Bovine Submaxillary Gland Research Articles

A head-to-head comparison of polymer interaction with mucin from porcine stomach and bovine submaxillary glands

Native mucus is heterogeneous, displays high inter-individual variation and is prone to changes during harvesting and storage. To overcome the lack of reproducibility and availability of native mucus, commercially available purified mucins, porcine gastric mucin (PGM) and mucin from bovine submaxillary gland (BSM), have been widely used. However, the question is to which extent the choice of mucin matters in studies of their interaction with polymers as their composition, structure and hence physicochemical properties differ. Accordingly, the interactions between PGM or BSM with two widely used polymers in drug delivery, polyethylene oxide and chitosan, was studied with orthogonal methods: turbidity, dynamic light scattering, and quartz crystal microbalance with dissipation monitoring. Polymer binding and adsorption to the two commercially available and purified mucins, PGM and BSM, is different depending on the mucin type. PEO, known to interact weakly with mucin, only displayed limited interaction with both mucins as confirmed by all employed methods. In contrast, chitosan was able to bind to both PGM and BSM. Interestingly, the results suggest that chitosan interacts with BSM to a greater extent than with PGM indicating that the choice of mucin, PGM or BSM, can affect the outcome of studies of mucin interactions with polymers.

Developing Method for Minor Acidic O-Glycan Analysis in Mucin and Cancer Cell Samples.

Minor acidic glycans, such as sulfated and phosphorylated glycans, constitute only a small fraction of biological glycome, making their analysis a considerable challenge. In this study, we developed a technique to analyze minor acidic O-glycans in biological samples. First, efficient reaction conditions for the release of O-glycans from the proteins were determined. Next, a high-throughput method was established for the recovery of minor acidic glycans using NH2 spin columns. The performance of the established method was evaluated using mucin samples, and sulfated O-glycans were successfully detected in bovine submaxillary gland mucin and porcine stomach mucin. We also analyzed the minor acidic O-glycans in cultured cancer cells. In addition to trifucosylated sulfated O-glycans and disulfated O-glycans, sulfated O-glycans with KDN were detected in LS174T cells. The relative amount of sulfated glycans in LS174T cells was almost 10-fold higher than that in the other cells. Moreover, a large polylactosamine-type sulfated O-glycan with a molecular weight >3500 was detected in MKN45 cells. Interestingly, phosphorylated ribose, possibly bound to serine/threonine, was observed in all the cells used in this study. Thus, our established analytical method allows for the analysis of minor acidic O-glycans that cannot be detected using existing glycomics methods.

Probing Relative Humidity Impact on Biological Protein Bovine Serum Albumin and Bovine Submaxillary Gland Mucin by Using Contact Resonance Atomic Force Microscopy.

In biomaterials, a substantial amount of research has been placed on the mechanical properties of biomolecules and their interactions with body fluids. Bovine serum albumin (BSA) is a widely studied model protein, while bovine submaxillary gland mucin (BSM) is another cow-derived protein frequently employed in research. Films were examined with contact resonance atomic force microscopy (CR-AFM), and the results showed that the mechanical characteristics of the films were affected by the relative humidity. We quantitatively analyze the viscoelasticity of these proteins after they have been subjected to humidity by measuring the resonance frequency and quality factor. The findings indicate that prolonged humidity exposure has a different effect on the mechanical properties of BSA and BSM films. The results show that after exposure to humidity, the resonance peaks of BSA shift to the left, indicating stiffness, while those of BSM shift to the right, indicating hydration. Moreover, BSM's hydration is caused by relative humidity, leading to a constant increase in resonance frequency and material softness. Contrarily, BSA showed a decrease in contact resonance frequency due to ongoing strain-induced deformation, indicating increased material stiffness. The findings have significance for the design and development of biomaterials for a variety of applications, such as the delivery of drugs, the engineering of tissue, and the development of biosensors. Our research demonstrates that CR-AFM has the potential to become a non-invasive and sensitive method that can be used to characterize the mechanical characteristics of biomolecules and their interactions with bodily fluids.

Features of the Synthesis of Extracellular Cytotoxic Lectin Bacillus subtilis IMV B-7724, Depending on the Cultivation Conditions in the Laboratory Fermenter

The level of oxygen mass transfer (KV) is an important parameter influencing the growth rate of aerobic microorganisms and the synthesis of metabolites. It is mainly determined by the agitation and the aeration rates in the fermenter. Aim. To study changes in pH, optical density (OD), and hemagglutinating (lectin) activity (HAA) of culture fluid (CF) of Bacillus subtilis strain IMV B-7724, a producer of extracellular cytotoxic lectin (ECL), during its cultivation in a laboratory fermenter at different agitation and aeration rates as well as to determine and compare the HAA, carbohydrate specifi city, and cytotoxic properties of the corresponding samples of the preparation isolated from CF. Methods. Batch antifoam-free fermentations were performed by culturing the strain in the modified Gause medium with galactose in two identical lab-scale fermenters with a working volume of 2.5 L at 37ºC for 48—72 h according to three fermentation variants. Variant 1: n — 400 rpm for the whole cultivation, the air supply to the CF — through a sparger at 0.1 vvm until the 39th h with further gradual decrease, KV — 4.2±0.3 g O2·L−1·h−1. Variant 2: n — 400 rpm for the first 24 h, then a gradual decrease to 200 rpm, air supply — through a sparger at 0.1 rpm for the first 12 h, followed by its switching into the fermenter free space, corresponding KV — from 4.2±0.3 to 0.3±0.1 g O2·L−1·h−1. Variant 3: n — 400 rpm and air supply to the fermenter free space during the whole cultivation, KV — 4.0±0.3 g O2·L−1·h−1. A number of biological properties of strain CF and isolated lectin samples were evaluated by biochemical, spectrophotometric, immunological, and culture methods. Statistical analysis was performed using Student’s t-test. Results. The maximum increase in the OD of CF relative to the initial values (28 and 21-fold) at the end of the period of the rapid growth of the strain (at 9th h), the μmax values of 0.33 and 0.41 h−1, and pH not lower than 6.7 and 6.3 units were observed for fermentation variants 1 and 2, respectively. In the case of variant 2, the HAA of CF reached 32 hemagglutinating units (HAU), and the samples isolated from it had a lectin activity of 512±64 HAU, whereas for variant 1 such values were lower:16 and 32±8 HAA, respectively; carbohydrate specificity of preparations to bovine submandibular gland mucin was the same, i.e. 0.2±0.1 mg/mL. In contrast to the above, a slower increase in the OD of the CF, a decrease in μmax, and significant acid formation (15-fold at the 9th h, 0.25 h−1, and pH decrease to 5.8 units, respectively) were observed for variant 3; in this case, the level of HAA of CF was minimal (2—4 HAU) and was absent in the corresponding isolated samples. The probable reason for such differences was the limited mass transfer in the CF due to the isolating effect of the foam layer on its surface formed as a result of intensive agitation. Conclusions. The rapid growth of the strain and an increase in the HAA of CF were observed during cultivation in a lab-scale fermenter by maintaining the maximum level of oxygen mass transfer with air supply into the CF through a sparger until the maximum OD was reached and the subsequent gradual decrease in the specifi ed level during further cultivation started.

O-acetylesterase activity of Bifidobacterium bifidum sialidase facilities the liberation of sialic acid and encourages the proliferation of sialic acid scavenging Bifidobacterium breve.

Bifidobacterium bifidum possesses two extracellular sialidases (SiaBb1 and SiaBb2) that release free sialic acid from mucin sialoglycans, which can be utilized via cross-feeding by Bifidobacterium breve that, otherwise, is prevented from utilizing this nutrient source. Modification of sialic acids with O-acetyl esters is known to protect mucin glycans from degradation by bacterial sialidases. Compared to SiaBb2, SiaBb1 has an additional O-acetylesterase (Est) domain. We aimed to elucidate the role of the SiaBb1 Est domain from B. bifidum in sialic acid cross-feeding within Bifidobacterium. Pre-treatment of mucin secreted from bovine submaxillary glands (BSM) using His6 -tagged-Est and -SiaBb2 released a higher amount of sialic acid compared to the pre-treatment by His6 -SiaBb2. Growth of B. breve increased with an increase in nanE expression when supplemented with both His6 -Est- and His6 -SiaBb2-treated BSM. These results indicate that the esterase activity of the SiaBb1 Est domain enhances the efficiency of SiaBb2 to cleave sialic acid from mucin. This free sialic acid can be utilized by coexisting sialic acid scavenging B. breve via cross-feeding. Here, we provide the molecular mechanism underlying the unique sialoglycan degradation property of B. bifidum which is mediated by the complementary activities of SiaBb1 and SiaBb2 in the context of sialic acid cross-feeding.

Covalently Cross-Linked Pig Gastric Mucin Hydrogels Prepared by Radical-Based Chain-Growth and Thiol-ene Mechanisms.

Mucin, a high molecular mass hydrophilic glycoprotein, is the main component of mucus that coats every wet epithelium in animals. It is thus intrinsically biocompatible, and with its protein backbone and the o-glycosidic bound oligosaccharides, it contains a plethora of functional groups which can be used for further chemical modifications. Here, chain-growth and step-growth (thiol-ene) free-radical cross-linked hydrogels prepared from commercially available pig gastric mucin (PGM) are introduced and compared as cost-efficient and easily accessible alternative to the more broadly applied bovine submaxillary gland mucin. For this, PGM is functionalized with photoreactive acrylate groups or allyl ether moieties, respectively. Whereas homopolymerization of acrylate-functionalized polymers is performed, for thiol-ene cross-linking, the allyl-ether-functionalized PGM is cross-linked with thiol-functionalized hyaluronic acid. Morphology, mechanical properties, and cell compatibility of both kinds of PGM hydrogels are characterized and compared. Furthermore, the biocompatibility of these hydrogels can be evaluated in cell culture experiments.

Mucoadhesion: mucin-polymer molecular interactions

Mucoadhesion, adhesion of a material to a mucous membrane or a mucus-covered surface, has been employed in drug delivery to prolong contact with adsorption sites and consequently a likely improvement of drug absorption. Mucoadhesion in the oral cavity also provides additional effects on tactile mouthfeel and extended flavor delivery, which impact consumer perception. The mechanisms behind mucoadhesion have not been well understood and there are contradictory literature results on the ranking of mucoadhesive properties of different polymers based on what in-vitro methods that are used. We herein examine the molecular interactions of different polymers with mucin from bovine submaxillary glands at pH 6.6 by using 1H NMR (Nuclear Magnetic Resonance) that provides atomically resolved information on conformational mobility of the mucin. Studying different types of polymers with different chemical structures and degrees of polymerization (DP), we can via the NMR linewidths and the signal intensities distinguish if the polymers interact with specific segments of the mucin or if they have a universal effect on the mobility of all the molecular segments of the mucin. The specific interaction sites on the mucin for positively charged polymer poly(ethyleneimine) are shown to be different from those for negatively and neutrally charged polymers. In addition, the interactions are also driven by the DP, the concentration of the polymers, and the dehydration. Deepened understanding of molecular effects of the different polymers on the mucin can therefore have strong impact on the development of mucoadhesive products in pharmaceutical and food applications. Finally, we raise awareness of the interpretation of rheological data in terms of mucoadhesion.

A thermodynamic theory of sorption in glassy polymers

Sorption of vapors by glassy polymers is known to have properties that are different from those typical for sorption by rubbery polymers or liquids. Despite an importance for many practical applications, this difference is still not fully understood. We developed a thermodynamic theory for description of penetrant activity in the glassy state. The presented approach considers the glass transition in terms of a second order phase transition and utilizes heat capacity changes for calculation of thermodynamic activity. The formalism developed here explains a deviation of penetrant activity in the glassy state from equilibrium values. Moreover, the sorption-desorption hysteresis can be explained in thermodynamic terms as well. We demonstrate that not only the heat capacity change of the polymer, but also the heat capacity change of the penetrant should be considered to understand the difference between three cases: sorption by equilibrium liquid, absorption by glass and desorption from glass.The theory presented here is tested on experimental data on water sorption by maltodextrin and bovine submaxillary gland mucin. A very good fitting of the experimental data for both sorption and desorption cases is demonstrated. The values of heat capacity increments of the components obtained by fitting the penetrant activity data can be used to get new insights into the non-equilibrium properties of glassy materials.

Induction of ciliary orientation by matrix patterning and characterization of mucociliary transport

Impaired mucociliary clearance (MCC) is a key feature of many airway diseases, including asthma, bronchiectasis, chronic obstructive pulmonary disease, cystic fibrosis, and primary ciliary dyskinesia. To improve MCC and develop new treatments for these diseases requires a thorough understanding of how mucus concentration, mucus composition, and ciliary activity affect MCC, and how different therapeutics impact this process. Although differentiated cultures of human airway epithelial cells are useful for investigations of MCC, the extent of ciliary coordination in these cultures varies, and the mechanisms controlling ciliary orientation are not completely understood. By introducing a pattern of ridges and grooves into the underlying collagen substrate, we demonstrate for the first time, to our knowledge, that changes in the extracellular matrix can induce ciliary alignment. Remarkably, 90% of human airway epithelial cultures achieved continuous directional mucociliary transport (MCT) when grown on the patterned substrate. These cultures maintain transport for months, allowing carefully controlled investigations of MCC over a wide range of normal and pathological conditions. To characterize the system, we measured the transport of bovine submaxillary gland mucin (BSM) under several conditions. Transport of 5% BSM was significantly reduced compared with that of 2% BSM, and treatment of 5% BSM with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) reduced viscosity and increased the rate of MCT by approximately twofold. Addition of a small amount of high-molecular-weight DNA increased mucus viscosity and reduced MCT by ∼75%, demonstrating that the composition of mucus, as well as the concentration, can have significant effects on MCT. Our results demonstrate that a simple patterning of the collagen substrate results in highly coordinated ciliated cultures that develop directional MCT, and can be used to investigate the mechanisms controlling the regulation of ciliary orientation. Furthermore, the results demonstrate that this method provides an improved system for studying the effects of mucus composition and therapeutic agents on MCC.

N-Glycan Modifications with Negative Charge in a Natural Polymer Mucin from Bovine Submaxillary Glands, and Their Structural Role.

Bovine submaxillary mucin (BSM) is a natural polymer used in biomaterial applications for its viscoelasticity, lubricity, biocompatibility, and biodegradability. N-glycans are important for mucin stability and function, but their structures have not been fully characterized, unlike that of O-glycans. In this study, BSM N-glycans were investigated using liquid chromatography-tandem mass spectrometry. The microheterogeneous structures of 32 N-glycans were identified, and the quantities (%) of each N-glycan relative to total N-glycans (100%) were obtained. The terminal N-acetylgalactosamines in 12 N-glycans (sum of relative quantities; 27.9%) were modified with mono- (10 glycans) and disulfations (2 glycans). Total concentration of all sulfated N-glycans was 6.1 pmol in BSM (20 µg), corresponding to 25.3% of all negatively charged glycans (sum of present N-glycans and reported O-glycans). No N-glycans with sialylated or phosphorylated forms were identified, and sulfate modification ions were the only negative charges in BSM N-glycans. Mucin structures, including sulfated N-glycans located in the hydrophobic terminal regions, were indicated. This is the first study to identify the structures and quantities of 12 sulfated N-glycans in natural mucins. These sulfations play important structural roles in hydration, viscoelasticity control, protection from bacterial sialidases, and polymer stabilization to support the functionality of BSM via electrostatic interactions.

Swelling of mucoadhesive electrospun chitosan/polyethylene oxide nanofibers facilitates adhesion to the sublingual mucosa

Mucoadhesive chitosan-based electrospun nanofibers are promising candidates for overcoming challenges associated with sublingual drug delivery, yet studies focusing on evaluating the mucoadhesive properties of nanofibers for sublingual administration are limited. The aim was to elucidate the mucoadhesive properties of chitosan/polyethylene oxide (PEO) nanofibers focusing on how the degree of deacetylation (DDA, 53–96 %) of chitosan influenced their morphological and mucoadhesive properties. The mechanism of mucoadhesion was explained by the intermolecular interactions of chitosan with mucin from bovine submaxillary glands using quartz-crystal microbalance with dissipation monitoring and by adhesion of the nanofibers to ex vivo porcine sublingual mucosa. An increase in chitosan DDA improved the morphological stability of the nanofibers in water, but did not contribute to altered mucoadhesive properties. This study demonstrates excellent mucoadhesive properties of chitosan/PEO nanofibers and shows that the strong mucoadhesiveness of the nanofibers is attributed to their swelling ability.

1,25(OH)D vitamin D promotes NOS2 expression in response to bacterial and viral PAMPs in primary bovine salivary gland fibroblasts.

The faecal-oral route is a predominant mode of infectious disease transmission and yet the immunology of the bovine oral cavity is poorly understood. The objectives of this study were to develop an in vitro cell model of bovine salivary gland cells and to characterize the role of vitamin D on the expression of innate immune genes induced by stimulation with bacterial and viral pathogen-associated molecular patterns (PAMPs). Submandibular glandular tissue was excised post-mortem, processed, cells isolated and cultured until confluency after which cells were incubated with the active form of vitamin D (1,25(OH)D) for 18h before stimulation with lipopolysaccharide (LPSμg/ml), lipoteichoic acid (LTAμg/ml) or polyinosinic:polycytidylic acid (poly I:C-20μg/ml) PAMPs for 6h and immune gene expression was assessed by Quantitative Real-Time PCR (RT-qPCR). RT-qPCR analysis of vimentin expression in cells derived from the bovine submandibular gland shows that cultured cells were fibroblast in origin. These cells significantly induce the pro-inflammatory cytokine IL1B, β-defensin and cathelicidin genes but these were not significantly altered in response to 1,25(OH)D. In contrast, 1,25(OH)D significantly up-regulates the expression of the NOS2 gene encoding iNOS in bovine submandibular stromal cells compared to EtOH (vehicle) control and this is a maintained response to all three bacterial and viral ligands. We have developed a new in vitro model to allow detailed investigations of mechanisms to enhance oral immunity in cattle. We show that these cells are fibroblast in nature, immunologically competent and vitamin D responsive. Their vitamin D-mediated enhancement of NOS2 expression warrants further investigation in saliva as a potential mechanism to boost oral immunity against infectious agents.

Binding and mucoadhesion of sulfurated derivatives of quaternary ammonium-chitosans and their nanoaggregates: An NMR investigation

The effect of insertion of SH and S-protected groups on the binding and mucoadhesion properties of quaternary ammonium-chitosans and their nanoparticulate forms has been investigated by NMR spectroscopy. Diclofenac sodium salt has been assumed as low molecular weight probe to detect the different binding behaviour of polymeric materials; mucin from bovine submaxillary glands was selected as the model protein for differentiating their mucoadhesion. NMR proton selective relaxation rates of the probe molecule were remarkably sensitive to the presence of very low amounts of sulfurated moieties. Impact of supramolecular aggregation in nanostructured species was demonstrated as well as the relevance of S-protection.

Impact of molecular linker size on physicochemical properties of assembled gold nanoparticle mono-/multi-layers and their applicability for functional binding of biomolecules

In this work the impact of molecular inter-linker size on gold nanoparticle (AuNP) mono-/multilayer structural properties, density and homogeneity has been investigated. These characteristics are of great importance for functional binding of biomolecules. Positively charged high or low molecular weight inter-linkers, poly-L-lysine (PLL) or N-(6-mercapto)hexylpyridinium (MHP), were used to attach negatively charged AuNPs on a planar gold surface as well as to further interlink into a multilayer structure via layer-by-layer deposition. The inter-particle interaction within the assembled AuNP films was adjusted by the ionic strength in the AuNPs dispersions. The AuNP layer density and structural/viscoelastic properties were evaluated by the quartz crystal microbalance with dissipation (QCM-D) technique. The validity of the commercial Voigt model, specifically developed for quantitative QCM-D data analysis of homogeneous viscoelastic films, was evaluated by a model independent analysis when comparing the assembled AuNP films with a homogeneous layer of a mucin from bovine submaxillary glands. Both AuNP mono- and multilayers, attached/interlinked via long flexible PLL molecules assembled to denser and more soft/viscous structures compared to those interlinked by short MHP compounds. Thus, PLL-interlinked AuNP mono-/multilayer structures were further investigated as a platform for laccase enzyme functional adsorption via qualitative assessment of bioelectrochemical characteristics of the enzyme.

De-O-Acetylation of mucin-derived sialic acids by recombinant NanS-p esterases of Escherichia coli O157:H7 strain EDL933

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 encodes the single chromosomal 9-O-acetylesterase NanS, and several copies of prophage-encoded 9-O-acetylesterases (NanS-p). These enzymes have recently been shown to cleave 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2) to yield de-O-acetylated Neu5Ac, the latter of which may serve as a carbon and/or nitrogen source.In the current study, we investigated the NanS- and NanS-p-mediated digestion of synthetic O-acetylated neuraminic acids and bovine submaxillary glands mucin (BSM)-derived O-acetylneuraminic acids by high-performance thin-layer chromatography (HPTLC) and nano electrospray ionization mass spectrometry (nanoESI MS). Initial HPTLC analyses showed the expected activity of NanS and NanS-p variants for Neu5,9Ac2. However, all tested enzymes were unable to de-O-acetylate 5-N-acetyl-4-O-acetylneuraminic acid (Neu5,4 Ac2) in our test system. The nanoESI MS analysis of neuraminic acids after treatment of BSM with NanS-p gave evidence that NanS-p variants of EHEC O157:H7 strain EDL933 cleave off O-acetyl groups from mono-, di-, and tri-O-acetylated Neu5Ac and N-glycolylneuraminic acid (Neu5Gc), regardless of the carbon positions C7, C8 or C9 of the acetate esters. This enzyme activity leads to neuraminidase-accessible Neu5Ac and Neu5Gc on mucin glycans.Moreover, we could demonstrate by HPTLC analyses that recombinant Bacteroides thetaiotaomicron sialidase (BTSA-His) was able to cleave Neu5Ac and Neu5,9Ac2 from BSM and that the combination of BTSA-His with both NanS-His and NanS-p-His derivatives enhanced the release of de-O-acetylated core Neu5Ac and Neu5Gc from mammalian mucin O-glycans.Growth experiments with EHEC wildtype strain EDL933, its nanS and nanS/nanS-p1a-p7 mutant and exogenous BTSA-His in BSM demonstrated that the presence of BTSA-His enhanced growth of EDL933 and the nanS deletion mutant but not the nanS/nanS-p1a-p7 mutant.Thus, we hypothesize that the expression of sialic acid O-acetylesterases with a broad specificity could be an advantage in competition with the gut microbiota for nutrients and facilitate EHEC colonization in the human large intestine.

Interactions of Fly Ash Particles with Mucin and Serum Albumin.

Fly ash particles can contribute to haze and adverse health outcomes. In this study, two mucins, one from bovine submaxillary glands (bovine submaxillary mucin, BSM) and one from porcine stomach (porcine gastric mucin), as well as bovine serum albumin (BSA), which served as the physical barriers against foreign substances entering the tissues and the blood protein, respectively, were chosen as models for the investigations of the interactions between the proteins and the fly ash particles. Their adsorption behaviors were studied using spectroscopy and a quartz crystal microbalance with a dissipation monitor (QCM-D). The results indicated that the fly ash particles can induce the loosening of mucins and BSA, probably via the formation of complexes. Further, the secondary structure of proteins changed in the presence of fly ash particles. The α-helix content decreased with an increasing fly ash particle concentration. The addition of fly ash particles into protein solutions led to fluorescence quenching, which suggested that there were interactions between these particles and the mucins and BSA. The association constants ( Ka) for BSM and BSA were 5.35 and 4.18 L/g, respectively. Furthermore, the results of QCM-D analyses showed that the amount decreased on the mucin surface but increased slightly on the BSA surface, which indicated that the fly ash particles disrupted the mucin layer upon adsorption. These findings provide clear evidence of the interactions between the fly ash particles and the mucins and BSA, which can lead to structural changes. This study contributes to a better understanding of the interactions and adsorptions of atmospheric particulate pollutants with the proteins in the human body.

Friction forces of saliva and red wine on hydrophobic and hydrophilic surfaces

The physical aspect of human oral astringency perception – the mouthfeel – of red wine has not been quantitatively studied in depth. In this study, the interfacial friction/lubrication properties of saliva (mucin from bovine submaxillary glands or human saliva) with red wines (cv. Cabernet sauvignon and Pinot noir) were measured with a surface force apparatus (SFA). In SFA measurements sliding occurs between smooth, undamaged surfaces with a well-defined contact area and film thickness. The surfaces were either hard, hydrophilic mica or soft, hydrophobic PDMS-coated mica which mimic in-mouth conditions. Saliva was a better lubricant than mucin with the soft, hydrophobic surfaces. In addition, saliva's lubricity was 2.5 times better on the soft hydrophobic surfaces than hard hydrophilic surfaces. The addition of red wine with saliva further decreased friction and improved lubrication. Surprisingly, the coefficient of friction measured for red wine with saliva as the lubricant was higher for Pinot noir than Cabernet sauvignon wine. The aggregation and precipitation of salivary proteins by tannins is well known. The lower friction of high tannin Cabernet sauvignon compared to lower tannin Pinot noir was attributed to exclusion of these aggregates and depletion of more polymeric and protein material from the interfacial sliding region.

Growth advantage of Escherichia coli O104:H4 strains on 5-N-acetyl-9-O-acetyl neuraminic acid as a carbon source is dependent on heterogeneous phage-Borne nanS-p esterases

Enterohemorrhagic E. coli (EHEC) are serious bacterial pathogens which are able to cause a hemorrhagic colitis or the life-threatening hemolytic-uremic syndrome (HUS) in humans. EHEC strains can carry different numbers of phage-borne nanS-p alleles that are responsible for acetic acid release from mucin from bovine submaxillary gland and 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2), a carbohydrate present in mucin. Thus, Neu5,9Ac2 can be transformed to 5-N-acetyl neuraminic acid, an energy source used by E. coli strains. We hypothesize that these NanS-p proteins are involved in competitive growth of EHEC in the gastrointestinal tract of humans and animals. The aim of the current study was to demonstrate and characterize the nanS-p alleles of the 2011 E. coli O104:H4 outbreak strain LB226692 and analyze whether the presence of multiple nanS-p alleles in the LB226692 genome causes a competitive growth advantage over a commensal E. coli strain.We detected and characterized five heterogeneous phage-borne nanS-p alleles in the genome of E. coli O104:H4 outbreak strain LB226692 by in silico analysis of its genome. Furthermore, successive deletion of all nanS-p alleles, subsequent complementation with recombinant NanS-p13-His, and in vitro co-culturing experiments with the commensal E. coli strain AMC 198 were conducted. We could show that nanS-p genes of E. coli O104:H4 are responsible for growth inhibition of strain AMC 198, when Neu5,9Ac2 was used as sole carbon source in co-culture. The results of this study let us suggest that multiple nanS-p alleles may confer a growth advantage by outcompeting other E. coli strains in Neu5,9Ac2 rich environments, such as mucus in animal and human gut.

A detection and quantification label-free tool to speed up downstream processing of model mucins.

Mucins are high-molecular weight glycoproteins (0.25–20 MDa) containing one or more domains that are heavily O-glycosylated. Their implications as targets for cancer treatment have increased the interest in these glycoproteins, mainly in the fields of vaccines and antibodies. However, mucins present high heterogeneity, posing challenges that affect purification processes and quality control analysis. In that sense, it is necessary to develop and improve downstream processes and analytical methods to characterize these products.Here a tool based on biolayer interferometry analysis to improve mucin’s detection and quantification in a fast, simple and label free-way is presented. Taking advantage of lectin recognition of mucins’ carbohydrate structures, several lectins were evaluated and immobilized on streptavidin biosensors. Different assay conditions were optimized and the most suitable lectin, Aleuria aurantia lectin (AAL), was selected. Bovine Submaxillary Gland and human MUC5B mucins were used as proof of concept and were successfully detected and quantified at different stages of purification. High sensitivity levels were achieved with LOD and LOQ of 3.8 μg mL-1 and 11.7 μg mL-1 for BSM, and 0.2 μg mL-1 and 0.6 μg mL-1 for MUC5B. AAL binding specificity was also confirmed with fucose competition assays. Our method represents an advance on mucins detection and quantification since the existing methods present several disadvantages for process development. Hereafter, it can be applied to the optimization of new or already established downstream processes for mucins’ purification.

Exploring gold nanoparticles interaction with mucins: A spectroscopic-based study

The interaction between two mucin types (mucin from porcine stomach – PGM and mucin from bovine submaxillary glands – BSM) and gold nanoparticles (GNPs) of various size (5, 20 and 40 nm) and functionalization (with cysteamine or thioglycolic acid) was studied under physiological conditions, in order to investigate the affinity of the nanoparticles to the proteins.Different methods are employed to monitor the interactions: UV–vis and fluorescence spectroscopy, fluorescence lifetime, circular dichroism and transmission electron microscopy. These studies have shown the formation of a complex between GNPs and both PGM and BSM.This aspect could be of great importance for the use of gold nanoparticles for biomedical purposes in those diseases where qualitative and quantitative mucin anomalies play an essential role in mucus composition and rheology.