Influence of sialic acid and bacterial sialidase on differential adhesion of Pseudomonas aeruginosa to epithelial cells

Epithelial cell lines from several tissues show a differential sensitivity to Pseudomonas aeruginosa adherence. A549 (lung), HepG2 (liver) and Caco-2 (colon) cells presented an adhesion index of about 3, 1.5 and 5 CFU/cell, respectively, whereas Mz-Ch cell lines (gallbladder cholangiocytes) presented adhesion indexes up to 35. These variations could be associated with the variable amount of sialic acid in cell surface glycoconjugates. Moreover, the presence of free sialic acid in culture media induces the secretion by P. aeruginosa of a sialidase which is able to hydrolyze glycoconjugate-linked sialic acid. As shown with A549 cells, this specific hydrolysis increases bacterial adhesion, probably by unmasking new binding sites onto the cell surface.     

Preferential Lectin Binding of Cancer Cells upon Sialic Acid Treatment Under Nutrient Deprivation

The terminal monosaccharide of glycoconjugates on a eukaryotic cell surface is typically a sialic acid (Neu5Ac). Increased sialylation usually indicates progression and poor prognosis of most carcinomas. Here, we utilize two human mammary epithelial cell lines, HB4A (breast normal cells) and T47D (breast cancer cells), as a model system to demonstrate differential surface glycans when treated with sialic acid under nutrient deprivation. Under a starved condition, sialic acid treatment of both cells resulted in increased activities of α2→3/6 sialyltransferases as demonstrated by solid phase assay using lectin binding. However, a very strong Maackia amurensis agglutinin I (MAL-I) staining on the membrane of sialic acid-treated T47D cells was observed, indicating an increase of Neu5Acα2→3Gal on the cell surface. To our knowledge, this is a first report showing the utility of lectins, particularly MAL-I, as a means to discriminate between normal and cancer cells after sialic acid treatment under nutrient deprivation. This method is sensitive and allows selective detection of glycan sialylation on a cancer cell surface.     

Expression and distribution of N-acetyl and N-glycolylneuraminic acids in secreted and cell-associated glycoconjugates by two human osteosarcoma cell lines

N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the dominant sialic acids (Sia) in mammals usually found in the non-reducing terminal of oligosaccharide side chains in glycoproteins and glycolipids. Their expression and distribution pattern have been correlated both with the malignant phenotype and tumor grade of human cancers. The aim of the present study was to determine by reversed-phase HPLC method the amounts of Neu5Ac and Neu5Gc as well as their distribution among the culture media and cell surface of MG-63 and Saos-2 human osteosarcoma cell lines of high and low metastatic potential. It was determined that MG-63 cells produce up to 5-fold more total sialic acid as compared with the Saos 2 cells. Neu5Ac accounts for ca 60% of the total sialic acids secreted by MG-63 cells, whereas Neu5Gc is the predominant sialic acid present on the MG-63 cell membrane. Saos 2 cells secrete considerable amounts of Neu5Ac to culture media. The obtained data indicate that the human osteosarcoma cells express both forms of Sia-containing glycoconjugates; the differences in the amounts of each of the two major Sia types and their distribution may be related to their differences in morphology and/or metastatic potentials.     

Surface physicochemical properties of Pseudomonas fluorescens and impact on adhesion and transport through porous media

The physiological state of an examined Pseudomonas fluorescens strain had a significant impact on its adhesion to glass surfaces and transport through glass-bead columns. In both batch and column studies collision efficiencies, , for exponential phase cells were much larger (≈2–3) than for stationary or decay phase cells (≈0.5–0.7). Centrifugation of exponential phase cells substantially reduced collision efficiencies (≈0.8). Over the examined range (0.02–0.2 M), ionic strength had no impact on cell attachment. The Lewis acid/base (A/B) character of the cell surface varied with physiological states: exponential phase cells exhibited larger values of the electron–donor parameter of the polar surface tension component, γ_S⁻, than stationary or decay phase cells, resulting in larger calculated cell hydrophilicities. A reduction in exponential phase cell ζ-potential was observed upon centrifugation. Traditional Derjaguin–Landau–Verwey–Overbeek (DLVO) interaction energy profiles (between cells and glass surfaces) indicated energy maxima of the order of 90–130kT, and secondary energy minima of less than 10kT. Extended DLVO modeling predicted infinite energy barriers attributable to repulsive A/B interactions, and similar magnitude secondary energy minima. A pseudo-chemical kinetic approach was used to calculate activation energies of adhesion from experimental collision efficiencies. Collision efficiencies were also predicted from a diffusion-governed mass transport model incorporating interacting force fields. Predicted energy barriers underestimated cell collision efficiencies, suggesting that secondary energy minimum interactions governed initial attachment of cells. The partial reversibility of adhesion upon ionic strength reduction supported the secondary minimum interaction hypothesis.     

GC/MS-based metabolomics reveals fatty acid biosynthesis and cholesterol metabolism in cell lines infected with influenza A virus

Metabolomics is the downstream of systems biology and has drawn significant interest for studying the metabolic networks from cells to organisms. To profile the metabolites in two different cell lines (A549 and AGS) infected with influenza A virus, gas chromatography coupled with mass spectrometry (GC/MS) was employed. Some differentiating metabolites in the cell lines were tentatively identified using reference library, interpreted and visualized by applying principal components analysis (PCA) and cluster heat map. Consequently, metabolic flux profiling allowed the differentiation of fatty acid biosynthesis and cholesterol metabolism during viral replication in the cell lines. The change in fatty acid turnover was also observed. Metabolomics investigation also revealed the different responses between A549 and AGS cell lines to the virus infection. From the pattern recognition results, AGS cell line might be more susceptible to influenza A virus. Regarding the fact that AGS is a poorly differentiated gastric adenocarcinoma cell line whereas A549 is a relatively differentiated lung tumor one, it is speculated that viral replication might be associated with the cell differentiations.     

Adaptive Flexible Sialylated Nanogels as Highly Potent Influenza A Virus Inhibitors

Flexible multivalent 3D nanosystems that can deform and adapt onto the virus surface via specific ligand–receptor multivalent interactions can efficiently block virus adhesion onto the cell. We here report on the synthesis of a 250 nm sized flexible sialylated nanogel that adapts onto the influenza A virus (IAV) surface via multivalent binding of its sialic acid (SA) residues with hemagglutinin spike proteins on the virus surface. We could demonstrate that the high flexibility of sialylated nanogel improves IAV inhibition by 400 times as compared to a rigid sialylated nanogel in the hemagglutination inhibition assay. The flexible sialylated nanogel efficiently inhibits the influenza A/X31 (H3N2) infection with IC₅₀ values in low picomolar concentrations and also blocks the virus entry into MDCK‐II cells.     

Modulating cell surface immunoreactivity by metabolic induction of unnatural carbohydrate antigens

BACKGROUND: Sialic acid is a component of many tumor-associated oligosaccharide antigens. The repertoire of sialic acids presented by cells can be expanded to include unnatural variants by intercepting the sialic acid biosynthetic pathway with unnatural precursors. We explored whether unnatural cell surface sialosides produced by metabolism can act as neo-antigens and modulate the immunogenicity of cells. RESULTS: Immunization of rabbits with synthetic conjugates of an unnatural sialic acid bound to keyhole limpet hemocyanin produced significant titers of antibodies that were specific for the structurally altered sialic acid. The antibodies recognized cells that were fed the unnatural biosynthetic precursor, and were capable of directing complement-mediated lysis. CONCLUSIONS: Structural alteration of sialic acids replaces a tolerized self-antigen with an antigenic determinant. Incorporation of unnatural sialosides into cell surface glycoconjugates through biosynthetic means can alter the immunoreactivity of cells, providing new possibilities for tumor immunotherapy.     

Microbial adhesion to zirconium alloys

We present data and analyses concerning the adhesion of clinically relevant Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa (bacteria) and Candida albicans (yeast) to Zircaloy-2 (Zry-2) and Zircadyne-705 (Zr705) surfaces. These zirconium-based materials are similar to those now being used in total hip and knee replacements. Here we study clinical strains of microbes under shaken and stationary exposure conditions, and their ability to adhere to Zr surfaces having different oxide thicknesses. We use X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), viable counts, endotoxin assays, and statistical analysis methods, and demonstrate a predictive model for microbial adhesion based on XPS data.     

Surface free energy effect on bacterial retention

Bacterial infection is one of the most frequent and severe complications in the long-term effectiveness of medical implants and devices, greatly increasing treatment cost and inconvenience to the patient. Surface physical and chemical properties are known to influence the extent and form of bacterial infection, although the exact correlation with specific properties is difficult due to the complexity of the system. One approach in the attempt to reduce the bacterial colonisation is to modify the surface energy and chemistry, so as to influence the interactions between the surface and the bacteria that come into contact with it. Five types of coatings were investigated in this study, together with silicone, and polished and non-polished stainless steel 316L. Surfaces were tested for retention of Pseudomonas aeruginosa AK1 after 1 h. A good correlation (>90%) was found between P. aeruginosa AK1 retention and total surface free energy, as well as its polar and dispersive components. The minimum level of P. aeruginosa AK1 retention was found for a range of total surface free energy in the range 20–27 mN/m.     

Infrared spectroscopy studies of cation effects on lipopolysaccharides in aqueous solution

The conformation of amphiphilic lipopolysaccharides (LPS) influences the behavior of free and cell-bound LPS in aqueous environments, including their adhesion to surfaces. Conformational changes in Pseudomonas aeruginosa serotype 10 LPS aggregates resulting from changes in solution pH (3, 6, and 9), ionic strength [I] 1, 10, and 100 mmol L⁻¹, and electrolyte composition (NaCl and CaCl₂) were investigated via attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy. ATR-FTIR data indicate that LPS forms more stable aggregates in NaCl relative to CaCl₂ solutions. Time- and cation-dependent changes in ATR-FTIR data suggest that LPS aggregates are perturbed by Ca²⁺ complexation at lipid A phosphoryl groups, which leads to reorientation of the lipid A at the surface of a ZnSe ATR internal reflection element (IRE). Polarized ATR-FTIR investigations reveal orientation of LPS dipoles approximately perpendicular to the IRE plane for both Na- and Ca-LPS. The results indicate that changes in solution chemistry strongly impact the conformation, intermolecular and interfacial behavior of LPS in aqueous systems.     

A simple flow cell system to evaluate in vitro bacterial adhesion on solids

A simple flow cell system was developed for studies of bacterial adhesion on hydrophobic or hydrophilic surfaces in the presence or absence of saliva. The system consists of flow cells made of borosilicate glass tubes (flow cell volume, 0.45 ml) that are connected to syringes mounted on an infusion pump. Hydrophilic or hydrophobic surfaces were obtained by treating the flow cells with acid or dichlorodimethylsilane, respectively. Liquids and bacterial suspensions were withdrawn directly into the flow cells and the flow rate was monitored through the infusion pump. The adhesion of ³H-labeled Streptococcus mutans cells to bare or saliva-coated surfaces was studied in this system by determining the radioactivity retained in the flow cells after rinsing with buffer at different flow rates. The results indicate that factors like the bacterial cell density, the surface properties, the presence of saliva pellicle and the rinsing flow rate significantly affect the adhesion of S. mutans. These results are in agreement with previous findings obtained with other flow cell systems. The present system has the advantages of using small volumes of liquids and allowing defined laminar flow conditions. Moreover, the entire cell, not only selected area spots, are used for the evaluation of bacterial adhesion. No part of the flow cell is re-used, thus avoiding the risk of contamination with other components. The system may therefore be suitable for assays testing the effect of different compounds on bacterial adhesion.     

Shear-flow induced detachment of Saccharomyces cerevisiae from stainless steel: influence of yeast and solid surface properties

The present study focused on the shear-induced detachment of Saccharomyces cerevisiae in adhesive contact with a 316L stainless steel surface using a shear stress flow chamber, with a view to determining the respective influence of the yeast surface properties and the support characteristics. The effect of cultivation of S. cerevisiae yeast cells on their subsequent detachment from the solid surface was particularly investigated. In order to elucidate the role of stainless steel, non-metallic supports were used as control, covering a broad range of surface properties such as surface free energy and roughness: polypropylene (hydrophobic), polystyrene (mildly hydrophobic, similar to stainless steel) and glass (hydrophilic). All materials were very smooth with respect to the size of yeast. First, experiments were carried out on two types of yeast cells, just rehydrated in saline solution, a biological model widely used in the literature. The influence of the ionic strength (1.5 and 150 mM NaCl) on glass and stainless steel was evaluated. Unlike on glass, no clear evidence was found for electrostatic repulsion with stainless steel since high adhesion was observed whatever the ionic strength. A lack of correlation in adhesion results was also obtained when considering the surface physico-chemical characteristics of type I (hydrophilic) and type II (hydrophobic) rehydrated cells and those of both polymers. It was postulated that unavoidable “sticky” compounds were present on the cell wall, which could not be completely removed during the successive washings of the rehydrated cell suspension before use. This could dramatically alter the yeast surface properties and modify the adhesion strength, thus clearly demonstrating the necessity to work with yeast coming from fresh cultures. Biologically active yeast cells were then used. Once cultured, type I- and type II-yeast cells were shown to exhibit the same hydrophilic properties. Regardless of the material used, for the same ionic strength (150 mM NaCl), yeast adhesion was drastically reduced compared to rehydrated yeast cells. Among all the materials tested, the specificity of 316L stainless steel was clearly established. Indeed, for glass and polymers, cell adhesion was substratum-dependent and driven by the balance between the Lifshitz-van der Waals and Lewis acid/base interactions. Despite nearly identical surface free energies for polystyrene and stainless steel, the metallic surface promoted a totally distinct behaviour which was characterized by a strong – although highly variable – yeast adhesion.     

In Vitro Effects of Papaverine on Cell Proliferation,Reactive Oxygen Species,and Cell Cycle Progression in Cancer Cells

Papaverine (PPV) is an alkaloid isolated from the Papaver somniferum. Research has shown that PPV inhibits proliferation. However, several questions remain regarding the effects of PPV in tumorigenic cells. In this study, the influence of PPV was investigated on the proliferation (spectrophotometry), morphology (light microscopy), oxidative stress (fluorescent microscopy), and cell cycle progression (flow cytometry) in MDA-MB-231, A549, and DU145 cell lines. Exposure to 150 μM PPV resulted in time- and dose-dependent antiproliferative activity with reduced cell growth to 56%, 53%, and 64% in the MDA-MB-231, A549, and DU145 cell lines, respectively. Light microscopy revealed that PPV exposure increased cellular protrusions in MDA-MB-231 and A549 cells to 34% and 23%. Hydrogen peroxide production increased to 1.04-, 1.02-, and 1.44-fold in PPV-treated MDA-MB-231, A549, and DU145 cells, respectively, compared to cells propagated in growth medium. Furthermore, exposure to PPV resulted in an increase of cells in the sub-G₁ phase by 46% and endoreduplication by 10% compared to cells propagated in growth medium that presented with 2.8% cells in the sub-G₁ phase and less than 1% in endoreduplication. The results of this study contribute to understanding of effects of PPV on cancer cell lines.     

Evaluation of photodynamic therapy (PDT) procedures using microfluidic system

A hybrid PDMS/glass microfluidic system for evaluation of the efficiency of photodynamic therapy is presented. 5-aminolevulinic acid (ALA) was used as a precursor of photosensitizer. The geometry of the microdevice presented in this paper enables to test different concentrations of the photosensitizer in a single assay. The viability of the A549 cells was determined 24 h after PDT procedure (irradiation with light which induced a photosensitizer accumulated in carcinoma cells, λ = 625 nm). The presented results confirmed the possibility to perform the photodynamic therapy process in vitro in microscale and the possibility to assess its effectiveness. Moreover, because two identical microstructures on a single chip were performed, the microchip can be used for examination simultaneously various cell lines (carcinoma and normal) or various photosensitizers.     

In vitro cytotoxic activities of (+)-usnic acid and (-)-usnic acid on V79, A549, and human lymphocyte cells and their non-genotoxicity on human lymphocytes

This study investigates cytotoxic and genotoxic activities of (+)-Usnic acid and (-)-usnic acid isolated from the lichen Ramalina farinacea and the lichen Cladonia foliacea, respectively. To determine the activities of these acids, we used the MTT assay on V79 (Chinese hamster lung fibroblast like) and A549 (human lung carcinoma epithelial like) cell lines and cytokinesis-blocked micronucleus (CBMN) assay in human lymphocytes in vitro. Our results suggest that both enantiomers of usnic acid are non-genotoxic shown by the absence of micronucleus induction in human lymphocytes and have significant cytotoxic and apoptotic effects to induce cell killing in cultured human lymphocytes, V79 and A549 cell lines. Even low doses of (+)-usnic acid showed high cytotoxic activity against cancerous cells. The MTT results and cell proliferation index (CPI) values based on the CBMN test results are found in good agreement.     

Steering Siglec–Sialic Acid Interactions on Living Cells using Bioorthogonal Chemistry

Sialic acid sugars that terminate cell‐surface glycans form the ligands for the sialic acid binding immunoglobulin‐like lectin (Siglec) family, which are immunomodulatory receptors expressed by immune cells. Interactions between sialic acid and Siglecs regulate the immune system, and aberrations contribute to pathologies like autoimmunity and cancer. Sialic acid/Siglec interactions between living cells are difficult to study owing to a lack of specific tools. Here, we report a glycoengineering approach to remodel the sialic acids of living cells and their binding to Siglecs. Using bioorthogonal chemistry, a library of cells with more than sixty different sialic acid modifications was generated that showed dramatically increased binding toward the different Siglec family members. Rational design reduced cross‐reactivity and led to the discovery of three selective Siglec‐5/14 ligands. Furthermore, glycoengineered cells carrying sialic acid ligands for Siglec‐3 dampened the activation of Siglec‐3⁺ monocytic cells through the NF‐κB and IRF pathways.     

Cisplatin-induced alteration on membrane composition of A549 cells revealed by ToF-SIMS

Cisplatin has been clinically used for treatment of solid tumors such as non–small-cell lung cancer for decades. However, tumor resistance may be acquired with losing the antitumor activity of cisplatin. As cellular membrane is the first barrier that cisplatin has to overcome before its further action inside the cells, the membrane composition must play a vital role in the cisplatin uptake and excretion, which further influences cisplatin sensitivity. In this work, we applied time-of-flight secondary ion mass spectrometry (ToF-SIMS) surface analysis combined with principle component analysis to distinguish the differences of cell membrane composition between non–small-cell lung cancer cells (A549) and its cisplatin resistant counterpart A549/DDP cells. The decreased phosphatidylcholine content and more abundant cholesterol were observed in the drug resistant cell surfaces, indicating the decreased membrane fluidity of A549/DDP cells. Moreover, we further compared membrane composition of A549 and A549/DDP cells after being treated with different concentrations of cisplatin. A higher composition level of proteins was discovered on all groups of A549/DDP cell membranes. The altered surface chemistry of cellular membranes induced by cisplatin indicates the significance of membrane structures in the drug resistance, which deserves further investigations to this regard.     

Physicochemical aspects of microbial adhesion — Influence of antibody adsorption on the deposition ofStreptococcus sobrinus in a parallel-plate flow chamber

Deposition of the oral bacteriumStreptococcus sobrinus HG977 onto glass (water contact angle 0°) and onto FEP-Teflon (fluoroethylenepropylene; water contact angle 110°) was studied in a parallel-plate flow chamber in the presence and absence of polyclonal antibodies (pAb) and monoclonal antibodies (mAbs) adsorbed onto the cells. The zeta potentials of the bacteria ranged from −7.1 to −8.5 mV at pH 6.8 and were not affected by the presence of pAb or mAbs. Hydrophobicity (by water contact angles) increased from 30° (no antibodies) to 88° in the presence of pAb adsorbed onto the bacterial cell surface. The untreatedS. sobrinus had a greater tendency to adhere to glass (44.5 × 10⁶ cm⁻²) than to FEP-Teflon (18.3 × 10⁶ cm⁻²), in accordance with thermodynamic modelling. After preincubation ofS. sobrinus with pAb, its clear preference for adhesion to glass disappeared as expected from its increased hydrophobicity. Although forS. sobrinus preincubated with OMVU10 no difference was found in hydrophobicity in comparison to the untreated bacteria, the number of bacteria adhering to glass decreased to 10.2 ¢ 10⁶ cm⁻². Formation of bacterial aggregates was found whenS. sobrinus, preincubated with pAb or OMVU10, adhered to glass and FEP. This was also observed when untreated bacteria adhered to glass coated with OMVU10, or to FEP coaled with OMVU10 or pAb. Adhesion in these experiments is therefore thought to occur via near-neighbour collection induced by the presence of pAb or mAbs. Low numbers of bacteria were removed from glass after draining the flow cell, whereas high numbers of untreated bacteria and bacteria preincubated with OMVU10 were removed from FEP.S. sobrinus cells preincubated with pAb were not removed but piled up. It was concluded that the adhesion of untreatedS. sobrinus andS. sobrinus preincubated with pAb is in accordance with thermodynamic modelling, based on the overall wettability of the cell surfaces, whereas the adhesion ofS. sobrinus preincubated with OMVU10 may be through localized interactions, not expressed in overall surface properties.     

Switchable Enzymatic Accessibility for Precision Cell-Selective Surface Glycan Remodeling

Precision cell-selective surface glycan remodeling is of vital importance for modulation of cell surface dynamics, tissue-specific imaging, and immunotherapy, but remains an unsolved challenge. Herein, we report a switchable enzymatic accessibility (SEA) strategy for highly specific editing of carbohydrate moieties of interest on the target cell surface. We demonstrate the blocking of enzyme in the inaccessible state with a metal-organic framework (MOF) cage and instantaneous switching to the accessible state through disassembly of MOF. We further show that this level of SEA regulation enables initial guided enzyme delivery to the target cell surface for subsequent cell-specific glycan remodeling, thus providing a temporally and spatially controlled tool for tuning the glycosylation architectures. Terminal galactose/N-acetylgalactosamine (Gal/GalNAc) remodeling and terminal sialic acid (Sia) desialylation have been precisely achieved on target cells even with other cell lines in close spatial proximity. The SEA protocol features a modular and generically adaptable design, a very short protocol duration (ca. 30 min or shorter), and a very high spatial resolving power (ability to differentiate immediately neighboring cell lines).     

咔咯铁(Ⅲ)配合物与DNA的作用及其抗肿瘤活性

本文合成了10-(4-吩噻嗪苯基)-5,15-二(五氟苯基)铁咔咯配合物(2-Fe)。 在H₂O₂存在下,配合物2-Fe能有效抑制人肺癌细胞(A549)增殖。 流式细胞术检测结果显示在H₂O₂和2-Fe共同作用下,A549细胞生长被阻滞在S期和G2期,且有63.76%发生细胞凋亡。 Hoechst-33342细胞核染色实验观察到A549细胞核呈典型的凋亡形态学变化,同时细胞线粒体膜电势发生明显下降。 2-Fe与小牛胸腺DNA(ct-DNA)以外部模式相结合,且能有效催化氧化断裂DNA,此过程中涉及的活性氧物种可能是单线态氧。