Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Influence of the position of charcoal air-filtration canisters on the efficacy of waste isoflurane scavenging as assessed in a randomized experiment.

OBJECTIVE To determine whether the position or elevation of charcoal air-filtration canisters would impact efficacy of waste anesthetic gas (WAG) scavenging. DESIGN Randomized experiment. SAMPLE 2 types of bottom-vented and 1 type of top-vented charcoal air-filtration canisters (n = 8 of each canister type/evaluation session). PROCEDURES Canisters were evaluated in a vertical or horizontal position at both low and high isoflurane gas flow rates in a modified Bain nonrebreathing circuit. Waste anesthetic gas concentrations were measured 2.54 cm from canister exhaust ports with an ambient air analyzer every 30 seconds for a maximum of 15 min/experimental condition. One type of bottom-vented canister was tested in a vertical position elevated above or suspended below the vaporizer at a high isoflurane flow rate and then a standard maintenance flow rate. RESULTS Position had no significant effect on WAG emission by any canister type at low isoflurane flow rates. Horizontally positioned bottom-vented canisters at the high isoflurane flow rate emitted significantly more WAG than vertically positioned canisters. Horizontally positioned top-vented canisters at high flow rates emitted significantly more WAG than vertically positioned canisters at the final 15-minute time point only. Cannister types differed significantly in intercanister variability. Canister elevation relative to the vaporizer had no impact on WAG scavenging efficacy. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that charcoal air-filtration canisters should be used in a vertical position when anesthetizing animals with the anesthetic delivery system used in this study.

Studying biomineralization pathways in a 3D culture model of breast cancer microcalcifications.

Microcalcifications serve as diagnostic markers for breast cancer, yet their formation pathway(s) and role in cancer progression are debated due in part to a lack of relevant 3D culture models that allow studying the extent of cellular regulation over mineralization. Previous studies have suggested processes ranging from dystrophic mineralization associated with cell death to bone-like mineral deposition. Here, we evaluated microcalcification formation in 3D multicellular spheroids, generated from non-malignant, pre-cancer, and invasive cell lines from the MCF10A human breast tumor progression series. The spheroids with greater malignancy potential developed necrotic cores, thus recapitulating spatially distinct viable and non-viable areas known to regulate cellular behavior in tumors in vivo. The spatial distribution of the microcalcifications, as well as their compositions, were characterized using nanoCT, electron-microscopy, and X-ray spectroscopy. Apatite microcalcifications were primarily detected within the viable cell regions and their number and size increased with malignancy potential of the spheroids. Levels of alkaline phosphatase decreased with malignancy potential, whereas levels of osteopontin increased. These findings support a mineralization pathway in which cancer cells induce mineralization in a manner that is linked to their malignancy potential, but that is distinct from physiological osteogenic mineralization.

A new method to characterize chemically and topographically nanopatterned surfaces.

Surface chemistry of topographically patterned grooved samples with ridges of 150 nm width, adsorbed with self-assembled monolayers (SAMs) of alkanethiols on gold, have been characterized by near edge X-ray absorption fine structure (NEXAFS) spectroscopy. Analysis reveals that NEXAFS may discriminate between different chemistries adsorbed to the tops, sidewalls and grooves of the patterns.

Nano- and microscale holes modulate cell-substrate adhesion, cytoskeletal organization, and -beta1 integrin localization in SV40 human corneal epithelial cells.

Human corneal epithelial cells (HCECs) interface with a basement membrane in vivo that possesses complex nanoscale topographic features. We report that synthetic substrates patterned with nano- and microscale holes differentially modulate the proliferation, shape and adhesion of SV40 human corneal epithelial cells (SV40-HCECs) as a function of feature size: 1) Cell proliferation was inhibited on nanoscale features (features size less than 800 nm in pitch) compared to microscale features or planar substrates in identical culture conditions. 2) Cells on nanoscale holes had a stellate morphology compared to those on microscale features that were more evenly spread. 3) Cells adhered more to nanoscale features than to microscale features when exposed to shear stress in a laminar flow chamber. Transmission electron microscopy showed that cells cultured on the 400 nm pitch patterns had longer and more numerous filopodia and retraction fibers than cells cultured on the 1600 nm pitch patterns. Immunogold labeling of -beta1 integrins revealed that these receptors were localized at the cell periphery and in the aforementioned cytoskeletal elements. Our findings indicate that surface discontinuities and the activation of mechanochemical cell signaling mechanisms may contribute to the observed responses exhibited by SV40-HCECs cultured on nano- and microscale topography.