Influence of Nanoscale Surface Roughness on Colloidal Force Measurements.

Forces between colloidal particles determine the performances of many industrial processes and products. Colloidal force measurements conducted between a colloidal particle AFM probe and particles immobilized on a flat substrate are valuable in selecting appropriate surfactants for colloidal stabilization. One of the features of inorganic fillers and extenders is the prevalence of rough surfaces-even the polymer latex particles, often used as model colloidal systems including the current study, have rough surfaces albeit at a much smaller scale. Surface roughness is frequently cited as the reason for disparity between experimental observations and theoretical treatment but seldom verified by direct evidence. This work reports the effect of nanoscale surface roughness on colloidal force measurements carried out in the presence of surfactants. We applied a heating method to reduce the mean surface roughness of commercial latex particles from 30 to 1 nm. We conducted force measurements using the two types of particles at various salt and surfactant concentrations. The surfactants used were pentaethylene glycol monododecyl ether, Pluronic F108, and a styrene/acrylic copolymer, Joncryl 60. In the absence of the surfactant, nanometer surface roughness affects colloidal forces only in high salt conditions when the Debye length becomes smaller than the surface roughness. The adhesion is stronger between colloids with higher surface roughness and requires a higher surfactant concentration to be eliminated. The effect of surface roughness on colloidal forces was also investigated as a function of the adsorbed surfactant layer structure characterized by AFM indentation and dynamic light scattering. We found that when the layer thickness exceeds the surface roughness, the colloidal adhesion is less influenced by surfactant concentration variation. This study demonstrates that surface roughness at the nanoscale can influence colloidal forces significantly and should be taken into account in colloidal dispersion formulations.

Membrane-directed molecular assembly of the neuronal SNARE complex.

Since the discovery and implication of N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full-length recombinant t-SNAREs and v-SNARE present in opposing liposomes, leading to the assembly of a t-/v-SNARE ring complex. Using high-resolution electron microscopy, the electron density maps and 3D topography of the membrane-directed SNARE ring complex was determined at nanometre resolution. Similar to the t-/v-SNARE ring complex formed when 50 nm v-SNARE liposomes meet a t-SNARE-reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t-SNARE-reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane-directed t-/v-SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome-reconstituted recombinant t-/v-SNARE proteins, and native v-SNARE present in isolated SV membrane, the membrane-directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.

Estrogen modulates intestinal mucus physiochemical properties and protects against oxidant injury.

BACKGROUND: The intestinal epithelial barrier and the intestinal mucus layer may be protective against trauma/hemorrhage shock-induced injury in females. This effect is related to estradiol (E2) concentrations and varies with the menstrual cycle. We examined the ability of E2 to impact the physiochemical properties of intestinal mucus and to protect against oxidant-related injury to the mucus and underlying intestinal epithelial barrier in an in vitro model. METHODS: Non-mucus-producing (HT29) and mucus-producing (HT29-MTX) intestinal epithelial cells (IECs) were exposed to E2 or no E2 for 3 days and then grown to confluence on transwell plates. Nonadherent and adherent mucus content was indexed by analysis of mucin using an enzyme-linked immunosorbent assay and mucus viscosity (cp) and elasticity (G') were determined by rheometry. In additional experiments, IEC groups were exposed to hydrogen peroxide and IEC apoptosis as well as permeability (fluorescein isothiocyanate-dextran) and oxidative damage determined by measuring lipid hydroperoxide and protein carbonyl content. RESULTS: There were nearly 50% increases in the mucin content of both the nonadherent and adherent mucus layer(s) in HT29-MTX cells exposed to estrogen. Estrogen treatment also resulted in a twofold and eightfold increase in mucus viscosity and elasticity versus HT29-MTX cells with no estrogen exposure, respectively. Oxygen radical damage to the mucus layer caused by H2O2 was significantly reduced by E2 compared with HT29-MTX + H2O2 without estrogen. Estrogen treatment resulted in significant reductions in both apoptosis and permeability seen after H2O2 challenge. CONCLUSION: The results of this study suggest that sex differences in gut barrier function following trauma/hemorrhage shock may in part be related to differences in intestinal mucus content and the resultant physiochemical and oxidant-resistant properties of the mucus layer.

Early tranexamic acid administration: A protective effect on gut barrier function following ischemia/reperfusion injury.

BACKGROUND: The mucus barrier is a critical component of the gut barrier and may be disrupted by pancreatic enzymes following trauma/hemorrhagic shock (T/HS). Luminal strategies against pancreatic enzyme activation or contact with the intestine are protective of the mucus layer and gut barrier integrity following T/HS. There is increasing evidence the use of tranexamic acid (TA) attenuates inflammatory responses in cardiac surgery and is readily absorbed from the gut. We therefore postulated that systemic administration of TA would attenuate mucus degradation and gut barrier failure following T/HS. This was studied in an in vitro model. METHODS: Confluent monolayers of HT29-MTX (mucus-producing clone) and Caco-2 cocultures were exposed to 90 minutes of hypoxia followed by reoxygenation (H/R), luminal trypsin (5 µM), or both treatment groups. In a subset of experiments, TA (40 µM or 150 µM) was added to the basal chamber (systemic side) of intestinal cell cultures immediately following the hypoxic period. Mucus barrier function was indexed by rheologic measurement of both mucus thickness and viscosity (G', dyne/cm) and oxidant stress. Intestinal cell barrier integrity was indexed by transepithelial electrical resistance, permeability to fluorescein isothiocyanate-dextran, and apoptosis by flow cytometry. RESULTS: Exposure to both trypsin and H/R of Caco-2/HT29-MTX cocultures led to the most severe effect on mucus barrier function. Administration of TA immediately following hypoxia abrogated the effects noted on mucus barrier function. The epithelial barrier was also most severely impacted by both trypsin and H/R. Addition of TA after the hypoxic event was shown to be protective. CONCLUSION: Intestinal mucus physiochemical properties and intestinal barrier function were most severely impacted by exposure to both trypsin (concentration related) and H/R. The "systemic" administration of TA immediately after the hypoxic period was protective and suggests an additional role for early administration of TA in trauma patients in shock.

Ca(2+) bridging of apposed phospholipid bilayers.

In an effort to provide insight into the mechanism of Ca(2+)-induced fusion of lipid vesicles, molecular dynamics simulations in the isobaric-isothermal ensemble are used to investigate interactions of Ca(2+) with apposed lipid bilayers in close proximity. Simulations reveal the formation of a Ca(2+)-phospholipid "anhydrous complex" between apposed bilayers, whereas similar calculations performed with Na(+) display only complexation between neighboring lipids within the same bilayer. The binding of Ca(2+) to apposed phospholipids brings large regions of the bilayers into close contact (<4 Å), displacing water from phospholipid head groups in the process and creating regions of local dehydration. Dehydration of the apposed bilayers leads to ordering of the phospholipid tails, which is partially disrupted by the presence of Ca(2+)-phospholipid bridges.

Ca2+-dimethylphosphate complex formation: providing insight into Ca2+-mediated local dehydration and membrane fusion in cells.

Earlier studies using X-ray diffraction, light scattering, photon correlation spectroscopy, and atomic force microscopy, strongly suggest that SNARE-induced membrane fusion in cells proceeds as a result of calcium bridging opposing bilayers. The bridging of phospholipid heads groups in the opposing bilayers by calcium leads to the release of water from hydrated Ca(2+) ions as well as the loosely coordinated water at PO-lipid head groups. Local dehydration of phospholipid head groups and the calcium, bridging opposing bilayers, then leads to destabilization of the lipid bilayers and membrane fusion. This hypothesis was tested in the current study by atomistic molecular dynamic simulations in the isobaric-isothermal ensemble using hydrated dimethylphosphate anions (DMP(-)) and calcium cations. Results from the study demonstrate, formation of DMP-Ca(2+) complexes and the consequent removal of water, supporting the hypothesis. Our study further demonstrates that as a result of Ca(2+)-DMP self-assembly, the distance between anionic oxygens between the two DMP molecules is reduced to 2.92A, which is in close agreement with the 2.8A SNARE-induced apposition established between opposing bilayers, reported earlier from X-ray diffraction measurements.

Organic nanostructures on silicon, created with semitransparent polystyrene spheres and 248 nm laser pulses.

Arrays of nanostructures are made starting with a template of close-packed, polystyrene spheres on a silicon surface. The spheres are either 1.091 or 2.99 µm in diameter (d) and are of polystyrene (PS). They are irradiated with a pulse of either 308 or 248 nm light to which they are transparent and semitransparent, respectively. A transparent sphere with d = 1.091 µm diameter concentrates incident light onto a small substrate area. As has been previously reported, that creates silicon nanobumps that rise from circular craters. At 248 nm and d = 2.99 µm, the light energy is mainly absorbed, destroys the sphere, and leaves a shrunken mass (typically about 500 nm wide and 100 nm high) of organic material that is probably polystyrene and its thermal degradation products. At 248 nm and d = 1.091 µm, the residual organic structures are on the order of 300 nm wide and 100 nm high. A distinctive feature is that these organic structures are connected by filaments that are on the order of 50 nm wide and 10 nm high. Filaments form because the close-packed PS spheres expand into each other during the early part of the laser pulse, and then, as the main structures shrink, their viscoelasticity leads to threads between them. Our results with 248 nm and d = 1.091 µm differ from those described by Huang et al with 248 nm and d = 1.0 µm. Future studies might include the further effect of wavelength and fluence upon the process as well the use of other materials and the replacement of nanospheres by other focusing shapes, such as ellipsoids or rods.