Best Poster Awards – Precision Health

140 researchers came together recently at the EMBL Advanced Training Centre in Heidelberg, Germany, for the EMBO Workshop: Precision Health: Molecular Basis, Technology and Digital Health (13 – 16 November 2019) to present and discuss the promises and challenges of precision health and the molecular insights necessary to enable a maintenance of wellness and prevention of disease.

Out of the posters presented, 4 were awarded a poster prize based on popular vote. Here we present the poster abstracts of four of the winners.

A computational modelling approach to characterizing postprandial glucose responses in individuals
Balazs Erdos from TiFN Wageningen and MaCSBio, Maastricht University, The Netherlands, PHOTO: Balazs Erdos

Balazs Erdos (1), (2)*, Bart van Sloun (1), (2), Shauna O’Donovan (2), Michiel Adriaens (2), Natal van Riel (3), Ellen Blaak (4), Ilja Arts (2)

The large variability in the dynamic properties of the postprandial glucose response curves in individuals suggest that it is not sufficient to use average values or single time point measures of postprandial glycemia in order to characterize individuals’ glycemic control. Instead, approaches that are capable of capturing the dynamic events are necessary. In this study, we develop personalized computational models based on ordinary differential equations, to describe the glucose and insulin dynamics of individuals in response to an oral glucose tolerance test. We observed that these personalized models are capable of capturing a wide range of glucose and insulin dynamics including normal, prediabetic and type 2 diabetic responses as well as responses from intermediate states.

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(1) TiFN, Wageningen, The Netherlands, (2) Maastricht Centre for Systems Biology (MaCSBio), Maastricht University, Maastricht, The Netherlands, (3) Dept. of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands, (4) Dept. of Human Biology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, The Netherlands

*E-mail: balazs.erdos@maastrichtuniversity.nl


Predict nephrotoxicity associated with cisplatin-based chemotherapy in testicular cancer patients

Sara Garcia (1), Jakob Lauritsen (2), Zeyu Zhang (3), Mikkel Bandak (2), Marlene Danner Dalgaard (1), Rikke Linnemann Nielsen (1), Gedske Daugaard (2), Ramneek Gupta (1)

In industrialized countries, testicular cancer (TC) is the most common solid tumor in men between 20 and 40 years old and besides being one of the most treatable types of cancer, the long-term side-effects of chemotherapy are worrisome, since they are largely irreversible. Their severity is normally related to the total amount of chemotherapy received, which makes that an important factor to a successful treatment. The standard treatment for TC is 3 cycles of cisplatin, etoposide and bleomycin (BEP), being that the number of cycles can vary between 4-5 or more if the prognosis of the patient is intermediate or poor. Some of the late side-effects include nephrotoxicity, which can be measured by the drop in glomerular filtration rate after the patient follows chemotherapy. Materials and Methods: Integrative machine learning models were built using a dataset of 400 Danish individuals in order to identify clinical and/or genomics features and classify patients at higher risk of developing nephrotoxicity given a treatment of BEP-cycles. Results: First, only clinical features, such as age at the time of treatment, dose of cisplatin, patient’s prognosis, and number of cycles, were considered, and relevant features were selected to use in the classifier (AUC 0.66, SD 0.02). The classifier was then optimized by adding genomics markers, which helped improving the prediction (AUC 0.75, SD 0.02). Conclusions: Therefore, it is proposed a machine learning algorithm which, by helping predicting nephrotoxicity in advance, can benefit to improve chemotherapy efficacy in TC patients. These data driven models can also be applicable to other cancers, such as ovarian, bladder, and lung cancer where more elderly patients are at risk of nephrotoxicity and identification upfront will have direct clinical implications.

Poster currently not available

(1) Technical University of Denmark, Denmark, (2) Copenhagen University Hospital, Denmark, (3) University of Chinese Academy of Sciences, China


Loss of N-glycanase 1 alters transcriptional and translational regulation
Petra Jakob from EMBL Heidelberg, Germany, PHOTO: Petra Jakob

Petra Jakob (1), William Mueller (1), Sandra Clauder-Münster (1), Han Sun (2), Sonja Ghidelli-Disse (3), Diana Ordonez (1), Markus Boesche (3), Markus Bantscheff (3), Paul Collier (1), Bettina Haase (1), Vladimir Benes (1), Malte Paulsen (1), Peter Sehr (1), Joe Lewis (1), Gerard Drewes (3), Lars Steinmetz (1)

N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

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(1) EMBL Heidelberg, Germany, (2) Stanford University, United States of America, (3) Cellzome, Germany


Data integration for prediction of weight loss in clinically controlled dietary trials

Rikke Linnemann Nielsen (1), Marianne Helenius (1), Sara Garcia (1), Henrik Munch Roager (2), Derya Aytan (3), Lea Benedicte Skov Hansen (1), Mads Vendelbo Lind (2), Josef Vogt (1), Marlene Danner Dalgaard (1), Martin I Bahl (3), Cecilia Bang Jensen (1), Rasa Muktupavela (1), Christina Warinner (4), Vincent Appel (5), Rikke Gøbel (5), Mette B Kristensen (2), Hanne Frøkjær (6), Morten H Sparholt (7), Anders F Christensen (7), Henrik Vestergaard (5), Torben Hansen (5), Karsten Kristiansen (6), Susanne Brix Pedersen (1), Thomas Nordahl Petersen (3), Lotte Lauritzen (2), Tine Rask Licht (3), Oluf Pedersen (5), Ramneek Gupta (1)

Diet is a key strategy in weight loss management. Advances in omics technologies research allow analyses of determinants of clinical interventions outcomes. We have previously reported diet-induced weight loss in non-diabetic middle-aged Danes in two clinically controlled dietary trials where the content of whole grain or gluten was changed. However, it remains elusive how predictable weight loss is at the individual level. We here classify weight loss responders and non-responders from the whole grain and gluten trials by integrating multi-omics data (host genetics, gut microbiome, urine metabolome) together with physiology and anthropometrics into random forest models. The most predictive models for weight loss included features of diet, gut microbial species and urine metabolites (ROC-AUC:0.84-0.88, model only with diet type ROC-AUC:0.62). Furthermore, we demonstrate that a model ensemble is robust to missing information of microbiome and metabolome profiles given features of physiology (including postprandial response), host genetics and transit-time (ROC-AUC:0.72).

Poster currently not available

(1) Technical University of Denmark, Denmark, (2) University of Copenhagen, National Food Institute, Technical University of Denmark, Denmark, (3) National Food Institute, Technical University of Denmark, Denmark, (4) Harvard University, United States of America, (5) The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark, (6) University of Copenhagen, Denmark, (7) Bispebjerg University Hospital, Denmark


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Best Poster Awards – Cancer Genomics

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

Jonas Demeulemeester is a postdoctoral researcher at the Francis Crick Insitute in UK. PHOTO: Jonas Demeulemeester

Authors: Jonas Demeulemeester (1), Stefan C. Dentro (2), Moritz Gerstung (2), Peter Van Loo (1)

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.

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(1) The Francis Crick Institute, United Kingdom, (2) EMBL-EBI, United Kingdom


The other award-winning poster was:

Understanding the early impact of activating PIK3CA mutation on cellular and genetic heterogeneity presented by Evelyn Lau, UCL Cancer Institute, United Kingdom


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Best Poster Awards – The Non-Coding Genome

Taking place for the third time,  the EMBO|EMBL Symposium: The Non-Coding Genome (16 – 19 October 2019) brought together 305 RNA experts to discuss the roles of non-coding RNAs in both prokaryotes and eukaryotes, gene regulation and function. 

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

Monah Abou Alezz is a Ph.D student in genetics, molecular and cellular biology at the University of Pavia, Italy. PHOTO: Monah Abou Alezz

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
DNA repair.

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MirGeneDB 2.0: The metazoan microRNA complement

Bastian Fromm is a Senior Researcher at Science for Life Laboratory, Stockholm University, Sweden. PHOTO: Bastian Fromm

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/.

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Fromm, B. et al. MirGeneDB 2.0: the metazoan microRNA complement. Nucleic Acids Research, gkz885, (2019), https://doi.org/10.1093/nar/gkz885

(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


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Best Poster Awards – Seeing is Believing

For the 5th time, the EMBL Advanced Training Centre played host to 466 researchers and imaging specialists at the EMBO|EMBL Symposium: Seeing is Believing – Imaging the Molecular Processes of Life (9 – 12 October 2019), where cutting-edge applications illustrated how imaging can answer biological questions and capture the dynamics of life. 

Out of the 248 posters presented, 2 stood out from the rest and were awarded a poster prize based on popular vote. Here we present the abstracts and posters of the winners.

CalQTrace: Simultaneous Calculation and Quantification of 100,000 immune activation Traces at single-cell resolution using CNN

Liliana Barbieri is a doctoral student at the Biomedical Imaging Doctoral Training Centre, University of Oxford, UK. PHOTO: Liliana Barbieri

Authors: Liliana Barbieri (1), Kseniya Korobchevskaya (2), Azeem Ahmad (3), Huw Colin-York (1), Aurelien Barbotin (4), Glykeria Karanika (1), Loic Peters (5), Isabela Pedroza-Pacheco (4), Angela Lee (1), Lena Cords (1), Anish Priyadarshi (3), Dominic Waithe (6), Jana Kohler (6), Christoffer Lagerholm (6), Balpreet Singh Ahluwalia (3), Marco Fritzsche (2)

Quantification of immune cell activation is essential to the understanding of their effector function. Tracing activation signatures like cellular calcium release and the expression of surface markers in response to activation signals allows the classification of the course of immune cell activation from early triggering events to late differentiation. However, robust quantitative platforms for such measurements represent a major challenge, restricting the analysis to small single-cell population or more recently to cell ensembles with high-dimensional parameter analysis tools. Here, we introduce a combination of a convolutional neural network-based CalQTrace (Calculation and Quantification of Trace) software, together with a Graphical User Interface, and an optical high-throughput light-sheet platform, allowing the simultaneous fully automated quantitation of immune cell activation traces of >100,000 live immune cells. CalQTrace enables user-independent statistically robust classification and quantification of multiple fluorescent activation markers including calcium, CD25+/- expression, and cell viability tracking single cells in space and time within a 5 mm x 5 mm large-field-of-view, opening-up unprecedented insights into physiological activation tracing in living immune cells.

(1) MRC Human Immunology Unit, University of Oxford, United Kingdom
(2) Kennedy Institute for Rheumatology, University of Oxford, United Kingdom
(3) The Arctic University of Norway, Norway
(4) University of Oxford, United Kingdom
(5) University College London, United Kingdom
(6) Weatherall Institute of Molecular Medicine, University of Oxford, United Kingdom

Poster currently not available


Bleaching-insensitive STED microscopy with exchangeable fluorescent probes

Mike Heilemann is a Principal Investigator at Johann Wolfgang Goethe-University, Frankfurt, Germany. PHOTO: Mike Heilemann

Authors: Christoph Spahn (1), Florian Hurter (1), Mathilda Glaesmann (1), Jonathan Grimm (2), Luke Lavis (2), Hans-Dieter Barth (1), Marko Lampe (3), Mike Heilemann (1)

Photobleaching affects image quality and resolution in fluorescence microscopy, and thus limits the extractable information. This is in particular relevant for super-resolution microscopy where typically high laser intensities are used. In order to minimize photobleaching, we repurposed the use of exchangeable fluorescent probes, as used in single-molecule localisation microscopy methods such as Point Accumulation for Imaging in Nanoscale Tomography (PAINT) [1], for STED microscopy. We demonstrate pseudo-permanent labeling of target structures and constant exchange of photobleached fluorophores. This concept allows for whole-cell, 3D, multi-color and live-cell STED microscopy [2]. Using transiently binding hydrophobic dyes and fluorophore-labeled major minor groove binders [3, 4], we visualised the nanostructure of chromatin, cell membranes and organelles in bacterial and mammalian cells in 3D. To expand the range of targets, we employed oligonucleotide-labeled antibodies that transiently bind fluorophore-labeled oligonucleotides, as used in single-molecule super-resolution imaging with DNA-PAINT [5], and demonstrate multi-color STED imaging.

References:
[1] Sharanov and Hochstrasser, PNAS 103 (50), 18911-18916 (2006)
[2] Spahn et al., Nano Letters 19 (1), 500-505 (2019)
[3] Lukinavičius et al., Nature Communications 6, 8497 (2015)
[4] Spahn et al., Scientific Reports 8, 14768 (2018)
[5] Schnitzbauer et al., Nature Protocols 12(6), 1198-1228 (2017)

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(1) Johann Wolfgang Goethe-University Frankfurt, Germany
(2) HHMI – Janelia Research Campus, United States of America
(3) EMBL Heidelberg, Germany


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Best Poster Awards – EMBO Workshop: Tools for Structural Biology of Membrane Proteins

183 researchers convened at the Centre for Structural Systems Biology (CSSB) in Hamburg, Germany, for the recent EMBO Workshop: Tools for Structural Biology of Membrane Proteins (7 – 9 October 2019) to present and discuss new technologies and approaches applied in studying membrane protein structure, dynamics and functions.

Out of the 82 posters presented, 6 were awarded a poster prize based on popular vote. Here we present the poster abstracts of four of the winners.

Structural insights into the role of the conserved ATPase component, EccC, in the mycobacterial T7SS

Katherine Beckham is a postdoctoral fellow in Matthias Wilmanns’ group at EMBL Hamburg. PHOTO: Katherine Beckham

Authors: Katherine Beckham (1), Luciano Ciccarelli (1), Mandy Rettel (2), Mikhail Savitski (2), Jan Kosinski (1), Annabel
Parret (1), Matthias Wilmanns (1)

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 [1]. 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 [2]. 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.

References:
[1] Houben, E. N. G., et al. Take five — Type VII secretion systems of Mycobacteria. Biochim. Biophys. Acta – Mol. Cell Res.1843, 1707–1716 (2014).
[2] 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).

(1) EMBL Hamburg, Germany, (2) EMBL Heidelberg, Germany

Poster currently not available


Dissection of protonation sites for antibacterial recognition and transport in QacA, a multidrug efflux transporter

Puja Majumder is a Ph.D student at the Indian Institute of Science. PHOTO: Puja Majumder

Authors: Puja Majumder (1), Shashank Khare (1), Arunabh Athreya (1), Nazia Hussain (1), Ashutosh Gulati (2), Aravind Penmatsa (1)

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.

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(1) Indian Institute of Science, India, (2) Stockholm University, Sweden


Biophysical analysis of circularized MSP nanodiscs for structural studies

Melina Daniilidis is a Ph.D student in Prof. Dr. Franz Hagn’s group at the Bavarian NMR Center (BNMRZ) of the Technical University of Munich. PHOTO: Melina Daniilidis

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

Dhenesh Puvanendran is a Ph.D student at the Institute of Physical and Chemical Biology in Paris, France. PHOTO: Dhenesh Puvanendran

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.

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The other award-winning posters are:

Novel antigen-binding chimeric proteins as tolls in crystallography and cryo-EM of membrane proteins presented by Thomasz Uchanski, Vrije Universiteit Brussel, Belgium

Cryo-Electron tomography of synaptic vesicle fusion junctions presented by Lucy Ginger, MRC Laboratory of Molecular Biology, United Kingdom


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