High resolution imaging of soft alginate hydrogels by atomic force microscopy.

This work explores the largely unknown surface microstructure and elastic modulus of soft calcium-alginate hydrogels (E = 100-4500 Pa) in their hydrated state by atomic force microscopy (AFM) in quantitative imaging mode. Alginate concentration influenced the surface topography with surface roughness measured to be 101 ± 6 nm and 57 ± 1 nm for 0.5 and 2.0% (w/v) alginate, respectively. The calculated range of pore sizes increased with decreasing alginate concentration, with radii smaller than 360 nm, 570 nm and 1230 nm for 2.0%, 1.0% and 0.5% alginate, respectively. Small changes in calcium concentration (from 20 to 25 mM, 1.5% alginate) did not induce changes in surface microstructure, although it increased the elastic modulus mean values and distribution. Introducing oxidized or peptide-grafted alginate in the gels resulted in rougher surfaces, larger pore sizes and lower elasticity than the respective hydrogels with no modified alginate.

Elucidating bacterial adhesion to mucosal surface by an original AFM approach.

BACKGROUND: Fish skin represents an ancient vertebrate mucosal surface, sharing characteristics with other mucosal surfaces including those of the intestine. The skin mucosa is continuously exposed to microbes in the surrounding water and is therefore important in the first line defense against environmental pathogens by preventing bacteria from accessing the underlying surfaces. Understanding the microbe-host interactions at the fish skin mucosa is highly relevant in order to understand and control infection, commensalism, colonization, persistence, infection, and disease. Here we investigate the interactions between the pathogenic bacteria Aeromonas salmonicida (A. salmonicida) and Yersinia ruckeri (Y. ruckeri), respectively, and the skin mucosal surface of Atlantic salmon fry using AFM force spectroscopy. RESULTS: The results obtained revealed that when retracting probes functionalized with bacteria from surfaces coated with immobilized mucins, isolated from salmon mucosal surfaces, rupture events reflecting the disruption of adhesive interactions were observed, with rupture strengths centered around 200 pN. However, when retracting probes functionalized with bacteria from the intact mucosal surface of salmon fish fry no adhesive interactions could be detected. Furthermore, rheological measurements revealed a near fluid-like behavior for the fish fry skin mucus. Taken together, the experimental data indicate that the adhesion between the mucin molecules within the mucous layer may be significantly weaker than the interaction between the bacteria and the mucin molecules. The bacteria, immobilized on the AFM probe, do bind to individual mucins in the mucosal layer, but are released from the near fluid mucus with little resistance upon retraction of the AFM probe, to which they are immobilized. CONCLUSION: The data provided in the current paper reveal that A. salmonicida and Y. ruckeri do bind to the immobilized mucins. However, when retracting the bacteria from intact mucosal surfaces, no adhesive interactions are detected. These observations suggest a mechanism underlying the protective function of the mucosal surface based on the clearing of potential threats by adhering them to loosely attached mucus that is subsequently released from the fish skin.

A Titratable Cell Lysis-on-Demand System for Droplet-Compartmentalized Ultrahigh-Throughput Screening in Functional Metagenomics and Directed Evolution.

Water-in-oil emulsion droplets are an attractive format for ultrahigh-throughput screening in functional metagenomics and directed evolution applications that allow libraries with more than 107 members to be characterized in a day. Single library members are compartmentalized in droplets that are generated in microfluidic devices and tested for the presence of target biocatalysts. The target proteins can be produced intracellularly, for example, in bacterial hosts in-droplet cell lysis is therefore necessary to allow the enzymes to encounter the substrate to initiate an activity assay. Here, we present a titratable lysis-on-demand (LoD) system enabling the control of the cell lysis rate in Escherichia coli. We demonstrate that the rate of cell lysis can be controlled by adjusting the externally added inducer concentration. This LoD system is evaluated both at the population level (by optical density measurements) and at the single-cell level (on single-cell arrays and in alginate microbeads). Additionally, we validate the LoD system by droplet screening of a phosphotriesterase expressed from E. coli, with cell lysis triggered by inducer concentrations in the µM range. The LoD system yields sufficient release of the intracellularly produced enzymes to bring about a detectable quantity of product (measured by fluorescence in flow cytometry of double emulsions), while leaving viable cells for the downstream recovery of the genetic material.

Insights into the roles of charged residues in substrate binding and mode of action of mannuronan C-5 epimerase AlgE4.

Mannuronan C-5 epimerases catalyze the epimerization of monomer residues in the polysaccharide alginate, changing the physical properties of the biopolymer. The enzymes are utilized to tailor alginate to numerous biological functions by alginate-producing organisms. The underlying molecular mechanism that control the processive movement of the epimerase along the substrate chain is still elusive. To study this, we have used an interdisciplinary approach combining molecular dynamics simulations with experimental methods from mutant studies of AlgE4, where initial epimerase activity and product formation were addressed with nuclear magnetic resonance spectroscopy, and characteristics of enzyme-substrate interactions were obtained with isothermal titration calorimetry and optical tweezers. Positive charges lining the substrate-binding groove of AlgE4 appear to control the initial binding of poly-mannuronate, and binding also seems to be mediated by both electrostatic and hydrophobic interactions. After the catalytic reaction, negatively charged enzyme residues might facilitate dissociation of alginate from the positive residues, working like electrostatic switches, allowing the substrate to translocate in the binding groove. Molecular simulations show translocation increments of two monosaccharide units before the next productive binding event resulting in mannuronate and guluronate (MG)-block formation, with the epimerase moving with its N-terminus towards the reducing end of the alginate chain. Our results indicate that the charge pair R343-D345 might be directly involved in conformational changes of a loop that can be important for binding and dissociation. The computational and experimental approaches used in this study complement each other, allowing for a better understanding of individual residues' roles in binding and movement along the alginate chains.

Exploring the effects of approach velocity on depletion force and coalescence in oil-in-water emulsions.

An emulsion is a thermodynamically unstable system consisting of at least two immiscible liquid phases, one of which is dispersed in the other in the form of droplets of varying size. Most studies on emulsions have focused on the behaviour of emulsion droplets with diameter from ∼50 µm and upwards. However, the properties of smaller droplets may be highly relevant in order to understand the behaviour of emulsions, including their performance in numerous applications within the fields of food, industry, and medical science. The relatively long life-time and small size of these droplets compared to other emulsion droplets, make them suited for optical trapping and micromanipulation technologies. Optical tweezers have previously shown potential in the study of stabilized emulsions. Here we employ optical tweezers to examine unstable oil-in-water emulsions to determine the effects of system parameters on depletion force and coalescence times.

Effect of design geometry, exposure energy, cytophilic molecules, cell type and load in fabrication of single-cell arrays using micro-contact printing.

In this study a range of factors influencing the fabrication of single-cell arrays (SCAs) are identified and investigated. Micro-contact printing was used to introduce spots coated with polyethyleneimine or Matrigel on glass surfaces pre-coated with polyethylene glycol. Unmodified E. coli, Synechococcus sp., Chlamydomonas reinhardtii as well as diverse mammalian cells including HUVEC, AAV293, U87, OHS, PC3, SW480, HepG2 and AY-27 were successfully immobilised onto the chemically coated spots. The developed SCAs show high cell viability and probability for capturing single-cells. A discrepancy between the size and shape of the squares described in the design file and the actual structures obtained in the final PDMS structure is characterised and quantified. The discrepancy is found to be depending on the exposure energy used in the photolithography process as well as the size of the squares and their separation distance as detailed in the design file. In addition to these factors, the effect of the cell density loaded onto the patterned surfaces is also characterised. The systematic characterisation of key parameters that need to be optimised prior to the fabrication of SCAs is essential in order to increase the efficiency and reproducibility of future fabrication of SCAs for single-cell studies.

The Role of Glycans in Bacterial Adhesion to Mucosal Surfaces: How Can Single-Molecule Techniques Advance Our Understanding?

Bacterial adhesion is currently the subject of increased interest from the research community, leading to fast progress in our understanding of this complex phenomenon. Resent research within this field has documented the important roles played by glycans for bacterial surface adhesion, either through interaction with lectins or with other glycans. In parallel with this increased interest for and understanding of bacterial adhesion, there has been a growth in the sophistication and use of sensitive force probes for single-molecule and single cell studies. In this review, we highlight how the sensitive force probes atomic force microscopy (AFM) and optical tweezers (OT) have contributed to clarifying the mechanisms underlying bacterial adhesion to glycosylated surfaces in general and mucosal surfaces in particular. We also describe research areas where these techniques have not yet been applied, but where their capabilities appear appropriate to advance our understanding.

Tn and STn are members of a family of carbohydrate tumor antigens that possess carbohydrate-carbohydrate interactions.

The mucin-type O-glycome in cancer aberrantly expresses the truncated glycans Tn (GalNAcα1-Ser/Thr) and STn (Neu5Acα2,6GalNAcα1-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, which may participate in oncogenesis.

Correction: Interactions between the breast cancer-associated MUC1 mucins and C-type lectin characterized by optical tweezers.

[This corrects the article DOI: 10.1371/journal.pone.0175323.].

Interactions between the breast cancer-associated MUC1 mucins and C-type lectin characterized by optical tweezers.

Carbohydrate-protein interactions govern many crucial processes in biological systems including cell recognition events. We have used the sensitive force probe optical tweezers to quantify the interactions occurring between MGL lectins and MUC1 carrying the cancer-associated glycan antigens mucins Tn and STn. Unbinding forces of 7.6 pN and 7.1 pN were determined for the MUC1(Tn)-MGL and MUC1(STn)-MGL interactions, at a force loading rate of ~40 pN/s. The interaction strength increased with increasing force loading rate, to 27 and 37 pN at a force loading rate of ~ 310 pN/s. No interactions were detected between MGL and MUC1(ST), a glycoform of MUC1 also expressed by breast carcinoma cells. Interestingly, this glycan (ST) can be found on proteins expressed by normal cells, although in this case not on MUC1. Additionally, GalNAc decorated polyethylene glycol displayed similar rupture forces as observed for MUC1(Tn) and MUC1(STn) when forced to unbind from MGL, indicating that GalNAc is an essential group in these interactions. Since the STn glycan decoration is more frequently found on the surface of carcinomas than the Tn glycan, the binding of MUC1 carrying STn to MGL may be more physiologically relevant and may be in part responsible for some of the characteristics of STn expressing tumours.

Interactions of mucins with the Tn or Sialyl Tn cancer antigens including MUC1 are due to GalNAc-GalNAc interactions.

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 (Galß1-3GalNAcα1-Ser/Thr) or ST antigen (NeuNAcα2-3Galß1-3GalNAcα1-Ser/Thr) failed to show self-interactions. However, in the case of ST-MUC1, self-interactions were observed after subsequent treatment with neuraminidase and ß-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.

Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy.

Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase-polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (xß), lifetimes in the absence of external perturbation (τ0) and free energies (ΔG#) were determined for the different epimerase-alginate complexes. This is the first determination of ΔG# for these complexes. The values determined were up to 8 kBT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the observed different affinities determined for AlgE4-polymannuronic acid (poly-M) and AlgE4-polyalternating alginate (poly-MG) macromolecular pairs these data give important contribution to the growing understanding of the mechanisms underlying the processive mode of these enzymes.

The Design of Simple Bacterial Microarrays: Development towards Immobilizing Single Living Bacteria on Predefined Micro-Sized Spots on Patterned Surfaces.

In this paper we demonstrate a procedure for preparing bacterial arrays that is fast, easy, and applicable in a standard molecular biology laboratory. Microcontact printing is used to deposit chemicals promoting bacterial adherence in predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion. Highly ordered arrays of immobilized bacteria were obtained using microcontact printed islands of polydopamine (PD) on glass surfaces coated with the antiadhesive polymer polyethylene glycol (PEG). On such PEG-coated glass surfaces, bacteria were attached to 97 to 100% of the PD islands, 21 to 62% of which were occupied by a single bacterium. A viability test revealed that 99% of the bacteria were alive following immobilization onto patterned surfaces. Time series imaging of bacteria on such arrays revealed that the attached bacteria both divided and expressed green fluorescent protein, both of which indicates that this method of patterning of bacteria is a suitable method for single-cell analysis.

The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL.

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galß1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar dead adhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.

Single molecule study of heterotypic interactions between mucins possessing the Tn cancer antigen.

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 (Galß1-3GalNAcαThr/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] Galß1-3GalNAcαSer/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 (xß) 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 that may be important to the role of the Tn antigen in cancer cells.

Structure-function relationships in glycopolymers: effects of residue sequences, duplex, and triplex organization.

The importance of residue sequence and duplex and triplex structures as basis for establishing molecular understanding of the structure-function relationships within glycopolymers is highlighted. The copolysaccharide alginate is the selected example for elucidating effects of residue sequence on functional properties like ionotropic gelation. Xanthan and comblike branched ß-D-glucans are used as examples of impact of duplex and triplex organization on global conformation and functional properties. Combined with further examples within self-interactions of mucins possessing different saccharide decorations, polyelectrolyte complexation and multilayer formation, the examples indicate that a molecular understanding of various properties related to impact of residue sequences, duplex, and triplex organization can be established. Strategies similar to those included in the highlighted examples, also combined with novel tools, for example single-molecule approaches, interrogated by combination of experimental and theoretical/numerical approaches, and investigated closer to the native biological state, are expected to further advance the field.

Higher order structures of a bioactive, water-soluble (1→3)-ß-D-glucan derived from Saccharomyces cerevisiae.

Water-soluble (1→3)-ß-D-glucans with 1,6-linked branches (SBG), originally isolated from the cell walls of Saccharomyces cerevisiae and partially depolymerised to a weight average degree of polymerisation (DP(w)) in the range 120-160 for optimal performance in wound healing applications, were studied by dynamic light scattering (DLS), SEC MALLS and AFM. Results indicate that dilute aqueous SBG solutions (1 µg/ml to 3 mg/ml) contain higher order structures with a very wide size distribution in water (10-500 nm), corresponding to a mixture of single chains, multi-chain aggregates including triple-stranded motifs, and particulate materials. The latter were enriched in longer chains compared to non-particulate fractions. The size distribution of SBG aggregates shifted to slightly lower values upon heating, but showed hysteresis upon cooling. AFM images prepared from very dilute aqueous solution (1-5 µg/ml) analysis showed by comparison to other (1→3)-ß-D-glucans that some of the structures were the triple helical species coexisting with larger aggregates and single chains, in contrast to carboxymethylated SBG, which contained predominantly single chains. The ability to control the aggregation behaviour of SBG enables tailoring of the physical, and possibly bioactive, properties of SBG preparations.

Enhanced self-association of mucins possessing the T and Tn carbohydrate cancer antigens at the single-molecule level.

Mucins are linear O-glycosylated glycoproteins involved in inflammation, cell adhesion, and tumorigenesis. Cancer-associated mucins often possess increased expression of the T (Galß1,3GalNAcαThr/Ser) and Tn (GalNAcαThr/Ser) cancer antigens, which are diagnostic markers for several cancers, including colon cancer. We have used AFM based single-molecule forced unbinding under near physiological conditions to investigate the self-interactions between porcine submaxillary mucin (PSM) as well as between PSM analogs possessing various carbohydrates including the T- and Tn-antigen. Distributions of unbinding forces and corresponding force loading rates were determined for force loading rates from 0.18 nN/s to 39 nN/s, and processed to yield most probable unbinding forces f* and lifetimes of the interactions. Parameter f* varied in the range 27 to 50 pN at force loading rates of about 2 nN/s among the various mucins. All mucin samples investigated showed self-interaction, but the tendency was greatest for PSM displaying only the Tn-antigen (Tn-PSM) or a mixture of Tn-, T-antigen, and the trisaccharide Fucα1,2Galß1,3GalNAc (Tri-PSM). Weaker self-interactions were observed for native PSM (Fd-PSM), which consists of a nearly equal mixture of the longer core 1 blood group A tetrasaccharide (GalNAcα1,3(Fucα1,2)Galß1,3GalNAcαSer/Thr) and Tn-antigen. The data are consistent with the truncated Tn and T glycans enhancing self-interaction of the mucins. These carbohydrate cancer antigens may, thus, play an active role in the disease by constitutively activating mucin and mucin-type receptors by self-association on cells.

Responsive hydrogels for label-free signal transduction within biosensors.

Hydrogels have found wide application in biosensors due to their versatile nature. This family of materials is applied in biosensing either to increase the loading capacity compared to two-dimensional surfaces, or to support biospecific hydrogel swelling occurring subsequent to specific recognition of an analyte. This review focuses on various principles underpinning the design of biospecific hydrogels acting through various molecular mechanisms in transducing the recognition event of label-free analytes. Towards this end, we describe several promising hydrogel systems that when combined with the appropriate readout platform and quantitative approach could lead to future real-life applications.

Single molecular pair interactions between hydrophobically modified hydroxyethyl cellulose and amylose determined by dynamic force spectroscopy.

Interactions among HMHEC (hydrophobically modified hydroxyethyl cellulose) and between HMHEC and amylose were investigated by means of dynamic force spectroscopy of single molecular pairs. The technique was realized using a scanning probe based platform, and the molecular pair interactions were investigated in aqueous solutions over a range of force loading rates. Both hydrophobic interactions among hydrophobe C(16) alkyl side chains in HMHEC and association between these hydrophobes in HMHEC and amylose showed a stretching type peak. The distribution analysis of rupture force based on Bell-Evans's model revealed that the peaks had a most probable rupture force ranging from 27 pN at a force loading rate r(f) = 0.43 nN/s to 125 pN at r(f) = 170 nN/s for HMHEC-HMHEC, and from 13 pN at r(f) = 0.20 nN/s to 34 pN at r(f) = 33.7 nN/s for HMHEC-amylose interactions. The distance of the energy barrier relative to the minimum, x(beta), and the apparent lifetime in the absence of external force, tau, were found to depend on the force loading rate, and the average values are estimated to be 0.99 nm and 0.89s for HMHEC-HMHEC and 0.31 nm and 0.075s for HMHEC-amylose interactions. The obtained data for these pairwise molecular interactions are underpinning the associative behavior of the macroscopic properties of aqueous solutions of these polysaccharides.