UNASSIGNED: Endometriosis presents diagnostic challenges, and there is a need for developing novel biomarkers with satisfactory specificity and sensitivity. Glycomics, exploring glycosylation changes in glycoproteins, offers potential solutions. The aim of this study was to analyze the carbohydrate-binding properties of IgG and IgM antibodies in the plasma and peritoneal fluid samples and to identify any differences in the presence and the specificities of anti-carbohydrate antibodies in the endometriosis patient and the controls.
UNASSIGNED: Multicenter study was conducted in Poland between 2018 and 2019. Plasma and peritoneal fluid samples were collected from women undergoing laparoscopic surgery. Endometriosis patients (n=8) and controls (n=8), matched for cycle phase and disease stage, were selected. The neoglycolipid-based oligosaccharide microarray system was used to investigate IgG and IgM antibody binding properties to glycan-related probes in biological materials.
UNASSIGNED: In peritoneal fluid samples, IgM binding to the following probes was significantly higher in endometriosis: GSC-915-4 (new), LNFP-I, NeuAcα-(6\')LNnO (F1), B-like decaosylceramide, log10(GM1-penta), and log10(GSC-915-5). In a control group higher IgG binding to log10(Orsay-5-AO) was observed. In plasma samples, endometriosis showed higher IgG binding to log10(NeuAcα-(6\')LNnO (F1)) and lower IgG binding to Gal2GlcNAc(1-3)-AO. After Benjamin-Hochberg correction, differences were not significant. Effect sizes highlighted some glycan probes in both plasma and peritoneal fluid. Strong correlations were observed among binding to certain glycan probes.
UNASSIGNED: This preliminary study suggests glycomics\' potential contribution to endometriosis diagnosis and understanding of its pathophysiology. Neoglycolipid-based microarrays hold promise for non-invasive endometriosis diagnostic tools. Further investigations with larger cohorts are warranted to validate these findings and explore potential correlations with antibody levels in plasma and peritoneal fluid. Glycomics emerges as a valuable diagnostic asset in endometriosis research.

All cells are decorated with complex carbohydrate structures called glycans that serve as ligands for glycan-binding proteins (GBPs) to mediate a wide range of biological processes. Understanding the specific functions of glycans is key to advancing an understanding of human health and disease. However, the lack of convenient and accessible tools to study glycan-based interactions has been a defining challenge in glycobiology. Thus, the development of chemical and biochemical strategies to address these limitations has been a rapidly growing area of research. In this review, we describe the use of glycosyltransferases (GTs) as versatile tools to facilitate a greater understanding of the biological roles of glycans. We highlight key examples of how GTs have streamlined the preparation of well-defined complex glycan structures through chemoenzymatic synthesis, with an emphasis on synthetic strategies allowing for site- and branch-specific display of glyco-epitopes. We also describe how GTs have facilitated expansion of glyco-engineering strategies, on both glycoproteins and cell surfaces. Coupled with advancements in bioorthogonal chemistry, GTs have enabled selective glyco-epitope editing of glycoproteins and cells, selective glycan subclass labeling, and the introduction of novel biomolecule functionalities onto cells, including defined oligosaccharides, antibodies, and other proteins. Collectively, these approaches have contributed great insight into the fundamental biological roles of glycans and are enabling their application in drug development and cellular therapies, leaving the field poised for rapid expansion.

Rapid dietary changes, such as switching from high-forage to high-grain diets, can modify the rumen microbiome and initiate gastrointestinal distress, such as bloating. In such cases, feed additives, including prebiotics and live microbials, can be used to mitigate these negative consequences. Bio-Mos® is a carbohydrate-based prebiotic derived from yeast cells that is reported to increase livestock performance. Here, the responses of rumen bacterial cells to Bio-Mos® were quantified, sorted by flow cytometry using fluorescently-labeled yeast mannan, and taxonomically characterized using fluorescence in situ hybridization and 16S rRNA sequencing. Further, to evaluate the effects of bovine-adapted Bacteroides thetaiotaomicron administration as a live microbial with and without Bio-Mos® supplementation, we analyzed microbial fermentation products, changes to carbohydrate profiles, and shifts in microbial composition of an in vitro rumen community. Bio-Mos® was shown to be an effective prebiotic that significantly altered microbial diversity, composition, and fermentation; while addition of B. thetaiotaomicron had no effect on community composition and resulted in fewer significant changes to microbial fermentation. When combined with Bio-Mos®, there were notable, although not significant, changes to major bacterial taxa, along with increased significant changes in fermentation end products. These data suggest a synergistic effect is elicited by combining Bio-Mos® and B. thetaiotaomicron. This protocol provides a new in vitro methodology that could be extended to evaluate prebiotics and probiotics in more complex artificial rumen systems and live animals.

Lung cancer is the leading cause of cancer death and non-small cell lung carcinoma (NSCLC) accounting for majority of lung cancers. Thus, it is important to find potential biomarkers, such as glycans and glycoproteins, which can be used as diagnostic tools against NSCLC. Here, the N-glycome, proteome, and N-glycosylation distribution maps of tumor and peritumoral tissues of Filipino lung cancer patients (n = 5) were characterized. We present several case studies with varying stages of cancer development (I-III), mutation status (EGFR, ALK), and biomarker expression based on a three-gene panel (CD133, KRT19, and MUC1). Although the profiles of each patient were unique, specific trends arose that correlated with the role of aberrant glycosylation in cancer progression. Specifically, we observed a general increase in the relative abundance of high-mannose and sialofucosylated N-glycans in tumor samples. Analysis of the glycan distribution per glycosite revealed that these sialofucosylated N-glycans were specifically attached to glycoproteins involved in key cellular processes, including metabolism, cell adhesion, and regulatory pathways. Protein expression profiles showed significant enrichment of dysregulated proteins involved in metabolism, adhesion, cell-ECM interactions, and N-linked glycosylation, supporting the protein glycosylation results. The present case series study provides the first demonstration of a multi-platform mass-spectrometric analysis specifically for Filipino lung cancer patients.

All living cells are coated with a diverse collection of carbohydrate molecules called glycans. Glycans are key regulators of cell behavior and important therapeutic targets for human disease. Unlike proteins, glycans are not directly templated by discrete genes. Instead, they are produced through multi-gene pathways that generate a heterogenous array of glycoprotein and glycolipid antigens on the cell surface. This genetic complexity has sometimes made it challenging to understand how glycosylation is regulated and how it becomes altered in disease. Recent years, however, have seen the emergence of powerful new functional genomics technologies that allow high-throughput characterization of genetically complex cellular phenotypes. In this review, we discuss how these techniques are now being applied to achieve a deeper understanding of glyco-genomic regulation. We highlight specifically how methods like ChIP-seq, RNA-seq, CRISPR genomic screening and scRNA-seq are being used to map the genomic basis for various cell-surface glycosylation states in normal and diseased cell types. We also offer a perspective on how emerging functional genomics technologies are likely to create further opportunities for studying cellular glycobiology in the future. Taken together, we hope this review serves as a primer to recent developments at the glycomics-genomics interface.

UNASSIGNED: Glycans play essential functional roles in the nervous system and their pathobiological relevance has become increasingly recognized in numerous brain disorders, but not fully explored in traumatic brain injury (TBI). We investigated longitudinal glycome patterns in patients with moderate to severe TBI (Glasgow Coma Scale [GCS] score ≤12) to characterize glyco-biomarker signatures and their relation to clinical features and long-term outcome.
UNASSIGNED: This prospective single-center observational study included 51 adult patients with TBI (GCS ≤12) admitted to the neurosurgical unit of the University Hospital of Pecs, Pecs, Hungary, between June 2018 and April 2019. We used a high-throughput liquid chromatography-tandem mass spectrometry platform to assess serum levels of N-glycans up to 3 days after injury. Outcome was assessed using the Glasgow Outcome Scale-Extended (GOS-E) at 12 months post-injury. Multivariate statistical techniques, including principal component analysis and orthogonal partial least squares discriminant analysis, were used to analyze glycomics data and define highly influential structures driving class distinction. Receiver operating characteristic analyses were used to determine prognostic accuracy.
UNASSIGNED: We identified 94 N-glycans encompassing all typical structural types, including oligomannose, hybrid, and complex-type entities. Levels of high mannose, hybrid and sialylated structures were temporally altered (p<0·05). Four influential glycans were identified. Two brain-specific structures, HexNAc5Hex3DeoxyHex0NeuAc0 and HexNAc5Hex4DeoxyHex0NeuAc1, were substantially increased early after injury in patients with unfavorable outcome (GOS-E≤4) (area under the curve [AUC]=0·75 [95%CI 0·59-0·90] and AUC=0·71 [0·52-0·89], respectively). Serum levels of HexNAc7Hex7DeoxyHex1NeuAc2 and HexNAc8Hex6DeoxyHex0NeuAc0 were persistently increased in patients with favorable outcome, but undetectable in those with unfavorable outcome. Levels of HexNAc5Hex4DeoxyHex0NeuAc1 were acutely elevated in patients with mass lesions and in those requiring decompressive craniectomy.
UNASSIGNED: In spite of the exploratory nature of the study and the relatively small number of patients, our results provide to the best of our knowledge initial evidence supporting the utility of glycomics approaches for biomarker discovery and patient phenotyping in TBI. Further larger multicenter studies will be required to validate our findings and to determine their pathobiological value and potential applications in practice.
UNASSIGNED: This work was funded by the Italian Ministry of Health (grant number GR-2013-02354960), and also partially supported by a NIH grant (1R01GM112490-08).

Reverse-phase solid-phase extraction (SPE) is regularly used for separating and purifying food-derived oligosaccharides and peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, the diversity in physicochemical properties of peptides may prevent the complete separation of the two types of analytes. Peptides present in the oligosaccharide fraction not only interfere with glycomics analysis but also escape peptidomics analysis. This work evaluated different SPE approaches for improving LC-MS/MS analysis of both oligosaccharides and peptides through testing on peptide standards and a food sample of commercial interest (proteolyzed almond extract). Compared with conventional reverse-phase SPE, mixed-mode SPE (reverse-phase/strong cation exchange) was more effective in retaining small/hydrophilic peptides and capturing them in the high-organic fraction and thus allowed the identification of more oligosaccharides and dipeptides in the proteolyzed almond extract, with satisfactory MS/MS confirmation. Overall, mixed-mode SPE emerged as the ideal method for simultaneously improving the identification of food-derived oligosaccharides and small peptides using LC-MS/MS analysis.

The Glyco@Expasy initiative was launched as a collection of interdependent databases and tools spanning several aspects of knowledge in glycobiology. In particular, it aims at highlighting interactions between glycoproteins (such as cell surface receptors) and carbohydrate-binding proteins mediated by glycans. Here, major resources of the collection are introduced through two illustrative examples centered on the N-glycome of the human Prostate Specific Antigen (PSA) and the O-glycome of human serum proteins. Through different database queries and with the help of visualization tools, this article shows how to explore and compare content in a continuum to gather and correlate otherwise scattered pieces of information. Collected data are destined to feed more elaborate scenarios of glycan function. Glycoinformatics introduced here is, therefore, proposed as a means to either strengthen, shape, or refute assumptions on the specificity of a protein glycome in a given context.

Despite remarkable progress in disease diagnosis and treatment, coronary heart disease (CHD) remains the number one leading cause of death worldwide. Many practical challenges still faced in clinical settings necessitates the pursuit of omics studies to identify alternative/orthogonal biomarkers, as well as to discover novel insights into disease mechanisms. Albeit relatively nascent as compared to the omics frontrunners (genomics, transcriptomics, and proteomics), omics beyond the central dogma (OBCD; e.g., metabolomics, lipidomics, glycomics, and metallomics) have undeniable contributions and prospects in CHD research. In this bibliometric study, we characterised the global trends in publication/citation outputs, collaborations, and research hotspots concerning OBCD-CHD, with a focus on the more prolific fields of metabolomics and lipidomics. As for glycomics and metallomics, there were insufficient publication records on their applications in CHD research for quantitative bibliometrics analysis. Thus, we reviewed their applications in health/disease research in general, discussed and justified their potential in CHD research, and suggested important/promising research avenues. By summarising evidence obtained both quantitatively and qualitatively, this study offers a first and comprehensive picture of OBCD applications in CHD, facilitating the establishment of future research directions.

Mucin-type O-glycosylation (O-glycans, O-glycome) is among the most biologically important post-translational modification in glycoproteins but O-glycan structural diversity and expression are poorly understood due to the inadequacy of current analytical methods. We recently developed a new tool termed cellular O-glycome reporter/amplification (CORA), which uses O-glycan precursors, benzyl-α-GalNAc (Bn-α-GalNAc) or azido-Bn-α-GalNAc (N3 -Bn-α-GalNAc), as surrogates of protein O-glycosylation. Living cells metabolically convert these precursors to all types of O-GalNAc glycans representative of the cells\' capabilities. The amplification and secretion of the O-glycome products greatly facilitates their analysis and functional studies. Here we describe protocols for analytical and preparative applications. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Cellular O-glycome reporter/amplification for the analysis of mucin-type O-glycans from living cells Basic Protocol 2: Preparation of cellular O-glycans from living cells for functional glycomics and glycan microarrays Basic Protocol 3: Conjugation of cellular O-glycans with a bifunctional fluorescent tag Basic Protocol 4: 2D-HPLC purification and MALDI-TOF/MS identification of individual PYAB-Bn-O-glycan.