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.

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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.

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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.

Whole genome sequencing of spermatocytic tumours

Speaker: Anne Goriely
Department: Josephine Nefkens Institute
Subject: Whole genome sequencing of spermatocytic tumours
Location: Erasmus MC
Date: 28-06-2017
Author: Renée van der Winden

Anne Goriely came to talk to us about her work on spermatocytic tumours. In her talk she first gave a lot of background information before going into detail about her latest findings. She started by giving us some information on spermatocytic tumours. These testicular tumours are very rare and mostly occur in older men. They are slow growing, but can become extremely large (3-30 cm in diameter). Luckily, the prognosis is usually very good. The cell of origin for these tumours is an adult spermatogonia. This is interesting, since the germline usually does not mutate because that is evolutionarily very disadvantageous. These mutations occur through a copy error during stem cell division and the mutation rate increases with age, which explains why these tumours are usually found in older men.

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Mutations found in relation to age (Goriely et al., 2017)

Next, Goriely brought up Apert syndrome. This disorder only has a paternal origin and the mutation causing it is spontaneous, which means it has to have occurred during spermatogenesis. It was found that the higher the age of the father, the more likely it was that the offspring would have the disorder. This is called the paternal age effect. Apert syndrome shares this feature with other disorders. It also turns out that only gain-of-function mutations are enriched with age. This can be beneficial for the current generation, but harmful for the next. That is why they are called ‘selfish mutations’.

Goriely’s lab searched for these selfish mutations in testis. To do so they made slices of testis and stained them. They found that there was a higher level of staining in some testicular tubes, which indicated mutated spermatogonia. These selfish clones can spread over large areas of the testis and different clones can be found in the same testis. It is even possible for mutated and non-mutated clones to be side by side in a tube. Some of these mutations are strong and thus lead to impaired spermatogenesis, but this is not the case for all mutations. It turns out that these selfish clones are more numerous with increasing age and that all men have them. In that sense they can be compared to moles on the skin.

Lastly, Goriely pointed out that when a distribution was made of the occurrence of spermatocytic tumours versus age, it turned out to be bimodal. Moreover, spermatocytic tumours are rare, while selfish mutations are common. This led to the belief that there might be a second source for these tumours. At this point whole genome sequencing was used to determine that aneuploidy occurs in the testis. Hypotheses connected to this finding are that aneuploidy comes before the selfish mutation and thus they might be passenger mutations. Thus aneuploidy might drive tumorigenesis. Moreover, aneuploidy can cause a gene imbalance, causing meiosis to fail so that the cell re-enters mitosis. This can lead to the giant tumours observed.

I thought this seminar was very easy to follow due to all the background information given. I liked the topic of the talk, but I do feel that for a large part of the talk we kept coming back to the same conclusion: that mutation rate goes up with age. I would be interested to see if the hypotheses given are true and what might then be done to help treatment of these tumours.

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.

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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.

Modeling of the ribosome and the RNA polymerase molecular motors

Speaker: Bahareh Shakiba
Department: Institute for Advanced Studies in Basic Sciences, Zanjan, Iran
Location: TU Delft
Date: July 12, 2017
Author: Teun Huijben

Bahareh has just finished her PhD and is now doing a Postdoc at the IASBS in Iran. In this talk she explained what she has studied during her PhD, in which she was interested in the two most important molecular motors present in the Central Dogma: the RNA polymerase and the ribosome. Both proteins can be seen as molecular motors since they use energy to move along a polymer (either DNA or mRNA). She was especially interested in the dynamics of these motors, so their pausing-behaviour and force-dependence.

The first part of the talk was about modeling the RNA polymerase (RNAP). Bahareh proposed a model to simulate the transcription steps using two components of the RNAP: the bridge helix and the trigger loop, which are both located very close the active site of RNAP (see Figure 1). The bridge helix promotes opening of the double stranded DNA helix and the trigger loop binds to the DNA and drags the DNA through the polymerase. The internal movement of components in the active site of RNAP during the transcription of a single nucleotide can be divided into multiple steps, which each having a energy barrier. Using the energy barriers calculated by other people, she computed the average duration of a pause during transcription. The average length of these pauses was the same as found in experiments.

 

ÈFigure 1: The bridge helix and the trigger loop are located near the active site of RNA polymerase. [M. Thomm, University of Regensburg]

However, I have some critical notes on this finding. First of all, she didn’t explain the model very well, so it was unclear to us in what way this model was new. Especially given the fact that many people do research on this subject and multiple models already exist. Besides that she didn’t mention the reaction rates she used in the simulations and where they came from.

Another important aspect is that she didn’t show the distribution of pauses. Normally it is very interesting to look at a histogram of pauses, to see how they are distributed, this can be Gaussian, exponential or Gamma, for example. Seeing the distribution will give a lot of information about the underlying processes and indicates if it is fair to simple take the average pausing time and compare this with experiments.

The second part of her presentation Bahareh talked about modeling ribosome dynamics. A ribosome is a protein complex that translated the mRNA to a protein. Bahareh studied how  mRNA-hairpins influence the processivity of the ribosome, since hairpins are formed in single stranded RNA and form roadblocks in front of the ribosome.

The active site of a ribosome has three free places to bind a tRNA molecule: the E- (exit), P- (polymer) and A-(active)-site (see Figure 2). The ribosome moves forward by first displacing the large subunit, followed by movement of the small subunit. If the mRNA in front of the ribosome has internally formed a hairpin, this can either result in necessary unwinding of the hairpin or a frameshift of the ribosome. The latter means that the ribosome temporarily detaches from the mRNA, resulting in errors in the produced protein.

hairpin

Figure 2: The active site of the ribosome has three active sites, the E-, P- and A-site. A tRNA loaded with an amino acid can bind to the ribosome and transfer its amino acid to the growing peptide chain. A hairpin in front of the ribosome. [Shakiba 2016, arXiv: 1607.0719v1]

There are currently two ideas of how ribosomes solve the hairpins. One states that the moving ribosome applies a force on the hairpin forcing it to unwind. The other theory states that the ribosome itself actively uses helicase activity to make the mRNA single stranded. Both theories are simulated in the model. Then she compared the model with experimental data of ribosomes translating mRNAs having hairpins, while applying a force on the mRNA. This comparison revealed that the model with ribosomes having helicase activity did the best job in explaining the experimental data. Giving the indication that ribosomes indeed have their own helicase activity.

As already stated in this report, the talk of Bahareh was quite hard to follow, especially because she didn’t explain the models very well. Therefore, the first part of her talk didn’t really impress me, since I didn’t see what was new and surprising in her model. However, the second part was easier to understand and got a clear, well described message.

Quo Vadis: Cees Dekker

Speaker: Cees Dekker
Department: Bionanoscience, TU Delft
Location: TU Delft
Date: July 6, 2017
Author: Teun Huijben

As start of the summer Quo Vadis of the Bionanoscience Department, Cees Dekker was invited to give a talk. Literally translated from Latin, Quo Vadis means: ’Where are you going?’. In light of this title Cees decided to give a summarizing talk about covering all the exciting research happening in his lab and what he hopes to achieve in the next years.

Since Cees has the largest lab of the department, of lot of different topics are studied in his group. The different topics can be roughly divided into three categories: developing novel nanotechnologies, studies on chromosomal organization and developing the synthetic cell. Of each of these subject he highlights some interesting ongoing studies and his vision on the future.

The first topic Cees elaborates on are the novel nanotechnological techniques his group is developing. The most important technique is the solid state nanopore, which is a very small hole in a thin membrane. When a voltage is applied over the chip, an ionic current starts running and DNA can be dragged through the pore, because of its overall negative charge. While translocating the pore, the DNA blocks the ionic current partly and the decrease in current can be measured. The nanopore technique can be used in different studies. Firstly, it may enable sequencing of DNA optically, when the pore is used in combination with plasmonic structures. The gold plasmonic structures create a high-energetic field trapping the DNA in the pore. In combination with Raman spectroscopy, chemical structures of the DNA can be deduced from the emitted radiation.

Secondly, nanopores are also useful in studying nuclear pore complexes. By covering the inside of the pore with nuclear pore proteins, the transport of proteins can be mimicked through this artificial pore. The advantage is that it can be done in vitro, instead of in living cells. At last, they are also trying to sequence proteins using biological nanopores.

The second main part of Cees’ talk was about the study of chromosome organization. For multiple years, his group is interested in the higher structures of DNA and how that structure is determined. An important part of chromosome structures are supercoils, in which DNA is coiled up to store it in a compact way and suppress transcription. The main question is whether these supercoils (or plectonemes) are sequence dependent. His group developed a new technique to study the coiling, and indeed the position of the supercoils is dependent on the sequence of the DNA. Modeling of the DNA gave the insight that certain sequences have an intrinsic curvature, and the model predicted which sequence will increase the probability of having a supercoil, since the tip of the supercoils needs Experiments with these sequence indeed showed a higher probability of having a supercoil at that position. This shows that the intrinsic curvature of the DNA determines higher structures of the chromatin, so the DNA sequence not only codes for proteins, but also for its own structure.

The last part of his talk was about making a synthetic cell, the ultimate dream of Cees. The goal is to create a vesicle (liposome) with a working division mechanism inside. One idea is to use the bacterial MinE-MinD oscillating proteins to position a FtsZ-ring in the middle of the cell and actively contract this ring to divide the cell. This idea will need some years to become reality, so right now his group is studying the different components of this idea separately and hopefully the first ’synthetic’ cell will be there is a few years.

What I found particularly nice about this talk was that Cees summarized all the research that is currently done in his large lab. In this way the department got a better idea of all the things happening within this part of Bionanoscience. Besides that, Cees is a very enthusiastic speaker what makes if nice to listen to.

Active polymer models for the 3D organization of chromosomes

Speaker: Johannes Nübler
Department: MIT
Subject: Active polymer models for the 3D organization of chromosomes
Location: TU Delft, A1.100
Date: 03-07-2017

Author: Kristian Blom

There it is, my final seminar report just before my trip to Japan starts. On the 3rd of July I visited a talk given by Johannes Nübler, postdoc at the MIT Mirny lab. Johannes works on the analysis of Hi-C and Micro-C data and modeling chromosomal organization in yeast, mouse, and human cells. His particular focus is on bridging large scale polymer models of chromatin with more fine-grained models of nucleosomes. He is interested in the role of active processes in chromatin organization, e.g. transcription and chromatin remodeling. In this talk we focused specifically on modelling Hi-C data.

The talk started with an introduction of Hi-C (High-throughput sequencing). With this method one can analyze the spatial organization of chromatin in a cell, by quantifying the number of interactions between genomic loci that are nearby in 3-D space, but may be separated by many nucleotides in the linear genome. A shortcoming of Hi-C is that when two sites are not in close contact, no additional information is provided about how far these sites are separated from each other. Hi-C data is usually visualized by a heatmap where the number of interactions between all the different locations of a genome is shown. In order to make sense of this data, a model has been created that simulates active polymer folding and recreates the experimentally obtained heatmaps. By comparing the experimental heatmaps with the modelled heatmaps, one can understand why certain features arise under specific circumstances.

Figure 1 – Top row, the update rules used in simulations: (A) a condensin extrudes a loop by moving the two ends along the chromosome in the opposite directions, (B) collision of condensins bound to chromosomes blocks loop extrusion on the collided sides, (C) a condensin spontaneously dissociates and the loop disassembles; (D) a condensin associates at randomly chosen site and starts extruding a loop. Bottom row, (E) we use polymer simulations to study how combined action of many loop extruding condensins changes the conformation of a long chromosome. Image from 10.7554/eLife.14864

After the introduction we looked at interaction heatmaps of entire chromosomes, characterized by repeating blocks of high/low interaction intensity in the vertical and horizontal direction. The blocks with high interaction intensity are called topologically contacted domains. A typical feature in these interaction heatmaps are the dense squares along the diagonal, which indicates that neighboring areas on the chromosome are relatively often in contact with each other. To simulate those features passive polymer folding was not enough, and therefore a loop extrusion model was introduced (Figure 1). In this model DNA folding arises due to an extrusion complex containing two subunits that attaches to the DNA, forming a small loop in the process. Thereafter the two subunits slide along the DNA in opposite directions, making the loop bigger. This mechanism actively folds the DNA, allowing far located parts on the sequence to come in closed contact with each other. During sliding the extrusion complex searches for a specific motif, which causes a protein called CTCF to bind to the DNA. When a subunit encounters a CTCF that is pointed towards the subunit, it will stop sliding. If the CTCF motif is pointed in the sliding direction of the subunit, it won’t be recognized and the subunit keeps sliding. The result of this behavior is that the pair of CTCF motifs at the end of a loop are pointing towards one another. One of the exciting results of the extrusion model is that loops formed by extrusion will be unknotted, allowing for easy access to the genetic information. Another very nice and cool result is that a collection of chromosomes that are intertwined will naturally segregate when extrusion takes place.

Although you had to have a bit of background knowledge in Hi-C to understand everything, I really liked the theoretical aspect of the talk. The fact that the model is very simple but produces very interesting results makes it a very powerful model. For the future it should be experimentally validated whether DNA looping is an active process involving some sort of extrusion enzyme, or a passive process that arises due to DNA-DNA interactions. For now it seems that the former comes closest to reality, but we have to wait for evidence to know the truth. Now it is time to go to Japan, cheers!

Disrupting ultrashort nucleic acid duplex with mechanical force

Speaker: Kevin Whitley

Department: University of Illionois at Urbana -Champain

Subject: Disrupting ultrashort nucleic acid duplex with mechanical force

Location: Bionanoscience Department, TU Delft

Date: 30-06-2017

Author: Mirte Golverdingen

Kevin Whitley did research to the hybridization kinetics of DNA under force. He tried to understand the transition state of nucleic acid hybridization. Hybridization is used for gene manipulation and targeting for DNA nanomachines, tweezers and bipadal walkers. Short strands (about 10 bp) show a ‘all of nothing’ behavior when hybridizing. Fist one or two base pairs bind and then all base pairs bind without stable intermediates. This describes a two-state reaction in a schematic energy landscape. The kinetics of any of these states is determined by the transition state between them. Whitley searched for the transition state between the bound and the unbound DNA.

To research the hybridization kinetics of the DNA the end-to-end extension of the DNA was researched. This is the length of the top of the energy to the bottom of the energy where the DNA is hybridized. Bell’s equation describes the energy barrier between the end-to-end extension. So, by measuring the rates of the Bell’s function you can calculate the end-to-end distance.

Whitley measured the hybridization rates by using optical beads that are bound to the DNA by double stranded handles and with a 9 nucleotides long middle strand. This short part is the binding site for short DNA. The distance between the two beads was approximately one. By using a flow chamber Whitley was able to look at oligo that were free in solution. He was able to measure the koff and kon rate under a constant amount of force. In this way, he could measure the moment a ss DNA oligomer binds to the middle strand by measuring the position of the bead. The ds DNA is stiffer than ss DNA, and therefore the position of the bead changes. By using this method, Whitley could measure the difference between ss and ds DNA up until a force of 12 pN.

By using Fleezers, which is a combination between fluorescence and optical tweezers, Whitley was able to label the oligomer with a fluorophore (see figure 1). In this way, also forces lower than 12 pN can be measured. Again, they calculated the binding and unbinding rates of the DNA. While the unbinding rate of the DNA goes up linearly when the force increases, the binding rate also goes up, however, not linearly. When the oligomer becomes longer, the unbinding rate goes down. For the binding rate no dependence on the length of the oligomer can be found.

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Figure 1: Measurement of single-oligonucleotide hybridization kinetics under force. (A) Schematic of the hybridization assay (not to scale). An engineered DNA molecule (red) containing a short, central ssDNA region flanked by long double-stranded DNA (dsDNA) handles is held under constant force by polystyrene beads (grey spheres) held in optical traps (orange cones). A fluorescence excitation laser (green cone) is focused on the central ssDNA region. Short oligonucleotides (blue) labeled with a Cy3 fluorophore at the 3΄ end (green disk) bind and unbind to the complementary ssDNA sequence in the center of the tethered DNA. The binding and unbinding is observed by the fluorescence emitted from the attached fluorophores.

Adapted from: Kevin D. Whitley, Matthew J. Comstock, Yann R. Chemla; Elasticity of the transition state for oligonucleotide hybridization. Nucleic Acids Res 2017; 45 (2): 547-555. doi: 10.1093/nar/gkw1173

When looking at Bell’s equation, the binding rate should be an exponential force-dependence, however, non-exponential behavior is reproducible across other conditions. The end-to-end distance is therefore not constant, and the Bells’ equation changes so an integral is used instead.  By calculating the unbound and bound extension by using the worm like chain model for double stranded DNA they were able to calculate the end-to-end extension. In this way, they also obtained the persistence length and contour length of the end-to-end extensions. He repeated these calculation for all different lengths of oligomers. The transitions state persistence length is comparable to ssDNA, however it is a bit stiffer.

The transition state is probably no random process, as simple polymer estimations predict a low probability of spontaneous alignments. The single strand that is a bit stiffer, as measured in the transition state, this could correspond to prearranging of the strands, prior to nucleation. Some enzymes can pre-organize mRNA so the transition state energy barrier becomes lower. Moreover, fast target finding is possible when there are preorganized bases by enzymes. In this way, they can speed hybridization up by pre-paying the entropy penalty.

Whitley told a clear story on obtaining the end-to-end distance of the DNA hybridization translation state. The combination of Optical Tweezers and fluorescence was very cool. Moreover, using the mathematics in combination with the biology inspired me as Nanobiology student.

Verrucomicrobia and the intestinal microbiome; the case of the Akkermansia muciniphila

Speaker:  Clara Belzer

Department: Laboratory of Microbiology, Wageningen University, The Netherlands

Subject: Verrucomicrobia and the intestinal microbiome; the case of the Akkermansia muciniphila

Location: BioTechnology department TU Delft

Date: 29-06-2017

Author: Mirte Golverdingen

To broaden my view of the studies done in the Applied Science building I visited a lunch lecture in the BioTechnology department. The microbiologist Clara Belzer from Wageningen University was the speaker and she spoke of the newly discovered Akkermansia muciniphila bacteria. Belzer and her group are interested in anaerobic micro-organisms that can degrade ligases and mucus in the gut. Verrucomicrobia is one of the phyla that lives in the gut, the bacteria Akkermansia muciniphila is one of the Verrucomicrobia and isolated by the group for a quest of new bacteria.

They discovered that the bacteria are very dominant along the mucus gut and an important player in human microbiota composition. The bacteria are a potential maker for healthy intestine and has an extraordinary mucus degrading capacity. The importance of the bacteria is shown in the fact that after a month after birth the bacteria can already be detected in the gut system. This is certainly not the case for all gut bacteria.

The human microbiota changes during life stages and perturbations, this is partly caused by the place of the bacteria on the body. However, over a life time the microbiota can also change due to physiological changes of the body by diet changes. Therefore, the change in microbiota can contribute to or starting diseases. Moreover, some microbiotia do not depend on food, they depend on mucus and mother milk.

Bacteria influence the host cell by a difference in glycal level. This has a microbial benefit; mucosal glycan foraging can solve the problem of the competition for glycans in the gut. For the host, the mucosal microbes can provide resistance against the colonization of pathogens. It directly inhibits the pathogens by production of antimicrobial compounds. Moreover, the depletion of nutrients modulates the immune response of the host.

In the gut, there are 5 major microbiological fila, some are mucus degraders and some are mucus binders. Mucus can be used as the source of growth because mucus glycans can be created by the bacteria. However, there are not only glucose molecules in these mucus glycans, there are also other more ‘awkward’ sugars which are hard to break down. These sugars need to be broken down by enzymes. The Akkermansia muciniphila is adapted to digest mucin, it contains a high number of genes that encodes for enzymes which can degrade mucin. None of all Verrucomicrobia have the same number of enzymes that are able to digest mucin as the A. muciniphila.  So, the A. muciniphila is really adapted to the work of mucin digestion.

So, to find out if A. muciniphila is also present in other animals, Belzer et al. went to the zoo to find the bacteria in mucus of mammals and non-mammals (see figure 1). The A. muciniphila found in these animals differ as the animals have different digestive physiologies, diets and mucus structure. Still, the bacteria were not very different, which is very remarkable. The only difference between the animals A. muciniphila was found in the antibiotic resistance and CRISPR Cas system, as the animals differ in defending to bacteria and viruses in their environment.

So, why are the bacteria so similar? This could be due to a spontaneous infection very recently, so the bacteria were not able to evolve since. The bacteria could also have a very stable genome that does not evolve. Or the bacteria are not specific on the type of mucus.  One genome, however, was different from the rest. The python strain of the A. muciniphila looks really different. Moreover, only a small number of all animals are tested on the A. muciniphila. Therefore, more different genomes could be discovered.

Belzer saw a similarity between organisms and the A. muciniphila system: As soon as the host was on a diet or starving, the A. muciniphila levels are going up. This is similar for all organisms and systems. This could indicate that A. muciniphila could be an obese related bacterium. Belzer moreover, showed that the A. muciniphila works as a growth support of beneficial microbes in the gut. Moreover, when the A. muciniphila levels rises in the gut, organisms were metabolic healthier.

It was very interesting to visit the BioTechonolgy department for this seminar. Some metabolic details were hard to follow for me; however, I was still able to follow her talk. I was surprised by the complex microbiota in the gut. The similarity of the A. muciniphila is very interesting and research for this bacteria in other animals can contribute to the understanding of this bacteria.

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Figure 1: Akkermansia muciniphila is universally distributed in intestinal tracts all over the animal kingdom. (a) Phylogenetic tree indicating the position of A. muciniphila among selected full-length 16S rRNA clones from mammalian gut samples. Red colored samples derive from human sources. Thermotoga thermarum is used as an outgroup. The tree was generated using the neighbor joining method. Full details and high-resolution information are provided in Supplementary Figure S1. (b) Schematic representation of the tree in (a) with the five different clades their position and similarity to A. muciniphila. (c) Taxonomic tree of mammals generated using iTol webtool from tree of life project using all available sequences from NCBI (Letunic and Bork). Animal silhouettes indicate single species as a representative of that order. When an animal species from the mammalian orders was positive for Akkermansia-like sequences the animal logo belonging to that order is colored red, when it was negative the animal logo is colored gray. No Akkermansia sequences have been reported yet in any of the animals belonging to the mammalian orders depicted in black.

Adapted from: Belzer, C., & De Vos, W. M. (2012). Microbes inside—from diversity to function: the case of Akkermansia. The ISME journal, 6(8), 1449-1458.

Structurally distinct periodic and dynamic chromatin domains revealed by super-resolution fluorescence microscopy

Speaker: Kirti Prakash

Department: Institute of BMolecular Biology, Mainz, Heidelberg University Heidelberg. Carnergie institution for science, Baltimore usa, current affiliation institute paris

Subject: Structurally distinct periodic and dynamic chromatin domains revealed by super-resolution fluorescence microscopy

Location: Bionanoscience Department, TU Delft

Date: 26-06-2017

Author: Mirte Golverdingen

A large spectrum of chromatin domains between DNA and the chromosome territory level remains unexplored.  We do understand the morphology of the DNA helix since the 1950’s and a decade ago Bolzer et al. showed the morphology of intra-chromatic departments of chromatins (see Figure 1).  However, the morphology and behavior of the region between single DNA morphology and intra-chromatic departments is still fairly unknown.  Genomic methods have already found a large spectrum of chromatin domains between the DNA and the chromosomal territory level, however their regulation is not known yet. To learn more of the regulation of gene expression, we have to obtain a better understanding of the unknown region.

 

Honours10

Figure 1: False color representation of all CTs visible in this mid-section after classification with the program goldFISH.

Adapted from: Bolzer, A., et al.  (2005). Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. PLoS biology, 3(5), e157.

Kirti Prakash studied these intermediate chromatin features by using single-molecule localization methods (SMLM). Classical microscopy methods, such as conventional light and electronic microscopy, are not able to identify intermediate chromatin features because of specificity and a poor resolution. Therefore, nanoscale imaging is required to describe chromatin complexity.

SMLM is the best method to study chromatin organization because it has the best resolution in a small area. From all SMLM methods, PALM/STROM has the best resolution and distance in xyz area. When asked if the time resolution had not to be taken into account, Prakash mentioned that time resolution is not important for the research he is doing. Moreover, he mentioned that the PALM/STORM microscope set-up technique is very simple, because no much alignment is needed.

However, the preparation of the buffers and fluorophores is very complicated, because the combination of buffer and fluorophores need to have a high photon expression number, prevents photo-bleaching and it has to blink for a long time. The search to a combination that has all three characteristics is time consuming and hard. Therefore, I think that PALM/STORM is not that better than other SMLM methods as Prakash suggested in his talk.

Dyes as Hoechst and DAPI undergo phot-conversion when binding to the minor DNA groove, therefore they are a good tool of chromatin studies for SMLM. The red shift of the fluorophore makes that you can exclusively excite the dyes that are bound to the DNA. This makes that the dyes do not bleach and that there are still a lot of signals. This high labeling density is required to study unknown structures.

According to Prakash, this method of direct DNA staining is better than previous methods based on antibody staining o histone proteins.  This is because you can have a 5 times higher labeling density. However, can we really compare the DNA-staining with histone labeling? Histones behave different from DNA itself, so are you not comparing apples with oranges?

By using the technique Prakash was able to study the hierarchical structure of the chromatin in a single experiment. He made 2D images of a 3D domain, so different in z-direction could not be measured. He showed that there were clusters of chromatin in the cell. However, is this really a fair conclusion if you have no idea of the clustering of chromatin in the z-direction.

By studying the clusters visualized, Prakash could research the chromatin complex morphology. He observed ring and rod like chromatin domains and displacement of active histone modification during stress. In mammalian cells, the chromatin complexes rearrange after stress. So, according to Prakash SMLM contributes to describe the spectrum of unknown chromatin domains between the nucleosome and chromosome level.

Prakash, however, did not convince me of the value of SMLM to the study of unknown chromatin domains. He compared the results of Histone labeling with his DNA staining assay, which was, I think, not fair. Moreover, his visualization of the chromatin complexes was in 2D without a verification that this 2D representation was valid. I think that a more validated study to chromatin complexes with this assay would have a better contribution to research on these complexes.