Formation of giant unilamellar vesicles from spin-coated lipid films by localized IR heating

We report a novel method for the generation of GUVs (generate unilamellar vesicles) from spin-coated lipid films by means of localized heating. This technique enables GUV formation from both charged and neutral lipid species, as well as from a complex lipid mixture, in various ionic strength conditions. Encapsulation was possible during and after GUV formation

Cellular communication via directed protrusion growth: Critical length-scales and membrane morphology

AbstractWe investigated the growth of cell protrusions from adherent HEK 293 cells and their capability to bridge cytophobic Teflon®  AF microgaps, establishing a critical length scale, beyond which cells cannot probe free space. For this purpose, we employed a photolithography-based surface fabrication strategy for producing micropatterned substrates composed of glass and the amorphous polymer Teflon®  AF. Cell protrusions growing from HEK 293 cells on these substrates were confined to extend on 2 μm wide glass lanes, intersected by Teflon®  AF microgaps of various lengths between 2 and 16 μm. After 24 hours of incubation, the frequency of cell protrusions crossing the gap was found to be strongly dependent on the gap size. Gaps which are greater than 4 μm were found to be increasingly difficult to cross. Cell extensions crossing the microgaps either appeared as nanosized connections, in approximately 30% of all observed cases, or as microsized connections. Molecular transport in the established cell-to-cell connection across the microgap was investigated by activation of TRPM8 ion channels followed by supply of Ca2+ to one of the connected cells. The diffusion of the Ca2+ ions was visualized by means of a cell-permeant pre-fluorescent dye. We observed both open- and closed-ended intercellular connections in both nano- and microsized cell protrusions

Heat-induced formation of single giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) are an excellent model system for the investigation of lipid membranes, the study of membrane proteins and ion channels in a biomimetic environment, and in the creation of artificial cells. Here, we describe a novel method for the preparation of GUVs from single multilamellar liposomes by means of directed infrared laser heating. Our method generates individual unilamellar vesicles at selected locations, not only from natural and artificial lipid mixtures containing negatively charged lipids, but also from preparations of single lipids, such as neutral phosphatidylethanolamine. The presented method provides a new efficient resource for giant vesicle research and offers an alternative to the electroformation and de/rehydration techniques

Millimetre-wave dielectric spectroscopy for cell analysis

A millimeter-wave sensor based on a CPW line has been designed and fabricated as a first prototype for impedance spectroscopy to be combined with a multifunctional micropipette for cell and membrane analysis. The first mm-wave measurement results show the sensitivity of the layout by distinguishing the cells from the media and monitoring the attachment process of the cells to the sensor surface. Measurements were performed on umbilical cord stem cells and cartilage thumb cells

Millimeter-wave sensor based on a λ/2-line resonator for identification and dielectric characterization of non-ionic surfactants

Studies of biological and artificial membrane systems, such as niosomes, currently rely on the use of fluorescent tags, which can influence the system under investigation. For this reason, the development of label-free, non-invasive detection techniques is of great interest. We demonstrate an open volume label-free millimeter-wave sensing platform based on a coplanar waveguide, developed for identification and characterization of niosome constituents. A design based on a λ/2-line resonator was used and on-wafer measurements of transmission and reflection parameters were performed up to 110 GHz. Our sensor was able to clearly distinguish between common niosome constituents, non-ionic surfactants Tween 20 and Span 80, measuring a resonance shift of 3 GHz between them. The complex permittivities of the molecular compounds have been extracted. Our results indicate insignificant frequency dependence in the investigated frequency range (3 GHz – 110 GHz). Values of permittivity around 3.0 + 0.7i and 2.2 + 0.4i were obtained for Tween 20 and Span 80, respectively

Label-free spatio-temporal monitoring of cytosolic mass, osmolarity, and volume in living cells

Microorganisms adapt their biophysical properties in response to changes in their local environment. However, quantifying these changes at the single-cell level has only recently become possible, largely relying on fluorescent labeling strategies. In this work, we utilize yeast (Saccharomyces cerevisiae) to demonstrate label-free quantification of changes in both intracellular osmolarity and macromolecular concentration in response to changes in the local environment. By combining a digital holographic microscope with a millifluidic chip, the temporal response of cellular water flux was successfully isolated from the rate of production of higher molecular weight compounds, in addition to identifying the produced compounds in terms of the product of their refractive index increment (dndc) and molar mass. The ability to identify, quantify and temporally resolve multiple biophysical processes in living cells at the single cell level offers a crucial complement to label-based strategies, suggesting broad applicability in studies of a wide-range of cellular processes

Maintenance of Large Subpopulations of Differentiated CD8 T-Cells Two Years after Cytomegalovirus Infection in Gambian Infants

BACKGROUND: In a previously published study, we found that large differentiated subpopulations of CD8 T-cells emerged rapidly after CMV infection in young infants and persisted throughout the following year. Here we describe a follow-up study conducted on the same infants to establish whether the differentiated subpopulations continued through the second year post-infection. METHODOLOGY / PRINCIPAL FINDINGS: CMV-specific cells identified using tetramers remained more activated and differentiated than the overall CD8 population. The large subpopulation of differentiated cytotoxic (CD28(-)CD62L(-)Bcl-2(low)CD95(+)perforin(+)) cells that emerged rapidly after infection remained stable after two years. No similar subpopulation was found in CMV-uninfected infants indicating that two years after infection, CMV remained a major factor in driving CD8 T-cell differentiation. Although markers of activation (CD45R0 and HLA-D) declined throughout the first year, HLA-D expression continued to decline during the second year and CD45R0 expression increased slightly. The age-related increase in IFNgamma response observed during the first year continued but was non-significant during the second year, indicating that the rate of functional improvement had slowed substantially. CONCLUSIONS / SIGNIFICANCE: The large differentiated subpopulations of CD8 T-cells that had emerged immediately after CMV infection persisted through the second year post-infection, while levels of activation and functional capacity remained fairly constant.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Radial Sizing of Lipid Nanotubes Using Membrane Displacement Analysis

We report a novel method for the measurement of lipid nanotube radii. Membrane translocation is monitored between two nanotube-connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. We elucidate nanotube radii, extracted from SPE vesicles, enabling quantification of membrane composition and lamellarity. Variances of nanotube radii were measured, showing a growth of 40-56 nm, upon increasing cholesterol content from 0 to 20%

Evolution of pathogenicity and sexual reproduction in eight Candida genomes

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.publishe