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This work focused on the synthesis, physicochemical characterization and biotechnological applications of new nanomaterials composed of sepiolite for gene transfer. Biohybrids were obtained of sepiolite nanofibers covered by nucleic acids (DNA and RNA molecules). They were characterized by novel techniques and procedures, identifying that the DNA gets adsorbed onto the surface of sepiolite nanofibers that were previously subjected to disaggregation procedures for a maximum fractionation. Such interaction is mediated by silanol groups evenly spaced on the outer surface of silicate particles, through electrostatic interactions, hydrogen bonds, cation bridges and Van der Waals forces. It was shown that sepiolite can be spontaneously internalized by eukaryotic cells in culture, and the mechanisms mediating such internalization were characterized, both endocytic and non-endocytic. Similarly, it was found that sepiolite nanofibers can be transferred between neighboring cells spontaneously, these particles displaying intrinsic fluorescence that facilitates its detection within the cell as gene transfer vector. The biohybrids were assayed with satisfactory results for the transfer of a plasmid DNA vector into cancer cell lines, and strategies were also established to significantly increase their transfection efficiency. These studies demonstrated for the first time the use of sepiolite nanofibers as a platform for transferring nucleic acids and potentially other types of molecules into mammalian cells, those molecules administered for functional or therapeutic purposes, among other applications. This work granted the Annual Award of the National Academy of Sciences of Cuba for the year 2016. [ABSTRACT FROM AUTHOR]

The ESCRT machinery (Endosomal Sorting Complex Required for Transport), consists of four multi-subunit complexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III, that play a role in the transport of ubiquitinated cargoes to intraluminal vesicles that bud from multivesicular bodies derived of endosomal compartments. These multivesicular bodies later fuse with lysosomes for degradation of the endocytosed cargoes or with the plasma membrane secreting intraluminal vesicles as a heterogeneous mixture of exosomes. Because Trypanosomatids, which are protozoan parasites that secrete extracellular vesicles and depends on endocytosis, here we analyzed the role of ESCRT complex in Trypanosoma cruzi, the agent of Chagas disease. Parasites expressing the Cas9 and T7 RNA polymerase were transfected with DNA fragments coding for two sgRNA that pair the VPS23 gene, a member of the ESCRT-I, together with DNA donor fragments to replace the entire gene by hygromycin and blasticidin resistance markers. Only one of the VPS23 alleles were replaced by one copy of the resistance markers, suggesting that VPS23 is essential for T. cruzi. Similar results were obtained for Leishmania mexicana. These partial knockouts significantly lower the receptor mediated endocytosis of transferrin, but not the fluid phase BSA-uptake in T. cruzi. In addition, they reduced the secretion of extracellular vesicles by epimastigotes. The partial knockout did not affect epimastigote proliferation and metacyclogenesis, and the resulting trypomastigotes were able to infect cells as the parental cell lines. We are currently analyzing whether the secretion of extracellular vesicles is also changed in trypomastigotes released from infected mammalian cells and the capacity of these parasites to promote infection. These results demonstrated the essential role of TcVPS23 by modulating endocytic and secretory activities in T. cruzi, and might be useful to understand the role of extracellular vesicles, know to play an important role in the parasite virulence. [ABSTRACT FROM AUTHOR]

La mastitis bovina provoca la inflamación de la glándula mamaria y es ocasionada principalmente por Staphylococcus aureus. Presenta una incidencia mayor durante el parto o después de la lactancia, periodos en que la prolactina (PRL) tiene función relevante, aunque se desconoce su participación en la infección. En este trabajo se evaluó in vitro, el efecto de la PRL bovina (1-50 ng/mL) sobre la endocitosis y proliferación intracelular de S. aureus (ATCC 27543) en células epiteliales mamarias bovinas (CEMB). Las CEMB tratadas durante 24 h con 5 ng/mL de bPRL incrementan ~3 veces la endocitosis de S. aureus, además de inducir la proliferación bacteriana. Este efecto fue revertido de manera específica tratando las CEMB con IgG anti-bPRL. Las CEMB tratadas con bPRL (5 ng/mL) presentaron mayor endocitosis de S. aureus (~3 veces), pero el número de células infectadas no se modificó. En conclusión, la presencia de bPRL favorece la endocitosis y la proliferación de S. aureus en las CEMB. [ABSTRACT FROM AUTHOR]