Publications by authors named "Daniel Spencer"

64 Publications

Interlaboratory evaluation of plasma N-glycan antennary fucosylation as a clinical biomarker for HNF1A-MODY using liquid chromatography methods.

Glycoconj J 2021 Mar 25. Epub 2021 Mar 25.

Ludger Ltd, Culham Science Centre, Oxfordshire, Abingdon, UK.

Antennary fucosylation alterations in plasma glycoproteins have been previously proposed and tested as a biomarker for differentiation of maturity onset diabetes of the young (MODY) patients carrying a functional mutation in the HNF1A gene. Here, we developed a novel LC-based workflow to analyze blood plasma N-glycan fucosylation in 320 diabetes cases with clinical features matching those at risk of HNF1A-MODY. Fucosylation levels measured in two independent research centers by using similar LC-based methods were correlated to evaluate the interlaboratory performance of the biomarker. The interlaboratory study showed good correlation between fucosylation levels measured for the 320 cases in the two centers with the correlation coefficient (r) of up to 0.88 for a single trait A3FG3S2. The improved chromatographic separation allowed the identification of six single glycan traits and a derived antennary fucosylation trait that were able to differentiate individuals carrying pathogenic mutations from benign or no HNF1A mutation cases, as determined by the area under the curve (AUC) of up to 0.94. The excellent (r = 0.88) interlaboratory performance of the glycan biomarker for HNF1A-MODY further supports the development of a clinically relevant diagnostic test measuring antennary fucosylation levels.
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http://dx.doi.org/10.1007/s10719-021-09992-wDOI Listing
March 2021

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin mediated clearance.

Haematologica 2021 Mar 25. Epub 2021 Mar 25.

Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland; National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland; National Coagulation Centre, St James's Hospital, Dublin.

Terminal sialylation determines plasma VWF half-life. A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we show that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF:Ag levels (p.
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http://dx.doi.org/10.3324/haematol.2020.274720DOI Listing
March 2021

Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes.

PLoS Negl Trop Dis 2021 Feb 2;15(2):e0009071. Epub 2021 Feb 2.

Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.
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http://dx.doi.org/10.1371/journal.pntd.0009071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880456PMC
February 2021

Assays for the identification and quantification of sialic acids: Challenges, opportunities and future perspectives.

Bioorg Med Chem 2021 Jan 21;30:115882. Epub 2020 Nov 21.

School of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, UK. Electronic address:

N-Acetyl neuraminic acid (sialic acid) is a monosaccharide generally found as the terminating unit on glycans, which in turn are found on the surface of cells and glycoproteins. These glycans aid in a variety of biological functions such as cell interactions and immune response. Sialic acid has been identified as a biomarker for cardiovascular disease, diabetes and a range of other inflammatory and degenerative conditions. It has also been identified as a marker for different types of cancer. Sialic acid levels vary depending on the level of inflammation present during the course of an inflammatory disease and it is overexpressed by tumours as a shield against the immune system. Since the discovery of sialic acid, numerous assays have been developed for the identification and quantification of different sialic acid derivative monosaccharides and these assays fall into four main groups: colorimetric, fluorometric, enzymatic and chromatographic/mass spectrometric, with much overlap between these. Given the importance of sialic acids in biological pathways, this review article critically appraises assays that are used to detect and quantify sialic acid and its derivatives. Thus it details the method, sensitivity, specificity and wider scope of a range of assays, and concludes by suggesting some future directions for assay development and application. In this way, insight is provided into assays that allow for the accurate quantitation of sialic acid in biological samples, which may facilitate identification of the roles of sialic acid in healthy and disease pathways.
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http://dx.doi.org/10.1016/j.bmc.2020.115882DOI Listing
January 2021

L1CAM as an E-selectin Ligand in Colon Cancer.

Int J Mol Sci 2020 Nov 5;21(21). Epub 2020 Nov 5.

Unidade de Ciências Biomoleculares Aplicadas (UCIBIO), Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLe) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 () coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLe in CRC. The SW620FUT6 cell line expressed high levels of sLe antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.
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http://dx.doi.org/10.3390/ijms21218286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672641PMC
November 2020

Allosterically Coupled Multisite Binding of Testosterone to Human Serum Albumin.

Endocrinology 2021 02;162(2)

Research Program in Men's Health: Aging and Metabolism, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

Human serum albumin (HSA) acts as a carrier for testosterone, other sex hormones, fatty acids, and drugs. However, the dynamics of testosterone's binding to HSA and the structure of its binding sites remain incompletely understood. Here, we characterize the dynamics of testosterone's binding to HSA and the stoichiometry and structural location of the binding sites using 2-dimensional nuclear magnetic resonance (2D NMR), fluorescence spectroscopy, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt partitioning, and equilibrium dialysis, complemented by molecular modeling. 2D NMR studies showed that testosterone competitively displaced 18-[13C]-oleic acid from at least 3 known fatty acid binding sites on HSA that also bind many drugs. Binding isotherms of testosterone's binding to HSA generated using fluorescence spectroscopy and equilibrium dialysis were nonlinear and the apparent dissociation constant varied with different concentrations of testosterone and HSA. The binding isotherms neither conformed to a linear binding model with 1:1 stoichiometry nor to 2 independent binding sites; the binding isotherms were most consistent with 2 or more allosterically coupled binding sites. Molecular dynamics studies revealed that testosterone's binding to fatty acid binding site 3 on HSA was associated with conformational changes at site 6, indicating that residues in in these 2 distinct binding sites are allosterically coupled. There are multiple, allosterically coupled binding sites for testosterone on HSA. Testosterone shares these binding sites on HSA with free fatty acids, which could displace testosterone from HSA under various physiological states or disease conditions, affecting its bioavailability.
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http://dx.doi.org/10.1210/endocr/bqaa199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774055PMC
February 2021

A fast impedance-based antimicrobial susceptibility test.

Nat Commun 2020 10 21;11(1):5328. Epub 2020 Oct 21.

Department of Electronics and Computer Science, and Institute for Life Science, University of Southampton, Hampshire, SO17 1BJ, UK.

There is an urgent need to develop simple and fast antimicrobial susceptibility tests (ASTs) that allow informed prescribing of antibiotics. Here, we describe a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material. The bacteria are incubated in the presence of an antibiotic for 30 min, and then approximately 10 cells are analysed one-by-one with microfluidic impedance cytometry for 2-3 min. The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window. The results are consistent with those obtained by classical broth microdilution assays for a range of antibiotics and bacterial species.
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http://dx.doi.org/10.1038/s41467-020-18902-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578651PMC
October 2020

Loss of ARNT in skeletal muscle limits muscle regeneration in aging.

FASEB J 2020 Dec 8;34(12):16086-16104. Epub 2020 Oct 8.

Division of Plastic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

The ability of skeletal muscle to regenerate declines significantly with aging. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a critical component of the hypoxia signaling pathway, was less abundant in skeletal muscle of old (23-25 months old) mice. This loss of ARNT was associated with decreased levels of Notch1 intracellular domain (N1ICD) and impaired regenerative response to injury in comparison to young (2-3 months old) mice. Knockdown of ARNT in a primary muscle cell line impaired differentiation in vitro. Skeletal muscle-specific ARNT deletion in young mice resulted in decreased levels of whole muscle N1ICD and limited muscle regeneration. Administration of a systemic hypoxia pathway activator (ML228), which simulates the actions of ARNT, rescued skeletal muscle regeneration in both old and ARNT-deleted mice. These results suggest that the loss of ARNT in skeletal muscle is partially responsible for diminished myogenic potential in aging and activation of hypoxia signaling holds promise for rescuing regenerative activity in old muscle.
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http://dx.doi.org/10.1096/fj.202000761RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756517PMC
December 2020

A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG.

Glycoconj J 2020 12 16;37(6):691-702. Epub 2020 Oct 16.

Ludger Ltd, Culham Science Centre, Abingdon, UK.

Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson's r correlation coefficient of 0.8208 between the two methods.
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http://dx.doi.org/10.1007/s10719-020-09953-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679266PMC
December 2020

Joint Bayesian Estimation of Voxel Activation and Inter-regional Connectivity in fMRI Experiments.

Psychometrika 2020 12 19;85(4):845-869. Epub 2020 Sep 19.

Department of Statistics, University of California, Santa Cruz, CA, USA.

Brain activation and connectivity analyses in task-based functional magnetic resonance imaging (fMRI) experiments with multiple subjects are currently at the forefront of data-driven neuroscience. In such experiments, interest often lies in understanding activation of brain voxels due to external stimuli and strong association or connectivity between the measurements on a set of pre-specified groups of brain voxels, also known as regions of interest (ROI). This article proposes a joint Bayesian additive mixed modeling framework that simultaneously assesses brain activation and connectivity patterns from multiple subjects. In particular, fMRI measurements from each individual obtained in the form of a multi-dimensional array/tensor at each time are regressed on functions of the stimuli. We impose a low-rank parallel factorization decomposition on the tensor regression coefficients corresponding to the stimuli to achieve parsimony. Multiway stick-breaking shrinkage priors are employed to infer activation patterns and associated uncertainties in each voxel. Further, the model introduces region-specific random effects which are jointly modeled with a Bayesian Gaussian graphical prior to account for the connectivity among pairs of ROIs. Empirical investigations under various simulation studies demonstrate the effectiveness of the method as a tool to simultaneously assess brain activation and connectivity. The method is then applied to a multi-subject fMRI dataset from a balloon-analog risk-taking experiment, showing the effectiveness of the model in providing interpretable joint inference on voxel-level activations and inter-regional connectivity associated with how the brain processes risk. The proposed method is also validated through simulation studies and comparisons to other methods used within the neuroscience community.
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http://dx.doi.org/10.1007/s11336-020-09727-0DOI Listing
December 2020

Mechanistic examination of C -C tyrosyl bond cleavage: Spectroscopic investigation of the generation of α-glycyl radical cations from tyrosyl (glycyl/alanyl)tryptophan.

J Mass Spectrom 2020 Jul 28;56(4):e4630. Epub 2020 Jul 28.

Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.

In this study, dissociative one-electron transfer dissociation of [Cu (dien)Y(G/A)W] [dien = diethylenetriamine; Y(G/A)W = tyrosyl (glycyl/alanyl)tryptophan] was used to generate the tripeptide radical cations [Y(G/A)W] ; subsequent loss of the Tyr side chain formed [G (G/A)W] . The π-centered species [YGW ] generated the α-centered species [G GW] through C -C bond cleavage, as revealed using infrared multiple photon dissociation (IRMPD) measurements and density functional theory (DFT) calculations. Comparisons of experimental and theoretical IR spectra confirmed that both the charge and spin densities of [Y(G/A)W ] were delocalized initially at the tryptophan indolyl ring; subsequent formation of the final [G (G/A)W] structure gave the highest spin density at the α-carbon atom of the N-terminal glycine residue, with a proton solvated by the first amide oxygen atom. The IRMPD mass spectra and action spectra of the [G (G/A)W] species were all distinctly different from those of their isomeric [G(G/A)W ] species. The mechanism of formation of the captodative [G (G/A)W] species-with the charge site separated from the radical site-from [Y(G/A)W ] has been elucidated. DFT calculations suggested that the C -C bond cleavage of the tyrosine residue in the radical cationic [Y(G/A)W ] precursor involves (a) through-space electron transfer between the indolyl and phenolic groups; (b) formation of proton-bound dimers through C -C cleavage of the tyrosine residue; and (c) a concerted proton rearrangement from the phenolic OH group to the carboxyl group and formation of the α-carbon-centered product [G (G/A)W] through hydrogen bond cleavage. The barriers for the electron transfer (a), the C -C cleavage (b), and the protonation rearrangement (c) were 12.8, 26.5, and 10.3 kcal mol , respectively.
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http://dx.doi.org/10.1002/jms.4630DOI Listing
July 2020

Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown.

Nat Commun 2020 08 11;11(1):4017. Epub 2020 Aug 11.

Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK.

The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.
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http://dx.doi.org/10.1038/s41467-020-17847-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419316PMC
August 2020

Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis.

Sci Rep 2020 07 31;10(1):12903. Epub 2020 Jul 31.

Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK.

During Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the host's immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC-MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with ManGlcNAc being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans.
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http://dx.doi.org/10.1038/s41598-020-69753-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395719PMC
July 2020

rPTMDetermine: A Fully Automated Methodology for Endogenous Tyrosine Nitration Validation, Site-Localization, and Beyond.

Anal Chem 2020 08 21;92(15):10768-10776. Epub 2020 Jul 21.

Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China.

We present herein rPTMDetermine, an adaptive and fully automated methodology for validation of the identification of rarely occurring post-translational modifications (PTMs), using a semisupervised approach with a linear discriminant analysis (LDA) algorithm. With this strategy, verification is enhanced through similarity scoring of tandem mass spectrometry (MS/MS) comparisons between modified peptides and their unmodified analogues. We applied rPTMDetermine to (1) perform fully automated validation steps for modified peptides identified from an database and (2) retrieve potential yet-to-be-identified modified peptides from raw data (that had been missed through conventional database searches). In part (1), 99 of 125 3-nitrotyrosyl-containing (nitrated) peptides obtained from a ProteinPilot search were validated and localized. Twenty nitrated peptides were falsely assigned because of incorrect monoisotopic peak assignments, leading to erroneous identification of deamidation and nitration. Five additional nitrated peptides were, however, validated after performing nonmonoisotopic peak correction. In part (2), an additional 236 unique nitrated peptides were retrieved and localized, containing 113 previously unreported nitration sites; 25 endogenous nitrated peptides with novel sites were selected and verified by comparison with synthetic analogues. In summary, we identified and confidently validated 296 unique nitrated peptides-collectively representing the largest number of endogenously identified 3-nitrotyrosyl-containing peptides from the cerebral cortex proteome of a model of stroke. Furthermore, we harnessed the rPTMDetermine strategy to complement conventional database searching and enhance the confidence of assigning rarely occurring PTMs, while recovering many missed peptides. In a final demonstration, we successfully extended the application of rPTMDetermine to peptides featuring tryptophan oxidation.
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http://dx.doi.org/10.1021/acs.analchem.0c02148DOI Listing
August 2020

Dissociative electron transfer of copper(ii) complexes of glycyl(glycyl/alanyl)tryptophan in vacuo: IRMPD action spectroscopy provides evidence of transition from zwitterionic to non-zwitterionic peptide structures.

Phys Chem Chem Phys 2020 Jun 3;22(23):13084-13091. Epub 2020 Jun 3.

Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.

We report herein the first detailed study of the mechanism of redox reactions occurring during the gas-phase dissociative electron transfer of prototypical ternary [Cu(dien)M]˙ complexes (M, peptide). The two final products are (i) the oxidized non-zwitterionic π-centered [M]˙ species with both the charge and spin densities delocalized over the indole ring of the tryptophan residue and with a C-terminal COOH group intact, and (ii) the complementary ion [Cu(dien)]. Infrared multiple photon dissociation (IRMPD) action spectroscopy and low-energy collision-induced dissociation (CID) experiments, in conjunction with density functional theory (DFT) calculations, revealed the structural details of the mass-isolated precursor and product cations. Our experimental and theoretical results indicate that the doubly positively charged precursor [Cu(dien)M]˙ features electrostatic coordination through the anionic carboxylate end of the zwitterionic M moiety. An additional interaction exists between the indole ring of the tryptophan residue and one of the primary amino groups of the dien ligand; the DFT calculations provided the structures of the precursor ion, intermediates, and products, and enabled us to keep track of the locations of the charge and unpaired electron. The dissociative one-electron transfer reaction is initiated by a gradual transition of the M tripeptide from the zwitterionic form in [Cu(dien)M]˙ to the non-zwitterionic M intermediate, through a cascade of conformational changes and proton transfers. In the next step, the highest energy intermediate is formed; here, the copper center is 5-coordinate with coordination from both the carboxylic acid group and the indole ring. A subsequent switch back to 4-coordination to an intermediate IM1, where attachment to GGW occurs through the indole ring only, creates the structure that ultimately undergoes dissociation.
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http://dx.doi.org/10.1039/d0cp02296aDOI Listing
June 2020

Fucosidases from the human gut symbiont Ruminococcus gnavus.

Cell Mol Life Sci 2021 Jan 24;78(2):675-693. Epub 2020 Apr 24.

The Gut Microbes and Health Institute Strategic Programme, Quadram Institute Bioscience, Norwich Research Park, Norwich, NR4 7UQ, UK.

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.
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http://dx.doi.org/10.1007/s00018-020-03514-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872956PMC
January 2021

A Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Assay for the Relative Quantitation of Antennary Fucosylated -Glycans in Human Plasma.

Front Chem 2020 28;8:138. Epub 2020 Feb 28.

Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands.

Changes in the abundance of antennary fucosylated glycans in human total plasma -glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However, it is challenging to address this important part of the human glycome. Most commonly, time-consuming chromatographic separations are performed to differentially quantify core and antenna fucosylation. Obtaining sufficient resolution for larger, more complex glycans can be challenging. We introduce a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) assay for the relative quantitation of antennary fucosylation in TPNG. -linked glycans are released from plasma by PNGase F and further treated with a core fucosidase before performing a linkage-informative sialic acid derivatization. The core fucosylated glycans are thus depleted while the remaining antennary fucosylated glycans are quantitated. Simultaneous quantitation of α2,3-linked sialic acids and antennary fucosylation allows an estimation of the sialyl-Lewis x motif. The approach is feasible using either ultrahigh-resolution Fourier-transform ion cyclotron resonance mass spectrometry or time-of-flight mass spectrometry. The assay was used to investigate changes of antennary fucosylation as clinically relevant marker in 14 colorectal cancer patients. In accordance with a previous report, we found elevated levels of antennary fucosylation pre-surgery which decreased after tumor resection. The assay has the potential for revealing antennary fucosylation signatures in various conditions including diabetes and different types of cancer.
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http://dx.doi.org/10.3389/fchem.2020.00138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059190PMC
February 2020

High-Speed Single-Cell Dielectric Spectroscopy.

ACS Sens 2020 02 17;5(2):423-430. Epub 2020 Feb 17.

Electronics and Computer Science, and Institute of Life Sciences University of Southampton , Southampton SO17 1BJ , U.K.

Single-cell impedance cytometry is a label-free analysis technique that is now widely used to measure the electrical properties of a cell and to differentiate different subpopulations. Current techniques are limited to measuring the impedance of a single cell at one or two simultaneous frequencies. Also, there are no methods that extrapolate the intrinsic electrical properties of single cells. We demonstrate a new approach that uses multifrequency impedance measurements to determine the complete intrinsic electrical properties of thousands of single cells at high throughput. The applicability of the method is demonstrated by measuring the properties of red blood cells and red cell ghosts, deriving the unique values of conductivity and permittivity of the membrane and cytoplasm for each individual cell.
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http://dx.doi.org/10.1021/acssensors.9b02119DOI Listing
February 2020

Method comparison for N-glycan profiling: Towards the standardization of glycoanalytical technologies for cell line analysis.

PLoS One 2019 7;14(10):e0223270. Epub 2019 Oct 7.

Ludger Ltd., Culham Science Centre, Abingdon, Oxfordshire, England, United Kingdom.

The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0223270PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779296PMC
March 2020

Characterization of sialidase and disruption of its role in host-pathogen interactions.

Microbiology (Reading) 2019 11;165(11):1181-1197

Integrated BioSciences, School of Clinical Dentistry, The University of Sheffield, 19 Claremont Crescent, Sheffield S10 2TA, UK.

Key to onset and progression of periodontitis is a complex relationship between oral bacteria and the host. The organisms most associated with severe periodontitis are the periodontal pathogens of the red complex: , and . These organisms express sialidases, which cleave sialic acid from host glycoproteins, and contribute to disease through various mechanisms. Here, we expressed and purified recombinant sialidase SiaPG (PG_0352) and characterized its activity on a number of substrates, including host sialoglycoproteins and highlighting the inability to cleave diacetylated sialic acids - a phenomenon overcome by the NanS sialate-esterase from . Indeed SiaPG required NanS to maximize sialic acid harvesting from heavily O-acetylated substrates such as bovine salivary mucin, hinting at the possibility of interspecies cooperation in sialic acid release from host sources by these members of the oral microbiota. Activity of SiaPG and was inhibited using the commercially available chemotherapeutic zanamivir, indicating its potential as a virulence inhibitor, which also inhibited sialic acid release from mucin, and was capable of inhibiting biofilm formation of on oral glycoprotein sources. Zanamivir also inhibited attachment and invasion of oral epithelial cells by and other periodontal pathogens, both in monospecies but also in multispecies infection experiments, indicating potential to suppress host-pathogen interactions of a mixed microbial community. This study broadens our understanding of the multifarious roles of bacterial sialidases in virulence, and indicates that their inhibition with chemotherapeutics could be a promising strategy for periodontitis therapy.
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http://dx.doi.org/10.1099/mic.0.000851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137779PMC
November 2019

OGT Controls the Expression and the Glycosylation of E-cadherin, and Affects Glycosphingolipid Structures in Human Colon Cell Lines.

Proteomics 2019 11 20;19(21-22):e1800452. Epub 2019 Aug 20.

Université de Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, F-59000, Lille, France.

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongst the phenotypic changes exhibited by tumor cells, a wide range of glycosylation has been reported for colon cancer-derived cell lines and CRC tissues. These aberrant modifications affect different aspects of glycosylation, including an increase in core fucosylation and GlcNAc branching on N-glycans, alteration of O-glycans, upregulated sialylation, and O-GlcNAcylation. Although O-GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O-GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches on three human colon cell lines (HT29, HCT116 and CCD841CoN), it is herein reported that silencing O-GlcNAc transferase (OGT, the sole enzyme driving O-GlcNAcylation), only slightly affects overall N- and O-glycosylation patterns. Interestingly, silencing of OGT in HT29 cells upregulates E-cadherin (a major actor of epithelial-to-mesenchymal transition) and changes its glycosylation. On the other hand, OGT silencing perturbs biosynthesis of glycosphingolipids resulting in a decrease in gangliosides and an increase in globosides. Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O-GlcNAcylation in colon cancer cells.
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http://dx.doi.org/10.1002/pmic.201800452DOI Listing
November 2019

Complex N-glycan breakdown by gut Bacteroides involves an extensive enzymatic apparatus encoded by multiple co-regulated genetic loci.

Nat Microbiol 2019 09 3;4(9):1571-1581. Epub 2019 Jun 3.

Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK.

Glycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp. have the capacity to utilize complex N-glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by Bacteroides thetaiotaomicron (Bt) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes from different families, including a CNG-specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of carbohydrate-active enzymes that have previously been implicated in the degradation of other classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche.
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http://dx.doi.org/10.1038/s41564-019-0466-xDOI Listing
September 2019

Improved and semi-automated reductive β-elimination workflow for higher throughput protein O-glycosylation analysis.

PLoS One 2019 17;14(1):e0210759. Epub 2019 Jan 17.

Ludger Ltd, Culham Science Centre, Abingdon, Oxfordshire, United Kingdom.

Protein O-glycosylation has shown to be critical for a wide range of biological processes, resulting in an increased interest in studying the alterations in O-glycosylation patterns of biological samples as disease biomarkers as well as for patient stratification and personalized medicine. Given the complexity of O-glycans, often a large number of samples have to be analysed in order to obtain conclusive results. However, most of the O-glycan analysis work done so far has been performed using glycoanalytical technologies that would not be suitable for the analysis of large sample sets, mainly due to limitations in sample throughput and affordability of the methods. Here we report a largely automated system for O-glycan analysis. We adapted reductive β-elimination release of O-glycans to a 96-well plate system and transferred the protocol onto a liquid handling robot. The workflow includes O-glycan release, purification and derivatization through permethylation followed by MALDI-TOF-MS. The method has been validated according to the ICH Q2 (R1) guidelines for the validation of analytical procedures. The semi-automated reductive β-elimination system enabled for the characterization and relative quantitation of O-glycans from commercially available standards. Results of the semi-automated method were in good agreement with the conventional manual in-solution method while even outperforming it in terms of repeatability. Release of O-glycans for 96 samples was achieved within 2.5 hours, and the automated data acquisition on MALDI-TOF-MS took less than 1 minute per sample. This largely automated workflow for O-glycosylation analysis showed to produce rapid, accurate and reliable data, and has the potential to be applied for O-glycan characterization of biological samples, biopharmaceuticals as well as for biomarker discovery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210759PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336230PMC
October 2019

Label-free enrichment of primary human skeletal progenitor cells using deterministic lateral displacement.

Lab Chip 2019 01;19(3):513-523

Faculty of Physical Sciences and Engineering, and Institute for Life Sciences, University of Southampton, SO17 1BJ, UK.

Skeletal stem cells (SSCs) are present in bone marrow (BM) and offer great potential for bone regenerative therapies. However, in the absence of a unique marker, current sorting approaches remain challenging in the quest for simple strategies to deliver SSCs with consistent regeneration and differentiation capacities. Microfluidics offers the possibility to sort cells marker-free, based on intrinsic biophysical properties. Recent studies indicate that SSCs are stiffer than leukocytes and are contained within the larger cell fraction in BM. This paper describes the use of deterministic lateral displacement (DLD) to sort SSCs based on cell size and stiffness. DLD is a technology that uses arrays of micropillars to sort cells based on their diameter. Cell deformation within the device can change the cell size and affect sorting - here evidenced using human cell lines and by fractionation of expanded SSCs. Following sorting, SSCs remained viable and retained their capacity to form clonogenic cultures (CFU-F), indicative of stem cell potential. Additionally, larger BM cells showed enhanced capacity to form CFU-F. These findings support the theory that SSCs are more abundant within the larger BM cell fraction and that DLD, or other size-based approaches, could be used to provide enriched SSC populations with significant implications for stem cell research and translation to the clinic.
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http://dx.doi.org/10.1039/c8lc01154kDOI Listing
January 2019

Transcriptional activation of fucosyltransferase (FUT) genes using the CRISPR-dCas9-VPR technology reveals potent N-glycome alterations in colorectal cancer cells.

Glycobiology 2019 02;29(2):137-150

Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Molecular Cell Biology and Immunology, Cancer Center Amsterdam, Amsterdam Infection & Immunity Institute, HZ Amsterdam, the Netherlands.

Aberrant fucosylation in cancer cells is considered as a signature of malignant cell transformation and it is associated with tumor progression, metastasis and resistance to chemotherapy. Specifically, in colorectal cancer cells, increased levels of the fucosylated Lewisx antigen are attributed to the deregulated expression of pertinent fucosyltransferases, like fucosyltransferase 4 (FUT4) and fucosyltransferase 9 (FUT9). However, the lack of experimental models closely mimicking cancer-specific regulation of fucosyltransferase gene expression has, so far, limited our knowledge regarding the substrate specificity of these enzymes and the impact of Lewisx synthesis on the glycome of colorectal cancer cells. Therefore, we sought to transcriptionally activate the Fut4 and Fut9 genes in the well-known murine colorectal cancer cell line, MC38, which lacks expression of the FUT4 and FUT9 enzymes. For this purpose, we utilized a physiologically relevant, guide RNA-based model of de novo gene expression, namely the CRISPR-dCas9-VPR system. Induction of the Fut4 and Fut9 genes in MC38 cells using CRISPR-dCas9-VPR resulted in specific neo-expression of functional Lewisx antigen on the cell surface. Interestingly, Lewisx was mainly carried by N-linked glycans in both MC38-FUT4 and MC38-FUT9 cells, despite pronounced differences in the biosynthetic properties and the expression stability of the induced enzymes. Moreover, Lewisx expression was found to influence core-fucosylation, sialylation, antennarity and the subtypes of N-glycans in the MC38-glycovariants. In conclusion, exploiting the CRISPR-dCas9-VPR system to augment glycosyltransferase expression is a promising method of transcriptional gene activation with broad application possibilities in glycobiology and oncology research.
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http://dx.doi.org/10.1093/glycob/cwy096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330019PMC
February 2019

High-throughput Serum -Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes.

Mol Cell Proteomics 2019 01 21;18(1):3-15. Epub 2018 Sep 21.

From the ‡Center for Proteomics and Metabolomics.

-Glycosylation is a fundamentally important protein modification with a major impact on glycoprotein characteristics such as serum half-life and receptor interaction. More than half of the proteins in human serum are glycosylated, and the relative abundances of protein glycoforms often reflect alterations in health and disease. Several analytical methods are currently capable of analyzing the total serum -glycosylation in a high-throughput manner.Here we evaluate and compare the performance of three high-throughput released -glycome analysis methods. Included were hydrophilic-interaction ultra-high-performance liquid chromatography with fluorescence detection (HILIC-UHPLC-FLD) with 2-aminobenzamide labeling of the glycans, multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) with 8-aminopyrene-1,3,6-trisulfonic acid labeling, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with linkage-specific sialic acid esterification. All methods assessed the same panel of serum samples, which were obtained at multiple time points during the pregnancies and postpartum periods of healthy women and patients with rheumatoid arthritis (RA). We compared the analytical methods on their technical performance as well as on their ability to describe serum protein -glycosylation changes throughout pregnancy, with RA, and with RA disease activity.Overall, the methods proved to be similar in their detection and relative quantification of serum protein -glycosylation. However, the non-MS methods showed superior repeatability over MALDI-TOF-MS and allowed the best structural separation of low-complexity -glycans. MALDI-TOF-MS achieved the highest throughput and provided compositional information on higher-complexity -glycans. Consequentially, MALDI-TOF-MS could establish the linkage-specific sialylation differences within pregnancy and RA, whereas HILIC-UHPLC-FLD and xCGE-LIF demonstrated differences in α1,3- and α1,6-branch galactosylation. While the combination of methods proved to be the most beneficial for the analysis of total serum protein -glycosylation, informed method choices can be made for the glycosylation analysis of single proteins or samples of varying complexity.
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http://dx.doi.org/10.1074/mcp.RA117.000454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317482PMC
January 2019

HappyTools: A software for high-throughput HPLC data processing and quantitation.

PLoS One 2018 6;13(7):e0200280. Epub 2018 Jul 6.

Research & Development, Ludger Limited., Abingdon, Oxfordshire, United Kingdom.

High-performance liquid chromatography (HPLC) is widely used for absolute quantitation. The advent of new columns and HPLC technology has enabled higher sample throughput, and hence, larger scale studies that perform quantitation on different sample types (e.g. healthy controls vs. patients with rheumatoid arthritis) using HPLC are becoming feasible. However, there remains a lack of methods that can analyse the increased number of HPLC samples. To address this in part, the modular toolkit HappyTools has been developed for the high-throughput targeted quantitation of HPLC measurements. HappyTools enables the user to create an automated workflow that includes retention time (tr) calibration, data extraction and the calculation of several quality criteria for data curation. HappyTools has been tested on a biopharmaceutical standard and previously published clinical samples. The results show comparable accuracy between HappyTools, Waters Empower and ThermoFisher Chromeleon. However, HappyTools offered superior precision and throughput when compared with Waters Empower and ThermoFisher Chromeleon. HappyTools is released under the Apache 2.0 license, both the source code and a Windows binary can be freely downloaded from https://github.com/Tarskin/HappyTools.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200280PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034860PMC
January 2019

Towards automation of glycomic profiling of complex biological materials.

Glycoconj J 2018 06 16;35(3):311-321. Epub 2018 Jun 16.

Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.

Glycosylation is considered one of the most complex and structurally diverse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious sample preparation steps. Therefore, in this study, we aim to improve sample preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein/protein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of samples proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.
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http://dx.doi.org/10.1007/s10719-018-9825-8DOI Listing
June 2018

GlycoStore: a database of retention properties for glycan analysis.

Bioinformatics 2018 09;34(18):3231-3232

Institute for Glycomics, Griffith University, Gold Coast, Australia.

Summary: GlycoStore is a curated chromatographic, electrophoretic and mass-spectrometry composition database of N-, O-, glycosphingolipid (GSL) glycans and free oligosaccharides associated with a range of glycoproteins, glycolipids and biotherapeutics. The database is built on publicly available experimental datasets from GlycoBase developed in the Oxford Glycobiology Institute and then the National Institute for Bioprocessing Research and Training (NIBRT). It has now been extended to include recently published and in-house data collections from the Bioprocessing Technology Institute (BTI) A*STAR, Macquarie University and Ludger Ltd. GlycoStore provides access to approximately 850 unique glycan structure entries supported by over 8500 retention positions determined by: (i) hydrophilic interaction chromatography (HILIC) ultra-high performance liquid chromatography (U/HPLC) and reversed phase (RP)-U/HPLC with fluorescent detection; (ii) porous graphitized carbon (PGC) chromatography in combination with ESI-MS/MS detection; and (iii) capillary electrophoresis with laser induced fluorescence detection (CE-LIF). GlycoStore enhances many features previously available in GlycoBase while addressing the limitations of the data collections and model of this popular resource. GlycoStore aims to support detailed glycan analysis by providing a resource that underpins current workflows. It will be regularly updated by expert annotation of published data and data obtained from the project partners.

Availability And Implementation: http://www.glycostore.org.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty319DOI Listing
September 2018

Engineering and stable production of recombinant IgE for cancer immunotherapy and AllergoOncology.

J Allergy Clin Immunol 2018 04 31;141(4):1519-1523.e9. Epub 2018 Jan 31.

St John's Institute of Dermatology, School of Basic & Medical Biosciences, King's College London, London, United Kingdom; NIHR Biomedical Research Centre at Guy's and St Thomas's Hospitals and King's College London, London, United Kingdom; Breast Cancer Now Unit, School of Cancer & Pharmaceutical Sciences, King's College London, Guy's Cancer Centre, London, United Kingdom. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2017.12.986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286379PMC
April 2018