Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days post-seeding to establish a stable and confluent cell monolayer, which is used in a single permeability assay during the period of monolayer stability (up to day 30). In this work, we characterize variations in the tightness of the cell monolayer over the stable time interval and evaluate the conditions required for their re-use in permeability assays. The monolayer integrity was assessed through TEER measurements and permeability of the paracellular marker Lucifer Yellow (LY), complemented with nuclei and ZO-1 staining for morphological studies and the presence of tight junctions. Over 150 permeability assays were performed, which showed that manipulation of the cell monolayer in the permeability assay may contribute significantly to the flux of LY, leading to Papp values that are dependent on the sampling duration. The assay also leads to a small decrease in the cell monolayer TEER, which is fully recovered when cell monolayers are incubated with culture media for two full days. When this procedure is followed, the cell monolayers may be used for permeability assays on days 22, 25, and 28, triplicating the throughput of this important assay.

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This work shows that under specific environmental conditions, H. pylori expresses a phase-variable cell-surface alpha-(1-->4)-glucose moiety.

Biological systems are the result of the interactions established among their many distinct molecules and molecular assemblies. The high concentration of small molecules dissolved in the aqueous media alter the water properties with important consequences in the interactions established. In this work, the effects of high concentrations of the disaccharide trehalose on the solubility of a homologous series of fluorescent amphiphiles (NBD-Cn, n=4-16) and on their interaction with a lipid bilayer and a serum protein is quantitatively characterized. Both kinetic and equilibrium aspects are reported for a better understanding of the effects observed.The aqueous solubility of the most hydrophobic amphiphiles (n ³ 8) is strongly increased by 1 M trehalose, while no significant effect is observed for the most polar amphiphile (n=4). This results from a decrease in the magnitude of the hydrophobic effect at conditions of molecular crowding. A small decrease is observed on the equilibrium association with serum albumin. This is most significant for amphiphiles with longer alkyl chains, in agreement with their increased solubility in the aqueous media containing trehalose.The effects on the association of the amphiphiles with lipid bilayers are influenced by both equilibrium and kinetic aspects. On one hand, the decreased magnitude of the hydrophobic effect leads to a decrease in the affinity of the amphiphiles towards the membrane. However, this tendency may be overbalanced by the effects on the kinetics of the interaction (insertion/desorption) due to the increase in the viscosity of the aqueous media.It is shown that the distribution of amphiphilic drugs in the crowded biological media is significantly different from that predicted from studies in dilute solutions and that the effects are dependent on solute's hydrophobicity.