Wageningen University & Research, The Netherlands
Viruses, plasmids, and transposons are selfish exogenous DNA elements that rely on invading a host for their replication, evolution, and survival . For prokaryotes (bacteria and archaea), these DNA elements pose a metabolic burden and can drive cell death. Prokaryotes have evolved various prokaryotic immune systems  that interfere with exogenous DNA after it entered the cell, including Restriction-Modification systems, prokaryotic Argonaute systems, and CRISPR-Cas systems. To escape interference, viruses evolved anti-immunity mechanisms , thereby driving an extreme diversification of prokaryotic immune systems [1, 4]. As a consequence, prokaryotic immune systems have distinct structural features and rely on diverse mechanisms for interfering with invading DNA. Many systems are extremely capable of recognizing specific DNA sequences, which has facilitated their repurposing as molecular tools that can be used for various applications including molecular cloning, genome editing, regulation of gene expression, and biosensors. In my group, we combine bacterial genetics, biochemistry, and X-ray crystallography to answer questions about diverse prokaryotic immune systems: how did they evolve, what is their functional role, on which mechanisms do they rely, and can we repurpose them as molecular tools?
 Koonin et al. (2017) Annu Rev Microbiol.  Van Houte et al. (2016) Microbiol Mol Biol Rev.  Stanley SY, Maxwell KL (2018) Annu Rev Genet.  Samson JE et al. (2013) Nat Rev Microbiol.
Department of Pathology, University of Cambridge, United Kingdom
RNA viruses are very diverse, have high mutational rates, and employ an enormous variety of molecular strategies to transcribe and translate their genes. While human-infecting viruses have been well-characterized, such viruses make up only a small proportion of natural RNA virus variation. To better understand the evolution of RNA viruses, and the molecular mechanisms that they employ, better methods are needed to identify divergent viruses infecting diverse host organisms. In this talk, I will cover a Hidden Markov Model profiles-based search for viral RNA-dependent RNA polymerases (RdRps) in publicly available transcriptomic databases. The polymerase is the only protein which is common to all RNA viruses. Therefore, we can at least partly uncover global RNA virus diversity by identification and comparison of RdRp sequences . When very divergent viruses are identified, we can propose new virus families  and potentially make predictions about their gene expression mechanisms. I will discuss the identification and analysis of over 10 000 RdRps (half of which are novel) as well as examples of unusual virus sequences. For example, in one of these, the virus RdRp is split between two ORFs that are predicted to be fused via alternative splicing. This is the first such case of splicing in RNA viruses, where it affects the core RdRp.
 Origins and Evolution of the Global RNA Virome. Wolf et al., 2018.  Polycipiviridae: a proposed new family of polycistronic picorna-like RNA viruses. Olendraite et al., 2017.
(1) Chemistry Research Laboratory, University of Oxford, United Kingdom
Metallo-β-lactamase (MBL) superfamily is widely spread in nature and is predicted to consist of >70000 proteins. Members of the MBL superfamily share a distinct αββα fold and bind one or two metal ions in their active sites . The name-lending and best studied members of the superfamily hydrolyse β-lactam antibiotics and contribute to the antimicrobial resistance including resistance to the last-resort antibiotics carbapenems. Other MBL superfamily members include small molecule hydrolases, DNA repair and RNA processing nucleases, and oxidoreductases . In addition to learning more about the enzymes and their unique functions, better understanding of the differences between the members of the MBL superfamily could help prevent off-target inhibition of host proteins of the same fold when targeting bacterial MBLs. Kinetic and structural studies were performed on Escherichia coli glyoxalase II (GloB), the second enzyme of the glyoxalase pathway responsible for the detoxification of methylglyoxal, a cytotoxic side product of several metabolic processes , and a new crystal structure of two different crystal forms of GloB has been solved. Biochemical and biophysical characterisation of an oxygenase member of the MBL superfamily cytidine monophosphate N-acetylneuraminic acid hydroxylase (CMAH) were performed. Human CMAH is catalytically inactive due to a 92 bp deletion which occurred 3 million years ago rendering humans unable to synthesise our own N-glycolylneuraminic acid, a mutation which likely had implications in the evolution of our species . The inactive human CMAH was studied together with its active mouse homologue.
 Bebrone C. Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochem Pharmacol. 2007;74(12):1686-701.  Sousa Silva M, Gomes RA, Ferreira AE, Ponces Freire A, Cordeiro C. The glyoxalase pathway: the first hundred years... and beyond. Biochem J. 2013;453(1):1-15.  Chou H-H, Hayakawa T, Diaz S, Krings M, Indriati E, Leakey M, Paabo S, Satta Y, Takahata N, Varki A. Inactivation of CMP-N-acetylneuraminic acid hydroxylase occurred prior to brain expansion during human evolution. Proc Natl Acad Sci U S A. 2002;99(18):11736-41.
(1) University of York, York, United Kingdom (2) Vernalis RnD Ltd, Cambridge, United Kingdom
Structural biology methods have determined 3D structures of many proteins describing their mechanism of action and providing insights into their biological function. However, the paradigm of ‘structure equals function’ has been changing since the identification of intrinsically disordered proteins (IDP) and regions (IDRs) that do not have a defined structure but can specifically interact with their partner proteins. The IDPs may constitute over 30% of the eukaryotic proteome and have been identified to be involved in various protein-protein interactions (PPIs) with potential therapeutic applications , . However, the identification of IDP binders has been challenging. Fragment-based lead discovery (FBLD) offers numerous advantages over conventional compound screening techniques, including larger chemical space . Fragments are more likely to bind to protein, though with lower affinity. This requires robust and sensitive biophysical approaches to detect and characterize such binding events. Our study has been focusing on characterizing the interactions between the monomeric form of the aggregation-prone tau K18 and various literature-reported compounds using a variety of biophysical techniques, including NMR, SPR and MST . In addition to this, multiple fragment screening approaches have also been attempted, including covalent fragments. The results of this project will contribute to a better understanding of IDP behaviour and IDP:ligand interaction mechanisms.
 C. J. Oldfield and A. K. Dunker, “Intrinsically disordered proteins and intrinsically disordered protein regions.,” Annu. Rev. Biochem., vol. 83, no. 1, pp. 553–84, 2014.  P. E. Wright and H. J. Dyson, “Intrinsically disordered proteins in cellular signalling and regulation,” Nat. Rev. Mol. Cell Biol., vol. 16, no. 1, pp. 18–29, 2014.  D. E. Scott, A. G. Coyne, S. A. Hudson, and C. Abell, “Fragment based approaches in drug discovery and chemical biology,” Biochemistry, vol. 51, pp. 4990–5003, 2012.  M. Pickhardt et al., “Identification of Small Molecule Inhibitors of Tau Aggregation by Targeting Monomeric Tau As a Potential Therapeutic Approach for Tauopathies.,” Curr. Alzheimer Res., vol. 12, no. 9, pp. 814–28, 2015.
Interdisciplinary Institute for Neuroscience, University of Bordeaux / CNRS, France
Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. I will present our recent methodological efforts 1) to construct a super-resolution platform for correlative live single-molecule imaging and STED microscopy, 2) to visualize the structural basis of astrocytic Ca2+ signals at tripartite synapses, and 3) to reveal the extracellular space in living brain tissue. Current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. Here, I will present a new method that combines 3D-STED, and its related SUSHI variant, with two popular SMLM techniques, sptPALM and uPAINT, on a single microscope platform. It is designed for rapid switching and precise alignment of the imaging modalities, enabling live nanoscale correlative analyses of protein dynamics within their changeable morphological cellular environment. Using 3D-STED microscopy, we resolved the spongiform domain of astrocytes and revealed new aspects of its morphology in brain slices and in vivo. We observed a reticular meshwork of nodes and shafts that featured rings of re-connecting astrocytic processes. The majority of dendritic spines were in contact with nodes, correlating in size with them. FRAP experiments and Ca2+ imaging showed that individual nodes were capable of biochemical compartmentalization and hosted highly localized Ca2+ transients. Our study reveals the nanoscale anatomical organization of astrocytes, identifying nodes as the functional astrocytic component of excitatory tripartite synapses, which may provide the anatomical basis for synapse-specific communication between neurons and astrocytes. We combined 3D-STED microscopy and fluorescent labeling of the extracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living brain slices. SUSHI enables quantitative analysis of ECS structure and produces sharp negative images of all cellular structures, providing an unbiased view of unlabeled brain cells with respect to their complete anatomical context in a live tissue setting.
 Arizono et al. Structural basis of astrocytic Ca2+ signals at tripartite synapses. Neuron (in revision)  Inavalli et al. A super-resolution platform for correlative live single-molecule imaging and STED microscopy. Nature Methods (2019)  Tønnesen et al. Super-resolution imaging of the extracellular space in living brain tissue. Cell (2018)  Pfeiffer et al. Chronic STED imaging reveals high turnover of dendritic spines in the hippocampus in vivo. eLife (2018) Chéreau et al. Super-resolution imaging reveals activity-dependent plasticity of axon morphology linked to changes in action potential conduction velocity. PNAS (2017)
(1) Hopp Children’s Cancer Center Heidelberg (KiTZ), Heidelberg, (2) KiTZ Clinical Trial Unit (ZIPO), Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Heidelberg, (3) Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, (4) Division of Biostatistics, German Cancer Research Center (DKFZ), Heidelberg, (5) Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, (6) Department of Pediatric Oncology, Hematology and Clinical Immunology, Consortium for Translational Cancer Research (DKTK), Düsseldorf University Hospital, Düsseldorf, (7) Division of Proteomics of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, (8) Faculty of Biosciences, Heidelberg University
Medulloblastoma (MB) is one of the most common malignant brain tumor in children. Patients with MYC-amplified Group 3 MBs exhibit poor survival rates even after intensive therapy. Surviving patients often suffer from long-term sequelae. This calls for new therapeutic strategies, e.g. targeted therapy. We have previously shown that MYC-amplified MBs are sensitive to HDAC inhibition. We here investigate possible combination treatment with HDACi entinostat and several PLK1 inhibitors. The drug interaction effect was determined by combination index calculation based on cell metabolic activity. Results were validated assessing cell viability, cell cycle and caspase-3 activity. The gene expression profile was analyzed in after single or combination treatment. We demonstrate that the MYC target gene PLK1 is significantly downregulated upon entinostat treatment, indicating that inhibition of HDACs and PLK1 could have synergistic effects. MYC-amplified cell lines were more sensitive to single treatments than non-amplified cell lines. PLK1i and HDACi interacted synergistically in clinically relevant concentrations in MYC-amplified cell lines. In addition to significant downregulation of MYC target genes, we also observed the loss of viability, caspase-3 activity induction and cell cycle arrest after combinatorial treatment. Finally, we demonstrated that volasertib and drug combination reduced MYC levels. In vivo examination of the drug combination is currently in progress. Our data suggests that MYC-amplification might serve as a predictive marker for PLK1i treatment in MB. The combination of HDACi and PLKi could be a candidate therapy for future clinical trials for MYC-amplified group 3 MB, and possibly other tumors harboring MYC amplification.
Chromatin labelling and imaging research group, Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
The ideal fluorescent probe for bioimaging is bright, absorbs light at long wavelengths (> 550 nm) and can be flexibly implemented in living cells and in vivo. Typically, such probe consists of a fluorophore connected via a linker to a targeting moiety. The availability of targeting ligands is assured by a large number of studies aiming at the development of inhibitors for a wide range of biomolecules. However, the design of synthetic, highly biocompatible fluorophores has proven to be extremely difficult and is lagging behind. Recently, silicon-rhodamine was identified as a far red dye that can be specifically coupled to proteins, lipids and nucleic acids using different techniques. Importantly, its high permeability and fluorogenic character permit imaging of proteins in living cells and tissues, while its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. Further investigations resulted in identification of cell-permeable fluorophores spanning the whole visible spectrum [1-7].
 Lukinavičius G. et al. (2013) Nat Chem 5(2): 132-139.  Lukinavičius G. et al. (2014) Nat Methods 11(7):731-3.  Lukinavičius G. et al. (2015) Nat Commun 6:8497.  Lukinavičius G. et al. (2016) JACS, 138(30):9365-8.  Butkevich, AN. et al. (2017) JACS 139(36):12378-12381.  Lukinavičius, G. et al. (2018) Chem Sci 9(13):3324-34.  Bucevičius J. et al (2019) Chem. Sci.,10, 1962-1970.
(1) Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania, (2) Faculty of Medicine, Vilnius University, Vilnius, Lithuania, (3) Research Unit of Medical Imaging, Physics and Technology, Faculty of Medicine, University of Oulu, Oulu, Finland, (4) Centre for Sport, Exercise and Osteoarthritis Research Versus Arthritis, Queen’s Medical Centre, Nottingham, United Kingdom, 5 (5) Sheik Salem Bin Mahfouz Scientific Chair for Treatment of Osteoarthritis With Stem Cells, King Abdulaziz University, Jeddah, Saudi Arabia
Articular cartilage is an avascular connective tissue, composed primarily of chondrocytes surrounded by a specialized extracellular matrix (ECM). Chondrocytes are responsible for the production of ECM proteins, and their metabolism is strongly dependent on intracellular calcium homeostasis. Nifedipine is an L-type calcium channel (CC) inhibitor, which prevents high pressure in blood vessels. Although both hypertension and osteoarthritis are most prevalent in older population with overweight and metabolic disorders, data on the potential correlations of these two diseases, especially concerning the effects of drugs for their treatment are still lacking. Alterations in chondrocyte metabolic pathways, as well as mechanotransduction and functions might be the outcomes of the extensive use of inhibitors of CC for the treatment of hypertension. The aim of this study is to analyze changes in human mesenchymal stem cell (MSC) and chondrocyte energy metabolism in cells incubated with nifedipine. Mitochondrial respiration and glycolysis were investigated in MSCs and chondrocytes by measuring cell oxygen consumption and extracellular acidification rates, cultivating the cells with nifedipine (10 µM) for 24 hours, using Agilent Seahorse. Our results demonstrate that nifedipine regulates both cell type energetic metabolism, as it downregulated mitochondrial respiration and ATP production in MSCs and chondrocytes. Moreover, nifedipine enhanced glycolytic capacity in chondrocytes, but not in MSCs suggesting that chondrocytes switch their metabolic pathways towards glycolytic. We conclude that nifedipine affects cell metabolic pathways by altering intracellular calcium concentrations.
1. Akkiraju, H.; Nohe, A. Role of chondrocytes in cartilage formation, progression of osteoarthritis and cartilage regeneration. J. Dev. Biol. 2015, 3, 177–192. 2. Matta, C. Calcium signalling in chondrogenesis implications for cartilage repair. Front. Biosci. 2013, S5, 305–324. 3. Littler, W.A.; Stallard, T.J.; Watson, R.D.S.; McLeay, R.A.B. The effect of nifedipine on arterial pressure and reflex cardiac control. Postgrad. Med. J. 1983, 59, 109–113. 4. Mobasheri, A.; Matta, C.; Uzielienè, I.; Budd, E.; Martín-Vasallo, P.; Bernotiene, E. The chondrocyte channelome: A narrative review. Jt. Bone Spine 2018, 1–7. 5. Uzieliene Ilona, Bernotas Paulius, Mobasheri Ali, Bernotiene Eiva. The Role of Physical Stimuli on Calcium Channels in Chondrogenic Differentiation of Mesenchymal Stem Cells. Int. J. Mol. Sci. 2018, 19, 2998.1§
Imperial College London, United Kingdom
In 2003 a ground-breaking idea was developed by Prof. Austin Burt, who suggested that mosquitoes could be engineered to contain a "gene drive" able to spread itself through natural populations of the malaria mosquito for sustainable vector control . Unlike other genetic modifications, gene drives spread by biasing their own inheritance and can be used to suppress vector populations to levels that no longer sustain disease transmission. Importantly, they do affect other mosquito species that do not transmit human disease. Recent advances in genome engineering, such as the incredible CRISPR revolution, have made gene drives a reality. In 2015 we demonstrated the first successful spread of a gene drive  but we predicted, and soon encountered, evolved resistance that could reverse its spread . We now present a suite of strategies to predict and mitigate resistance, and demonstrate the first gene drive to successfully spread through, and eliminate, entire populations of caged mosquitoes . Incredibly, the population modification took just 7-11 generations (<10 months) and required only a single release of 25% gene drive mosquitoes. There are technical, social and political challenges ahead but gene drives may help in the final push towards the eradication of malaria and, perhaps, other vector-borne diseases.
 Burt (2003) Site-specific selfish genes as tools for the control and genetic engineering of natural populations. Proc Biol Sci. 270(1518): 921–928.  Hammond et al. (2016) A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nature biotechnology 34 (1), 78.  Hammond et al. (2017) The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito. PLoS genetics 13 (10), e1007039.  Kyrou & Hammond et al. (2018) A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nature biotechnology 36 (11), 1062
(1) European Molecular Biology Laboratory (EMBL), Germany (2) Heidelberg University, Germany
In order to maintain genome integrity and stability, eukaryotic cells have developed a number of sophisticated mechanisms termed as DNA damage response (DDR). Even though DDR is a well-studied process, most of the studies focus on following nuclear events, while the response and effects of DNA damage events on the cytoplasmic organelles remain poorly understood. Nevertheless, a small number of studies over the last few years shed some light on the cytoplasmic processes, suggesting that there are elaborate mechanisms at play in response to DNA damage, particularly at the Golgi level. In parallel, a screen carried out in our lab identified 15 DDR related proteins which are localised specifically at the Golgi and the nucleus. Further analysis of one of the hits Rad51c, for the first time, show an active role for the Golgi in DDR, in particular, homologous recombination (HR) DNA repair pathway. This led us to the question: Does the Golgi play an active role in other DDR pathways? In this study, we propose that the Golgi is important for the activation and regulation of various DDR pathways. To test this hypothesis, we started by probing the identified dual-localising proteins by applying DNA damaging agents, inducing various types of DNA lesions. These experiments revealed that several of our candidates responded to DNA damage through a shift in localisation either from the Golgi to the nucleus or from the nucleus to the Golgi. These preliminary results already suggest that the Golgi could play an active role in DDR regulation for other types of DNA lesions. As we further characterise these candidates, our ultimate goal is to elucidate the interplay between the Golgi complex and DNA damage response.
(1) National Cancer Institute, Vilnius (2) State Research Institute Centre for Innovative Medicine, Vilnius (3) Vilnius University Life Sciences Center, Vilnius
Doxorubicin (DOX) is one of anthracycline antibiotics, widely used for mono or combined treatment of various cancers, in particular breast, ovarian, prostate, brain, bladder and lung cancers. The drug is also included to the treatment regimens for acute lymphoblastic and acute myeloblastic leukemia and Hodgkin lymphoma . Previous studies showed that the treatment involving combination of DOX and IL-2 resulted in elimination the syngeneic EL4 lymphoma in C57BL/6 mice in 50-80% of cases . The eradication was related to T cell mediated specific anti-lymphoma response and specific immune memory. DOX may affect immune responses by reducing tumor burden and selectively modifying regulatory T cells and/or macrophage activity. Although the primary mechanism of DOX role in inhibition of both DNA and RNA synthesis by intercalating within DNA base pairs is well-known , the drug contribution to host immune response efficacy is not clear. The aim of this study was to investigate DOX uptake in EL4 lymphoma cells and in host lymphocytes in the peritoneal cavity of C57BL/6NCr mice in vivo as well as to analyze its role in immune response during tumor growth and chemotherapy. For the experiments C57BL/6NCr mice were inoculated intraperitoneally (IP) with 5∗10^4 EL4 lymphoma cells on Day 0 followed by single IV DOX injection on Day 5. Cellular DOX content was determined by both HPLC and Flow Cytometry (FCM) methods. FCM analysis of peritoneal exudate cells (PEC) from naïve C57BL/6NCr and EL4 lymphoma cells demonstrated differences in light scatter characteristics. Both specific cell subsets (lymphoma-associated lymphocytes or tumor cells) may be clearly distinguishable on scatter in subsequent analyses. Cellular composition analysis after EL4 implantation demonstrated the 8-fold increase, in absolute numbers, in accumulation of host lymphocytes in the peritoneal cavity of tumor bearing animals. This finding correlates with other studies which demonstrate the relation between tumor regression and accumulation of inflammatory cells. However, we demonstrated no drug-induced elevation of cells after Dox administration. EL4 lymphoma cells were shown to take up higher amounts of Dox as well as showed greater drug sensitivity compared to host lymphocytes. Since inflammatory cells are not affected negatively by DOX and remain functionally active during chemotherapy, they may play important role in antitumor response and may be effectively engaged in combination treatment with chemotherapeutic agents. We also applied both HPLC and FCM methods to measure Dox uptake in vivo and in vitro. The high correlation (r2=0.90) between results of these methods was noticed. FCM was shown to be useful for heterogenous cell populations where both inflammatory and neoplastic cells can be distinguished due to different light scatter characteristics. On the other hand, HPLC detects only one peak associated with only authentic Dox but not with metabolites. The combination of both FCM and HPLC may be successfully applied in experimental lymphoma model.
1. Tacar O, Sriamornsak P, Dass CR. Doxorubicin:an update of anticancer molecular action, toxicity and novel drug delivery systems. J Pharm Pharmacol. 2013; 65(2):157-70. 2. Ewens A1, Luo L, Berleth E, Alderfer J, Wollman R, Hafeez BB, Kanter P, Mihich E, Ehrke MJ. Doxorubicin plus interleukin-2 chemoimmunotherapy against breast cancer in mice. Cancer Res. 2006; 66(10):5419-26. 3. Yang F, Teves SS, Kemp CJ, Henikoff S. Doxorubicin, DNA torsion, and chromatin dynamics. Biochim Biophys Acta. 2014; 1845(1): 84–89.
University of Kent, UK Berkeley Lights, USA Fujifilm Diosynth, UK University of Kent, UK
Currently, most of the best selling drugs worldwide are biologics manufactured in mammalian cell systems . Recently, there has been a surge in next generation single cell technologies to dramatically empower industrial drug discovery and cell line development efforts . One such platform is the optofluidic Berkeley Lights Beacon  system which allows the culturing of cells in microfluidics pens with the ability to monitor their bioproduction characteristics over time. We report here the first independent academic effort to test this platform head-to-head against an established cell line development workflow to estimate what impact this technology can have in terms of development timelines, improving recombinant drug yields and expanding production capacity. We also consider the paradigm shift in regulating biologic manufacture resulting from ability of these high-throughput technologies to provide unprecedented amounts of bioproduction characteristic data at a single cell level. We propose that the observed levels of biological noise exhibited by genetically identical cells challenges existing dogma that monoclonality is an essential criterion for commercial manufacture process.
 Walsh, G. Biopharmaceutical benchmarks 2018. Nat. Biotechnol. 36, 1136–1145 (2018).  Coluccio, M. L. et al. Microfluidic platforms for cell cultures and investigations. Microelectron. Eng. 208, 14–28 (2019).  https://www.berkeleylights.com/products-services/beacon
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