Publications by authors named "Radka Saldova"

63 Publications

5-AZA-dC induces epigenetic changes associated with modified glycosylation of secreted glycoproteins and increased EMT and migration in chemo-sensitive cancer cells.

Clin Epigenetics 2021 Feb 12;13(1):34. Epub 2021 Feb 12.

GlycoScience Group, the National Institute for Bioprocessing, Research and Training (NIBRT), Fosters Avenue, Mount Merrion, Blackrock, Co Dublin, Ireland.

Background: Glycosylation, one of the most fundamental post-translational modifications, is altered in cancer and is subject in part, to epigenetic regulation. As there are many epigenetic-targeted therapies currently in clinical trials for the treatment of a variety of cancers, it is important to understand the impact epi-therapeutics have on glycosylation.

Results: Ovarian and triple negative breast cancer cells were treated with the DNA methyltransferase inhibitor, 5-AZA-2-deoxycytidine (5-AZA-dC). Branching and sialylation were increased on secreted N-glycans from chemo-sensitive/non-metastatic cell lines following treatment with 5-AZA-dC. These changes correlated with increased mRNA expression levels in MGAT5 and ST3GAL4 transcripts in ovarian cancer cell lines. Using siRNA transient knock down of GATA2 and GATA3 transcription factors, we show that these regulate the glycosyltransferases ST3GAL4 and MGAT5, respectively. Moreover, 5-AZA-dC-treated cells displayed an increase in migration, with a greater effect seen in chemo-sensitive cell lines. Western blots showed an increase in apoptotic and senescence (p21) markers in all 5-AZA-dC-treated cells. The alterations seen in N-glycans from secreted glycoproteins in 5-AZA-dC-treated breast and ovarian cancer cells were similar to the N-glycans previously known to potentiate tumour cell survival.

Conclusions: While the FDA has approved epi-therapeutics for some cancer treatments, their global effect is still not fully understood. This study gives insight into the effects that epigenetic alterations have on cancer cell glycosylation, and how this potentially impacts on the overall fate of those cells.
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http://dx.doi.org/10.1186/s13148-021-01015-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881483PMC
February 2021

N-Linked glycosylation profiles of therapeutic induced senescent (TIS) triple negative breast cancer cells (TNBC) and their extracellular vesicle (EV) progeny.

Mol Omics 2021 02 16;17(1):72-85. Epub 2020 Dec 16.

UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin (UCD), Dublin, Ireland.

Triple negative breast cancer (TNBC) has poor clinical outcomes and limited treatment options. Chemotherapy, while killing some cancer cells, can result in therapeutic-induced-senescent (TIS) cells. Senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Recently, N- and O-linked glycosylation alterations have been associated with senescence. We aimed to profile the N-linked glycans of whole cells, membrane, cytoplasm and EVs harvested from TIS TNBC cells and to compare these to results from non-senescent cells. TIS was induced in the Cal51 TNBC cells using the chemotherapeutic agent paclitaxel (PTX). Ultra-performance liquid chromatography (UPLC) analysis of exoglycosidase digested N-linked glycans was carried out on TIS compared to non-treated control cells. LC-Mass spectrometry (MS) analysis of the N-linked glycans and lectin blotting of samples was carried out to confirm the UPLC results. Significant differences were found in the N-glycan profile of the Cal51 membrane, cytoplasm and EV progeny of TIS compared to non-senescent cells. Protein mass spectrometry showed that the TIS cells contain different glycan modifying enzymes. The lectin, calnexin demonstrated a lower kDa size (∼58 kDa) in TIS compared to control cells (∼90 kDa) while Galectin 3 demonstrated potential proteolytic cleavage with 32 kDa and ∼22 kDa bands evident in TIS compared to non-senescent control cells with a major 32 kDa band only. TIS CAL51 cells also demonstrated a reduced adhesion to collagen I compared to control non-senescent cells. This study has shown that therapeutic-induced-senescent TNBC cells and their EV progeny, display differential N-glycan moieties compared to non-senescent Cal51 cells and their resultant EV progeny. For the future, N-glycan moieties on cancer senescent cells and their EV progeny hold potential for (i) the monitoring of treatment response as a liquid biopsy, and (ii) cancer senescent cell targeting with lectin therapies.
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http://dx.doi.org/10.1039/d0mo00017eDOI Listing
February 2021

Glycosylation in Indolent, Significant and Aggressive Prostate Cancer by Automated High-Throughput -Glycan Profiling.

Int J Mol Sci 2020 Dec 3;21(23). Epub 2020 Dec 3.

NIBRT GlycoScience Group, National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, A94 X099 Co. Dublin, Ireland.

The diagnosis and treatment of prostate cancer (PCa) is a major health-care concern worldwide. This cancer can manifest itself in many distinct forms and the transition from clinically indolent PCa to the more invasive aggressive form remains poorly understood. It is now universally accepted that glycan expression patterns change with the cellular modifications that accompany the onset of tumorigenesis. The aim of this study was to investigate if differential glycosylation patterns could distinguish between indolent, significant, and aggressive PCa. Whole serum -glycan profiling was carried out on 117 prostate cancer patients' serum using our automated, high-throughput analysis platform for glycan-profiling which utilizes ultra-performance liquid chromatography (UPLC) to obtain high resolution separation of -linked glycans released from the serum glycoproteins. We observed increases in hybrid, oligomannose, and biantennary digalactosylated monosialylated glycans (M5A1G1S1, M8, and A2G2S1), bisecting glycans (A2B, A2(6)BG1) and monoantennary glycans (A1), and decreases in triantennary trigalactosylated trisialylated glycans with and without core fucose (A3G3S3 and FA3G3S3) with PCa progression from indolent through significant and aggressive disease. These changes give us an insight into the disease pathogenesis and identify potential biomarkers for monitoring the PCa progression, however these need further confirmation studies.
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http://dx.doi.org/10.3390/ijms21239233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730228PMC
December 2020

Quantitative levels of serum N-glycans in type 1 diabetes and their association with kidney disease.

Glycobiology 2020 Nov 27. Epub 2020 Nov 27.

MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK.

We investigated associations of quantitative levels of N-glycans with haemoglobin A1c (HbA1c), renal function and renal function decline in type 1 diabetes. We measured 46 total N-glycan peaks (GPs) on 1565 serum samples from the Scottish Diabetes Research Network Type 1 Bioresource Study (SDRNT1BIO) and a pool of healthy donors. Quantitation of absolute abundance of each GP used 2AB-labelled mannose-3 as standard. We studied cross-sectional associations of GPs and derived measures with HbA1c, albumin/creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR), and prospective associations with incident albuminuria and final eGFR. All GPs were 1.4 to 3.2 times more abundant in SDRTN1BIO than in the healthy samples. Absolute levels of all GPs were slightly higher with higher HbA1c, with strongest associations for triantennary trigalactosylated disialylated, triantennary trigalactosylated trisialylated structures with core or outer arm fucose, and tetraantennary tetragalactosylated trisialylated glycans. Most GPs showed increased abundance with worsening ACR. Lower eGFR was associated with higher absolute GP level, most significantly with biantennary digalactosylated disialylated glycans with and without bisect, triantennary trigalactosylated trisialylated glycans with and without outer arm fucose, and core fucosylated biantennary monogalactosylated monosialylated glycans. Although several GPs were inversely associated prospectively with final eGFR, cross-validated multivariable models did not improve prediction beyond clinical covariates. Elevated HbA1c is associated with an altered N-glycan profile in type 1 diabetes. Although we could not establish GPs to be prognostic of future renal function decline independently of HbA1c, further studies to evaluate their impact in the pathogenesis of diabetic kidney disease are warranted.
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http://dx.doi.org/10.1093/glycob/cwaa106DOI Listing
November 2020

Characterisation of the main PSA glycoforms in aggressive prostate cancer.

Sci Rep 2020 11 4;10(1):18974. Epub 2020 Nov 4.

Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, C/Maria Aurèlia Capmany 40, 17003, Girona, Spain.

Serum levels of prostate specific antigen (PSA) are commonly used for prostate cancer (PCa) detection. However, their lack of specificity to distinguish benign prostate pathologies from PCa, or indolent from aggressive PCa have prompted the study of new non-invasive PCa biomarkers. Aberrant glycosylation is involved in neoplastic progression and specific changes in PSA glycosylation pattern, as the reduction in the percentage of α2,6-sialic acid (SA) are associated with PCa aggressiveness. In this study, we have characterised the main sialylated PSA glycoforms from blood serum of aggressive PCa patients and have compared with those of standard PSA from healthy individuals' seminal plasma. PSA was immunoprecipitated and α2,6-SA were separated from α2,3-SA glycoforms using SNA affinity chromatography. PSA N-glycans were released, labelled and analysed by hydrophilic interaction liquid chromatography combined with exoglycosidase digestions. The results showed that blood serum PSA sialylated glycoforms containing GalNAc residues were largely increased in aggressive PCa patients, whereas the disialylated core fucosylated biantennary structures with α2,6-SA, which are the major PSA glycoforms in standard PSA from healthy individuals, were markedly reduced in aggressive PCa. The identification of these main PSA glycoforms altered in aggressive PCa opens the way to design specific strategies to target them, which will be useful to improve PCa risk stratification.
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http://dx.doi.org/10.1038/s41598-020-75526-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643140PMC
November 2020

Deep phenotyping classical galactosemia: clinical outcomes and biochemical markers.

Brain Commun 2020 29;2(1):fcaa006. Epub 2020 Jan 29.

Division of Metabolic Disorders, Department of Pediatrics, Emma Children's Hospital, Amsterdam, UMC, University of Amsterdam, Amsterdam, the Netherlands.

Early diagnosis and dietary treatment do not prevent long-term complications, which mostly affect the central nervous system in classical galactosemia patients. The clinical outcome of patients is highly variable, and there is an urgent need for prognostic biomarkers. The aim of this study was first to increase knowledge on the natural history of classical galactosemia by studying a cohort of patients with varying geno- and phenotypes and second to study the association between clinical outcomes and two possible prognostic biomarkers. In addition, the association between abnormalities on brain MRI and clinical outcomes was investigated. Classical galactosemia patients visiting the galactosemia expertise outpatient clinic of the Amsterdam University Medical Centre were evaluated according to the International Classical Galactosemia guideline with the addition of an examination by a neurologist, serum immunoglobulin G -glycan profiling and a brain MRI. The biomarkers of interest were galactose-1-phosphate levels and -glycan profiles, and the clinical outcomes studied were intellectual outcome and the presence or absence of movement disorders and/or primary ovarian insufficiency. Data of 56 classical galactosemia patients are reported. The intellectual outcome ranged from 45 to 103 (mean 77 ± 14) and was <85 in 62%. Movement disorders were found in 17 (47%) of the 36 tested patients. In females aged 12 years and older, primary ovarian insufficiency was diagnosed in 12 (71%) of the 17 patients. Significant differences in -glycan peaks were found between controls and patients. However, no significant differences in either -glycans or galactose-1-phosphate levels were found between patients with a poor (intellectual outcome < 85) and normal intellectual outcome (intellectual outcome ≥ 85), and with or without movement disorders or primary ovarian insufficiency. The variant patients detected by newborn screening, with previously unknown geno- and phenotypes and currently no long-term complications, demonstrated significantly lower galactose-1-phospate levels than classical patients ( < 0.0005). Qualitative analysis of the MRI's demonstrated brain abnormalities in 18 of the 21 patients, more severely in patients with a lower intellectual outcome and/or with movement disorders. This study demonstrates a large variability in clinical outcome, which varies from a below average intelligence, movement disorders and in females primary ovarian insufficiency to a normal clinical outcome. In our cohort of classical galactosemia patients, galactose-1-phosphate levels and -glycan variations were not associated with clinical outcomes, but galactose-1-phosphate levels did differentiate between classical and variant patients detected by newborn screening. The correlation between brain abnormalities and clinical outcome should be further investigated by quantitative analysis of the MR images. The variability in clinical outcome necessitates individual and standardized evaluation of all classical galactosemia patients.
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http://dx.doi.org/10.1093/braincomms/fcaa006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425409PMC
January 2020

Current Methods for the Characterization of -Glycans.

J Proteome Res 2020 10 19;19(10):3890-3905. Epub 2020 Sep 19.

NIBRT GlycoScience Group, National Institute for Bioprocessing, Research and Training, Blackrock, Dublin, Ireland.

Glycosylation is crucial in cellular metabolism and survival. Of interest is the role of -linked and -linked glycans in disease states. Robust analytical methods must be defined to identify suitable glycan biomarkers and glyco-therapeutics. Fortunately, in -glycan analysis, a universal enzyme exists to deglycosylate a variety of common-core structures from proteins for analysis using mass spectrometric and fluorescence techniques. Unfortunately, for their -linked counterparts, no such enzyme exists. Furthermore, -glycan heterogeneity is vast due to the lack of a common glycan core, making analysis challenging. As such, chemical methods are used to liberate -glycans, however, often to the detriment of the glycan's structure due to "peeling" reactions. This review outlines approaches for -glycan release and downstream glycomic and glycoproteomic analysis.
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http://dx.doi.org/10.1021/acs.jproteome.0c00435DOI Listing
October 2020

Hypoxia Alters Epigenetic and -Glycosylation Profiles of Ovarian and Breast Cancer Cell Lines .

Front Oncol 2020 29;10:1218. Epub 2020 Jul 29.

GlycoScience Group, The National Institute for Bioprocessing Research and Training (NIBRT), Dublin, Ireland.

Glycosylation is one of the most fundamental post-translational modifications. Importantly, glycosylation is altered in many cancers. These alterations have been proven to impact on tumor progression and to promote tumor cell survival. From the literature, it is known that there is a clear link between chemoresistance and hypoxia, hypoxia and epigenetics and more recently glycosylation and epigenetics. Our objective was to investigate these differential parameters, in an model of ovarian and breast cancer. Ovarian (A2780, A2780cis, PEO1, PEO4) and triple negative breast cancer (TNBC) (MDA-MB-231 and MDA-MB-436) cells were exposed to differential hypoxic conditions (0.5-2% O) and compared to normoxia (21% O). Results demonstrated that in hypoxic conditions some significant changes in glycosylation on the secreted -glycans from the ovarian and breast cancer cell lines were observed. These included, alterations in oligomannosylated, bisected glycans, glycans with polylactosamine extensions, in branching, galactosylation and sialylation in all cell lines except for PEO1. In general, hypoxia exposed ovarian and TNBC cells also displayed increased epithelial to mesenchymal transition (EMT) and migration, with a greater effect seen in the 0.5% hypoxia exposed samples compared to 1 and 2% hypoxia ( ≤ 0.05). SiRNA transient knock down of transcription factors resulted in a decrease in the expression of glycosyltransferases and , which are responsible for sialylation and branching, respectively. These glycan changes are known to be integral to cancer cell survival and metastases, suggesting a possible mechanism of action, linking GATA2 and 3, and invasiveness of both ovarian and TNBC cells .
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http://dx.doi.org/10.3389/fonc.2020.01218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405916PMC
July 2020

Region-Specific Characterization of -Glycans in the Striatum and Substantia Nigra of an Adult Rodent Brain.

Anal Chem 2020 10 14;92(19):12842-12851. Epub 2020 Sep 14.

GlycoScience Group, National Institute for Bioprocessing Research and Training (NIBRT), Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin A94X099, Ireland.

-glycan alterations in the nervous system can result in different neuropathological symptoms such as mental retardation, seizures, and epilepsy. Studies have reported the characterization of -glycans in rodent brains, but there is a lack of spatial resolution as either the tissue samples were homogenized or specific proteins were selected for analysis of glycosylation. We hypothesize that region-specific resolution of -glycans isolated from the striatum and substantia nigra (SN) can give an insight into the establishment and pathophysiological degeneration of neural circuitry in Parkinson's disease. Specific objectives of the study include isolation of -glycans from the rat striatum and SN; reproducibility, resolution, and relative quantitation of -glycome using ultra-performance liquid chromatography (UPLC), weak anion exchange-UPLC, and lectin histochemistry. The total -glycomes from the striatum and SN were characterized using database mining (GlycoStore), exoglycosidase digestions, and liquid chromatography-mass spectrometry. It revealed significant differences in complex and oligomannose type -glycans, sialylation (mono-, di-, and tetra-), fucosylation (tri-, core, and outer arm), and galactosylation (di-, tri-, and tetra-) between striatum and SN -glycans with the detection of phosphorylated -glycans in SN which were not detected in the striatum. This study presents the most comprehensive comparative analysis of relative abundances of -glycans in the striatum and SN of rodent brains, serving as a foundation for identifying "brain-type" glycans as biomarkers or therapeutic targets and their modulation in neurodegenerative disorders.
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http://dx.doi.org/10.1021/acs.analchem.0c01206DOI Listing
October 2020

Maternal and infant factors that shape neonatal gut colonization by bacteria.

Expert Rev Gastroenterol Hepatol 2020 Aug 30;14(8):651-664. Epub 2020 Jun 30.

APC Microbiome Ireland, National University of Ireland , Cork, Ireland.

Introduction: Early life is a critical developmental window coinciding with the establishment of a community of neonatal gut microbes which are vitally important for immune development. The composition of this microbial community is affected by multiple factors.

Areas Covered: The effect of pre-pregnancy and pregnancy maternal health, maternal nutrition, pregnancy disorders such as gestational diabetes, maternal antibiotic usage, delivery mode, infant feeding, and infant antibiotic usage on gut microbial composition are outlined along with the potential impact of associated microbiota differences on infant health.

Expert Opinion: Recent developments in understanding what shapes our microbiota indicates that the greatest impact on infant gut microbiota composition during the first year of life is seen with the mode of delivery, infant diet, and infant antibiotic usage. Current data is insufficient to fully establish the role of apparently less important factors such as maternal health on microbiota development although their impact is likely smaller. Technological advances will allow for improved understanding of underlying mechanisms by which specific microbes impact on infant health, which in time will enable full appreciation of the role of the gut microbiota in early life development.
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http://dx.doi.org/10.1080/17474124.2020.1784725DOI Listing
August 2020

Can a probiotic supplement in pregnancy result in transfer to the neonatal gut: A systematic review.

Acta Obstet Gynecol Scand 2020 10 30;99(10):1269-1277. Epub 2020 May 30.

UCD Perinatal Research Center, School of Medicine, University College Dublin, National Maternity Hospital, Dublin, Ireland.

Introduction: The establishment of the neonatal gut microbiome is a crucial step that may have lifelong health implications. We aimed to systematically review evidence on maternal probiotic supplementation during pregnancy and vertical transfer of the corresponding strain to the infant gut.

Material And Methods: Medline, CINAHL, Embase, Web of Science, and OVID were searched from inception to September 2018. Studies of maternal probiotic supplementation for a minimum duration of 2 weeks and analyses of neonatal stool samples were included. The primary outcome was presence of the specific probiotic strain in the infant stool. Electronic databases were searched for relevant studies and references were cross-checked. Risk of bias among included studies was assessed and data were extracted independently by two authors.

Results: Three studies were included in the review. Only one study was identified involving prenatal maternal probiotic supplementation alone. Neonatal colonization with the maternally administered probiotic was not demonstrated but supplementation with the probiotic influenced levels of a bacterial strain other than that found in the probiotic product. The other two studies identified included both prenatal and postnatal supplementation of either mother or infant. All three studies reported employing strain-specific isolation methodology to isolate the supplemented bacterial strain in infant stool but none used whole metagenome shotgun sequencing.

Conclusions: Few studies investigating transfer of a specific probiotic bacterial strain from mother to infant were identified, showing inconclusive evidence of vertical transfer.
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http://dx.doi.org/10.1111/aogs.13899DOI Listing
October 2020

The association between the maternal diet and the maternal and infant gut microbiome: a systematic review.

Br J Nutr 2020 Mar 4:1-29. Epub 2020 Mar 4.

UCD Perinatal Research Centre, School of Medicine, University College Dublin, National Maternity Hospital, Dublin, Ireland.

During pregnancy, changes occur to influence the maternal gut microbiome, and potentially the fetal microbiome. Diet has been shown to impact the gut microbiome. Little research has been conducted examining diet during pregnancy with respect to the gut microbiome. To meet inclusion criteria, dietary analyses must have been conducted as part of the primary aim. The primary outcome was the composition of the gut microbiome (infant or maternal), as assessed using culture-independent sequencing techniques. This review identified seven studies for inclusion, five examining the maternal gut microbiome and two examining the fetal gut microbiome. Microbial data were attained through analysis of stool samples by 16S rRNA gene-based microbiota assessment. Studies found an association between the maternal diet and gut microbiome. High-fat diets (% fat of total energy), fat-soluble vitamins (mg/day) and fibre (g/day) were the most significant nutrients associated with the gut microbiota composition of both neonates and mothers. High-fat diets were significantly associated with a reduction in microbial diversity. High-fat diets may reduce microbial diversity, while fibre intake may be positively associated with microbial diversity. The results of this review must be interpreted with caution. The number of studies was low, and the risk of observational bias and heterogeneity across the studies must be considered. However, these results show promise for dietary intervention and microbial manipulation in order to favour an increase of health-associated taxa in the gut of the mother and her offspring.
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http://dx.doi.org/10.1017/S0007114520000847DOI Listing
March 2020

Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy.

Sci Rep 2020 01 30;10(1):1464. Epub 2020 Jan 30.

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

Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77-81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fucosylation mediated more efficient ADCC and clearance than anti-D Ig. Galactosylation of B mAb-Ds was 57-83% but 15-58% for rodent mAb-Ds. HH mAb-Ds had non-human sugars. These data reveal high galactosylation like anti-D Ig (>60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis.
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http://dx.doi.org/10.1038/s41598-019-57393-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992666PMC
January 2020

IgG Fc glycosylation as an axis of humoral immunity in childhood.

J Allergy Clin Immunol 2020 02 24;145(2):710-713.e9. Epub 2019 Oct 24.

Division of Immunology, Boston Children's Hospital, Boston, Mass; Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Boston, Mass. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2019.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010538PMC
February 2020

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Authors:
Maria Lorna A De Leoz David L Duewer Adam Fung Lily Liu Hoi Kei Yau Oscar Potter Gregory O Staples Kenichiro Furuki Ruth Frenkel Yunli Hu Zoran Sosic Peiqing Zhang Friedrich Altmann Clemens Gru Nwald-Grube Chun Shao Joseph Zaia Waltraud Evers Stuart Pengelley Detlev Suckau Anja Wiechmann Anja Resemann Wolfgang Jabs Alain Beck John W Froehlich Chuncui Huang Yan Li Yaming Liu Shiwei Sun Yaojun Wang Youngsuk Seo Hyun Joo An Niels-Christian Reichardt Juan Echevarria Ruiz Stephanie Archer-Hartmann Parastoo Azadi Len Bell Zsuzsanna Lakos Yanming An John F Cipollo Maja Pucic-Bakovic Jerko Štambuk Gordan Lauc Xu Li Peng George Wang Andreas Bock René Hennig Erdmann Rapp Marybeth Creskey Terry D Cyr Miyako Nakano Taiki Sugiyama Pui-King Amy Leung Paweł Link-Lenczowski Jolanta Jaworek Shuang Yang Hui Zhang Tim Kelly Song Klapoetke Rui Cao Jin Young Kim Hyun Kyoung Lee Ju Yeon Lee Jong Shin Yoo Sa-Rang Kim Soo-Kyung Suh Noortje de Haan David Falck Guinevere S M Lageveen-Kammeijer Manfred Wuhrer Robert J Emery Radoslaw P Kozak Li Phing Liew Louise Royle Paulina A Urbanowicz Nicolle H Packer Xiaomin Song Arun Everest-Dass Erika Lattová Samanta Cajic Kathirvel Alagesan Daniel Kolarich Toyin Kasali Viv Lindo Yuetian Chen Kudrat Goswami Brian Gau Ravi Amunugama Richard Jones Corné J M Stroop Koichi Kato Hirokazu Yagi Sachiko Kondo C T Yuen Akira Harazono Xiaofeng Shi Paula E Magnelli Brian T Kasper Lara Mahal David J Harvey Roisin O'Flaherty Pauline M Rudd Radka Saldova Elizabeth S Hecht David C Muddiman Jichao Kang Prachi Bhoskar Daniele Menard Andrew Saati Christine Merle Steven Mast Sam Tep Jennie Truong Takashi Nishikaze Sadanori Sekiya Aaron Shafer Sohei Funaoka Masaaki Toyoda Peter de Vreugd Cassie Caron Pralima Pradhan Niclas Chiang Tan Yehia Mechref Sachin Patil Jeffrey S Rohrer Ranjan Chakrabarti Disha Dadke Mohammedazam Lahori Chunxia Zou Christopher Cairo Béla Reiz Randy M Whittal Carlito B Lebrilla Lauren Wu Andras Guttman Marton Szigeti Benjamin G Kremkow Kelvin H Lee Carina Sihlbom Barbara Adamczyk Chunsheng Jin Niclas G Karlsson Jessica Örnros Göran Larson Jonas Nilsson Bernd Meyer Alena Wiegandt Emy Komatsu Helene Perreault Edward D Bodnar Nassur Said Yannis-Nicolas Francois Emmanuelle Leize-Wagner Sandra Maier Anne Zeck Albert J R Heck Yang Yang Rob Haselberg Ying Qing Yu William Alley Joseph W Leone Hua Yuan Stephen E Stein

Mol Cell Proteomics 2020 01 7;19(1):11-30. Epub 2019 Oct 7.

Mass Spectrometry Data Center, Biomolecular Measurement Division, Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau Drive Gaithersburg, Maryland 20899.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.
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http://dx.doi.org/10.1074/mcp.RA119.001677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944243PMC
January 2020

A Robust and Versatile Automated Glycoanalytical Technology for Serum Antibodies and Acute Phase Proteins: Ovarian Cancer Case Study.

Mol Cell Proteomics 2019 11 30;18(11):2191-2206. Epub 2019 Aug 30.

NIBRT GlycoScience Group, National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland, A94X099.

The direct association of the genome, transcriptome, metabolome, lipidome and proteome with the serum glycome has revealed systems of interconnected cellular pathways. The exact roles of individual glycoproteomes in the context of disease have yet to be elucidated. In a move toward personalized medicine, it is now becoming critical to understand disease pathogenesis, and the traits, stages, phenotypes and molecular features that accompany it, as the disruption of a whole system. To this end, we have developed an innovative technology on an automated platform, "GlycoSeqCap," which combines -glycosylation data from six glycoproteins using a single source of human serum. Specifically, we multiplexed and optimized a successive serial capture and glycoanalysis of six purified glycoproteins, immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA), transferrin (Trf), haptoglobin (Hpt) and alpha-1-antitrypsin (A1AT), from 50 μl of human serum. We provide the most comprehensive and in-depth glycan analysis of individual glycoproteins in a single source of human serum to date. To demonstrate the technological application in the context of a disease model, we performed a pilot study in an ovarian cancer cohort ( = 34) using discrimination and classification analyses to identify aberrant glycosylation. In our sample cohort, we exhibit improved selectivity and specificity over the currently used biomarker for ovarian cancer, CA125, for early stage ovarian cancer. This technology will establish a new state-of-the-art strategy for the characterization of individual serum glycoproteomes as a diagnostic and monitoring tool which represents a major step toward understanding the changes that take place during disease.
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http://dx.doi.org/10.1074/mcp.RA119.001531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823853PMC
November 2019

Circulating Markers of Inflammation Persist in Children and Adults With Giant Aneurysms After Kawasaki Disease.

Circ Genom Precis Med 2019 04 7;12(4):e002433. Epub 2019 Mar 7.

University of California San Diego School of Medicine (J.O., C.S., S.H., A.M.K., L.B.D., A.H.T., J.C.B.).

Background: The sequelae of Kawasaki disease (KD) vary widely with the greatest risk for future cardiovascular events among those who develop giant coronary artery aneurysms (CAA). We sought to define the molecular signature associated with different outcomes in pediatric and adult KD patients.

Methods: Molecular profiling was conducted using mass spectrometry-based shotgun proteomics, transcriptomics, and glycomics methods on 8 pediatric KD patients at the acute, subacute, and convalescent time points. Shotgun proteomics was performed on 9 KD adults with giant CAA and matched healthy controls. Plasma calprotectin was measured by ELISA in 28 pediatric KD patients 1 year post-KD, 70 adult KD patients, and 86 healthy adult volunteers.

Results: A characteristic molecular profile was seen in pediatric patients during the acute disease, which resolved at the subacute and convalescent periods in patients with no coronary artery sequelae but persisted in 2 patients who developed giant CAA. We, therefore, investigated persistence of inflammation in KD adults with giant CAA by shotgun proteomics that revealed a signature of active inflammation, immune regulation, and cell trafficking. Correlating results obtained using shotgun proteomics in the pediatric and adult KD cohorts identified elevated calprotectin levels in the plasma of patients with CAA. Investigation of expanded pediatric and adult KD cohorts revealed elevated levels of calprotectin in pediatric patients with giant CAA 1 year post-KD and in adult KD patients who developed giant CAA in childhood.

Conclusions: Complex patterns of biomarkers of inflammation and cell trafficking can persist long after the acute phase of KD in patients with giant CAA. Elevated levels of plasma calprotectin months to decades after acute KD and infiltration of cells expressing S100A8 and A9 in vascular tissues suggest ongoing, subclinical inflammation. Calprotectin may serve as a biomarker to inform the management of KD patients following the acute illness.
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http://dx.doi.org/10.1161/CIRCGEN.118.002433DOI Listing
April 2019

Circulating Truncated Alpha-1 Antitrypsin Glycoprotein in Patient Plasma Retains Anti-Inflammatory Capacity.

J Immunol 2019 04 22;202(8):2240-2253. Epub 2019 Feb 22.

Irish Centre for Genetic Lung Disease, Department of Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland.

Alpha-1 antitrypsin (AAT) is an acute phase protein that possesses immune-regulatory and anti-inflammatory functions independent of antiprotease activity. AAT deficiency (AATD) is associated with early-onset emphysema and chronic obstructive pulmonary disease. Of interest are the AATD nonsense mutations (termed null or Q0), the majority of which arise from premature termination codons in the mRNA coding region. We have recently demonstrated that plasma from an AATD patient homozygous for the Null Bolton allele ( ) contains AAT protein of truncated size. Although the potential to alleviate the phenotypic consequences of AATD by increasing levels of truncated protein holds therapeutic promise, protein functionality is key. The goal of this study was to evaluate the structural features and anti-inflammatory capacity of Q0-AAT. A low-abundance, truncated AAT protein was confirmed in plasma of a Q0-AATD patient and was secreted by patient-derived induced pluripotent stem cell-hepatic cells. Functional assays confirmed the ability of purified Q0-AAT protein to bind neutrophil elastase and to inhibit protease activity. Q0-AAT bound IL-8 and leukotriene B, comparable to healthy control M-AAT, and significantly decreased leukotriene B-induced neutrophil adhesion ( = 0.04). Through a mechanism involving increased mRNA stability ( = 0.007), ataluren treatment of HEK-293 significantly increased mRNA expression ( = 0.03) and Q0-AAT truncated protein secretion ( = 0.04). Results support the rationale for treatment with pharmacological agents that augment levels of functional Q0-AAT protein, thus offering a potential therapeutic option for AATD patients with rare mutations of similar theratype.
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http://dx.doi.org/10.4049/jimmunol.1801045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452030PMC
April 2019

Resident bacteria in breast cancer tissue: pathogenic agents or harmless commensals?

Discov Med 2018 09;26(142):93-102

UCD School of Medicine, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, UCD, Belfield, Dublin, Ireland.

Breast cancer is the second most common cancer in women. Recent evidence identifies a unique microbiome in breast tissue; a site previously thought to be sterile. The identification that this microbiome varies considerably from healthy subjects to cancer patients has prompted investigations into the role of specific bacterial species in oncogenesis. Indeed, certain bacteria have been shown to aid cancer development in vitro by promoting genomic instability, invasion, and chemotherapy resistance. However, the in vivo role of the breast microbiome in cancer appears to be more complex, involving numerous interactions between its constituent species and host cells. As such, reduced abundances of species which exert a protective effect against oncogenesis have come into focus and there is an emerging consensus that states of microbial dysbiosis, in which the normal balance of bacterial species is altered, can contribute to the development of cancer. This review summarizes the findings to date from the available literature pertaining to the microbiome in breast cancer and outlines areas worthy of further investigation.
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September 2018

Integrating biomarkers across omic platforms: an approach to improve stratification of patients with indolent and aggressive prostate cancer.

Mol Oncol 2018 09 7;12(9):1513-1525. Epub 2018 Aug 7.

UCD School of Medicine, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland.

Classifying indolent prostate cancer represents a significant clinical challenge. We investigated whether integrating data from different omic platforms could identify a biomarker panel with improved performance compared to individual platforms alone. DNA methylation, transcripts, protein and glycosylation biomarkers were assessed in a single cohort of patients treated by radical prostatectomy. Novel multiblock statistical data integration approaches were used to deal with missing data and modelled via stepwise multinomial logistic regression, or LASSO. After applying leave-one-out cross-validation to each model, the probabilistic predictions of disease type for each individual panel were aggregated to improve prediction accuracy using all available information for a given patient. Through assessment of three performance parameters of area under the curve (AUC) values, calibration and decision curve analysis, the study identified an integrated biomarker panel which predicts disease type with a high level of accuracy, with Multi AUC value of 0.91 (0.89, 0.94) and Ordinal C-Index (ORC) value of 0.94 (0.91, 0.96), which was significantly improved compared to the values for the clinical panel alone of 0.67 (0.62, 0.72) Multi AUC and 0.72 (0.67, 0.78) ORC. Biomarker integration across different omic platforms significantly improves prediction accuracy. We provide a novel multiplatform approach for the analysis, determination and performance assessment of novel panels which can be applied to other diseases. With further refinement and validation, this panel could form a tool to help inform appropriate treatment strategies impacting on patient outcome in early stage prostate cancer.
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http://dx.doi.org/10.1002/1878-0261.12348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120220PMC
September 2018

N-glycan signatures identified in tumor interstitial fluid and serum of breast cancer patients: association with tumor biology and clinical outcome.

Mol Oncol 2018 06 14;12(6):972-990. Epub 2018 May 14.

Danish Cancer Society Research Center, Genome Integrity Unit, Breast Cancer Biology Group, Copenhagen, Denmark.

Particular N-glycan structures are known to be associated with breast malignancies by coordinating various regulatory events within the tumor and corresponding microenvironment, thus implying that N-glycan patterns may be used for cancer stratification and as predictive or prognostic biomarkers. However, the association between N-glycans secreted by breast tumor and corresponding clinical relevance remain to be elucidated. We profiled N-glycans by HILIC UPLC across a discovery dataset composed of tumor interstitial fluids (TIF, n = 85), paired normal interstitial fluids (NIF, n = 54) and serum samples (n = 28) followed by independent evaluation, with the ultimate goal of identifying tumor-related N-glycan patterns in blood of patients with breast cancer. The segregation of N-linked oligosaccharides revealed 33 compositions, which exhibited differential abundances between TIF and NIF. TIFs were depleted of bisecting N-glycans, which are known to play essential roles in tumor suppression. An increased level of simple high mannose N-glycans in TIF strongly correlated with the presence of tumor infiltrating lymphocytes within tumor. At the same time, a low level of highly complex N-glycans in TIF inversely correlated with the presence of infiltrating lymphocytes within tumor. Survival analysis showed that patients exhibiting increased TIF abundance of GP24 had better outcomes, whereas low levels of GP10, GP23, GP38, and coreF were associated with poor prognosis. Levels of GP1, GP8, GP9, GP14, GP23, GP28, GP37, GP38, and coreF were significantly correlated between TIF and paired serum samples. Cross-validation analysis using an independent serum dataset supported the observed correlation between TIF and serum, for five of nine N-glycan groups: GP8, GP9, GP14, GP23, and coreF. Collectively, our results imply that profiling of N-glycans from proximal breast tumor fluids is a promising strategy for determining tumor-derived glyco-signature(s) in the blood. N-glycans structures validated in our study may serve as novel biomarkers to improve the diagnostic and prognostic stratification of patients with breast cancer.
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http://dx.doi.org/10.1002/1878-0261.12312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983225PMC
June 2018

Expression, Purification, and Biochemical Characterization of Human Afamin.

J Proteome Res 2018 03 20;17(3):1269-1277. Epub 2018 Feb 20.

Vitateq Biotechnology GmbH , A-6020 Innsbruck, Austria.

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.
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http://dx.doi.org/10.1021/acs.jproteome.7b00867DOI Listing
March 2018

Erratum: Improvement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes: Erratum.

Theranostics 2018 1;8(3):746-748. Epub 2018 Jan 1.

Biochemistry and Molecular Biology Unit. Department of Biology, University of Girona, Campus Montilivi, 17071, Girona, Spain.

[This corrects the article DOI: 10.7150/thno.15226.].
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http://dx.doi.org/10.7150/thno.23906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771090PMC
January 2018

Serum N-glycome alterations in breast cancer during multimodal treatment and follow-up.

Mol Oncol 2017 10 24;11(10):1361-1379. Epub 2017 Jul 24.

NIBRT GlycoScience Group, National Institute for Bioprocessing Research and Training, Dublin, Ireland.

Using our recently developed high-throughput automated platform, N-glycans from all serum glycoproteins from patients with breast cancer were analysed at diagnosis, after neoadjuvant chemotherapy, surgery, radiotherapy and up to 3 years after surgery. Surprisingly, alterations in the serum N-glycome after chemotherapy were pro-inflammatory with an increase in glycan structures associated with cancer. Surgery, on the other hand, induced anti-inflammatory changes in the serum N-glycome, towards a noncancerous phenotype. At the time of first follow-up, glycosylation in patients with affected lymph nodes changed towards a malignant phenotype. C-reactive protein showed a different pattern, increasing after first line of neoadjuvant chemotherapy, then decreasing throughout treatment until 1 year after surgery. This may reflect a switch from acute to chronic inflammation, where chronic inflammation is reflected in the serum after the acute phase response subsides. In conclusion, we here present the first time-course serum N-glycome profiling of patients with breast cancer during and after treatment. We identify significant glycosylation changes with chemotherapy, surgery and follow-up, reflecting the host response to therapy and tumour removal.
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http://dx.doi.org/10.1002/1878-0261.12105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623820PMC
October 2017

Glycosylation engineering of therapeutic IgG antibodies: challenges for the safety, functionality and efficacy.

Protein Cell 2018 01 8;9(1):47-62. Epub 2017 Jun 8.

NIBRT GlycoScience Group, National Institute for Bioprocessing Research and Training, Mount Merrion, Blackrock, Dublin 4, Ireland.

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex-type oligosaccharide attached to Asn297 of the Fc is essential for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that generate structural heterogeneity (glycoforms) exhibit unique biological activities. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for the development and quality control of therapeutic antibodies, and glycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosylation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibilities for the design of novel antibody therapeutics. Furthermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosynthases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as next-generation therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety, functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.
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http://dx.doi.org/10.1007/s13238-017-0433-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777974PMC
January 2018

Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics.

Methods 2017 03 8;116:63-83. Epub 2016 Nov 8.

NIBRT GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland. Electronic address:

This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose β-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose β-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product.
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http://dx.doi.org/10.1016/j.ymeth.2016.11.002DOI Listing
March 2017

Cause of cancer and chronic inflammatory diseases and the implications for treatment.

Authors:
Radka Saldova

Discov Med 2016 09;22(120):105-119

NIBRT GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Ave., Mount Merrion, Blackrock, Dublin 4, Ireland.

Many pathogens exist in metabolically inactive, non-culturable, cell-wall-deficient (CWD) forms that allow them to survive in conditions not conducive for growth. These forms were found in both cancer and chronic inflammatory autoimmune diseases. This review presents several novel concepts about how chronic inflammatory response and cancer develops from CWD infection, involvement and role of the immune system and other 'omics' systems and how to better diagnose, treat and even cure these conditions. This concept shows that CWD forms of intracellular microbes could also be evolutionary advantageous spreading through the host without using the classical replication route.
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September 2016

Epigenetic regulation of glycosylation and the impact on chemo-resistance in breast and ovarian cancer.

Epigenetics 2016 Dec 30;11(12):845-857. Epub 2016 Sep 30.

a NIBRT GlycoScience Group , The National Institute for Bioprocessing Research and Training , Mount Merrion, Blackrock, Dublin , Ireland.

Glycosylation is one of the most fundamental posttranslational modifications in cellular biology and has been shown to be epigenetically regulated. Understanding this process is important as epigenetic therapies such as those using DNA methyltransferase inhibitors are undergoing clinical trials for the treatment of ovarian and breast cancer. Previous work has demonstrated that altered glycosylation patterns are associated with aggressive disease in women presenting with breast and ovarian cancer. Moreover, the tumor microenvironment of hypoxia results in globally altered DNA methylation and is associated with aggressive cancer phenotypes and chemo-resistance, a feature integral to many cancers. There is sparse knowledge on the impact of these therapies on glycosylation. Moreover, little is known about the efficacy of DNA methyltransferase inhibitors in hypoxic tumors. In this review, we interrogate the impact that hypoxia and epigenetic regulation has on cancer cell glycosylation in relation to resultant tumor cell aggressiveness and chemo-resistance.
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http://dx.doi.org/10.1080/15592294.2016.1241932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5193495PMC
December 2016

Comprehensive N-Glycan Profiling of Avian Immunoglobulin Y.

PLoS One 2016 26;11(7):e0159859. Epub 2016 Jul 26.

NIBRT GlycoScience Group, National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Dublin 4, Ireland.

Recent exploitation of the avian immune system has highlighted its suitability for the generation of high-quality, high-affinity antibodies to a wide range of antigens for a number of therapeutic and biotechnological applications. The glycosylation profile of potential immunoglobulin therapeutics is species specific and is heavily influenced by the cell-line/culture conditions used for production. Hence, knowledge of the carbohydrate moieties present on immunoglobulins is essential as certain glycan structures can adversely impact their physicochemical and biological properties. This study describes the detailed N-glycan profile of IgY polyclonal antibodies from the serum of leghorn chickens using a fully quantitative high-throughput N-glycan analysis approach, based on ultra-performance liquid chromatography (UPLC) separation of released glycans. Structural assignments revealed serum IgY to contain complex bi-, tri- and tetra-antennary glycans with or without core fucose and bisects, hybrid and high mannose glycans. High sialic acid content was also observed, with the presence of rare sialic acid structures, likely polysialic acids. It is concluded that IgY is heavily decorated with complex glycans; however, no known non-human or immunogenic glycans were identified. Thus, IgY is a potentially promising candidate for immunoglobulin-based therapies for the treatment of various infectious diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0159859PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961449PMC
July 2017