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I am interested in cancer heterogeneity, why some cells respond to therapy while others do not, thus contributing to development of resistance and metastasis. In particular, I am intrigued about the complex effects of transcription factors, which are required for normal physiology of the mammary gland and are also implicated in tumorigenesis and development of resistance to therapy in breast cancer.
Why did you choose to become a scientist?
When I was young I had a variety of interests – psychology, physics, art, biology… However, I was intrigued by science and anything related to DNA and its regulation. Then I did my PhD at EMBL Heidelberg (Gene Expression Programme) and discovered the opportunities in research for identifying problems, looking for solutions and the thrill of finding some of the answers…and I was hooked!
Where do you see this field heading in the future?
Despite significant progress in cancer research and clinical advances, breast cancer still is the most commonly diagnosed cancer – one in eight women will develop this disease during their lifetime – and it claims the lives of more women than any other cancer, plus men can also get breast cancer. This highlights the unmet clinical need for improved strategies for prevention, early detection and more efficient and specific treatments in order to accelerate progress and help more patients survive the disease.
One of the features that characterises breast cancer is its heterogeneity, both among patients and within each patient tumor. This heterogeneity is found at molecular, phenotypic and functional levels, complicating diagnosis and challenging approaches to therapy. Currently, huge efforts are dedicated to understanding this heterogeneity at all levels, including at single-cell resolution, which is anticipated to open new possibilities for more efficient and specific anti-cancer therapies.
How has training influenced your career?
Doing my PhD at EMBL marked the way I envision science, and this vision was reinforced and developed further at UCSF. Science can – and SHOULD – be fun. Later on, funding struggles and the current publishing madness have somehow taken a toll on the fun element, so I just have to remind myself sometimes that science is still exciting!
Some of the techniques practiced at this course are specific for the mammary gland and thus it provides a solid base for researchers starting in this field. In addition, there is a significant emphasis on imaging and comparison of mouse and human studies, the two major systems for looking at normal physiology and cancer research that, when combined, offer great insights into this heterogeneous disease. In addition, having the opportunity to work alongside other trainees contributes to the establishment of a network that may be helpful in the future. Cancer is a very complex problem, and having collaborators with different views and expertise will be very useful in your career.
The 4th EMBL Conference: Cancer Genomics (4 – 6 November 2019) brought together over 240 scientists in the field of cancer research to present the latest findings in cancer functional genomics, systems biology, cancer immunogenomics and epigenomics, as well as their translation and clinical impact.
123 posters were presented at the two poster sessions, out of which two were selected as the winners by popular vote.
Infinite sites violations during tumour evolution reveal local mutational determinants
The infinite sites model of molecular evolution requires that every base in the genome is mutated at most once. It is a cornerstone of (tumour) phylogenetic analysis, and is often implied when calling, phasing and interpreting variants or studying the mutational landscape as a whole. It is unclear however, whether this assumption holds in practice for bulk tumour samples. Here we provide frameworks to model and detect infinite sites violations, identifying 24,459 in total, including 6 candidate biallelic driver events, in 700 bulk tumour samples (26.3%) from the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes project. Violations generally occur at mutational hotspots and their frequency and type can accurately be predicted from the overall mutation spectrum. In melanoma, their local sequence context evidences how not only ETS, but also NFAT-family transcription factor binding creates hotspots for UV-induced cyclobutane pyrimidine dimer formation. In colorectal adenocarcinoma, violations reveal hypermutable special cases of the trinucleotide mutational contexts identified in POLE-mutant tumours. Taken together, we reveal the infinite sites model breaks down at the bulk level for a considerable fraction of tumours. These results warrant a careful evaluation of current pipelines relying on the validity of the infinite sites assumption, especially when scaling up to larger sets of mutations and lineages in the future.
Meet Pavel Baranov, Professor of Biomolecular Informatics at the University College Cork, Ireland. Pavel’s research group focuses on the understanding of how proteins are synthesised and how their synthesis is regulated.
Why did you choose to become a scientist?
When I was a toddler, I wanted to be a firefighter. Within a couple of years, I decided that being an astronaut would be more fun. A few more years passed, and I began to dream of becoming a scientist. I guess at that point I stopped growing and started living my dream.
What is your research focus?
My research group studies RNA translation. Translation is at the core of biology. Cells spend most of their energy on protein synthesis and the ribosome is the most abundant molecular machine in almost all cells. Ribosomal RNAs and tRNAs are the most conserved molecules across all kingdoms of life, and it is now apparent that proteins evolved earlier than DNA. Life as we know it relies on two main type of molecules not found outside of living systems – nucleic acids and proteins. It is the process of translation that connects these two chemistries together. I could hardly think of a more fundamental, interesting and challenging cellular process than translation.
Where do you see this field heading in the future?
As translation brings two chemistries together it is far more complex than other molecular process such as transcription and replication. Because of its complexity and the lack of tools to study it, studying translation is very challenging. The tools are now being developed, e.g. variations of ribosome profiling techniques, real-time single molecule imaging, cryo-EM microscopy, etc. The main change that I foresee is that translation will draw the attention of many more biomedical researchers, for better or worse.
What is your number one tip for people looking for scientific training?
Independent practice is the key in my opinion. After taking a course you may get the impression that you can do something, but it could be a false impression – you don’t really know if you can unless you have done it.
If you weren’t a scientist, what would you be?
Science is not a job for me, it is a dream. If I were not able to make my living as a researcher, I would have to find something else to make earnings, but I would not give up on my scientific interests.
The invention of ribosome profiling is the most significant development in the field of protein synthesis since the deciphering of the ribosome 3D structure. Ribosomal profiling is a popular technique for measuring the rate of translation in addition to measuring RNA levels, but this was somewhat possible even before. The unique ability of ribosome profiling is the detection of which open reading frames are being translated in RNA. The application of ribosome profiling revealed that even in eukaryotes the same mRNA molecule is often used for making more than one polypeptide, and that our current knowledge of the human genome protein coding repertoire is still far from complete. In addition to detecting translated frames, ribosome profiling could be used to detect ribosome pauses. We recently learned that such pauses could be used to regulate gene expression and other biological processes. This course will provide trainees with everything what is needed for mastering this powerful technology, from hands-on experience in generating ribosome profiling data to bioinformatics analysis and the use of public data resources.
A total of 189 posters were presented, from which two were singled out as the winners by popular vote.
Characterization of the genomic and splicing features of long non-coding RNAs using bioinformatics approaches
Authors: Monah Abou Alezz, Ludovica Celli, Giulia Belotti, Silvia Bione, Institute of Molecular Genetics L. L Cavalli-Sforza – National Research Council, Italy
Recent developments in deep sequencing approaches have simulated the continuous discovery of a significantly large number of novel long non-coding RNA (lncRNA) genes loci in the genomes. Long non-coding RNAs are recognized as a new class of regulatory molecules despite very little is known about their functions in the cellular processes. Due to their overall low expression level and tissue-specificity, the identification and annotation of lncRNA genes still remains challenging. The characterization of lncRNAs’ features is crucial to understand and get functional insights on their mechanisms of action. We exploited recent annotations by the GENCODE compendium to characterize the genomic and splicing features of long non-coding genes, in comparison to protein-coding ones, in the human and mouse genome by using bioinformatics approaches. Our analysis highlighted differences between the two classes of genes in terms of gene architecture regarding exons and introns length, GC-content, and the combinatorial patterns of chromatin marks and states. Moreover, significant differences in the splice sites usage were observed between long non-coding and protein-coding genes. While the frequency of non-canonical GC-AG splice junctions represents about 0.8% of total splice sites in protein-coding genes, we identified a remarkable enrichment of the GC-AG splice sites in long non-coding genes, both in human (3.0%) and mouse (1.9%). In addition, we identified peculiar characteristics of the GC-AG introns in terms of donor and acceptor splice sites strength, poly-pyrimidine tract, intron length, and a positional bias of GC-AG junctions being enriched in the first intron. Genes containing at least one GC-AG intron were found conserved in many species across large evolutionary distances, more prone to alternative splicing and a functional analysis pointed toward their enrichment in specific biological processes such as
Authors: Bastian Fromm (1), Diana Domanska (2), Eirik Hoye (3), Vladimir Ovchinnikov (4), Wenjing Kang (5), Ernesto Aparicio-Puerta (6), Morten Johansen (7), Kjersti Flatmark (3), Anthony Mathelier (8), Hovig
Eivind (3), Michael Hackenberg (6), Marc Friedländer (5), Kevin Peterson (9)
Non-coding RNAs (ncRNA) have gained substantial attention due to their roles in human disorders and animal development. microRNAs (miRNAs) are unique within this class as they are the only ncRNAs with individual gene sequences conserved across the animal kingdom. Bona fide miRNAs can be clearly distinguished from the myriad small RNAs generated in cells by a set of unique criteria. Unfortunately, recognition and utilization of these clear and mechanistically well understood features is not a common practice. We addressed this by extensively expanding our curated miRNA gene database MirGeneDB to 45 organisms that represent the breadth of Metazoa. By consistently annotating and naming more than 11,000 miRNA genes in these organisms, we show that previous miRNA annotations contained not only many false positives, but surprisingly many false negatives as well. Indeed, curated miRNA complements of closely related organisms are very similar and can be used to reconstruct evolution of miRNA genes, families and biogenesis across more than 1 billion years of evolution. MirGeneDB represents a robust platform for providing deeper and more significant insights into the biology of miRNAs, possible sources of mis-regulation, and evolutionary mechanisms. MirGeneDB is publicly and freely available under http://mirgenedb.org/.
(1) Science for Life Laboratory, Sweden (2) Department of Informatics, University of Oslo, Oslo, Norway (3) Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway (4) School of Life Sciences, Faculty of Health and Life Sciences, University of Nottingham, United Kingdom (5) Stockholm University, SciLifeLab, Sweden (6) Department of Genetics, Faculty of Sciences, University of Granada, Granada, Spain (7) Institute for Medical Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway (8) Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo, Oslo, Norway (9) Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, United States of America
Mycobacteria have a unique membrane structure with a complex hydrophobic outer-membrane rich in mycolic acids. To transport substances across this impermeable barrier, mycobacteria rely on a highly specialised translocation machinery – the Type VII secretion system (T7SS). Pathogenic mycobacteria encode up to five distinct T7SSs ESX-1 to 5 . Our previous work characterised the structure of the of the inner-membrane complex of the ESX-5 T7SS from Mycobacterium xenopi using negative stain electron microscopy, revealing a hexameric 1.8 MDa complex comprising the four conserved core components: EccB5, EccC5, EccD5 and EccE5 . The large cytosolic domain of EccC5, an FtsK/SpoE-like ATPase, is absent in our current EM map due to its conformational flexibility, which may be required to accommodate a range of protein substrates. Our current work aims to understand the role of EccC5 in secretion. In isolation this component can oligomerise into a hexameric ring-like conformation, as observed for other ATPases in this family. In addition, chemical cross-linking of the ESX-5 complex coupled with mass spectrometry (XL-MS) supports the oligomerisation of EccC5 in the secretion complex, suggesting that it may form a channel or ‘translocation tunnel’. Thus, we propose that EccC5 may exist in two conformational states: an extended, flexible monomeric state and a more compact hexameric state. Using an integrative structural biology approach, we are combining structures of isolated proteins derived from X-ray crystallography and electron microscopy studies with XL-MS data. Together these data aim to further elucidate the secretion pathway across the mycobacterial cell envelope.
 Houben, E. N. G., et al. Take five — Type VII secretion systems of Mycobacteria. Biochim. Biophys. Acta – Mol. Cell Res.1843, 1707–1716 (2014).
 Beckham, K. S. H. et al. Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis. Nat. Microbiol.2, 17047 (2017).
Emergence of multidrug-resistance poses serious threat to the society. One of the effective way by which bacteria gain drug resistance is through active efflux of antibiotics and other antibacterial compounds using multidrug efflux transporters. Among the battery of efflux pumps present in pathogenic bacteria, our work is focused on QacA, a drug-proton anitiporter (DHA) with 14-transmembrane helices that provide resistance to methicillin resistant Staphylococcus aureus (MRSA) strain, with homologs present in other pathogenic organisms. QacA is a highly promiscuous transporter, capable of effluxing diverse array of monovalent and divalent cationic antibacterial compounds and dyes. This study using a homology model, dissects the role of six protonatable residues present in the transport vestibule of QacA. Systematic mutagenesis resulted in identification of D34 (TM1) and E407 (TM13) as crucial residues and D323 (TM10) and D411 (TM13) as conditional residues needed for transport process of QacA. Whole cells, inside-out vesicles, substrate-induced proton release and microscale thermophoresis based assays were used to investigate the transport and binding properties of the transporter and its mutants. The activity of purified protein was checked with reconstituted QacA in a proteoliposome using substrate-induced proton transport assay. We identify two sites, D34 and D411 playing vital role in recognition of most of the substrates tested while E407 facilitates substrate efflux as a protonation site. It was also observed that E407 has an additional role as a recognition site for the transport of dequalinium, a divalent quaternary ammonium compound. These observations rationalize the promiscuity at the residue level of QacA for diverse substrates. The study identifies the role of acidic residues in QacA with implications for substrate recognition, promiscuity and processive transport in multidrug efflux transporters, related to QacA.
(1) Indian Institute of Science, India, (2) Stockholm University, Sweden
Biophysical analysis of circularized MSP nanodiscs for structural studies
Authors: Melina Daniilidis (1), Ralf Stehle (1), Franz Hagn (1,2)
Structure and dynamics of membrane proteins are crucial aspects for understanding functional properties of this protein class. Unfortunately, stabilizing them in their isolated form is still difficult. By incorporating membrane proteins into nanodiscs, they can be studied in a native-like
environment using biochemical and structural methods. However, thermal and long-term stability of small nanodiscs limit these studies and make it difficult to carry out nuclear magnetic resonance spectroscopy (NMR) measurements at elevated temperatures. Circularized membrane scaffold proteins (MSPs) produced via split-inteins have been shown to be more stable and homogenous than their linear counterparts. However, their biophysical properties, as well as suitability for membrane protein insertion and structural studies have not yet been assessed in a systematic manner. Thus, we examined circular and linear nanodiscs of varying size using several biophysical methods. An important issue for NMR structural studies is that the size and shape of circular nanodiscs do not expand above the phase transition temperature, increasing their homogeneity and reducing their size as compared to linear nanodiscs at high temperatures. 1H,15N-TROSY experiments could demonstrate that circular MSP1D1 nanodiscs with incorporated VDAC-1 are stable at higher temperatures, making it possible to obtain high-resolution NMR spectra of superior quality. Furthermore, NMR relaxation experiments were carried out to compare rotational correlation times of VDAC-1 in circular and linear nanodiscs, respectively. Despite the higher molecular weight, the circular nanodiscs showed lower rotational correlation times, which corroborated the biophysical results on the temperature-dependecy of the nanodisc diameter and homogeneity. The presented data demonstrate that these very stable circularized MSPs are well applicable to the study of membrane proteins in a lipid environment by NMR, but also other structural methods like electron microscopy.
(1) Technical University of Munich, Germany, (2) Helmholtz Zentrum München, Germany
Poster currently not available
Reconstitution of the activity of RND efflux pumps into proteoliposomes
Authors: Dhenesh Puvanendran, Quentin Cece, Martin Picard, IBPC, France
Efflux pumps are the major systems in bacterial resistance against antibiotics. They are classified by the energy needed to be active (ATP hydrolysis or ion counter-transport). Efflux pumps from the RND (Resistance, Nodulation, and cell Division) family use a proton gradient to be active and are composed of three proteins: a membrane fusion protein (MFP) and a transporter (RND) in the inner membrane, and an Outer Membrane Factor (OMF) localized in the outer membrane. We focus on the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa.The overall goal of my research is to measure in vitro the velocity of transport by efflux pumps. To that end, we reconstitute MexA and MexB as one population of proteoliposome, and OprM as another population of proteoliposome. The whole tripartite pump forms upon association of the respective populations of liposomes. The proof of concept of this method has already been described, leading to a qualitative monitoring of transport. We now work at defining a reconstitution procedure amenable to now quantify the rate of transport. To do so we take extreme care to precisely determine the efficiency of protein reconstitution and the type of lipids component used to perform liposomes. I will present the roadmap towards the rational, step-by-step, reconstitution of the MexA-MexB-OprM efflux pump as well as the methodologies that are undertaken to measure the velocity of transport, and possible perspectives regarding the screening of efflux pump inhibitors.