Effect of Loading Method on a Peptide Substrate Reporter in Intact Cells [post-print]

Studies of live cells often require loading of exogenous molecules through the cell membrane; however, effects of loading method on experimental results are poorly understood. Therefore, in this work, we compared three methods for loading a fluorescently labeled peptide into cells of the model organism Dictyostelium discoideum. We optimized loading by pinocytosis, electroporation, and myristoylation to maximize cell viability and characterized loading efficiency, localization, and uniformity. We also determined how the loading method affected measurements of enzyme activity on the peptide substrate reporter using capillary electrophoresis. Loading method had a strong effect on the stability and phosphorylation of the peptide. The half-life of the intact peptide in cells was 19 ± 2, 53 ± 15, and 12 ± 1 min, for pinocytosis, electroporation, and myristoylation, respectively. The peptide was phosphorylated only in cells loaded by electroporation. Fluorescence microscopy suggested that the differences between methods were likely due to differences in peptide localization

Chemical evolution of atmospheric organic carbon over multiple generations of oxidation

The evolution of atmospheric organic carbon as it undergoes oxidation has a controlling influence on concentrations of key atmospheric species, including particulate matter, ozone and oxidants. However, full characterization of organic carbon over hours to days of atmospheric processing has been stymied by its extreme chemical complexity. Here we study the multigenerational oxidation of α-pinene in the laboratory, characterizing products with several state-of-the-art analytical techniques. Although quantification of some early generation products remains elusive, full carbon closure is achieved (within measurement uncertainty) by the end of the experiments. These results provide new insights into the effects of oxidation on organic carbon properties (volatility, oxidation state and reactivity) and the atmospheric lifecycle of organic carbon. Following an initial period characterized by functionalization reactions and particle growth, fragmentation reactions dominate, forming smaller species. After approximately one day of atmospheric aging, most carbon is sequestered in two long-lived reservoirs—volatile oxidized gases and low-volatility particulate matter.National Science Foundation (U.S.). Graduate Research Fellowship Program (AGS-PRF 1433432)National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant AGS-1536939)National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant AGS-1537446)National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant AGS-1536551