Glycan variation and evolution in the eukaryotes.

In this review, we document the evolution of common glycan structures in the eukaryotes, and illustrate the considerable variety of oligosaccharides existing in these organisms. We focus on the families of N- and O-glycans, glycosphingolipids, glycosaminoglycans, glycosylphosphatidylinositol (GPI) anchors, sialic acids (Sias), and cytoplasmic and nuclear glycans. We also outline similar and divergent aspects of the glycans during evolution within the groups, which include inter- and intraspecies differences, molecular mimicry, viral glycosylation adaptations, glycosyltransferase specificity relating to function, and the natural dynamism powering these events. Finally, we present an overview of the patterns of glycosylation found within the groups comprising the Eukaryota, namely the Deuterostomia, Fungi, Viridiplantae, Nematoda, and Arthropoda.

Divalent Naphthalene Diimide Ligands Display High Selectivity for the Human Telomeric G-quadruplex in K+ Buffer.

Selective G-quadruplex ligands offer great promise for the development of anti-cancer therapies. A novel series of divalent cationic naphthalene diimide ligands that selectively bind to the hybrid form of the human telomeric G-quadruplex in K+ buffer are described herein. We demonstrate that an imidazolium-bearing mannoside-conjugate is the most selective ligand to date for this quadruplex against several other quadruplex and duplex structures. We also show that a similarly selective methylpiperazine-bearing ligand was more toxic to HeLa cancer cells than doxorubicin, whilst exhibiting three times less toxicity towards fetal lung fibroblasts WI-38.

Lactose as a "Trojan horse" for quantum dot cell transport.

A series of glycan-coated quantum dots were prepared to probe the effect of glycan presentation in intracellular localization in HeLa and SV40 epithelial cells. We show that glycan density mostly impacts on cell toxicity, whereas glycan type affects the cell uptake and intracellular localization. Moreover, we show that lactose can act as a "Trojan horse" on bi-functionalized QDs to help intracellular delivery of other non-internalizable glycan moieties and largely avoid the endosomal/lysosomal degradative pathway.

Understanding Ocular Surface Inflammation in Tears Before and After Autologous Cultivated Limbal Epithelial Stem Cell Transplantation.

INTRODUCTION: We aimed to determine the expression of inflammatory cytokines in the tears of patients with unilateral total limbal stem cell deficiency (TLSCD) caused by chemical burns before and after autologous cultivated limbal epithelial stem cell transplantation (CLET). METHODS: Tear samples were collected from both eyes of 23 patients with unilateral TLSCD and 11 healthy controls, at fixed timepoints before and after CLET. Dissolved molecules were extracted from Schirmer's strips using a standardised method and analysed on an array plate of ten inflammatory cytokines (V-Plex Proinflammatory Panel 1 Human Kit, MSD). RESULTS: IL1ß expression was significantly elevated in the TLSCD eye compared with the unaffected eye at baseline (p < 0.0001) but decreased to normal 3 months post-CLET (p = 0.22). IL6 and IL8 were unaffected at baseline but significantly elevated in the TLSCD eyes at 1 month post-CLET (p = 0.001 and p < 0.0001, respectively). IL6 returned to normal at 3 months and IL8 at 6 months post-CLET. There was a significant renewed increase in IL1ß, IL6 and IL8 expression at 12 months post-CLET (p < 0.0001, p = 0.0001 and p = 0.0003, respectively). IFNγ, IL10 and IL12p70 expression were significantly reduced in both eyes of patients with unilateral TLSCD at all timepoints. CONCLUSION: IL1ß is a specific marker of inflammation in TLSCD eyes that could be therapeutically targeted pre-CLET to improve stem cell engraftment. At 12 months post-CLET the spike in levels of IL1ß, IL6 and IL8 coincides with cessation of topical steroids, suggesting ongoing subclinical inflammation. We therefore recommend not discontinuing topical steroid treatment in cases where penetrating keratoplasty is indicated; however, further investigation is needed to ascertain this. TRIAL REGISTRATION: European Union Drug Regulating Authorities Clinical Trials Database (EuDRACT 2011-000608-16); ISRCTN (International Standard Randomised Controlled Trial Number (isrctn51772481).

Trefoil factor family peptides are increased in the saliva of children with mucositis.

BACKGROUND: Mucositis is a painful ulcerative condition of the oral cavity and gastrointestinal tract, occurring in association with chemotherapy and radiotherapy regimes. Trefoil factor family peptides (TFF, trefoil peptides), present in saliva, contribute to epithelial restitution and repair and are therefore potentially important in the healing phase of mucositis. This study aimed to assess any changes in the levels of trefoil peptides in oncology patients with and without mucositis. METHODS: Saliva was collected from healthy children, pretreatment oncology patients, neutropenic patients on treatment with no oral disease and mucositic patients. TFF1, 2 and 3 were quantified using ELISA. RESULTS: In healthy children TFF2 and 3 were positively correlated with age (r = 0.454, p = 0.01 for TFF2; r = 0.410, p = 0.05 for TFF3 Spearman rank correlation). TFF3 was higher in mucositis compared to all other groups. A linear regression prediction model indicated that TFF3, but not TFF1 and TFF2, was significantly different in mucositic and healthy controls, suggesting an altered pattern of trefoil peptide secretion (p = 0.021). CONCLUSIONS: This study is the first to focus on trefoil peptides in paediatric saliva. It shows the correlation between TFF2, TFF3 and age in healthy children. Paediatric mucositis disease occurs in the presence of increased concentrations and an altered pattern of trefoil peptides.

Selective photothermal killing of cancer cells using LED-activated nucleus targeting fluorescent carbon dots.

The development of effective theranostic probes in cancer therapy is hampered due to issues with selectivity and off-target toxicity. We report the selective LED-photothermal ablation of cervical (HeLa) cancer cells over human dermal fibroblasts (HDF) using a new class of green-emissive fluorescent carbon dots (FCDs). The FCDs can be easily prepared in one pot using cheap and commercial starting materials. Physico-chemical characterization revealed that a surface coating of 2,5-deoxyfructosazine on a robust amorphous core gives rise to the nanomaterial's unique properties. We show that intracellular uptake mostly involves passive mechanisms in combination with intracellular DNA interactions to target the nucleus and that cancer cell selective killing is likely due to an increase in intracellular temperature in combination with ATP depletion, which is not observed upon exposure to either the "naked" core FCDs or the surface components individually. The selectivity of these nanoprobes and the lack of apparent production of toxic metabolic byproducts make these new nanomaterials promising agents in cancer therapy.

Clinical tests for successful contact lens wear: relationship and predictive potential.

PURPOSE: Although comfort is important for contact lens wearers, common clinical tests can fail to predict patients' symptoms. Lid wiper epitheliopathy (LWE) and lid parallel conjunctival folds (LIPCOF) are related to dry eye symptoms in lens wearers. This study investigates the predictive value of LWE and LIPCOF as objective measures of discomfort, and their relation to the ocular surface in soft contact lens wearers. METHODS: Subjects were classified as symptomatic or asymptomatic, using the Contact Lens Dry Eye Questionnaire (CLDEQ). Pre-lens tear break-up time (PLBUT), limbal and bulbar hyperaemia, corneal staining, LWE and LIPCOF were assessed in the right eyes of 61 (23 M, 38 F; mean age 32.1 years; range = 18 to 55) experienced contact lens wearers. Differences between groups, and relationships between LWE, LIPCOF (nasal, temporal and sum) and objective signs were examined using non-parametric analyses. The positive and negative predictive values for symptoms of each objective measure were calculated. RESULTS: Thirty eight subjects were classified as asymptomatic, 23 symptomatic. LWE and LIPCOF severity scores were significantly increased in symptomatic patients (U-test, p < 0.03), while no significant differences were found between groups for PLBUT, corneal staining or hyperaemia (0.29 < p < 0.88). Significant positive correlations were found between LWE and LIPCOF scores (temporal r = 0.67, p < 0.001; nasal r = 0.39, p < 0.001), and between LWE and hyperaemia (bulbar, r = 0.28, p < 0.001; limbal r = 0.36, p < 0.001). Age and gender were different in the two groups (p < 0.05). The predictive value of temporal LIPCOF was positive = 56.9%, negative = 77.1% with a cutoff value of > or =2 (PPV/NPV/cutoff value), of nasal LIPCOF 70.7%/75.0%/> or =1, of LIPCOF Sum 79.8%/86.5%/> or =2, and of LWE 53.1%/81.1%/> or =1. CONCLUSIONS: Contact lens wearers with dryness symptoms exhibit significantly more LWE and LIPCOF, but not increased corneal staining, bulbar hyperaemia or decreased PLBUT. LWE and LIPCOF are significantly correlated: this may reflect their common frictional origin. LIPCOF Sum severity scores appear to be most predictive for symptoms.

Mucins and ocular signs in symptomatic and asymptomatic contact lens wear.

PURPOSE: Lid wiper epitheliopathy (LWE) and lid parallel conjunctival folds (LIPCOF) are related to dry eye symptoms in contact lens wearers. Both clinical signs are assumed to be related to mechanical forces during blinking. As the mucus layer is a protector of the ocular surface tissue, this study investigates whether any alterations of mucins are detectable comparing symptomatic and asymptomatic soft contact lens wearers. METHODS: Comfort was evaluated using the Contact Lens Dry Eye Questionnaire. Corneal staining, LWE, and LIPCOF were assessed in the right eyes of 50 (19 men, 31 women; mean age, 32.1 +/- 11.4 years) experienced lens wearers. The tear film was sampled using Schirmer strips pressed onto the temporal conjunctiva and from harvested contact lenses. Mucins were assessed in dot-blots and Western blots after electrophoresis on 1% agarose or 4 to 12% NuPAGE Gels. Non-parametric analyses were used to study differences between groups and correlations between objective tests, mucins, and symptoms. RESULTS: Thirty-one subjects were classified asymptomatic and 19 symptomatic by the questionnaire. LWE and LIPCOF were significantly increased in the symptomatic group (p < 0.035). MUC5AC reactivity was significantly decreased in symptomatics (p = 0.050). MUC4 was correlated to temporal LIPCOF and LWE, (r = -0.47 and -0.46; p < 0.01). MUC16 and MUC5AC correlated with corneal staining (0.36 < r < 0.53; p < 0.04). CONCLUSIONS: Symptomatic contact lens wearers exhibit significantly more LWE and LIPCOF, and decreased MUC5AC reactivity. LWE and LIPCOF are significantly correlated; this may reflect their common frictional origin. Increased friction might follow from insufficient mucins, or an altered composition of the resident mucins at the ocular surface. In this study, we show that decreased mucin production is associated with the severity of LWE and LIPCOF.

Practical Three-Minute Synthesis of Acid-Coated Fluorescent Carbon Dots with Tuneable Core Structure.

We report a one-pot, three-minute synthesis of carboxylic acid-decorated fluorescent carbon dots (COOH-FCDs) with tuneable core morphology dependent on the surface passivating agent. Mechanism investigations highlighted the presence of key pyrazine and polyhydroxyl aromatic motifs, which are formed from the degradation of glucosamine in the presence of a bifunctional linker bearing acid and amine groups. The novel COOH-FCDs are selective Fe3+ and hemin sensors. Furthermore, the FCDs are shown to be non-toxic, fluorescent bioimaging agents for cancer cells.

Quantum Dot-Based Probes for Labeling and Imaging of Cells that Express Matrix Metalloproteinases.

The practical synthesis of novel multivalent fluorescent quantum-dot-based probes to target cellular matrix metalloproteinases (MMPs) (MT-MMPs) is reported. We show that these probes, which are decorated with a nanomolar water-soluble MMP inhibitor, can be used to label preferentially the surface of cancer cells that are known to express MMPs while no binding was observed on cells that do not.

Mucins and TFF peptides of the tear film and lacrimal apparatus.

The three-dimensional organization of the tear film, which is produced and drained by the different structures of the ocular adnexa, is essential for maintainance and protection of the ocular surface. This is facilitated by a class of large, highly glycosylated, hydrophilic glycoproteins, the mucins, which are usually expressed in association with a class of peptides having a well-defined, structurally conserved trefoil domain, the mammalian trefoil factor family (TFF) peptides. In this review, the latest information regarding mucin and TFF peptide function and regulation in the human lacrimal system, the tear film and the ocular surface is summarized with regard to mucous epithelia integrity, rheological and antimicrobial properties of the tear film and tear outflow, age-related changes and certain disease states such as dry eye, dacryostenosis and dacryolith formation.

Three-minute synthesis of sp3 nanocrystalline carbon dots as non-toxic fluorescent platforms for intracellular delivery.

A one-pot, three-minute, gram-scale synthesis of novel sp3-nanocrystalline, water-soluble, and fluorescent carbon dots (FCDs), from simple and cheap sugar starting materials is described. Mechanism studies showed that NH2-FCD formation proceeds via a crucial imine intermediate derived from reaction between a sugar hemiacetal and an amine. Moreover, we successfully demonstrate the utility of lactose functionalized FCDots (Lac-FCDots) as non-toxic fluorescent intracellular delivery vehicles.

Imidazolium-tagged glycan probes for non-covalent labeling of live cells.

Selective, bioorthogonal and fast labeling of glycoconjugates in living cells is a major challenge for synthetic and cellular biology. Here we report the use imidazolium tagged-mannosamine derivative (ITag-Man) for the non-covalent, rapid and site-specific labeling of sialic acid containing glycoproteins using commercial N-nitrilotriacetate fluorescent reagents in a range of cell lines.

Patterns of mucin adherence to contact lenses.

PURPOSE: Contact lens wear alters the preocular fluid through factors that include tear deposits on the lens. In the current study, lens-adherent material was extracted to assess whether contact lenses sample mucins from the preocular fluid. METHODS: Discarded extended-wear contact lenses were collected from patients with no ocular surface disease. Mucins were extracted in guanidine hydrochloride (GuHCl) with protease inhibitors. After the supernatant was removed, the extraction was repeated twice with the addition of 10 mM dithiothreitol, making a total of three extractions. Mucins were isolated by cesium chloride (CsCl) gradient centrifugation and size fractionated on Sepharose CL2B. Charge distribution was analyzed on ion-exchange chromatography with a lithium perchlorate (LiClO(4)) gradient. RESULTS: Contact lens-adherent mucins comprised soluble mucins and mucins that required solubilization by (repeated) dithiothreitol treatment. MUC1, MUC4, MUC2, and MUC5AC mucins eluted mainly at low buoyant densities in extractions from lenses worn long term without disinfection and at successively higher buoyant densities from monthly disposable contact lenses. Mucins with little negative charge, which were observed in all extractions, and very highly negatively charged species, present in the second and third extractions from contact lenses, had no equivalents in tissue-extracted mucins. CONCLUSIONS: Mucins adhering to contact lenses are altered forms of intracellular mucins. Different degrees of adherence of mucins to contact lenses may occur, either because of mucin characteristics or after mucin complexation with adherent materials. In the context of good contact lens hygiene, their presence may offer some protection from toxicants in the tear film, because mucins could function as acceptors for charged moieties such as free radicals.

Characterization of mucins in human lacrimal sac and nasolacrimal duct.

PURPOSE: Mucins are polymers that may reduce drag and enhance tear outflow. Mucin expression and distribution in human efferent tear ducts were tested in the physiological state, and potential differences in the expression pattern were investigated in the presence of primary acquired dacryostenosis (PANDO). METHODS: Expression of mucins in human lacrimal sac and nasolacrimal ducts was monitored by reverse transcription-polymerase chain reaction analysis. The presence and distribution of MUC1, -2, -4, -5AC, -5B, -6, and -7 in epithelia of the efferent tear duct passage are assessed with antisera to mucin peptide cores. Twenty normal tissues from cadavers and surgical specimens from 20 patients with PANDO were tested. RESULTS: mRNAs for all mucins investigated were detected in healthy human lacrimal sacs and nasolacrimal ducts. MUC6 mRNA was detected in only about half of the investigated samples. A reduced level of MUC2, -5AC, and -5B mRNAs was observed in PANDO. Immunohistochemistry revealed MUC2 in goblet cells and single epithelial cells. Both MUC5AC and -5B were detected in goblet cells forming intraepithelial mucous glands. MUC7 was present only in columnar epithelial cells of the efferent tear duct system. No immunoreactivity was observed with antibodies against MUC1, -4, and -6 peptide cores. CONCLUSIONS: Human efferent tear ducts express and produce a broad spectrum of mucins that is partly comparable with that in the conjunctiva and the salivary glands. The mucin diversity of the efferent tear ducts could enhance tear transport and antimicrobial defense. Reduced levels of mucin mRNA in a nonfunctioning though patent segment of the lacrimal passage, which is associated with epiphora, suggests that mucins ease tear flow through the efferent tear ducts.

Molecular adsorption: early stage surface exploration.

In this study, the atomic force microscope has been employed in force spectroscopy mode to gain information on the interaction between long mucin molecules and a positively charged surface during the first few seconds of interaction. Recent studies have revealed that negatively charged mucin molecules introduced to a positively charged surface are kinetically trapped and bind very rapidly, assuming non-equilibrium conformations. This systematic study of surface dwell times has revealed that significant differences exist in mucin adsorption during the first three seconds of introduction to the surface and provides direct evidence of molecular rearrangement for several seconds before trapping occurs. Limited interactions were recorded at dwell times of less than one second, with increased molecular rearrangement observed between 1.5 and 2.25 s. Increasing the surface dwell time beyond this critical limit caused rupture of the tip-tethered mucin molecules during the retract cycle of the cantilever. All subsequent recorded events, at increased dwell times up to 3s, revealed events at much reduced distances from the point of contact between the mucin functionalised-cantilever and the positively charged surface.

Optimisation of sample preparation methods for air imaging of ocular mucins by AFM.

The addition of cations to the imaging buffer for AFM has been previously shown to improve the binding of biological molecules to mica. Investigations were carried out to find the concentration of NiCl(2) required to immobilize mucin molecules on a freshly cleaved mica surface, for imaging using intermittent contact in air. Drop-deposition of samples prepared in HEPES buffer with 1, 2 and 5mM NiCl(2) revealed the sensitivity of the mucin molecules to salt. Dialysis of the mucin solutions dramatically reduced the amount of salt present and allowed single molecules to be imaged, revealing a variation in thickness along their length. Spray deposition of the same mucin solutions produced single molecules that, although less affected by co-adsorbed salt, showed a degree of self-folding. This shows the sensitive balance between HEPES and NiCl(2) required for successful imaging of the sub-molecular features of individual mucin molecules.

Still confused about rose bengal?

We have surveyed the ophthalmic literature of the last five years in an attempt to evaluate the use and usefulness of Rose Bengal staining as an aid to differential diagnosis in dry eyes. Included both as a criterion and as an adjunct measure of disease progression, Rose Bengal scores of patients with different dry eye conditions overlap, sometimes to a considerable extent. A mechanistic link between staining with this dye and disease etiology is unlikely; however, Rose Bengal could be a surrogate marker of changes in ocular surface physiology. The question whether the extent and pattern of staining with Rose Bengal provide the clinician with information not available from other tests, and in particular from fluorescein staining of the ocular surface, has to be answered positively, though the nature of this information is not clearly understood. A more widespread recognition that Rose Bengal is not a vital dye is necessary in order not to bias the interpretation of experimental results.

Bacterial invasion of HT29-MTX-E12 monolayers: effects of human breast milk.

AIM: The supramucosal gel, crucial for gut barrier function, might be compromised in necrotizing enterocolitis (NEC). Breast milk is associated with a reduced incidence of NEC. We compared the effects of human breast milk (BM) versus a neonatal formula, Nutriprem 1 (FF), on adherence, internalisation, and penetration of NEC-associated Escherichia coli through monolayers of mucus producing intestinal cells, HT29-MTX-E12 (E12). METHODS: E12 cells were grown to confluence on membranes permeable to bacteria. E. coli, reference strain and isolated from a NEC-affected intestine, were cultured in LB broth, labelled with fluorescein and biotinylated. Bacteria were suspended in tissue culture medium (TC) or mixtures of TC with BM or FF and applied to the E12 cultures. Bacterial numbers were assessed by fluorescence. DyLight 650-labelled neutravidin, which cannot cross cell membrane, evaluated extracellular bacteria. Fluorescence of basolateral medium was measured to quantify translocation. Bacterial concentrations were compared using the Mann Whitney U test. RESULTS: After 1h exposure, E12 cultures adhered or internalised more NEC-derived bacteria than standard strain E. coli and more suspended in FF than BM (P<0.001). A greater proportion of NEC-derived bacteria internalised when suspended in TC or BM. In FF, the NEC-derived strain internalised least. More translocation occurred in BM incubations compared to FF in the first 1-4h: NEC-E. coli less than the reference strain. After 24h translocated bacterial populations were equal. CONCLUSION: In this pilot study, breast milk was associated with relatively less adhesion and internalisation of NEC-associated E. coli to mucus covered E12s compared to formula milk.

Molecular weight distribution analysis by ultracentrifugation: adaptation of a new approach for mucins.

Mucins are the key macromolecular component of mucus, nature's natural lubricant, and one of the most important physical properties is their molecular weight distribution. A new approach for polydisperse polymers was recently published based on sedimentation velocity in the analytical ultracentrifuge and converts a distribution of sedimentation coefficient g(s) vs. s plot into a distribution of molecular weight utilising the power-law or scaling relationship between the sedimentation coefficient and molecular weight, s=κsMw(b) where s is the sedimentation coefficient, Mw is the weight average molecular weight and κs and b are characteristic coefficients related to conformation. We investigate the possibility of using a large database of previously published values of s an M to define κs and b for both aqueous solution and aqueous solution supplemented by 6M guanidine hydrochloride (a solvent which helps to minimise sample degradation). These values are then applied to a study of the molecular weight distributions of preparations of human gastric mucin in the different solvents and at different stages of purification.