In trypanosomatids, all mRNAs are processed via trans-splicing, although cis-splicing also occurs. In trans-splicing, a common small exon, the spliced leader (SL), which is derived from a small SL RNA species, is added to all mRNAs. Sm and Lsm proteins are core proteins that bind to U snRNAs and are essential for both these splicing processes. In this study, SmD3- and Lsm3-associated complexes were purified to homogeneity from Leishmania tarentolae. The purified complexes were analyzed by mass spectrometry, and 54 and 39 proteins were purified from SmD3 and Lsm complexes, respectively. Interestingly, among the proteins purified from Lsm3, no mRNA degradation factors were detected, as in Lsm complexes from other eukaryotes. The U1A complex was purified and mass spectrometry analysis identified, in addition to U1 small nuclear ribonucleoprotein (snRNP) proteins, additional co-purified proteins, including the polyadenylation factor CPSF73. Defects observed in cells silenced for U1 snRNP proteins suggest that the U1 snRNP functions exclusively in cis-splicing, although U1A also participates in polyadenylation and affects trans-splicing. The study characterized several trypanosome-specific nuclear factors involved in snRNP biogenesis, whose function was elucidated in Trypanosoma brucei. Conserved factors, such as PRP19, which functions at the heart of every cis-spliceosome, also affect SL RNA modification; GEMIN2, a protein associated with SMN (survival of motor neurons) and implicated in selective association of U snRNA with core Sm proteins in trypanosomes, is a master regulator of snRNP assembly. This study demonstrates the existence of trypanosomatid-specific splicing factors but also that conserved snRNP proteins possess trypanosome-specific functions.
Silk Fibroin (SF) has been extensively studied for various applications due to its impressive mechanical properties and biocompatibility. Recently, SF based-particles have been proposed as controlled drug delivery systems. A new and efficient method was developed to prepare SF nanoparticles (SF-NPs) by high pressure homogenization (HPH) emulsification, in oil-in-water emulsions (o/w). During the NPs production by HPH emulsification process, the secondary SF structure changed from random-coil conformation to a more stable structure, β-sheets. To improve even more the NPs stability over time the effect of various surfactants was studied, namely poloxamer 407, transcutol, tween 80 and sodium dodecyl sulfate, in which SF nanoemulsions with 1% of transcutol demonstrated lower diameters and better polydispersity values during the 4 weeks of evaluation. The drug incorporation efficiency and release of SF-NPs was assessed using orange IV dye as model-drug. The influence of a human protease (human neutrophil elastase) on orange IV release profile was also evaluated. The encapsulation of orange IV effectively stabilized the size and size distribution of the SF-NPs over time, being evident the conformational change to β-sheets. SF-NPs encapsulated with orange IV had a formation and encapsulation efficiency of 67% and 91%, respectively, with a controlled release over time. The stability and release profile induced by the SF-NPs enhances its potential for various applications, including biomedical.
In the present work we used sonochemically prepared proteinaceous BSA spheres as a novel RNA-delivery system. The preparation of RNA-loaded BSA spheres was accomplished using an environmental friendly method termed the "ultrasonic emulsification method". It was demonstrated that ultrasonic waves do not cause the RNA chains to degrade and the RNA molecules remain untouched. The BSA-RNA complex was successfully introduced into mammalian (human) U2OS osteosarcoma cells and Trypanosoma brucei parasites. Using PVA coating of the RNA-BSA spheres we have achieved a significant increase in the number of microspheres penetrating mammalian cells. The mechanism of RNA encapsulation and the structure of the RNA-BSA complex are reported.
Silk-based matrix was produced for delivery of a model anticancer drug, methotrexate (MTX). The calculation of net charge of silk fibroin and MTX was performed to better understand the electrostatic interactions during matrix formation upon casting. Silk fibroin films were cast at pH 7.2 and pH 3.5. Protein kinase A was used to prepare phosphorylated silk fibroin. The phosphorylation content of matrix was controlled by mixing at specific ratios the phosphorylated and unphosphorylated solutions. In vitro release profiling data suggest that the observed interactions are mainly structural and not electrostatical. The release of MTX is facilitated by use of proteolytic enzymes and higher pHs. The elevated β-sheet content and crystallinity of the acidified-cast fibroin solution seem not to favor drug retention. All the acquired data underline the prevalence of structural interactions over electrostatical interactions between methotrexate and silk fibroin.
Silk fibroin (SF) is a natural biopolymer that has been extensively studied in various applications due to its impressive mechanical properties and biocompatibility. Recently, SF‐based particles have been proposed as controlled drug delivery systems. A new and efficient method to prepare SF microemulsions (SF‐MEs) was developed by oil‐in‐water emulsions using high‐pressure homogenization to promote emulsification. During SF‐ME production, the secondary structure of SF changed to a more stable conformation (from random coil to β‐sheets), thus allowing the formation of small and stable (140.7 ± 1.9 nm; polydispersity index, 0.25) SF microparticles (SF‐MPs). The efficiency of SF‐MP formation was 60%. Orange IV was used as a model compound for incorporation and release studies, although its incorporation into the SF‐MEs significantly improved particle size and size distribution over at least 4 wk compared to traditional stabilizers (e.g., poloxamer 407, transcutol, Tween 80, and SDS). This should be a call of attention when using dyes as model compounds since they can influence particle properties and lead to misinterpretation of the results. Orange IV showed an incorporation efficiency of 91% and a controlled release over time. Stable SF‐MP formulations, further enhanced by orange IV incorporation, provide an innovative method with potential application in pharmaceutical development due to its associated high biocompatibility and release profile.
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