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Find video protocols related to scientific articles indexed in Pubmed.
Cytoadhesion of P. falciparum-infected erythrocytes to chondroitin-4-sulfate is cooperative and shear-enhanced.
Blood
PUBLISHED: 10-30-2014
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Infections with the human malaria parasite Plasmodium falciparum during pregnancy can lead to severe complications for both mother and child, resulting from the cytoadhesion of parasitized erythrocytes in the intervillous space of the placenta. Cytoadherence is conferred by the specific interaction of the parasite-encoded adhesin VAR2CSA with chondroitin-4-sulfate (CSA) present on placental proteoglycans. CSA presented elsewhere in the microvasculature does not afford VAR2CSA-mediated cytoadhesion of parasitized erythrocytes. To address the placenta-specific binding tropism we have investigated the effect of the receptor/ligand arrangement on cytoadhesion, using artificial membranes with different CSA spacing intervals. We found that cytoadhesion is strongly dependent on the CSA distance, with half maximal adhesion occurring at a CSA distance of 9 ± 1 nm at all hydrodynamic conditions. Moreover, binding to CSA was cooperative and shear stress-induced. These findings suggest that the CSA density, together with allosteric effects in VAR2CSA, aid in discriminating between different CSA milieus.
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Counteracting the inhibitory effect of proteins towards lung surfactant substitutes: a fluorocarbon gas helps displace albumin at the air/water interface.
Chem. Commun. (Camb.)
PUBLISHED: 08-21-2014
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Perfluorohexane gas lowers the kinetic barrier that opposes the displacement of albumin by dipalmitoylphosphatidylcholine at the air/water interface submitted to sinusoidal oscillations at frequencies in the range of those encountered in respiration.
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Cyclic phosphatidic acid treatment suppress cuprizone-induced demyelination and motor dysfunction in mice.
Eur. J. Pharmacol.
PUBLISHED: 03-04-2014
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Multiple sclerosis is a chronic demyelinating disease of the central nervous system leading to progressive cognitive and motor dysfunction, which is characterized by neuroinflammation, demyelination, astrogliosis, loss of oligodendrocytes, and axonal pathologies. Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. In this study, we investigated the effects of cPA on cuprizone-induced demyelination, which is a model of multiple sclerosis. Mice were fed a diet containing 0.2% cuprizone for 5 weeks, which induces severe demyelination, astrocyte and microglial activation, and motor dysfunction. Simultaneous administration of cPA effectively attenuated cuprizone-induced demyelination, glial activation, and motor dysfunction. These data indicate that cPA may be a useful treatment to reduce the extent of demyelination and the severity of motor dysfunction in multiple sclerosis. cPA is a potential lead compound in the development of drugs for the treatment of this devastating disease.
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Quantitative determination of lateral concentration and depth profile of histidine-tagged recombinant proteins probed by grazing incidence X-ray fluorescence.
J Phys Chem B
PUBLISHED: 04-23-2013
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We have demonstrated that the complementary combination of grazing incidence X-ray fluorescence (GIXF) with specular X-ray reflectivity (XRR) can be used to quantitatively determine the density profiles of Ni(2)(+) ions complexed with chelator headgroups as well as S atoms in recombinant proteins anchored to lipid monolayers at the air/water interface. First, we prepared phospholipid monolayers incorporating chelator lipid anchors at different molar fractions at the air/water interface. The fine-structures perpendicular to the global plane of monolayers were characterized by XRR in the presence of Ni(2)(+) ions, yielding the thickness, roughness, and electron density of the stratified lipid monolayers. X-ray fluorescence intensities from Ni K? core levels recorded at the incidence angles below and above the critical angle of total reflection allow for the determination of the position and lateral density of Ni(2)(+) ions associated with chelator headgroups with a high spatial accuracy (±5 Å). The coupling of histidine-tagged Xenopus cadherin 11 (Xcad-11) can also be identified by changes in the fines-structures using XRR. Although fluorescence intensities from S K? level were much weaker than Ni K? signals, we could detect the location of S atoms in recombinant Xcad-11 proteins.
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Morphology and adhesion strength of myoblast cells on photocurable gelatin under native and non-native micromechanical environments.
J Phys Chem B
PUBLISHED: 04-08-2013
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We have quantitatively determined how the morphology and adhesion strength of myoblast cells can be regulated by photocurable gelatin gels, whose mechanical properties can be fine-tuned by a factor of 10(3) (0.1 kPa ? E ? 140 kPa). The use of such gels allows for the investigation of mechanosensing of cells not only near the natural mechanical microenvironments (E ~ 10 kPa) but also far below and beyond of the natural condition. Optical microscopy and statistical image analysis revealed that myoblast cells sensitively adopt their morphology in response to the substrate elasticity at E ~ 1-20 kPa, which can be characterized by the significant changes in the contact area and order parameters of actin cytoskeletons. In contrast, the cells in contact with the gels with lower elastic moduli remained almost round, and the increase in the elasticity beyond E ~ 20 kPa caused no distinct change in morphology. In addition to the morphological analysis, the adhesion strength was quantitatively evaluated by measuring the critical detachment pressure with an aid of intensive pressure waves induced by picosecond laser pulses. This noninvasive technique utilizing extremely short pressure waves (pulse time width ~100 ns) enables one to determine the critical pressure for cell detachment with reliable statistics while minimizing the artifacts arising from the inelastic deformation of cells. The adhesion strength also exhibited a transition from weak adhesion to strong adhesion within the same elasticity range (E ~ 1-20 kPa). A clear correlation between the cell morphology and adhesion strength suggests the coupling of the strain of the substrate and the mechanosensors near focal adhesion sites.
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Grazing-incidence neutron-induced fluorescence probes density profiles of labeled molecules at solid/liquid interfaces.
Langmuir
PUBLISHED: 03-14-2013
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We report on the use of characteristic prompt ?-fluorescence after neutron capture induced by an evanescent neutron wave to probe densities and depth profiles of labeled molecules at solid/liquid interfaces. In contrast to classical scattering techniques and X-ray fluorescence, this method of "grazing-incidence neutron-induced fluorescence" combines direct chemical specificity, provided by the label, with sensitivity to the interface, inherent to the evanescent wave. We demonstrate that the formation of a supported lipid membrane can be quantitatively monitored from the characteristic fluorescence of (157)Gd(3+) ions bound to the headgroup of chelator lipids. Moreover, we were able to localize the (157)Gd(3+) ions along the surface normal with nanometer precision. This first proof of principle with a well-defined model system suggests that the method has a great potential for biology and soft matter studies where spatial resolution and chemical sensitivity are required.
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Cell differentiation of pluripotent tissue sheets immobilized on supported membranes displaying cadherin-11.
PLoS ONE
PUBLISHED: 02-12-2013
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Investigating cohesive tissue sheets in controlled cultures still poses a challenge since the complex intercellular interactions are difficult to mimic in in vitro models. We used supported lipid membranes functionalized by the adhesive part of the extracellular domain of the cell adhesion molecule cadherin-11 for the immobilization of pluripotent tissue sheets, the animal cap isolated from Xenopus laevis blastula stage embryos. Cadherin-11 was bound via histidine tag to lipid membranes with chelator head groups. In the first step, quantitative functionalization of the membranes with cadherin-11 was confirmed by quartz crystal microbalance and high energy specular X-ray reflectivity. In the next step, animal cap tissue sheets induced to neural crest cell fate were cultured on the membranes functionalized with cadherin-11. The adhesion of cells within the cohesive tissue was significantly dependent on changes in lateral densities of cadherin-11. The formation of filopodia and lamellipodia in the cohesive tissue verified the viability and sustainability of the culture over several hours. The expression of the transcription factor slug in externally induced tissue demonstrated the applicability of lipid membranes displaying adhesive molecules for controlled differentiation of cohesive pluripotent tissue sheets.
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Physical interactions of fish protamine and antisepsis peptide drugs with bacterial membranes revealed by combination of specular x-ray reflectivity and grazing-incidence x-ray fluorescence.
Phys Rev E Stat Nonlin Soft Matter Phys
PUBLISHED: 01-17-2013
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As a defined model of outer membranes of gram negative bacteria, we investigated the interaction of monolayers of lipopolysacchrides from Salmonella enterica rough strains R90 (LPS Ra) with natural and synthetic peptides. The fine structures perpendicular to the membrane plane and the ion distribution near the interface were determined by specular x-ray reflectivity (XRR) and grazing-incidence x-ray fluorescence (GIXF) in the presence and absence of divalent cations. The unique combination of XRR and GIXF allows for the quantitative identification of different modes of interactions in a high spatial resolution, which cannot be assessed by other experimental methods. Natural fish protamine disrupts the stratified membrane structures in the absence of Ca(2+) ions, while staying away from the membrane surface in the presence of Ca(2+) ions. In contrast, synthetic antisepsis peptide Pep 19-2.5 weakly adsorbs to the membrane and stays near the uncharged sugar units even in the absence of Ca(2+). In the presence of Ca(2+), Pep 19-2.5 can reach the negatively charged inner core without destroying the barrier capability against ions.
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First order melting transitions of highly ordered dipalmitoyl phosphatidylcholine gel phase membranes in molecular dynamics simulations with atomistic detail.
J Chem Phys
PUBLISHED: 08-10-2011
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Molecular dynamics simulations with atomistic detail of the gel phase and melting transitions of dipalmitoyl phosphatidylcholine bilayers in water reveal the dependency of many thermodynamic and structural parameters on the initial system ordering. We quantitatively compare different methods to create a gel phase system and we observe that a very high ordering of the gel phase starting system is necessary to observe behavior which reproduces experimental data. We performed heating scans with speeds down to 0.5 K/ns and could observe sharp first order phase transitions. Also, we investigated the transition enthalpy as the natural intrinsic parameter of first order phase transitions, and obtained a quantitative match with experimental values. Furthermore, we performed systematic investigations of the statistical distribution and heating rate dependency of the microscopic phase transition temperature.
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Membrane adhesion via homophilic saccharide-saccharide interactions investigated by neutron scattering.
Biophys. J.
PUBLISHED: 03-11-2011
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Solid-supported membrane multilayers doped with membrane-anchored oligosaccharides bearing the LewisX motif (Le(X) lipid) were utilized as a model system of membrane adhesion mediated via homophilic carbohydrate-carbohydrate interactions. Specular and off-specular neutron scattering in bulk aqueous electrolytes allowed us to study multilayer structure and membrane mechanics at full hydration at various Ca(2+) concentrations, indicating that membrane-anchored Le(X) cross-links the adjacent membranes. To estimate forces and energies required for cross-linking, we theoretically modeled the interactions between phospholipid membranes and compared this model with our experimental results on membranes doped with Le(X) lipids. We demonstrated that the bending rigidity, extracted from the off-specular scattering signals, is not significantly influenced by the molar fraction of Le(X) lipids, while the vertical compression modulus (and thus the intermembrane confinement) increases with the molar fraction of Le(X) lipids.
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Dissipative structure formation in lipid/lipopolymer monolayers.
J Phys Chem B
PUBLISHED: 02-22-2011
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We study the formation of dissipative microstructures in monomolecular films of surfactant mixtures, which occur near the three-phase contact line during Langmuir-Blodgett transfer onto a solid substrate. Continuous stripes parallel to the transfer direction are generated over several centimeters, indicating the phase separation of phospholipids and lipids with polymer head groups (lipopolymers). The systematic variation of transfer conditions revealed that transfer speed and subphase viscosity determine the stripe-to-stripe distance from several micrometers to submicrometers. To account for the physical mechanism of such pattern formation, we characterize the local film thickness and the membrane composition in the vicinity of the three-phase contact line using imaging ellipsometry and fluorescence microscopy. At relatively slow rates of substrate lifting, the power law exponent that we found between the interstripe distance and the transfer speed suggests that the stripe formation is due to spinodal decomposition, which can be accounted under the framework of the Cahn-Hilliard equation, whereas at relatively high rates, the distance is found to be proportional to the substrate speed, suggesting a dominant effect of the shear force on the stripe formation.
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Physical chemistry of biological interfaces: generic and specific roles of soft interlayers.
Chem Asian J
PUBLISHED: 02-05-2011
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Nature defines the spatial boundaries between different phases using membranes, and the interfacial interactions are mediated by soft biopolymer interlayers that contain various carbohydrates. This Review provides a comprehensive overview on the interplay of generic and specific interactions at cell-cell and cell-tissue interfaces. A focus will be put on the combination of defined model systems, experimental techniques in real- and reciprocal space, and theoretical simulations.
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Quantitative evaluation of mechanosensing of cells on dynamically tunable hydrogels.
J. Am. Chem. Soc.
PUBLISHED: 01-10-2011
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Thin hydrogel films based on an ABA triblock copolymer gelator [where A is pH-sensitive poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) and B is biocompatible poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC)] were used as a stimulus-responsive substrate that allows fine adjustment of the mechanical environment experienced by mouse myoblast cells. The hydrogel film elasticity could be reversibly modulated by a factor of 40 via careful pH adjustment without adversely affecting cell viability. Myoblast cells exhibited pronounced stress fiber formation and flattening on increasing the hydrogel elasticity. As a new tool to evaluate the strength of cell adhesion, we combined a picosecond laser with an inverted microscope and utilized the strong shock wave created by the laser pulse to determine the critical pressure required for cell detachment. Furthermore, we demonstrate that an abrupt jump in the hydrogel elasticity can be utilized to monitor how cells adapt their morphology to changes in their mechanical environment.
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Quantitative determination of ion distributions in bacterial lipopolysaccharide membranes by grazing-incidence X-ray fluorescence.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-04-2010
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A model of the outer membrane of Gram-negative bacteria was created by the deposition of a monolayer of purified rough mutant lipopolysaccharides at an air/water interface. The density profiles of monovalent (K(+)) and divalent (Ca(2+)) cations normal to the lipopolysaccharides (LPS) monolayers were investigated using grazing-incidence X-ray fluorescence. In the absence of Ca(2+), a K(+) concentration peak was found in the negatively charged LPS headgroup region. With the addition of CaCl(2), Ca(2+) ions almost completely displaced K(+) ions from the headgroup region. By integrating the experimentally reconstructed excess ion density profiles, we obtained an accurate measurement of the effective charge density of LPS monolayers. The experimental findings were compared to the results of Monte Carlo simulations based on a coarse-grained minimal model of LPS molecules and showed excellent agreement.
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Equivalent aqueous phase modulation of domain segregation in myelin monolayers and bilayer vesicles.
Biophys. J.
PUBLISHED: 04-15-2010
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Purified myelin can be spread as monomolecular films at the air/aqueous interface. These films were visualized by fluorescence and Brewster angle microscopy, showing phase coexistence at low and medium surface pressures (<20-30 mN/m). Beyond this threshold, the film becomes homogeneous or not, depending on the aqueous subphase composition. Pure water as well as sucrose, glycerol, dimethylsulfoxide, and dimethylformamide solutions (20% in water) produced monolayers that become homogeneous at high surface pressures; on the other hand, the presence of salts (NaCl, CaCl(2)) in Ringers and physiological solution leads to phase domain microheterogeneity over the whole compression isotherm. These results show that surface heterogeneity is favored by the ionic milieu. The modulation of the phase-mixing behavior in monolayers is paralleled by the behavior of multilamellar vesicles as determined by small-angle and wide-angle x-ray scattering. The correspondence of the behavior of monolayers and multilayers is achieved only at high surface pressures near the equilibrium adsorption surface pressure; at lower surface pressures, the correspondence breaks down. The equilibrium surface tension on all subphases corresponds to that of the air/alkane interface (27 mN/m), independently on the surface tension of the clean subphase.
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Crucial roles of charged saccharide moieties in survival of gram negative bacteria against protamine revealed by combination of grazing incidence x-ray structural characterizations and Monte Carlo simulations.
Phys Rev E Stat Nonlin Soft Matter Phys
PUBLISHED: 04-02-2010
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Grazing incidence x-ray scattering techniques and Monte Carlo (MC) simulations are combined to reveal the influence of molecular structure (genetic mutation) and divalent cations on the survival of gram negative bacteria against cationic peptides such as protamine. The former yields detailed structures of bacterial lipopolysaccharide (LPS) membranes with minimized radiation damages, while the minimal computer model based on the linearized Poisson-Boltzmann theory allows for the simulation of conformational changes of macromolecules (LPSs and peptides) that occur in the time scale of ms. The complementary combination of the structural characterizations and MC simulation demonstrates that the condensations of divalent ions (Ca2+ or Mg2+) in the negatively charged core saccharides are crucial for bacterial survival.
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Modulation of substrate-membrane interactions by linear poly(2-methyl-2-oxazoline) spacers revealed by X-ray reflectivity and ellipsometry.
Chemphyschem
PUBLISHED: 10-07-2009
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Hydrated polymer interlayers between planar lipid membranes and solid substrates provide a water reservoir and thus maintain a finite membrane-substrate distance. Linear polymer spacers attached to lipid head groups (lipopolymer tethers) can be used as a defined model of oligo- and polysaccharides covalently anchored on cell surfaces (glycocalyx). They can offer a unique advantage over membranes physisorbed on polymer films (called polymer-cushioned membranes), owing to their ability to control both the length and density of polymer chains. In this study, a lipopolymer tether composed of a stable ether lipid moiety and a hydrophilic poly(2-methyl-2-oxazoline) spacer with a length of 60 monomer units is used to fabricate supported membranes by the successive deposition of proximal (lower) and distal (upper) leaflets. Using specular X-ray reflectivity and ellipsometry, we systematically investigate how the lateral density of polymer chains influences the membrane-substrate interactions. The combination of two types of reflectivity techniques under various conditions enables the calculation of quantitative force-distance relationships. Such artificial membrane systems can be considered as a half-model of cell-cell contacts mediated via the glycocalyx, which reveals the influence of polymer chain density on the interplay of interfacial forces at biological interfaces.
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Calcium ions induce collapse of charged O-side chains of lipopolysaccharides from Pseudomonas aeruginosa.
J R Soc Interface
PUBLISHED: 07-15-2009
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Lipopolysaccharide (LPS) monolayers deposited on planar, hydrophobic substrates were used as a defined model of outer membranes of Pseudomonas aeruginosa strain dps 89. To investigate the influence of ions on the (out-of-plane) monolayer structure, we measured specular X-ray reflectivity at high energy (22 keV) to ensure transmission through water. Electron density profiles were reconstructed from the reflectivity curves, and they indicate that the presence of Ca(2+) ions induces a significant change in the conformation of the charged polysaccharide head groups (O-side chains). Monte Carlo simulations based on a minimal computer model of LPS molecules allow for the modelling of 100 or more molecules over 10(-3) s and theoretically explained the tendency found by experiments.
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Native supported membranes on planar polymer supports and micro-particle supports.
J. Struct. Biol.
PUBLISHED: 03-31-2009
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To bridge soft biological materials and hard inorganic materials is an interdisciplinary scientific challenge. Despite of experimental difficulties, the deposition of native biological membranes on supports is a straightforward strategy. This review provides an overview of advances in the fabrication and characterization of native biological membranes on planar polymer supports and micro-particles.
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Mechanical properties of interacting lipopolysaccharide membranes from bacteria mutants studied by specular and off-specular neutron scattering.
Phys Rev E Stat Nonlin Soft Matter Phys
PUBLISHED: 03-11-2009
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Specular and off-specular neutron scattering are used to study the influence of molecular chemistry (mutation) on the intermembrane interactions and mechanical properties of the outer membrane of Gram-negative bacteria consisting of lipopolysaccharides (LPSs). For this purpose, solid-supported multilayers of mutant LPS membranes are deposited on silicon wafers and hydrated either at defined humidity or in bulk buffers. The planar sample geometry allows to identify out-of-plane and in-plane scattering vector components. The measured two-dimensional reciprocal space maps are simulated with membrane displacement correlation functions determined by two mechanical parameters (vertical compression modulus and bending rigidity) and an effective cutoff radius for the membrane fluctuation wavelength. Experiments at controlled humidity enable one to examine the influence of the disjoining pressure on the saccharide-mediated intermembrane interactions, while experiments in bulk buffers (i.e., in the absence of an external osmotic stress) reveal the effect of divalent cations on LPS membranes, highlighting the role of divalent cations in the survival mechanism of bacteria in the presence of antimicrobial molecules.
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Covalent modification of chitin with silk-derivatives acts as an amphiphilic self-organizing template in nacre biomineralisation.
J. Struct. Biol.
PUBLISHED: 02-12-2009
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Molluscs have a well-deserved reputation for being expert mineralizers of various shell types such as nacre. Nacre is defined as regularly arranged layers and stacks of approximately 0.5 microm thick aragonite platelets that are extracellularly formed within a complex mixture of organic matrix. The control of species-specific layer thickness by the animal is still enigmatic. Despite the recent findings on the periodic layer-by-layer structures of chitin layers and silk-like protein layers in nacre-type biominerals, little is known about how the interface is defined between two different layers. In this paper, we demonstrate the presence of covalently attached, hydrophobic amino acid side chains in the chitin matrix in the bivalve mollusc Mytilus galloprovincialis by the combination of infrared spectroscopy and mass spectroscopy. The accumulation of the modified chitin matrix at the interface is quantified by the critical aggregate concentration of the purified chitin matrix, which is approximately an order of magnitude smaller than that of pure chitin. Our finding suggests an active role of such chemically modified chito-oligosaccharides in the creation of a defined interface and guidance of the periodic matrix textures, which would result in unique material properties of natural mollusc shells.
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Quantitative determination of the lateral density and intermolecular correlation between proteins anchored on the membrane surfaces using grazing incidence small-angle X-ray scattering and grazing incidence X-ray fluorescence.
J Chem Phys
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As a physical model of the surface of cells coated with densely packed, non-crystalline proteins coupled to lipid anchors, we functionalized the surface of phospholipid membranes by coupling of neutravidin to biotinylated lipid anchors. After the characterization of fine structures perpendicular to the plane of membrane using specular X-ray reflectivity, the same membrane was characterized by grazing incidence small angle X-ray scattering (GISAXS). Within the framework of distorted wave Born approximation and two-dimensional Percus-Yevick function, we can analyze the form and structure factors of the non-crystalline, membrane-anchored proteins for the first time. As a new experimental technique to quantify the surface density of proteins on the membrane surface, we utilized grazing incidence X-ray fluorescence (GIXF). Here, the mean intermolecular distance between proteins from the sulfur peak intensities can be calculated by applying Abele?s matrix formalism. The characteristic correlation distance between non-crystalline neutravidin obtained by the GISAXS analysis agrees well with the intermolecular distance calculated by GIXF, suggesting a large potential of the combination of GISAXS and GIXF in probing the lateral density and correlation of non-crystalline proteins displayed on the membrane surface.
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Intraoperative fluorescence lymphography using indocyanine green in a patient with chylothorax after esophagectomy: report of a case.
Surg. Today
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We report a case of chylothorax treated successfully by a new diagnostic tool: indocyanine green (ICG) fluorescence lymphography. The patient, a 65-year-old man with adenocarcinoma of the esophagogastric junction, underwent radical esophagectomy, which was followed by the development of chylothorax. On postoperative day 10, we performed transabdominal ligation of the thoracic duct. During the re-operation, we injected ICG into the mesentery of the small bowel. We then performed mass ligation of the tissue right and dorsal of the aorta, including the thoracic duct, after which a near-infrared camera system revealed a fluorescent stripe on the caudal part of the ligation. The remnant thoracic duct appeared to be dilated as a result of lymphatic stasis. The patient was discharged 35 days after his initial surgery. We report this case to demonstrate the usefulness of intraoperative ICG lymphography as a tool to identify and confirm ligation of the thoracic duct transabdominally.
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Spatio-temporal patterns of pancreatic cancer cells expressing CD44 isoforms on supported membranes displaying hyaluronic acid oligomers arrays.
PLoS ONE
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In this paper, we designed a quantitative model of biological membranes by the deposition of planar lipid membranes on solid substrates (called supported membranes), and immobilized biotinylated oligomers of hyaluronic acid (oligo-HA, 6-8 disaccharide units in length) to the membrane surface via neutravidin cross-linkers. By controlling the self-assembly of biotinylated lipid anchors, the mean distance between the oligo-HA molecules on the membrane could be controlled to nm accuracy. The adhesion and motility of pancreatic adenocarcinoma cells expressing different splice variants of the HA-binding cell surface receptor CD44 on these surfaces were investigated quantitatively. The combination of label-free, time-lapse imaging of living cells and statistical analysis suggests that the static morphology (global shape and cytoskeleton remodeling) of cells, their stochastic morphological dynamics, and the probability of directed motion reflect the metastatic behaviour of the cancer cells.
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Quantitative evaluation of adhesion of osteosarcoma cells to hydrophobic polymer substrate with tunable elasticity.
J Phys Chem B
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We investigated a potential application of hydrophobic poly(n-butyl acrylate) networks (cPnBA) as substrates with tunable elasticity for culturing, maintenance, and regulation of human osteosarcoma cells (U2OS). Nanoindentation experiments with an atomic force microscope revealed that the mechanical properties of cPnBA films are maintained under aqueous conditions, confirming that the substrate elasticity can be controlled simply by the degree of cross-linking, independent from the culture medium. We found that the adhesion U2OS cells to cPnBA substrates could be improved by surface treatments such as oxgen plasma and serum proteins. To determine the strength of cell adhesion, the critical pressure to detach cells from cPnBA substrates was measured using a shock wave induced by an intensive picosecond laser pulse. A monotonic increase in the cell adhesion strength in accordance with the substrate elasticity demonstrated the potential of intrinsically hydrophobic cPnBA as a new class of substrate material with tunable mechanical properties that are not influenced by the culture medium.
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Functional expression of Ca²? dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum.
Biochem. Biophys. Res. Commun.
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In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.