Extrusion of hydrated lipid suspensions is frequently employed to produce vesicles of uniform size, and the resulting vesicles are often reported to be unilamellar. We describe a method for the quantitative fluorescence image analysis of individual vesicles to obtain information on the size, lamellarity, and structure of vesicles produced by extrusion. In contrast to methods for bulk analysis, fluorescence microscopy provides information about individual vesicles, rather than averaged results, and heterogeneities in vesicle populations can be characterized. Phosphatidylcholine vesicles containing small fractions of biotin-modified phospholipid and fluorescently labeled 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) phospholipid were immobilized through biotin-avidin-biotin binding to the surface of a biotin-modified glass coverslip. Biotin was attached to the surface in a mixed cyano-terminated silane monolayer. Initial fluorescence intensities for each immobilized vesicle were recorded, and a solution of membrane impermeable quencher was passed through the flow cell to quench the fluorescence of the outer layer. Fluorescence from individual vesicles was measured by fitting the spots to 2-dimensional Gaussian functions. The integrated signals under the peaks yielded a pre- and postquench intensity. From the fractional loss of intensity, the number and structure of the bilayers in individual vesicles could be quantified; the results showed that extruded vesicles exhibit a distribution of size, lamellarity, and structure.
Bilateral free flap breast reconstruction can be performed safely despite an increase in operative time when compared with unilateral reconstruction.
The addition of trivalent chromium, Cr(III), reagents to peptide solutions can increase the intensity of doubly protonated peptides, [M + 2H]2+, through electrospray ionization (ESI). Three model heptapeptides were studied: neutral (AAAAAAA), acidic (AAEEEAA), and basic (AAAKAAA). The neutral and acidic peptides form almost no 2+ ions in the absence of Cr(III). Twenty Cr(III) complexes were used as potential enhanced protonation reagents, including 11 complexes with nonlabile ligands and nine with labile ligands. The complexes that provide the most abundant [M + 2H]2+, the greatest [M + 2H]2+ to [M + H]+ ratio, and the cleanest mass spectra are [Cr(H2O)6](NO3)3·3H2O and [Cr(THF)3]Cl3. Anions in Cr(III) reagents can also affect the intensity of [M + 2H]2+ and the [M + 2H]2+ to [M + H]+ ratio through cation‐anion interactions. The influence of anions on the extent of peptide protonation follows the trend ClO4− ˃ SO42− ˃ Br− ˃ Cl− ˃ F− ≈ NO3−. Solvent effects and complexes with varying number of water ligands were investigated to study the importance of water in enhanced protonation. Aqueous solvent systems and Cr(III) complexes that have at least one bound water ligand in solution must be used for successful increase in the intensity of [M + 2H]2+, which indicates that water is involved in the mechanism of Cr(III)‐induced enhanced protonation. The ESI source design is also important because no enhanced protonation was observed using a Z‐spray source. The current results suggest that this Cr(III)‐induced effect occurs during the ESI desolvation process.
This integrated drug delivering nerve guide simplifies the design process and provides increased versatility for releasing a variety of different growth factors. This innovative device has the potential for broad applicability and allows for easier customization to change the type of drugs and dosage of individual drugs without devising a completely new biomaterial-drug conjugate each time.
A range of carbene structures and their adducts with one another and with a selection of small-molecule electrophiles and nucleophiles were examined at the composite correlated molecular orbital theory G3MP2 level to explore ground-state "carbenic" structures, their stabilities, and reactivities. Differences between carbene general classification as a singlet electrophilic carbene or singlet nucleophilic carbene and their given reactivity are discussed. A key quantity is the carbon−carbon bond dissociation energy for carbene dimers or the carbene-adduct dissociation energy for other species. The carbene dimer bond dissociation energies span a wide range from 10 to 170 kcal/mol. The hydrogenation energies and singlet−triplet splitting were found to correlate best with the carbene's self-dimerization energy, whereas other descriptors do not. The proton and fluoride affinities of the carbenes alone prove inadequate for classifying reactivity among classes of carbenes. The self-dimerization bond dissociation energy, hydrogenation energy, and singlet−triplet splitting of various carbenes, despite sometimes large differences in proton affinity and other indicators of reactivity, provide usable metrics to correlate substantial amounts of thermodynamic and kinetic (reactivity) information regarding these structures.
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