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Aberrant expression of glycans, i.e., oligosaccharide moiety covalently attached to proteins or lipids, is characteristic of various cancers, including urothelial ones. The binding of lectins to glycans is classified as molecular recognition, which makes lectins a strong tool for understanding their role in developing diseases. Here, we present a quantitative approach to tracing glycan–lectin interactions in cells, from the initial to the steady phase of adhesion. The cell adhesion was measured between urothelial cell lines (non-malignant HCV29 and carcinoma HT1376 and T24 cells) and lectin-coated surfaces. Depending on the timescale, single-cell force spectroscopy, and adhesion assays conducted in static and flow conditions were applied. The obtained results reveal that the adhesion of urothelial cells to two specific lectins, i.e., phytohemagglutinin-L and wheat germ agglutinin, was specific and selective. Thus, these lectins can be applied to selectively capture, identify, and differentiate between cancer types in a label-free manner. These results open up the possibility of designing lectin-based biosensors for diagnostic or prognostic purposes and developing strategies for drug delivery that could target cancer-associated glycans. [ABSTRACT FROM AUTHOR]

Scleroglucan is a (1,3)-β-D-glucan polysaccharide produced by the fungus Sclerotium. Dissolved in water at room temperature it adopts a linear, rigid, triple helical structure. Gelation of scleroglucan can be obtained by Schiff-base formation between partly periodate oxidized scleroglucan and the primary amine groups of chitosan. The scleraldehyde-chitosan gels exhibit a collapse transition when exposed to volume fractions of isopropanol, Wp, larger than 65%. The aim of the present study is to provide structural information concerning the local polymer distribution and the collapse transition in (1,3)-β-D-glucan-chitosan gels. Small angle x-ray scattering was used to investigate solutions and gels of scleroglucan in water, as well as in an aqueous mixture containing 65% isopropanol. The results reveal that in aqueous solution, the polysaccharide scleroglucan chains have an approximately cylindrical cross section of external diameter close to 17 Å. The gels display the same local structure, but form clusters on a longer distance scale. For the collapsed gels in the water-isopropanol mixture, partial phase separation occurs in which ordered domains of approximate size of 110 Å develop. This study indicates that local ordering in liquid-crystalline-type domains is a possible molecular mechanism contributing to the collapse of gels composed of semiflexible polymers. The triple helical structure of the molecule appears not to be conserved in the majority phase in this solvent, but it is conserved in the liquid crystalline domains. [ABSTRACT FROM AUTHOR]

The mucin-type O-glycome in cancer aberrantly expresses the truncated glycans Tn (GalNAca1-Ser/Thr) and STn (Neu5Aca2,6GalNAca1-Ser/Thr). However, the role of Tn and STn in cancer and other diseases is not well understood. Our recent discovery of the self-binding properties (carbohydrate-carbohydrate interactions, CCIs) of Tn (Tn-Tn) and STn (STn-STn) provides a model for their possible roles in cellular transformation. We also review evidence that Tn and STn are members of a larger family of glycan tumor antigens that possess CCIs, whichmay participate in oncogenesis. [ABSTRACT FROM AUTHOR]

The molecular mechanism(s) underlying the enhanced self-interactions of mucins possessing the Tn (GalNAcα1-Ser/Thr) or STn (NeuNAcα2-6GalNAcα1-Ser/Thr) cancer markers were investigated using optical tweezers (OT). The mucins examined included modified porcine submaxillary mucin containing the Tn epitope (Tn-PSM), ovine submaxillary mucin with the STn epitope (STn-OSM), and recombinant MUC1 analogs with either the Tn and STn epitope. OT experiments in which the mucins were immobilized onto polystyrene beads revealed identical self-interaction characteristics for all mucins. Identical binding strength and energy landscape characteristics were also observed for synthetic polymers displaying multiple GalNAc decorations. Polystyrene beads without immobilized mucins showed no self-interactions and also no interactions with mucin-decorated polystyrene beads. Taken together, the experimental data suggest that in these molecules, the GalNAc residue mediates interactions independent of the anchoring polymer backbone. Furthermore, GalNAc–GalNAc interactions appear to be responsible for self-interactions of mucins decorated with the STn epitope. Hence, Tn-MUC1 and STn-MUC1 undergo self-interactions mediated by the GalNAc residue in both epitopes, suggesting a possible molecular role in cancer. MUC1 possessing the T (Galβ1-3GalNAcα1-Ser/Thr) or ST antigen (NeuNAcα2-3Galβ1-3GalNAcα1-Ser/Thr) failed to show self-interactions. However, in the case of ST-MUC1, self-interactions were observed after subsequent treatment with neuraminidase and β-galactosidase. This enzymatic treatment is expected to introduce Tn-epitopes and these observations thus further strengthen the conclusion that the observed interactions are mediated by the GalNAc groups. [ABSTRACT FROM AUTHOR]

Currently there are limitations to gelation strategies to form ionically crosslinked hydrogels, derived in particular from a lack of control over the release kinetics of crosslinking ions, which severely restrict applications. To address this challenge, we describe a new approach to form hydrogels of ionotropic polymers using competitive displacement of chelated ions, thus making specific ions available to induce interactions between polymer chains and form a hydrogel. This strategy enables control of ion release kinetics within an aqueous polymer solution and thus control over gelation kinetics across a wide range of pH. The described technique simplifies or facilitates the use of ionotropic hydrogels in a range of applications, such as 3D printing, microfluidic-based cell encapsulation, injectable preparations and large scale bubble and solid free mouldable gels. We investigate a range of chelator-ion combinations and demonstrate this powerful method to form hydrogels across a wide range of pH and µm–cm length scales. We highlight our findings by applying this gelation strategy to some of the more challenging hydrogel application areas using alginate and polygalacturonate as model polymer systems. [ABSTRACT FROM AUTHOR]

Alginate is used extensively in microfluidic devices to produce discrete beads or fibres at the microscale. Such structures may be used to encapsulate sensitive cargoes such as cells and biomolecules. On chip gelation of alginate represents a significant challenge since gelling kinetics or physicochemical conditions are not biocompatible. Here we present a new method that offers a hitherto unprecedented level of control over the gelling kinetics and pH applied to the encapsulation of a variety of cells in both bead and fibre geometries. This versatile approach proved straightforward to adjust to achieve appropriate solution conditions required for implementation in microfluidic devices and resulted in highly reliable device operation and very high viability of several different encapsulated cell types for prolonged periods. We believe this method offers a paradigm shift in alginate gelling technology for application in microfluidics. [ABSTRACT FROM AUTHOR]

Toll-like receptor 4 ( TLR4) is responsible for the immediate response to Gram-negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal- MyD88 [Mal (MyD88-adaptor-like) - MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro-inflammatory cytokines while TRAM-TRIF [TRAM (TRIF-related adaptor molecule)-TRIF (TIR-domain-containing adapter-inducing interferon-β)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide ( LPS) stimulation. We found that LPS induced a CD14-dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy ( TIRF) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14/ LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373-CD14. These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment ( ERC) and to early sorting endosomes. Moreover, CD14/ LPS but not TRAM was immobilized on RAB11A-positive endosomes, which indicates that TRAM and CD14/ LPS can independently be recruited to endosomes. [ABSTRACT FROM AUTHOR]

Mucins are linear, heavily O-glycosylated proteins with physiological roles that include cell signaling, cell adhesion, inflammation, immune response and tumorgenesis. Cancer-associated mucins often differ from normal mucins by presenting truncated carbohydrate chains. Characterization of the binding properties of mucins with truncated carbohydrate side chains could thus prove relevant for understanding their role in cancer mechanisms such as metastasis and recognition by the immune system. In this work, heterotypic interactions of model mucins that possess the Tn (GalNAcαThr/ Ser) and T (Galβ1-3GalNAcαThr/Ser) cancer antigens derived from porcine submaxillary mucin (PSM) were studied using atomic force microscopy. PSM possessing only the Tn antigen (Tn-PSM) was found to bind to PSM analogs possessing a combination of T, Tn and STn antigens as well as biosynthetic analogs of the core 1 blood group A tetrasaccharide (GalNAcα1-3[Fucα1-2] Galβ1-3GalNAcαSer/Thr). The rupture forces for the heterotypic interactions ranged from 18- to 31 pN at a force-loading rate of -0.5 nN/s. The thermally averaged distance from the bound complex to the transition state (xβ) was estimated to be in the range 0.37-0.87 nm for the first barrier of the Bell Evans analysis and within 0.34-0.64 nm based on a lifetime analysis. These findings reveal that the binding strength and energy landscape for heterotypic interactions of Tn-PSM with the above mucins, resemble homotypic interactions of Tn-PSM. This suggests common carbohydrate epitope interactions for the Tn cancer antigen with the above mucin analogs, a finding thatmay be important to the role of the Tn antigen in cancer cells. [ABSTRACT FROM AUTHOR]

Aqueous chitosan possesses attractive interaction capacities with various molecular groups that can be involved in hydrogen bonds and electrostatic and hydrophobic interactions. In the present paper, we report on the direct determination of chitosan-mucin molecular pair interactions at various solvent conditions as compared to alginate-mucin interactions. Two chitosans of high molecular weight with different degrees of acetylation--thus possessing different solubility profiles in aqueous solution as a function of pH and two alginates with different fractions of a-guluronic acid were employed. The interaction properties were determined through a direct unbinding assay at the single-molecular pair level using an atomic force microscope. When probed against immobilized mucin, both chitosans and alginates revealed unbinding profiles characteristic of localized interactions along the polymers. The interaction capacities and estimated parameters of the energy landscapes of the pairwise chitosan-mucin and alginate-mucin interactions are discussed in view of possible contributions from various fundamental forces. Signatures arising both from an electrostatic mechanism and hydrophobic interaction are identified in the chitosan-mucin interaction properties. The molecular nature of the observed chitosan-mucin and alginate-mucin interactions indicates that force spectroscopy provides fundamental insights that can be useful in understanding the surface binding properties of other potentially mucoadhesive polymers. [ABSTRACT FROM AUTHOR]

The specific interaction between human Toll-like receptor 9 (TLR9)-ectodomain (ECD)-fusion protein and immunostimulatory CpG-DNA was measured using force spectroscopy. Flexible tethers were used between receptors and surface as well as between DNA and atomic force microscope tip to make efficient recognition studies possible. The molecular recognition forces detected are in the range of 50 to 150 ± 20 pN at the used force-loading rates, and the molecular interaction probability was much reduced when the receptors were blocked with free CpG-DNA. A linear increase of the unbinding force with the logarithm of the loading rate was found over the range 0.1 to 30 nN/s. This indicates a single potential barrier characterizing the energy landscape and no intermediate state for the unbinding pathway of CpG-DNA from the TLR9-ECD. Two important kinetic parameters for CpG-DNA interaction with TLR9-ECD were determined from the force-loading rate dependency: an off-rate of koff = 0.14 ± 0.10 s-1 and a binding interaction length of xβ = 0.30 ± 0.03 nm, which are consistent with literature values. Various models for the molecular interaction of this innate immune receptor binding to CpG-DNA are discussed. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Mixing oppositely charged polyelectrolytes in aqueous solutions leads to the spontaneous formation of polyelectrolyte complexes. Here, we characterize the interaction between xanthan of two different chain lengths, a tri-glucosamine and a chitosan polymer by isothermal titration calorimetry (ITC). Analysis of the experimental thermodynamic data assuming a single set of identical sites indicated both enthalpic and entropic contributions to the overall interaction in the interaction between xanthan and tri-glucosamine. The relative contribution of entropy compared to enthalpy was found to be largest for the shortest chain length of xanthan. Using a chitosan polymer instead of tri-glucosamine gave rise to two different stages in the interaction process. A model where the first stage of the ITC curve represent an initial polyelectrolyte complexation stage followed by aggregation on further titration of chitosan to the xanthan is suggested. Ultrastructure images by applying atomic force microscopy at some selected extents of titration are consistent with the two-stage interpretation of the thermodynamic data. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 1-10, 2012. [ABSTRACT FROM AUTHOR]

Functionalized sensor surfaces combined with microfluidic channels are becoming increasingly important in realizing efficient biosensing devices applicable to small sample volumes. Relaxing the limitations imposed by laminar flow of the microfluidic channels by passive mixing structures to enhance analyte mass transfer to the sensing area will further improve the performance of these devices. In this paper, we characterize the flow performance in a group of microfluidic flow channels with novel double curved passive mixing structures (DCMS) fabricated in the ceiling. The experimental strategy includes confocal imaging to monitor the stationary flow patterns downstream from the inlet where a fluorophore is included in one of the inlets in a Y-channel microfluidic device. Analyses of the fluorescence pattern projected both along the channel and transverse to the flow direction monitored details in the developing homogenization. The mixing index (MI) as a function of the channel length was found to be well accounted for by a double-exponential equilibration process, where the different parameters of the DCMS were found to affect the extent and length of the initial mixing component. The range of MI for a 1 cm channel length for the DCMS was 0.75–0.98, which is a range of MI comparable to micromixers with herringbone structures. Overall, this indicates that the DCMS is a high performing passive micromixer, but the sensitivity to geometric parameter values calls for the selection of certain values for the most efficient mixing. [ABSTRACT FROM AUTHOR]