Structural and stochastical modelling of possible contaminant pathways below nuclear installations

Structural and stochastical modelling of possible contaminant pathways below nuclear installations 1Richard Haslam, 1Stuart Clarke, 1Peter Styles & 2Clive Auton 1Earth Sciences and Geography, School of Physical and Geographical Sciences, Keele University, Keele, Staffordshire, ST5 5BG, United Kingdom 2British Geological Survey, Murchison House, West Mains Road, Edinburgh, EH9 3LA, United Kingdom Dounreay Nuclear Power station is situated on the northern coast of Caithness, Scotland on complex normally faulted Devonian sedimentary rocks with a thin, intermittent cover of superficial deposits, predominantly comprising glacial tills of varying provenance. Bedrock structure, fracture patterns and the relationships between bedrock and the superficial deposits have a considerable impact on the transmissivity of any possible contaminants. Consequently, an understanding of the bedrock-superficial boundary and how fractures and faults influence and control the transport of fluids are a key concern. The principal aims of this work are to gain an understanding of the processes and controls on fluid flow pathways within such complex geological terrains, and develop methods of stochastatically evaluating likely contamination transport within the subsurface. This work focuses on the near-surface bedrock geology and superficial deposits. The near surface geology of the Dounreay site comprises cyclic sequences of lacustrine rocks; their cyclicity has enabled a reference stratigraphy to be created and correlated across the site. This stratigraphy, the coastal exposures and the extensive amount of borehole data available, provide a unique opportunity to construct and constrain a three-dimensional bedrock model; the interpretive element of which has been robustly test using structural restoration techniques. In the bedrock of Dounreay, three principal fracture sets have been identified. The first two sets of fractures are approximately orthogonal, trending north-northwest and west-southwest respectively; they represent the regional fracture set. It is proposed that these fractures where produced during loading and burial of the Devonian sediments. The final fracture set is predominantly parallel to bedding of the laminated sediments; it gives the Caithness Flagstones their ‘flaggy’ nature. The regional fracture sets are approximately constant over the site area and vary little with depth, whereas the bedding parallel fracture set shows a marked decrease in the number of fractures per meter with depth, on a logarithmic trend. This relationship is also visible in the Rock Quality Designation (RQD) values and hydraulic conductivity data from boreholes. It follows that the bedding parallel fractures are the major controlling factor of flow in the shallow subsurface and that the RQD values can be used as a proxy for fracture density. RQD values are a commonly collected during borehole drilling and the relationship between hydraulic conductivity and RQD values offer a method for stochastically populating a 3D geological model with hydraulic conductivity values. Current geological interpretations of the superficial deposits are based primarily on their genesis. Consequently, subdivisions based on the origin of the sediments do not relate directly to their fluid transmissivity. The superficial deposits generally have a very low hydraulic conductivity, compared to that of the bedrock, impeding the flow of water from the surface to the groundwater system at depth. A combination of driller’s description and comparisons of grain-size distribution enables subdivisions of the Quaternary strata to be established based on their properties instead of their genesis. These properties can then be stochastically interpolated throughout the 3D geological model. This work provides a framework from which likely contamination scenarios can be modelled, both in the well-constrained subsurface of Dounreay, and at other nuclear installations where the nature of the subsurface is less well constrained

Choice of Host Cell Line Is Essential for the Functional Glycosylation of the Fc Region of Human IgG1 Inhibitors of Influenza B Viruses

Abs are glycoproteins that carry a conserved N-linked carbohydrate attached to the Fc whose presence and fine structure profoundly impacts on their in vivo immunogenicity, pharmacokinetics, and functional attributes. The host cell line used to produce IgG has a major impact on this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. In this study, we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the human endothelial kidney 293-F or Chinese hamster ovary–K1 systems. We show that the types of N-linked glycans between matched pairs of Fc mutants vary greatly and in particular, with respect, to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B–mediated agglutination of human erythrocytes when expressed in Chinese hamster ovary–K1, but not in human endothelial kidney 293-F cells

Designing an artificial Golgi reactor for cell-free glycosylation

Glycosylation of therapeutically relevant proteins such as monoclonal antibodies (mAbs), is critical as it can offer increased drug efficiency, efficacy and half-life. Therefore, the production of modern biotherapeutics focuses on controlling the protein glycosylation profile using various methods. Currently, the dominating method is the traditional cell-line engineering of host cells such as mammalian cells. The main goal is to produce mAbs with a human-like glycosylation pattern. However, this approach often struggles due to high sensitivity to the fermentation environment making it difficult to scale up and control. The latter can lead to structural heterogeneity amongst the products which can be immunogenic. In addition to the in vivo methods, there are many in vitro techniques such as chemoselective or enzymatic glycosylation. However, they are often limited by the difficult implementation and, as before, product heterogeneity due to lack of control over the enzymatic reactions. In line with the need to control glycosylation in the production of therapeutic proteins, we propose an artificial Golgi reactor for in vitro glycosylation. By expressing selected glycosyltransferases and immobilizing them on solid supports we can achieve sequential enzymatic reactions required for protein glycosylation. The spatial separation will allow strict control over the reaction conditions while addressing enzyme promiscuity. Both should enhance product quality. Furthermore, we aim to perform a single-step glycosyltransferases purification/immobilization. Thanks to that, as well as the modularity of our design, the proposed system would be more sustainable and easily tailored for each application, thus producing any desired glycoform to homogeneity. A detailed mathematical approach to design and optimisation of the proposed artificial Golgi reactor focusing on mAb therapeutics has been published [1]. The authors report an optimisation of the reactor design and operational parameters that directs the whole process towards the desired glycan structure. In this research project, we have achieved expression and in vivo biotinylation of Nicotiana Tabacum GnTI (NtGnTI) and human GalT in E. coli. The biotinylated enzymes were successfully bound to streptavidin beads and used for artificial glycan synthesis. NtGnTI and GalT reacted in a sequential fashion to produce the glycan GalGlcNAcMan5GlcNAc2, as confirmed with MALDI/TOF MS analysis. In the future, we aim in extending the pathway of immobilized enzymes thus demonstrating the importance of this novel platform for in vitro glycosylation. References: [1] Klymenko, O. V., Shah, N., Kontoravdi, C., Royle, K. E. & Polizzi, K. M. Designing an Artificial Golgi reactor to achieve targeted glycosylation of monoclonal antibodies. AIChE J. 62, 2959–2973 (2016)

Role of galectin-glycan circuits in reproduction: from healthy pregnancy to preterm birth (PTB)

Growing evidence suggests that galectins, an evolutionarily conserved family of glycan-binding proteins, fulfill key roles in pregnancy including blastocyst implantation, maternal-fetal immune tolerance, placental development, and maternal vascular expansion, thereby establishing a healthy environment for the growing fetus. In this review, we comprehensively present the function of galectins in shaping cellular circuits that characterize a healthy pregnancy. We describe the current understanding of galectins in term and preterm labor and discuss how the galectin-glycan circuits contribute to key immunological pathways sustaining maternal tolerance and preventing microbial infections. A deeper understanding of the glycoimmune pathways regulating early events in preterm birth could offer the broader translational potential for the treatment of this devastating syndrome

Role of galectin-glycan circuits in reproduction: from healthy pregnancy to preterm birth (PTB)

Growing evidence suggests that galectins, an evolutionarily conserved family of glycan-binding proteins, fulfill key roles in pregnancy including blastocyst implantation, maternal-fetal immune tolerance, placental development, and maternal vascular expansion, thereby establishing a healthy environment for the growing fetus. In this review, we comprehensively present the function of galectins in shaping cellular circuits that characterize a healthy pregnancy. We describe the current understanding of galectins in term and preterm labor and discuss how the galectin-glycan circuits contribute to key immunological pathways sustaining maternal tolerance and preventing microbial infections. A deeper understanding of the glycoimmune pathways regulating early events in preterm birth could offer the broader translational potential for the treatment of this devastating syndrome.Fil: Blois, Sandra M.. Experimental and Clinical Research Center; Alemania. Charité-Universitätsmedizin Berlin; Alemania. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Verlohren, Stefan. Charité-Universitätsmedizin Berlin; AlemaniaFil: Wu, Gang. Imperial College London; Reino UnidoFil: Clark, Gary. University of Missouri; Estados UnidosFil: Dell, Anne. Imperial College London; Reino UnidoFil: Haslam, Stuart M.. Imperial College London; Reino UnidoFil: Barrientos, Gabriela Laura. Hospital Aleman. Laboratorio de Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Glycoproteomic studies of IgE from a novel hyper IgE syndrome linked to PGM3 mutation

International audienceGlycans serve as important regulators of antibody activities and half-lives. IgE is the most heavily glycosylated antibody, but in comparison to other antibodies little is known about its glycan structure function relationships. We therefore describe the site specific IgE glycosylation from a patient with a novel hyper IgE syndrome linked to mutations in PGM3, which is an enzyme involved in synthesizing UDP-GlcNAc, a sugar donor widely required for glycosylation. A two-step method was developed to prepare two IgE samples from less than 1 mL of serum collected from a patient with PGM3 mutation and a patient with atopic dermatitis as a control subject. Then, a glycoproteomic strategy was used to study the site-specific glycosylation. No glycosylation was found at Asn264, whilst high mannose glycans were only detected at Asn275, tri-antennary glycans were exclusively observed at Asn99 and Asn252, and non-fucosylated complex glycans were detected at Asn99. The results showed similar glycosylation profiles between the two IgE samples. These observations, together with previous knowledge of IgE glycosylation, imply that IgE glycosylation is similarly regulated among healthy control, allergy and PGM3 related hyper IgE syndrome

Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis

Essential biological systems employ self-correcting mechanisms to maintain cellular homeostasis. Mammalian cell function is dynamically regulated by the interaction of cell surface galectins with branched N-glycans. Here we report that N-glycan branching deficiency triggers the Golgi to generate bioequivalent N-glycans that preserve galectin-glycoprotein interactions and cellular homeostasis. Galectins bind N-acetyllactosamine (LacNAc) units within N-glycans initiated from UDP-GlcNAc by the medial-Golgi branching enzymes as well as the trans-Golgi poly-LacNAc extension enzyme β1,3-N-acetylglucosaminyltransferase (B3GNT). Marginally reducing LacNAc content by limiting N-glycans to three branches results in T-cell hyperactivity and autoimmunity; yet further restricting branching does not produce a more hyperactive state. Rather, new poly-LacNAc extension by B3GNT maintains galectin binding and immune homeostasis. Poly-LacNAc extension is triggered by redistribution of unused UDP-GlcNAc from the medial to trans-Golgi via inter-cisternal tubules. These data demonstrate the functional equivalency of structurally dissimilar N-glycans and suggest a self-correcting feature of the Golgi that sustains cellular homeostasis

Immobilized enzyme cascade for targeted glycosylation

Glycosylation is a critical post-translational protein modification that affects folding, half-life and functionality. Glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. We describe a platform for sequential glycosylation reactions for tailored sugar structures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro enabled by immobilized enzymes produced with a one-step immobilization/purification method. We reconstruct a reaction cascade mimicking a glycosylation pathway where promiscuity naturally exists to humanize a range of proteins derived from different cellular systems, yielding near-homogeneous glycoforms. Immobilized β-1,4-galactosyltransferase is used to enhance the galactosylation profile of three IgGs, yielding 80.2–96.3% terminal galactosylation. Enzyme recycling is demonstrated for a reaction time greater than 80 h. The platform is easy to implement, modular and reusable and can therefore produce homogeneous glycan structures derived from various hosts for functional and clinical evaluation.UKRI Engineering and Physical Sciences Research Council (EP/N509486/1)UKRI Engineering and Physical Sciences Research Council (EP/K038648/1, EP/H04986X/1 and EP/K038648/1)UK Research and Innovation ‘Global Challenges Research Fund’ BB/P02789X/1)UK Biotechnology and Biological Sciences Research Counci

Enhanced Aromatic Sequons Increase Oligosaccharyltransferase Glycosylation Efficiency and Glycan Homogeneity

SummaryN-Glycosylation plays an important role in protein folding and function. Previous studies demonstrate that a phenylalanine residue introduced at the n-2 position relative to an Asn-Xxx-Thr/Ser N-glycosylation sequon increases the glycan occupancy of the sequon in insect cells. Here, we show that any aromatic residue at n-2 increases glycan occupancy in human cells and that this effect is dependent upon oligosaccharyltransferase substrate preferences rather than differences in other cellular processing events such as degradation or trafficking. Moreover, aromatic residues at n-2 alter glycan processing in the Golgi, producing proteins with less complex N-glycan structures. These results demonstrate that manipulating the sequence space surrounding N-glycosylation sequons is useful both for controlling glycosylation efficiency, thus enhancing glycan occupancy, and for influencing the N-glycan structures produced