The alteration and role of glycoconjugates in Alzheimer's disease.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by abnormal protein deposition. With an alarming 30 million people affected worldwide, AD poses a significant public health concern. While inhibiting key enzymes such as ß-site amyloid precursor protein-cleaving enzyme 1 and γ-secretase or enhancing amyloid-ß clearance, has been considered the reasonable strategy for AD treatment, their efficacy has been compromised by ineffectiveness. Furthermore, our understanding of AD pathogenesis remains incomplete. Normal aging is associated with a decline in glucose uptake in the brain, a process exacerbated in patients with AD, leading to significant impairment of a critical post-translational modification: glycosylation. Glycosylation, a finely regulated mechanism of intracellular secondary protein processing, plays a pivotal role in regulating essential functions such as synaptogenesis, neurogenesis, axon guidance, as well as learning and memory within the central nervous system. Advanced glycomic analysis has unveiled that abnormal glycosylation of key AD-related proteins closely correlates with the onset and progression of the disease. In this context, we aimed to delve into the intricate role and underlying mechanisms of glycosylation in the etiopathology and pathogenesis of AD. By highlighting the potential of targeting glycosylation as a promising and alternative therapeutic avenue for managing AD, we strive to contribute to the advancement of treatment strategies for this debilitating condition.

Semisynthetic glycoconjugates as potential vaccine candidates against Haemophilus influenzae type a.

Glycoconjugate vaccines are based on chemical conjugation of pathogen-associated carbohydrates with immunogenic carrier proteins and are considered a very cost-effective way to prevent infections. Most of the licensed glycoconjugate vaccines are composed of saccharide antigens extracted from bacterial sources. However, synthetic oligosaccharide antigens have become a promising alternative to natural polysaccharides with the advantage of being well-defined structures providing homogeneous conjugates. Haemophilus influenzae (Hi) is responsible for a number of severe diseases. In recent years, an increasing rate of invasive infections caused by Hi serotype a (Hia) raised some concern, because no vaccine targeting Hia is currently available. The capsular polysaccharide (CPS) of Hia is constituted by phosphodiester-linked 4-ß-d-glucose-(1→4)-d-ribitol-5-(PO4→) repeating units and is the antigen for protein-conjugated polysaccharide vaccines. To investigate the antigenic potential of the CPS from Hia, we synthesized related saccharide fragments containing up to five repeating units. Following the synthetic optimization of the needed disaccharide building blocks, they were assembled using the phosphoramidite approach for the installation of the phosphodiester linkages. The resulting CPS-based Hia oligomers were conjugated to CRM197 carrier protein and evaluated in vivo for their immunogenic potential, showing that all glycoconjugates were capable of raising antibodies recognizing Hia synthetic fragments.

Antigenic determinants driving serogroup-specific antibody response to Neisseria meningitidis C, W, and Y capsular polysaccharides: Insights for rational vaccine design.

Meningococcal glycoconjugate vaccines sourced from capsular polysaccharides (CPSs) of pathogenic Neisseria meningitidis strains are well-established measures to prevent meningococcal disease. However, the exact structural factors responsible for antibody recognition are not known. CPSs of Neisseria meningitidis serogroups Y and W differ by a single stereochemical center, yet they evoke specific immune responses. Herein, we developed specific monoclonal antibodies (mAbs) targeting serogroups C, Y, and W and evaluated their ability to kill bacteria. We then used these mAbs to dissect structural elements responsible for carbohydrate-protein interactions. First, Men oligosaccharides were screened against the mAbs using ELISA to select putative lengths representing the minimal antigenic determinant. Next, molecular interaction features between the mAbs and serogroup-specific sugar fragments were elucidated using STD-NMR. Moreover, X-ray diffraction data with the anti-MenW CPS mAb enabled the elucidation of the sugar-antibody binding mode. Our findings revealed common traits in the epitopes of all three sialylated serogroups. The minimal binding epitopes typically comprise five to six repeating units. Moreover, the O-acetylation of the neuraminic acid moieties was fundamental for mAb binding. These insights hold promise for the rational design of optimized meningococcal oligosaccharides, opening new avenues for novel production methods, including chemical or enzymatic approaches.

Exploring the diverse biological significance and roles of fucosylated oligosaccharides.

Long since, carbohydrates were thought to be used just as an energy source and structural material. However, in recent years, with the emergence of the field of glycobiology and advances in glycomics, much has been learned about the biological role of oligosaccharides, a carbohydrate polymer containing a small number of monosaccharides, in cell-cell interaction, signal transduction, immune response, pathogen adhesion processes, early embryogenesis, and apoptosis. The function of oligosaccharides in these processes is diversified by fucosylation, also known as modification of oligosaccharides. Fucosylation has allowed the identification of more than 100 different oligosaccharide structures that provide functional diversity. ABO blood group and Lewis antigens are among the best known fucosyl-linked oligosaccharides. In addition, the antigens in the ABO system are composed of various sugar molecules, including fucosylated oligosaccharides, and Lewis antigens are structurally similar to ABO antigens but differ in the linkage of sugars. Variation in blood group antigen expression affects the host's susceptibility to many infections. However, altered expression of ABO and Lewis antigens is related with prognosis in carcinoma types. In addition, many pathogens recognize and bind to human tissues using a protein receptor with high affinity for the fucose molecule in glycoconjugates, such as lectin. Fucosylated oligosaccharides also play vital roles during fertilization and early embryogenesis. Learning and memory-related processes such as neurite growth, neurite migration, and synapse formation seen during the development of the brain, which is among the first organs to develop in embryogenesis, are regulated by fucosylated oligosaccharides. In conclusion, this review mentions the vital roles of fucosylated oligosaccharides in biology, drawing attention to their importance in the development of chemical tools to be used in function analysis and the investigation of various therapeutic targets.

Tailoring Metallosupramolecular Glycoassemblies for Enhancing Lectin Recognition.

Multivalency is a fundamental principle in nature that leads to high-affinity intermolecular recognition through multiple cooperative interactions that overcome the weak binding of individual constituents. For example, multivalency plays a critical role in lectin-carbohydrate interactions that participate in many essential biological processes. Designing high-affinity multivalent glycoconjugates that engage lectins results in systems with the potential to disrupt these biological processes, offering promising applications in therapeutic design and bioengineering. Here, a versatile and tunable synthetic platform for the synthesis of metallosupramolecular glycoassemblies is presented that leverages subcomponent self-assembly, which employs metal ion templates to generate complex supramolecular architectures from simple precursors in one pot. Through ligand design, this approach provides precise control over molecular parameters such as size, shape, flexibility, valency, and charge, which afforded a diverse family of well-defined hybrid glyconanoassemblies. Evaluation of these complexes as multivalent binders to Concanavalin A (Con A) by isothermal titration calorimetry (ITC) demonstrates the optimal saccharide tether length and the effect of electrostatics on protein affinity, revealing insights into the impact of synthetic design on molecular recognition. The presented studies offer an enhanced understanding of structure-function relationships governing lectin-saccharide interactions at the molecular level and guide a systematic approach towards optimizing glyconanoassembly binding parameters.

Chemical Resolution of an Epitope Recognized by Blood Group Antibodies Capable of Binding Both A and B Red Blood Cells.

The high specificity of human antibodies to blood group A and B antigens is impressive, especially when considering the structural difference between these antigens (tetrasaccharides) is a NHAc versus a OH-group on the terminal monosaccharide residue. It is well established that in addition to anti-A and anti-B there is a third antibody, anti-A,B capable of recognizing both A and B antigens. To analyze this AB specificity, we synthesized a tetrasaccharide, where the NHAc of the A antigen was replaced with NH2. This NH2-group was used to attach the glycan to an affinity resin, creating an AB-epitope (ABep) adsorbent where the critical site for recognition by A and B antibodies was not accessible, while the rest of the (conformationally compact) tetrasaccharide remained accessible. Anti-ABep antibodies were isolated from blood group O donors and found to have expected A,B-specificity against immobilized and red cell bound synthetic antigens, including ABep, and were able to agglutinate both A and B red cells. The amount of these anti-ABep (anti-A,B) antibodies found in the blood of group O donors was comparable to levels of anti-A and anti-B found in group B and A individuals. Using STD-NMR the location for the AB-epitope on the tetrasaccharide was found.

Nonreducing Sugar Scaffold Enables the Development of Immunomodulatory TLR4-specific LPS Mimetics with Picomolar Potency.

Innate immune defense mechanisms against infection and cancer encompass the modulation of pattern recognition receptor (PRR)-mediated inflammation, including upregulation of various transcription factors and the activation of pro-inflammatory pathways important for immune surveillance. Dysfunction of PRRs-mediated signaling has been implicated in cancer and autoimmune diseases, while the overactivation of PRRs-driven responses during infection can lead to devastating consequences such as acute lung injury or sepsis. We used crystal structure-based design to develop immunomodulatory lipopolysaccharide (LPS) mimetics targeting one of the ubiquitous PRRs, toll-like receptor 4 (TLR4). Taking advantage of an exo-anomeric conformation and specific molecular shape of synthetic nonreducing ß,ß-diglucosamine, which was investigated by NMR, we developed two sets of Lipid A mimicking glycolipids capable of either potently activating innate immune responses or inhibiting pro-inflammatory signaling. Stereoselective 1,1'-glycosylation towards fully orthogonally protected nonreducing GlcNß(1↔1')ßGlcN followed by stepwise assembly of differently functionalised phosphorylated glycolipids provided biologically active molecules that were evaluated for their ability to trigger or to inhibit cellular innate immune responses. Two LPS mimetics, identified as potent TLR4-specific inducers of the intracellular signaling pathways, serve as vaccine adjuvant- and immunotherapy candidates, while anionic glycolipids with TLR4-inhibitory potential hold therapeutic promise for the management of acute or chronic inflammation.

Structure of the type 38 Streptococcus pneumoniae capsular polysaccharide.

Streptococcus pneumoniae is one of the globally important encapsulated human pathogens and more than 100 different serotypes have been identified. Despite very extensive genetic and immune-serological studies, the capsular polysaccharide repeating unit structure of several serotypes has not been determined yet, including the type 38 (type 38 in Danish nomenclature; type 71 in US nomenclature). Physicochemical data revealed that type 38 polysaccharide is composed of a pentasaccharide repeat unit →3)-[ß-D-Galf(1 â†’ 2)]-ß-D-GalpA6(L-Ser)-(1 â†’ 3)-α-D-GlcpNAc-(1 â†’ 3)-α-D-Sugp-(1 â†’ 4)-α-D-Galp(2OAc)-(1 â†’ . The polysaccharide is O-acetylated at position C2 of the α-Gal residue at approximately (68-87 %) of the repeat units.

Semisynthetic Glycoconjugate Vaccine Candidates against Cryptococcus neoformans.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, which poses a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semisynthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semisynthetic glycoconjugate vaccines contain an identical synthetic decasaccharide (M2 motif) antigen. This antigen is present in serotype A strains, which constitute 95% of the clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity toward M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced weakly opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). These findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. This antigen could serve as a component in a multivalent GXM motif vaccine.

Rutinosides-derived from Sarocladium strictum 6-O-α-rhamnosyl-ß-glucosidase show enhanced anti-tumoral activity in pancreatic cancer cells.

BACKGROUND: Low targeting efficacy and high toxicity continue to be challenges in Oncology. A promising strategy is the glycosylation of chemotherapeutic agents to improve their pharmacodynamics and anti-tumoral activity. Herein, we provide evidence of a novel approach using diglycosidases from fungi of the Hypocreales order to obtain novel rutinose-conjugates therapeutic agents with enhanced anti-tumoral capacity. RESULTS: Screening for diglycosidase activity in twenty-eight strains of the genetically related genera Acremonium and Sarocladium identified 6-O-α-rhamnosyl-ß-glucosidase (αRßG) of Sarocladium strictum DMic 093557 as candidate enzyme for our studies. Biochemically characterization shows that αRßG has the ability to transglycosylate bulky OH-acceptors, including bioactive compounds. Interestingly, rutinoside-derivatives of phloroglucinol (PR) resorcinol (RR) and 4-methylumbelliferone (4MUR) displayed higher growth inhibitory activity on pancreatic cancer cells than the respective aglycones without significant affecting normal pancreatic epithelial cells. PR exhibited the highest efficacy with an IC50 of 0.89 mM, followed by RR with an IC50 of 1.67 mM, and 4MUR with an IC50 of 2.4 mM, whereas the respective aglycones displayed higher IC50 values: 4.69 mM for phloroglucinol, 5.90 mM for resorcinol, and 4.8 mM for 4-methylumbelliferone. Further, glycoconjugates significantly sensitized pancreatic cancer cells to the standard of care chemotherapy agent gemcitabine. CONCLUSIONS: αRßG from S. strictum transglycosylate-based approach to synthesize rutinosides represents a suitable option to enhance the anti-proliferative effect of bioactive compounds. This finding opens up new possibilities for developing more effective therapies for pancreatic cancer and other solid malignancies.

Impact of Protein Nanoparticle Shape on the Immunogenicity of Antimicrobial Glycoconjugate Vaccines.

Protein self-assembling nanoparticles (NPs) can be used as carriers for antigen delivery to increase vaccine immunogenicity. NPs mimic the majority of invading pathogens, inducing a robust adaptive immune response and long-lasting protective immunity. In this context, we investigated the potential of NPs of different sizes and shapes-ring-, rod-like, and spherical particles-as carriers for bacterial oligosaccharides by evaluating in murine models the role of these parameters on the immune response. Oligosaccharides from Neisseria meningitidis type W capsular polysaccharide were conjugated to ring-shape or nanotubes of engineered Pseudomonas aeruginosa Hemolysin-corregulated protein 1 (Hcp1cc) and to spherical Helicobacter pylori ferritin. Glycoconjugated NPs were characterized using advanced technologies such as High-Performance Liquid Chromatography (HPLC), Asymmetric Flow-Field Flow fractionation (AF4), and Transmission electron microscopy (TEM) to verify their correct assembly, dimensions, and glycosylation degrees. Our results showed that spherical ferritin was able to induce the highest immune response in mice against the saccharide antigen compared to the other glycoconjugate NPs, with increased bactericidal activity compared to benchmark MenW-CRM197. We conclude that shape is a key attribute over size to be considered for glycoconjugate vaccine development.

Targeting Pivotal Hallmarks of Cancer for Enhanced Therapeutic Strategies in Triple-Negative Breast Cancer Treatment-In Vitro, In Vivo and Clinical Trials Literature Review.

This literature review provides a comprehensive overview of triple-negative breast cancer (TNBC) and explores innovative targeted therapies focused on specific hallmarks of cancer cells, aiming to revolutionize breast cancer treatment. TNBC, characterized by its lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), presents distinct features, categorizing these invasive breast tumors into various phenotypes delineated by key elements in molecular assays. This article delves into the latest advancements in therapeutic strategies targeting components of the tumor microenvironment and pivotal hallmarks of cancer: deregulating cellular metabolism and the Warburg effect, acidosis and hypoxia, the ability to metastasize and evade the immune system, aiming to enhance treatment efficacy while mitigating systemic toxicity. Insights from in vitro and in vivo studies and clinical trials underscore the promising effectiveness and elucidate the mechanisms of action of these novel therapeutic interventions for TNBC, particularly in cases refractory to conventional treatments. The integration of targeted therapies tailored to the molecular characteristics of TNBC holds significant potential for optimizing clinical outcomes and addressing the pressing need for more effective treatment options for this aggressive subtype of breast cancer.

Exploring glycans as vital biological macromolecules: A comprehensive review of advancements in biomedical frontiers.

This comprehensive review delves into the intricate landscape of glycans and glycoconjugates, unraveling their multifaceted roles across diverse biological dimensions. From influencing fundamental cellular processes such as signaling, recognition, and adhesion to exerting profound effects at the molecular and genetic levels, these complex carbohydrate structures emerge as linchpins in cellular functions and interactions. The structural diversity of glycoconjugates, which can be specifically classified into glycoproteins, glycolipids, and proteoglycans, underscores their importance in shaping the architecture of cells. Beyond their structural roles, these molecules also play key functions in facilitating cellular communication and modulating recognition mechanisms. Further, glycans and glycoconjugates prove invaluable as biomarkers in disease diagnostics, particularly in cancer, where aberrant glycosylation patterns offer critical diagnostic cues. Furthermore, the review explores their promising therapeutic applications, ranging from the development of glycan-based nanomaterials for precise drug delivery to innovative interventions in cancer treatment. This review endeavors to comprehensively explore the intricate functions of glycans and glycoconjugates, with the primary goal of offering valuable insights into their extensive implications in both health and disease. Encompassing a broad spectrum of biological processes, the focus of the review aims to provide a comprehensive understanding of the significant roles played by glycans and glycoconjugates.

Investigation on production and reaction conditions of sucrose synthase based glucosylation cascade towards flavonoid modification.

Enzyme-based glycosylation is of great interest in the context of natural products decoration. Yet, its industrial application is hindered by optimisation difficulties and hard-to-standardise productivities. In this study, five sugar nucleotide-dependent glucosyltransferases from different origins (bacterial, plant and fungal) were coupled with soy sucrose synthase (GmSuSy) to create a set of diverse cascade biocatalysts for flavonoid glucosylation, which evaluation brought new insights into the field. Investigations into co-expression conditions and reaction settings enabled to define optimal induction temperature (25 °C) and uridine diphosphate (UDP) concentration (0.5 mM) for all tested pairs of enzymes. Moreover, the influence of pH and substrate concentration on the monoglucosylated product distribution was detected and analysed. The utilisation of crude protein extracts as a cost-effective source of catalysts unveiled their glycosidase activity against flavonoid glucosides, resulting in decreased productivity, which, to our knowledge, has not previously been discussed in such a context. Additionally, examination of the commercially available EziG immobilisation resins showed that selection of suitable carrier for solid catalyst production can be problematic and not only enzyme's but also reagent's properties have to be considered. Flavonoids, due to their complexation and hydrophobic properties, can adsorb on different types of surfaces, including divalent metal ions required for IMAC based immobilisation, necessitating detailed examination of the resins while the catalysis design.

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.

Nematicidal activity of paucimannose-type glycoconjugates from acacia honey.

Natural honey contains glycoconjugates as minor components. We characterized acacia honey glycoconjugates with molecular masses in the range of 2-5 kDa. The glycoconjugates were separated by RP-HPLC into three peaks (termed RP-2-5 k-I, RP-2-5 k-II, and RP-2-5 k-III) which demonstrated paralyzing effects on the model nematode C. elegans (ED50 of 50 ng glycoconjugates/µL). To examine molecular mechanisms underlying the nematicidal effects of honey glycoconjugates, expressional analyses of genes that are essential for the growth, development, reproduction, and movement of C. elegans were carried out. Quantitative PCR-based assays showed that these molecules moderately regulate the expression of genes involved in the citric acid cycle (mdh-1 and idhg-1) and cytoskeleton (act-1 and act-2). MALDI-ToF-MS/MS analysis of RP-HPLC peaks revealed the presence of paucimannose-like N-glycans which are known to play important roles in invertebrates e.g., worms and flies. These findings provided novel information regarding the structure and nematicidal function of honey glycoconjugates.

A Review of the Leishmanicidal Properties of Lectins.

Lectins are proteins widely distributed among plants, animals and microorganisms that have the ability to recognize and interact with specific carbohydrates. They have varied biological activities, such as the inhibition of the progression of infections caused by fungi, bacteria, viruses and protozoa, which is related to the interaction of these proteins with the carbohydrates present in the cell walls of these microorganisms. Leishmaniasis are a group of endemic infectious diseases caused by protozoa of the genus Leishmania. In vitro and in vivo tests with promastigotes and amastigotes of Leishmania demonstrated that lectins have the ability to interact with glycoconjugates present on the cell surface of the parasite, it prevents their development through various mechanisms of action, such as the production of ROS and alteration of membrane integrity, and can also interact with defense cells present in the human body, thus showing that these molecules can be considered alternative pharmacological targets for the treatment of leishmaniasis. The objective of the present work is to carry out a bibliographic review on lectins with leishmanicidal activity, emphasizing the advances and perspectives of research in this theme. Through the analysis of the selected studies, we were able to conclude that lectins have great potential for inhibiting the development of leishmaniasis. However, there are still few studies on this subject.

Anionic extracellular polymeric substances extracted from seawater-adapted aerobic granular sludge.

Anionic polymers, such as heparin, have been widely applied in the chemical and medical fields, particularly for binding proteins (e.g., fibroblast growth factor 2 (FGF-2) and histones). However, the current animal-based production of heparin brings great risks, including resource shortages and product contamination. Recently, anionic compounds, nonulosonic acids (NulOs), and sulfated glycoconjugates were discovered in the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS). Given the prevalence of anionic polymers, in marine biofilms, it was hypothesized that the EPS from AGS grown under seawater condition could serve as a raw material for producing the alternatives to heparin. This study aimed to isolate and enrich the anionic fractions of EPS and evaluate their potential application in the chemical and medical fields. The AGS was grown in a lab-scale reactor fed with acetate, under the seawater condition (35 g/L sea salt). The EPS was extracted with an alkaline solution at 80 °C and fractionated by size exclusion chromatography. Its protein binding capacity was evaluated by native gel electrophoresis. It was found that the two highest molecular weight fractions (438- > 14,320 kDa) were enriched with NulO and sulfate-containing glycoconjugates. The enriched fractions can strongly bind the two histones involved in sepsis and a model protein used for purification by heparin-column. These findings demonstrated possibilities for the application of the extracted EPS and open up a novel strategy for resource recovery. KEY POINTS: • High MW EPS from seawater-adapted AGS are dominant with sulfated groups and NulOs • Fifty-eight percent of the EPS is high MW of 68-14,320 kDa • EPS and its fractions can bind histones and fibroblast growth factor 2.

Beyond Volatile Phenols: An Untargeted Metabolomic Approach to Revealing Additional Markers of Smoke Taint in Grapevines (Vitis vinifera L.) cv. Merlot.

When bushfires occur near wine regions, vineyards are frequently exposed to environmental smoke, which can negatively affect grapes and wine. For evaluating the severity of smoke exposure, volatile phenols and their glycosides are commonly used as biomarkers of smoke exposure. While critical to refining smoke taint diagnostics, few studies have comprehensively assessed the compositional impact of smoke exposure of grapes. In this study, Merlot grapevines were exposed to smoke post-véraison, with grapes being sampled both pre-smoke exposure and repeatedly post-smoke exposure, for analysis by liquid chromatography-high-resolution mass spectrometry. Volatile phenol glycosides were detected in control and smoke-affected grapes at ≤22 µg/kg and up to 160 µg/kg, respectively. The metabolite profiles of control and smoke-affected grapes were then compared using an untargeted metabolomics approach and compounds differentiating the sample types tentatively identified. The results demonstrate the presence of novel phenolic glycoconjugates as putative metabolites from environmental smoke together with stress-related grapevine metabolites and highlight the need to further characterize the consequences of grapevine smoke exposure with respect to the regulation of abiotic stress and plant defense mechanisms.

Unique architecture of microbial snottites from a methane driven biofilm revealed by confocal microscopy.

Microbial biofilms occur in many shapes and different dimensions. In natural and semi-artificial caves they are forming pendulous structures of 10 cm and more. In this study a methane driven microbial community of a former medicinal spring was investigated. The habitat was completely covered by massive biofilms and snottites with a wobbly, gelatinous appearance. By using fluorescence techniques in combination with confocal laser scanning microscopy the architecture of these so far unknown snottites was examined. The imaging approaches applied comprised reflection of geogenic and cellular origin, possible autofluorescence, nucleic acid staining for bacterial cells, protein staining for bacteria and extracellular fine structures, calcofluor white for ß 1 → 3, ß 1 → 4 polysaccharide staining for possible fungi as well as lectin staining for the extracellular biofilm matrix glycoconjugates. The results showed a highly complex, intricate structure with voluminous, globular, and tube-like glycoconjugates of different dimensions and densities. In addition, filamentous bacteria seem to provide additional strength to the snottites. After screening with all commercially available lectins, by means of fluorescence lectin barcoding and subsequent fluorescence lectin binding analysis, the AAL, PNA, LEA, and Ban lectins identified α-Fuc, ß-Gal, ß-GlcNAc, and α-Man with α-Fuc as a major component. Examination of the outer boundary with fluorescent beads revealed a potential outer layer which could not be stained by any of the fluorescent probes applied. Finally, suggestions are made to further elucidate the characteristics of these unusual microbial biofilms in form of snottites. RESEARCH HIGHLIGHTS: The gelatinous snottites revealed at the microscale a highly complex structure not seen before. The extracellular matrix of the snottite biofilm was identified as clusters of different shape and density. The matrix of snottites was examined by taking advantage of 78 fluorescently-labeled lectins. The extracellular matrix glycoconjugates of snottites identified comprised: α-Fuc, ß-Gal, ß-GlcNAc, and α-Man. Probing the snottite outer surface indicated an additional unknown stratum.