The Pathways Traveled, Structural Studies of Mononegales Virus Assembly

Speaker: Elizabeth R Wright
Department: Department of Pediatrics, Emory University
Subject: The Pathways Traveled, Structural Studies of Mononegales Virus Assembly
Location:
Delft University of Technology
Date: 19 May 2017
Author: Romano van Genderen

Professor Wright started by giving us an overview of different size scales and the techniques used when studying life on that scale. One thing she pointed at is that electron microscopy, her field of interest, is improving on two different terrains. It can now both make pictures of smaller and smaller structures, but also on a bigger scale.

Afterwards, she discusssed the advantages of Cryo-EM. She told us that since it does not use any staining methods, it can measure a specimen in its native state, unlike techniques such as crystallography. Also, there are few to no artifacts visible from the preparation of the specimen.

Next, she showed us the recent advances in the field of Cryo-EM. We now can not only prepare better samples due to techniques like active substrates, but also gather better data thanks to phase plates and process the data more efficiently, not only because of more processing power of contemporary computers, but also because of advantages in auto-segmentation algorithms. But according to her, the most important improvement was the invention of direct electron detectors. These DDCs are far more useful than the previously used CCD cameras, which turn electrons into photons that are then counted. This conversion leads to loss of signal. These new cameras therefore give better signals, and can also be used to record videos in real-time with framerates up to fifty frames per second.

Her lab is currently using these new tools to their best abilities, leading to research on correlative microscopy, overlaying EM and fluorescence images to better locate compounds in the cell. They also study enveloped viruses, the topic of the rest of her talk.

She started by explaining the structure of such a virus, a completely new topic to me. To be specific, she studied paramyxo and pneumoviruses. These viruses have glycoproteins on their surface, a lipid envelope, matrix proteins and a nucleocapsid protein that surrounds and protects their RNA genome. These viruses are very common, for example the common measles belongs to this class.

Paramyxo_virion.jpg

Figure 1: The structure of a myoxivirus

This class has commonly been regarded as hard to purify. Firstly, people are unsure whether or not it still looks like its native configuration after purification. Also, there are a lot of artifacts and damage introduced by current purification methods. And to finish things off, you also only get very small numbers of virus particles back from it.

This is why professor Wright wanted to improve this method. She used a method currently used in protein purification, using nickel-NTA along with histadine tags binding to it. But in her case she incorporated the nickel-NTA into the cryo-EM grid and added HIS tags to the surface glycoproteins. This makes the grid attract the viruses and leads to far better yields and also removes a lot of artifacts from the sample.

Next, she wanted to study the interior of these viruses during virus assembly and release. There were two common hypotheses on the location of the membrane protein during these processes. One says that the matrix protein covers the inside of the capsid. The other says that it forms a protective layer around the nucleocapsid protein for even more protection. Using cryo-EM, she was able to directly see the matrix protein, and that it does not cover the nucleocapsid protein.

Also, she saw a mesh of fusion proteins, proteins that play a role in binding to the host. These proteins form a two-layered lattice, but there is something weird about this lattice. It seems to have a hole in it. This hole does seem to be the same size as the protein on the host’s surface, suggesting that this a binding pocket.

The final part of her talk made the topic a bit more practical. She talked about how her techniques were used to discover more about the virus known as RSV. This virus causes asthma in newborns and there is currently no vaccine known against it. Her research found that this virus is filamentous when secreted. Also, that its structure can be discovered in large detail by using cryo-EM. One peculiar fact she found was that the RSV-F fusion protein forms a hexamer-of-trimers when in its pre-fusion form. This knowledge can be used to develop a vaccine for this virus.

I did really enjoy the first half of the talk, where the techniques and their advantages were discussed. I did notice a lack of disadvantages, a fact that I find very suspicious to say the least…

On the other hand, the second half was not that interesting, because the main points got drowned in all the details about the virus and its shape. Also, the images were not understandable, even when she told what we were supposed to see. But perhaps that was the fault of the lighting or the screen in the room.

New chemical therapeutics of genetic disease by manipulating the transcriptome

Speaker: Masatoshi Hagiwara
Department: Developmental biology
Subject: New chemical therapeutics of genetic disease by manipulating the transcriptome 
Location: Erasmus MC Rotterdam
Date: 3 April 2017
Author: Carolien Bastiaanssen

Professor Hagiwara leads a research group at Kyoto University Graduate School of Medicine Japan. His long-lasting dream is to cure genetic diseases with the compounds he and his colleagues develop.  Nowadays, with the availability of CRISPR-Cas9 as a genome editing technology, it has become relatively easy to manipulate DNA. However, before this discovery it was easier to manipulate RNA than DNA using small and cheap molecules. The research of professor Hagiwara is therefore focused on compounds that influence the splicing of RNA and that can be used in splicing therapies for genetic diseases.

In order to study the effect of different compounds on the splicing of pre-mRNA, professor Hagiwara and his colleagues developed a way to visualize alternative splicing. As a model organism the nematode C. elegans was used with egl-15 as the model gene. This gene encodes a fibroblast growth factor receptor and alternative splicing gives rise to two different isomers containing one of the mutually exclusive exons 5B and 5A. The first isomer, EGL-15(5B),  is essential for viability and the second isomer, EGL-15(5A), is involved in the migration of sex myoblasts. Depending on which exon is present, cells express either GFP or RFP (Figure 1). After this first success professor Hagiwara and his colleagues also developed reporters to for alternative splicing dependent on the developmental stage. Furthermore they succeeded in expressing their reporter system in mice and in mammalian cells.

 alternative splicing reporterFigure 1: Alternative splicing reporter in C. elegans. A) The construct for the alternative-splicing reporter. B) Transgenic C. elegans expressing the aforementioned reporter. From left to right: RFP, GFP, the first two merged, and differential interference contrast (DIC) image. C) Close up of the vulva showing that the vulval muscles express E5A-RFP and not E5B-GFP. Adapted from: Kuroyanagi, H. et al. Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo. Nat Meth 3, 909–915 (2006).

Once they were able to visualize alternative splicing, professor Hagiwara and his colleagues tried to find compounds that could correct for aberrant splicing in order to treat patients with for example familial dysautonomia (FD). This is a hereditary disease caused by mutations in the IkB kinase complex-associated protein (IKAP) gene. In these patients exon 20 is skipped, especially in neurons, and this results in a truncated protein product. FD patients could benefit from a treatment that stimulates exon 20 inclusion. Using a reporter similar to the one shown above, professor Hagiwara and colleagues tested all kinds of compounds in their chemical libraries. One of these compounds increased exon 20 inclusion in cells of FD patients. They named the compound rectifier of aberrant splicing or in short RECTAS. This compound was shown to be effective in cells from FD patients, future studies on FD mouse models are now required to get RECTAS towards clinical trials.

RECTASFigure 2: RECTAS is a small molecule that rescues aberrant splicing in FD cells A) Structure of RECTAS B) Cells that lack exon 20 (thus FD phenotype) express RFP and wildtype cells that do have exon 20 express GFP. DMSO is a negative control and kinetin is a positive control. RECTAS rescues aberrant splicing in FD cells and it does so to a larger extent than kinetin.  C) Quantification of GFP/RFP ratios after treatment with RECTAS or kinetin. Lower concentrations of RECTAS were required to obtain the same effect that was achieved with higher concentrations of kinetin. Source: Yoshida, M. et al. Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia. Proc. Natl. Acad. Sci.  112, 2764–2769 (2015).

Another example of a disease where patients can benefit from splicing therapy is Duchenne muscular dystrophy (DMD). This fatal disease is caused by a mutation in the dystrophin gene that results in a lack of the dystrophin protein. The mutation introduces a frameshift, thereby creating a premature stop codon. Thus no dystrophin protein is produced. A milder phenotype of DMD is Becker muscular dystrophy (BMD). Patients with this phenotype also have a mutation in the dystrophin gene, however this mutation does not cause a shift of the reading frame.  Instead the mutation promotes skipping of an exon. Although part of the protein is missing, it is still partially functional therefore BMD patients show less severe symptoms than DMD patients. Thus by treating DMD patients with a compound that causes the mutated exon to be skipped, the symptoms can be drastically reduced. Professor Hagiwara and colleagues found such a compound. Its name is TG003 and it stimulates skipping of  the mutated exon while it does not affect the wildtype exon. More importantly, TG003 did not affect the splicing patterns of the other exons in the dystrophin gene.

All in all, Professor Hagiwara showed that splicing therapies with small molecules can be used to treat FD and Duchenne muscular dystrophy patients. These results are promising, not only for these groups of patients but in the future splicing therapies with small molecules can potentially be used for other genetic diseases as well. Professor Hagiwara tried to explain everything clearly, yet due to his heavy accent I had to pay very close attention to follow the talk. However his passion for his work was obvious and the promising results are of interest for multiple groups at the Erasmus MC who might want to use splicing therapy for the patients they try to help.

 

 

 

Information processing in neural and gene regulatory networks

Speaker: Gašper Tkačik
Department:
IST Austria
Subject:
Information processing in neural and gene regulatory networks
Location: A1.100 TU Delft
Date: 22-03-2017

Author: Kristian Blom

On the 22nd of March I visited a seminar given by Gašper Tkačik, a theoretical physicist who is interested in using statistical physics and information theory to explain phenomena related to the cell. The most fundamental principle that underlies all the research that dr. Tkačik conducts is that information processing networks have evolved or adapted to maximize the information transmitted from their inputs to the outputs, given the biophysical noise and resource constraints.

Dr. Tkačik showed us multiple examples of his research during his talk. For now I’d like to focus on the most interesting one (from my point of view), which is about reading the positional code in early development. It is commonly known that a morphogen gradient in early development generates different cell types in distinct spatial orders. This is called the French flag model. Despite decades of biological study, a quantitative answer to how much appositional information there is in an expression pattern remained unanswered. Therefore Dr. Tkačik to look at the French flag model from an information theory point of view and asked the following question: How much information is there in spatial patterns of gene expression? Using the gap genes in the Drosophila embryo he measured the amount of information in bits. I will now discuss shortly how one can measure the information contained in gap genes.

Figure 1: Normalized dorsal profiles of fluorescence intensity, which we identify as Hb expression level g, from 24 embryos selected in a 38- to 48-min time interval after the beginning of nuclear cycle 14. Considering all points with g = 0.1, 0.5, or 0.9 (Left) , yields conditional distributions with probability densities P(x|g) (Right). Note that these distributions are much more sharply concentrated than the uniform distribution P(x) shown in light gray. Image adapted from: Dubuis, J.O.; Tkačik, G.; Wieschaus, E.F.; Gregor, T; Bialek, W. PNAS, 2013, 110 (41), pp 16301-16308

We start by looking at the early stages of Drosphila development. At this stage most cells are similar in morphology, so we do not have any information about the position of cell when we neglect gene expression information. Mathematically we can say that the position of the cell is drawn from a distribution of possibilities P(x). If we know take into account the gene expression levels, our uncertainty in position is reduced.  Looking specifically at the expression levels of the hunchback gap (Hb) gene (figure 1), one can see that a certain expression level (g) is not a unique indicator for the position of the cell along the posterior/anterior axis.  Instead there is a range of positions that have the value g. Let P(x|g) be the conditional probability that a cell with expression level g is located at position x.

We define the entropy  of our two probability distributions as:

The information gain due to an observation of the hg expression level at on cell is now given by

From this point I will leave the mathematical expressions as it is, but I challenge you to get a firm understanding of why the final expression represents the information gain. After a small adaption to the final formula, Dr. Tkačik  used that result to make a ‘’direct’’ measurement of the amount of information carried in the gap genes. Using this method he found that individual genes carry almost two bits of information. In the extension of this result he also found that four gap genes carry enough information to define a cell’s location within an error bar of ~1% along the anterior/posterior axis of the embryo. How cool is that!

Although the talk went a bit fast, the content was really good. During the talk I was reminded of the lectures we had during evolutionary & developmental biology (evodevo), since it was this course where I got familiar with the gap genes in drosophila development. Therefore I decided to inform one of the evodevo teachers with the content of this talk, because it might be of good use in the future for them. Although it sounds a bit cliché, afterwards I was again (it happens on a regular basis) astonished by the fact that nanobiology is a really strong field of science. What Dr. Tkačik did fits very well into our program because he used mathematics, especially information theory, to understand why those gap genes function the way they do. For me it was really a wakeup call to keep questioning myself: Why? If one keeps asking this again and again, I think at some point you will find yourself in the fields of mathematics and physics where the answer will be waiting for you to be found.

Big data analysis for precision medicine in dementia

Speaker:              Wiro Niessen

Department:      Biomedical image analysis

Location:            TU Delft

Date:                    17-5-2017

Author:                Katja Slangewal

Are doctors in the near future being replaced by technology? Approximately five years ago scientists were still laughing at people working at neural networks. However, at the moment neural networks can process more and more data in a short amount of time. Together with a huge progress in imaging, this revolution in machine intelligence makes the first question a reasonable one. According to Dr. Wiro Niessen artificial intelligence is still complementary to humans, he sees a bright future in which human and computer will work together.

Artificial intelligence keeps surprising people. It is already clear that many (repetitive kinds of jobs) will soon be taken over by machines. However, at the moment machines are also capable of making music and paintings (figure 1). These are things which were long considered to be limited to humans. In the late 1980s computers were already capable of beating humans in a game of chess. Challenges were designed to come up with algorithms for other games. For instance, the game GO, a very complex Chinese board game, was a subject of focus. Scientists predicted artificial intelligence to beat humans around the year 2025. However, this already happened last year (2016). One interesting thing Niessen mentioned during his talk is the difference between human and computer intelligence. During the game of GO the computer program, based on neural networks, learned through experience. So it was very unexpected when the computer made a move which was never made by humans before. In the end the move turned out to be truly smart 5 or 6 turns later. This means that the computer was capable of looking ahead in the game. So computers are getting better and better. This is not only useful in games, but also in the medical field.

seminar 10.png

Figure 1: Machines can learn styles from various painters and apply them to any picture. In small are paintings from famous painters. In large is the top left picture remade to the different styles. https://motherboard.vice.com/en_us/article/artificial-intelligence-can-now-paint-like-arts-greatest-masters

Niessen and his colleagues are focusing on early prognosis of dementia. They showed that several markers of the disease already show an elevated level long before the onset of the disease. To find the markers visible before the start of a disease is a difficult thing to do. This is why a population study was started in 1990 in Rotterdam. During this study people regularly undergo various tests and over the years it becomes clear how their health develops. During the study, risk factors (genetic markers, lifestyle, smoking, etc.) are being linked to an outcome (dementia, stroke, etc.). Next, the brain of the patients can be traced back to look for markers in morphology, volume, lesions and more. Niessen and his group have found patterns in the brain images linked to a disease. These patterns are visible long before other symptoms are starting. So, they use the data about several markers to write algorithms which will predict the chance of getting dementia or a stroke.  One example which has been determined during the Rotterdam study, is the shrinking rate of the brain and its standard deviation. Programs and information like this will help radiologists to diagnose patients.

At the moment Niessen and his colleagues are connecting SNPs to imaging phenotypes (like brain morphology or volume). This way they found certain SNPs that are correlated with the volume of the hippocampus. These SNPs are being put in a library accessible online. So, to conclude, computers are getting smarter and smarter. They will take over repetitive jobs and they will also be useful in more complex kinds of work. In the medical field Niessen expects computers to shift the focus from curing diseases to preventing diseases.

Dr. Wiro Niessen gave an interesting talk. It keeps surprising me how much computers are already capable of. I had no idea computers can paint quite good already. I also liked to hear about the possibilities of combining biological knowledge about the brain with writing algorithms to predict diseases. A lot of different research areas are combined here, which is of course interesting for nanobiologists.