In humans, pregnancy lasts around nine months. In mice, only 20 days, and in rhinoceroses, as long as 17 months. Although many mammalian species go through the same stages during embryo development, the speed of development differs substantially across animals. Another example of an event that differs in time across species is the formation of the vertebrate body axis, the spine. The formation of the body segments that will give rise to the vertebrae and ribs, called somites, is controlled by a mechanism called segmentation clock. The segmentation clock is a group of genes that oscillates. Each oscillation controls the formation of a pair of somites. The frequency of the oscillations differs across species, taking two to three times longer in humans compared to mice.

 The segmentation clock is a convenient system to study differences in species, and the Ebisuya group has been studying it for a long time, recently revealing that thedifferences in biochemical reaction speeds are responsible for the differences in the mouse and human clocks. However, in order to establish whether this is a general principle of development, researchers needed to broaden the species that have been studied, which up to now has been relatively limited to human and mouse.

Now, researchers from the Ebisuya Group have recapitulated in the lab the segmentation clock of four novel mammalian species, in addition to mouse and human: marmoset, rabbit, cattle and rhinoceros. This work has been done in collaboration with research groups based in Europe, Japan and the United States.

What is a stem cell zoo?

A stem cell zoo is like a library of stem cells from several species to study and compare different developmental events. The collaboration group collected embryonic stem cells and induced pluripotent stem cells from marmoset, rabbit, cattle and rhinoceros, which added to the already existing library of human and mouse. This diverse sampling of species is unprecedented for developmental studies, and aims to constitute a platform for comparison of developmental processes.

“We wanted to create a platform of cells from several mammalian species to study why their developmental time is different. We wanted to have as wide a range as possible, so we chose species with body weights spanning from 50 grams to 2 tonnes, gestation lengths from 20 days to 17 months, and three different evolutionary histories or phylogenies: Primates (human and marmoset), Glires (mouse and rabbit) and Ungulates (cattle and rhino).” said Jorge Lázaro, pre-doctoral student at Ebisuya Group and first author of the paper.

The group focused on studying the differences in the segmentation clock of the four new species. They applied experimental protocols to differentiate the embryonic and induced pluripotent stem cells into pre-somitic mesoderm like cells, the cells that will give rise to the spine, ribs and skeleton muscles.

 “Our stem cell zoo serves as an ideal platform to investigate the cause of interspecies differences in the segmentation clock period, as well as to determine whether there is any general relationship between segmentation tempo and the characteristics of the organism.” said Miki Ebisuya, Group Leader at EMBL Barcelona and at the Cluster of Excellence Physics of Life, TU Dresden.

Correlating the segmentation clock

The gestation length, as well as many other bodily parameters are known to scale with the animal body weight. Larger species tend to have a longer gestation period. The group thus hypothesized that the differences in the segmentation clock could be related to body weight. However, surprisingly they found no correlation between the average body weight of each of the species and its segmentation clock period. Similarly, the gestation length did not correlate with the segmentation clock period.

Instead, the group found that the segmentation clock period was highly correlated with the duration of embryogenesis. Embryogenesis is the time between fertilisation until the end of organogenesis, when all organs are formed in an embryo. This could mean that the segmentation clock can serve as a good system to understand how general embryonic developmental time is established across species.

Furthermore, the group found that the three different evolutionary histories – Primates, Glires and Ungulates –, corresponded with slow, fast and intermediate segmentation clock periods respectively, pointing to a relation between developmental tempo and evolutionary groups.

In previous studies, the Ebisuya group already found thatbiochemical reaction speeds scale with the segmentation clock period. However, those studies focused on mice and human. The group has now extended the species under study and has confirmed that the four new mammals also show differences in the biochemical reactions speeds, correlating very well with the segmentation clock period. That indicates that changes in the biochemical rates might be a general mechanism to control developmental tempo.

Moreover, they found that genes related to biochemical processes show an expression pattern that correlates with the segmentation clock period, providing a concrete clue for a potential molecular mechanism underlying the differences in developmental speeds across species.

“Our aim is to keep adding species in our stem cell zoo,” said Ebisuya. “If we want to confirm whether the findings of our research could constitute a universal principle of mammalian development, we need to expand the zoo and include a wider range of species and phylogenies.”

In the current study published in Cell Stem Cell, the group focused on the segmentation clock, but the stem cell zoo approach opens the possibility to study other biological times such as the heart rate or the lifespan. The more researchers know about how biological time works, the more they might be able to control it. For example, in the field of organoids, if one could accelerate the time required to develop organoids, it could speed up regenerative medicine studies.

“Another aspect that I really like about the stem cell zoo is the possibility to learn from different species outside of human and mouse,” said Lázaro. “Many animals have particular features that make them interesting to study, but due to practical or ethical reasons we don’t have access to them in the lab. Features like for example the size of a rhino, or the long neck of giraffes. Who knows, perhaps in our next project we can use stem cells to try to understand how do giraffes develop their long neck – and longer somites!”

New EMBL research, published recently in Cell Genomics, indicates that long-read genomic sequencing can reveal important patterns of chromosomal structural rearrangement that had previously eluded the more predominant short-read sequencing used in cancer genomics.

A collaboration co-led by EMBL Heidelberg, the German Cancer Research Center (DKFZ), and EMBL-EBI researchers applied new technologies to harness long-read sequencing in a way that could potentially be applied to clinical settings.

 Research Summary:

• New EMBL research shows that long-read genomic sequencing seems to detect some DNA mutations better than short-read genomic sequencing.
• Using Oxford Nanopore long-read sequencing, the scientists sequenced a primary childhood brain tumour known as a medulloblastoma, uncovering a novel mutation pattern.
• Collaboration between EMBL’s bioinformaticians, genomic biologists, and clinicians from the German Cancer Research Centre (DKFZ) allowed them to devise methods to identify and characterise DNA mutations and epigenetic patterns in data.  

Researchers from the European Molecular Biology Laboratory (EMBL) in Heidelberg and the Spanish National Cancer Research Centre (CNIO) have found that:

• A genetic signature of 27 microorganisms in stool defines the high-risk population for pancreatic ductal adenocarcinoma, the most common pancreatic cancer, and could be used for early detection of the disease.

• This is the most comprehensive study of the influence of the microbiome, the microorganisms that live alongside cells in the human body, on pancreatic cancer.

• Late diagnosis of the disease and the absence of efficient screening tests are some of the causes of the high lethality of this type of cancer.

• CNIO and EMBL have patented the results to develop a rapid, non-invasive and affordable diagnostic kit for pancreatic cancer from stool samples.

 Researchers from the Bork group at EMBL Heidelberg, working together with a European consortium involving more than twenty European institutes, have now shown that many commonly used drugs have powerful effects on our gut microbes. These include drugs used to treat cardiometabolic disorders and antibiotics. The results were published in the journal Nature. 

The gut microbiome consists of billions of microorganisms essential to the body’s normal functioning.

“We analysed the effects of 28 different drugs and several drug combinations,” explained Peer Bork, Director of Scientific Activities at EMBL Heidelberg, “Many drugs negatively impact the composition and state of the gut bacteria, but others, including aspirin, can have a positive influence on the gut microbiome. We found that drugs can have a more pronounced effect on the host microbiome than disease, diet, and smoking combined.”

While the negative and lasting impact of antibiotics on gut bacteria is already well-known, this study showed that such effects likely accumulate over time. “We found that the gut microbiome of patients taking multiple courses of antibiotics over five years became less healthy. That included signs indicating antimicrobial resistance,” said co-first author of the study Sofia Forslund, a former postdoctoral fellow in the Bork group and now group leader at the Max Delbrück Center for Molecular Medicine (MDC), Berlin.

 “We wanted to disentangle the effect that diseases have on host microbiomes from the effect of medications, particularly in patients taking more than one drug at the same time,” said co-first author Maria Zimmermann-Kogadeeva, group leader and former postdoc at EMBL Heidelberg. “Being part of the MetaCardis consortium enabled us to use multi-omics data from more than 2000 patients with cardiometabolic diseases,” she added. The large cohort also allowed the researchers to establish that the dosage of drugs prescribed also has a significant effect on the level of impact on the microbiome.

 “We know that the microbiome can reflect the status of a patient’s health and provide a range of biomarkers to assess the severity of diseases. What is often overlooked, however, is that the medication used to treat a disease also affects the state of the microbiome,” added Rima Chakaroun, one of the lead authors of the study and a clinician scientist at the University of Leipzig Medical Center. Dr Chakaroun is currently a postdoctoral fellow at the Wallenberg Laboratory, University of Gothenburg.

By developing a statistical approach that accounts for the effects of multiple confounding factors, the researchers could tease out the effects of drugs and disease separately. “We now have a robust methodological framework that makes it possible to get rid of many of the standard errors,” said Professor Bork. “That allowed us to show that medication can mask the signatures of disease and conceal potential biomarkers or therapeutic targets.”

 The researchers are hopeful that these results can provide knowledge that could potentially help in drug repurposing as well as in planning individualised treatment and prevention strategies.

 The study combined the insight, knowledge and approaches of experts in six countries. “It was very motivating to work with an interdisciplinary team of clinicians, bioinformaticians, and computational systems biologists to advance our understanding of molecular interactions in cardiometabolic disease,” said Dr Zimmermann-Kogadeeva.

Tuberculosis is caused by infection with Mycobacterium tuberculosis: a bacterium that infects human lungs and other organs by using complex molecular machineries. These include protein complexes known as type VII secretion systems, which enable M. tuberculosis to release molecules into its host, which disarm and ultimately kill the infected human cell. Five such secretion systems, labelled ESX-1 to ESX-5, are found among M. tuberculosis and other closely related mycobacteria, many of which are pathogenic. Without them, the bacteria are unable to infect human cells.

The Wilmanns group at EMBL Hamburg has been using high-resolution structural biology to study mycobacterial proteins for the last two decades. The molecular understanding of the bacterial machinery used to infect cells resulted in collaborations with industry to develop new drugs against tuberculosis. In their most recent study, they determined the molecular structure of the secretion system ESX-5 to a high level of detail. They saw that the core of ESX-5 is built of 30 protein units, which form a dynamic membrane pore to allow secretion of proteins that enable the bacterium to survive and multiply inside human cells. Knowledge of the ESX-5 structure at high-resolution is essential to target specific sites with small-molecule drugs.

“Our new structure of the ESX-5 secretion complex provides deep insight into a major sluice gate that separates the inner of these bacteria from the outer host environment. Opening this gate allow the pathogen to spit out its deadly weapons to infect humans to develop tuberculosis. We can use this structure as a toolbox with literally thousands of potential drug targets. This will open an entirely new field of studies on tuberculosis,” says Matthias Wilmanns, who leads the study. Kate Beckham, who developed an innovative way to isolate ESX-5, adds: “The central pore we saw in ESX-5 could serve as a new drug target. Blocking it could prevent infection with pathogenic mycobacteria.”

The study could also help scientists to develop new vaccines for tuberculosis. The widely used Bacillus Calmette–Guérin (BCG) vaccine, which has its 100th anniversary this year, is based on a strain of mycobacterium that has lost its ability to cause disease because of a defect in the ESX-1 system. However, as BCG vaccination offers insufficient protection and is most effective in young infants only, so alternative vaccines are urgently needed. Due to its close structural and functional relation with ESX-1, targeting the ESX-5 secretion system might spur the development of new vaccines that could complement or replace those currently used.

Determining the molecular structure of ESX-5 was particularly challenging because of its large size and complexity. No single structural biology method can provide the full picture. In this case, the key to success was using integrative structural biology, in which data obtained using different methods –cryo-electron microscopy, X-ray crystallography, mass spectrometry and computational methods – were used jointly to create a coherent model.

“Eighteen months ago, solving this structure looked like mission: impossible,” says Matthias Wilmanns. “We managed to put the puzzle pieces together because each team member contributed unique expertise. To solve the complete structure, we collaborated with Jan Kosinski’s group at EMBL Hamburg and the Centre for Structural Systems Biology, which provided necessary expertise in integrative structural biology. We also received great help from our colleagues at EMBL Heidelberg, who performed cryo-electron microscopy experiments.”

This study illustrates some of EMBL’s approaches to life science research in its forthcoming scientific programme, Molecules to Ecosystems 2022–2026. As part of this programme, EMBL will take an interdisciplinary approach to understanding the molecular basis of life in the context of environmental changes. This will provide translational potential to support advances in human and planetary health.

EMBL’s approach, including this study, is aligned with the collaborative efforts of other research groups and institutions from Northern Germany working together at the Centre for Structural Systems Biology.

The ability of SARS-CoV-2 to infect cells depends on interactions between the viral spike protein and the human cell surface protein ACE2. To enable the virus to hook onto the cell surface, the spike protein binds ACE2 using three finger-like protrusions, called the receptor binding domains (RBDs). Blocking the RBDs therefore has the potential to stop the virus from entering human cells. This can be done using antibodies.

Nanobodies, small antibodies found in camels and llamas, are promising as tools against viruses due to their high stability and small size. Although obtaining them from animals is time consuming, technological advances now allow for rapid selection of synthetic nanobodies, called sybodies. A technology platform to select sybodies from large synthetic libraries was recently developed in the lab of Markus Seeger at the University of Zurich, and made available for this study.

In search of the best sybody against SARS-CoV-2

EMBL Hamburg’s Christian Löw group searched through the existing libraries to find sybodies that could block SARS-CoV-2 from infecting human cells. First, they used the viral spike protein’s RBDs as bait to select those sybodies that bind to them. Next, they tested the selected sybodies according to their stability, effectiveness, and the precision of binding. Among the best binders, one called sybody 23 turned out to be particularly effective in blocking the RBDs.

To learn exactly how sybody 23 interacts with the viral RBDs, researchers in the group of Dmitri Svergun at EMBL Hamburg analysed the binding of sybody 23 to the RBDs by small-angle X-ray scattering. In addition, Martin Hällberg at CSSB and Karolinska Institutet used cryo-EM to determine the structure of the full SARS-CoV-2 spike bound to sybody 23. The RBDs switch between two positions: in the ‘up’ position the RBDs poke out, ready to bind ACE2; in the ‘down’ position they are furled to hide from the human immune system. The molecular structures revealed that sybody 23 binds RBDs in both ‘up’ and ‘down’ positions, and blocks the areas where ACE2 would normally bind. This ability to block RBDs regardless of their position might explain why sybody 23 is so effective.

Finally, to test if sybody 23 can neutralise a virus, the group of Ben Murrell at Karolinska Institutet used a different virus, called a lentivirus, modified such that it carried SARS-CoV-2’s spike protein on its surface. They observed that sybody 23 successfully disabled the modified virus in vitro. Additional tests will be necessary to confirm whether this sybody could stop SARS-CoV-2 infection in the human body.

Scientific collaboration during lockdown

“The collaborative spirit has been enormous in these times, and everybody was motivated to contribute,” says Christian Löw, one of the lead scientists in the study. The researchers started the project as soon as they received approval from EMBL leadership to reopen their laboratories during the COVID-19 lockdown. They managed to select the candidate sybodies and perform the analyses in just a few weeks.

“Getting the results so quickly was only possible because the methodologies we used had already been established for other research projects unrelated to SARS-CoV-2. Developing these tools would have taken significantly more time and resources,” says Löw.

The results of this project hold out the promise of a potential way to treat COVID-19. In future work, the scientists will perform further analyses to confirm whether sybody 23 could be an effective COVID-19 treatment.

Links:

• Löw group
• EMBL Hamburg
• Karolinska Institutet
• Centre for Structural Systems Biology
• EMBL’s contribution to fighting the coronavirus pandemic
• Exploring synthetic antibodies to stop coronavirus

 Source Articles
Tânia F. et al. Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2. Nature Communications, published on 4 November 2020
DOI: 10.1038/s41467-020-19204

Dilated cardiomyopathy (DCM) is a leading cause of heart failure, affecting 1 in 250 people. The disease is characterised by an increase in size of the left ventricle of the heart. The stretched heart muscle is then unable to pump blood as effectively, which can lead to irregular heartbeat, heart valve problems, and ultimately heart failure. As the leading cause of heart failure, DCM is the most common reason for carrying out a heart transplant, which is only offered in end-stage heart failure when all other treatment options and lifestyle changes have failed. Despite years of work to improve patient survival after transplantation, the 10-year survival rate is still only 50%.

“Currently DCM is treated with medicines used for heart failure in general, their function being to lower strain on the heart. Patient health would be significantly improved with targeted treatment options prior to the need for a heart transplant,” explains Lars Steinmetz, from the European Molecular Biology Laboratory in Heidelberg and Stanford University. “We need therapeutic strategies that target the cause of the disease in a personalised medicine approach.” The new study by the Steinmetz group, in cooperation with Mark Mercola’s lab at Stanford University, provides fresh insight into this deadly disease, hinting at potential new treatment possibilities.

The researchers had the unique opportunity to study a single family with inherited DCM to understand the cause of their disease. Because it’s an inherited disease, studying the genome, or complete set of DNA and genes, can provide important information on the mutation causing the disease. By studying a family, the researchers were able to look for differences in the regions of the genome that carry instructions for making proteins, in family members who had died with a diagnosis of DCM, had been diagnosed with DCM, or were unaffected by DCM. This comparison allowed them to find a single mutation (P633L) in a gene coding for a protein called RBM20, which was disease causing. Although the mutation was previously unknown, changes in RBM20 are known to cause a severe form of inherited DCM that is often associated with early onset of end-stage heart failure.

“When we started the project, we wanted to identify the mutation causing the disease in this family,” explains Steinmetz, who is also the founder of the Steinmetz Cardiomyopathy Fund, by which most of the study was financed. Francesca Briganti, from the Mercola Lab, adds: “When we found the new mutation, we had to demonstrate that this is indeed the pathogenic mutation – there were already over 30 genes linked to the disease beforehand. We did this by showing that the mutation causes splicing and cellular contraction defects using in vitro cell models.”

The researchers used a combination of patient-derived cells and genome-edited cells. This process of genome editing involved making specific changes to the DNA of the cells, to introduce the RBM20 mutation (P633L) into patient-derived cells known as induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). This gave the researchers the chance to understand how the mutation was causing DCM and also to propose a potential treatment option. “We were lucky. We searched the database and found a fitting compound. Going from finding the disease gene to finding the potential solution overnight was easy with the open databases at EMBL’s European Bioinformatics Institute,” says Steinmetz.

The team identified a chemical called all-trans retinoic acid (ATRA) as a potential treatment of DCM. ATRA regulates RBM20, and can partially fix the defects in the altered cells. ATRA is a drug used for the treatment of acne and a type of leukaemia called acute promyelocytic leukaemia. In this case, Steinmetz reasoned that increasing expression of RBM20 might overcome the insufficient expression of this protein that is seen in patients who have one functioning and one mutated copy of the gene.

“This is a promising result in approaching RBM20-deficient DCM,” explains Steinmetz. “In addition, the general approach and the strategy we used in this study could work for a number of other dominant diseases!”

Links

• Steinmetz Group
• Mercola Lab at Stanford
• Stanford University
• Steinmetz Cardiomyopathy Fund

Source Article
Lars Steinmetz et al. iPSC modelling of RBM20-deficient DCM identifies upregulation of RBM20 as a therapeutic strategy. Cell, published 8 September 2020
DOI: 10.1016/j.celrep.2020.108117

The organisation of cells into specific compartments is critical for their function. For instance, by separating the nucleus from the cytoplasm, the nuclear envelope prevents premature translation of immature RNAs.

During mitosis, however, the nuclear envelope disassembles, allowing large cytoplasmic components such as ribosomes to mix with nuclear material. When the nuclear envelope reassembles following mitosis, these cytoplasmic components must once again be removed. “The nuclear envelope can contribute to this by actively importing or exporting substrates up to a certain size, but it was not clear what happens with very large cytoplasmic components”, says Mina Petrovic, from the Gerlich lab at IMBA – the Institute of Molecular Biotechnology – and joint first author of the study.

The research team from IMBA and EMBL have now shown that large components such as ribosomes are in fact removed from the forming nucleus before the nuclear envelope is assembled again. This exclusion process requires the protein Ki-67, which was the focus of an earlier publication in Nature by Sara Cuylen-Häring, group leader at EMBL Heidelberg and the other joint first author of this study.

In this older study it was discovered that Ki-67 was responsible for keeping chromosomes separated in early stages of mitosis by acting as a surfactant. “Remarkably, we have now found that it changes its properties at the end of mitosis and performs the opposite function, namely clustering of chromosomes,” said Cuylen-Häring. By coming together into a dense cluster at the end of cell division, chromosomes are able to exclude large cytoplasmic components before the nuclear envelope reforms.

“This collaborative work shows how a single protein can dynamically change the surface properties of chromosomes,” explains Alberto Hernandez Armendariz, PhD student in the Cuylen-Häring group. “Ultimately, this facilitates effective compartmentalisation of key processes within the cell.”

Links

• IMBA – Institute for Molecular Biotechnology
• Cuylen-Häring group

Source Articles
Daniel Gerlich et al. Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly. Nature, published on 02 September 2020. DOI: 10.1038/s41586-020-2672-3

Sara Cuylen-Häring et al. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes. Nature, published 29 June 2016. DOI: 10.1038/nature18610

About IMBA
IMBA – Institute of Molecular Biotechnology – is one of the leading biomedical research institutes in Europe focusing on cutting-edge stem cell technologies, functional genomics, and RNA biology. IMBA is located at the Vienna BioCenter, the vibrant cluster of universities, research institutes and biotech companies in Austria. IMBA is a subsidiary of the Austrian Academy of Sciences, the leading national sponsor of non-university academic research. The stem cell and organoid research at IMBA is being funded by the Austrian Federal Ministry of Science and the City of Vienna.