Explore the vast range of titles available.

In the era of precision medicine, the tailoring of cancer treatment is increasingly important as we transition from organ‐based diagnosis towards a more comprehensive and patient‐centric molecular diagnosis. This is particularly the case for high‐grade serous adenocarcinomas of the ovary and peritoneum, which are commonly diagnosed at an advanced stage, and collectively treated and managed similarly. We characterized the N‐ and O‐glycome of serous ovarian (OC) and peritoneal cancer (PC) tissues using PGC‐LC‐ESI‐IT‐MS/MS profiling and validated the discriminatory glycans and their corresponding glyco‐gene expression levels using cell lines and transcriptomic data from 232 patients. Overall, the N‐ and O‐glycan repertoires of both cancer types were found to comprise mostly of α2,6‐sialylated glycan structures, with the majority of N‐glycans displaying the biantennary mono‐ and disialylation as well as bisecting‐type biantennary glycans. The MS profiling by PGC‐LC also revealed several glycan structural isomers that corresponded to LacdiNAc‐type (GalNAcβ1‐4GlcNAc) motifs that were unique to the serous ovarian cancers and that correlated with elevated gene expression of B4GALNT3 and B4GALNT4 in patients with serous cancer. Statistical evaluation of the discriminatory glycans also revealed 13 N‐ and 3 O‐glycans (P < 0.05) that significantly discriminated tumour‐sampling sites, with LacdiNAc‐type N‐glycans (m/z 1205.02− and m/z 1059.42−) being associated with ovarian‐derived cancer tissue and bisecting GlcNAc‐type (m/z 994.92−) and branched N‐glycans (m/z 1294.02− and m/z 1148.42−) upregulated at the metastatic sites. Hence, we demonstrate for the first time that OC and PC display distinct molecular signatures at both their glycomic and transcriptomic levels. These signatures may have potential utility for the development of accurate diagnosis and personalized treatments. We characterized the glycomic profiles of serous ovarian and peritoneal cancer cells and tissues and their corresponding glyco‐gene expression levels. The profiling revealed sugar motifs that were unique to serous ovarian cancers and correlated with elevated gene expression of the synthetic enzymes. We demonstrate for the first time that ovarian and peritoneal cancers display distinct molecular signatures with potential utility for the development of accurate diagnosis and personalized treatments.

Applications of nanoparticles (NPs) in the life sciences require control over their properties in protein-rich biological fluids, as an NP quickly acquires a layer of proteins on the surface, forming the so-called “protein corona” (PC). Understanding the composition and kinetics of the PC at the molecular level is of considerable importance for controlling NP interaction with cells. Here, we present a systematic study of hard PC formation on the surface of upconversion nanoparticles (UCNPs) coated with positively-charged polyethyleneimine (PEI) and negatively-charged poly (acrylic acid) (PAA) polymers in serum-supplemented cell culture medium. The rationale behind the choice of UCNP is two-fold: UCNP represents a convenient model of NP with a size ranging from 5 nm to >200 nm, while the unique photoluminescent properties of UCNP enable direct observation of the PC formation, which may provide new insight into this complex process. The non-linear optical properties of UCNP were utilised for direct observation of PC formation by means of fluorescence correlation spectroscopy. Our findings indicated that the charge of the surface polymer coating was the key factor for the formation of PC on UCNPs, with an ensuing effect on the NP–cell interactions.

Hematogenous metastasis limits the survival of colorectal cancer (CRC) patients. Here, we illuminated the roles of CD44 isoforms in this process. Isoforms 3 and 4 were predominantly expressed in CRC patients. CD44 isoform 4 indicated poor outcome and correlated with epithelial–mesenchymal transition (EMT) and decreased oxidative phosphorylation (OxPhos) in patients; opposite associations were found for isoform 3. Pan‐CD44 knockdown (kd) independently impaired primary tumor formation and abrogated distant metastasis in CRC xenografts. The xenograft tumors mainly expressed the clinically relevant CD44 isoforms 3 and 4. Both isoforms were enhanced in the paranecrotic, hypoxic tumor regions but were generally absent in lung metastases. Upon CD44 kd, tumor angiogenesis was increased in the paranecrotic areas, accompanied by reduced hypoxia‐inducible factor‐1α and CEACAM5 but increased E‐cadherin expression. Mitochondrial genes and proteins were induced upon pan‐CD44 kd, as were OxPhos genes. Hypoxia increased VEGF release from tumor spheres, particularly upon CD44 kd. Genes affected upon CD44 kd in xenografts specifically overlapped concordantly with genes correlating with CD44 isoform 4 (but not isoform 3) in patients, validating the clinical relevance of the used model and highlighting the metastasis‐promoting role of CD44 isoform 4. Pan‐CD44 knockdown decreases spontaneous metastasis in human colorectal cancer xenograft models. Concurrent intratumoral gene expression alterations significantly correlate with genes differentially regulated among CD44 isoform 4 (but not isoform 3) high vs. low patients (TCGA). The corresponding gene sets and pathways include epithelial–mesenchymal transition, angiogenesis, and OxPhos. CD44 isoform 4 (but not isoform 3) correlates with poor patient outcomes.

Recent developments in spatial proteomics have paved the way for retrospective in situ mass spectrometry (MS) analyses of formalin-fixed paraffin-embedded clinical tissue samples. This type of analysis is commonly referred to as matrix-assisted laser desorption/ionization (MALDI) imaging. Recently, formalin-fixed paraffin-embedded MALDI imaging analyses were augmented to allow in situ analyses of tissue-specific N -glycosylation profiles. In the present study, we outline an improved automated sample preparation method for N -glycan MALDI imaging, which uses in situ PNGase F-mediated release and measurement of N -linked glycans from sections of formalin-fixed murine kidney. The sum of the presented data indicated that N -glycans can be cleaved from proteins within formalin-fixed tissue and characterized using three strategies: (i) extraction and composition analysis through on-target MALDI MS and liquid chromatography coupled to electrospray ionization ion trap MS; (ii) MALDI profiling, where N -glycans are released and measured from large droplet arrays in situ; and (iii) MALDI imaging, which maps the tissue specificity of N -glycans at a higher resolution. Thus, we present a complete, straightforward method that combines MALDI imaging and characterization of tissue-specific N -glycans and complements existing strategies. Graphical Abstract MALDI imaging MS of N-linked glycans released from formalin-fixed paraffin-embedded murine kidney sections. Ion intensity maps for (Hex) 2 (HexNAc) 3 (Deoxyhexose) 3 +(Man) 3 (GlcNAc) 2 ( m/z 2304.932, red), (Hex) 6 +(Man) 3 (GlcNAc) 2 ( m/z 1905.742, green) and (Hex) 2 (HexNAc) 2 +(Man) 3 (GlcNAc) 2 ( m/z 1663.756, blue)

Polysialic acid (polySia), a long homopolymer of 2,8-linked sialic acids, is abundant in the embryonic brain and is restricted largely in adult brain to regions that exhibit neurogenesis and structural plasticity. In the central nervous system (CNS), polySia is highly important for cell-cell interactions, differentiation, migration and cytokine responses, which are critical neuronal functions regulating intercellular interactions that underlie immune signalling in the CNS. In recent reports, a metabolite of morphine, morphine-3-glucuronide (M3G), has been shown to cause immune signalling in the CNS. In this study, we compared the effects of neurite growth factor (NGF), lipopolysaccharide (LPS) and M3G exposure on the expression of polySia in PC12 cells using immunocytochemistry and Western blot analysis. PolySia was also extracted from stimulated cell proteins by endo-neuraminidase digestion and quantitated using fluorescent labelling followed by HPLC analysis. PolySia expression was significantly increased following NGF, M3G or LPS stimulation when compared with unstimulated cells or cells exposed to the TLR4 antagonist LPS-RS. Additionally, we analyzed the effects of test agent exposure on cell migration and the oxidative stress response of these cells in the presence and absence of polySia expression on their cell surface. We observed an increase in oxidative stress in cells without polySia as well as following M3G or LPS stimulation. Our study provides evidence that polySia expression in neuronal-like PC12 cells is influenced by M3G and LPS exposure alike, suggestive of a role of TLR4 in triggering these events.

Multidrug-resistant strains of Neisseria gonorrhoeae are emerging worldwide, and novel treatment and prevention strategies are needed. Glycans are ubiquitously expressed by all human cells and can be specifically targeted by pathogens to facilitate association with host cells. Here we identify and characterize the N. gonorrhoeae host-glycan binding profile (glycointeractome), which revealed numerous interactions, including high-affinity binding to mannosyl glycans. We identify gonococcal potential mannose-binding proteins and show that N. gonorrhoeae uses mannosyl glycans expressed on the surface of cervical and urethral epithelia to facilitate adherence. Furthermore, a mannose-binding lectin or a mannoside compound was able to reduce this adherence. By characterizing the glycointeractome of N. gonorrhoeae, we were able to elucidate a novel mechanism used by this important pathogen to interact with human cells, and this interaction could be exploited to develop novel therapeutics to treat antibiotic-resistant gonorrhea. Neisseria gonorrhoeae is a significant threat to global health for which a vaccine and novel treatment options are urgently needed. Glycans expressed by human cells are commonly targeted by pathogens to facilitate interactions with the host, and thus characterization of these interactions can aid identification of bacterial receptors that can be exploited as vaccine and/or drug targets. Using glycan array analysis, we identified 247 specific interactions between N. gonorrhoeae and glycans representative of those found on human cells. Interactions included those with mannosylated, fucosylated, and sialylated glycans, glycosaminoglycans (GAGs), and glycans terminating with galactose (Gal), N -acetylgalactosamine (GalNAc), and N -acetylglucosamine (GlcNAc). By investigating the kinetics of interactions with selected glycans, we demonstrate that whole-cell N. gonorrhoeae has a high affinity for mannosylated glycans (dissociation constant [ K D ], 0.14 to 0.59 μM), which are expressed on the surface of cervical and urethral epithelial cells. Using chromatography coupled with mass spectrometric (MS) analysis, we identified potential mannose-binding proteins in N. gonorrhoeae . Pretreatment of cells with mannose-specific lectin (concanavalin A) or free mannose competitor (α-methyl- d -mannopyranoside) substantially reduced gonococcal adherence to epithelial cells. This suggests that N. gonorrhoeae targets mannosyl glycans to facilitate adherence to host cells and that mannosides or similar compounds have the potential to be used as a novel treatment option for N. gonorrhoeae . IMPORTANCE Multidrug-resistant strains of Neisseria gonorrhoeae are emerging worldwide, and novel treatment and prevention strategies are needed. Glycans are ubiquitously expressed by all human cells and can be specifically targeted by pathogens to facilitate association with host cells. Here we identify and characterize the N. gonorrhoeae host-glycan binding profile (glycointeractome), which revealed numerous interactions, including high-affinity binding to mannosyl glycans. We identify gonococcal potential mannose-binding proteins and show that N. gonorrhoeae uses mannosyl glycans expressed on the surface of cervical and urethral epithelia to facilitate adherence. Furthermore, a mannose-binding lectin or a mannoside compound was able to reduce this adherence. By characterizing the glycointeractome of N. gonorrhoeae, we were able to elucidate a novel mechanism used by this important pathogen to interact with human cells, and this interaction could be exploited to develop novel therapeutics to treat antibiotic-resistant gonorrhea.

Human CD52 is a small glycopeptide (12 amino acid residues) with one linked glycosylation site at asparagine 3 (Asn3) and several potential glycosylation serine/threonine sites. Soluble CD52 is released from the surface of activated T cells and mediates immune suppression via its glycan moiety. In suppressing activated T cells, it first sequesters the pro-inflammatory high mobility group Box 1 (HMGB1) protein, which facilitates its binding to the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor. We aimed to identify the features of CD52 glycan that underlie its bioactivity. Analysis of native CD52 purified from human spleen revealed extensive heterogeneity in glycosylation and multi-antennary sialylated glycans with abundant polyLacNAc extensions, together with mainly di-sialylated glycosylation type structures. Glycomic (porous graphitized carbon-ESI-MS/MS) and glycopeptide (C8-LC-ESI-MS) analysis of recombinant soluble human CD52-immunoglobulin Fc fusion proteins revealed that CD52 bioactivity was correlated with a high abundance of tetra-antennary α-2,3/6 sialylated glycans. Removal of α-2,3 sialylation abolished bioactivity, which was restored by re-sialylation with α-2,3 sialyltransferases. When glycoforms of CD52-Fc were fractionated by anion exchange MonoQ-GL chromatography, bioactive fractions displayed mainly tetra-antennary, α-2,3 sialylated glycan structures and a lower relative abundance of bisecting GlcNAc structures compared to non-bioactive fractions. In addition, glycan core type-2 di-sialylated structures at Ser12 were more abundant in bioactive CD52 fractions. Understanding the structural features of CD52 glycan required for its bioactivity will aid its development as an immunotherapeutic agent.

Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure–function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N - and O -glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data. Figure ᅟ

Every living cell is covered with a dense and complex layer of glycans on the cell surface, which have important functions in the interaction between cells and their environment. Glycosphingolipids (GSLs) are glycans linked to lipid molecules that together with sphingolipids, sterols, and proteins form plasma membrane lipid rafts that contribute to membrane integrity and provide specific recognition sites. GSLs are subdivided into three major series (globo-, ganglio-, and neolacto-series) and are synthesized in a non-template driven process by enzymes localized in the ER and Golgi apparatus. Altered glycosylation of lipids are known to be involved in tumor development and metastasis. Metastasis is frequently linked with reversible epithelial-to-mesenchymal transition (EMT), a process involved in tumor progression, and the formation of new distant metastatic sites (mesenchymal-to-epithelial transition or MET). On a single cell basis, cancer cells lose their epithelial features to gain mesenchymal characteristics via mechanisms influenced by the composition of the GSLs on the cell surface. Here, we summarize the literature on GSLs in the context of reversible and cancer-associated EMT and discuss how the modification of GSLs at the cell surface may promote this process.

Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.