Replication Protein A Presents Canonical Functions and Is Also Involved in the Differentiation Capacity of Trypanosoma cruzi

Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi

Protein encapsulation in SBA-15 with expanded pores

This work reports the encapsulation of proteins with different molecular weights into SBA-15 ordered mesoporous silica, a potential immunological adjuvant. The Human Gammaglobulin G (HGG) and Bovine Serum Albumin (BSA) proteins were incorporated into the mesoporous silica with expanded pores. A structure swelling agent, triisopropylbenzene (TIPB), was used in the synthesis process, promoting an increase of the average pore diameter and a more disordered pore network, as revealed by nitrogen adsorption isotherm (NAI) and small angle X-ray scattering (SAXS) data. SAXS measurements were also performed to obtain the overall size of the studied proteins. The results showed that both proteins have dimensions that would allow their encapsulation inside the pores of SBA-15. The HGG and BSA proteins were dissolved in phosphate buffered saline (PBS) solutions before encapsulation. It was evidenced the filling of the micropores by the PBS solution and a larger variation in pore volume and surface area for the material with higher mean pore diameter, which was also confirmed by the modeling of SAXS data. It was not observed any significant difference in the SAXS and NAI results of both proteins, indicating that the immunogens could be encapsulated in the silica macroporosity, obstructing the mesopore entrances. (C) 2016 Elsevier Inc. All rights reserved.

Large unilamellar vesicles as trehalose-stabilised vehicles forvaccines storage time and in vivo studies

Liposomes, as a pharmaceutical formulation must display a long shelf life. The recombinant heat-shock protein fromMycobacterium leprae (18-kDa hsp) or its N-acylated derivative, when entrapped within or externally associated with largeunilamellar vesicles, acts as a T-epitope source. Freeze-fracture electron microscopy shows unequivocally that trehaloseavoids aggregation and fusion of these vesicles. Formulations containing trehalose retained up to 98% of the entrappedprotein. The highest antibody level is obtained with formulations containing trehalose. The adjuvant effect depends on theliposomal membrane integrity.