Hybrid scaffolds from natural and synthetic polymers have been widely used due to the complementary nature of their physical and biological properties. The aim of the present study, therefore, has been to analyze in vivo a bilayer scaffold of poly(lactide-co-glycolide) (PLGA)/fibrin electrospun membrane and fibrin hydrogel layer on a rat skin model. Fibroblasts were cultivated in the fibrin hydrogel layer and keratinocytes on the electrospun membrane to generate a skin substitute. The scaffolds without and with cells were tested in a full-thickness wound model in Wistar Kyoto rats. The histological results demonstrated that the scaffolds induced granulation tissue growth, collagen deposition and epithelial tissue remodeling. The wound-healing markers showed no difference in scaffolds when compared with the positive control. Activities of antioxidant enzymes were decreased concerning the positive and negative control. The findings suggest that the scaffolds contributed to the granulation tissue formation and the early collagen deposition, maintaining an anti-inflammatory microenvironment.
Background:
Mucopolysaccharidosis type I (MPS I) is an inherited disorder caused by α-L-iduronidase (IDUA) deficiency. The available treatments are not effective in improving all signs and symptoms of the disease.
Objective:
: In the present study, we evaluated the transfection efficiency of repeated intravenous administrations of cationic nanoemulsions associated with the plasmid pIDUA (containing IDUA gene).
Methods:
Cationic nanoemulsions were composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(amino[polyethylene glycol]-2000) (DSPE-PEG), 1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP), medium chain triglycerides, glycerol, and water and were prepared by high-pressure homogenization and were repeatedly administered to MPS I mice for IDUA production and gene expression.
Results:
A significant increase in IDUA expression was observed in all organs analyzed, and IDUA activity tended to increase with repeated administrations when compared to our previous report, when mice received a single administration of the same dose. In addition, GAGs were partially cleared from organs, as assessed through biochemical and histology analyzes. There was no presence of inflammatory infiltrate, necrosis, or signs of increase in apoptosis. Furthermore, immunohistochemistry for CD68 showed reduced presence of macrophage cells in treated than in untreated MPS I mice.
Conclusion:
These set of results suggest that repeated administrations can improve transfection efficiency of cationic complexes without significant increase in toxicity in the MPS I murine model.
In bacteria, the biosynthesis of the cofactor flavin adenine dinucleotide (FAD), important in many physiological responses, is catalyzed by the bifunctional enzyme FAD synthase (FADSyn) which converts riboflavin into FAD by both kinase and adenylylation activity. The in silico 3D structure of a putative FADSyn from Mycoplasma hyopneumoniae (MhpFADSyn), the etiological agent of enzootic pneumonia was already reported, nevertheless, the in vitro functional characterization was not yet demonstrated. Our phylogenetic analysis revealed that MhpFADSyn is close related to the bifunctional FADSyn from Corynebacterium ammoniagenes. However, only the domain related to adenylylation was assigned by InterPro database. The activity of MhpFADSyn was evaluated through in vitro enzymatic assays using cell extracts from IPTG-inducible heterologous expression of MhpFADSyn in Escherichia coli. The flavoproteins were analyzed by HPLC and results showed that IPTG-induced cell lysate resulted in the formation of twofold increased amounts of FAD if compared to non IPTG-induced cells. Consumption of riboflavin substrate was also threefold greater in IPTG-induced lysate compared to non IPTG-induced cell extract. Thus, the recombinant MhpFADSyn protein could be associated to FAD biosynthesis. These findings contribute to expand the range of potential drug targets in diseases control and unveil metabolic pathways that could be attribute to mycoplasmas.
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