Fluorescent Lectin Research Articles

Carbohydrate-Binding Mechanism of the Coagulant Lectin from Moringa oleifera Seeds (cMoL) Is Related to the Dimeric Protein Structure

This study characterized the binding mechanisms of the lectin cMoL (from Moringa oleifera seeds) to carbohydrates using spectroscopy and molecular dynamics (MD). The interaction with carbohydrates was studied by evaluating lectin fluorescence emission after titration with glucose or galactose (2.0–11 mM). The Stern–Volmer constant (Ksv), binding constant (Ka), Gibbs free energy (∆G), and Hill coefficient were calculated. After the urea-induced denaturation of cMoL, evaluations were performed using fluorescence spectroscopy, circular dichroism (CD), and hemagglutinating activity (HA) evaluations. The MD simulations were performed using the Amber 20 package. The decrease in Ksv revealed that cMoL interacts with carbohydrates via a static mechanism. The cMoL bound carbohydrates spontaneously (ΔG < 0) and presented a Ka on the order of 102, with high selectivity for glucose. Protein–ligand complexes were stabilized by hydrogen bonds and hydrophobic interactions. The Hill parameter (h~2) indicated that the binding occurs through the cMoL dimer. The loss of HA at urea concentrations at which the fluorescence and CD spectra indicated protein monomerization confirmed these results. The MD simulations revealed that glucose bound to the large cavity formed between the monomers. In conclusion, the biotechnological application of cMoL lectin requires specific methods or media to improve its dimeric protein structure.

Multiscale Imaging to Monitor Functional SHED-Supported Engineered Vessels.

Regeneration of orofacial tissues is hampered by the lack of adequate vascular supply. Implantation of in vitro engineered, prevascularized constructs has emerged as a strategy to allow the rapid vascularization of the entire graft. Given the angiogenic properties of dental pulp stem cells, we hereby established a preclinical model of prevascularized constructs loaded with stem cells from human exfoliating deciduous teeth (SHED) in a 3-dimensional-printed material and provided a functional analysis of their in vivo angiogenesis, vascular perfusion, and permeability. Three different cell-loaded collagen hydrogels (SHED-human umbilical vein endothelial cell [HUVEC], HUVEC with SHED-conditioned medium, and SHED alone) were cast in polylactic acid (PLA) grids and ectopically implanted in athymic mice. At day 10, in vivo positron emission tomography (PETscan) revealed a significantly increased uptake of radiotracer targeting activated endothelial cells in the SHED-HUVEC group compared to the other groups. At day 30, ex vivo micro-computed tomography imaging confirmed that SHED-HUVEC constructs had a significantly increased vascular volume compared to the other ones. Injection of species-specific lectins analyzed by 2-photon microscopy demonstrated blood perfusion of the engineered human vessels in both prevascularized groups. However, in vivo quantification showed increased vessel density in the SHED-HUVEC group. In addition, coinjection of fluorescent lectin and dextran revealed that prevascularization with SHED prevented vascular leakage, demonstrating the active role of SHED in the maturation of human-engineered microvascular networks. This preclinical study introduces a novel PLA prevascularized and implantable construct, along with an array of imaging techniques, to validate the ability of SHED to promote functional human-engineered vessels, further highlighting the interest of SHED for orofacial tissue engineering. Furthermore, this study validates the use of PETscan for the early detection of in vivo angiogenesis, which may be applied in the clinic to monitor the performance of prevascularized grafts.

Recombinant thrombomodulin and recombinant antithrombin attenuate pulmonary endothelial glycocalyx degradation and neutrophil extracellular trap formation in ventilator-induced lung injury in the context of endotoxemia

BackgroundVascular endothelial damage is involved in the development and exacerbation of ventilator-induced lung injury (VILI). Pulmonary endothelial glycocalyx and neutrophil extracellular traps (NETs) are endothelial protective and damaging factors, respectively; however, their dynamics in VILI and the effects of recombinant thrombomodulin and antithrombin on these dynamics remain unclear. We hypothesized that glycocalyx degradation and NETs are induced by VILI and suppressed by recombinant thrombomodulin, recombinant antithrombin, or their combination.MethodsVILI was induced in male C57BL/6J mice by intraperitoneal lipopolysaccharide injection (20 mg/kg) and high tidal volume ventilation (20 mL/kg). In the intervention groups, recombinant thrombomodulin, recombinant antithrombin, or their combination was administered at the start of mechanical ventilation. Glycocalyx degradation was quantified by measuring serum syndecan-1, fluorescence-labeled lectin intensity, and glycocalyx-occupied area in the pulmonary vascular lumen. Double-stranded DNA in the bronchoalveolar fluid and fluorescent areas of citrullinated histone H3 and myeloperoxidase were quantified as NET formation.ResultsSerum syndecan-1 increased, and lectin fluorescence intensity decreased in VILI. Electron microscopy revealed decreases in glycocalyx-occupied areas within pulmonary microvessels in VILI. Double-stranded DNA levels in the bronchoalveolar lavage fluid and the fluorescent area of citrullinated histone H3 and myeloperoxidase in lung tissues increased in VILI. Recombinant thrombomodulin, recombinant antithrombin, and their combination reduced glycocalyx injury and NET marker levels. There was little difference in glycocalyx injury and NET makers between the intervention groups.ConclusionVILI induced glycocalyx degradation and NET formation. Recombinant thrombomodulin and recombinant antithrombin attenuated glycocalyx degradation and NETs in our VILI model. The effect of their combination did not differ from that of either drug alone. Recombinant thrombomodulin and antithrombin have the potential to be therapeutic agents for biotrauma in VILI.

Aptamer biorecognition and gold nanoshell-mediated fluorescence quenching for glycoprotein analysis and breast cancer diagnosis

In recent years, glycoproteins have gained increasing attention due to their pivotal roles in cellular chemistry and signal transduction. However, the limited availability of rapid and wash-free analytical tools has impeded their utilization as circulating biomarkers for disease diagnosis in clinical settings. To address this challenge, we developed a gold nanoshell-mediated method for analyzing glycoprotein composition directly in native biofluids. Initially, glycoproteins are captured at the micelle surface through aptamer-protein interactions and were labeled with fluorescent lectin. Subsequently, the fluorescence of the lectin is locally quenched by the in-situ formation of gold nanoshell on micelle surface, while free-floating lectin remains unresponsive throughout this process. After optimizing the HAuCl4 dosage for gold nanoshell formation, we achieved rapid and strong fluorescence quenching against fluorescent lectin captured at micelle surface. Using this method, we analyzed the glycan compositions of CEA, HER2 and MUC1 with fluorescent ConA, Jacalin, LCA and PHA. The analyzing results demonstrated that our method can discriminate between glycoprotein compositions of plasma samples obtained from breast cancer patients and those from healthy donors.

Neuraminidase-3 Inhibition Improves Vascular Function in Diabetic Mice

In type 2 diabetes (T2D), endothelial dysfunction and arterial stiffening play a significant role in the development of cardiovascular disease. A central mediator of vascular dysfunction is degradation of the endothelial glycocalyx, a semipermeable mechanosensitive structure that separates the endothelium from the flow of blood and shear forces associated with it. Neuraminidase, a sialidase that cleaves sialic acid from the terminal branches of glycoproteins and glycolipids, is implicated in glycocalyx degradation. However, its role in mediating vascular dysfunction in the setting of T2D remains relatively unexplored. Herein, we tested the hypothesis that pharmacological inhibition of neuraminidase-3 (Neu3i) improves glycocalyx integrity, ameliorates endothelial dysfunction, and reduces arterial stiffness in diabetes. In support of this hypothesis, we report that, relative to vehicle-treated cells, endothelial cells exposed to Neu3i for 24hr have increased glycocalyx components including sialic acid and N-acetylglucosamine (GlcNAc), one of the amino sugars comprising the N-acetyllactosaminoglycans chains within the glycocalyx, as assessed with fluorescent lectin staining using MAA/Mal I+2 and WGA, respectively. We further show that treatment of diabetic ( i.e., db/db) mice with Neu3i for 28 days reduced aortic pulse wave velocity, with no change in blood pressure, when compared to vehicle-treated mice. Neu3i also reduced the stiffness of aortic explants, as assessed via atomic force microscopy. Excised arteries were also assessed for flow-mediated dilation and vascular stiffness using pressure myography. Diabetic mice treated with Neu3i had increased flow-mediated dilation and reduced incremental modulus of elasticity, indicative of improved endothelial function and reduced arterial stiffness. Collectively, these data support the hypothesis that Neu3 is a potential therapeutic target for the amelioration of vascular dysfunction in T2D. National Institutes of Health grant: R01HL153264 to LM-L and JP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Biomarker-based human and animal sperm phenotyping: the good, the bad and the ugly†.

Conventional, brightfield-microscopic semen analysis provides important baseline information about sperm quality of an individual; however, it falls short of identifying subtle subcellular and molecular defects in cohorts of "bad," defective human and animal spermatozoa with seemingly normal phenotypes. To bridge this gap, it is desirable to increase the precision of andrological evaluation in humans and livestock animals by pursuing advanced biomarker-based imaging methods. This review, spiced up with occasional classic movie references but seriously scholastic at the same time, focuses mainly on the biomarkers of altered male germ cell proteostasis resulting in post-testicular carryovers of proteins associated with ubiquitin-proteasome system. Also addressed are sperm redox homeostasis, epididymal sperm maturation, sperm-seminal plasma interactions, and sperm surface glycosylation. Zinc ion homeostasis-associated biomarkers and sperm-borne components, including the elements of neurodegenerative pathways such as Huntington and Alzheimer disease, are discussed. Such spectrum of biomarkers, imaged by highly specific vital fluorescent molecular probes, lectins, and antibodies, reveals both obvious and subtle defects of sperm chromatin, deoxyribonucleic acid, and accessory structures of the sperm head and tail. Introduction of next-generation image-based flow cytometry into research and clinical andrology will soon enable the incorporation of machine and deep learning algorithms with the end point of developing simple, label-free methods for clinical diagnostics and high-throughput phenotyping of spermatozoa in humans and economically important livestock animals.

Xylene versus Isopropanol for Paraffin Wax Processing of Lung Tissue

The microscopic observation of lung tissue is challenging due to its fragile nature. Xylene and isopropanol are common tissue-clearing reagents used before paraffin embedding, yet no studies have compared these two reagents in lung tissue processing. Due to the well-known health risks xylene could introduce to operators, as well as its environmental hazards, it has long been desired that a less harmful alternative to xylene with the same staining effects be introduced. Thus, we systematically assessed the efficacy of isopropanol as a substitution for xylene. Lung tissue obtained from diseased donors and explanted lungs from recipients were processed simultaneously using either xylene or isopropanol prior to paraffin embedding. Scoring of the overall staining quality after H&E staining, along with the ease of sectioning, was compared systematically. Fluorescent staining was performed to explore alveolar morphology and the overall lectin fluorescence signal between groups. To understand differences in antibody staining, the signal-to-noise ratio (SNR) of smooth muscle actin (SMA) and elastin was examined. No difference was observed with regard to ease of sectioning, staining quality, alveolar circularity, alveolar wall thickness or the SNR between slides processed with xylene or isopropanol. This study demonstrated comparable outcomes of isopropanol and xylene in lung tissue processing, suggesting isopropanol as a more favorable, operator- and environment-friendly substitute for xylene with regards to tissue processing.

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 β 1 → 3, β 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, β-Gal, β-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, β-Gal, β-GlcNAc, and α-Man. Probing the snottite outer surface indicated an additional unknown stratum.

Exogenous progesterone during in vitro fertilization improves developmental competence of partially cumulus-denuded bovine oocytes

The progesterone (P4) secreted by cumulus cells has gained attention for its role as a possible physiological inducer of sperm acrosome exocytosis. In mammals, it is generally accepted that fertilization rates of oocytes without cumulus are markedly low. This study assessed the integrity of capacitated bovine sperm acrosome when exposed to increasing concentrations of P4, and evaluated whether exogenous P4 during in vitro fertilization (IVF) increases the developmental competence of partially cumulus-denuded oocytes in serum-free conditions. After a 4-h capacitation induction, sperm were incubated with increasing concentrations of P4 (0, 0.1, 10 and 100 μM) and evaluated for viability, caspase activation and acrosome status at three different times (4, 5, and 22 h), including the capacitation induction period. Progesterone induced sperm acrosomal exocytosis without compromising sperm viability or activating sperm caspases. Sperm undergoing acrosome reaction exhibited three differential Concanavalin A patterns, corresponding to early, intermediate and late acrosomal exocytosis. The percentage of these patterns significantly increased over time, regardless of P4 concentration, except for those spermatozoa with late acrosomal exocytosis, which only showed an increase at 22 h of incubation. After incubation for 1 h with 100 μM P4, spermatozoa showing intermediate acrosomal exocytosis significantly increased. At 22 h of incubation, the pattern corresponding to early acrosomal exocytosis evidenced a dose-dependent increase. However, prematurely high levels of acrosome reaction induced by 100 μM P4 led to inefficient IVF outcomes (P < 0.05). Therefore, IVF trials with partially cumulus-denuded oocytes were carried out with lower P4 concentrations (0, 0.1, 1, 5, 10 μM). Cleavage rate significantly increased at 1 μM P4, which translated to increased total embryo production after 7 days of in vitro culture (P < 0.05). Significantly higher percentages of expanded blastocysts were observed at both 1 μM and 10 μM P4 as compared to the other experimental conditions. In conclusion, the different patterns of acrosomal exocytosis identified over time by incubation of live sperm with a fluorescent lectin revealed the existence of sperm subpopulations heterogeneous in their physiological states. Moreover, exogenous P4 at 1 μM during IVF improved the developmental competence of partially cumulus-denuded oocytes in serum-free conditions.

Optimizing Lectin Staining Methodology to Assess Glycocalyx Composition of Legionella-Infected Cells

Introduction: Legionella is a gram-negative bacterium that replicates intracellularly within macrophages. Legionella utilizes effector proteins to hijack ER-Golgi vesicle trafficking to sustain proliferation in its intracellular niche. Legionella has a considerable influence on O-glycosylation but not N-glycosylation events in the Golgi of infected cells. This research aims to optimize the use of fluorescent lectins, which are proteins that bind carbohydrates, to effectively label host-cell glycocalyx during Legionella infection. Methods: Epifluorescence imaging or flow cytometry were used to optimize the lectin staining methodology. We noted that the most effective conditions for lectin-labeling were when live HeLa cells were incubated with lectins diluted in Hank’s balanced salt solution (HBSS) with 3% Bovine serum albumin (BSA) for 10-30 minutes at 4 °C. Results: Incubating suspended cells with lectins necessitated smaller lectin concentrations, whereas lectin labeling of adherent cells required considerably larger concentrations. Wheat germ agglutinin (WGA) lectin mean fluorescence intensity (MFI) was concentration-dependent, but Concanavalin A (ConA) and Maclura pomifera (MPA) MFIs did not alter substantially with increasing lectin concentrations. Discussion: The optimal lectin concentration required was lectin-specific and based on whether the lectin fluorescence was assessed using flow cytometry or epifluorescence. Furthermore, the use of phosphate-buffered saline (PBS) for lectin dilution, cell permeabilization for intracellular labelling, and incubation of lectins in fixed cells reduced productive labelling of lectins on cell surfaces because it inhibited the lectin's ability to effectively bind the associated carbohydrate structure. Conclusion: Further research using diverse lectins on U937 macrophages is necessary to reach a definitive conclusion on the effect of Legionella on the overall host-cell glycocalyx composition during infection of these relevant immune cells.

Cerebrovascular glycocalyx damage and microcirculation impairment in patients with temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is increasingly associated with blood-brain barrier dysfunction and microvascular alterations, yet the pathophysiological link is missing. An important barrier function is exerted by the glycocalyx, a gel-like layer coating the endothelium. To explore such associations, we used intraoperative videomicroscopy to quantify glycocalyx and microcirculation properties of the neocortex and hippocampus of 15 patients undergoing resective brain surgery as treatment for drug-resistant TLE, and 15 non-epileptic controls. Fluorescent lectin staining of neocortex and hippocampal tissue was used for blood vessel surface area quantification. Neocortical perfused boundary region, the thickness of the glycocalyx’ impaired layer, was higher in patients (2.64 ± 0.52 µm) compared to controls (1.31 ± 0.29 µm), P < 0.01, indicative of reduced glycocalyx integrity in patients. Moreover, erythrocyte flow velocity analysis revealed an impaired ability of TLE patients to (de-)recruit capillaries in response to changing metabolic demands (R2 = 0.75, P < 0.01), indicating failure of neurovascular coupling mechanisms. Blood vessel quantification comparison between intraoperative measurements and resected tissue showed strong correlation (R2 = 0.94, P < 0.01). This is the first report on in vivo assessment of glycocalyx and microcirculation properties in TLE patients, confirming the pivotal role of cerebrovascular changes. Further assessment of the cerebral microcirculation in relation to epileptogenesis might open avenues for new therapeutic targets for drug-resistant epilepsy.

Interplay between Furin and Sialoglycans in Modulating Adeno-Associated Viral Cell Entry.

Adeno-associated viruses (AAVs) are small, helper-dependent, single-stranded DNA viruses that exploit a broad spectrum of host factors for cell entry. During the course of infection, several AAV serotypes have been shown to transit through the trans-Golgi network within the host cell. In the current study, we investigated whether the Golgi-localized, calcium-dependent protease furin influences AAV transduction. While CRISPR/Cas9-mediated knockout (KO) of the Furin gene minimally affected the transduction efficiency of most recombinant AAV serotypes tested, we observed a striking increase in transgene expression (~2 log orders) for the African green monkey isolate AAV4. Interrogation of different steps in the infectious pathway revealed that AAV4 binding, uptake, and transcript levels are increased in furin KO cells, but postentry steps such as uncoating or nuclear entry remain unaffected. Recombinant furin does not cleave AAV4 capsid proteins nor alter cellular expression levels of essential factors such as AAVR or GPR108. Interestingly, fluorescent lectin screening revealed a marked increase in 2,3-O-linked sialoglycan staining on the surface and perinuclear space of furin KO cells. The essential nature of increased sialoglycan expression in furin KO cells in enhancing AAV4 transduction was further corroborated by (i) increased transduction by the closely related isolates AAVrh.32.33 and sea lion AAV and (ii) selective blockade or removal of cellular 2,3-O-linked sialoglycans by specific lectins or neuraminidase, respectively. Based on the overall findings, we postulate that furin likely plays a key role in regulating expression of cellular sialoglycans, which in turn can influence permissivity to AAVs and possibly other viruses. IMPORTANCE Adeno-associated viruses (AAVs) are a proven recombinant vector platform for gene therapy and have demonstrated success in the clinic. Continuing to improve our knowledge of AAV-host cell interactions is critical for improving the safety and efficacy. The current study dissects the interplay between furin, a common intracellular protease, and certain cell surface sialoglycans that serve as viral attachment factors for cell entry. Based on the findings, we postulate that differential expression of furin in host cells and tissues is likely to influence gene expression by certain recombinant AAV serotypes.

Effects of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Infection on the Surface Glycoprofiling of Porcine Pulmonary Microvascular Endothelial Cells.

Previously, our study has demonstrated that porcine pulmonary microvascular endothelial cells (PPMVECs) were susceptible to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and produced a significant non-specific immune response to it. The significance of microvascular endothelial glycocalyx is increasingly attracting attention, and its rich carbohydrate components are not only important signaling molecules, but also remarkably influence the signaling of most proteins. Comprehending changes in the carbohydrate chains contributes to understanding cell functions. This study aimed to reveal the effects of HP-PRRSV infection on the surface carbohydrate chains of PPMVECs. PPMVECs were isolated and cultured in vitro and infected with HP-PRRSV HN and JXA1 strains. Scanning electron microscopy analysis indicated that at 48 h post-infection, some broken holes were in their cell membranes, and that the surface fibrous glycocalyx was obviously reduced or even disappeared. Lectin microarray analysis indicated that the fluorescence intensities of 8 and 7 lectin sites were significantly changed by the HP-PRRSV HN and JXA1 strains, respectively, among which there were 6 common lectin sites. The up-regulation of common lectins (RCA-I, LEL, and STL) and the down-regulation of common lectins (LCA, DSA, and PHA-E) were confirmed by lectin fluorescence staining and lectin flow cytometry, respectively. Together, the results show that the HP-PRRSV infection can induce the glycocalyx disruption of PPMVECs and their surface glycoprofiling changes, and that the poly-N-acetyllactosamine and complex N-glycan are the main up-regulated and down-regulated carbohydrate chains, respectively. Our findings may provide insights into revealing the pathogenesis of HP-PRRSV from the perspective of glycobiology.

Suitability of lectin binding studies for the characterization of redox-active microbial environmental biofilms

Aquatic environmental microbial biofilms grow in a broad range of redox environments from oxic to methanogenic, and they often also establish internal redox gradients. In technical applications, biofilms are also subjected to controlled redox conditions. Studies on biofilms often make use of fluorescence microscopic imaging techniques together with lectin binding analysis to gain insights into structure, composition, and functions of the biofilms. Here we studied the direct influence of redox potentials on fluorescence lectin binding analyses (FLBA) for two commonly used lectin-fluorophore conjugates. An effect of the electrical potential on signal intensity was observed and found to be statistically significant. The signal intensity changes however, remained within the range of a few percent total. A significant drop in intensity was only observed for extremely oxidizing potentials, typically not found under environmental conditions. Our results showed that the fluorophore itself and not the lectin binding to the respective glycoconjugate causes fluorescence changes. The two tested lectin-fluorophores are shown to be suitable for studying the distribution and composition of EPS in environmental biofilms or technical applications and under varying redox conditions.

Modification of Morphology and Glycan Pattern of the Oviductal Epithelium of Baboon Papio hamadryas during the Menstrual Cycle.

Simple SummaryThe oviduct or Fallopian tube is the anatomical region where fertilization and early embryonic development take place. The morphology and activity of the epithelial cells are under the control of the sex hormone levels. The non-ciliated cells secrete oviduct-specific estrogen-dependent glycoproteins whose glycan component, despite its key role in the oviductal function, has not yet been studied in baboon species. This study examined the morphology and the glycan composition of the oviductal epithelium of the baboon Papio hamadryas during the menstrual cycle. Different segments of the laparoscopically removed oviducts from healthy adult females during the follicular, preovulatory, and luteal phases were processed. The stage of the menstrual cycle was based on the sex hormone levels and the vaginal cytology features. The morphological and glycopattern analyses revealed that in the epithelium lining, all the oviductal segments were taller, more differentiated, and richer in glycoconjugates during the preovulatory phase than in the follicular and luteal phases. A region-specific glycosylation pattern was also detected. These results provide an insight into the molecular differences occurring in the oviductal regions during the phases of the menstrual cycle of the baboon oviduct, which is a primate phylogenetically close to humans and whose reproductive tract is similar to that of women and influenced by the same hormonal events.The mammalian oviduct is a highly specialized structure where fertilization and early embryonic development occur. Its mucosal epithelium is involved in maintaining and modulating a dynamic intraluminal fluid. The oviductal epithelium consists of ciliated and non-ciliated (secretory) cells whose differentiation and activity are sex hormone-dependent. In this study, we investigated for the first time both the morphology and the glycan composition of baboon oviductal epithelium during the menstrual cycle. Oviducts were laparoscopically removed from 14 healthy adult female Papio hamadryas whose menstrual cycle phase was assessed based on the sex hormone levels and the vaginal cytology features. Histological investigations were carried out on fimbriae, infundibulum, ampulla, and isthmus separately fixed in 4% (v/v) paraformaldehyde, embedded in paraffin wax, and stained with hematoxylin-eosin for morphological analyses and using a panel of nine fluorescent lectins for glycoconjugate characterization. The histomorphological analysis revealed that in the entire oviduct (i) the ciliated and non-ciliated cells were indistinguishable during the follicular and luteal phases, whereas they were highly differentiated during the preovulatory phase when the non-ciliated cells exhibited apical protrusions, (ii) the epithelium height was significantly higher in the preovulatory phase compared to other menstrual phases, and (iii) the number of ciliated cells significantly (p ≤ 0.05) increased from the fimbriae to the infundibulum and progressively reduced in the other oviductal segments with the lower presence of ciliated cells in the isthmus. The glycan characterization revealed a complex and region-specific composition during the different phases of the menstrual cycle. It can be summarized as follows: (i) high-mannosylated N-linked glycans (Con A reactivity) were present throughout the oviductal epithelium during the entire menstrual cycle and characteristically in the apical protrusions of non-ciliated cells of the ampulla during the preovulatory phase; (ii) sialoglycans with α2,3-linked sialic acids (MAL II binding) were expressed along the entire oviductal surface only during the preovulatory phase, whereas α2,6-linked ones (SNA affinity) were also detected in the surface of the luteal phase, although during the preovulatory phase they were characteristically found in the glycocalyx of the isthmus cilia, and O-linked sialoglycans with sialic acids linked to Galβl,3GalNAc (T antigen) (KsPNA) and terminal N-acetylgalactosamine (Tn antigen) (KsSBA) were found in the entire oviductal surface during all phases of the menstrual cycle; (iii) GalNAc terminating O-linked glycans (HPA staining) were mainly expressed in the entire oviducts of the luteal and preovulatory phases, and characteristically in the apical protrusions of the isthmus non-ciliated cells of the preovulatory phase; and (iv) fucosylated glycans with α1,2-linked fucose (LTA reactivity) occurred in the apical surface of fimbriae during the luteal phase, whereas α1,3/4-linked fucose (UEA I binders) were present in the apical protrusions of the ampulla non-ciliated cells and in the apical surface of isthmus during the preovulatory phase as well as in the isthmus apical surface of follicular-phase oviducts. These results demonstrate for the first time that morphological and glycan changes occur in the baboon oviductal epithelium during the menstrual cycle. Particularly, the sex hormone fluctuation affects the glycan pattern in a region-specific manner, probably related to the function of the oviductal segments. The findings add new data concerning baboons which, due to their anatomical similarity to humans, make an excellent model for female reproduction studies.

Bone marrow mesenchymal stromal cell-derived extracellular matrix displays altered glycosaminoglycan structure and impaired functionality in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematologic malignancies characterized by clonal hematopoiesis, one or more cytopenias such as anemia, neutropenia, or thrombocytopenia, abnormal cellular maturation, and a high risk of progression to acute myeloid leukemia. The bone marrow microenvironment (BMME) in general and mesenchymal stromal cells (MSCs) in particular contribute to both the initiation and progression of MDS. However, little is known about the role of MSC-derived extracellular matrix (ECM) in this context. Therefore, we performed a comparative analysis of in vitro deposited MSC-derived ECM of different MDS subtypes and healthy controls. Atomic force microscopy analyses demonstrated that MDS ECM was significantly thicker and more compliant than those from healthy MSCs. Scanning electron microscopy showed a dense meshwork of fibrillar bundles connected by numerous smaller structures that span the distance between fibers in MDS ECM. Glycosaminoglycan (GAG) structures were detectable at high abundance in MDS ECM as white, sponge-like arrays on top of the fibrillar network. Quantification by Blyscan assay confirmed these observations, with higher concentrations of sulfated GAGs in MDS ECM. Fluorescent lectin staining with wheat germ agglutinin and peanut agglutinin demonstrated increased deposition of N-acetyl-glucosamine GAGs (hyaluronan (HA) and heparan sulfate) in low risk (LR) MDS ECM. Differential expression of N-acetyl-galactosamine GAGs (chondroitin sulfate, dermatan sulfate) was observed between LR- and high risk (HR)-MDS. Moreover, increased amounts of HA in the matrix of MSCs from LR-MDS patients were found to correlate with enhanced HA synthase 1 mRNA expression in these cells. Stimulation of mononuclear cells from healthy donors with low molecular weight HA resulted in an increased expression of various pro-inflammatory cytokines suggesting a contribution of the ECM to the inflammatory BMME typical of LR-MDS. CD34+ hematopoietic stem and progenitor cells (HSPCs) displayed an impaired differentiation potential after cultivation on MDS ECM and modified morphology accompanied by decreased integrin expression which mediate cell-matrix interaction. In summary, we provide evidence for structural alterations of the MSC-derived ECM in both LR- and HR-MDS. GAGs may play an important role in this remodeling processes during the malignant transformation which leads to the observed disturbance in the support of normal hematopoiesis.

Fluorescence lectin binding analysis of carbohydrate components in dental biofilms grown in situ in the presence or absence of sucrose.

Carbohydrate components, such as glycoconjugates and polysaccharides, are constituents of the dental biofilm matrix that play an important role in biofilm stability and virulence. Exopolysaccharides in Streptococcus mutans biofilms have been characterized extensively, but comparably little is known about the matrix carbohydrates in complex, in situ-grown dental biofilms. The present study employed fluorescence lectin binding analysis (FLBA) to investigate the abundance and spatial distribution of glycoconjugates/polysaccharides in biofilms (n=306) from 10 participants, grown in situ with (SUC) and without (H2O) exposure to sucrose. Biofilms were stained with 10 fluorescently labeled lectins with different carbohydrate specificities (AAL, ABA, ASA, HPA, LEA, MNA-G, MPA, PSA, VGA and WGA) and analyzed by confocal microscopy and digital image analysis. Microbial composition was determined by 16S rRNA gene sequencing. With the exception of ABA, all lectins targeted considerable matrix biovolumes, ranging from 19.3% to 194.0% of the microbial biovolume in the biofilms, which illustrates a remarkable variety of carbohydrate compounds in in situ-grown dental biofilms. MNA-G, AAL, and ASA, specific for galactose, fucose, and mannose, respectively, stained the largest biovolumes. AAL and ASA biovolumes were increased in SUC biofilms, but the difference was not significant due to considerable biological variation. SUC biofilms were enriched in streptococci and showed reduced abundances of Neisseria and Haemophilus spp., but no significant correlations between lectin-stained biovolumes and bacterial abundance were observed. In conclusion, FLBA demonstrates the presence of a voluminous biofilm matrix comprising a variety of different carbohydrate components in complex, in situ-grown dental biofilms.

Legionella pneumophila Infection of Human Macrophages Retains Golgi Structure but Reduces O-Glycans.

Legionella pneumophila is an accidental pathogen that replicates intracellularly within the Legionella-containing vacuole (LCV) in macrophages. Within an hour of infection, L. pneumophila secretes effectors to manipulate Rab1 and intercept ER-derived vesicles to the LCV. The downstream consequences of interrupted ER trafficking on the Golgi of macrophages are not clear. We examined the Golgi structure and function in L. pneumophila-infected human U937 macrophages. Intriguingly, the size of the Golgi in infected macrophages remained similar to uninfected macrophages. Furthermore, TEM analysis also did not reveal any significant changes in the ultrastructure of the Golgi in L. pneumophila-infected cells. Drug-induced Golgi disruption impacted bacterial replication in human macrophages, suggesting that an intact organelle is important for bacteria growth. To probe for Golgi functionality after L. pneumophila infection, we assayed glycosylation levels using fluorescent lectins. Golgi O-glycosylation levels, visualized by the fluorescent cis-Golgi lectin, Helix pomatia agglutinin (HPA), significantly decreased over time as infection progressed, compared to control cells. N-glycosylation levels in the Golgi, as measured by L-PHA lectin staining, were not impacted by L. pneumophila infection. To understand the mechanism of reduced O-glycans in the Golgi we monitored UDP-GalNAc transporter levels in infected macrophages. The solute carrier family 35 membrane A2 (SLC35A2) protein levels were significantly reduced in L. pneumophila-infected U937 and HeLa cells and L. pneumophila growth in human macrophages benefitted from GalNAc supplementation. The pronounced reduction in Golgi HPA levels was dependent on the translocation apparatus DotA expression in bacteria and occurred in a ubiquitin-independent manner. Thus, L. pneumophila infection of human macrophages maintains and requires an intact host Golgi ultrastructure despite known interference of ER–Golgi trafficking. Finally, L. pneumophila infection blocks the formation of O-linked glycans and reduces SLC35A2 protein levels in infected human macrophages.

Matrix glycoconjugate characterization in multispecies biofilms and bioaggregates from the environment by means of fluorescently-labeled lectins.

Environmental biofilms represent a complex mixture of different microorganisms. Their identity is usually analyzed by means of nucleic acid-based techniques. However, these biofilms are also composed of a highly complex extracellular matrix produced by the microbes within a particular biofilm system. The biochemical identity of this extracellular matrix remains in many cases an intractable part of biofilms and bioaggregates. Consequently, there is a need for an approach that will give access to the fully hydrated structure of the extracellular matrix or at least a major part of it. A crucial compound of the matrix identified as carbohydrate-based polymers represents major structural and functional constituents. These glycoconjugates can be characterized by using fluorescently-labeled lectins in combination with confocal laser scanning microscopy. The lectin approach is defined previously, as fluorescence lectin barcoding (FLBC) and fluorescence lectin-binding analysis (FLBA), where FLBC is equal to the screening of a particular sample with all the commercially available lectins and FLBA is the actual analysis of the matrix throughout an experiment with a selected panel of lectins. As the application of immune-based techniques in environmental biofilm systems is impossible, the lectin approach is currently the only option for probing lectin-specific glycoconjugates in complex biofilms and bioaggregates. From all the commercially available lectins tested, the lectins such as AAL, HAA, WGA, ConA, IAA, HPA, and LEA showed the highest binding efficiency. Furthermore, 20 of the overall lectins tested showed an intermediate signal intensity, nevertheless very useful for the assessment of matrix glycoconjugates. With the data compiled, we shall virtually shed more light on the dark matter of the extracellular matrix and their 3-dimensional distribution in environmental biofilm systems. The results will be helpful in future studies with a focus on the extracellular matrix glycoconjugates present in environmental microbial communities.

Evaluation of Cell Models to Study Monocyte Functions in PMM2 Congenital Disorders of Glycosylation.

Congenital disorders of glycosylation (CDG) are inherited metabolic diseases characterized by mutations in enzymes involved in different steps of protein glycosylation, leading to aberrant synthesis, attachment or processing of glycans. Recently, immunological dysfunctions in several CDG types have been increasingly documented. Despite these observations, detailed studies on immune cell dysfunction in PMM2-CDG and other CDG types are still scarce. Studying PMM2-CDG patient immune cells is challenging due to limited availability of patient material, which is a result of the low incidence of the disease and the often young age of the subjects. Dedicated immune cell models, mimicking PMM2-CDG, could circumvent many of these problems and facilitate research into the mechanisms of immune dysfunction. Here we provide initial observations about the immunophenotype and the phagocytic function of primary PMM2-CDG monocytes. Furthermore, we assessed the suitability of two different glycosylation-impaired human monocyte models: tunicamycin-treated THP-1 monocytes and PMM2 knockdown THP-1 monocytes induced by shRNAs. We found no significant differences in primary monocyte subpopulations of PMM2-CDG patients as compared to healthy individuals but we did observe anomalous surface glycosylation patterns in PMM2-CDG patient monocytes as determined using fluorescent lectin binding. We also looked at the capacity of monocytes to bind and internalize fungal particles and found a slightly increased uptake of C. albicans by PMM2-CDG monocytes as compared to healthy monocytes. Tunicamycin-treated THP-1 monocytes showed a highly decreased uptake of fungal particles, accompanied by a strong decrease in glycosylation levels and a high induction of ER stress. In contrast and despite a drastic reduction of the PMM2 enzyme activity, PMM2 knockdown THP-1 monocytes showed no changes in global surface glycosylation levels, levels of fungal particle uptake similar to control monocytes, and no ER stress induction. Collectively, these initial observations suggest that the absence of ER stress in PMM2 knockdown THP-1 cells make this model superior over tunicamycin-treated THP-1 cells and more comparable to primary PMM2-CDG monocytes. Further development and exploitation of CDG monocyte models will be essential for future in-depth studies to ultimately unravel the mechanisms of immune dysfunction in CDG.