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Synthetic glycobiology

Scientific meeting

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Starts:

October
082018

09:00

Ends:

October
092018

17:00

Location

Kavli Royal Society Centre, Chicheley Hall, Newport Pagnell, Buckinghamshire, MK16 9JJ

Overview

Theo Murphy international scientific meeting organised by Professor Bruce Turnbull, Dr Anne Imberty and Professor Ola Blixt.

Lectin from Ralstonia solanacearum with carbohydrate ligands. Credit: Serge Pérez and Anne Imberty, graphics by SweetUnityMol (Pérez et al., Glycobiology 2015)

Cell surface carbohydrates mediate diverse biological processes including fertilisation, inflammation and invasion by pathogens. Our understanding of glycobiology has advanced remarkably over recent years, raising the prospect of applying the methods of synthetic biology to reengineer these systems for new and useful purposes. This meeting will bring together chemists, biologists and physicists to define the emerging field of synthetic glycobiology.

The schedule of talks and speaker abstracts and biographies are available below. Recorded audio of the presentations is also available below. Meeting papers will be published in a future issue of Interface Focus.

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Schedule of talks

08 October

Session 1 09:00-12:00

Synthetic glycobiology building blocks from proteins to protocells

4 talks Show detail Hide detail

Chairs

Professor Bruce Turnbull, University of Leeds, UK

09:10-09:35 Lectin engineering: possibilities and perspectives

Professor Jun Hirabayashi, National Institute of Advanced Industrial Science and Technology, Japan

Abstract

Lectins are a wide group of sugar-binding proteins occurring in all kinds of organisms including animals, plants, bacteria, fungi and even viruses. According to a recent report, the number of lectin scaffolds (~Pfam), of which 3D-structures are known and sugar-binding functions have been confirmed in literature, exceeds 50, which is far beyond our image in the 20th century. This fact suggests that new lectins will be discovered either by a conventional screening approach or just by chance like in the case of POMGnT1 stem region. It is also expectable that new lectin domains are generated in the future of evolution, although such an attempt has never been done at an experimental level. Based on the current states of the art, various ways of lectin engineering are available, by which lectin specificity and/or stability can be improved with the known lectin scaffold. However, the above observation implies that any other protein scaffold, which has never been described as lectin, is entitled to acquire a sugar-binding function. In this presentation, possible approaches and new items to create sugar-binding properties of synthetic peptides will be described.

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09:35-09:45 Discussion

09:45-10:10 Computer-aided engineering of reprogrammed carbohydrate-active enzymes for biotechnological applications

Dr Isabelle André, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, France

Abstract

Combined with chemical synthesis, the use of biocatalysts holds great potential to open the way to molecular diversity. Nonetheless, the lack of appropriate enzymatic tools with requisite properties has hampered extensive exploration of chemo-enzymatic routes to complex carbohydrates. To circumvent this limitation, protein engineering has proven to be very efficient to tailor enzymes with novel substrate specificities. However, the outcome of protein engineering strategies strongly depends on our understanding of enzyme catalysis and our comprehension of the inter-relationships between protein structure, activity and dynamics. Herein, Dr André reports the latest work of her laboratory in the development of 'programmed' chemo-enzymatic pathways that take advantage of both knowledge-based and computer-aided enzyme engineering to produce complex microbial cell-surface oligosaccharides entering in the composition of multivalent vaccines to prevent shigellosis.

This lecture will cover and discuss recent developments of Dr André's laboratory.

This work was partially funded by the French National Research Agency (Project GLUCODESIGN ANR-08-PCVI-0002-02; Project CARBUNIVAX ANR-15-CE07-0019-01).

Show speakers

10:10-10:20 Discussion

10:20-10:50 Coffee

10:50-11:15 Non-canonical amino acids as building blocks for lectins

Dr Birgit Wiltschi, Austrian Centre of Industrial Biotechnology, Austria

Abstract

Lectins are carbohydrate binding proteins with a high specificity for their target ligands. They play diverse roles in cellular recognition and signalling processes, as well as in infections and cancer metastasis. Due to their high specificity, lectins find application in biotechnology and medicine for eg blood group typing, purification of glycoproteins or -lipids and as markers, that target cancer cells. In some applications, lectins must be immobilised on a solid support for purification processes or they have to be conjugated with other molecules. However, traditional conjugation reactions are unspecific and in most cases the site of conjugation cannot be pre-defined. Therefore, Dr Wiltschi and her group devised lectins containing non-canonical amino acids with bioorthogonal reactive handles, with which these can be conjugated with other molecules in a pre-defined manner. As a proof of principle, the group conjugated these lectins with small molecules and other lectins. The conjugate lectins might be useful for any process, where lectins shall be conjugated with another module in a pre-defined and site-specific manner.

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11:15-11:25 Discussion

11:25-11:50 Lectin-mediated protocell crosslinking and fusion

Professor Winfried Römer, University of Freiburg, Germany

Abstract

Synthetic membrane systems are extremely useful for the better understanding of complex cellular structures and processes. The engineering of proto-tissues from proto-cells through lectin-glycan interactions represents an important step towards synthetic minimal tissues.

As a first step, Professor Römer's group succeeded in integrating natural and synthetic glyco-modules into giant unilamellar vesicles, which then were specifically recognized by lectins. In a second step, multivalent lectins with opposing carbohydrate binding sites triggered the crosslinking of glycan-functionalised vesicles. The crosslinking process drives the progression from contact puncta into elongated proto-cellular junctions, which form the vesicles into polygonal clusters resembling tissues. Due to their carbohydrate specificity, different lectins can be engaged in parallel with both natural and synthetic glyco-conjugates to generate complex interfaces with distinct lectin domains. In addition, the formation of proto-cellular junctions can be combined with adhesion to a functionalised support by other ligand-receptor interactions to render increased stability against fluid flow.

The group also assembled proto-cells to proto-tissues by employing a chimeric, bispecific lectin, with two rationally oriented and distinct recognition surfaces (from A Imberty). This lectin, coined Janus lectin in allusion to the two-faced roman god, is able to bind independently to both fucosylated and sialylated glyco-conjugates.
Moreover, the group could demonstrate that a synthetic lectin complex (from B Turnbull) consisting of 2-3 cholera toxin B-subunits induces hemifusion and fusion events, and also leakage or rupture of vesicles.

Show speakers

11:50-12:00 Discussion

12:00-14:10 Lunch, followed by poster session

Session 2 14:10-18:00

Engineering novel polysaccharide biomaterials

4 talks Show detail Hide detail

Chairs

Professor Ola Blixt, University of Copenhagen, Denmark

14:10-14:35 Nanoglycobiology based on the S-layer system

Professor Christina Schäffer, Universität für Bodenkultur Wien, Austria

Abstract

Molecular self-assembly systems that exploit the manufacturing precision of biological systems are valuable building blocks in synthetic glycobiology. Two-dimensional crystalline-cell surface (S-) layers have evolved in bacteria as a stable cell surface display system for protein epitopes and attached glycans. S-layers are formed by self-assembly of constituting monomers and can be utilised as nanometer-scaled patterning elements in bottom-up approaches.

By combining S-layer-inherent features with the Campylobacter jejuni glycosylation machinery and E. coli O-antigen biosynthesis, respectively, the group created in a proof-of-principle study, novel biocomposites with regular display of non-native carbohydrates. The glyco-engineered S-layer protein self-assembly system is an attractive approach for periodic and controllable display of glycans and offers a valuable alternative to chemical coupling of glycans. The incorporation of (re)engineered carbohydrates into the S-layer system provides a platform for addressing various questions in the fields of basic and applied research, relating to the areas of biomimetics, drug targeting, vaccine design, or diagnostics.

In nanoglycobiology approaches, the group is taking advantage of carbohydrate-active enzymes of bacterial origin, which have unsurpassed diversity in substrate and linkage specificities. This offers new possibilities for engineering of synthetic glycoconjugates, such as equipping these with sialic acid mimics.

Key to exploiting the synthetic biology potential of S-layers is the understanding of their three-dimensional structure, and, for cellular approaches, also the structural basis of cell wall anchoring. The group has recently obtained first molecular insights into how the S-layer monomers interact with their cell wall glycopolymer ligand, thereby unravelling a new building block for synthetic glycobiology approaches.

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14:35-14:45 Discussion

14:45-15:10 Sugary coats: biosyntheses of cell surface complex carbohydrates

Professor Jochen Zimmer, University of Virginia School of Medicine, USA

Abstract

Complex carbohydrates are frequently deposited on the cell surface to form cell walls, capsules, or 3-dimensional biofilm matrices. Often, the high molecular weight polymers are synthesised intracellularly and transported across at least one biological membrane by dedicated translocation machineries. Professor Zimmer's group is interested in how polysaccharides are assembled and deposited on the cell surface. In particular, cellulose is a major biofilm component of many Enterobacteriaceae and secreted during its synthesis by the membrane-embedded cellulose synthase, a processive glycosyltransferase. Using the tools of structural and molecular biology, the group delineated the mechanism by which the enzyme elongates a cellulose polymer and translocates it across the membrane through a pore formed by its own transmembrane region. The group is also interested in O antigens, which constitute the variable region of lipopolysaccharides in the outer membrane of Gram-negative bacteria. The polymers are synthesised inside the cell on a lipid anchor and transported to the periplasm by the WzmWzt ABC transporter. Crystal structures of the transporter in nucleotide-free and ATP-bound states suggest a processive translocation mechanism through a channel formed by the transporter. Molecular dynamics simulations suggest that membrane lipids are a fundamental part of the transporter’s function by sealing its channel in a resting state.

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15:10-15:20 Discussion

15:20-15:50 Tea

15:50-16:15 Getting a grip on glycans in guts and roots with chemical probes

Dr Tom Wennekes, Utrecht University, The Netherlands

Abstract

The complex layer of glycans called the glycocalyx that covers almost all cells on earth plays an essential role in many inter- and intracellular processes. This layer is also the primary biomolecular boundary encountered by symbiotic and pathogenic microbes when residing in or on their host or trying to infect it. Glycans thus dominate the microbiota-host interface and they are thus ideally positioned to modulate these interactions between the host and its microbiota at the molecular-level. Both plants and humans have a strikingly similar interaction with bacteria when looking at plant roots and the human gut that in both cases is crucial for host health. The aim of Dr Wennekes' research group is to develop smart glycan-based chemical probes, arrays and glycotherapeutic leads to study and perturb the activity of specific glycan subtypes and associated glycowriter (glycosyltransferases), reader (glycan-binding proteins) and eraser (glycosidases) proteins in healthy and pathological microbial host-guest interactions.

In this talk Dr Wennekes will discuss three research projects in his group where they investigate glycan readers and writers that are directly or indirectly involved in human-bacteria and plant-bacteria interactions. 1) Towards a glycoarray chip for the rapid diagnosis of Campylobacter jejuni induced cross-reactive antibodies in Guillain-Barré syndrome patient subtypes. 2) Capture and growth of (probiotic) gut microbes on glycan and antibody functionalised porous aluminum oxide microarray chips. 3) Metabolic labeling of glycans in Arabidopsis thaliana.

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16:15-16:25 Discussion

16:25-16:50 Bespoke glycan-presenting surfaces for molecular, supramolecular and cellular interaction studies

Dr Ralf Richter, University of Leeds, UK

Abstract

Polysaccharides of the glycosaminoglycan (GAGs) family are ubiquitously present at the cell surface and in extracellular matrix, and crucial for matrix assembly, cell-cell and cell-matrix communication. In all these contexts, the supramolecular presentation of GAG chains, on their own or along with other matrix components, is functionally important. Controlling and characterising the supramolecular organization of GAGs, however, poses significant challenges in vivo and in vitro. In this talk Dr Richter shall present methods developed in the lab to create biomimetic surfaces that display GAGs in well-defined yet tunable organizations, and illustrate how these surfaces can be used to provide new insights into fundamental biological questions. Examples that will be covered include the ‘super’selective discrimination of cells by the density of their surface receptors, the formation of chemokine gradients in the context of directed cell migration, and the selective adhesion of circulating cells to the inner wall of blood vessels.

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16:50-17:00 Discussion

17:00-18:00 Poster session

09 October

Session 3 09:00-11:50

Remodelling glycosylation from model systems to living cells

4 talks Show detail Hide detail

Chairs

Dr Anne Imberty, Centre National de la Recherche Scientifique, France

09:00-09:25 Glycan antigen decorated Giant Unilamellar Vesicles and live cells reveals distinct interactions with pathogens

Professor Ola Blixt, University of Copenhagen, Denmark

Abstract

Cellular membranes comprise the physical barrier of a cell towards the environment performing a variety of functions, including communication with other cells or pathogens. A large portion of the cell surface constitutes of complex glycans, many with specific adhesion properties. To elucidate such interactions various assay techniques are utilised, a majority based on flat rigid surfaces such as ELISA and microarray glass slides. Spherical cell-size Giant Unilamellar Vesicles (GUVs) are increasingly used as cell models to study membrane interactions. The group has developed a fully synthetic method to produce fluorescently labelled cholesterylated glycopeptides for efficient incorporation into GUV membranes, forming an artificial glycocalyx. With this system the group has begun evaluating membrane receptor interactions with pathogens such as the Malaria infected red blood cells and intact viruses.

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09:25-09:35 Discussion

09:35-10:00 A glycosylation programme activated via membrane trafficking controls tissue invasion and remodelling

Dr Frederic Bard, IMCB, A*Star, Singapore

Abstract

GalNac type O-glycosylation, contrary to N-glycosylation, is typically started in the Golgi apparatus. The polypeptide N-acetyl-Galactosamyl transferases (GALNTs) are initiating the pathway by generating the Tn glycan. GALNTs act on thousands of sites in cell surface and secreted proteins. GALNTs can be relocated from Golgi to the ER upon Src activation, a process called GALA. Dr Bard finds that GALA stimulates the glycosylation of about 20% of detectable glycosites. Roughly 200 proteins and 800 glycosites are strongly affected. Many of these proteins have been implicated in tumorigenesis. One of them is the metalloprotease MMP14, whose glycosylation strongly increases activity. GALA also affects the ER resident protein Calnexin, which is found to also be required for ECM degradation. In a mouse model of liver tumour, stimulating GALA leads to a strong acceleration of tumour progression, while inhibiting it blocks it. In sum, it is proposed that GALA is a glycosylation program that controls matrix degradation and tissue invasion and is required for tissue remodelling during tumour growth.

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10:00-10:10 Discussion

10:10-10:40 Coffee

10:40-11:05 Chemical tools for engineering glycan interactions at the cell-matrix interface

Professor Kamil Godula, University of California, San Diego, USA

Abstract

Glycans (also known as carbohydrates, saccharides or, simply, sugars) are among the most intriguing carriers of biological information in living systems. The structures of glycans not only convey the cells’ physiological state, but also regulate cellular communication and responses by engaging receptors on neighbouring cells and in the extracellular matrix. Despite their structural complexity, individual glycans rarely engage their protein partners with high affinity. Yet, glycans modulate biological processes with exquisite selectivity and specificity. To correctly evaluate glycan interactions and their biological consequences, one needs to look beyond individual glycan structures and consider the entirety of the cell-surface landscape. There, glycans are presented on protein scaffolds, or are linked directly to membrane lipids, forming a complex, hierarchically organised network with specialised functions, called the glycocalyx. Professor Godula's research program focuses on the development of nanoscale glycomaterials, which can mimic the various components of the glycocalyx, together with chemical methods for cell surface engineering to reveal how the presentation of glycans within the glycocalyx can influence their biological functions. In this presentation, Professor Godula will describe the recent efforts in this area, placing emphasis on the applications of glycomaterials to provide new insights into the mechanisms through which glycans mediate cellular differentiation and host-pathogen interactions.

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11:05-11:15 Discussion

11:15-11:40 Revealing glycan-mediated interactions through photocrosslinking

Professor Jennifer Kohler, University of Texan Southwestern Medical Center, USA

Abstract

Glycan-mediated interactions play critical roles in diverse biology processes. However, glycan-mediated interactions are often low-affinity and transient, impeding their characterisation by traditional biochemistry methods. Using synthetic sugar analogs, Professor Kohler's group has been able to incorporate a photoactivatable function group into glycoconjugates in living cells. Professor Kohler will present data demonstrating that both photocrosslinking GlcNAc and sialic acid can be incorporated into cell surface glycoconjugates in place of natural sugars. Cells metabolically labelled with photocrosslinking sugars can be subjected to UV irradiation, resulting in covalently crosslinking between glycoconjugates and neighbouring molecules. These crosslinked complexes can be characterised by standard biochemistry methods, including immunoblot and mass spectrometry-based proteomics analysis. Professor Kohler will also describe how the group used photocrosslinking sugar technology to determine that cholera toxin subunit B (CTB) recognises fucosylated glycoproteins on the surface of human colonic epithelial cells. The results of these experiments have led to the discovery that fucosylated molecules function in host cell intoxication, and to new fucose-based strategies that can competitively inhibit CTB binding to host cells and potentially block host cell intoxication. Metabolically incorporated photocrosslinking sugars can potentially be used to capture and define many molecular recognition events in which glycans play a critical role.

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11:40-11:50 Discussion

11:50-13:10 Lunch

Session 4 13:10-17:00

Synthetic glycobiology for diagnostic and therapeutic applications

5 talks Show detail Hide detail

Chairs

Professor Bruce Turnbull, University of Leeds, UK

13:10-13:35 Identification of glycosylated biomarkers in cancer and new approaches for enhancing responsiveness to anti-cancer treatments

Dr Miriam Dwek, University of Westminster, UK

Abstract

Dr Dwek will describe studies undertaken using lectins to identify proteins showing alterations in O-linked glycosylation in cancer. The strategies taken to identify proteins with altered glycosylation in cancer in both colorectal and breast cancer validated using tissue samples from patients and highlighting commonalities of glycosylation changes in both tumour types will be described. Serum tests for cancer are often based on glycoproteins but in the majority of tests the protein levels are monitored whilst glycosylation is ignored. A glycoproteomic approach was used to identify serum proteins with altered glycosylation in metastatic breast cancer. This was accompanied by development of a rapid screening method with a lectin-based recognition system. In this manner, serum biomarkers were identified – this will be described with respect to VE-cadherin – a biomarker for metastatic oestrogen receptor positive breast cancer. Glycosidases have been identified as mediators of cancer cell invasion and glycosidase inhibitors have emerged as potential compounds for prevention of cancer cell invasion. The pivotal role of the glycocalyx as a barrier, preventing cancer drug interaction at the cell surface, has led to development of a novel biosensor system for studying cell-protein interactions; this will be described alongside recent approaches for enhancing interaction between the drug trastuzumab (Herceptin) and HER2 in breast cancer. The importance of glycosylation in modulating the responsiveness of cancer cells to treatments as well novel approaches to enhancing cancer treatments will be discussed.

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13:35-13:45 Discussion

13:45-14:10 Specific lectin-based protein glycoprofiling for diagnostics

Dr Jan Tkac, Slovak Academy of Sciences, Slovakia

Abstract

In this contribution Dr Tkac will describe an alternative to mass spectrometry-based analysis of glycans by the use of lectins for development of various types of bioanalytical devices. Application of lectins allows us to analyse glycans of the glycoproteins directly on an intact protein backbone. There is no need to release glycans from the proteins, which significantly simplifies assay procedure. The devices developed so far are based on electrochemical biosensors, fluorescent lectin microarrays and lectin-based ELLA (Enzyme-linked lectin assays) like platform of analysis. The most recent approach involves the use of magnetic nanoparticles in combination of ELLA for highly sensitive assay format requiring small sample consumption. The potential of lectin-based glycan analysis will be provided by the use of the bioanalytical devices for analysis of serum samples from people who have prostate cancer, breast cancer, rheumatoid arthritis and chronic lymphocytic leukaemia. The clinical potential of magnetic ELLA for diagnostics of prostate cancer will be discussed.

The last part of the presentation will show application of glycan biosensors with an immobilised Tn antigen to detect antibodies. Two different approaches were applied for immobilisation of a small glycan either directly to a modified surface (2D biosensor) or to a layer of human serum albumin formed on the surface (3D biosensor) and these two approaches are compared.

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14:10-14:20 Discussion

14:20-14:45 Targeting gangliosides using synthetic toxins for pain management

Professor Bazbek Davletov, University of Sheffield, UK

Abstract

Professor Davletov will present the evidence showing that a chimera made of cholera and botulinum toxins gives long-lasting pain relief in rats without adverse effects and, in time, could replace opioid drugs as a safe and effective way of treating chronic pain. Cholera toxin is known to target GM1 gangliosides present on neurons and the cholera ganglioside binding subunit has been used widely as a strong neuronal tracer. The group deconstructed the botulinum (‘Botox’) molecule, which blocks neuromuscular junctions, and reassembled it with the ganglioside-binding part of the cholera toxin to make Cho-Bot – a compound which successfully targets pain neurons. Injected under the skin just once, Cho-Bot relieves chronic pain induced by a chemotherapeutic drug in a rodent model. It doesn’t affect muscles like the Botox used to reduce wrinkles but it does block nerve pain for up to four months without affecting normal pain responses. Thus Cho-Bot could avoid the adverse events of tolerance and addiction often associated with repeated opioid drug use. Chronic pain of ‘moderate to severe’ intensity is widespread affecting 7.8 million people in the UK and 19% of adult Europeans. Opioids like morphine are considered to be the gold standard for pain relief but there is little evidence that their long-term use is effective in treating chronic pain.

In summary, to prepare Cho-Bot the group developed a molecular Lego system which allows us to link the botulinum 'warhead' to a navigation molecule, in this case, the ganglioside binding cholera binding subunit, allowing the creation of widely desired long-lasting pain killer without the side effects of opioids.

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14:45-14:55 Discussion

14:55-15:25 Tea

15:25-15:50 Targeting cancer with modified viruses

Professor Rita Gerardy-Schahn, Hannover Medical School, Germany

Abstract

Conditionally replicating (oncolytic) adenoviruses are promising tools for therapy of solid tumours but their wildtype tropism lacks effective recognition and binding to tumour cells. Genetic modification of the adenoviral fibers has proven difficult since frequently used tumour ligands such as scFvs or TCRs are usually incompatible with synthesis and assembly of adenoviral capsid proteins. In this study, Professor Gerardy-Schahn utilised endosialidase NF (endoNF), the trimeric tailspike protein of bacteriophage K1F, to replace the entire knob part of adenoviral fiber and to facilitate oncolytic adenovirus infection of tumours expressing the cell surface antigen polysialic acid (polySia). For this purpose, the group exchanged the fiber gene locus of the oncolytic adenovirus hTert-Ad by genes for fiber-endoNF fusion protein comprising the fiber capsid anchoring domain and an N-terminally truncated form of endoNF. Functional incorporation of chimeric fibers into the capsids of viral progeny was demonstrated by electron microscopic images of viral particles and detection of endoNF activity on isolated capsids. Chimeric viruses effectively infected cells in a polySia-dependent manner as shown by in an isogenic assay using HEK293 cells with or without polySia expression. Interestingly, the group found that the ability of endoNF to degrade polySia was essential for infection. Investigations on the oncolytic properties in comparison with the maternal virus hTertAd showed significantly improved oncolysis in a polysialic acid-expressing human neuroblastoma cell line and effective inhibition of tumour growth in subcutaneous neuroblastoma in nude mice. In summary, the group's engineered oncolytic adenoviruses with a genetically stable tropism towards polysia-expressing tumours represent promising tools for the treatment of clinically relevant cancers such as small cell lung cancer and neuroblastoma.

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15:50-16:00 Discussion

16:00-17:00 Panel discussion

Synthetic glycobiology

Theo Murphy international scientific meeting organised by Professor Bruce Turnbull, Dr Anne Imberty and Professor Ola Blixt.

Kavli Royal Society Centre, Chicheley Hall Newport Pagnell Buckinghamshire MK16 9JJ
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