Detecting normal and cancer skin cells via glycosylation and adhesion signatures: A path to enhanced microfluidic phenotyping.

Recruiting circulating cells based on interactions between surface receptors and corresponding ligands holds promise for capturing cells with specific adhesive properties. Our study investigates the adhesion of skin cells to specific lectins, particularly focusing on advancements in lectin-based biosensors with diagnostic potential. We explore whether we can successfully capture normal skin (melanocytes and keratinocytes) and melanoma (WM35, WM115, WM266-4) cells in a low-shear flow environment by coating surfaces with lectins. Specifically, we coated surfaces with Dolichos biflorus (DBA) and Maackia Amurensis (MAL) lectins, which were used to detect and capture specific skin cells from the flow of cell mixture. Alterations in glycan expression (confirmed by fluorescent microscopy) demonstrated that DBA binds predominantly to normal skin cells, while MAL interacts strongly with melanoma cells. Assessing adhesion under static and dynamic low-shear stress conditions (up to 30 mPa) underscores the reliability of DBA and MAL as markers for discriminating specific cell type. Melanocytes and keratinocytes adhere to DBA-coated surfaces, while melanoma cells prefer MAL-coated surfaces. A comprehensive analysis encompassing cell shape, cytoskeleton, and focal adhesions shows the independence of our approach from the inherent characteristics of cells, thus demonstrating its robustness. Our results carry practical implications for lectin-biosensor designs, emphasizing the significance of glycan-based discrimination of pathologically altered cells. Combined with microfluidics, it demonstrates the value of cell adhesion as a discriminant of cancer-related changes, with potential applications spanning diagnostics, therapeutic interventions, and advanced biomedical technologies.

Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors.

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents.

Omentin-1 May Be One Treatment Factor for Intravenous Thrombolysis of Acute Cerebral Infarction Through the Inhibition of NLRP3 Ubiquitination by AMPK Function: Preliminary Findings.

BACKGROUND: Acute cerebral infarction (ACI) is a common neurological disease that is associated with high morbidity, disability and mortality rates. At present, antiplatelet therapy is a necessary treatment for ACI. The present study aimed to investigate the effects of omentin-1 on the intravenous thrombolysis of ACI. OBJECTIVE: The present study aimed to investigate the effects of omentin-1 on the intravenous thrombolysis of ACI. MATERIAL AND METHODS: The mouse model of ACI was induced using male C57BL/6 mice through middle cerebral artery occlusion (MCAO). Meanwhile, the murine BV2 microglial cells were pretreated with 0.1 mg/ml of lipopolysaccharide (LPS), and then induced with 2 mM of adenosine triphosphate (ATP). RESULTS: The omentin-1 mRNA expression in patients receiving intravenous thrombolysis for ACI was down-regulated compared with the normal group. Additionally, the serum level of omentin-1 was negatively correlated with National Institute of Health Stroke Scale (NIHSS) score or serum level of IL-1ß or MMP-2 in patients receiving intravenous thrombolysis for ACI. Meanwhile, the serum mRNA expression of omentin-1 was positively correlated with Barthel index or high-sensitivity C-reactive protein (hs-CRP) in patients undergoing intravenous thrombolysis for ACI. As observed from the in vitro model, Omentin-1 reduced inflammation, promoted cell growth, alleviated ROS-induced oxidative stress, and enhanced AMPK activity through activating NLRP3 ubiquitination. Omentin-1 presented ACI in the mouse model of ACI. Regulating AMPK activity contributed to controlling the effects of Omentin-1 on the in vitro model. CONCLUSIONS: Omentin-1 reduced neuroinflammation and ROS-induced oxidative stress in the mouse model of ACI, which was achieved by inhibiting NLRP3 ubiquitination through regulating AMPK activity. Therefore, omentin-1 may serve as a treatment factor for the intravenous thrombolysis of ACI in further clinical application.

The expression characteristic and prognostic role of Siglec-15 in lung adenocarcinoma.

Sialic acid-binding immunoglobulin-like lectin-15 (Siglec-15) has been identified as an immune suppressor and a promising candidate for immunotherapy of cancer management. However, the association between Siglec-15 expression and clinicopathological features of lung adenocarcinoma (LUAD), especially the prognostic role, is not fully elucidated. In this present study, a serial of bioinformatics analyses in both tissue and cell levels were conducted to provide an overview of Siglec-15 expression. Real-time quantitative PCR (qPCR) test, western blotting assay, and immunohistochemistry (IHC) analyses were conducted to evaluate the expression of Siglec-15 in LUAD. Survival analysis and Kaplan-Meier curve were employed to describe the prognostic parameters of LUAD. The results of bioinformatics analyses demonstrated the up-regulation of Siglec-15 expression in LUAD. The data of qPCR, western blotting, and IHC analyses further proved that the expression of Siglec-15 in LUAD tissues was significantly increased than that in noncancerous tissues. Moreover, the expression level of Siglec-15 protein in LUAD was substantially associated with TNM stage. LUAD cases with up-regulated Siglec-15 expression, positive N status, and advance TNM stage suffered a critical unfavorable prognosis. In conclusion, Siglec-15 could be identified as a novel prognostic biomarker in LUAD and targeting Siglec-15 may provide a promising strategy for LUAD immunotherapy.

Ficolin-A induces macrophage polarization to a novel pro-inflammatory phenotype distinct from classical M1.

BACKGROUND: Macrophages are key inflammatory immune cells that orchestrate the initiation and progression of autoimmune diseases. The characters of macrophage in diseases are determined by its phenotype in response to the local microenvironment. Ficolins have been confirmed as crucial contributors to autoimmune diseases, with Ficolin-2 being particularly elevated in patients with autoimmune diseases. However, whether Ficolin-A stimulates macrophage polarization is still poorly understood. METHODS: We investigated the transcriptomic expression profile of murine bone marrow-derived macrophages (BMDMs) stimulated with Ficolin-A using RNA-sequencing. To further confirm a distinct phenotype activated by Ficolin-A, quantitative RT-PCR and Luminex assay were performed in this study. Additionally, we assessed the activation of underlying cell signaling pathways triggered by Ficolin-A. Finally, the impact of Ficolin-A on macrophages were investigated in vivo through building Collagen-induced arthritis (CIA) and Dextran Sulfate Sodium Salt (DSS)-induced colitis mouse models with Fcna-/- mice. RESULTS: Ficolin-A activated macrophages into a pro-inflammatory phenotype distinct to LPS-, IFN-γ- and IFN-γ + LPS-induced phenotypes. The transcriptomic profile induced by Ficolin-A was primarily characterized by upregulation of interleukins, chemokines, iNOS, and Arginase 1, along with downregulation of CD86 and CD206, setting it apart from the M1 and M2 phenotypes. The activation effect of Ficolin-A on macrophages deteriorated the symptoms of CIA and DSS mouse models, and the deletion of Fcna significantly alleviated the severity of diseases in mice. CONCLUSION: Our work used transcriptomic analysis by RNA-Seq to investigate the impact of Ficolin-A on macrophage polarization. Our findings demonstrate that Ficolin-A induces a novel pro-inflammatory phenotype distinct to the phenotypes activated by LPS, IFN-γ and IFN-γ + LPS on macrophages.

A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

Probing the expression and adhesion of glycans involved in Helicobacter pylori infection.

Helicobacter pylori infects approximately half the human population and has an unusual infective niche of the human stomach. Helicobacter pylori is a major cause of gastritis and has been classified as a group 1 carcinogen by the WHO. Treatment involves triple or quadruple antibiotic therapy, but antibiotic resistance is becoming increasingly prevalent. Helicobacter pylori expresses certain blood group related antigens (Lewis system) as a part of its lipopolysaccharide (LPS), which is thought to assist in immune evasion. Additionally, H. pylori LPS participates in adhesion to host cells alongside several adhesion proteins. This study profiled the carbohydrates of H. pylori reference strains (SS1 and 26695) using monoclonal antibodies (mAbs) and lectins, identifying interactions between two carbohydrate-targeting mAbs and multiple lectins. Atomic force microscopy (AFM) scans were used to probe lectin and antibody interactions with the bacterial surfaces. The selected mAb and lectins displayed an increased adhesive force over the surface of the curved H. pylori rods. Furthermore, this study demonstrates the ability of anti-carbohydrate antibodies to reduce the adhesion of H. pylori 26695 to human gastric adenocarcinoma cells via AFM. Targeting bacterial carbohydrates to disrupt crucial adhesion and immune evasion mechanisms represents a promising strategy for combating H. pylori infection.

Maackia amurensis seed lectin (MASL) and soluble human podoplanin (shPDPN) sequence analysis and effects on human oral squamous cell carcinoma (OSCC) cell migration and viability.

Maackia amurensis lectins serve as research and botanical agents that bind to sialic residues on proteins. For example, M. amurensis seed lectin (MASL) targets the sialic acid modified podoplanin (PDPN) receptor to suppress arthritic chondrocyte inflammation, and inhibit tumor cell growth and motility. However, M. amurensis lectin nomenclature and composition are not clearly defined. Here, we sought to definitively characterize MASL and its effects on tumor cell behavior. We utilized SDS-PAGE and LC-MS/MS to find that M. amurensis lectins can be divided into two groups. MASL is a member of one group which is composed of subunits that form dimers, evidently mediated by a cysteine residue in the carboxy region of the protein. In contrast to MASL, members of the other group do not dimerize under nonreducing conditions. These data also indicate that MASL is composed of 4 isoforms with an identical amino acid sequence, but unique glycosylation sites. We also produced a novel recombinant soluble human PDPN receptor (shPDPN) with 17 threonine residues glycosylated with sialic acid moieties with potential to act as a ligand trap that inhibits OSCC cell growth and motility. In addition, we report here that MASL targets PDPN with very strong binding kinetics in the nanomolar range. Moreover, we confirm that MASL can inhibit the growth and motility of human oral squamous cell carcinoma (OSCC) cells that express the PDPN receptor. Taken together, these data characterize M. amurensis lectins into two major groups based on their intrinsic properties, clarify the composition of MASL and its subunit isoform sequence and glycosylation sites, define sialic acid modifications on the PDPN receptor and its ability to act as a ligand trap, quantitate MASL binding to PDPN with KD in the nanomolar range, and verify the ability of MASL to serve as a potential anticancer agent.

The correlation between human seminal plasma sialoproteins and ejaculate parameters.

Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics.

LectoScape: A Highly Multiplexed Imaging Platform for Glycome Analysis and Biomedical Diagnosis.

Glycosylation, a fundamental biological process, involves the attachment of glycans to proteins, lipids, and RNA, and it plays a crucial role in various biological pathways. It is of great significance to obtain the precise spatial distribution of glycosylation modifications at the cellular and tissue levels. Here, we introduce LectoScape, an innovative method enabling detailed imaging of tissue glycomes with up to 1 µm resolution through image mass cytometry (IMC). This method utilizes 12 distinct, nonoverlapping lectins selected via microarray technology, enabling the multiplexed detection of a wide array of glycans. Furthermore, we developed an efficient labeling strategy for these lectins. Crucially, our approach facilitates the concurrent imaging of diverse glycan motifs, including N-glycan and O-glycan, surpassing the capabilities of existing technologies. Using LectoScape, we have successfully delineated unique glycan structures in various cell types, enhancing our understanding of the glycan distribution across human tissues. Our method has identified specific glycan markers, such as α2,3-sialylated Galß1, 3GalNAc in O-glycan, and terminal GalNAc, as diagnostic indicators for cervical intraepithelial neoplasia. This highlights the potential of LectoScape in cancer diagnostics through the detection of abnormal glycosylation patterns.

The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

Targeting SIGLEC15 as an emerging immunotherapy for anaplastic thyroid cancer.

Anaplastic thyroid carcinoma (ATC) is the most aggressive subtype of thyroid cancer with few effective therapies. Though immunotherapies such as targeting PD-1/PD-L1 axis have benefited patients with solid tumor, the druggable immune checkpoints are quite limited in ATC. In our study, we focused on the anti-tumor potential of sialic acid-binding Ig-like lectins (Siglecs) in ATC. Through screening by integrating microarray datasets including 216 thyroid-cancer tissues and single-cell RNA-sequencing, SIGLEC family members CD33, SIGLEC1, SIGLEC10 and SIGLEC15 were significantly overexpressed in ATC, among which SIGLEC15 increased highest and mainly expressed on cancer cells. SIGLEC15high ATC cells are characterized by high expression of serine protease PRSS23 and cancer stem cell marker CD44. Compared with SIGLEC15low cancer cells, SIGLEC15high ATC cells exhibited higher interaction frequency with tumor microenvironment cells. Further study showed that SIGLEC15high cancer cells mainly interacted with T cells by immunosuppressive signals such as MIF-TNFRSF14 and CXCL12-CXCR4. Notably, treatment of anti-SIGLEC15 antibody profoundly increased the cytotoxic ability of CD8+ T cells in a co-culture model and zebrafish-derived ATC xenografts. Consistently, administration of anti-SIGLEC15 antibody significantly inhibited tumor growth and prolonged mouse survival in an immunocompetent model of murine ATC, which was associated with increase of M1/M2, natural killer (NK) cells and CD8+ T cells, and decrease of myeloid-derived suppressor cells (MDSCs). SIGLEC15 inhibited T cell activation by reducing NFAT1, NFAT2, and NF-κB signals. Blocking SIGLEC15 increased the secretion of IFN-γ and IL-2 in vitro and in vivo. In conclusion, our finding demonstrates that SIGLEC15 is an emerging and promising target for immunotherapy in ATC.

A Complex-Type N-Glycan-Specific Lectin Isolated from Green Alga Halimeda borneensis Exhibits Potent Anti-Influenza Virus Activity.

Marine algal lectins specific for high-mannose N-glycans have attracted attention because they strongly inhibit the entry of enveloped viruses, including influenza viruses and SARS-CoV-2, into host cells by binding to high-mannose-type N-glycans on viral surfaces. Here, we report a novel anti-influenza virus lectin (named HBL40), specific for complex-type N-glycans, which was isolated from a marine green alga, Halimeda borneensis. The hemagglutination activity of HBL40 was inhibited with both complex-type N-glycan and O-glycan-linked glycoproteins but not with high-mannose-type N-glycan-linked glycoproteins or any of the monosaccharides examined. In the oligosaccharide-binding experiment using 26 pyridylaminated oligosaccharides, HBL40 only bound to complex-type N-glycans with bi- and triantennary-branched sugar chains. The sialylation, core fucosylation, and the increased number of branched antennae of the N-glycans lowered the binding activity with HBL40. Interestingly, the lectin potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells at IC50 of 8.02 nM by binding to glycosylated viral hemagglutinin (KD of 1.21 × 10-6 M). HBL40 consisted of two isolectins with slightly different molecular masses to each other that could be separated by reverse-phase HPLC. Both isolectins shared the same 16 N-terminal amino acid sequences. Thus, HBL40 could be useful as an antivirus lectin specific for complex-type N-glycans.

Serum Clusterin Concentration and Its Glycosylation Changes as Potential New Diagnostic Markers of SARS-CoV-2 Infection and Recovery Process.

COVID-19 is an infectious disease caused by the SARS-CoV-2 virus. Glycoprotein clusterin (CLU) has many functions such as phagocyte recruitment, complement system inhibition, apoptosis inhibition, hormone and lipid transport, as well as in the immune response. The study aimed to assess the changes in CLU concentrations and the profile and degree of CLU glycosylation between patients with severe COVID-19, convalescents, and healthy subjects (control). The profile and degree of serum CLU N-glycosylation were analyzed using lectin-ELISA with specific lectins. CLU concentrations were significantly lower and relative reactivities of CLU glycans with SNA (Sambucus nigra agglutinin) were significantly higher in severe COVID-19 patients in comparison to convalescents and the control group. The relative reactivities of CLU glycans with MAA (Maackia amurensis agglutinin), together with relative reactivity with LCA (Lens culinaris agglutinin), were also significantly higher in patients with severe COVID-19 than in convalescents and the control group, but they also significantly differed between convalescents and control. The development of acute inflammation in the course of severe COVID-19 is associated with a decrease in CLU concentration, accompanied by an increase in the expression of α2,3-linked sialic acid, and core fucose. Both of these parameters can be included as useful glycomarkers differentiating patients with severe COVID-19 from convalescents and the control group, as well as convalescents and healthy subjects.

Omentin associates with serum metabolite profiles indicating lower diabetes risk: KORA F4 Study.

INTRODUCTION: Circulating omentin levels have been positively associated with insulin sensitivity. Although a role for adiponectin in this relationship has been suggested, underlying mechanisms remain elusive. In order to reveal the relationship between omentin and systemic metabolism, this study aimed to investigate associations of serum concentrations of omentin and metabolites. RESEARCH DESIGN AND METHODS: This study is based on 1124 participants aged 61-82 years from the population-based KORA (Cooperative Health Research in the Region of Augsburg) F4 Study, for whom both serum omentin levels and metabolite concentration profiles were available. Associations were assessed with five multivariable regression models, which were stepwise adjusted for multiple potential confounders, including age, sex, body mass index, waist-to-hip ratio, lifestyle markers (physical activity, smoking behavior and alcohol consumption), serum adiponectin levels, high-density lipoprotein cholesterol, use of lipid-lowering or anti-inflammatory medication, history of myocardial infarction and stroke, homeostasis model assessment 2 of insulin resistance, diabetes status, and use of oral glucose-lowering medication and insulin. RESULTS: Omentin levels significantly associated with multiple metabolites including amino acids, acylcarnitines, and lipids (eg, sphingomyelins and phosphatidylcholines (PCs)). Positive associations for several PCs, such as diacyl (PC aa C32:1) and alkyl-alkyl (PC ae C32:2), were significant in models 1-4, whereas those with hydroxytetradecenoylcarnitine (C14:1-OH) were significant in all five models. Omentin concentrations were negatively associated with several metabolite ratios, such as the valine-to-PC ae C32:2 and the serine-to-PC ae C32:2 ratios in most models. CONCLUSIONS: Our results suggest that omentin may influence insulin sensitivity and diabetes risk by changing systemic lipid metabolism, but further mechanistic studies investigating effects of omentin on metabolism of insulin-sensitive tissues are needed.

Targeted and Self-Adjuvated Nanoglycovaccine Candidate for Cancer Immunotherapy.

Advanced-stage solid primary tumors and metastases often express mucin 16 (MUC16), carrying immature glycans such as the Tn antigen, resulting in specific glycoproteoforms not found in healthy human tissues. This presents a valuable approach for designing targeted therapeutics, including cancer glycovaccines, which could potentially promote antigen recognition and foster the immune response to control disease spread and prevent relapse. In this study, we describe an adjuvant-free poly(lactic-co-glycolic acid) (PLGA)-based nanoglycoantigen delivery approach that outperforms conventional methods by eliminating the need for protein carriers while exhibiting targeted and adjuvant properties. To achieve this, we synthesized a library of MUC16-Tn glycoepitopes through single-pot enzymatic glycosylation, which were then stably engrafted onto the surface of PLGA nanoparticles, generating multivalent constructs that better represent cancer molecular heterogeneity. These glycoconstructs demonstrated affinity for Macrophage Galactose-type Lectin (MGL) receptor, known to be highly expressed by immature antigen-presenting cells, enabling precise targeting of immune cells. Moreover, the glycopeptide-grafted nanovaccine candidate displayed minimal cytotoxicity and induced the activation of dendritic cells in vitro, even in the absence of an adjuvant. In vivo, the formulated nanovaccine candidate was also nontoxic and elicited the production of IgG specifically targeting MUC16 and MUC16-Tn glycoproteoforms in cancer cells and tumors, offering potential for precise cancer targeting, including targeted immunotherapies.

Selective Quantification of Bacteria in Mixtures by Using Glycosylated Polypyrrole/Hydrogel Nanolayers.

Here, we present a covalent nanolayer system that consists of a conductive and biorepulsive base layer topped by a layer carrying biorecognition sites. The layers are built up by electropolymerization of pyrrole derivatives that either carry polyglycerol brushes (for biorepulsivity) or glycoside moieties (as biorecognition sites). The polypyrrole backbone makes the resulting nanolayer systems conductive, opening the opportunity for constructing an electrochemistry-based sensor system. The basic concept of the sensor exploits the highly selective binding of carbohydrates by certain harmful bacteria, as bacterial adhesion and infection are a major threat to human health, and thus, a sensitive and selective detection of the respective bacteria by portable devices is highly desirable. To demonstrate the selectivity, two strains of Escherichia coli were selected. The first strain carries type 1 fimbriae, terminated by a lectin called FimH, which recognizes α-d-mannopyranosides, which is a carbohydrate that is commonly found on endothelial cells. The otherE. coli strain was of a strain that lacked this particular lectin. It could be demonstrated that hybrid nanolayer systems containing a very thin carbohydrate top layer (2 nm) show the highest discrimination (factor 80) between the different strains. Using electrochemical impedance spectroscopy, it was possible to quantify in vivo the type 1-fimbriated E. coli down to an optical density of OD600 = 0.0004 with a theoretical limit of 0.00005. Surprisingly, the selectivity and sensitivity of the sensing remained the same even in the presence of a large excess of nonbinding bacteria, making the system useful for the rapid and selective detection of pathogens in complex matrices. As the presented covalent nanolayer system is modularly built, it opens the opportunity to develop a broad band of mobile sensing devices suitable for various field applications such as bedside diagnostics or monitoring for bacterial contamination, e.g., in bioreactors.

Infection with different Neospora caninum strains causes differences in the glycosylation pattern in the uteri and placentae of Neospora caninum-infected heifers.

Neospora caninum is an obligate intracellular parasite that causes abortion in ruminants. Different strains produce differences in the severity of disease outcomes. These differences may cause physiological or pathological changes in cells, modifying the intercellular interactions and intracellular transport pathways that could be evidenced by identifying the terminal sugars. This study aimed to characterize the oligosaccharide pattern in the bovine placenta and uterus after infection with tachyzoites of three different strains of N. caninum (Nc-1, Nc-6 Argentina and Nc Spain-7) during early gestation. Fourteen heifers were inoculated intravenously on day 70 of gestation with 2 × 108 N. caninum tachyzoites and samples of placentae and uteri were analysed by histology and lectin histochemistry. In the infected groups, severe placentitis was associated with changes in lectin binding in the vascular endothelium by Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA) and Ricinus communis I (RCA-I) lectins, in the epithelial cells of the endometrial glands by RCA-I, Dolichos biflorus agglutinin (DBA), succinylated wheat germ agglutinin, peanut agglutinin (PNA), concanavalin-A (CON-A), LCA, PSA and Phaseolus vulgaris erythroagglutinin (PHA-e), and in the trophoblast layer by PNA, CON-A, LCA, PSA, PHA-e, soybean agglutinin, RCA-I, DBA and Bandieraea simplicifolia agglutinin (BSA-I). The results suggest that N. caninum causes changes in the glycosylation pattern in the maternofetal interface tissues and might cause abortions in early gestation due to changes in the cellular structure of the placenta.

Site-specific immobilization of the endosialidase reveals QSOX2 is a novel polysialylated protein.

Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN on streptavidin-coated magnetic beads. We showed that immobilizing EndoN allows for effective removal of the enzyme from samples, while retaining hydrolase activity. We used the same strategy to immobilize the polySia lectin EndoNDM, which enabled the enrichment of polysialylated proteins from complex mixtures such as serum for their identification via mass spectrometry. We used this methodology to identify a novel polysialylated protein, QSOX2, which is secreted from the breast cancer cell line MCF-7. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.

Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.