In an attempt to dissect endocytosis in Nicotiana tabacum L. pollen tubes, two different probes – positively or negatively charged nanogold – were employed. The destiny of internalized plasma membrane domains, carrying negatively or positively charged residues, was followed at the ultrastructural level and revealed distinct endocytic pathways. Time-course experiments and electron microscopy showed internalization of subapical plasma-membrane domains that were mainly recycled to the secretory pathway through the Golgi apparatus and a second mainly degradative pathway involving plasma membrane retrieval at the tip. In vivo time-lapse experiments using FM4-64 combined with quantitative analysis confirmed the existence of distinct internalization regions. Ikarugamycin, an inhibitor of clathrin-dependent endocytosis, allowed us to further dissect the endocytic process: electron microscopy and time-lapse studies suggested that clathrin-dependent endocytosis occurs in the tip and subapical regions, because recycling of positively charged nanogold to the Golgi bodies and the consignment of negatively charged nanogold to vacuoles were affected. However, intact positively charged-nanogold transport to vacuoles supports the idea that an endocytic pathway that does not require clathrin is also present in pollen tubes.

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The teratogenic potential of commercially available copper oxide (CuO), titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) was evaluated using the standardized FETAX test. After characterization of NP suspensions by TEM, DLS and AAS, histopathological screening and advanced confocal and energy-filtered electron microscopy techniques were used to characterize the induced lesions and to track NPs in tissues. Except for nCuO, which was found to be weakly embryolethal only at the highest concentration tested, the NPs did not cause mortality at concentrations up to 500 mg/L. However, they induced significant malformation rates, and the gut was observed to be the main target organ. CuO NPs exhibited the highest teratogenic potential, although no specific terata were observed. ZnO NPs caused the most severe lesions to the intestinal barrier, allowing NPs to reach the underlying tissues. TiO₂ NPs showed mild embryotoxicity, and it is possible that this substance could be associated with hidden biological effects. Ions from dissolved nCuO contributed greatly to the observed embryotoxic effects, but those from nZnO did not, suggesting that their mechanisms of action may be different.

In this paper, the preparation and characterization of oxygen barrier pullulan sodium montmorillonite (Na+-MMT) nanocomposite coatings are presented for the first time. Full exfoliation of platelets during preparation of the coating water dispersions was mediated by ultrasonic treatment, which turned out to be a pivotal factor in the oxygen barrier performance of the final material even at high relative humidity (RH) conditions [oxygen permeability coefficients ∼1.43 ± 0.39 and 258.05 ± 13.78 mL·μm·m–2·(24 h)−1·atm–1 at 23 °C and 0% RH and 70% RH, respectively]. At the micro- and nanoscale, the reasons are discussed. The final morphology of the coatings revealed that clay lamellae were stacked on top of one another, probably due to the forced confinement of the platelets within the coating thickness after solvent evaporation. This was also confirmed by modeling the experimental oxygen permeability data with the well-known Nielsen and Cussler permeation theoretical models, which suggested a reasonable aspect ratio (α) of ∼100. Electron microscopic analyses also disclosed a peculiar cell-like arrangement of the platelets. The stacking of the clay lamellae and the cell-like arrangement create the excellent oxygen barrier properties. Finally, we demonstrated that the slight haze increase in the bionanocomposite coating materials arising from the addition of the clays depends on the clay concentration but not so much on the sonication time, due to the balance of opposite effects after sonication (an increase in the number of scattering centers but a reduction in their size).