Nano-scaled properties of the amyloid life-cycle

Speaker: Wei-Feng Xue

Department: School of Biosciences, University of Kent

Subject: Nano-scaled properties of the amyloid life-cycle

Location: TU Delft

Date: 12-05-16

Author: Hielke Walinga

When people first discovered the cause of the mad-cow disease, they thought it was some kind of black magic in nature. It was caused by a misfolded protein that could convert other proteins into a misfolded protein as well. They called them prions. These proteins were insoluble and aggregated after their replication. It was a kind of life that was even stranger than viruses, something that was already discussed as something that cannot be classified as life anymore. Now, it becomes clear that other diseases might as well be prion linked diseases, like Alzheimer, Parkinson, and Diabetes type II. Researching the properties of these proteins, and its non-toxic variant the amyloid, becomes therefore more and more important.

The presenter of this talk is Wei-Feng Xue and he researched the nano-scale properties of the amyloid life-cycle. Xue explained that amyloids could be a potential nanomaterial because of its mechanical strength and elasticity, but in his presentation he primarily focusses on the disease linked properties of the amyloids, or the prions.

Amyloids are just fibers (in fact they are stalked cross beta sheets) and the list of functional amyloids in organisms grows and grows. If we could know more about its general life-cycle we might discover what makes some amyloids become the toxic prion variant. The prion is in fact just a transmissible amyloid (you can get infected for example by eating infected food). Our focus on prions should therefore be directed to its propagation and transmission.

The life-cycle of amyloids can be divided in three parts. First there is the nucleation; this is the forming of the first amyloids. When the amyloids are formed, they will grow by recruiting more proteins on them. This is the elongation. Then there is the fragmentation; this is the dividing of the amyloids in multiple pieces. This process goes largely together with elongation. What’s more, Xue believes that in the beginning there is a period of largely elongation, and after that there is a large period which is dominated by fragmentation. After that, steady state should be achieved, but, this is just a prediction.

Nucleation is a rather slow process. There is large energy barrier that needs to be crossed and this just takes a long time. That’s also the reason why Alzheimer, Parkinson, and Diabetes type II are largely found by older people. Other infectious prion diseases might originally have arisen by spontaneous nucleation, but when spread they will be seeded. Like for example when you eat infected food. In the lab, nucleation is sped up by increasing the concentration or by a special solution.

The length of the amyloid largely reflects the properties of it. Longer fibers are much more stable, although in live you will find decreasing lengths. In the disease’s perspective, fragmentation is good; because it increases net growth and increases the toxicity (short fibrils disrupt the membrane). It should be interested how this fragmentation really works, and how the chaperones of the cell react on these amyloids.

I think this research gives great new insights in diseases and might reveal more diseases which are caused by amyloids or which symptoms can be explained by amyloids. In a project for my Bachelor I found for example that Huntington disease patients certain proteins that are linked to prions have an increased concentration. This might explain certain symptoms of the disease.

amyloid_lifetimeImage 1: The predicted life-cycle of an amyloid. The average length vs. the time; log-log scale. Blue is dominated by elongation, red is dominated by fragmentation, green is the steady state. (Source: Xue, Wei-Feng, and Sheena E. Radford. “An imaging and systems modeling approach to fibril breakage enables prediction of amyloid behavior.” Biophysical journal 105.12 (2013): 2811-2819.)

Nano-scale Properties of the Amyloid Life-cycle

Speaker: Wei-Feng Xue

Department: School of Biosciences, University of Kent

Subject: Nano-scale Properties of the Amyloid Life-cycle

Location: TU Delft, Bionanoscience, Kavli Institute of Nanoscience

Date: 12-05-2016

Author: Mirte Golverdingen

Xue started his talk by introducing amyloid and prions. Amyloid and prions are important biological structures. Amyloids are fibrillar structures that are formed from proteins or peptides. They are only around 10 nm width, up top micrometers long. Amyloids are associated with diseases such as type II diabetes, Alzheimer’s disease and Parkinson’s disease. Prions are transmissible amyloid associated with diseases such as mad-cow disease, scrapie and Variant Creutzfeldt-Jakob disease.

However, Amyloids are also potential candidate as high-performance nano-material. The fibers have interesting mechanical and elastic properties. Xue and his group try to understand these properties.

The Amyloid fiber structure involves multiple β-sheets that run parallel to the fiber axis, with individual β-strands perpendicular to the fiber axis. However, this is only concluded from models, the actual structure is not precisely known yet. Amyloid fibers are very tightly controlled and their functions are more and more discovered.

The biophysics of the amyloid fibers is a very slow and complex process. All the amyloid has the same free energy; this makes them very hard to distinguish. The free energy barrier is very high, so it is very hart to cross the border and to form filaments. The kinetics of amyloid forming is very slow.

Xue sees the aggregation pathway of amyloid forming as a life cycle. His research focusses on this life cycle of amyloid assembly. To start this cycle prima
ry nucleation occurs, this is de novo formation of amyloid fibers. After that, secondary nucleation and fragmentation occurs. So this pathway results in fragments that on their turn can form assemblies again. This life cycle, therefore, is also a positive feedback loop.  See figure 1.

Figure 1. 
Schematic illustration of the lifecycle of amyloid. (Circles) Soluble monomeric protein. (Parallelograms) Monomeric units in the amyloid cross-β conformation. (Colored arrows) Main processes in amyloid assembly. (Red arrows) Primary nucleation, which may occur as homogeneous nucleation in solution or heterogeneous nucleation at interfaces. (Purple arrow) Secondary nucleation, which may occur as heterogeneous nucleation at surfaces presented by preformed aggregates. (Blue and orange arrows) Key nonnucleation based processes elongation growth, and breakage division, respectively. (Single arrowsmay represent multiple consecutive steps; arrow thickness symbolizes relative rates involved in the processes.) Wei-Feng Xue, Nucleation: The Birth of a New Protein Phase, Biophysical Journal, Volume 109, Issue 10, 17 November 2015, Pages 1999-2000, ISSN 0006-3495,

Xue and his lab are also interested in the structure of the fibers, however, not in the atomic level structure. The mesoscopic level is more interesting because the amyloid fibril size and shape defines their biological activities. Amyloid can form a lot of different fibers, from curly to clumps, networks and combinations of them. However, amyloid fibers are never branched. The amyloid structure is a stable structure. However, in the lab, you sometimes give a kickstart to the reaction. This results in a condition that changes the binding. So then the amyloid structure can become less stable. Moreover, if you remove chaperones and other control molecules of the amyloid fiber polymerization, the amyloid fibers also will become less stable. 

The properties of amyloid fibers, like size distribution, persistence length and force resistance are important in the interaction of the fibers. However, it is not yet known how they interact with each other and surfaces. Xue uses the AFM technique to scan the surface of the fibers. Using this method makes that individual amyloid particles can be studied in great detail. He uses a program called Trace-Y in Matlab to trace the polymers. In this way he can use the raw data to make models of the folding of the fibers.  

This models are, however, not enough to answer questions like: ‘how does de novo amyloid formation involves stochastic nucleation?’ and ‘Can we prevent amyloid to form de novo in the first place?’. Xue and his group use theoretical models to show how they think amyloid nucleation works. These models are then tested on the data. They for example showed, when looking at the concentration of the monomer, that decreasing the monomer concentration results in a longer forming time of the amyloid fibers.

The fibril fragmentation of the amyloid growth and the activity of the fibrils are also very interesting. Fragmentation controls the number and length of amyloid particles; it therefore also controls the fibrils’ environment. Fibril fragmentation accelerates amyloid growth. Moreover, Xue showed that the length of the fibrils interacts different with the cell than longer fibers. Short particles make the membrane bend and destroyed while long fibers do not have this effect. This shows that structures with the same atomic structure can have a different effect when they have different length scales.

This talk highlighted the complexity of one small compartment of the cell. Moreover, it showed the importance of the mesoscopic level. Even if molecules have the same atomic structure, their behavior can be different because of their behavior on the mesoscopic level. During Xue’s talk it became clear that there is still a lot of research needed to fully understand the behavior of small structures.




Dynamics of translation of single mRNA molecules in vivo

  • Speaker:         Marvin Tanenbaum
  • Department:  Hubrecht institute Utrecht
  • Subject:         Dynamics of translation of single mRNA molecules in vivo
  • Location:       TU Delft
  • Date:             11-05-2016
  • Author:         Katja Slangewal

The fate and function of a cell depend among others on the protein expression control. The lifetime of mRNA is approximately 10 hours and the lifetime of proteins is approximately 20 hours. This means that every protein is renewed each day. These numbers stress the importance of ribosomes. This is why Marvin Tanenbaum and his group explore the ribosome thoroughly. They want to know the speed, variability, noise and error rate of this complex molecular machine.

The variation in gene expression can be seen in western blots of several proteins over time. There is a large difference in expression of certain proteins between cells in the G1 phase and cells in the M phase for instance. However, these experiments involve a lot of averaging over different cells. Single cells can behave very different. They are all stochastic processes, so even single cells differ a lot over time. The question is: how do you assure, on single cell basis that a cell behaves as it is supposed to?

Before it is possible to answer this question, it is important to know how you observe single cell gene expression. The first approach included using GFP as a tag. Unfortunately a problem occurred. This problem was the maturation time of GFP. It takes approximately 5-60 minutes before GFP becomes visible. This time is much larger than the translation time, which means you cannot see the protein being translated. Additionally GFP has a very weak signal. So Marvin and his colleagues searched for more options.

They found a solution in using secondary tags. They made sure that GFP was abound and could bind to the SunTag (SuperNova tag) added to the protein. This technique enabled to see the translation from the start. The SunTag could bind many GFP’s causing very bright blobs. Since there are many ribosomes at each mRNA strand, it was not yet possible to track a single mRNA strand.

Figure 1: The SunTag labeled with GFP. This tag helped the Tanenbaum group to see single ribosomes and to see the transcriptional activation [1].

Next, they added not only a SunTag to the gene, but also a mcherry tag. The last one was added after the stop-codon. So now it was possible to follow a single mRNA molecule. Then the task was to find the ribosome speed. This was done by blocking translation at once. No new ribosomes were able to bind to the mRNA strand and the remaining ones were slowly removed. The decrease in GFP signal allowed calculating the ribosome rate, which appeared to be 2-5 codons per second. Marvin has the opinion that this is a large variation, since the speed was measured over ~1000 steps. The variation could be because ribosomes get stuck sometimes. However, this needs to be validated with future research.

Finally, Marvin showed how they accomplished to follow a single ribosome. They used a clever trick for that. By adding a certain structure at the start of a sequence the mRNA forms a structure. Many ribosomes fall off after bumping in this structure, but rarely a ribosome is able to stay on the mRNA. If that is the case, you can observe a single ribosome.

I think Marvin gave a clear talk. The structure was quite nice. He clearly stressed the problems they encountered and the solutions they found for them. It was nice to see the progress in his research. He started his talk very calm and I was afraid it would get boring. Luckily Marvin became more and more enthusiastic as he went on with the talk. All together I liked the talk and I learned new things, like the fact that GFP has a maturation time.

[1] Tanenbaum M.E., Gilbert L.A., Qi L.S., Weissman J.S., Vale R.D. (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:3 635-646

ITOP – easy and efficient intracellular delivery of macromolecules

SURE symposium ‘Science Fiction: The impact of scientific research on modern society’

Speaker:     Prof. Dr. Niels Geijsen (Hubrecht Institute Utrecht)
Subject:      ITOP – novel opportunities for safe and efficient gene repair
Location:    Erasmus MC Rotterdam
Date:           Friday, 13.05.2016

Author: Edgar Schönfeld

Introducing a new protein into a cell can be done by transfecting the cells of interest with the appropriate DNA sequence. Direct delivery of protein into cells, however, is more challenging. An example of a currently used protein delivery system are cell-penetrating peptides (CPPs). Currently, it seems that a protein that is fused to a CPP can easily penetrate cell membranes of all types of cells. Yet, such a fusion can negatively influence the protein’s function.
When Prof. Geijsen was thinking about alternative ways of intracellular protein delivery, he found inspiration in the membrane-penetration mechanism of the Anthrax toxin (Fig.1). The Anthrax toxin consists of three proteins: two inducers of apoptosis and one protein (PA83) which binds to the cell membrane and assembles into a simple core complex, allowing the other two proteins to enter the cell.
Fig.1: The 3 Anthrax toxins are Edema Factor (EF), Lethal Factor (LF) and PA83. PA83 binds the Anthrax Toxin Receptor (ATR), reacts with Furin and gives rise to PA63. PA63 forms a heptameric complex that eneables EF and LF to enter the cytoplasm. [1]

Geijsen experimented with the PA protein in an attempt to guide Oct4 into the cytoplasm of cells. After 9 months of ongoing research, however, he was not unamused when he realized that Oct4 also enters the cytoplasm if the PA protein is omitted. Geijsen’s first thought was that he wasted 9 months of research. Then it dawned upon him that the composition of the buffer might have enabled Oct4 to enter the cells. He tested his idea by repeating the experiment several times, each time omitting only one ingredient of the buffer. It became evident that NaCl, as well as a substance named “non-detergent Sulfobetaine-201” (NDSB-201, full name: 3-(1-Pyridino)-1-propane sulfonate), were essential for successful cell membrane penetration. Further investigations revealed that the NaCl-induced hypertonicity triggers macropinocytosis, a process in which cells take up a certain amount of their surrounding fluid, including solute molecules. In this case, NaCl causes the cells to take up Oct4 and NDSB-201, which is encapsulated in intracellular vesicles in the process. NDSB-201 has the ability to dissolve these vesicles, thereby releasing the vesicular contents into the cytoplasm.
Geijsen and colleagues chose the name “iTOP” for their newly developed system, which stands for “induced transduction by osmocytosis and propanebetaine” (Fig.2).
Fig.2: Scheme of the iTOP system. NaCl hypertonicity cause macropinocytosis. Consequently the protein of interest and NDSB-201 are taken up in vesicles. In the cytoplasm, NDSB-201 dissolves the vesicles, which thereby releases the protein of interest.[2]
They went one step further and demonstrated that this system can be employed for super efficient intracellular delivery of the Cas9 protein and its guide RNA. In particular, they targeted the DPH7 gene in a leukemia cell line. If both DPH7 alleles are knocked-out, a cell becomes resistant to diphtheria-toxin-mediated cell death. Geijsen and colleagues found that after 2 rounds of treatment with the diphtheria toxin none of the control cells survived, but 70% of the cells targeted by iTOP-CRISPR/Cas9. Seventy percent are remarkable, given that two alleles have to be knocked out parallelly. Later, it was found that the remaining 30% were also mutated at the DPH7 loci, but the particular mutations did not shield them from DPH7 mediated cell death. For this reason, Geijsen concluded in his talk that he actually achieved a 100% efficiency in targeting the DPH7 gene.
All in all, the iTOP method is simple and efficient and does not seem to disrupt cellular processes. I think it will find wide application in experiments in which abrupt introduction of a protein is more desirable than expressing it via a cloning.



[2] D’Astolfo DS, Pagliero RJ, Pras A, Karthaus WR, Clevers H, Prasad V, Lebbink RJ, Rehmann H, Geijsen N. Efficient intracellular delivery of native proteins. Cell. 2015 Apr 23;161(3):674-90


Biology in the context of boundary: Genome organization and Pattern formation

  • Speaker:           Fabai Wu
  • Department:     BioNanoScience
  • Subject:            Biology in the context of boundary: Genome organization and pattern formation
  • Location:          TU Delft
  • Date:                11-05-2016
  • Author:             Katja Slangewal

Fabai Wu starts his talk with the plant Brunsvigia, which lives in the dry areas of Africa. When, after several years, the rain starts falling, the plant starts growing and its branch breaks of the roots. Because of the spherical shape, the plant starts rolling in the wind until its seeds can grow new plants far away. Wu wonders why there are almost no animals using a rolling strategy instead of walking. Clear is that walking appeared evolutionary preferable over rolling. This can be traced back to the complex interaction between the genotype and the phenotype. The development from a relatively simple embryo to a complex organism lies within these interactions.  The appearance of asymmetry for instance depends on the interaction of several chemicals. Even the simplest cases with two chemicals can form patterns. Wu is interested in these self-organized patterns and in the regulation of the transfer of information between genotype and phenotype. A better understanding of these processes will lead to a better understanding of the emergence of life. Today, he tells more about self-organized patterns and genome organization in the boundary of biology.

seminar 5 dna

Figure 1: E. coli DNA needs extra forces to fit into the small cells. [1]

Wu has been using E. coli and its Min system to learn more about genome organization and self-organized patterns. The usual day of an E. coli bacterium exists of growing and dividing over and over
again. To have an idea of the size: an E. coli bacterium is on average 2-6μm long and it has a diameter of 1μm. Its genome consists of 4,6Mbp and if it were stretched out, it would be 1,5m long. To fit this large molecule into the small bacterium extra forces are needed. The question was, whether these forces mainly come from intra-nucleoid interactions or from boundary constraints. So far, it is not possible to see the DNA structure within E. coli with imaging techniques.  This was solved by making more space in the bacterium. The genes encoding for division were knocked out so that E. coli cannot divide. Also, they made sure that the cell lost its rod-shape. After approximately 1,5 hour the bacteria had grown enough to see the circular shape of the DNA. This showed that the chromosome is pushed to a helical form due to a small cell-width.

Next, E. coli was allowed to grow only in one direction. This maintained the rod-shape of the bacterium. The DNA was not allowed to divide. Wu found that until a certain point the DNA does not expand further, although the bacteria are still growing.  After knocking down Fis, a protein able to link different parts of the genome together, the DNA was able to expand further than the wild-type. This suggests the boundary constraints are not the only important forces for fitting the genome into the small bacteria, also nucleoid interactions play a role.

Finally Wu talked about self-organized patterns. The Min system is an oscillating system which prevents the Z ring for forming outside mid-cell. In 30 seconds the Min proteins travel from pole to pole. Wu made nanostructures in different forms, like squares, triangles and circles. The structures were filled with E. coli bacteria that were not able to divide, so they could occupy the entire area. They looked at the Min oscillations in all the forms. They also looked at the same experiment at different scales. It appeared that the Min system searches for a symmetry axis. First it was thought this should be the longest axis, because this is the case in E. coli. However, the experiment showed that the Min system also oscillated around the shortest axis in some cases. The similarity between all the axes is the length. The E. coli Min system prefers a symmetry axis between 3 and 6μm. This agrees with the length E. coli. Wu and his team were also able to effectively predict the experiments with computational models. So to conclude: biology works through self-organization, both in genome organization and pattern formation.
seminar 5 min

Figure 2: The oscillating Min system in different shapes. Left, the shapes are shown. Then, some snap-shots at different time points are shown. Next, the average intensity is visible. The green arrows show the symmetry axis. And on the right all the axes of each shape are shown. [2]

Fabai Wu gave an interesting talk. First I didn’t grasp the subject. The story about the Brunsvigia plant was also interesting, I didn’t know this survival tactic yet, but I found it hard to see the link between the plant and the Min system in E. coli. The rest of the story was quite nice. He explained clearly the results and reasoning behind the conclusions. I already read the article Wu and his colleges wrote about the oscillations of the Min system in different shapes, since my Honours Project is also about the Min system. So my background knowledge for this talk was quite good. Sometimes this was a shame because he was telling a lot of things I already knew. But it was nice to hear about the process behind an article as well. This gave the article an extra dimension.

[1] Griswold, A. (2008) Genome packaging in prokaryotes: the circular chromosome of E. coli. Nature Education 1(1):57.

[2] Wu, F., van Schie, B.G.C, Keymer, J.E., Dekker, C. (2015) Symmetry and scale orient Min protein patterns in shaped bacterial sculptures. Nature nanotechnology. 10: 719-726.

Nano-scale properties of the amyloid life-cycle

Speaker:          Wei-Feng Xue
Department:   School of Biosciences, University of Kent
Subject:          Nano-scale properties of the amyloid life-cycle
Location:        TU Delft
Date:              12-05-2016

Author: Carolien Bastiaanssen

Amyloids are protein aggregates that often form a fibrillar structure with a width of ~10 nm. They come in a variety sizes, ranging from ~10 nm to several μm in length. Xue refers to this length scale as the mesoscopic scale. Some amyloids have a functional role, but they are the subject of many studies because they are involved in multiple diseases such as Alzheimer’s disease, Parkinson’s disease and type II diabetes mellitus. Some amyloids are transmissible, in this case they are called prions. Examples of diseases where prions are involved are Creutzfeldt-Jakob disease and scrapie. Nowadays there is no sharp boundary anymore between amyloids and prions due to the realization that many amyloids that were thought not to be transmissible now appear to be transmissible. One of the questions that Xue wants to answer is what makes an amyloid transmissible.

Fibrils of different lengths. (Top: EM images, bottom: AFM images)
                            Fibrils of different lengths. (Top: EM images, bottom: AFM images) [1]

Before being able to answer this question, Xue needs to understand how amyloids assemble. This is a slow and complex process, which is often compared with a life cycle. When monomers are put into solution it takes quite some time before they nucleate. This waiting time is called the lag phase. Next an exponential growth phase follows, until a plateau is reached. The long lag time causes diseases such as Alzheimer’s disease to primarily affect the elderly. However, in case of prion diseases the long lag time is skipped because of the transmission of preformed polymers. After polymers have formed, they can fragment and new polymers can grow from these fragments. This process is called seeding. Many question remain, regarding the life cycle of amyloids. It is for example still unclear how monomers form seeds and what the exact molecular structure is of these seeds.

In order to gain more knowledge about amyloids, Xue studies their mechanical properties. He uses atomic force microscopy (AFM) to characterize the size distribution, persistence length and force resistance of amyloids. AFM has the advantage that it allows the study of individual amyloid particles in great detail. Because they analyze many samples, Xue and his team developed a tool (trace-y) to automatically trace amyloid fibrils.

One of the experiments Xue and his team performed, involved measuring the length of amyloids and then studying their interaction with membranes. They found that long fibrils are less harmful for cells than short fibrils. They measured a cell viability of 82% for long fibrils and 36% for short fibrils. The reason why shorter fibrils have a higher cytotoxicity than longer fibrils is still a matter of debate. Research suggests that the ends of the fibrils interact with the membrane and cause damage, in this case a more fragmented amyloid would indeed be more lethal to cells than one large amyloid. Therefore, Xue would not pursue a treatment of e.g. Alzheimer’s disease which is aimed at dissolving prions. He would opt for stabilizing the prions that are there already or in case you can start treatment early, prevent amyloids from forming at all.

The crucial part of the amyloid life cycle is the fragmentation process. Therefore it is of large interest to Xue. Many aspects of this process still require research to elucidate the underlying mechanisms. It is for example unknown what triggers fibrils to break and how stable different amyloids are. Xue and his team for example study the stability of fibrils towards breakage using AFM and they check whether the obtained data fit with the theoretical model they developed.

All together, it was a very interesting talk by Xue. Although I did not have any prior knowledge on amyloids, everything was clear to me. Xue was very enthusiastic and I could sense the passion he has for his work. It was nice to see that in this research physics and biology come together and this a good example of a field of research in which a Nanobiologist could be of good use.

1. W. F. Xue et al., Fibril fragmentation enhances amyloid cytotoxicity. J. Biol. Chem. 284, 34272-34282 (2009).

Nano-scaled properties of the Amyloid life-cycle

  • Speaker:           Wei-Feng Xue
  • Department:     School of Biosciences, University of Kent
  • Subject:            Nano-scaled properties of the amyloid life-cycle
  • Location:          TU Delft
  • Date:                12-05-2016 
  • Author:             Katja Slangewal

It is well known that viruses and bacteria can cause diseases. However, they are not the only disease causing agents. Type II diabetes, Alzheimer, Parkinson and Mad-cow disease are for instance associated with amyloids and prions. Amyloids and prions are aggregates of proteins and/or peptides. Prions are usually seen as the transmissible variant of amyloids. However, Xue showed that the border between them gets more blurry over time.

Amyloids and prions both form fibrils with a diameter of approximately 10nm and a length between several nanometers and hundreds of micrometers. Later it will be shown that the length has a large influence on the biological activity. Since amyloids are shown to have an influence at several diseases, it is important to learn more about these bio-structures. Xue and his team want to find the growth rate, deposition and cytotoxicity of amyloids.  He also wants to know more about the propagation and transmission of prions.
seminar 4 amyloids

Figure 1: Amyloids have a large size range. A) shows the oligomers before polymerization. B) shows short fibrils. C) shows long fibrils. [1]

The first thing Xue showed was the rate of assembly of amyloids. This appeared to be very slow and complex. The polymerization of amyloids has a very high energy barrier. This is somehow good news for elderly people who are being infected with an amyloid causing Alzheimer. The time it takes for the amyloids to form enough fibrils to be harmful is probably longer than the time the elderly people still have left. However, this is only true when the fibrils have to form from scratch. When you are infected with preformed fibrils, the long lag phase is skipped and the amyloids can grow faster. This is called seeding.

Although amyloids are not considered to be alive, they have something like a life cycle. They can form de novo by nucleation. After growing, they can fragmentation can occur. The smaller polymers can grow again until a next fragmentation. It appears that fragmentation accelerates the growth of amyloids. Two samples were tested, one with large amyloids and one with small amyloids. It appeared that the small amyloids grow quicker than the larger ones. Also, the small ones appeared to be more harmful to membranes. This was shown by measuring the membrane disruption for both samples (figure 1). Next, the amyloids were added to tumor cells.  The viability of the tumor cells was measured. For the large amyloids the viability was 82% and for the small amyloids it was 36%. Finally, the stability was measured by applying a mechanical force to the amyloids. The results show that the longer fibrils fragmentize easier and shorter fibrils are more stable.
seminar 4 liposomes

Figure 2: Left) shows liposomes and short fibrils. The arrowheads show disrupted liposomes. Right) shows liposomes and long fibrils. Almost no disruption of liposomes was observed. [2]

Summarizing the results, Xue proposed a model which shows the ‘life’ of an amyloid. First the amyloid grows for a long time, than it fragmentizes before it reaches a steady state. The growth and fragmentizing phase take around 109 seconds, which equals approximately 30 years. The experiments showed that the amyloids are most dangerous when they are short.

The last experiment Xue described was about the transmissibility of prions. He used synthetic prions that were not capable of causing harm and introduced them to yeast. These prions had never seen yeast before, but they were able to induce a certain phenotype when infecting them. This phenotype showed the transmissibility. It appeared that the smallest fibrils had the highest transmissibility.

So amyloids and prions can cause severe diseases. This means it is necessary to study their properties. The biological activity of the amyloids is dependent on the length of the fibrils. It was shown that the smaller the fibrils the more harm they can induce. Also, the transmissibility of prions increases when the length decreases. More research has to be done in order to find a way to prevent amyloid/prion based diseases.

Wei-Feng Xue gave a nice talk. He was very enthusiastic about his work and he was smiling during the entire talk. This made his talk very lively. He clearly stated the importance of his work and the explanation of the results was also very good. Every graph was discussed properly. He didn’t go to deep into the methods he used. This made it easier for me to understand everything he was saying. I think this talk was the first talk in which I didn’t feel like missing a large part of the background information. I liked the talk, but I don’t think I want to do research to amyloids or prions myself. I like living and more complex cells better than these fibrils.

[1]: A. Relini, N. Marano, A. Gliozzi (2014) Misfolding of amyloidogenic proteins and their interactions with membranes. Biomolecules. 4(1): 20-55

[2]: L. Milanesi et al. (2012) Direct three-dimensional visualization of membrane disruption by amyloid fibrils. PNAS. 109(50): 20455-20460

Nanoscale properties of the amyloid lifecycle

BN Seminars

Speaker: Wei-Feng Xue (School of Biosciences, University of Kent, UK)

Subject: Nanoscale properties of the amyloid life-cycle

Location: TU Delft

Date: Thursday, 12.05.2016, 16:00-17:00

Author: Edgar Schönfeld

Fig.1: Amyloid fibrils imaged by an atomic force microscope [1]

Amyloids are misfolded proteins that self-assemble into filaments (fibrils), typically 10 nm wide and several micrometers long (Fig.1). While amyloid fibrils are typically associated with a variety of diseases, there is growing evidence for functional roles in cellular processes for some kinds of amyloid fibrils. Amyloids are thought to be the cause of Alzheimer’s and Parkinson disease. The extracellular amyloid deposits are thought to be cytotoxic. Prions are transmissible amyloids, mainly being known for their role in mad-cow disease and Kreutzfeld-Jakob syndrome. However, according to Xue, the border between amyloids and prions blurs increasingly, as amyloids with prion-like properties are being found.
Xue investigates why some amyloids are transmissible, while others are not, and what makes some amyloids more cytotoxic than others. To address these questions, Xue examines the structural properties of amyloid fibrils at the mesoscopic scale (nm-µm), as the size and shape of the fibers in this spatial regime determines their function. Within the last 10 years Xue characterized amyloid fibrils in terms of nucleation- and fragmentation behavior, stability and the effects of fibril length. He was able to relate these quantities to the cytotoxicity and transmissibility of amyloid fibrils.
In fact, amyloid fibers that are a 100% identical in the atomic structure of their components can take greatly different shapes. Yet, structural properties common to all amyloid fibrils are their corkscrew-shape and the fact that they are are built up of beta-sheets running perpendicular to the fibril’s main axis. The formation of these fibers can be described by the amyloid life-cycle (Fig.2), a term that not all of Xue’s colleagues agree with.



Schematic illustration of the lifecycle of amyloid. (Circles) Soluble monomeric ...
Fig.2: The amyloid life cycle (red: primary nucleation, purple: secondary nucleation, blue: elongation growth, orange: breakage division) [2]

The initial step of the lifecycle is the primary nucleation, meaning that monomers come together to form a sequence of connected components. The secondary nucleation refers to the further elongation of this basic polypeptide. The reason that this distinction is made is that primary nucleation has to overcome a large energy barrier. For this reason, it proceeds so slowly that one can say that people in their 80’s who do not have Alzheimer’s yet will never get it. If one were to measure fibril concentration in solution over time after addition of the amyloid building blocks, one can distinguish two possible outcomes: either a lag phase followed by a steep increase in fibril concentration, or an immediate increase in concentration that levels off over time. The latter observation is associated with secondary nucleation, which is associated with a lower energy barrier and therefore occurs faster.
There are studies out there claiming that Alzheimers is transmissible. In these studies, amyloid fibrils were directly transmitted from a diseased to a healthy animal. According to Xue, the reason why those animals subsequently develop Alzheimer’s is that the transmitted fibril fragments can be seen as ‘seeds’, only requiring secondary nucleation to form fibrils. The next step in the amyloid life-cycle is fragmentation. In Xue’s lab fibrils of different sizes were analyzed under the microscope. They found that cytotoxicity is mediated through the interaction of the terminal ends of the fragmented fibrils with cell membranes. Thus, the more fragmented a fibril is, the more damage it can cause to a cell. For this reason, Xue says that Alzheimer therapies aimed at dissolving amyloids may actually be counterproductive. It is, however, unclear at this point what gives the terminal ends of the fragments the ability to disrupt membranes. Xue has ambitious goals with regard to research on amyloids, and his contagious enthusiasm leaves no doubt he will be successful in doing so.
Research on amyloids will at some point benefit patients. For the case of Alzheimer’s, Xue suggests that, given his findings, preventing fragmentation of already existing fibrils could slow down further neurodegeneration.



[2] Xue WF. Nucleation: The Birth of a New Protein Phase. Biophys J. 2015 Nov 17;109(10):1999-2000

Nano-scale Properties of the Amyloid Life-Cycle

Speaker: Wei-Feng Xue
Department: School of Biosciences, University of Kent
Subject: Nano-scale Properties of the Amyloid Life-Cycle
Location: TU Delft
Date: May 16, 2016

author: Teun Huijben

Amyloid are fibrillar structures that have a width of approximately ten nanometers and are formed from proteins or peptides. They can be made from a wide variety of monomers, can differ a lot in length and can have many different functions. People started doing research on amyloid when they found out that prions (transmissible amyloid) had a lot to do with diseases like Scrapie, Creutzfeldt-Jakob and the Mad Cow Disease. These days still a lot of research in going on in this field, not only to get a better understanding of the structure and function of amyloid, but also because of its interesting mechanical properties like strength and elasticity.

As already stated, there are many different types of amyloid and they can be formed by the assembly of a wide variety of monomers. This is mainly the reason why its function is not well understood, because amyloid has so many different forms. A common thing in the structure of the fibrils is that they exist of many β-sheets that are connected in a parallel fashion orthogonal to the propagation axis of the fibrils. But knowing that this molecular structure determines the shape and properties of the complex, this is not the area of interest for Wei-Feng Xue. Who is doing research on amyloid for over 10 years and is currently working at the University of Kent (UK).

Mr. Xue is more interested in how amyloid is formed, how it forms toxic structures and how it is nucleated, growing and fragmented. After doing a lot of research on amyloid his current hypothesis is that amyloid has a certain life-cycle (see figure 1 below). This life-cycle starts with the primary nucleation of monomers to form short fibrils. This short fibrils grow by elongation and can later be fragmented into shorter pieces that can spread and elongate to form new fibrils. He is fascinated by the great distribution in length of amyloid fibers and what this variety has for influence on its function. A lot of different aspects of amyloid are studied in his lab and he gave us a brief overview of things that are going on  there.

Schermafbeelding 2016-05-12 om 21.21.02figure 1: suggested life-cycle of Amyloid. The circles represent monomers and the arrows represent steps in the life-cycle. The cycle starts with nucleation (red and purple), then the fibrils are elongated (blue) and fragmented (yellow) to start new fibrils. 

Amyloid fibers are twisted and one thing they are looking at is what the precise structure is and what influences this structure. With atomic force microscopy (AFM) pictures are made of the fibrils and this data is analyzed by home-made MatLab software. From this data they make models of the structure and how the fibrils are twisted around each other.

Another major theme in his research on amyloid is the fragmentation of fibrils. When the fibrils have some length they get fragmented into shorter pieces and these pieces can grow into new fibrils. Their hypothesis is that fragmentation speeds up the growing of fibrils, because other experiments have shows that short fibrils elongate faster than long fibrils. To do in vitro experiments on fragmentation a solution of fibrils is mixed with a magnetic stirrer that brutally breaks the fibrils in pieces. When more force was applied, the average length of fibrils got shorter and the length distribution more narrow. This indicates that short fibrils are more stable. Now they are testing different types of amyloid and plot persistence length versus fragmentation constant. They hope to see distinct groups, what will give more insight in the mechanical properties and functions of the fibrils. The same things are measured in vivo, but then enzymes are used to cut the fibrils instead of using brute force.

The most recent thing they are doing right now is producing synthetic prions and insert them into yeast cells. Then with a colorimetric assay is tested if the prion is in its toxic prion-form or not. The goal is to find characteristics of amyloid fibrils that make them more likely to become a dangerous prion and getting more insight in the toxicity of amyloid fibers. And I think this is very necessary to do, because still a lot is unclear about prions; how they cause diseases and how to prevent them from doing that.

A link between our diet and colorectal cancer

JNI Oncology Lectures

Speaker: Leonard Augenlicht (Albert Einstein College of Medicine, Bronx, NY USA)

Subject: Nutrient genetic interactions in sporadic intestinal cancer

Location: Erasmus MC Rotterdam

Date: Wednesday, 04.05.2016, 11:00-12:00

Author: Edgar Schönfeld

In his talk, Leonard Augenlicht elucidated the influence of our diet on the occurrence of colon cancer.
Worldwide, colon cancer is the 3rd most common cancer in men and the 2nd most common cancer in women. However, it is much more prevalent in western and developed countries than in the rest of the world. The highest estimated incident rate is found for Australia (>30 per 100,000), while Western Africa exhibits the lowest incident rate worldwide (ca 4 per 100,000) [1]. Twin studies, as well as studies on migrating populations, have confirmed that environmental factors contribute far more to the occurrence of colorectal cancer than genetic factors. Prof. Augenlicht estimated that we could reduce the incidence of colorectal cancer by 90% if we are able to determine the environmental factors that contribute to this malignancy.
Fig.1: Incident rates of colorectal cancer worldwide, expressed in cases per 100 000 [1]

In the vast majority of cases, colorectal cancer is initiated by a mutation in the tumor suppressor gene APC. Only 1-2% of all cases occur through an inherited APC mutation (1 wild type and 1 mutated copy of the gene) while sporadic colorectal cancer accounts for circa 90% of all cases (patients are born with 2 wild-type APC alleles). In the latter case, the first allele is hit on average at the age of 40 years, while the second hit usually occurs at the age of 50-60. The central question is: How do environmental factors increase the probability of such a hit?

To examine the impact of our diet, Augenlicht and colleagues fed mice on the “New Western-style Diet” (NWD). The nutrient composition is inspired by the average USA human diet, which is high in fat and low in fibers, calcium, and vitamin D [2]. They observed that 20% of the mice developed 1-2 tumors at circa 2/3 of their lifetime. That makes it actually the only mouse model of truly sporadic colorectal cancer. In fact, the NWD amplifies tumor development in all mouse genetic models. The diet does not seem to cause mutations directly, but to rather influence gene regulation: In the inner layer of the gastrointestinal tract (mucosa), the diet perturbs cell maturation, alters the expression of lineage-specific cell markers and elevates Wnt signaling in the villi and crypts, just to name some important examples. Intriguingly, all these effects can be reversed by a “rescue diet”, having raised levels of calcium and vitamin D. In addition, the stem cells in the intestinal crypts (Lgr5+ cells) show a high expression of the vitamin D receptor. Those cells, in turn, are known to initiate colorectal tumor formation (an APC mutation in a Lgr5+ cell will result in a tumor). This led Augenlicht to take a closer look at Lgr5+ cells in mice fed with the NWD, using a technique called lineage tracing (see my last post, or this review on lineage tracing: The constant renewal of the inner layer of the gastrointestinal tract is fueled by Lgr5+ stem cells which reside at the bottom of the intestinal crypts. These cells constantly produce new cells, which subsequently move towards the top of the crypt. However, this process is highly compromised in mice fed on western diet compared to mice fed on a control diet. Inspired by these findings, the researchers fed mice a normal diet, in which only vitamin D levels were decreased. Again, compromised cell migration was observed. Last but not least they used tamoxifen to knock out vitamin D receptors in Lgr5+ cells only, observing the exact same effect (Fig.2).


lineage tracing
Fig.1: Lineage tracing of Lgr5⁺ cells in the intestinal crypts after tamoxifen administration (Red=marker for Lgr5⁺, AIN76=control diet, NWD1=western diet as described in this post, NWD2=NWD1 with elevated levels of vitamin D (“rescue diet”)) [3]

In conclusion, vitamin D is a key factor in determining Lgr5+ cell function. This finding may also explain why animals fed on western diets often loose hair, as Lgr5+ stem cells also reside in the hair follicles. Prof. Augenlicht’s research on the impact of the western diet is ongoing. Recent findings suggest that the western diet actuates a metabolic switch, causing cells in the mucosa to favor glycolysis over the citric acid cycle for generating energy.
With more research to come in the following years, I am curious to see how these scientific findings will influence our culinary preferences.

How should we adjust our diet in order to minimize the risk of colorectal cancer? According to the US National Cancer Institute “There is no reliable evidence that a diet started in adulthood that is low in fat and meat and high in fiber, fruits, and vegetables reduces the risk of CRC by a clinically important degree” (“PDQ (Physician Data Query) – NCI’s comprehensive source of cancer information”, Status as of 11.02.2016) [4]. The same report states “The evidence is inadequate to determine whether calcium supplementation reduces the risk of CRC”. I conclude that we have to wait until we know more. Until then, try to avoid the usual risk factors such as smoking, excessive alcohol use, and obesity.

1: GLOBOCAN 2012: estimated cancer incidence mortality and prevalence worldwide in 2012, Factsheet Colorectal Cancer, World Health Organization (

2: Newmark HL, Yang K, Kurihara N, Fan K, Augenlicht LH, Lipkin M. Western-style
diet-induced colonic tumors and their modulation by calcium and vitamin D in
C57Bl/6 mice: a preclinical model for human sporadic colon cancer.
Carcinogenesis. 2009 Jan;30(1):88-92.

3: Peregrina K, Houston M, Daroqui C, Dhima E, Sellers RS, Augenlicht LH. Vitamin
D is a determinant of mouse intestinal Lgr5 stem cell functions. Carcinogenesis.
2015 Jan;36(1):25-31.

4: “Colorectal Cancer Prevention–Health Professional Version (PDQ®)”(Status as of 11.02.2016)